Relaxin (RLX) is a pleiotropic peptide hormone with marked renal vasodilatory actions which are physiologically important during pregnancy. RLX also has potent antifibrotic actions and is being tested therapeutically in various fibrotic diseases including chronic kidney disease (CKD). Since renal vasodilation may expose the glomerulus to increased blood pressure (PGC), which exacerbates progression of CKD, we assessed the glomerular hemodynamic actions of RLX when administered acutely (75 min; 0.89 µg/100g body weight/hr, iv) and chronically (1.5µg/100g body weight/hr, sc). Both acute and chronic RLX produced marked renal vasodilation and increased renal plasma flow (RPF) in euvolemic, anesthetized male rats. GFR also increased with RLX but the magnitude of the rise was much less than the increase in RPF, due to concomitant falls in filtration fraction (FF). The fall in FF was the result of significant falls in glomerular blood pressure (PGC) which occurred despite a slight rise in mean arterial blood pressure (MAP) with acute RLX, and no net change in MAP with chronic RLX. This fall in PGC occurred because of the "in-series" arrangement of the afferent and efferent arteriolar resistance vessels (RA and RE) which can regulate PGC independently of MAP. With both acute and chronic RLX RE relaxed to a greater extent than RA, thus producing falls in PGC. Based on this finding, RLX has a beneficial hemodynamic impact on the kidney, which together with the antifibrotic actions suggest a strong therapeutic potential for use in CKD.
Skeletal muscle insulin resistance is a hallmark of Type II Diabetes (T2DM) and may be exacerbated by protein modifications by methylglyoxal (MG), known as dicarbonyl stress. The glyoxalase enzyme system composed of glyoxalase 1/2 (GLO1/GLO2) is the natural defense against dicarbonyl stress, yet its protein expression, activity and regulation remain largely unexplored in skeletal muscle. Therefore, this study investigated dicarbonyl stress and the glyoxalase enzyme system in the skeletal muscle of subjects with T2DM (age: 56 ± 5 yrs; BMI: 32 ± 2 kg/m2) compared to lean healthy control subjects (LHC; age: 27 ± 1 yrs; BMI: 22 ± 1 kg/m2). Skeletal muscle biopsies obtained from the vastus lateralis at basal and insulin-stimulated states of the hyperinsulinemic (40 mU/m2/min) -euglycemic (5 mM) clamp were analyzed for proteins related to dicarbonyl stress and glyoxalase biology. At baseline, T2DM had increased carbonyl stress and lower GLO1 protein expression (-78.8%, p<0.05), which inversely correlated with BMI, percent body fat and HOMA-IR while positively correlating with clamp derived glucose disposal rates. T2DM also had lower NRF2 protein expression (-31.6%, p<0.05), which is a positive regulator of GLO1, while Keap1 protein expression, a negative regulator of GLO1, was elevated (207%, p<0.05). Additionally, insulin stimulation during the clamp had a differential effect on NRF2, Keap1 and MG-modified protein expression. These data suggest that dicarbonyl stress and the glyoxalase enzyme system are dysregulated in T2DM skeletal muscle and may underlie skeletal muscle insulin resistance. Whether these phenotypic differences contribute to the development of T2DM warrants further investigation.
The extracellular matrix (ECM) modulates brain maturation and plays a major role in regulating neuronal plasticity during critical periods of development. We examined: 1) whether there is a critical postnatal period of ECM expression in brainstem cardio-respiratory control regions; and 2) if the attenuated hypoxic ventilatory response (HVR) following neonatal sustained (5 days) hypoxia exposure (SH, 11% O2, 24hrs/day) is associated with altered ECM formation. The nucleus tractus solitarius (nTS), dorsal motor nucleus of the vagus (DMNV), the hypoglossal motor nucleus (XII), the cuneate nucleus (CN) and area postrema (AP) were immunofluorescently processed for aggrecan and Wisteria floribunda (WFA) agglutinin, a key proteoglycan of the ECM and the perineuronal net. From postnatal age 5 days (P5), aggrecan and WFA expression increased postnatally in all regions. Aggrecan expression in the nTS, a region that integrates and receives afferent inputs from the carotid body, increased abruptly between P10-15 followed by a distinct and transient plateau between P15-20. WFA expression in the nTS exhibited an analogous transient plateau, but it occurred earlier (plateauing between P10-P15). SH exposure between this period (P11-15) attenuated the HVR (assessed at P16) and increased aggrecan (but not WFA) expression in the nTS, DMNV and AP. An intracisternal micro-injection of chondroitinase ABC (ChABC), an enzyme that digests chondroitin sulfate proteoglycans, rescued the HVR and the increased aggrecan expression. These data indicate there are important stages of ECM formation that take place in key brainstem respiratory neural control regions that appear to be associated with a heightened vulnerability to hypoxia.
Recent studies demonstrate that maternal hypertension during pregnancy sensitizes an angiotensin (ANG) II-induced increase in blood pressure (BP) in adult male offspring that was associated with upregulation of mRNA expression of several renin-angiotensin-aldosterone system (RAAS) components and NADPH oxidase in the lamina terminalis (LT) and paraventricular nucleus (PVN). The purpose of the present study was to test whether there are sex differences in the maternal hypertension-induced sensitization of ANG II hypertension, and whether sex hormones are involved in the sensitization process. Male offspring of hypertensive dams showed an enhanced hypertensive response to systemic ANG II when compared to male offspring of normotensive dams and to female offspring of either normotensive or hypertensive dams. Castration did not alter the hypertensive response to ANG II in male offspring. Intact female offspring had no upregulation of RAAS components and NADPH oxidase in the LT and PVN. Whereas, ovariectomy (OVX) upregulated mRNA expression of several RAAS components and NADPH oxidase in these nuclei and induced a greater increase in the pressor response to ANG II in female offspring of hypertensive dams compared to female offspring of normotensive dams. This enhanced increase in BP was partially attenuated by E2 replacement in the OVX offspring of hypertensive dams. The results suggest that maternal hypertension induces a sex-specific sensitization of ANG II-induced hypertension and mRNA expression of brain RAAS and NADPH oxidase in offspring. Female offspring are protected from maternal hypertension-induced sensitization of ANG II hypertension and female sex hormones are partially responsible for this protective effect.
To assess the existence of central amino acid sensing systems in fish we carried out two experiments in rainbow trout. In the first one, we injected ICV two different branched-chain amino acids (BCAA), leucine and valine, and assessed food intake up to 48 h later. Leucine decreased and valine increased food intake. In a second experiment, 6h after similar ICV treatment we determined changes in parameters related to putative amino acid sensing systems. Amino acid sensing systems respond to leucine in hypothalamus and telencephalon, and to valine in telencephalon. The decreased food intake observed in fish treated ICV with leucine could relate to changes in mRNA abundance of hypothalamic neuropeptides (POMC, CART, NPY and AgRP). These in turn could relate to amino acid sensing systems present in the same area, related to BCAA and glutamine metabolism, as well as mechanistic target of rapamycin (mTOR), taste receptors and general control non-derepressible 2 (GCN2) kinase signalling. The treatment with valine did not affect amino acid sensing parameters in hypothalamus. These responses are comparable to those characterized in mammals. However, clear differences arise when comparing rainbow trout and mammals, in particular with respect to the clear orexigenic effect of valine, which could relate to the finding that valine partially stimulated two amino acid sensing systems in telencephalon. Another novel result is the clear effect of leucine on telencephalon, in which amino acid sensing systems, but not neuropeptides, were activated as in hypothalamus
Nitrite (NO2-) causes vasodilation in mammals due to the formation of (nitric oxide) NO by endogenous NO2- reduction in the vascular wall. In this study, we determined if a similar mechanism operates in amphibians. Dual-wire myography of the iliac artery from Rhinella marina showed that applied NO2- caused a concentration-dependent vasodilation in normoxia (21% O2; EC50 438 µM). Hypoxia (0.63% O2) significantly increased the maximal dilation to NO2- by 5 % (P = 0.0398). The addition of oxyhemoglobin significantly increased the EC50 (P = 0.0144; EC50 2236 µM), but did not affect the maximal vasodilation. In contrast, partially deoxygenated hemoglobin (90% desaturation) did not affect the EC50 (P = 0.1189) but significantly (P = 0.0012) increased the maximal dilation to NO2- by 11%. The soluble guanylyl cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) completely abolished the response to NO2- (P < 0.0001), and of the nitric oxide synthase inhibitors, only vinyl-L-NIO (P = 0.0028) significantly reduced the NO2- vasodilation. The xanthine oxidoreductase inhibitor, allopurinol (P = 0.927), the NO-scavenger C-PTIO (P = 0.478), and disruption of the endothelium (P = 0.094) did not affect the NO2- vasodilation. Incubation of iliac arteries with 1 mM NO2- did not a cause a change in cGMP concentration (P = 0.407). Plasma NO2- was found to be 0.86 ± 0.20 µmol.L-1, while nitrate (NO3-) was 19.55 ± 2.55 µmol.L-1. Both cygb and ngb mRNAs were expressed in the iliac artery and it is possible that these globins facilitate NO2- reduction in hypoxia. In addition, NO2- intracellular disproportionation processes could be important in the generation of NO from NO2-.
In pathological populations, elevated sympathetic activity is associated with increased activity of individual sympathetic neurons. We used custom action potential detection software to analyze multi-unit sympathetic activity in 18 normotensive pregnant women (third trimester; 33 ± 5 weeks) and 19 non-pregnant women at rest and a subset (10 and 13 respectively) during a cold pressor challenge. Although the number of action potentials per burst, and number of active amplitude based "clusters" were not different between groups the total number of sympathetic action potentials per minute was higher in pregnant women at rest. Individual clusters were active predominately once per burst suggesting they represent single neurons. Action potentials occurred in closer succession in normotensive pregnant (inter-spike interval 36 ± 10 ms) versus non-pregnant women (50 ± 27 ms; P<0.001) at rest. Pregnant women had a lower total peripheral resistance (11.7 ± 3.0 mmHg/L/min) than non-pregnant women (15.1 ± 2.7 mmHg/L/min; P<0.001) indicating a blunted neurovascular transduction. The cold pressor reduced the number of action potentials per burst in both groups due to shortening of the R-R interval in conjunction with increased burst frequency, total neural firing per minute was unchanged. Thus, elevated sympathetic activity during normotensive pregnancy is specific to increased incidence of multi-unit bursts. This is likely due to decreased central gating of burst output as opposed to generalized increases in central drive. These data also reinforce the concept that pregnancy appears to be the only healthy state of chronic sympathetic hyperactivity that we are aware of.
Moderate acute intermittent hypoxia (mAIH) elicits a form of respiratory motor plasticity known as phrenic long-term facilitation (pLTF). Preconditioning with modest protocols of chronic intermittent hypoxia enhances pLTF, demonstrating pLTF metaplasticity. Since "low dose" protocols of repetitive acute intermittent hypoxia show promise as a therapeutic modality to restore respiratory (and non-respiratory) motor function in clinical disorders with compromised breathing, we tested the hypothesis that preconditioning with a mild repetitive AIH (rAIH) protocol enhances pLTF and hypoglossal (XII) LTF, and whether the enhancement is regulated by glycolytic flux. In anesthetized, paralyzed and ventilated adult, male Lewis rats, mAIH (3, 5 minute episodes of 10% O2) elicited pLTF (pLTF at 60 min post-mAIH: 49 ± 5% baseline). rAIH pre-conditioning (10, 5 minute episodes of 11% O2/day, 5 min normoxic intervals, 3x per week, 4 weeks) significantly enhanced pLTF (100 ± 16% baseline). XII LTF was unaffected by rAIH. When glycolytic flux was inhibited with 2-deoxy-D-glucose (2-DG) administered via drinking water (~80mg/kg/day), pLTF returned to normal levels (58 ± 8% baseline); 2-DG had no effect on pLTF in normoxia pre-treated rats (59 ± 7% baseline). In ventral cervical spinal homogenates (C4/5), rAIH increased iNOS mRNA versus normoxic controls, an effect blocked by 2-DG. However, there were no detectable rAIH or 2-DG effects on several molecules associated with phrenic motor plasticity, including 5-HT2A, 5-HT7, BDNF, TrkB or VEGF mRNA. We conclude that modest but prolonged rAIH elicits pLTF metaplasticity, and that a drug (2DG) known to inhibit glycolytic flux blocks pLTF enhancement.
The recruitment of thermoeffectors, including thermoregulatory behavior, relative to changes in body temperature has not been quantified in humans. We tested the hypothesis that changes in skin blood flow, behavior, and sweating or metabolic rate are initiated with increasing changes in mean skin temperature (Tskin) in resting humans. While wearing a water perfused suit, twelve healthy young adults underwent heat (HEAT) and cold (COLD) stress that induced gradual changes in Tskin. Subjects controlled the temperature of their dorsal neck to their perceived thermal comfort. Thus, neck skin temperature provided an index of thermoregulatory behavior. Neck skin temperature, Tskin, core temperature (Tcore), metabolic rate, sweat rate, and non-glabrous skin blood flow were measured continually. Data were analyzed using segmental regression analysis, providing an index of thermoeffector activation relative to changes in Tskin. In HEAT, increases in skin blood flow were observed with the smallest elevations in Tskin (P<0.01). Thermal behavior was initiated with an increase in Tskin of 2.4 ± 1.3°C (P=0.04), while sweating was observed with further elevations in Tskin (3.4 ± 0.5°C, P=0.04), which coincided with increases in Tcore (P=0.98). In COLD, reductions in skin blood flow occurred with the smallest decrease in Tskin (P<0.01). Thermal behavior was initiated with a Tskin decrease of 1.5 ± 1.3°C, while metabolic rate (P=0.10) and Tcore (P=0.76) did not change throughout. These data indicate that autonomic and behavioral thermoeffectors are recruited in coordination with one another and likely in an orderly manner relative to the comparative physiological cost.
Chronic stress detrimentally affects animal health and homeostasis, with somatic growth, and thus skeletal muscle, being particularly affected. A detailed understanding of the underlying endocrine and molecular mechanisms of how chronic stress affects skeletal muscle growth remains lacking. To address this issue, the present study assessed primary (plasma cortisol), secondary (key components of the GH/IGF system, muscular proteolytic pathways and apoptosis), and tertiary (growth performance) stress responses in fine flounder (Paralichthys adspersus) exposed to crowding chronic stress. Levels of plasma cortisol, glucocorticoid receptor 2 (gr2), and its target genes (klf15 and redd1) mRNA increased significantly only at 4wk of crowding (p<0.05). The components of the GH/IGF system, including ligands, receptors, and their signaling pathways, were significantly downregulated at 7wk of crowding (p<0.05). Interestingly, chronic stress upregulated the ubiquitin-proteasome pathway and the intrinsic apoptosis pathways at 4wk (p<0.01), whereas autophagy was only significantly activated at 7wk (p<0.05), meanwhile the ubiquitin-proteasome and the apoptosis pathways returned to control levels. Overall growth was inhibited in fish in the 7wk chronic stress trial (p<0.05). In conclusion, chronic stress directly affects muscle growth and downregulates the GH/IGF system, an action through which muscular catabolic mechanisms are promoted by two different and non-overlapping proteolytic pathways. These findings provide new information on molecular mechanisms involved in the negative effects that chronic stress has on muscle anabolic/catabolic signaling balance.
Females born growth restricted have poor adult bone health. Stress exposure during pregnancy increases risk of pregnancy complications. We determined whether maternal stress exposure in growth restricted females exacerbates long-term maternal and offspring bone phenotypes. On gestational day 18, bilateral uterine vessel ligation (Restricted) or sham (Control) surgery was performed in WKY rats. At 4 months, Control and Restricted females were mated and allocated to Unstressed or Stressed pregnancies. Stressed pregnancies had physiological measurements performed, Unstressed females were not handled. After birth, mothers were aged to 13 months. Second-generation (F2) offspring generated 4 experimental groups; Control-Unstressed, Restricted-Unstressed, Control-Stressed and Restricted-Stressed. F2 offspring were studied at postnatal day 35 (PN35), 6, 12 and 16months. Peripheral quantitative computed tomography (pQCT) was performed on maternal and F2 offspring femurs. Restricted females, irrespective of stress during pregnancy, had decreased endosteal circumference, bending strength and increased osteocalcin concentrations after pregnancy at 13 months. F2 offspring of Stressed mothers were born lighter. F2 male offspring from Stressed pregnancies had decreased trabecular content at 6 months and decreased endosteal circumference at 16 months. F2 female offspring from growth restricted mothers had reduced cortical thickness at PN35 and reduced endosteal circumference at 6 months. At 12 months, females from Unstressed Restricted and Stressed Control mothers had decreased trabecular content. Low birth weight females had long-term bone changes, highlighting programming effects on bone health. Stress during pregnancy did not exacerbate these programmed effects. Male and female offspring responded differently to maternal growth restriction and stress, indicating gender-specific programming effects.
Systemic hypertension, preeclampsia and pulmonary arterial hypertension (PAH) are diseases of high blood pressure in the systemic or pulmonary circulation. Beyond the well-defined contribution of more traditional pathophysiological mechanisms, such as changes in the renin-angiotensin-aldosterone system, to the development of these hypertensive disorders, there is substantial clinical evidence supporting an important role for inflammation and immunity in the pathogenesis of each of these three conditions. Over the last decade, work in small animal models, bearing targeted deficiencies in specific cytokines or immune cell subsets, has begun to clarify the immune-mediated mechanisms that drive changes in vascular structure and tone in hypertensive disease. By summarizing the clinical and experimental evidence supporting a contribution of the immune system to systemic hypertension, preeclampsia and PAH, the current review highlights the cellular and molecular pathways that are common to all three hypertensive disorders. These mechanisms are centred on an imbalance in CD4+ helper T cell populations, defined by excessive Th17 responses and impaired Treg activity, as well as the excessive activation or impairment of additional immune cell types, including macrophages, dendritic cells, CD8+ T cells, B cells and Natural Killer cells. The identification of common immune mechanisms in systemic hypertension, preeclampsia and PAH raises the possibility of new therapeutic strategies that target the immune component of hypertension across multiple disorders.
Our previous study demonstrated that connexin (Cx)43 participates in the regulation of vascular permeability in severe sepsis. Whether OPN is involved in Cx43 regulation of vascular permeability after sepsis and the relationship with tight-junction proteins are not clear. In this study, we investigated the role of zo-1 and clauidn-5 in Cx43 regulation of vascular permeability and its relationship to OPN. The expression of zo-1 and claudin-5 were decreased in CLP rats and LPS-treated pulmonary vein VECs. Cx43 over-expressed lentivirus induced the degradation of zo-1 and claudin-5, while Cx43 RNAi lentivirus abrogate the degradation of zo-1 and cludin-5 induced by LPS. The vascular permeability and expression of OPN were significantly increased in CLP rats and LPS-treated pulmonary vein VECs. Silencing OPN by OPN RNAi lentivirus inhibited vascular hyper-permeability induced by LPS. Over-expressed Cx43 lentivirus increased the expression of OPN and vascular permeability, and down-regulated the expressions of zo-1 and claudin-5 in pulmonary vein VECs. Silencing OPN inhibited the effects of Cx43 over-expressed lentivirus on down-regulation of zo-1 and claudin-5 and vascular hyper-permeability in pulmonary vein VECs. Transfection of specific double-stranded RNA targeting to β-catenin and T-cell factor-4 (Tcf-4) abolished the up-regulation of OPN induced by Cx43 over-expression. These results suggest that OPN participates in the regulation of vascular permeability by Cx43 after sepsis. Cx43 up-regulation of OPN is via Tcf-4/β-catenin transcription pathway, OPN increases vascular permeability by decreasing the expressions of the tight junction proteins zo-1 and claudin-5.
Perinatal exposures are associated with altered risks of childhood allergy. Human studies and our previous work suggest restricted growth in utero (IUGR) is protective against allergic disease. The mechanisms are not clearly defined but reduced fetal abundance and altered metabolism of methyl donors are hypothesized as possible underlying mechanisms. We therefore examined whether late gestation maternal dietary methyl donor and co-factor supplementation of the placentally-restricted (PR) sheep pregnancy would reverse allergic protection in progeny. Allergic outcomes were compared between progeny from control pregnancies (CON, n=49), from PR pregnancies without intervention (PR, n=28), and from PR pregnancies where the dam was fed a methyl donor plus co-factor supplement from day 120 of pregnancy until delivery (PR+METHYL, n=25). Both PR and PR+METHYL progeny were smaller than CON; supplementation did not alter birth size. PR was protective against cutaneous hypersensitivity responses to ovalbumin (OVA, P < 0.01 in singletons). Cutaneous hypersensitivity responses to OVA in PR+METHYL progeny were intermediate to and not different from responses of CON and PR sheep. Cutaneous hypersensitivity responses to house dust mite did not differ between treatments. In singleton progeny, upper dermal mast cell density was greater in PR+METHYL than PR or CON (each P < 0.05). The differences in the cutaneous allergic response were not explained by treatment effects on circulating immune cells or antibodies. Our results suggest that mechanisms underlying in utero programming of allergic susceptibility by IUGR and methyl donor availability may differ, and imply that late gestation methyl donor supplementation may increase allergy risk.
The carotid body chemoreceptors are activated during hypoglycemia and contribute to glucoregulation during prolonged exercise in dogs. Low dose intravenous infusions of dopamine have been shown to blunt the activation of the carotid body chemoreceptors during hypoxia. Therefore, we tested the hypotheses that dopamine would blunt glucoregulatory responses and attenuate plasma glucose during prolonged aerobic exercise in healthy participants. Twelve healthy participants completed two randomized exercise sessions at 65% peak oxygen consumption for up to 120 minutes. Saline was infused during one exercise session and dopamine (2 g/kg/min) was infused during the other session. Arterial plasma glucose, growth hormone, glucagon, cortisol, norepinephrine, and epinephrine were measured every 10 minutes. Exercise duration during dopamine infusion was 107 ± 6 minutes and 119 ± 0.8 minutes during saline infusion. Glucose area under the curve during exercise was lower during dopamine (9,821 686 vs. 11,194 395 a.u.; P = 0.016). The ratio of circulating growth hormone to glucose and the ratio of glucagon to glucose were greater during dopamine (P = 0.045 and P = 0.037, respectively). These results indicate that the infusion of dopamine during aerobic exercise impairs glucoregulation. This suggests that the carotid body chemoreceptors contribute to glucoregulation during prolonged exercise in healthy exercise trained humans.
Mitochondria are hypothesized to display a biphasic response to reactive oxygen species (ROS) exposure. In this study, we evaluated the time course changes in mitochondrial performance and oxidative stress in house mice following X-irradiation. Forty-eight mice were equally divided among six groups, including a non-irradiated control and 5 experimental groups that varied in time between X-ray exposure and euthanasia (1 hour, 1, 4, 7, or 10 days after X-irradiation). We measured parameters associated with mitochondrial respiratory function and ROS emission from isolated liver and skeletal muscle mitochondria and levels of oxidative damage and antioxidants in liver, skeletal muscle, and heart tissues. Mitochondrial function dropped initially after X-irradiation but recovered quickly and was elevated 10 days after the exposure. Hydrogen peroxide production, lipid peroxidation, and protein carbonylation showed inverse U-shaped curves, with levels returning to control or lower than control 10 days after X-irradiation. Enzymatic antioxidants and markers for mitochondrial biogenesis exhibited tissue specific response after irradiation. These data provide the first chronological description of the mitohormetic response after a mild dosage of irradiation and highlights the protective response that cells display to ROS exposure. This study also provides valuable information and application for future mitochondrial and oxidative stress studies in numerous physiological settings.
Compared to fishmeal and fish oil, plant ingredients differ not only in the protein content, amino acid and fatty acid profiles but are also devoid of cholesterol, the major component of cell membrane and precursor of several bioactive compounds. Based on these nutritional characteristics, plant-based diets can affect fish physiology and cholesterol metabolism. In order to investigate the mechanisms underlying cholesterol homeostasis, rainbow trout were fed from 1g body weight during six months with a totally plant-based diet (V), a marine diet (M) and a marine restricted diet (MR) with feed intake adjusted to that of the V group. The expression of genes involved in cholesterol synthesis, esterification, excretion, bile acid synthesis and cholesterol efflux was measured in liver. Results showed that genes involved in cholesterol synthesis were up-regulated in trout fed V diet whereas expression of genes related to bile acid synthesis (cyp7a1) and cholesterol elimination (abcg8) were reduced. Feeding trout with V diet also enhanced the expression of srebp-2 while reducing that of lxrα and miR-223. Overall, these data suggested that rainbow trout coped with the altered nutritional characteristics and absence of dietary cholesterol supply by increasing cholesterol synthesis and limiting cholesterol efflux through molecular mechanisms involving at least srebp-2, lxrα and miR-223. However, plasma and body cholesterol levels in trout fed V diet were lower compared to fish fed marine diet raising the question of the role of cholesterol in the negative effect of plant based-diet on growth.
Dysregulation of sodium (Na+) balance is a major cause of hypertensive cardiovascular disease. The current dogma is that interstitial Na+ readily equilibrates with plasma and that renal excretion and reabsorption is sufficient to regulate extracellular fluid volume and control blood pressure. These ideas have been recently challenged by the discovery that Na+ accumulates in tissues without commensurate volume retention and activates immune cells, leading to hypertension and autoimmune disease. However, objections have been raised to this new paradigm, with some investigators concerned about where and how salt is stored in tissues. Further concerns also include how Na+ is mobilized from tissue stores, and how it interacts with various organ systems to cause hypertension and end-organ damage. This review assesses these two paradigms of Na+ regulation in the context of inflammation-mediated hypertension and cardiovascular disease pathogenesis. I also highlight future perspectives and important gaps in our understanding of how Na+ is linked to inflammation and hypertension. Understanding mechanisms of salt and body fluid regulation is the sine qua non of research efforts to identify therapeutic targets for hypertension and cardiovascular disease.
This study aimed to examine, at the level of the active muscles, whether the plateau in oxygen (O2) extraction normally observed near the end of a ramp incremental (RI) exercise test to exhaustion, is caused by the achievement of an upper limit in O2 extraction. 11 healthy men (27.3 ± 3.0 years; 81.6 ± 8.1 kg; 183.9 ± 6.3 cm) performed a RI cycling test to exhaustion. O2 extraction of the vastus lateralis (VL) was measured continuously throughout the test using the near-infrared spectroscopy (NIRS)-derived deoxygenated hemoglobin [HHb] signal. A leg blood flow occlusion was performed at rest (LBFOCC 1) and immediately post the RI test (LBFOCC 2). The [HHb] values during the resting occlusion (108.1 ± 21.7%) (LBFOCC 1) and the peak values during exercise (100 ± 0%) ([HHb]PLATEAU) were significantly greater than those observed at baseline (bsln) (0.84 ± 10.6% at bsln 1 and 0 ± 0% at bsln 2) (p < 0.05). No significant difference was found between LBFOCC 1 and [HHb]PLATEAU (p > 0.05) nor between the bsln measures (p > 0.05). [HHb] values at LBFOCC 2 (130.5 ± 19.7%) were significantly greater than all other time points (p < 0.05). These results support the existence of an O2 extraction reserve in the VL muscle at the end of a RI cycling test and suggest that the observed plateau in the [HHb] signal towards the end of a RI test is not representative of an upper limit in O2 extraction.
The roles of nitric oxide synthase (NOS), reactive oxygen species (ROS) and angiotensin II type 1 receptor (AT1R) activation in regulating cutaneous vasodilation and sweating during prolonged (≥60 min) exercise are currently unclear. Moreover, it remains to be determined whether fluid replacement (FR) modulates the above thermoeffector responses. To investigate, eleven young men completed 90-min of continuous moderate intensity (46% of VO2peak) cycling performed at a fixed rate of metabolic heat production of 600 W (No-FR condition). On a separate day, participants completed a second session of the same protocol while receiving FR to offset sweat losses (FR condition). Cutaneous vascular conductance (CVC) and local sweat rate (LSR) were measured at four intradermal microdialysis forearm sites perfused with: (1) lactated Ringer (Control); (2) 10 mM NG-nitro-L-arginine methyl ester (LNAME, NOS inhibition); (3) 10 mM ascorbate (non-selective anti-oxidant); or (4) 4.34 nM Losartan (AT1R inhibition). Relative to Control (71% CVCmax at both time points), CVC with Ascorbate (80% and 83% CVCmax) was elevated at 60- and 90-min of exercise during FR (both P<0.02) but not at any time during No-FR (all P>0.31). In both conditions, CVC was reduced at end exercise with LNAME (60% CVCmax; both P<0.02), but was not different relative to Control at the Losartan site (76% CVCmax; both P>0.19). LSR did not differ between sites in either condition (all P>0.10). We conclude that NOS regulates cutaneous vasodilation but not sweating, irrespective of FR, and that ROS influence cutaneous vasodilation during prolonged exercise with FR.
This study tested the hypothesis that sacral neuromodulation, i.e. electrical stimulation of afferent axons in sacral spinal root, can block pudendal afferent inhibition of the micturition reflex. In α-chloralose anesthetized cats, pudendal nerve stimulation (PNS) at 3-5 Hz was used to inhibit bladder reflex activity while the sacral S1 or S2 dorsal root was stimulated at 15-30 Hz to mimic sacral neuromodulation and to block the bladder inhibition induced by PNS. The intensity threshold (T) for PNS or S1/S2 dorsal root stimulation (DRS) to induce muscle twitch of anal sphincter or toe was determined. PNS at 1.5-2T intensity inhibited the micturition reflex by significantly (p<0.01) increasing bladder capacity to 150-170% of control capacity. S1 DRS alone at 1-1.5T intensity did not inhibit bladder activity, but completely blocked PNS inhibition and restored bladder capacity to control level. At higher intensity (1.5-2T), S1 DRS alone inhibited the micturition reflex and significantly increased bladder capacity to 135.8±6.6% of control capacity. However, the same higher intensity S1 DRS applied simultaneously with PNS, suppressed PNS inhibition and significantly (p<0.01) reduced bladder capacity to 126.8±9.7% of control capacity. S2 DRS at both low (1T) and high (1.5-2T) intensity failed to significantly reduce PNS inhibition. PNS and S1 DRS did not change the amplitude and duration of micturition reflex contractions, but S2 DRS at 1.5-2T intensity doubled the duration of the contraction and increased bladder capacity. These results are important for understanding the mechanisms underlying sacral neuromodulation of non-obstructive urinary retention in Fowler's syndrome.
The three AKT kinases are related proteins that are essential for normal growth and metabolic regulation, and are implicated as key signaling mediators in many physiological and patho-physiological processes. Each AKT is activated by common biochemical signals that act downstream of growth factor and hormone receptors via phosphatidylinositol-3 kinase, and each controls several downstream pathways. The importance of AKT actions in human physiology is strengthened by the rarity of modifying mutations in their genes, and by the devastating impact caused by these mutations on growth and development, and in disorders such as cancer. Recent advances in genomics present unique opportunities for enhancing our understanding of human physiology and disease predisposition through the lens of population genetics, and the availability of DNA sequence data from 60,706 people in the Exome Aggregation Consortium has prompted this analysis. Results reveal a cohort of potential missense and other alterations in the coding regions of each AKT gene, but with nearly all changes being uncommon. The total number of different alleles per gene varied over an ~3-fold range, from 52 for AKT3 to 158 for AKT2, with variants distributed throughout all AKT protein domains. Previously characterized disease-causing mutations were found rarely in the general population. In contrast, a fairly prevalent amino acid substitution in AKT2 appears linked to increased predisposition for type-2 diabetes. Further analysis of variant AKT molecules as identified here will provide opportunities to understand the intricacies of AKT signaling and actions at a population level in human physiology and pathology.
Aim: Blood lactate increases during incremental exercise at high-intensity workloads and limited exercise capacity is a characteristic of obese animals. This study examined whether blood lactate changes in response to incremental exercise is disrupted in obese animals. Muscular and hepatic proteins that are critical in lactate metabolism were also investigated. Methods: Rats were randomized to either standard chow (control) or high fat diet (HFD) groups. All animals underwent an incremental treadmill test after 14 weeks of diet intervention. Blood lactate levels were measured before and after the treadmill test. Activities of mitochondrial oxidative phosphorylation and glycolysis were examined in muscle tissues. Proteins in the liver and skeletal muscles that participate in the turnover of blood lactate were determined by Western blot. Results: Running time in the incremental treadmill test decreased in the HFD group, and blood lactate accumulated faster in these animals than in the control group. Animals with HFD had a decreased level of hepatic monocarboxylate transporter 2, the protein responsible for blood lactate uptake in the liver. Skeletal muscles of animals with HFD showed greater glycolytic activity and decreased content of lactate dehydrogenase B, which converts lactate to pyruvate. Conclusion: Blood lactate accumulated faster during incremental exercise in obese animals and associated with their decreased exercise performance. Changes in the metabolic pattern of muscles and changes of liver and muscle proteins associated with lactate utilization likely contribute to the abnormal response of blood lactate to incremental exercise in obese animals.
Ghrelin secretion has been associated with increased caloric intake and adiposity. The expressions of ghrelin and its receptor (GHS-R1a) in pancreas have raised the interest about the role of ghrelin in glucose homeostasis. Most of the studies showed that ghrelin promoted hyperglycaemia and inhibited insulin secretion. This raised the interest in using GHS-R1a antagonists as therapeutic targets for type 2 diabetes. Available data of GHS-R antagonists are on a short-term basis. Moreover, the complexity of GHS-R1a signalling makes it difficult to understand the mechanism of action of GHS-R1a antagonists. This study examined the possible effects of long-term treatment with a GHS-R1a antagonist, [D-Lys3]-GHRP-6 on glucose homeostasis, food intake and indirect calorimetric parameters in non-obese diabetic MKR mice. Our results showed that [D-Lys3]-GHRP-6 (200nmol/mouse), reduced pulsatile growth hormone secretion and body fat mass as expected, but worsened glucose and insulin intolerances and increased cumulative food intake unexpectedly. In addition, a significant increase in blood glucose and decreases in plasma insulin and c-peptide levels were observed in MKR mice following long-term [D-Lys3]-GHRP-6 treatment, suggested a direct inhibition of insulin secretion. Immunofluorescence staining of pancreatic islets showed a proportional increase in somatostatin positive cells and a decrease in insulin positive cells in [D-Lys3]-GHRP-6 treated mice. Furthermore, [D-Lys3]-GHRP-6 stimulated food intake on long-term treatment via reduction of POMC gene expression and antagonized GH secretion via reduced GHRH gene expression in hypothalamus. These results demonstrate that [D-Lys3]-GHRP-6 is not completely opposite to ghrelin and may not be a treatment option for type 2 diabetes.
Background: Obesity is a common co-morbidity of COPD and has been associated with worse outcomes. However, it is unknown whether the interaction between obesity and COPD modulates diaphragm shape and consequently its function. The body mass index has been used as a correlate of obesity. We tested the hypothesis that the shape of the diaphragm muscle and size of the ring of its insertion in Non-COPD and COPD subjects are modulated by BMI. Methods: We recruited 48 COPD patients with post-bronchiodilator FEV1/FVC < 0.7 and 29 age-matched smoker/ex-smoker control (Non-COPD) subjects, who underwent chest CT at lung volumes ranging from FRC to TLC. We then computed maximum principle diaphragm curvature in the midcostal region of the left hemidiaphragm at end of inspiration during quiet breathing (EI) and at total lung capacity (TLC). Results: The radius of maximum curvature of diaphragm muscle increased with BMI in both COPD and Non-COPD subjects. The size of diaphragm ring of insertion on the chest wall also increased significantly with increasing BMI. Surprisingly, COPD severity did not appear to cause significant alteration in diaphragm shape except in normal weight subjects at TLC. Conclusion: Our data uncovered important factors such as BMI, the size of the diaphragm ring of insertion, and disease severity that modulate the structure of the ventilatory pump in Non-COPD and COPD subjects.
The ability of many reptilian hemoglobins (Hbs) to form high-molecular weight polymers, albeit known for decades, has not been investigated in detail. Given that turtle Hbs often contain a high number of cysteine (Cys), potentially contributing to the red blood cell defense against reactive oxygen species, we have examined whether polymerization of Hb could occur via intermolecular disulfide bonds in red blood cells of freshwater turtle Trachemys scripta, a species that is highly tolerant of hypoxia and oxidative stress. We find that one of the two Hb isoforms of the hemolysate, HbA, is prone to polymerization in vitro into linear flexible chains of different size that are visible by electron microscopy, but not the HbD isoform. Polymerization of purified HbA is favored by hydrogen peroxide, a main cellular reactive oxygen species and a thiol oxidant, and inhibited by thiol reduction and alkylation, indicating that HbA polymerization is due to disulfide bonds. By using mass spectrometry, we identify Cys5 of the αA subunit of HbA as specifically responsible for forming disulfide bonds between adjacent HbA tetramers. Polymerization of HbA does not affect oxygen affinity, cooperativity and sensitivity to the allosteric cofactor ATP, indicating that HbA is still fully functional. Polymers also form in T. scripta blood after exposure to anoxia but not normoxia, indicating that they are of physiologically relevance. Taken together, these results show that HbA polymers may form during oxidative stress and that Cys5αA of HbA is a key element of the antioxidant capacity of turtle red blood cells.
Vital parameters of living organisms exhibit circadian rhythmicity. Although rats are nocturnal animals, most of the studies involving rats are performed during the day. The objective of this study was to examine the circadian variability of the body temperature responses to methamphetamine (Meth). Body temperature was recorded in male Sprague-Dawley rats received Meth (1 or 5 mg/kg) or saline at 10am or 10pm. The baseline body temperature at night was 0.8ºC higher than during the day. Both during the day and at night 1 mg/kg of Meth induced monophasic hyperthermia. However, the maximal temperature increase at night was 50% smaller than during the daytime. Injection of 5 mg/kg of Meth during the daytime caused a delayed hyperthermic response. In contrast, the same dose at night produced responses with a tendency towards a decrease of body temperature. Using mathematical modeling, we previously showed that the complex dose-dependence of the daytime temperature responses to Meth results from an interplay between inhibitory and excitatory drives. Here, using the model, we explain the suppression of the hyperthermia in response to Meth at night. First, we found that the baseline activity of the excitatory drive is greater at night. It appears partially saturated, and, thus, is additionally activated by Meth to a lesser extent. Therefore, the excitatory component causes lesser hyperthermia or becomes overpowered by the inhibitory drive in response to the higher dose. Second, at night the injection of Meth results in reduction of the equilibrium body temperature leading to gradual cooling counteracting hyperthermia.
Muscle contraction requires the physiology to adapt rapidly to meet the surge in energy demand. To investigate the shift in metabolic control, especially between oxygen and metabolism, researchers often depend on near infrared spectroscopy (NIRS) to measure non-invasively the tissue O2. Because NIRS detects the overlapping myoglobin (Mb) and hemoglobin (Hb) signals in muscle, interpreting the data as an index of cellular or vascular O2 requires deconvoluting the relative contribution. Currently, many in the NIRS field ascribe the signal to Hb. In contrast, 1H NMR has only detected the Mb signal in contracting muscle, and comparative NIRS and NMR experiments indicate a predominant Mb contribution. The present study has examined the question of the NIRS signal origin by measuring simultaneously the 1H, 31P NMR, and NIRS signals in finger flexor muscles during the transition from rest to contraction, recovery, ischemia, and reperfusion. The experiment results confirm a predominant Mb contribution to the NIRS signal from muscle. Given the NMR and NIRS corroborated changes in the intracellular O2, the analysis shows that at the onset of muscle contraction, O2 declines immediately and reaches new steady states as contraction intensity rises. Moreover, lactate formation increases even under quite aerobic condition.
Care of premature infants often requires parental and caregiver separation particularly during hypoxic and hypothermic episodes. We have established a neonatal rat model of human prematurity involving maternal-neonatal separation and hypoxia with spontaneous hypothermia prevented with external heat. Adults previously exposed to these neonatal stressors show a sex difference in the insulin and glucose response to arginine stimulation suggesting a state of insulin resistance. The current study used this cohort of adult rats to evaluate insulin resistance (Homeostatic Model Assessment of Insulin Resistance [HOMA-IR]), plasma adipokines (reflecting insulin resistance states), and testosterone. The major findings were that daily maternal-neonatal separation led to an increase in body weight and HOMA-IR in adult male and female rats and increased plasma leptin in adult male rats only; neither prior neonatal hypoxia (without or with body temperature control) nor neonatal hypothermia altered subsequent adult HOMA-IR or plasma adiponectin. Adult male-female differences in plasma leptin were lost with prior exposure to neonatal hypoxia or hypothermia; male-female differences in resistin were lost in the adults with prior neonatal hypoxia allowing spontaneous hypothermia Exposure of neonates to daily hypoxia while preventing spontaneous hypothermia led to a decrease in plasma testosterone in adult male rats. We conclude that neonatal stressors result in subsequent adult sex-dependent increases in insulin resistance and adipokines, and that our rat model of prematurity with hypoxia with the prevention of hypothermia alters adult testosterone dynamics.
In addition to their intended clinical actions, all general anesthetic agents in common use have detrimental intra- and post-surgical side effects on organs and systems including the heart. The major cardiac side effect of anesthesia is bradycardia, which increases the probability of insufficient systemic perfusion during surgery. These side effects also occur in all vertebrate species so far examined, but the underlying mechanisms are not clear. The zebrafish heart is a powerful model for studying cardiac electrophysiology, employing the same pacemaker system and neural control as do mammalian hearts. In this study isolated zebrafish hearts were significantly bradycardic during exposure to the vapor anesthetics sevoflurane (SEVO), desflurane (DES) and isoflurane (ISO). Bradycardia induced by DES and ISO continued during pharmacological blockade of the intracardiac portion of the autonomic nervous system, but the chronotropic effect of SEVO was eliminated during blockade. Bradycardia evoked by vagosympathetic nerve stimulation was augmented during DES and ISO exposure; nerve stimulation during SEVO exposure had no effect. Together these results support the hypothesis that the cardiac chronotropic effect of SEVO occurs via a neurally mediated mechanism while DES and ISO act directly upon cardiac pacemaker cells via an as yet unknown mechanism.
Funny current (If), formed by hyperpolarization-activated HCN channels, is supposed to be crucial for membrane clock regulating the cardiac pacemaker mechanism. We examined the presence and activity of HCN channels in the brown trout (Salmo trutta fario) sinoatrial (SA) pacemaker cells, and their putative role in heart rate (fH) regulation. Six HCN transcripts (HCN1, HCN2a, HCN2ba, HCN2bb, HCN3 and HCN4) were expressed in the brown trout heart. The total HCN transcript abundance was 4.0 and 4.9 times higher in SA pacemaker tissue than in atrium and ventricle, respectively. In the SA pacemaker HCN3 and HCN4 were the main isoforms representing 35.8±2.7% and 25.0±1.5%, respectively, of the total HCN transcripts. Only a small If with a mean current density of -1.2±0.37 pA/pF at -140 mV was found in 4 pacemaker cells out of 16 spontaneously beating cells examined, despite the optimization of recording conditions for If activity. If was not found in any of the 24 atrial myocytes and 21 ventricular myocytes examined. HCN4 co-expressed with MiRP1 β-subunit in CHO cells, generated large If currents. In contrast, HCN3 (+MiRP1) failed to produce If in the same expression system. Cs+ (2 mM), which blocked 84±12% of the native If, reversibly reduced fH 19.2±3.6% of the excised multicellular pacemaker tissue from 53±5 to 44±5 beats per minute (P<0.05). However, this effect was probably due to the reduction of IKr, which was also inhibited (63.5±4.6%) by Cs+. These results strongly suggest that fH regulation in the brown trout heart is largely independent on If.
Cardiovascular adjustments to exercise, resulting in increased blood pressure (BP) and heart rate (HR), occur in response to activation of several neural mechanisms - the exercise pressor reflex, central command, and the arterial baroreflex. Neural inputs from these feedback and feedforward mechanisms integrate in the cardiovascular control centers in the brain stem, and modulate sympathetic and parasympathetic neural outflow, resulting in the increased BP and HR observed during exercise. Another specific consequence of the central neural integration of these inputs during exercise is increased sympathetic neural outflow directed to the kidneys, causing renal vasoconstriction, a key reflex mechanism involved in blood flow redistribution during increased skeletal muscle work. Studies in humans have shown that muscle mechanoreflex activation inhibits cardiac vagal outflow, decreasing the sensitivity of baroreflex control of HR. Metabolite sensitization of muscle mechanoreceptors can lead to reduced sensitivity of baroreflex control of HR, with thromboxane being one of the metabolites involved, via greater inhibition of cardiac vagal outflow without affecting baroreflex control of BP or baroreflex resetting. Muscle mechanoreflex activation appears to play a predominant role in causing renal vasoconstriction, both in isolation and in the presence of local metabolites. Limited investigations in older adults and patients with cardiovascular-related disease have provided some insight into how the influence of muscle mechanoreflex activation on baroreflex function and renal vasoconstriction is altered in these populations. However, future research is warranted to better elucidate the specific effect of muscle mechanoreflex activation on baroreflex and neurovascular responses with aging and cardiovascular-related disease.
A reduction in central blood volume can lead to cardiovascular decompensation (i.e., failure to maintain blood pressure). Cooling the forehead and cheeks using ice water raises blood pressure. Therefore, face cooling (FC) could be used to mitigate decreases in blood pressure during central hypovolemia. Purpose: We tested the hypothesis that FC during central hypovolemia induced by lower body negative pressure (LBNP) would increase blood pressure. Methods: Ten healthy participants (22±2 years, 3 women) completed two randomized LBNP trials on separate days. Trials began with 30 mmHg of LBNP for 6 minutes. Then, a 2.5 L plastic bag of ice water (0±0°C) (LBNP+FC) or thermoneutral water (34±1°C) (LBNP+Sham) was placed on the forehead, eyes, and cheeks during 15 minutes of LBNP at 30 mmHg. Results: Forehead temperature was lower during LBNP+FC vs. LBNP+Sham with the greatest difference at 21 minutes of LBNP (11.1±1.6 vs. 33.9±1.4°C, P < 0.001). Mean arterial pressure was greater during LBNP+FC vs. LBNP+Sham with the greatest difference at 8 minutes of LBNP (98±15 vs. 80±8 mmHg, P < 0.001). Cardiac output was higher during LBNP+FC vs. LBNP+Sham with the greatest difference at 18 minutes of LBNP (5.9±1.4 vs. 4.9±1.0 L/min, P = 0.005). Forearm cutaneous vascular resistance was greater during LBNP+FC vs. LBNP+Sham with the greatest difference at 15 minutes of LBNP (7.2±3.4 vs. 4.9±2.7 mmHg/PU, P < 0.001). Conclusion: Face cooling during LBNP increases blood pressure through increases in cardiac output and vascular resistance.
Fungal diseases of wildlife typically manifest as superficial skin infections but can have devastating consequences for host physiology and survival. White-nose syndrome (WNS) is a fungal skin disease that has killed millions of hibernating bats in North America since 2007. Infection with the fungus Pseudogymnoascus destructans causes bats to rewarm too often during hibernation, but the cause of increased arousal rates remains unknown. Based on data from studies of captive and free-living bats, two mechanistic models have been proposed to explain disease processes in WNS. Key predictions of both models are that WNS-affected bats will show (1) higher metabolic rates during torpor (TMR), and (2) higher rates of evaporative water loss (EWL). We collected bats from a WNS-negative hibernaculum, inoculated one group with P. destructans and sham-inoculated a second group as controls. After four months of hibernation, we used respirometry to measure TMR and EWL. Both predictions were supported and our data suggest that infected bats were more affected by variation in ambient humidity than controls. Furthermore, disease severity, as indicated by the area of the wing with UV fluorescence, was positively correlated with EWL but not TMR. Our results provide the first direct evidence that heightened energy expenditure during torpor, and higher EWL, independently contribute to WNS pathophysiology with implications for the design of potential treatments for the disease.
Remote ischemic preconditioning (RIPC) can attenuate tissue damage sustained by ischemia-reperfusion injury. Blood flow restriction exercise (BFRE) restricts blood flow to exercising muscles. We implemented a novel approach to BFRE with cyclical bouts of blood flow restriction-reperfusion, reflecting the RIPC model. A concern about BFRE, however, is potential amplification of the exercise pressor reflex, which could be unsafe in at-risk populations. We hypothesized that cyclical BFRE would elicit greater increases in sympathetic outflow and arterial pressure than conventional exercise (CE), performed at the same relative intensity. We also assessed the cerebrovascular responses, due to potential implementation of BFRE in stroke rehabilitation. Fourteen subjects performed treadmill exercise at 65-70% HRmax with and without intermittent BFR (4x5-min intervals of bilateral thigh-cuff pressure followed by 5-min reperfusion periods). Mean arterial pressure (MAP), plasma norepinephrine (NE), and middle and posterior cerebral artery velocities (MCAv and PCAv) were compared between trials. As expected, BFRE elicited higher [NE] compared to CE (1249±170 vs 962±114 pg/ml; P=0.06). Unexpectedly, however, there were no differences in MAP between conditions (overall P=0.33), and MAP was 4-5 mmHg lower with BFRE vs. CE during the reperfusion periods (P≤0.05 for reperfusion periods 3 and 4). There were no differences in MCAv or PCAv between trials (P≥0.22), suggesting equivalent cerebro-metabolic demand. The exaggerated sympatho-excitatory response with BFRE was not accompanied by higher MAP, likely due to the cyclical reperfusions. This cyclical BFRE paradigm could be adapted to cardiac- or stroke-rehabilitation, where exercising patients could benefit from the cardio- and cerebro-protection associated with RIPC.
Remote ischemic preconditioning (RIPC) is characterized by the cyclical application of limb blood flow restriction and reperfusion, and has been shown to protect vital organs during a subsequent ischemic insult. Blood flow restriction exercise (BFRE) similarly combines bouts of blood flow restriction with low-intensity exercise and thus could potentially emulate the protection demonstrated by RIPC. One concern with BFRE, however, is the potential for an augmented rise in sympathetic outflow, due to greater activation of the exercise pressor reflex. Due to the use of lower workloads, however, we hypothesized that BFRE would elicit an attenuated increase in sympathetic outflow (assessed via plasma norepinephrine (NE) and mean arterial pressure (MAP)), and middle cerebral artery velocity (MCAv) when compared with conventional exercise (CE). Fifteen subjects underwent two leg-press exercise interventions: 1.BFRE-220 mmHg bilateral thigh occlusion at 20% 1 rep-max (1RM), and; 2.CE-65% 1RM without occlusion. Each condition consisted of 4 x 5-min cycles of exercise, with 3 x 10-reps in each cycle. 5-min of rest and reperfusion (for BFRE) followed each cycle. MAP increased with exercise (P<0.001), and was 4-5 mmHg higher with CE vs. BFRE (P≤0.09). Mean MCAv also increased with exercise (P<0.001) and was higher with CE compared to BFRE during the first bout of exercise only (P=0.07). Plasma [NE] increased with CE only (P<0.001), and was higher than BFRE throughout exercise (P≤0.02). The attenuated sympathetic response combined with similar cerebrovascular responses suggest that cyclical BFRE could be explored as an alternative to CE in the clinical setting.
The large doses of vitamin C and E and β-carotene used to reduce reactive oxygen species (ROS) production and oxidative damages in cancerous tissue, have produced disappointing and contradictory results. This therapeutic conundrum was attributed to the double-faced role of ROS, notably, their ability to induce either proliferation or apoptosis of cancer cells. However, for a ROS-inhibitory approach to be effective, it must targets ROS when they induce proliferation rather than apoptosis. Based on recent advances in redox biology, this review underlined a differential regulation of pro- and antioxidant system, respective to the stage of cancer. At early precancerous and neoplastic stages, antioxidant activity decreases and ROS appear to promote cancer initiation via inducing oxidative damage and base pair substitution mutations in pro-oncogenes and tumor suppressor genes, such as RAS and TP53, respectively. Whereas in late stages of cancer progression, tumor cells escape apoptosis by producing high levels of intracellular antioxidants, like NADPH and GSH, via the pentose phosphate pathway to buffer the excessive production of ROS and related intra-tumor oxidative injuries. Therefore, antioxidants should be prohibited in patients with advanced stages of cancer and/or undergoing anticancer therapies. Interestingly, the biochemical and biophysical properties of some polyphenols allow them to selectively recognize tumor cells. This characteristic was exploited to design and deliver nanoparticles coated with low doses of polyphenols and containing chemotherapeutic drugs into tumor-bearing animals. First results are encouraging, which may revolutionize the conventional use of antioxidants in cancer.
Obesity-induced vascular dysfunction involves pathological remodeling of the visceral adipose tissue (VAT) and increased inflammation. Our previous studies showed that arginase 1 (A1) in endothelial cells (EC) is critically involved in obesity-induced vascular dysfunction. We tested the hypothesis that EC A1 activity also drives obesity-related VAT remodeling and inflammation. Our studies utilized wild-type and EC-A1 knockout (KO) mice made obese by high fat/high sucrose (HFHS) diet. HFHS diet induced increases in body weight, fasting blood glucose, and VAT expansion. This was accompanied by increased arginase activity and A1 expression in vascular EC and increased expression of tumor necrosis factor α (TNFα), monocyte chemoattractant protein-1 (MCP-1), interleukin-10 (IL-10), vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) mRNA and protein in both VAT and EC. HFHS also markedly increased circulating inflammatory monocytes and VAT infiltration by inflammatory macrophages, while reducing reparative macrophages. Additionally, adipocyte size and fibrosis increased and capillary density decreased in VAT. These effects of HFHS, except for weight gain and hyperglycemia, were prevented or reduced in mice lacking EC-A1 or treated with the arginase inhibitor ABH (2-(S)-amino-6-boronohexanoic acid). In mouse aortic EC, exposure to high glucose (25 mM) and Na palmitate (200 μM) reduced NO production, increased A1, TNFα, VCAM-1, ICAM-1 and MCP-1 mRNA, and monocyte adhesion. Knockout of EC-A1 or ABH prevented these effects. Summary- HFHS diet-induced VAT inflammation is mediated by EC A1 expression/activity. Limiting arginase activity is a possible therapeutic means of controlling obesity-induced vascular and VAT inflammation.
Background: Visceral adipose tissue (VAT) and subcutaneous adipose tissue (SCAT) have different structures and metabolic functions, and play different roles in the regulation of mammal systemic endocrine. However, little is known about morphology and physiological and metabolic functions between VAT and SCAT in fish. Methods: We compared the morphological, physiological and biochemical characteristics of VAT and SCAT in Nile Tilapia and measured their functions in energy intake flux, lipolytic ability, and gene expression patterns. Results: SCAT contained more large adipocytes and non-adipocytes than VAT in Nile tilapia. VAT had a higher lipid and was the primary site for lipid deposition. Conversely, SCAT had higher hormone-induced lipolytic activity. Furthermore, SCAT had a higher percentage of monounsaturated and lower polyunsaturated fatty acids than VAT. SCAT had higher mitochondrial DNA, gene expression for fatty acid β-oxidation, adipogenesis and brown adipose tissue characteristics, but it also had a lower gene expression for inflammation and adipocyte differentiation than VAT. Conclusions: SCAT and VAT have different morphological structures, as well as physiological and metabolic functions in fish. VAT is the preferable lipid deposition tissue, whereas SCAT exhibits higher lipid catabolic activity than VAT. General Significance: The physiological functions of SCAT in fish are commonly overlooked. The present study indicates SCAT has specific metabolic characteristics that differ VAT. The differences between VAT and SCAT should be considered in future metabolism studies using fish as models, either in biomedical or aquaculture studies.
Inactivity, obesity, and insulin resistance are significant risk factors for the development of Alzheimer's disease (AD). Several studies have demonstrated that diet induced obesity, inactivity, and insulin resistance exacerbates neuropathological hallmarks of AD. The aggregation of beta-amyloid peptides is one of these hallmarks. Beta-site amyloid precursor protein cleaving enzyme 1 (BACE1) is the rate-limiting enzyme in amyloid precursor protein (APP) processing, leading to beta-amyloid peptide formation. Understanding how BACE1 content and activity is regulated is essential for establishing therapies aimed at reducing and/or slowing the progression of AD. Exercise training has proven to reduce the risk of AD as well as decrease beta-amyloid production and BACE1 content and/or activity. However, these long-term interventions also result in improvements in adiposity, circulating metabolites, glucose tolerance, and insulin sensitivity making it difficult to determine the direct effects of exercise on brain APP processing. This review highlights this large void in our knowledge and aims discusses our current understanding the direct of effect of exercise on beta-amyloid production. We have concentrated on the central role that brain-derived neurotrophic factor (BDNF) may play in mediating the direct effects of exercise on reducing brain BACE1 content and activity as well as beta-amyloid production. Future studies should aim to generate a greater understanding of how obesity and exercise can directly alter APP processing and AD related pathologies. This knowledge could provide evidence-based hypotheses for designing therapies to reduce the risk of AD and dementia.
Taste stimuli have a temperature that can stimulate thermosensitive neural machinery in the mouth during gustatory experience. Although taste and oral temperature are sometimes discussed as different oral sensory modalities, there is a body of literature that demonstrates temperature is an important component and modulator of the intensity of gustatory neural and perceptual responses. Available data indicate that the influence of temperature on taste, herein referred to as "thermogustation", can vary across taste qualities, can also vary among stimuli presumed to share a common taste quality, and is conditioned on taste stimulus concentration, with neuronal and psychophysical data revealing larger modulatory effects of temperature on gustatory responding to weakened taste solutions compared to concentrated. What is more, thermogustation is evidenced to involve interplay between mouth and stimulus temperature. Given these and other dependencies, identifying principles by which thermal input affects gustatory information flow in the nervous system may be important for ultimately unravelling the organization of neural circuits for taste and defining their involvement with multisensory processing related to flavor. Yet thermal effects are relatively understudied in gustatory neuroscience. Major gaps in our understanding of the mechanisms and consequences of thermogustation include delineating supporting receptors, the potential involvement of oral thermal and somatosensory trigeminal neurons in thermo-gustatory interactions, and the broader operational roles of temperature in gustatory processing. This review will discuss these and other issues in the context of the literature relevant to understanding thermogustation.
Remote and selective spatiotemporal control of the activity of neurons to regulate behavior and physiological functions has been a long-sought goal in system neuroscience. Identification and subsequent bioengineering of light-sensitive ion channels (e.g., channelrhodopsins, halorhodopsin and archaerhodopsins) from the bacteria has made it possible to utilize light to artificially modulate neuronal activity, namely optogenetics. Recent advance in genetics has also allowed development of novel pharmacological tools to selectively and remotely control neuronal activity using engineered G-protein coupled receptors which can be activated by otherwise inert drug-like small molecules such as the Designer Receptors Exclusively Activated by Designer Drug (DREADD) - a form of chemogenetics. The cutting-edge optogenetics and pharmacogenetics are powerful tools in neuroscience which allow selective and bidirectional modulation of the activity of defined populations of neurons with unprecedented specificity. These novel toolboxes are enabling significant advances in deciphering how the nervous system works and its influence on various physiological processes in health and disease. Here, we discuss the fundamental elements of optogenetics and chemogenetics approaches and some of the applications that yielded significant advances in various areas of neuroscience and beyond.
Modern dairy cows meet the energy demand of early lactation by calling on hormonally-driven mechanisms to increase the use of lipid reserves. In this context, we recently reported that fibroblast growth factor-21 (FGF21), a hormone required for efficient use of lipid reserves in rodents, is upregulated in periparturient dairy cows. Increased plasma FGF21 in early lactation coincides with elevated circulating concentrations of glucagon (GCG) and non-esterified fatty acids (NEFA). To assess the relative contribution of these factors in regulating FGF21, two experiments were performed in energy sufficient, non-pregnant, non-lactating dairy cows. In the first study, cows were injected with saline or GCG every 8 h over 72 h. GCG increased hepatic FGF21 mRNA by an average of 5-fold over matched controls but had no effect on plasma FGF21. In the second study, cows were infused and injected with saline, infused with intralipid and injected with saline or infused with intralipid and injected with GCG. Infusions and injections were respectively administered IV over 16 hours and SC every 8 h. Intralipid infusion increased plasma NEFA from 92 to 550 µM within 3 h and increased plasma FGF21 from 1.3 ng/ml to >11 ng/ml 6 h later; FGF21 mRNA increased by 34-fold in liver but remained invariant in adipose tissue. GCG injections during the intralipid infusion had no additional effects on plasma NEFA, liver FGF21 mRNA or plasma FGF21. These data implicate plasma NEFA as a key factor triggering hepatic production and increased circulating concentrations of FGF21 in early lactation.
Ischemic Heart Disease, in the absence of obstructive coronary artery disease, is prevalent in women, and constitutes a major risk factor for developing major adverse cardiovascular events, including myocardial infarction, stroke, and heart failure. For decades, diagnosis was considered benign and often minimized; however, it is now known that this etiology caries much risk and is a significant burden to the health care system. This review summarizes the current state-of-knowledge on non-obstructive ischemic heart disease (NOIHD), the association between NOIHD and left ventricular diastolic dysfunction, potential links between NOIHD and the development of heart failure with preserved ejection fraction (HFpEF), and therapeutic options and knowledge gaps for patients living with NOIHD.
BACKGROUND: Endothelial function and arterial stiffness are known to be altered in pre-eclamptic pregnancies. Previous studies have shown conflicting results regarding the best technique for assessing vascular function in pregnancy. In this study, we made a comprehensive evaluation of in vivo vascular function (including flow-mediated dilatation (FMD), peripheral arterial tonometry (PAT), and arterial stiffness) in pre-eclamptic patients and compared them to normal pregnancies. In addition, we assessed the relation between vascular function and systemic inflammation. METHODS: Fourteen patients with pre-eclampsia (PE) and 14 healthy pregnant controls were included. Endothelial function was determined by FMD and PAT, arterial stiffness by carotid-femoral pulse wave velocity (cfPWV) and augmentation index (AIx). Systemic inflammation was assessed using mean platelet volume (MPV) and neutrophil-leucocyte ratio (NLR). RESULTS: The reactive hyperemia index (RHI), assessed using PAT is decreased at the third trimester in comparison to the first trimester in a normal uncomplicated pregnancy (p=0.001). Arterial stiffness is significantly higher in PE versus normal pregnancy (p<0.001). Endothelial function obtained by FMD is deteriorated in PE versus normal pregnancy (p=0.015), while endothelial function assessment by PAT is improved in PE versus normal pregnancy (p=0.001). Systemic inflammation (MPV, NLR) increases during normal pregnancy. CONCLUSIONS: Flow mediated dilation and peripheral arterial tonometry are disturbed in pre-eclampsia. Endothelial function assessed by FMD and PAT show distinct results. This may indicate that measurements with FMD and PAT reflect different aspects of endothelial function and that PAT should not be used as a substitute for FMD as measure of endothelial function in pregnancy.
Apolipoprotein AIV (ApoAIV) and cholecystokinin (CCK) are well-known satiating signals that are stimulated by fat consumption. Peripheral ApoAIV and CCK interact to prolong satiating signals. In the present study, we hypothesized that ApoAIV and CCK control energy homeostasis in response to high-fat diet feeding. To test this hypothesis, energy homeostasis in ApoAIV and CCK double knockout (ApoAIV/CCK-KO), ApoAIV knockout (ApoAIV-KO), and CCK knockout (CCK-KO) mice were monitored. When animals were maintained on a low-fat diet, ApoAIV/CCK-KO, ApoAIV-KO, and CCK-KO mice had comparable energy intake and expenditure, body weight, fat mass, fat absorption, and plasma parameters relative to the controls. In contrast, these KO mice exhibited impaired lipid transport to epididymal fat pads in response to intraduodenal infusion of dietary lipids. Furthermore, ApoAIV-KO mice had upregulated levels of CCK receptor 2 (CCK2R) in the small intestine while ApoAIV/CCK-KO mice had upregulated levels of CCK2R in the brown adipose tissue. After 20 weeks of a high-fat diet, ApoAIV-KO and CCK-KO mice had comparable body weight and fat mass, as well as lower energy expenditure at some time points. However, ApoAIV/CCK-KO mice exhibited reduced body weight and adiposity relative to wild-type mice, despite having normal food intake. Furthermore, ApoAIV/CCK-KO mice displayed normal fat absorption and locomotor activity, as well as enhanced energy expenditure. These observations suggest that mice lacking ApoAIV and CCK have reduced body weight and adiposity, possibly due to impaired lipid transport and elevated energy expenditure.
Maltodextrins, such as Maltrin and Polycose, are glucose polymer mixtures of varying chain lengths that are palatable to rodents. Although glucose and other sugars activate the T1R2+T1R3 "sweet" taste receptor, recent evidence from T1R2 or T1R3 knockout (KO) mice suggests that maltodextrins, despite their glucose polymer composition, activate a separate receptor mechanism to generate a taste percept qualitatively distinguishable from that of sweeteners. However, explicit discrimination of maltodextrins from prototypical sweeteners has not yet been psychophysically tested in any murine model. Therefore, mice lacking T1R2+T1R3 and wild-type controls were tested in a two-response taste discrimination task to determine if maltodextrins are 1) detectable when both receptor subunits are absent, and 2) perceptually distinct from that of sucrose irrespective of viscosity, intensity, and hedonics. Most KO mice displayed similar Polycose sensitivity as controls. However, some KO mice were only sensitive to the higher Polycose concentrations, implicating potential allelic variation in the putative polysaccharide receptor or downstream pathways unmasked by the absence of T1R2+T1R3. Varied Maltrin and sucrose concentrations of approximately matched viscosities were then presented to render the oral somatosensory features, intensity, and hedonic value of the solutions irrelevant. While both genotypes competently discriminated Maltrin from sucrose, performance was apparently driven by the different orosensory percepts of the two stimuli in control mice and the presence of a Maltrin but not sucrose orosensory cue in KO mice. These data support the proposed presence of an orosensory receptor mechanism that gives rise to a qualitatively distinguishable sensation from that of sucrose.
To elucidate the role of leptin in acute systemic inflammation, we investigated how its infusion at low, physiologically relevant doses affects the responses to bacterial lipopolysaccharide (LPS) in rats subjected to 24 h of food deprivation. Leptin was infused subcutaneously (0-20 μg/kg/h) or intracerebroventricularly (0-1 μg/kg/h). Using hypothermia and hypotension as biomarkers of systemic inflammation, we identified the phase extending from 90 to 240 min post-LPS as the most susceptible to modulation by leptin. In this phase, leptin suppressed the rise in plasma TNF-α, and accelerated the recoveries from hypothermia and hypotension. Suppression of TNF-α was not accompanied by changes in other cytokines or prostaglandins. Leptin suppressed TNF-α when infused peripherally, but not when infused into the brain. Importantly, the leptin dose that suppressed TNF-α corresponded to the lowest dose that limited food consumption; this dose elevated plasma leptin within the physiological range (to 5.9 ng/ml). We then conducted in-vitro experiments to investigate whether an action of leptin on macrophages could parallel our in-vivo observations. The results revealed that, when sensitized by food deprivation, LPS-stimulated peritoneal macrophages can be inhibited by leptin at concentrations that are lower than those reported to promote cytokine release. It is concluded that physiological levels of leptin do not exert a pro-inflammatory effect, but rather an anti-inflammatory effect involving selective suppression of TNF-α via an action outside the brain. The mechanism of this effect might involve a previously unrecognized, suppressive action of leptin on macrophage subpopulations sensitized by food deprivation, but future studies are warranted.
Rainbow trout (Oncorhynchus mykiss) confined in pairs form social hierarchies in which subordinate fish typically experience fasting and high circulating cortisol levels, resulting in low growth rates. The present study investigated the role of AMP-activated protein kinase (AMPK) in mediating metabolic adjustments associated with social status in rainbow trout. After 3 d of social interaction, liver AMPK activity in subordinate trout was significantly higher than that of dominant or sham (fish handled in the same fashion as paired fish but held individually) trout. Elevated liver AMPK activity in subordinate fish likely reflected a significantly higher ratio of phosphorylated AMPK (phospho-AMPK) to total AMPK protein, which was accompanied by significantly higher AMPKα1 relative mRNA abundance. Liver ATP and creatine phosphate (CrP) concentrations in subordinate fish also were elevated, perhaps as a result of AMPK activity. Sham fish that were fasted for 3 d exhibited effects parallel to those of subordinate fish, suggesting that low food intake was an important trigger of elevated AMPK activity in subordinate fish. Effects on white muscle appeared to be influenced by the physical activity associated with social interaction. Overall, muscle AMPK activity was significantly higher in dominant and subordinate trout than sham fish. Muscle phospho-AMPK:total AMPK protein abundance was highest in subordinate fish, while muscle AMPKα1 relative mRNA abundance was elevated by social dominance. Muscle ATP and CrP concentrations were high in dominant and subordinate fish at 6 h of interaction, decreasing significantly thereafter. Collectively, the findings of the present study support a role for AMPK in mediating the liver and white muscle metabolic adjustments associated with social hierarchy formation in rainbow trout.
Oxytocin (OT) administration elicits weight loss in diet-induced obese (DIO) rodents, nonhuman primates and humans by both reducing energy intake and increasing energy expenditure (EE). Although the neurocircuitry underlying these effects remains uncertain, OT neurons in the paraventricular nucleus are positioned to control both energy intake and sympathetic nervous system outflow to interscapular brown adipose tissue (IBAT) through projections to both the hindbrain nucleus of the solitary tract and spinal cord. The current work was undertaken to examine whether central OT increases BAT thermogenesis, whether this effect involves hindbrain OT receptors (OTRs), and whether such effects are associated with sustained weight loss following chronic administration. To assess OT-elicited changes in BAT thermogenesis, we measured the effects of intracerebroventricular administration of OT on IBAT temperature (TIBAT) in both rats and mice. Because fourth ventricular (4V) infusion targets hindbrain OTRs, whereas third ventricular (3V) administration targets both forebrain and hindbrain OTRs, we compared responses to OT following chronic 3V infusion in DIO rats and mice with chronic 4V infusion in DIO rats. We report that chronic 4V infusion of OT into two distinct rat models recapitulates the effects of 3V OT to ameliorate diet-induced obesity by reducing fat mass. While reduced food intake contributes to this effect, our finding that 4V OT also increases BAT thermogenesis suggests that increased EE may contribute as well. These findings collectively support the hypothesis that in DIO rats, OT action in the hindbrain evokes sustained weight loss by both reducing energy intake and increasing BAT thermogenesis.
In obesity the increased O2 cost of breathing negatively affects the O2 cost of exercise and exercise tolerance. The purpose of the study was to determine whether, in obese adolescents, the addition of respiratory muscle endurance training (RMET) (isocapnic hyperpnea) to a standard body mass reduction program decreases the O2 cost of exercise and perceived exertion and increases exercise tolerance. 9 male obese adolescents (16.0±1.4 years [x±SD], body mass 114.4±22.3 kg) underwent 3 weeks of RMET (5 days/week); 8 age-and sex- matched obese adolescents underwent the standard body mass reduction program (CTRL). Before and after interventions patients performed on a cycle ergometer: incremental exercise; 12-min constant work-rate exercises (CWR) at 65% and 120% of gas exchange threshold (GET) determined before the intervention. Breath-by-breath pulmonary ventilation (V'E) and O2 uptake (V'O2), heart rate (HR) and ratings of perceived exertion for dyspnea/respiratory discomfort (RPER) and leg effort (RPEL) were determined. Body mass decreased (by ~3.0 kg) after both RMET and CTRL (P=0.001; GLM for repeated measures). Peak O2 and peak work rate were not affected by both interventions. During CWR<GET no changes were observed after both interventions. During CWR>GET the O2 cost of cycling (P=0.014), the slope of V'O2 vs. time (P=0.012), RPER (P=0.012), RPEL (P=0.016) and HR (P=0.001) decreased following RMET but not following CTRL, whereas V'E did not change. In obese adolescents RMET, superimposed on a standard body mass reduction program, lowered the O2 cost of cycling and perceived exertion during constant heavy-intensity exercise.
We performed a prospective, longitudinal study of pregnant women presenting to their first obstetrics visits to characterize the changes in spot urine protein-to-creatinine (UPCR) and albumin-to-creatinine ratios (UACR) in normotensive pregnancies, as well as identify clinical characteristics associated with isolated proteinuria and preeclampsia. We measured spot urinary albumin, protein, and creatinine at the first prenatal visit, end of the second trimester, and at delivery. In normotensive pregnancy (n=142), we found that from the beginning of pregnancy to delivery, UACR increased by a median (interquartile range (IQR)) of 14.7 mg/g Cr (3.74-51.8) and UPCR by 60 mg/g Cr (30-130) (p<0.001 for both changes). Isolated proteinuria (defined as UPCR > 300 mg/g Cr in the absence of hypertension) was identified in 19 /142 (13.4%) normotensive pregnancies. Increases in systolic and diastolic blood pressure from early pregnancy to delivery, and increases in UACR from early to mid-pregnancy were associated with isolated proteinuria at delivery. Twelve women developed preeclampsia. Nulliparity, early and mid-pregnancy diastolic blood pressures were strongly associated with the development of preeclampsia, but early changes in UACR were not. In conclusion, women who develop isolated proteinuria at delivery have a larger increase in blood pressure than women without proteinuria and have a 'microalbuminuric' phase earlier in gestation, unlike women who develop preeclampsia. These findings suggest a different mechanism of urine protein excretion in women with isolated proteinuria as compared to women with preeclampsia, where proteinuria has a more abrupt onset.
High protein diet (HPD) curtails obesity and/or fat mass but it is unknown whether it reverses neuroinflammation, altered glucose levels, cholecystokinin (CCK) sensitivity and gut microbiome in rats fed a Western diet (WD)-induced obesity (DIO). Male rats fed a WD (high fat and sugar) for 12 weeks were switched to a HPD for 6 weeks. Body composition, food intake, meal pattern, sensitivity to intraperitoneal CCK-8S, blood glucose, brain signaling, and cecal microbiota were assessed. Compared to normal diet, WD increased body weight (9.3%) and fat mass (73.4%). CCK-8S (1.8 or 5.2 nmol/kg) did not alter food intake and meal pattern in DIO rats. Switching to a HPD for six weeks reduced fat mass (15.7%) with a non-significantly reduced body weight gain, normalized blood glucose and decreased feeding after CCK-8S. DIO rats on the WD or switched to a HPD showed comparable microbial diversity. However, in HPD vs WD rats, there was enrichment of 114 operational taxonomic units (OTUs) and depletion of 188 OTUs. Of those, Akkermansia muciniphila (enriched on a HPD), an unclassified Clostridiales, a member of the RF39 order, and a Phascolarctobacterium were significantly associated with fat mass. The WD increased cytokine expression in the hypothalamus and dorsal medulla that was unchanged by switching to HPD. These data indicate that HPD reduces body fat and restores glucose homeostasis and CCK sensitivity while not modifying brain inflammation. In addition, expansion of cecal Akkermansia muciniphila correlated to fat mass loss may represent a potential peripheral mechanism of HPD beneficial effects.
Arterial stiffness and cardiac function are important predictors of cardiovascular events in patients with hypertension even with adequate blood pressure (BP) control. We evaluated whether a direct renin inhibitor, aliskiren, reduces arterial stiffness and modulates left ventricular function compared with a diuretic, hydrochlorothiazide, in elderly hypertensives. Twenty one hypertensives [67±14 (SD) yrs] were randomly assigned to receive 6-month aliskiren (n=11) or hydrochlorothiazide (n=10) based therapy. We assessed β-stiffness of the local arteries, arterial elastance (Ea), and echocardiographic variables including early (E) and late (A) mitral inflow velocity, deceleration time of E, early (E') and late (A') diastolic mitral annular velocity, and left ventricular end-systolic elastance (Ees) before and after treatment. BP similarly decreased (P<0.001) after both therapies. β-stiffness of the carotid artery decreased after aliskiren, but increased after hydrochlorothiazide treatment (aliskiren: 6.42±2.34 pre vs. 5.07±1.29 post; hydrochlorothiazide: 5.05±1.78 vs. 7.25±2.68, P=0.001 for interaction). β-stiffness of the femoral and radial arteries were not different after either treatment. Different from aliskiren, E decreased (73±16 vs. 67±14 cm/s, P=0.026) and the deceleration time prolonged (218±40 vs. 236±35 ms, P=0.032) after hydrochlorothiazide therapy, while the E/A, and E' remained unchanged after both treatments. Ea and Ees decreased after aliskiren therapy (both P<0.05), while the Ea/Ees (ventricular-arterial coupling) was maintained after both treatments. Thus, aliskiren decreased the stiffness of carotid artery and left ventricular end-systolic elastance with maintenance of ventricular-arterial coupling without any effects on diastolic filling, while hydrochlorothiazide increased carotid arterial stiffness and slowed early diastolic filling in elderly hypertensives.
Hindlimb skeletal muscle stretch (i.e., selective activation of the muscle mechanoreflex) in decerebrate rats evokes reflex increases in blood pressure and sympathetic nerve activity. Bradykinin has been found to sensitize mechano-gated channels through a bradykinin B2 receptor-dependent mechanism. Moreover, bradykinin B2 receptor expression on sensory neurons is increased following chronic femoral artery ligation in the rat (a model of simulated peripheral artery disease). We tested the hypothesis that, in decerebrate, unanesthetized rats, the injection of bradykinin into the arterial supply of a hindlimb would acutely augment (i.e., sensitize) the increase in blood pressure and renal sympathetic nerve activity (RSNA) during hindlimb muscle stretch to a greater extent in rats with a ligated femoral artery than in rats with freely perfused femoral arteries. The pressor response during static hindlimb muscle stretch was compared before and after the hindlimb arterial injection of 0.5 µg of bradykinin. The injection of bradykinin itself increased blood pressure to a greater extent in "ligated" rats (n=10) than in "freely perfused" rats (n=10). The increase in blood pressure during hindlimb muscle stretch, however, was not different before compared to after bradykinin injection in either freely perfused (control: 14±2, post-bradykinin: 15±2 mmHg, p=0.62) or ligated (control: 15±3, post-bradykinin: 14±2 mmHg, p=0.80) rats. Likewise, the increase in RSNA during stretch was not different before compared to after bradykinin injection in either group of rats. We conclude that bradykinin does not acutely sensitize the pressor response during hindlimb skeletal muscle stretch in either freely perfused or ligated decerebrate rats.
Long-term studies have found low carbohydrate diets are more effective for weight loss than calorie restricted diets in the short-term, but equally or only marginally more effective in the long-term. Low carbohydrate diets have been linked to reduced glycogen stores and increased feelings of fatigue. We propose that reduced physical activity in response to lowered glycogen explains the diminishing weight loss advantage of low carbohydrate compared to low calorie diets over longer time scales. We explored this possibility by feeding adult Drosophila melanogaster either a standard or low carbohydrate diet for nine days and measured changes in metabolic rate, glycogen stores, activity, and body mass. We hypothesized that a low carbohydrate diet would cause a reduction in glycogen stores that recovers over time, reduced physical activity, and an increase in resting metabolic rate. The low carbohydrate diet was found to reduce glycogen stores, which recovered over time. Activity was unaffected by diet but the low carbohydrate group experienced a reduction in metabolic rate. We conclude that metabolic depression could explain the decreased effectiveness of low carbohydrate diets over time and recommend further investigation of long-term metabolic effects of dietary interventions and a greater focus on physiological plasticity within the study of human nutrition.
Radiation exposure accelerates the onset of age-related diseases such as diabetes, cardiovascular disease, and neoplasia and thus lends insight into in vivo mechanisms common to these disorders. Fibrosis and extracellular matrix (ECM) remodeling, which occur with aging, overnutrition, and following irradiation, are both risk factors for type-2 diabetes mellitus (T2DM) development. Our prior work demonstrated that monkeys with whole-body radiation exposure five to nine years prior had an increased incidence of skeletal muscle insulin resistance and T2DM. We hypothesized that irradiation-induced fibrosis alters muscle architecture predisposing irradiated animals to insulin resistance and overt diabetes. Rhesus macaques (Macaca mulatta; n=7-8/group) grouped as non-irradiated age-matched controls (NonRad-CTL), irradiated non-diabetic monkeys (Rad-CTL) and irradiated monkeys that subsequently developed diabetes (Rad-DM) were compared. Prior radiation exposure resulted in persistent skeletal muscle ECM changes including a relative overabundance of collagen IV and a trend of increased transforming growth factor beta 1 (TGFβ1). Notably, preservation of microvascular markers differentiated the irradiated diabetic and non-diabetic groups. Rad-DM had lower microvascular density, plasma nitrate, and heat shock protein 90 levels compared to Rad-CTL. These results are consistent with a protective effect of abundant microvasculature in maintaining glycemic control within radiation-induced fibrotic muscle.
The neuropeptide, pituitary adenylate cyclase-activating polypeptide (PACAP), has emerged as a principal and rate-limiting regulator of physiological stress responses in adult rodents and has been implicated in SIDS (Sudden Infant Death Syndrome). Recent studies show that PACAP plays a role in neonatal cardiorespiratory responses to hypoxia, hypercapnia and hypothermia, but not hyperthermia, which is often associated with SIDS. Here we tested the hypothesis that, consistent with a role in SIDS, PACAP is involved in regulating the neonatal cardiorespiratory responses to severe heat. To address this, we studied the cardiorespiratory physiology of conscious neonatal PACAP-null and wild-type mice at ambient temperatures of 32°C (baseline) and 40°C (heat stress), using head-out plethysmography and surface ECG electrodes. We also assessed body surface temperature, to give an indication of cutaneous heat loss. Our results show that wild-type neonatal mice respond to heat stress by increasing ventilation (P=0.007) and associated expired CO2 (P=0.041), heart rate (P<0.001) and cutaneous heat loss (P<0.001). In PACAP-null neonates, this heat response is impaired, as indicated by a decrease in ventilation (P=0.04) and associated expired CO2 (P=0.006), and a blunted increase in heart rate (P=0.001) and cutaneous heat loss (P=0.0002). In addition, heart rate variability at baseline in PACAP-null neonates was lower than in wild-type controls (P<0.01). These results suggest that during heat stress, PACAP is important for neonatal cardiorespiratory responses that help regulate body temperature. Abnormal PACAP regulation could, therefore, contribute to neonatal disorders in which the autonomic response to heat stress is impaired, such as SIDS.
Angiotensin II acts via two main receptors within the CNS, with the type 1A receptor (AT1AR) most widely expressed in adult neurons. Activation of the AT1R in the nucleus of the solitary tract (NTS), the principal nucleus receiving central synapses of viscerosensory afferents, modulates cardiovascular reflexes. Expression of the AT1R occurs in high density within the NTS of most mammals, including humans, but the fundamental electrophysiological and neurochemical characteristics of the AT1AR-expressing NTS neurons are not known. To address this, we have used a transgenic mouse, in which the AT1AR promoter drives expression of green fluorescent protein (GFP). Approximately one third of AT1AR-expressing neurons express the catecholamine-synthetic enzyme, tyrosine hydroxylase (TH) and a sub-population of these stained for the transcription factor Phox2b. A third group, comprising approximately two thirds of the AT1AR-expressing NTS neurons showed Phox 2b-immunoreactivity alone. A fourth group in the ventral subnucleus expressed neither TH nor Phox2b. In whole cell recordings from slices in vitro, AT1AR-GFP neurons exhibited voltage-activated potassium currents, including the transient outward current and the M-type potassium current. In two different mouse strains, both AT1AR-GFP neurons and TH-GFP neurons showed similar AT1AR-mediated depolarizing responses to superfusion with angiotensin II. These data provide a comprehensive description of AT1AR-expressing neurons in the NTS and increase our understanding of the complex actions of this neuropeptide in the modulation of viscerosensory processing.
Hypertension is a prevalent pathology that increases risk for numerous cardiovascular diseases. Because the etiology of hypertension varies across patients, specific and effective therapeutic approaches are needed. The role of renal sympathetic nerves is established in numerous forms of hypertension, but their contribution to salt-sensitivity and interaction with factors such as endothelin-1 are poorly understood. Rats deficient of functional ETB receptors (ETB-def) on all tissues except sympathetic nerves are hypertensive and exhibit salt-sensitive increases in blood pressure. We hypothesized that renal sympathetic nerves contribute to hypertension and salt-sensitivity in ETB-def rats. The hypothesis was tested through bilateral renal sympathetic nerve denervation and measuring blood pressure during normal (0.49% NaCl) and high (4.0% NaCl) salt diets. Denervation reduced mean arterial pressure in ETB-def rats compared to sham-operated controls by 12 ± 3 mmHg; however, denervation did not affect the increase in blood pressure following two weeks of high salt diet (+19 ± 3 vs. +16 ± 3 mmHg relative to normal salt diet; denervated vs. sham, respectively). Denervation reduced cardiac sympathetic to parasympathetic tone (LF/HF) during normal salt diet and vasomotor LF/HF tone during high salt diet in ETB-def rats. We conclude that the renal sympathetic nerves contribute to the hypertension but not to salt sensitivity of ETB-def rats.
Placental hypoxia is associated with maternal hypertension, placental insufficiency and fetal growth restriction. In the pregnant guinea pig, prenatal hypoxia during early gestation inhibits cytotrophoblast invasion of spiral arteries, increases maternal blood pressure and induces fetal growth restriction. This study evaluated the impact of chronic maternal hypoxia on fetal heart structure with 4D echocardiography with spatio-temporal image correlation and tomographic ultrasound (STIC-TUI) and uterine and umbilical artery resistance/pulsatility indices and fetal heart function using pulsed wave Doppler ultrasound. Pregnant guinea pigs were exposed to either normoxia (N=7) or hypoxia (10.5%O2; N=9) at 28-30d gestation and studied at term. Fetal heart structure and outflow tracts were evaluated in the four chamber view. Fetal heart diastolic function was assessed by E/A ratios of both ventricles and systolic function by the myocardial performance index (or Tie) of left ventricles of normoxic (N=21) and hypoxic (N=17) fetuses. There were no structural abnormalities in fetal hearts. However, hypoxia induced asymmetric fetal growth restriction and increased the relative placental weight compared to normoxic controls. Hypoxia increased Doppler resistance/pulsatility of uterine but not umbilical arteries, had no effect on the Tie index, and increased the E/A ratio in left but not right ventricles. Thus, early and prolonged hypoxia increases uterine artery resistance, stimulates placental compensation, and generates fetal growth restriction at term. Further, the enhanced cardiac diastolic filling with no changes in systolic function or umbilical artery resistance suggests that the fetal guinea pig undergoes a compensated, adaptive response of the systemic circulation to prolonged hypoxia exposure.
The beneficial effects of angiotensin converting enzyme (ACE) inhibitors and angiotensin II (AII) receptor antagonists in patients with heart failure secondary to reduced ejection fraction (HFrEF) are felt to result from prevention of the adverse effects of AII on systemic afterload and renal homeostasis. However, AII can activate the sympathetic nervous system and part of the beneficial effects of ACE inhibitors and AII antagonists may result from their ability to inhibit such activation. We examined the acute effects of the ACE inhibitor captopril (25 mg, n = 9) and the AII receptor antagonist losartan (50 mg, n = 10) on hemodynamics as well as total body and cardiac norepinephrine spillover in patients with chronic HFrEF. Hemodynamic and neurochemical measurements were made at baseline and at 1, 2 and 4 hours after oral dosing. Administration of both drugs caused significant reductions in systemic arterial, cardiac filling and pulmonary artery pressures (P < 0.05 vs baseline). There was no significant difference in the magnitude of those hemodynamic effects. Plasma concentrations of AII were significantly decreased by captopril and increased by losartan (P < 0.05 vs baseline for both). Total body sympathetic activity increased in response to both captopril and losartan (P < 0.05 vs baseline for both), however there was no change in cardiac sympathetic activity in response to either drug. The results of the current study do not support the hypothesis that the acute inhibition of the renin angiotensin system has sympathoinhibitory effects in patients with chronic HFrEF.
Normobaric hypoxic conditioning (HC) denotes exposure to hypoxia at rest (passive) or combined with exercise (active). HC has been applied acutely (single exposure) and chronically (repeated exposure) to obese populations for managing/increasing cardio-metabolic health and weight loss. Cardio-metabolic health and weight loss responses of obese populations in response to passive and active HC are unclear. A systematic search for articles published between 2000-2017 was carried out. Studies investigating the effects of HC for improving cardio-metabolic health and weight loss of obese populations were included. Studies investigated passive (n = 7; 5 animal, 2 humans), active (n = 4; all humans) and a combination of (n = 4; 3 animal, 1 human) HC to an inspired oxygen fraction between 4.8-15.0%, during a single session and daily sessions per week, lasting between 5 days and 8 months. Passive HC could reduce insulin concentrations (-37-22%) and increase energy expenditure (+12-16). Active HC may reduce body weight (-4-2%) and blood pressure (-8-3%). Inconclusive findings exist in determining the impact of acute and chronic HC on markers of triglycerides, cholesterol levels and fitness capacity. Studies that included animal models involved exposure to severe levels of hypoxia (inspired oxygen fraction of 5.0%; simulated altitude >10,000 m) that are not suitable for human populations. HC demonstrated positive findings in relation to insulin and energy expenditure, and body weight and blood pressure, for improving the cardio-metabolic health and body weight management of obese populations. Responses of plasma biomarkers to passive and active HC in humans is warranted.
We investigated the underlying molecular mechanisms by which post-exercise cold-water immersion (CWI) may alter key markers of mitochondrial biogenesis following both a single session and six weeks of sprint interval training (SIT). Nineteen males performed a single SIT session, followed by one of two 15-min recovery conditions: cold-water immersion (COLD; 10°C) or a passive room-temperature control (CON; 23°C). Sixteen of these participants also completed six weeks SIT, each session followed immediately by their designated recovery condition. Four muscle biopsies were obtained in total, three during the single SIT session (pre-exercise, post-recovery, and 3 h post-recovery), and one 48h after the last SIT session. Following a single SIT session, phosphorylated (p-) AMPK, p-p38 MAPK, p-p53 and PGC1α mRNA were all increased (P < 0.05). Post-exercise CWI had no effect on these responses. Consistent with the lack of a response following a single session, regular post-exercise CWI had no effect on PGC-1α or p53 protein content. Six weeks of SIT increased peak aerobic power, VO2peak, maximal uncoupled respiration (complexes I and II), and 2-km time-trial performance (P < 0.05). However, regular CWI had no effect on changes in these markers, consistent with the lack of response in the markers of mitochondrial biogenesis. While these observations suggest CWI is not detrimental to endurance adaptations following six weeks of SIT, they question whether post-exercise CWI is an effective strategy to promote mitochondrial biogenesis and improvements in endurance performance.
Hypertension is a complex disease affecting 78 million adults in the United States. The etiology of essential hypertension is unknown and current experimental models do not recapitulate all behavioral and physiological characteristics of the pathology. Researchers should assess the translational capacity of these models and look to other animal models for the discovery of new therapies. Chlorocebus aethiops sabaeus, the African Green Monkey (AGM), is a nonhuman primate that develops spontaneous hypertension and is a novel translational model for the study of hypertension and associated diseases. In a group of 424 adult AGMs, 37% (157/424) exhibited systolic blood pressures (SBP) >140 mmHg (SBP: 172.0±2.2 mmHg) and were characterized as hypertensive (HT). 44% (187/424) were characterized as normotensive with SBP <120 mmHg (NT, SBP: 99.6±1.0 mmHg) and the remaining 18% (80/424) as borderline hypertensive (BHT, SBP: 130.6±0.6 mmHg). Compared to NT animals, HT AGMs are older (8.7±0.6 vs 12.4±0.7 years, p<0.05) with elevated heart rates (121.3±1.91 vs 34.3±2.1 BPM, p<0.05). BHT animals had average heart rates of 138.2±3.1 BPM (p<0.05 vs. NT) and were 11.00±0.9 years old. NT and HT animals had similar levels of angiotensinogen gene expression, plasma renin activity, and renal cortical renin content (p>0.05). HT monkeys exhibit renal vascular remodeling (wall/lumen ratio NT 0.11±0.01 vs HT 0.15±0.02, p<0.05) and altered glomerular morphology (Bowman's capsular space: NT 30.9±1.9% vs HT 44.4±3.1%, p<0.05). The hypertensive AGM is an animal model that is highly similar to humans and may help identify novel, effective targets for the treatment of hypertension.
The objective of the present investigation was to determine whether energy restriction (ER) influences expression of skeletal muscle specific microRNA in circulation (c-myomiR), and if changes in c-myomiR are associated with rates of whole-body protein synthesis. Sixteen older (64 ± 2 yrs) overweight (28.5 ± 1.2 kg·m-2) males enrolled in this 35-day controlled feeding trial. A 7-day weight maintenance (WM) period was followed by 28 days of 30% ER. Whole-body protein turnover was determined from 15N-glycine enrichments in 24-hr urine collections, and c-myomiR (miR-1-3p, miR-133a-3p, miR-133b, and miR-206) expressions were assessed from serum samples using RT-qPCR at the conclusions of WM and ER. Participants lost 4.4 ± 0.3 kg body mass during ER (P < 0.05). Following 28 days of ER miR-133a and miR-133b expression was upregulated (P < 0.05) compared to WM. When all four c-myomiR were grouped as a c-myomiR Score (sum of the median fold change of all myomiR), overall expression of c-myomiR was higher (P < 0.05) at ER versus WM. Backward linear regression analysis of whole-body protein synthesis, breakdown, and carbohydrate, fat and protein oxidation determined protein synthesis to be the strongest predictor of c-myomiR Score. An inverse association (P < 0.05) was observed with ER c-myomiR Scores and whole-body protein synthesis (r = -0.729, r2 = -0.530). Findings from the present investigation provide evidence that upregulation in c-myomiR expression profiles in response to short-term ER are associated with lower rates of whole-body protein synthesis.
Amphibian neuromuscular junctions (NMJs) are composed of hundreds of neurotransmitter release sites which exhibit non-uniform transmitter release probabilities and demonstrated seasonal modulation. We examined whether recruitment of release sites is variable when the extracellular calcium concentration ([Ca2+]o) is increased in the wet and dry seasons. The amount of transmitter released from the entire nerve terminal increases by approximately the 4th-power as [Ca2+]o is increased. Toad (Bufo marinus) NMJs were visualised using DiOC2(5)-fluorescence and focal loose patch extracellular recordings were used to record the end-plate currents (EPCs) from small groups of release sites. Quantal content (e), average probability of quantal release (pe) and the number of active release sites (ne) were determined for different [Ca2+]o. Our results indicated that the recruitment of quantal release sites with increasing [Ca2+]o differs spatially (between different groups of release sites) and also temporally (in different seasons). These differences were reflected by the non-uniform alterations in pe and ne. Most release site groups demonstrated an increase in both pe and ne when [Ca2+]o increased. In approximately 30% of release site groups examined, pe decreased while ne increased only during the active period (wet season). Although the dry season induced parallel right shift in the quantal release versus extracellular calcium concentration, when compared to the wet season, the dependence of quantal content on [Ca2+]o was not changed. These results demonstrate the flexibility, reserve and adaptive capacity of neuromuscular junctions in maintaining appropriate levels of neurotransmission.
Skeletal muscle stem cells play a critical role in regeneration of myofibers. We have previously demonstrated that chronic binge alcohol (CBA) markedly attenuates myoblast differentiation potential and myogenic gene expression. Muscle specific microRNAs are implicated in regulating myogenic genes. The aim of this study was to determine whether myoblasts isolated from asymptomatic CBA administered SIV-infected macaques treated with antiretroviral therapy (ART) showed similar impairments and if so, to elucidate potential underlying mechanisms. Myoblasts were isolated from muscle at 11 months post-SIV infection from CBA/SIV and from time matched sucrose-treated SIV-infected (SUC/SIV) macaques and controls. Myoblasts isolated from SUC/SIV and CBA/SIV animals had a significant decrease in myoblast differentiation and myogenic gene expression compared to myoblasts from controls. SIV and CBA decreased muscle specific, miR-206, in plasma and muscle and SIV decreased miR-206 expression in myoblasts, with no statistically significant changes in other muscle specific microRNAs. This was associated with an observed significant increase in histone deacetylase 4 (HDAC4) and decrease in myogenic enhancer factor 2C (MEF2C) expression in CBA/SIV muscle. Transfection with miR-206 inhibitor decreased myotube differentiation, increased expression of HDAC4, and decreased MEF2C, suggesting a critical role of miR-206 in myogenesis. Moreover, HDAC4 was confirmed as a direct miR-206 target. These results support a mechanistic role for decreased miR-206 in suppression of myoblast differentiation resulting from chronic alcohol and SIV infection. The parallel changes in skeletal muscle and circulating levels of miR-206 warrant studies to establish the possible use of plasma miR-206 as an indicator of impaired muscle function.
The long-term consequences of early life nicotine exposure are poorly defined. Approximately 8-10% of women report smoking during pregnancy, and this may promote aberrant development in the offspring. To this end, we investigated potential enduring effects of perinatal nicotine exposure on murine sleep and affective behaviors in adulthood (~13-15 weeks of age) in C57Bl6j mice. Mothers received a water bottle containing 200 µg/ml nicotine bitartrate dihydrate in 2% w/v saccharin or pH-matched 2% saccharin with 0.2% (v/v) tartaric acid throughout pregnancy and prior to weaning. Upon reaching adulthood, offspring were tested in the open field and elevated plus maze, as well as the forced swim and sucrose anhedonia tests. Nicotine-exposed male (but not female) mice reduced mobility in the open field, but no differences were observed in anxiety-like or depressive-like responses. Upon observing this male-specific phenotype, we further assessed sleep-wake states via wireless EEG/EMG telemetry. Following baseline recording, we assessed whether mice exposed to nicotine altered their homeostatic response to 5 hours of total sleep deprivation, and whether nicotine influenced responses to a powerful somnogen (i.e., lipopolysaccharides; LPS). Males exposed to perinatal nicotine decreased the percent time spent awake and increased time in non-rapid eye movement (NREM) sleep, without changes to REM sleep. Nicotine-exposed males also displayed exaggerated responses (increased time asleep and NREM spectral power) to sleep deprivation. Nicotine-exposed animals additionally had blunted EEG slow-wave responses to LPS administration. Together, our data suggest that perinatal nicotine exposure has long-lasting effects on normal sleep and homeostatic sleep processes into adulthood.
Although interleukin (IL)-33, a member of the IL-1 cytokine family, plays pro-inflammatory roles in immune cells as an "alarmin", little is known regarding the biological actions of IL-33 on vascular endothelial cells. To investigate the effects of IL-33 on vascular endothelial cells, we first screened the IL-33-regulated proteins in human umbilical vein endothelial cells (HUVECs) using a dot blot array, and observed that IL-33 markedly increased growth-regulated oncogene (GRO)-α, a chemokine that is also known as CXCL1. Real time reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) demonstrated that IL-33 induced the GRO-α expression and secretion in HUVECs in a dose- and a time-dependent manner. Western immunoblot assay revealed that IL-33 activated the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 and c-Jun N-terminal kinase (JNK). In addition, translocation of nuclear factor-kappa B (NF-B) p65 to the nucleus of HUVECs was observed by IL-33 stimulation. Further, treatment with pharmacological inhibitors against ERK1/2 (PD98059), JNK (SP600125), or NF-B (BAY11-7085) significantly suppressed IL-33-induced GRO-α gene expression and secretion from HUVECs. Moreover, immunohistochemical staining demonstrated that IL-33 and GRO-α co-expressed in the endothelium of human carotid atherosclerotic plaque. Taken together, the present study indicates that IL-33 localized in the human atherosclerotic plaque increases GRO-α mRNA expression and protein secretion via activation of ERK1/2, JNK, and NF-B in HUVECs, suggesting that IL-33 plays an important role in the pathophysiology and development of atherosclerosis.
Heart rate variability (HRV) has become an important clinical marker of cardiovascular health and a research measure for the study of the cardiac conduction system and its autonomic controls. While zebrafish (Danio rerio) is an ideal vertebrate model for understanding heart development, HRV has only recently been investigated in this system. We have previously demonstrated that nkx2.5 and nkx2.7, two homologues of Nkx2-5 expressed in zebrafish cardiomyocytes, play vital roles in maintaining cardiac chamber-specific characteristics. Given observed defects in ventricular and atrial chamber identities in nkx2.5-/- embryos coupled with conduction system abnormalities in murine models of Nkx2.5 insufficiency, we postulated that reduced HRV would serve as a marker of poor cardiac health in nkx2.5 mutants and in other zebrafish models of human congenital heart disease. Using live video image acquisition, we derived beat-to-beat intervals to compare HRV in wild-type and nkx2.5-/- embryos. Our data illustrate that the nkx2.5 loss-of-function model exhibits increased heart rate and decreased HRV when compared to wild type during embryogenesis. These findings validate HRV analysis as a useful quantitative tool for assessment of cardiac health in zebrafish and underscore the importance of nkx2.5 in maintaining normal heart rate and HRV during early conduction system development.
Glucose is a crucial substrate essential for cell survival and function. Changes in glucose levels impact neuronal activity and glucose deprivation increases feeding. Several brain regions have been shown to respond to glucoprivation, including the nucleus of the solitary tract (NTS) in the brainstem. The NTS is the primary site in the brain that receives visceral afferent information from the gastrointestinal tract. The catecholaminergic (CA) subpopulation within the NTS modulates many homeostatic functions including cardiovascular reflexes, respiration, food intake, arousal and stress. However, it is not known if they respond to changes in glucose. Here we determined whether NTS-CA neurons respond to changes in glucose concentration and the mechanism involved. We found that decreasing glucose concentrations from 5mM to 2mM to 1mM, significantly decreased action potential firing in a cell-attached preparation, while increasing it back to 5 mM increased the firing rate. This effect was dependent on glutamate release from afferent terminals and required presynaptic 5-HT3Rs. Decreasing the glucose concentration also decreased both basal and 5-HT3R-agonist induced increase in the frequency of spontaneous glutamate inputs onto NTS-CA neurons. Low glucose also blunted 5-HT induced inward currents in nodose ganglia neurons, which are the cell bodies of vagal afferents. The effect of low glucose in both nodose ganglia cells and in NTS slices was mimicked by the glucokinase inhibitor glucosamine. This study suggests that NTS-CA neurons are glucosensing through a presynaptic mechanism that is dependent on vagal glutamate release, 5-HT3R activity, and glucokinase.
This study investigated the ability to sustain quadriceps central motor drive while subjected to localized heat and metaboreceptive feedback from the contralateral leg. Eight active males each completed two counter-balanced trials, in which muscle temperature (Tm) of a single-leg (TEMP-LEG) was altered to 29.4 (COOL) or 37.6°C (WARM), while the contralateral leg (CL-LEG) remained thermoneutral; 35.3 and 35.2°C Tm in COOL and WARM respectively. To activate metaboreceptive feedback, participants first performed one 120-s isometric maximal voluntary contraction (MVC) of the knee extensors in the TEMP-LEG, immediately followed by post-exercise muscle ischemia (PEMI) via femoral blood flow occlusion. To assess central motor drive of a remote muscle group immediately following PEMI, another 120-s MVC was subsequently performed in the CL-LEG. Voluntary muscle activation (VA) was assessed using the twitch interpolation method. Perceived mental effort and limb discomfort were also recorded. In a cooled muscle, a significant increase in mean force output and mean VA (force, p<0.001; VA, p<0.05) as well as a significant decrease in limb discomfort (p<0.05) occurred during the sustained MVC in the TEMP-LEG. However, no differences between Tm were observed in mean force output, mean VA or limb discomfort during the sustained MVC in the CL-LEG (Force, p=0.33; VA, p>0.68, limb discomfort, p=0.73). The present findings suggest that elevated local Tsk and Tm can increase limb discomfort and decrease central motor drive, but this does not limit systemic motor activation of a thermoneutral muscle group.
Adaptations to heat and hypoxia are typically studied in isolation, but are often encountered in combination. Whether the adaptive response to multiple stressors affords the same response as when examined in isolation is unclear. We examined: i) the influence of overnight moderate normobaric hypoxia on the time course and magnitude of adaption to daily heat exposure; ii) whether heat acclimation (HA) was ergogenic and if this was influenced by an additional hypoxic-stimulus. Eight males (VO2max=58.5[8.3] mL·kg-1·min-1) undertook two 11-day HA programmes (balanced-crossover design), once with overnight normobaric hypoxia (8[1] h per night; 10 nights; FIO2=0.156; SpO2=91[2]% [HAHyp]) and once without (HACon). Days 1, 6, 11 were exercise-heat stress tests (HST [40°C, 50% RH]); days 2-5 and 7-10 were isothermal-strain (target rectal temperature [Tre] ~38.5°C), exercise-heat sessions. A graded exercise test and 30-minute cycle trial were undertaken pre, post and 14-days after HA in temperate-normoxia (22°C, 55% RH; FIO2=0.209). HA was evident on day 6 (e.g. reduced Tre, mean skin temperature [Tsk], heart rate, sweat [Na+], P<0.05) with additional adaptations on day 11 (further reduced Tsk, heart rate). HA increased plasma volume (+5.9[7.3]%) and erythropoietin concentration (+1.8[2.4] mIU/mL); tHbmass was unchanged. Peak power output (+12[20] W), lactate threshold (+15[18] W) and work done (+12[20] kJ) increased following HA. The additional hypoxic-stressor did not affect these adaptations. In conclusion, a separate moderate overnight normobaric hypoxic-stimulus does not affect the time-course or magnitude of HA. Performance may be improved in temperate-normoxia following HA, but this is unaffected by an additional hypoxic stressor.
Some studies have observed a functional relationship between sweating and skin blood flow. However, the implications of this relationship during physiologically-relevant conditions remain unclear. We manipulated sudomotor activity through changes in sweating efficiency to determine if parallel changes in vasomotor activity are observed. Eight young males completed two trials at 36°C and two trials at 42°C. During these trials, air temperature remained constant while ambient vapor pressure increased from 1.6 to 5.6 kPa over 2 hours. Forced airflow across the skin was used to create conditions of high (HiSeff) or low (LoSeff) sweating efficiency. Local sweat rate (LSR), local skin blood flow (SkBF), as well as mean skin and esophageal temperatures were measured continuously. It took longer for LSR to increase during HiSeff at 36°C (HiSeff: 99 ± 11 vs. LoSeff: 77 ± 11 min, P<0.01) and 42°C (HiSeff: 72 ± 16 vs. LoSeff: 51 ± 15 min, P<0.01). In general, an increase in LSR preceded the increase in SkBF when expressed as ambient vapor pressure and time for all conditions (P<0.05). However, both responses were activated at a similar change in mean body temperature (average across all trials, LSR: 0.26 ± 0.15 vs. SkBF: 0.30 ± 0.18°C, P=0.26). These results demonstrate that altering the point at which local sweat rate is initiated during heat exposure is paralleled by similar shifts for the increase in SkBF. However, local sweat production occurs before an increase in SkBF, suggesting that SkBF is not necessarily a pre-requisite for sweating.
Background/Aims: Patients with chronic kidney disease (CKD) commonly complain upper gastrointestinal (GI) symptoms, especially anorexia. Hemodialysis (HD) has been noted to improve GI symptoms; however, underlying mechanisms are unclear. This study was designed 1) to study effects of HD on GI symptoms and gastric slow waves; 2) to investigate possible roles of ghrelin and glucagon-like peptide-1 (GLP-1). Methods: This study recruited 13 healthy controls, 20 CKD patients without HD (CKD group) and 18 CKD patients with HD (HD group). Dyspeptic symptoms, autonomic functions, gastric slow waves, and plasma level of ghrelin and GLP-1 were analyzed. Results: 1) The CKD patients with HD showed markedly lower scores of anorexia (0.6±0.2 vs. 3.2±0.4, P<0.001) compared to patients without HD. 2) The CKD group but not HD group showed a significant reduction (25.6%) in the percentage of normal gastric slow waves, compared to controls. 3)The CKD group exhibited a significantly lower ghrelin level compared to the HD group (26.8±0.9ng/L vs. 34.1±2.3ng/L, P<0.02) and a higher GLP-1 level(29.4±2.8 pmol/L vs. 20.0±2.1pmol/L, P<0.05) compared to controls. Moreover, the percentage of normal slow waves was positively correlated with ghrelin (r=0.385, P=0.019) but negatively correlated with GLP-1 (r=-0.558, P<0.001) in all CKD patients. Conclusions: Hemodialysis improves upper GI symptoms and gastric slow waves in CKD patients. An increase in ghrelin and a decrease in GLP-1 might be involved in the HD-induced improvement in gastric slow waves.
Mammalian hibernators, such as golden-mantled ground squirrels (Callospermophilus lateralis; GMGS), cease to feed while reducing metabolic rate and body temperature during winter months, surviving exclusively on endogenous fuels stored prior to hibernation. We hypothesized that mitochondria, the cellular sites of oxidative metabolism, undergo tissue-specific seasonal adjustments in carbohydrate and fatty acid utilization to facilitate or compliment this remarkable phenotype. To address this, we performed high-resolution respirometry of mitochondria isolated from GMGS liver, heart, skeletal muscle, and brown adipose tissue (BAT) sampled during summer (active), fall (prehibernation), and winter (hibernation) seasons using multi-substrate titration protocols. Mitochondrial phospholipid composition was examined as a postulated intrinsic modulator of respiratory function across tissues and seasons. Respirometry revealed seasonal variations in mitochondrial oxidative phosphorylation capacity, substrate utilization, and coupling efficiency that reflected the distinct functions and metabolic demands of the tissues they support. A consistent finding across tissues was a greater influence of fatty acids (palmitoylcarnitine) on respiratory parameters during the prehibernation and hibernation seasons. In particular, fatty acids had a greater suppressive effect on pyruvate-supported oxidative phosphorylation in heart, muscle and liver mitochondria, and enhanced uncoupled respiration in BAT and muscle mitochondria in the colder seasons. Seasonal variations in the mitochondrial membrane composition reflected changes in the supply and utilization of polyunsaturated fatty acids, but were generally mild and inconsistent with functional variations. In conclusion, mitochondria respond to seasonal variations in physical activity, temperature and nutrient availability in a tissue-specific manner that compliment circannual shifts in the bioenergetic and thermoregulatory demands of mammalian hibernators.
Fish routinely experience environmental hypoxia and have evolved various strategies to tolerate this challenge. Given the key role of the corticotropin-releasing factor (CRF) system in coordinating the response to stressors and its cardioprotective actions against ischemia in mammals, we sought to characterize the cardiac CRF system in zebrafish and its role in hypoxia tolerance. We established that all genes of the CRF system, the ligands CRFa, CRFb, urotensin 1 (UTS1) and urocortin 3 (UCN3), the two receptor subtypes (CRFR1 and CRFR2), and the binding protein (CRFBP) are expressed in the heart of zebrafish: crfr1 > crfr2 = crfbp > crfa > ucn3 > crfb > uts1. In vivo, exposure to 5% O2 saturation for 15 min and 90 min recovery resulted in 4-5 fold increases in whole heart crfb and ucn3 mRNA levels but did not affect the gene expression of other CRF system components. In vitro, as assessed by monitoring caspase 3 activity and the number of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells, pre-treatment of excised whole hearts with CRF or UCN3 for 30 min prevented the increase in apoptosis associated with exposure to 1% O2 saturation for 30 min and 24 h recovery. Lastly, addition of the non-selective CRF receptor antagonist, αh-CRF(9-41), prevented the cytoprotective effects of CRF. We show that the CRF system is expressed in fish heart, is up-regulated by hypoxia, and is cytoprotective. These findings identify a novel role for the CRF system in fish and a new strategy to tolerate hypoxia.
Resistance breathing improves tolerance to central hypovolemia induced by lower body negative pressure (LBNP), but this is not related to protection of anterior cerebral blood flow (indexed by mean middle cerebral artery velocity, MCAv). We hypothesized that inspiratory resistance breathing improves tolerance to central hypovolemia by maintaining cerebral oxygenation (ScO2), and protecting cerebral blood flow in the posterior cerebral circulation (indexed by posterior cerebral artery velocity, PCAv). Eight subjects (4M/4F) completed two experimental sessions of a presyncopal-limited LBNP protocol (3 mmHg/min onset rate) with and without (Control) resistance breathing via an impedance threshold device (ITD). ScO2 (via near-infrared spectroscopy), MCAv and PCAv (both via transcranial Doppler ultrasound), and arterial pressure (via finger photoplethysmography) were measured continuously. Hemodynamic responses were analyzed between the Control and ITD condition at baseline (T1) and the time representing 10-s prior to presyncope in the Control condition (T2). While breathing on the ITD increased LBNP tolerance from 1506±75 s to 1704±88 s (P=0.003), both mean MCAv and mean PCAv were similar between conditions at T2 (P≥0.46), and decreased by the same magnitude with and without ITD breathing (P≥0.53). ScO2 also decreased by approximately 9% with or without ITD breathing at T2 (P=0.972), and there were also no differences in deoxygenated (dHb) or oxygenated hemoglobin (HbO2) between conditions at T2 (P≥0.43). There was no evidence that protection of regional cerebral blood velocity (i.e., anterior or posterior cerebral circulation), nor cerebral oxygen extraction played a key role in the determination of tolerance to central hypovolemia with resistance breathing.
Consumption of a high-fat high-fructose diet (western diet, [WD]) promotes vascular stiffness, a critical factor in the development of cardiovascular disease (CVD). Obese and diabetic women exhibit greater arterial stiffness than men, which contributes to the increased incidence of CVD in women. Further, high-fructose diets result in elevated plasma concentrations of uric acid via xanthine oxidase (XO) activation, and are also associated with vascular stiffness. In turn, uric acid levels lead to vascular stiffness and CVD in women. However, the mechanisms by which fructose contributes to vascular stiffness in females remain to be fully uncovered. We examined the role of XO inhibition on vascular stiffness in female C57BL/6J mice fed a WD or regular chow for 16 weeks. WD feeding resulted in increased arterial stiffness, measured by atomic force microscopy in aortic explants (16.19±1.72 vs 5.21±0.54 kPa, p<0.05), as well as abnormal aortic endothelium-dependent and independent vasomotor responses. XO inhibition using allopurinol (widely utilized in the clinical setting) significantly improved vasomotor responses and stiffness (16.9±0.50 vs 3.44± 0.50 kPa, p<0.05), while simultaneously lowering serum uric acid levels (0.55±0.98 vs 0.21±0.04 mg/dL, p<0.05). Also, allopurinol improved WD-induced markers of fibrosis and oxidative stress in aortic tissue, as analyzed by immunohistochemistry and transmission electronic microscopy. Collectively, these results demonstrate that XO inhibition protects against WD-induced vascular oxidative stress, fibrosis, impaired vasorelaxation and aortic stiffness in females. Further, excessive oxidative stress resulting from XO activation appears to play a key role in mediating vascular dysfunction induced by chronic exposure to WD in females.
Acetaminophen (APAP) given during chronic exercise reduces skeletal muscle collagen and cross-linking in rats. We propose that the effect of APAP on muscle ECM may, in part, be mediated by dysregulation of the balance between matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs). The purpose of this study was to evaluate the impact of APAP consumption during acute resistance exercise (RE) on several regulators of the ECM in human skeletal muscle. In a double-blinded, placebo-controlled, randomized cross-over design, recreationally active men (n=8, 25±2yr) performed two trials of knee extension. Placebo (PLA) or APAP (1000mg/6h) was given for 24 hours prior to and immediately following RE. Vastus lateralis biopsies were taken at baseline, 1-hr, and 3-hr post-RE. RT-qPCR was used to determine differences in mRNA expression. MMP-2, COL1A1, and COL3A1 mRNA expression were not altered by exercise or APAP (p>0.05). When compared to PLA, TIMP-1 expression was lower at 1-hr post-RE during APAP conditions but greater than PLA at 3-hr post-RE (p<0.05). MMP-9 expression and protein levels were elevated at 3-hr post-RE independent of treatment (p<0.05). LOX expression was greater at 3-hr post-RE during APAP consumption (p<0.05) when compared to PLA. MMP-2 and TIMP-1 protein were not altered by RE or APAP (p>0.05). Phosphorylation of ERK1/2 and p38-MAPK increased (p<0.05) with RE but were not influenced by APAP. Our findings do not support our hypothesis and suggest that short-term APAP consumption prior to RE has a small impact on the measured ECM molecules in human skeletal muscle following acute RE.
Insulin stimulates skeletal muscle glucose uptake via activation of the protein kinase B/Akt (Akt) pathway. Recent studies suggest that insulin down-regulates AMP-activated protein kinase (AMPK) activity via Ser485/491 phosphorylation of the AMPK α-subunit. Thus, lower blood insulin concentrations may induce AMPK signal activation. Acute exercise is one method to stimulate AMPK activation; however, no study has examined the relationship between blood insulin levels and acute resistance exercise-induced AMPK pathway activation. Based on previous findings, we hypothesized that the acute resistance exercise-induced AMPK pathway activation would be augmented by disruptions in insulin secretion through a decrease in AMPKα Ser485/491 inhibitory phosphorylation. To test the hypothesis, 10-week-old male Sprague-Dawley rats were administered the toxin streptozotocin (STZ; 55 mg/kg) to destroy the insulin secreting β-cells. Three-day post-injection, the right gastrocnemius muscle from STZ and control rats was subjected to resistance exercise by percutaneous electrical stimulation. Animals were sacrificed 0, 1, or 3 h later; activation of the Akt/AMPK and downstream pathways in the muscle tissue were analyzed by western blotting and real-time PCR. Notably, STZ rats showed a significant decrease in basal Akt and AMPKα Ser485/491 phosphorylation. However, substantial exercise caused increases in both AMPKα Thr172 and acetyl-CoA carboxylase (ACC) Ser79 phosphorylation. Although no significant impact on resistance exercise-induced Akt pathway activation or glucose uptake was found, resistance exercise–induced PGC-1α gene expression was augmented by STZ treatment. Collectively, these data suggest that circulating insulin levels may regulate acute resistance exercise-induced AMPK pathway activation and AMPK-dependent gene expression relating to basal AMPKα Ser485/491 phosphorylation.
Hagfish consume carrion, potentially exposing them to hypoxia, hypercarbia, and high environmental ammonia (HEA). We investigated branchial and cutaneous ammonia handling strategies by which Pacific hagfish (Eptatretus stoutii) tolerate and recover from high ammonia loading. Hagfish were exposed to HEA (20 mmol L-1) for 48 h to elevate plasma total ammonia (TAmm) levels before placement into divided chambers for a 4 h recovery period in ammonia-free seawater where ammonia excretion (JAmm) was measured independently in the anterior and posterior compartments. Localized HEA exposures were also conducted by subjecting hagfish to HEA in either the anterior or posterior compartments. During recovery, HEA-exposed animals increased JAmm in both compartments, with the posterior compartment comprising ~20% of the total JAmm compared to ~11% in non-HEA exposed fish. Plasma TAmm increased substantially when whole hagfish, and the posterior regions, were exposed to HEA. Alternatively, plasma TAmm did not elevate following anteriorly-localized HEA exposure. JAmm was concentration-dependent (0.05-5 mmol L-1) across excised skin patches at up to 8-fold greater rates than in skin sections that were excised from HEA-exposed hagfish. Skin excised from more posterior regions displayed greater JAmm than those from more anterior regions. Immunohistochemistry with hagfish-specific anti-rhesus glycoprotein type c (α-hRhcg; ammonia transporter) antibodies was characterized by staining on the basal aspect of hagfish epidermis while Western blotting demonstrated greater expression of Rhcg in more posterior skin sections. We conclude that cutaneous Rhcg proteins are involved in cutaneous ammonia excretion by Pacific hagfish, and that this mechanism could be particularly important during feeding.
Abrupt cessation of chronic alcohol consumption triggers signaling cascades that harm vulnerable brain regions and produce neurobehavioral deficits. We have demonstrated that a program of intermittent, normobaric hypoxia training (IHT) in rats prevents neurobehavioral impairment resulting from abrupt ethanol withdrawal (EW). Moreover, EW induced expression of stress-activated protein kinase p38 and presenilin 1 (PS1), the -secretase component that produces the neurotoxic amyloid-β (Aβ) peptides, Aβ40 and Aβ42. We tested the hypotheses that (1) IHT limits EW-induced activation of the p38-PS1 axis, thereby attenuating -secretase activation and Aβ accumulation, and (2) EW disables heat shock protein 25 (HSP25), a p38 substrate, molecular chaperone and antioxidant, and provokes protein carbonylation in a manner suppressed by IHT. Adult male rats completed two cycles of 4 wk ethanol diet (0 or 6.5% w/v) and 3 wk EW. A 20 d IHT program of cyclic, 5-8 min exposures to 9.5-10% FIO2 was administered during the first EW phase. PS1, phosphorylated p38 (P-p38) and HSP25 were analyzed by immunoblot, PS1 messenger RNA by quantitative polymerase chain reaction, protein carbonyl content by spectrometry, and Aβ40 and Aβ42 contents by enzyme-linked immunosorbent assay, in prefrontal cortical extracts. IHT attenuated the EW-associated increases in PS1, P-p38, Aβ40, Aβ42 and protein carbonyl contents, but not that of PS1 messenger RNA, while preserving functionally competent HSP25 dimers in EW rats. Collectively, these findings demonstrate that IHT attenuates EW-activation of the p38-PS1--secretase axis, thereby dampening Aβ accumulation, and prevents EW-induced oxidative protein damage.
Intrauterine growth restriction (IUGR) is associated with persistent metabolic complications, but information is limited for IUGR infants. We determined glucose-stimulated insulin secretion (GSIS) and insulin sensitivity in young lambs with placental insufficiency-induced IUGR. Lambs with hyperthermia-induced IUGR (n=7) were compared to control lambs (n=8). GSIS was measured at 8±1 days of age, and at 15±1 days body weight-specific glucose utilization rates were measured with radiolabeled D-glucose during a hyperinsulinemic-euglycemic clamp (HEC). IUGR lambs weighed 23% less (P<0.05) than controls at birth. Fasting plasma glucose and insulin concentrations were not different between IUGR and controls for either study. First-phase insulin secretion was enhanced 2.3-fold in IUGR lambs compared to controls. However, second-phase insulin concentrations, glucose-potentiated arginine-stimulated insulin secretion, and β-cell mass were not different, indicating that IUGR β-cells have an intrinsic enhancement in acute GSIS. Compared to controls, IUGR lambs had higher body weight-specific glucose utilization rates and greater insulin sensitivity at fasting (1.6-fold) and hyperinsulinemic periods (2.4-fold). Improved insulin sensitivity for glucose utilization was not due to differences in skeletal muscle insulin receptor and glucose transporters 1 and 4 concentrations. Plasma lactate concentrations during HEC were elevated in IUGR lambs compared to controls, but no differences were found for glycogen content or citrate synthase activity in liver and muscle. Greater insulin sensitivity for glucose utilization and enhanced acute GSIS in young lambs are predicted from fetal studies, but may promote conditions that exaggerate glucose disposal and lead to episodes of hypoglycemia in IUGR infants.
A sedentary lifestyle is a major risk factor for cardiovascular disease, and both conditions are associated with overactivity of the sympathetic nervous system. Ongoing discharge of sympathetic nerves is regulated by the rostral ventrolateral medulla (RVLM), which in turn is modulated by the primary excitatory and inhibitory neurotransmitters glutamate and -amino-butyric acid (GABA), respectively. We reported previously that sedentary conditions enhance GABAergic modulation of sympathoexcitation in the RVLM, despite overall increased sympathoexcitation. Thus, the purpose of this study was to test the hypothesis that sedentary conditions increase responsiveness to GABA in RVLM. Male Sprague-Dawley rats performed either chronic wheeling running or remained sedentary for 12-15 weeks. Animals were instrumented to perform RVLM microinjections under Inactin anesthesia while recording mean arterial pressure (MAP) and splanchnic sympathetic nerve activity (SSNA). Unilateral microinjections of GABA (30 nl, 0.3-600 mM) into the RVLM produced dose-dependent decreases in MAP and SSNA; however, no group differences were observed. Inhibition of the contralateral RVLM (muscimol, 2 mM, 90 nl) caused decreases in MAP and SSNA that were not different between groups but enhanced decreases in SSNA to GABA in sedentary rats only. In sinoaortic denervated rats, GABA microinjections prior to or after inhibition of the contralateral RVLM caused decreases in MAP and SSNA that were not different between groups. Results suggest that contralateral RVLM plays an important role in buffering responses to inhibition of the ipsilateral RVLM under sedentary conditions. Enhanced sympathoinhibition may act to reduce already elevated sympathetic nervous system activity following sedentary conditions.
Direct intracerebroventricular injection of angiotensin II causes increases in blood pressure, salt and water intake, presumably mimicking an effect mediated by an endogenous mechanism. The subfornical organ (SFO) is a potential source of cerebrospinal fluid (CSF) angiotensin-I (Ang-I) and angiotensin-II (Ang-II), and thus we hypothesized that the SFO has a secretory function. Endogenous levels of angiotensinogen (AGT) and renin are very low in the brain. We therefore examined the immunohistochemical localization of angiotensin peptides and AGT in the SFO, and AGT in the CSF in two transgenic models over-expressing either human AGT (A+ mice), or both human AGT and human renin (SRA mice) in the brain. Measurements were made at baseline and following volumetric depletion of CSF. Ultrastructural analysis with immunoelectron microscopy revealed that superficially located Ang-I/Ang-II and AGT immunoreactive cells in the SFO were vacuolated and opened directly into the ventricle. Withdrawal of CSF produced an increase in AGT in the CSF which was accompanied by a large decline in AGT immunoreactivity within SFO cells. Our data provide support for the hypothesis that the SFO is a secretory organ releasing AGT and possibly Ang-I/Ang-II into the ventricle at least under conditions when renin-angiotension system genes are overexpressed in mice.
Muscle metaboreflex activation during submaximal dynamic exercise, increases arterial pressure primarily via increases in cardiac output as there is little systemic vasoconstriction. Indeed, in normal animals we have often shown a small, but significant, peripheral vasodilation during metaboreflex activation, which is mediated, at least in part, by release of epinephrine and activation of vascular β2 receptors. We tested whether this vasodilation is in part due to increased release of nitric oxide caused by the rise in cardiac output eliciting endothelium-dependent flow-mediated vasodilation. The muscle metaboreflex was activated via graded reductions in hindlimb blood flow during mild exercise with and without nitric oxide synthesis blockade (L-nitroarginine methyl ester (L-name); 5mg/kg). We assessed the role of increased cardiac output in mediating peripheral vasodilation via the slope of the relationship between the rise in non-ischemic vascular conductance (conductance of all vascular beds excluding hindlimbs) vs. the rise in cardiac output. L-name increased mean arterial pressure at rest and during exercise. The metaboreflex-induced increases in mean arterial pressure were unaltered by L-name; whereas, the increases in cardiac output and non-ischemic vascular conductance were attenuated. However, the slope of the relationship between non-ischemic vascular conductance and cardiac output was not affected by L-name indicating that the rise in cardiac output did not elicit vasodilation via increased release of nitric oxide. Thus, although nitric oxide is intrinsic to the vascular tonus, endothelial-dependent flow-mediated vasodilation plays little role in the small peripheral vasodilation observed during muscle metaboreflex activation.
Autophagy plays a major role in podocytes health and disease. P62, also known as Sequestosome-1, is a marker for autophagic activity and is required for the formation and degradation of ubiquitnated protein by autophagy. Knockout of p62 enhanced extracellular signal-regulated kinases (ERK1/2) activity. (Pro)renin receptor (PRR) is expressed in podocytes where it contributes to the homeostasis of these cells. The influence of autophagy on (Pro)renin receptor (PRR) expression is unknown. We hypothesized that in podocytes, upregulation of autophagic activity increases PRR expression via reduction of p62 and stimulation of ERK1/2 signaling pathway. Cultured mouse podocytes were treated with the autophagy activators, rapamycin or Earles's balanced salt solution (EBSS), for 48 hours. Both rapamycin and EBSS significantly decreased p62 protein levels, increased ERK1/2 activation by phosphorylating pTpY185/187 and increased mRNA and protein expressions of PRR. Utilizing confocal microscopy, demonstrated that rapamycin and EBSS significantly decreased p62/SQSTM1, and increased PRR protein expressions. Similarly, by enhancing autophagic activity by transfection with autophagy related 5 (ATG5) cDNA or ATG7 cDNA, results similar to those observed with rapamycin and EBSS treatments were produced. Inhibition of autophagic flux with bafilomycin A1 reversed the effects of rapamycin. ERK1/2 inhibitor U0126 significantly attenuated mRNA and protein expressions of PRR in podocytes treated with rapamycin. In conclusion, upregulation of autophagy enhanced PRR expression through reduction of p62 and stimulation of ERK1/2 activity signaling pathway.
The guinea pig is an alternate small animal model for the study of metabolism, including insulin sensitivity. However, only one study to date has reported the use of the hyperinsulinemic-euglycemic clamp (HEC) in anaesthetized animals in this species, and the dose-response has not been reported. We therefore characterized the dose-response curve for whole-body glucose uptake using recombinant human insulin in the adult guinea pig. Inter-species comparisons with published data showed species differences in maximal whole body responses (guinea pig human < rat < mouse), and the insulin concentrations at which half-maximal insulin responses occurred (guinea pig > human rat > mouse). In subsequent studies, we used concomitant D-[3-3H]-glucose infusion to characterize insulin sensitivities of whole body glucose uptake, utilization, production, storage and glycolysis in young adult guinea pigs at human insulin doses that produced ~half (7.5 mU.min-1.kg-1) and near-maximal whole body responses (30 mU.min-1.kg-1). Although human insulin infusion increased rates of glucose utilization (up to 68%) and storage, and at high concentrations increased rates of glycolysis in females, glucose production was only partially suppressed (~23%), even at high insulin doses. Fasting glucose, metabolic clearance of insulin and rates of glucose utilization, storage and production during insulin stimulation were higher in female than male guinea pigs (P<0.05), but insulin sensitivity of these and whole body glucose uptake did not differ between sexes. This study establishes a method for measuring partitioned glucose metabolism in chronically catheterized conscious guinea pigs, allowing studies of regulation of insulin sensitivity in this species.
In skeletal muscle, resting intracellular Ca2+ concentration ([Ca2+]i) homeostasis is exquisitely regulated by Ca2+ transport across the sarcolemmal, mitochondrial and sarcoplasmic reticulum (SR) membranes. Of these three systems, the relative importance of the mitochondria in [Ca2+]i regulation remains poorly understood in in vivo skeletal muscle. We tested the hypothesis that the capacity for Ca2+ uptake by mitochondria is a primary factor in determining [Ca2+]i regulation in muscle at rest and following contractions. Tibialis anterior muscle of anesthetized PGC-1α overexpressing (PGC-1α OE, increased mitochondria model) and wild-type littermates (WT) mice were exteriorized in vivo and loaded with Fura-2 AM and Rhod-2 AM. Ca2+ buffering and mitochondrial [Ca2+] were evaluated at rest and during recovery from fatiguing tetanic contractions. In addition, the effects of pharmacological inhibition of SR (thapsigargin) and mitochondrial (FCCP) function were examined at rest. [Ca2+]i in WT remained elevated for the entire post-contraction recovery period but in PGC-1α OE [Ca2+]i returned to resting baseline. Thapsigargin immediately and substantially increased resting [Ca2+]i in WT whereas in PGC-1α OE this effect was delayed and markedly diminished. FCCP abolished this improvement of [Ca2+]i regulation in PGC-1α OE. Mitochondrial [Ca2+] accumulation was observed in PGC-1α OE following contractions and thapsigargin treatment. In the SR PGC-1α OE down-regulated SERCA1 (Ca2+ uptake) and PV (Ca2+ buffering) protein levels whilst mitochondrial Ca2+ uptake-related proteins (Mfn1, Mfn2 and MCU) were up-regulated. These data demonstrate a heretofore unappreciated role for skeletal muscle mitochondria in [Ca2+]i regulation in vivo following fatiguing tetanic contractions and at rest.
Endothelin-1 (ET-1) contributes to age-related endothelial dysfunction in men via the ETA receptor. However, there are sex differences in the ET-1 system, and ETB receptors are modulated by sex hormones. The purpose of this study was to test the hypothesis that ETB receptors contribute to impaired vasodilatory function in postmenopausal women (PMW). We measured flow-mediated dilation (FMD) using ultrasound, and cutaneous nitric oxide-mediated vasodilation during local heating (42°C) via laser Doppler flowmetry in 18 young (YW; 22±1 years) and 16 PMW (56±1 years). Cutaneous microdialysis perfusions of lactated Ringer's (control), an ETB receptor antagonist (BQ-788, 300nM), and an ETA receptor antagonist (BQ-123, 500 nM), were done through separate fibers, followed by perfusions of sodium nitroprusside (28mM) and local heating to 43°C (max). Cutaneous vascular conductance (CVC) was calculated as cutaneous blood flow/MAP, and expressed as a percent of maximal dilation. FMD (YW: 7.5±0.5 vs. PMW: 5.6±0.6 %) and cutaneous vasodilation (YW: 93±2 vs. PMW: 83±4 CVC % max) were lower in PMW (both P<0.05). Blockade of ETB receptors decreased cutaneous vasodilation in YW (87±2 CVC % max; P<0.05 vs. control), but increased vasodilation in PMW (93±1 CVC % max; P<0.05 vs. control). ETA receptor blockade had minimal effect in YW (92±1 CVC % max), but increased cutaneous vasodilation in PMW (91±2 CVC % max; P<0.05 vs. control). In conclusion, ETB receptors mediate vasodilation in YW, but this effect is lost after menopause. Impaired vasodilatory function in PMW is due in part to a loss of ETB mediated dilation.
Using red knots (Calidris canutus) as a model, we determined how changes in mass and metabolic activity of organs relate to temperature-induced variation in metabolic performance. In cold-acclimated birds, we expected large muscles and heart, improved oxidative capacity and lipid transport, and we predicted that this would explain variation in maximal thermogenic capacity (Msum). We also expected larger digestive and excretory organs in these birds and predicted that this would explain variation in basal metabolic rate (BMR). Knots kept at 5°C were 20% heavier and maintained 1.5 times more body fat than individuals kept at thermoneutrality (25°C). Cold-acclimated birds also had a BMR up to 32% higher and a Msum 16% higher than birds at 25°C. Organs were larger in the cold, with muscles and heart being 9-20% heavier and digestive and excretory organs being 21-36% larger than at thermoneutrality. Rather than the predicted digestive and excretory organs, the cold-induced increase in BMR correlated with changes in mass of the heart, pectoralis and carcass. Msum varied positively with the mass of the pectoralis, supracoracoideus and heart, highlighting the importance of muscles and cardiac function in cold endurance. Cold-acclimated knots also expressed upregulated capacity for lipid transport across mitochondrial membranes in their pectoralis and leg muscles, higher lipid catabolism capacity in their pectoralis muscles and elevated oxidative capacity in their liver and kidney. These adjustments may have contributed to BMR through changes in metabolic intensity. Our results also suggest indirect constraints on thermogenic capacity through limited cardiac capacity.
In spite of recent advances on the knowledgement of the neural control of cardiovascular function, the cause of sympathetic overactivity in neurogenic hypertension remains unknown. Studies from our laboratory point out that rats submitted to chronic intermittent hypoxia (CIH), an experimental model of neurogenic hypertension, present changes in the central respiratory network that impact the pattern of sympathetic discharge and the levels of arterial pressure. In addition to the fine coordination of respiratory muscle contraction and relaxation, essential for O2 and CO2 pulmonary exchanges, neurons of the respiratory network are precisely connected to the neurons controlling the sympathetic activity in the brainstem. This respiratory-sympathetic neuronal interaction provides adjustments in the sympathetic outflow to the heart and vasculature during each respiratory phase, according to the metabolic demands. Herein we report that CIH-induced sympathetic overactivity and mild hypertension are associated with increased frequency discharge of ventral medullary pre-sympathetic neurons. We also describe that their increased frequency discharge is dependent on synaptic inputs, mostly from neurons of the brainstem respiratory network, rather than to changes in their intrinsic electrophysiological properties. In perspective, we are taking into consideration the possibility that changes in the central respiratory rhythm/pattern generator contribute to increased sympathetic outflow and the development of neurogenic hypertension. Our experimental evidence provides support to the hypothesis that changes in the coupling of respiratory and sympathetic networks might be one of the unrevealed secrets of neurogenic hypertension in rats.
Glomerular damage is common in preeclampsia (PE), but the extent and etiology of tubular injury are not well understood. The aim of this study was to evaluate tubular injury in patients with PE and to assess whether it predates clinical disease. We performed a prospective cohort study of 315 pregnant women who provided urine samples at the end of the second trimester and at delivery. This analysis included women who developed PE (n=15), gestational hypertension (GH) (n=14), and normotensive controls (NC) (n=44). Urinary markers of tubular injury, alpha-1 microglobulin (A1M), retinol-binding protein (RBP), kidney-injury molecule-1 (KIM1), complement C5b-9, tissue inhibitor metalloproteinase-2 (TIMP-2), and insulin-like growth factor binding protein-7 (IGFBP-7) were measured by enzyme-linked immunosorbent assay (ELISA) and reported in relation to urine creatinine concentration. Second trimester concentrations of all markers were similar among groups. At delivery, A1M concentrations were higher in the PE group than in the GH and NC groups, as an A1M/creatinine ratio >13 (66.7%, 8.3%, and 35%, respectively; p=0.01). Concentrations of C5b-9 were higher in the PE group than in GH and NC groups (medians 9.85 ng/mg, 0.05 ng/mg, and 0.28 ng/mg, respectively; p=0.003). KIM1, RBP, TIMP-2, and IGFBP-7 concentrations did not differ among groups at delivery. In conclusion, proximal tubular dysfunction, as assessed by A1M and C5b-9, developed during the interval between the end of the second trimester and delivery in patients with PE. However, this was not matched by abnormalities in markers previously associated with tubular cell injury (KIM-1, IGFBP-7 and TIMP-2).
We herein investigated the effects of face/head and whole body cooling during passive heat stress on human somatosensory processing recorded by somatosensory-evoked potentials (SEPs) at C4' and Fz electrodes. Fourteen healthy subjects received a median nerve stimulation at the left wrist. SEPs were recorded at normothermic baseline (Rest), when esophageal temperature had increased by ~1.2 °C (Heat stress: HS) during passive heating, face/head cooling during passive heating (face/head cooling: FHC), and after HS (whole body cooling: WBC). The latencies and amplitudes of P14, N20, P25, N35, P45, and N60 at C4' and P14, N18, P22, and N30 at Fz were evaluated. Latency indicated speed of the subcortical and cortical somatosensory processing, while amplitude reflected the strength of neural activity. Blood flow in the internal and common carotid arteries (ICA and CCA, respectively) and psychological comfort were recorded in each session. Increases in esophageal temperature due to HS significantly decreased the amplitude of N60, psychological comfort, and ICA blood flow in the HS session, and also shortened the latencies of SEPs (all, p<0.05). While esophageal temperature remained elevated, FHC recovered the peak amplitude of N60, psychological comfort, and ICA blood flow toward pre-heat baseline levels as well as WBC. However, the latencies of SEPs did not recover in the FHC and WBC sessions. These results suggest that impaired neural activity in cortical somatosensory processing during passive HS was recovered by FHC, whereas conduction velocity in the ascending somatosensory input was accelerated by increases in body temperature.
Maternal high-fat diet (HFD) consumption during pregnancy decreased fetal body weight and impacted development of hypothalamic melanocortin neural circuitry in nonhuman primate offspring. We investigated whether these impairments during gestation persisted in juvenile offspring and examined the interaction between maternal and early postnatal HFD consumption. Adult dams consumed either a control diet (CTR, 15% calories from fat) or a high saturated-fat diet (HFD, 37% calories from fat) during pregnancy. Offspring were weaned onto a CTR or HFD at approximately 8 months of age. Offspring from HFD-fed dams displayed early catch-up growth and elevated body weight at 6 and 13 months of age. Maternal and postnatal HFD exposure reduced the amount of agouti-related peptide fibers in the paraventricular nucleus of the hypothalamus. Postnatal HFD consumption also decreased the amount of agouti-related peptide fibers in the arcuate nucleus of the hypothalamus. Postnatal HFD was associated with decreased food intake and increased activity. These results support and extend our previous findings of maternal diet effects on fetal development and reveal, for the first time in a nonhuman primate model, that maternal HFD-induced disturbances in offspring body weight regulation extended past gestation into the juvenile period. Maternal HFD consumption increases the risk for offspring developing obesity, with the developmental timing of HFD exposure differentially impacting the melanocortin system and energy balance regulation. The present findings provide translational insight into human clinical populations, suggesting that profound health consequences may await individuals later in life following intrauterine and postnatal HFD exposure.
Aerobic exercise has a positive impact on animals by enhancing skeletal muscle function and locomotor performance. Responses of skeletal muscle to exercise involve changes in energy metabolism, calcium handling, and the composition of contractile protein isoforms, which together influence contractile properties. Histone deacetylases (HDAC) can cause short-term changes in gene expression, and may thereby mediate plasticity in contractile properties of skeletal muscle in response to exercise. The aim of this project was to determine (in zebrafish, Danio rerio) the traits that mediate inter-individual differences in sustained and sprint performance, and to determine whether inhibiting class I and II HDACs mediates exercise-induced changes in these traits. High sustained performers had greater aerobic metabolic capacity (citrate synthase [CS] activity), calcium handling capacity (sarco/endoplasmic reticulum ATPase [SERCA] activity), and slow contractile protein concentration (slow myosin heavy chain [MHC]) compared to low performers. High sprint performers had lower CS activity and slow MHC concentrations compared to low performers, but there were no significant differences in lactate dehydrogenase activity or fast MHC concentrations. Four weeks of aerobic exercise training increased sustained performance, CS activity, SERCA activity, and slow MHC concentration. Inhibiting class I and II HDACs increased slow MHC concentration in untrained fish but not in trained fish. However, inhibiting HDACs reduced SERCA activity, which was paralleled by a reduction in sustained and sprint performance. The regulation of muscle phenotypes by HDACs could be a mechanism underlying the adaptation of locomotor performance to different environmental conditions, and may therefore be of therapeutic and ecological significance.
Hypoxia results in decreased arterial PO2, arterial chemoreflex activation, and compensatory increases in breathing, sympathetic outflow, and neuroendocrine secretions, including increased secretion of vasopressin (AVP), corticotropin releasing hormone (CRH), adrenocorticotropin hormone (ACTH), and corticosterone. In addition to a brainstem pathway including the nucleus tractus solitarius (nTS) and the rostral ventrolateral medulla (RVLM), medullary pathways to the paraventricular nucleus of the hypothalamus (PVN) contribute to chemoreflex responses. Experiments evaluated activation of specific cell phenotypes within the PVN following an acute hypoxic stimulus (AH, 2 hr, 10% O2) in conscious rats. Retrograde tracers (from spinal cord and RVLM) labeled pre-sympathetic (PreS) neurons; immunohistochemistry (IHC) identified AVP- and CRH-IR cells; Fos- IR was an index of neuronal activation. Hypoxia activated AVP-IR (~ 6%) and CRH-IR (~15%) cells, but not PreS cells in the PVN, suggesting that sympathoexcitation during moderate AH is mediated mainly by a pathway that does not include PreS neurons in the PVN. Approximately 14 to 17% of all PVN cell phenotypes examined expressed nNOS-IR. AH activated only nNOS-negative AVP-IR neurons. In contrast ~ 23% of activated CRH-IR neurons in the PVN contained nNOS. In the median eminence, CRH-IR terminals were closely opposed to tanycyte processes and endfeet (vimentin-IR) in the external zone, where vascular NO participates in tanycyte retraction to facilitate neuropeptide secretion into the pituitary portal circulation. Results are consistent with an inhibitory role of NO on AVP and PreS neurons in the PVN, and an excitatory role of NO on CRH secretion in the PVN and median eminence.
The purpose of the present study was to characterize the progression of red blood cell volume (RBCV) expansion and potential volumetric and endocrine regulators of erythropoiesis during endurance training (ET). Nine healthy untrained volunteers (age=27±4 years) underwent supervised ET consisting of 3-4 x 60 min cycle ergometry sessions per week for 8 weeks. Plasma volume (PV), RBCV and overnight fasting hematological markers were determined prior to and at weeks 2, 4 and 8 of ET. In addition, plasma erythropoietin (EPO), cortisol, copeptin and pro-atrial natriuretic peptide concentrations were measured during a 3-hour morning period at baseline and post-exercise at weeks 1 and 8. PV increased from baseline (2405±335 ml) at weeks 2, 4 and 8 (+374±194, +505±156, +341±160 ml, respectively, P<0.001). Increases in RBCV from baseline (1737±442 ml) were manifest at week 4 (+109±114 ml, P=0.030) and week 8 (+205±109 ml, P=0.001). Overnight fasting plasma EPO concentration increased from baseline (11.3±4.8 mIU•ml-1) at week 2 (+2.5±2.8 mIU•ml-1, P=0.027) and returned to baseline concentration at weeks 4 and 8. Higher 3-hour-post-exercise EPO concentration was observed at week 1 (11.6 mIU•ml-1) compared with week 8 (8.4±3.9 mIU•ml-1, P=0.009) and baseline (9.0±4.2 mIU•ml-1, P=0.019). Linear relationships between EPO concentration and hematocrit (β =-56.2, P < 0.001) and cortisol (β=0.037, P<0.001) were detected throughout the ET intervention. In conclusion, ET leads to mild transient increases in circulating EPO concentration concurring with early PV expansion and lowered hematocrit, preceding gradual RBCV enhancement.
During both dynamic (e.g. endurance) and static (e.g. strength) exercise there are exaggerated cardiovascular responses in hypertension. This includes greater increases in blood pressure, heart rate, and efferent sympathetic nerve activity than in normals. Two of the known neural factors which contribute to this abnormal cardiovascular response are the exercise pressor reflex (EPR) and functional sympatholysis. The EPR originates in contracting skeletal muscle and reflexly increases sympathetic efferent nerve activity to the heart and blood vessels as well as decreases parasympathetic efferent nerve activity to the heart. These changes in autonomic nerve activity cause an increase in blood pressure, heart rate, left ventricular contractility, and vasoconstriction in the arterial tree. However, arterial vessels in the contracting skeletal muscle have a markedly diminished vasoconstrictor response. The markedly diminished vasoconstriction in contracting skeletal muscle has been termed functional sympatholysis. It has been shown in hypertension that there is an enhanced EPR, including both its mechanoreflex and metaboreflex components, and an impaired functional sympatholysis. These conditions set up a positive feedback or vicious cycle situation which causes a progressively greater decrease in the blood flow to the exercising muscle. Thus, these two neural mechanisms contribute significantly to the abnormal cardiovascular response to exercise in hypertension. In addition, exercise training in hypertension decreases the enhanced EPR, including both mechanoreflex and metaboreflex function, and improves the impaired functional sympatholysis. These two changes, caused by exercise training, improve the muscle blood flow to exercising muscle and cause a more normal cardiovascular response to exercise in hypertension.
Reflex renal vasoconstriction occurs during exercise, and renal vasoconstriction in response to upper-limb muscle mechanoreflex activation has been documented. However, the renal vasoconstrictor response to muscle mechanoreflex activation originating from lower limbs, with and without local metabolite accumulation, has not been assessed. Eleven healthy young subjects (26 ± 1y; 5 men) underwent two trials involving 3-min passive calf muscle stretch (mechanoreflex) during 7.5-min lower-limb circulatory occlusion (CO). In one trial, 1.5-min 70% maximal voluntary contraction isometric calf exercise preceded CO to accumulate metabolites during CO and stretch (mechanoreflex and metaboreflex; 70% trial). A control trial involved no exercise before CO (mechanoreflex alone; 0% trial). Beat-to-beat renal blood flow velocity (RBFV; Doppler ultrasound), mean arterial blood pressure (MAP; photoplethysmographic finger cuff), and heart rate (electrocardiogram) were recorded. Renal vascular resistance (RVR), an index of renal vasoconstriction, was calculated as MAP/RBFV. All baseline cardiovascular variables were similar between trials. Stretch increased RVR and decreased RBFV in both trials (change from CO with stretch: RVR - 0% trial = 10 ± 2 %, 70% trial = 7 ± 3 %; RBFV - 0% trial = -3.8 ± 1.1 cm.sec-1, 70% trial = -2.7 ± 1.5 cm.sec-1; P < 0.05 for RVR and RBFV). These stretch-induced changes were of similar magnitudes in both trials, e.g. with and without local metabolite accumulation, as well as when thromboxane production was inhibited. These findings suggest that muscle mechanoreflex activation via passive calf stretch causes renal vasoconstriction, with and without muscle metaboreflex activation, in healthy humans.
Background: Calcium binding protein, spermatid specific 1 (CABS1) is expressed in human submandibular gland and has an anti-inflammatory motif similar to that in submandibular rat 1 (SMR1) in rats. Here we investigate CABS1 in human saliva and its association with psychological and physiological distress and inflammation in humans. Method: Volunteers participated across three studies: weekly baseline measures; a psychosocial speech and mental arithmetic stressor under evaluative threat; and during academic exam stress. Salivary samples were analyzed for CABS1 and cortisol. Additional measures included questionnaires of perceived stress and negative affect; exhaled nitric oxide; respiration and cardiac activity; lung function; salivary and nasal inflammatory markers. Results: We identified a CABS1 immunoreactive band at 27 kDa in all participants, and additional molecular weight forms in some participants. Temporal stability of the 27 kDa band was satisfactory (rtt=.62 - .86). Acute stress increased intensity of 18, 27, and 90 kDa bands; 27 kDa increases were associated with more negative affect and lower heart rate, sympathetic activity, respiration rate, and minute ventilation. In both acute and academic stress, changes in 27 kDa were positively associated with salivary cortisol. The 27 kDa band was also positively associated with VEGF and salivary LTB4 levels. Participants with low molecular weight CABS1 bands showed reduced habitual stress and negative affect in response to acute stress. Conclusion: CABS1 is readily detected in human saliva and is associated with psychological and physiological indicators of stress. The role of CABS1 in inflammatory processes, stress, and stress resilience requires careful study.
Introduction: Women with a history of preeclampsia (PE) have an increased risk to develop cardiovascular and renal diseases later in life, but the mechanisms underlying this effect are unknown. In rats, we assessed whether placental ischemia results in long-term effects on the maternal cardiovascular and renal systems using the Reduced Uterine Perfusion Pressure (RUPP) model for PE. Methods: Sprague-Dawley rats received either a SHAM or RUPP operation at gestational day 14. The rats were followed for eight weeks after delivery (SHAM n=12, RUPP n=21) at which time mean arterial pressure (MAP; conscious), 24-hour albuminuria, GFR (transcutaneous, FITC-sinistrin), and cardiac function (Vevo 770 system) were assessed. Subsequently, all rats were sacrificed for mesenteric artery vasorelaxation and histology of heart and kidney. Results: At eight weeks after delivery, there was no difference in MAP and albuminuria. However, RUPP rats showed a significantly reduced GFR [2.61 ± 0.53 vs 3.37 ± 0.74ml/min; p=0.01]. Ultrasound showed comparable cardiac structure, but RUPP rats had a lower left ventricular ejection fraction (62 ± 7 vs 69 ± 10%; p=0.04). Heart and kidney histology was not different between SHAM or RUPP rats. Furthermore, there were no differences in endothelial dependent or independent vasorelaxation. Conclusions: We show that exposure to placental ischemia in rats is accompanied by functional disturbances in maternal renal and cardiac function eight weeks after a preeclamptic pregnancy. However, these changes were not dependent on differences in blood pressure, small artery vasorelaxation, or cardiac and renal structure at this time point postpartum.
Diabetes remains one of the leading causes of morbidity and mortality worldwide, affecting an estimated 422 million adults. In the U.S. it is predicted that 1 in every 3 children born as of 2000 will suffer from diabetes in their lifetime. Type 2 diabetes results from combinatorial defects in pancreatic beta cell glucose-stimulated insulin secretion and in peripheral glucose uptake. Both processes, insulin secretion and glucose uptake, are mediated by exocytosis proteins, SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complexes, Sec1/Munc18 (SM), and Double C2-domain protein B (DOC2B). Increasing evidence links deficiencies in these exocytosis proteins to diabetes in rodents and humans. Given this, emerging studies aimed at restoring and/or enhancing cellular levels of certain exocytosis proteins point to promising outcomes in maintaining functional beta cell mass and enhancing insulin sensitivity. In doing so, new evidence also shows that enhancing exocytosis protein levels may promote health span and longevity, and may also harbor anti-cancer and anti-Alzheimer's disease capabilities. Herein we present a comprehensive review of the described capabilities of certain exocytosis proteins and how these might be targeted for improving metabolic dysregulation.
The adipocyte derived hormone leptin is a peripheral signal that informs the brain about the metabolic status of an organism. Although traditionally viewed as an appetite suppressing hormone, studies in the past decade have highlighted the role of leptin in energy expenditure. Leptin has been shown to increase energy expenditure in particular through its effects on the cardiovascular system and brown adipose tissue (BAT) thermogenesis via the hypothalamus. The current review summarizes the role of leptin signaling in various hypothalamic nuclei and its effects on the sympathetic nervous system to influence blood pressure, heart rate and brown adipose tissue thermogenesis. Specifically, the role of leptin signaling on three different hypothalamic nuclei, the dorsomedial hypothalamus, the ventromedial hypothalamus, and the arcuate nucleus, is reviewed. It is known that all these brain regions influence the sympathetic nervous system activity and thereby regulate BAT thermogenesis and the cardiovascular system. Thus, the current work focuses on how leptin signaling in specific neuronal populations within these hypothalamic nuclei influences certain aspects of energy expenditure.
Exposure to glucocorticoids in utero is associated with changes in organ function and structure in the adult. The aims of this study were to characterize the effects of antenatal exposure to glucocorticoids on glucose handling and the role of adipose tissue. Pregnant sheep received betamethasone (Beta, 0.17 mg/kg) or vehicle (V) 24-h apart at 80-days gestation and allowed to deliver at term. At 9 months, male and female offspring were fed at either 100% of nutritional allowance (lean) or ad libitum for 3 months (obese). At one year they were instrumented under general anesthesia. Glucose tolerance was evaluated using a bolus of glucose (0.25 g/kg). Adipose tissue was harvested after euthanasia to determine mRNA expression levels of angiotensinogen (AGT), angiotensin converting enzyme (ACE) 1, ACE2, and peroxisome proliferator-activated receptor (PPAR). Data are expressed as Mean±SEM and analyzed by ANOVA. Sex, obesity and Beta exposure had significant effects on glucose tolerance and mRNA expression. Beta impaired glucose tolerance in lean females but not males. Superimposed obesity worsened the impairment in females and unmasked the defect in males. Beta increased ACE1 mRNA in females and males and AGT in females only (p<0.05 by Three-way ANOVA). Obesity increased AGT in females but had no effect on ACE1 in either males or females. PPAR mRNA exhibited a significant sex (F=42.8; p<0.01) and obesity (F=6.9; p<0.05) effect and was higher in males (p<0.01 by Three-way ANOVA). We conclude that adipose tissue may play an important role in the sexually dimorphic response to antenatal glucocorticoids.
Cerebrovascular CO2 reactivity is affected by nitric oxide (NO). We tested the hypothesis that sildenafil selectively potentiates NO-cGMP signaling affects CO2 reactivity. Fourteen healthy males (34±2 years) were enrolled in the study. BP, ECG, velocity of cerebral blood flow (CBF, measured by TCD), end tidal CO2 (EtCO2) were assessed at baseline (CO2 ~ 39 mm Hg), during hyperventilation (CO2 ~ 24 mm Hg), hypercapnia (CO2 ~ 46 mm Hg), boluses of phenylephrine (25-200 µg) and during graded head-up tilting (HUT). Measurements were repeated one hour after taking 100 mg sildenafil. Results showed that sildenafil did not affect resting BP, HR, CBF peak and mean velocities, eCVR (mean BP/mean CBF), breath/min, and EtCO2: 117±2/67±3 mm Hg, 69±3 bpm, 84±5 and 57±4 cm/sec, 1.56±0.1 mmHg/cm*sec, 14±0.5 breaths/min and 39±0.9 mm Hg, respectively. Sildenafil increased and decreased the hypercapnia-induced in CBF and eCVR, respectively. Sildenafil also attenuated the decrease in peak velocity of CBF, 25±2% vs. 20±2% (p<0.05), and increased the eCVR, 2.5±0.2% vs. 2±0.2% (p<0.03) during hyperventilation. Sildenafil did not affect CBF despite significant increases in the eCVRs that were elicited by phenylephrine and HUT. This investigation suggests that sildenafil, which potentiates the NO-cGMP signaling, seems to affect the cerebrovascular CO2 reactivity without affecting the static and dynamic pressure-dependent mechanisms of cerebrovascular autoregulation.
Recent evidence suggests that endocannabinoids acting via cannabinoid CB1 receptors may modulate vascular responses of various vasoconstrictors in the rodent systemic vasculature. The aim of the study was to investigate whether endocannabinoids modulate the contractile responses evoked by a thromboxane A2 analog (U46619), angiotensin II (Ang II), serotonin (5-HT) and phenylephrine which stimulate distinct Gq/11-protein coupled receptors (TP, AT1, 5-HT2 and α1-adrenergic) in isolated endothelium-intact human (hPAs) and rat pulmonary arteries (rPAs). The CB1 receptor antagonist AM251 (1 μM) and diacylglycerol lipase (2-arachidonoylglycerol synthesis enzyme) inhibitor RHC80267 (40 μM) enhanced contractions induced by U46619 in hPAs and rPAs, and by Ang II in rPAs, in an endothelium-dependent manner. AM251 did not influence vasoconstrictions induced by 5-HT or phenylephrine in rPAs. The monoacylglycerol lipase (2-arachidonoylglycerol degradation enzyme) inhibitor JZL184 (1 μM), but not the fatty acid amide hydrolase (anandamide degradation enzyme) inhibitor URB597 (1 μM) attenuated contractions evoked by U46619 in hPAs and rPAs, and Ang II in rPAs. 2-arachidonoylglycerol concentration-dependently induced relaxation of hPAs, which was inhibited by endothelium denudation or AM251 and enhanced by JZL184. Expression of CB1 receptors was confirmed in hPAs and rPAs using Western blotting and immunohistochemistry. The present study shows the protective interaction between the endocannabinoid system and vasoconstriction to U46619 and Ang II in the human and rat pulmonary circulation. U46619 and Ang II may stimulate rapid endothelial release of endocannabinoids (mainly 2-arachidonoylglycerol), leading to CB1 receptor-dependent and/or -independent vasorelaxation, which in the negative feedback mechanism reduces later agonists-induced vasoconstriction.
The risk of cognitive decline in diabetes (Type 1 and Type 2) is significantly greater compared to normoglycemic patients, and the risk of developing dementia in diabetic patients is doubled. The etiology for this is likely multifactorial, but one mechanism that has gained increasing attention is decreased cerebral perfusion as a result of cerebrovascular dysfunction. The innate immune system has been shown to play a role in diabetic vascular complications, notably through Toll-like receptor (TLR) stimulated release of pro-inflammatory cytokines and chemokines that leads to vascular damage. TLR2 has been implicated in playing a crucial role in the development of diabetic microvascular complications such as nephropathy, and thus we hypothesized that TLR2-mediated cerebrovascular dysfunction leads to decreased CBF and cognitive impairment in diabetes. Knockout of TLR2 conferred protection from impaired CBF in early-stage diabetes and from hyperperfusion in long-term diabetes, prevented the development of endothelium dependent vascular dysfunction in diabetes, created a hyperactive and anxiolytic phenotype, and protected against diabetes induced impairment of long term hippocampal- and prefrontal cortex- mediated fear learning. In conclusion, these findings support the involvement of TLR2 in the pathogenesis of diabetic vascular disease and cognitive impairment.
Thermal tolerance in fish may be related to an oxygen limitation of cardiac function. While the hearts of some fish species receive oxygenated blood via a coronary circulation, the influence of this oxygen supply on thermal tolerance and cardiac performance during warming remain unexplored. Here, we analyzed the effect in vivo of acute warming on coronary blood flow in adult sexually mature rainbow trout (Onchorhynchus mykiss), and the consequences of chronic coronary ligation on cardiac function and thermal tolerance in juvenile trout. Coronary blood flow at 10°C was higher in females than males (0.56±0.08 vs. 0.30±0.08 ml min-1 g-1 ventricle), and averaged 0.47±0.07 ml min-1 g-1 ventricle across sexes. Warming increased coronary flow in both sexes until 14°C, at which it peaked and plateaued at 0.78±0.1 and 0.61±0.1 ml min-1 g-1 ventricle in females and males, respectively. Thus, the scope for increasing coronary flow was 101% in males, but only 39% in females. Coronary ligated juvenile trout exhibited elevated heart rate across temperatures, reduced Arrhenius breakpoint temperature for heart rate (23.0 vs. 24.6°C) and reduced upper critical thermal maximum (25.3 vs. 26.3°C). To further analyze the effects of coronary flow restriction on cardiac rhythmicity, electrocardiogram characteristics were determined before and after coronary occlusion in anaesthetized trout. Occlusion resulted in reduced R wave amplitude and an elevated S-T segment indicating myocardial ischemia, while heart rate was unaffected. This suggests that the tachycardia in ligated trout across temperatures in vivo was mainly to compensate for reduced cardiac contractility to maintain cardiac output. Moreover, our findings show that coronary flow increases with warming in a sex-specific manner. This may improve whole animal thermal tolerance, presumably by sustaining cardiac oxygenation and contractility at high temperatures.
To maintain core body temperature in mammals, the normal CNS thermoregulatory reflex networks produce an increase in brown adipose tissue (BAT) thermogenesis in response to skin cooling, and an inhibition of the sympathetic outflow to BAT during skin rewarming. In contrast, these normal thermoregulatory reflexes appear to be inverted in hibernation/torpor: thermogenesis is inhibited during exposure to a cold environment, allowing dramatic reductions in core temperature and metabolism, and thermogenesis is activated during skin rewarming, contributing to a return of normal body temperature. Here we describe two unrelated experimental paradigms in which rats, a non-hibernating/torpid species, exhibit a "Thermoregulatory Inversion", characterized by an inhibition of BAT thermogenesis in response to skin cooling, and a switch in the gain of the skin cooling reflex transfer function from negative to positive values. Either transection of the neuraxis immediately rostral to the dorsomedial hypothalamus in anesthetized rats, or activation of A1 adenosine receptors within the CNS of free-behaving rats produces a state of thermoregulatory inversion, in which skin cooling inhibits BAT thermogenesis, leading to hypothermia, and skin warming activates BAT, supporting an increase in core temperature. These results reflect the existence of a novel neural circuit that mediates inverted thermoregulatory reflexes, and suggests a pharmacologic mechanism through which a deeply hypothermic state can be achieved in non-hibernating/torpid mammals, possibly including humans.
This study tested the hypothesis that varying degrees of hemodynamic fluctuations seen after birth following immediate cord clamping were related to development of asphyxia with longer cord clamp-to-ventilation intervals, resulting in higher perinatal circulating levels of the catecholamines norepinephrine (NE) and epinephrine (Epi), and thus increased heart rate, blood pressures and cardiac contractility after birth. Anesthetized preterm fetal lambs were instrumented with 1) aortic (AoT) and pulmonary trunk (PT) micromanometers to obtain pressures and the maximal rate of pressure rise (dP/dtmax) as a surrogate measure of ventricular contractility, and 2) an AoT catheter to obtain samples for blood gas and catecholamine analyses. After delivery, immediate cord clamping was followed by ventilation 40sec (n=7), 60sec (n=8), 90sec (n=9) or 120sec later (n=8), with frequent blood sampling performed before and after ventilation. AoT O2 content fell rapidly after immediate cord clamping (P<0.001), with an asphyxial state evident at ≥60sec. Plasma NE and Epi levels increased progressively with longer cord clamp-to-ventilation intervals, with an exponential relation between falling AoT O2 content and rising catecholamines (R2=0.64-0.67). Elevated circulating catecholamines persisted for some minutes after ventilation onset, with post-birth surges in heart rate, AoT and PT pressures, and AoT and PT dP/dtmax linearly related to loge of catecholamine levels (R2=0.41-0.54, all P<0.001). These findings suggest that 1) a greater degree of asphyxia-induced sympathoadrenal activation (reflected in elevated circulating catecholamine levels) occurs with longer intervals between immediate cord clamping and subsequent ventilation, and 2) this activation is a major determinant of hemodynamic fluctuations evident with birth.
Despite the effects of insulinopenia in type 1 diabetes and evidence that insulin stimulates multiple renal sodium transporters, it is not known whether normal variation in plasma insulin regulates sodium homeostasis physiologically. This study tested whether the normal postprandial increase in plasma insulin significantly attenuates renal sodium and volume losses. Rats were instrumented with chronic artery and vein catheters, housed in metabolic cages, and connected to hydraulic swivels. Measurements of urine volume and sodium excretion (UNaV) over 24 hrs and the 4-hr postprandial period were made in Control (C) rats and Insulin-Clamped (IC) rats in which the postprandial increase in insulin was prevented. 24-hr urine volume (36±3 vs. 15±2 ml/day) and UNaV (3.0±0.2 vs. 2.5±0.2 mmol/day) were greater in the IC compared to C rats, respectively. Four hrs after rats were given a gel meal, blood glucose and urine volume were greater in IC rats, but UNaV decreased. To simulate a meal while controlling blood glucose, C and IC rats received a glucose bolus that yielded peak increases in blood glucose that were not different between groups. Urine volume (9.7±0.7 vs. 6.0±0.8 ml/4-hrs) and UNaV (0.50±0.08 vs. 0.20±0.06 mmol/4-hrs) were greater in the IC vs. C rats, respectively, over the 4 hr test. These data demonstrate that the normal increase in circulating insulin in response to hyperglycemia may be required to prevent excessive renal sodium and volume losses, and suggest that insulin may be a physiologic regulator of sodium balance.
Heat stress evokes significant increases in muscle sympathetic nerve activity (MSNA) in healthy individuals. The MSNA response to heat stress in chronic heart failure (CHF) is unknown. We hypothesized that the MSNA response to heat stress is attenuated in CHF. Passive whole body heating was applied with water-perfused suits on 13 patients (61 ± 2 years) with stable class II-III CHF, 12 age-matched (62 ± 2 years) healthy subjects, and 14 young (24 ± 1 years) healthy subjects. Mild heating (i.e. increases in skin temperature Tsk ~2-4 °C, internal temperature Tcore <0.3 °C), significantly decreased MSNA in CHF patients, but did not significantly alter the MSNA in the age-matched and young healthy subjects. Heat stress (i.e. Tsk ~4 °C and Tcore ~0.6 °C) raised MSNA in the age-matched (32.9 ± 3.2 to 45.6 ± 4.2 bursts/min; P < 0.001) and young (14.3 ± 1.7 to 26.3 ± 2.4 bursts/min; P < 0.001) controls, but not in CHF (46.2 ± 5.3 to 50.5 ± 5.3 bursts/min; P = 0.06). The MSNA increase by the heat stress in CHF (4.2 ± 2.0 bursts/min) was significantly less than those seen in the age-matched (12.8 ± 1.7 bursts/min, P < 0.05) and young (12.0 ± 2.7 bursts/min, P < 0.05) control groups. These data suggest that the MSNA response to heat stress is attenuated in CHF patients. We speculate that the attenuated MSNA response to heat stress may contribute to impaired cardiovascular adjustments in CHF in a hot environment.
Carnitine Palmitoyltransferase 1 (CPT1) is essential for the transport of long chain fatty acids into the mitochondria for oxidation. Recently, it was reported that decreased CPT1b mRNA in adipose tissue was a contributing factor for obesity in rats. We therefore closely examined the expression level of Cpt1 in adipose tissue from mice, rats, and humans. Cpt1a is the predominate isoform in adipose tissue from all three species. Rat white adipose tissue has a moderate amount of Cpt1b mRNA, but it is very minor compared to Cpt1b expression in muscle. Total CPT1 activity in adipose tissue is also minor relative to other tissues. Both Cpt1a and Cpt1b mRNA were increased in gonadal fat but not inguinal fat by diet-induced obesity in mice. We also measured CPT1a and CPT1b expression in subcutaneous adipose tissue from human subjects with a wide range of BMI. Interestingly, CPT1a expression positively correlated with BMI (R=0.46), but there was no correlation with CPT1b (R=0.04). Our findings indicate that white adipose tissue fatty acid oxidation capacity is minor compared to metabolically active tissues. Further, given the already low abundance of Cpt1b in white adipose tissue, it is unlikely that decreases in its expression can quantitatively decrease whole body energy expenditure enough to contribute to an obese phenotype.
The oviduct of Chinese brown frog (Rana dybowskii) expands specifically during pre-hibernation instead of in the breeding period. In this study, we investigated the expression of leptin receptor (Ob-Rb) in Rana dybowskii oviduct during the breeding period and pre-hibernation. Histologically, the oviduct of Rana dybowskii consists of glandular cells, tubule lumen and epithelial cells. The oviductal weight and pipe diameter also revealed significant differences, which were higher in pre-hibernation than that of the breeding period. Ob-Rb was observed in stromal cells of oviductal tissue in both the breeding period and pre-hibernation. The mean protein and mRNA levels of the Ob-Rb were significantly higher in pre-hibernation as compared to the breeding period. In addition, oviductal content of leptin was also higher in pre-hibernation than that of the breeding period. These results suggested that oviduct of Rana dybowskii might be a target organ of leptin, and leptin may play an autocrine/paracrine role mediated by Ob-Rb in regulating the oviductal hypertrophy during pre-hibernation.
Reperfusion is essential for ischemic tissue survival, but causes additional damage to the endothelium (i.e. ischemia-reperfusion [IR] injury). Ischemic preconditioning (IPC) refers to short repetitive episodes of ischemia that can protect against IR. However, IPC efficacy attenuates with older age. Whether physical inactivity contributes to the attenuated efficacy of IPC to protect against IR injury in older humans is unclear. We tested the hypotheses that lifelong exercise training relates to 1) attenuated endothelial IR and 2) maintained IPC efficacy that protects veteran athletes against endothelial IR. In 18 sedentary male individuals (SED, <1 exercise hour/week for >20 years, 63±7 years) and 20 veteran male athletes (ATH, >5 exercise hours/week for >20 years, 63±6 years), we measured brachial artery endothelial function with flow-mediated dilation (FMD) before and after IR. We induced IR by 20-minutes of ischemia followed by 20-minutes of reperfusion. Randomized over 2 days, participants underwent either 35-minute rest or IPC (3 cycles of 5-minutes cuff inflation to 220 mmHg with 5-minutes of rest) before IR. In SED, FMD decreased after IR (median [interquartile range]): (3.0% [2.0-4.7] to 2.1% [1.5-3.9], P=0.046) and IPC did not prevent this decline (4.1% [2.6-5.2] to 2.8% [2.2-3.6],P=0.012). In ATH, FMD was preserved after IR (3.0% [1.7-5.4] to 3.0% [1.9-4.1], P=0.82) and when IPC preceded IR (3.2% [1.9-4.2] to 2.8% [1.4-4.6],P=0.18). These findings indicate that lifelong exercise training is associated with increased tolerance against endothelial IR. These protective, preconditioning effects of lifelong exercise against endothelial ischemia-reperfusion may contribute to the cardio-protective effects of exercise training.
Sex-specific differences in renin-angiotensin-system (RAS) and arterial pressure have been evidenced in many mammals including spontaneously hypertensive rats (SHRs). Although SHRs have been used extensively as a leading experimental model of hypertension, effects of sex-specific differences in RAS on aortic function and related cardiac remodeling during aging and hypertension have not been documented in detail. We examined structural and functional changes in aorta and heart of female and male SHRs at ages of 5, 14, 29 and 36-weeks. SHRs of both sexes were hypertensive from 14-weeks. Aortic endothelial dysfunction and fibrosis, left ventricular (LV) hypertrophy and cardiac fibrosis was evident at the age of 29-weeks in male SHRs, but first appeared only at the age of 36-weeks in female SHRs. There was a pronounced delay of matrix metalloproteinase-2 activity in aorta and heart of female SHRs, which was associated with preservation of 40 % more elastin and less extensive cardiac fibrosis than in males. At 5, 29 and 36-weeks of age female SHRs showed higher levels of aortic and myocardial AT2R and MasR mRNA and decreased ANGII-mediated aortic constriction. While female SHRs had increased relaxation to AT2R stimulation at 5 and 29-weeks compared to males, this difference disappeared at 36-weeks of age. This study documents sex-specific differences in the temporal progression of aortic dysfunction and LV hypertrophy in SHRs which are independent of arterial pressure and are apparently mediated by higher AT2R expression in the heart and aorta of female SHRs.
An important adaptive feature of heat acclimation (AC) is the induction of cross-tolerance against novel stressors (HACT). Reprogramming of gene expression leading to enhanced innate cytoprotective features by attenuating damage and/or enhancing the response of "help" signals, plays a pivotal role. HIF-1α, constitutively upregulated by AC (1mo, 34°C), is a crucial transcription factor in this program, although its specific role is as yet unknown. By using a rat AC model we studied the impact of disrupting HIF-1α transcriptional activation [(HIF-1α:HIF-1β dimerization blockade by Acriflavine (4mg/kg b.wgt, ip)], on its mitochondrial gene targets (PDK1, LON, COX4 isoforms) in the HACT rat heart. Physiological measures of cardiac HACT were infarct size following ischemia/reperfusion and time to rigor contracture during hypoxia in cardiomyocytes. We show that HACT requires transcriptional activation of HIF-1α throughout the course of AC and that this activation is accompanied by two metabolic switches: (i) profound upregulation of PDK1, which reduces pyruvate entry into the mitochondria, consequently increasing glycolytic lactate production; (ii) remodeling of the COX4 isoform ratio, inducing hypoxic-tolerant COX4.2 dominance, optimizing electron transfer and possibly ATP production during the ischemic and hypoxic insults. LON and COX4.2 transcript upregulation accompanied this shift. Loss of HACT despite elevated expression of the cytoprotective protein HSP72 concomitantly with disrupted HIF-1α dimerization suggests that HIF-1α is essential for HACT. The role of a PDK1 metabolic switch is well known in hypoxia acclimation, but not in the AC model and its ischemic setting. Remodeling of COX4 isoforms by environmental acclimation is a novel finding.
Recent clinical trials in patients with drug-resistant hypertension indicate that electrical activation of the carotid sinus baroreflex (baroreflex activation therapy) can reduce arterial pressure (AP) for more than a year. To examine whether the electrical stimulation from one baroreflex system impedes normal short-term AP regulation via another unstimulated baroreflex system, we electrically stimulated the left aortic depressor nerve (ADN) while estimating the dynamic characteristics of the carotid sinus baroreflex in anesthetized normotensive Wistar-Kyoto (WKY, n=8) rats and spontaneously hypertensive rats (SHR, n=7). Isolated carotid sinus regions were perturbed for 20 min using a Gaussian white noise signal with a mean of 120 mmHg for WKY and 160 mmHg for SHR. Tonic ADN stimulation (2 Hz, 10 V, 0.1-ms pulse width) decreased mean sympathetic nerve activity (73.4±14.0 vs. 51.6±11.3 arbitrary units in WKY, P = 0.012; and 248.7±33.9 vs. 181.1±16.6 arbitrary units in SHR, P = 0.018) and mean AP (90.8±6.6 vs. 81.2±5.4 mmHg in WKY, P=0.004; and 128.6±9.8 vs. 114.7±10.3 mmHg in SHR, P = 0.009). The slope of dynamic gain in the neural arc transfer function from carotid sinus pressure to sympathetic nerve activity was not different between trials with and without the ADN stimulation (12.55±0.93 vs. 13.03±1.28 dB/decade in WKY, P = 0.542; and 17.37±1.01 vs. 17.47±1.64 dB/decade in SHR, P = 0.946). These results indicate that the tonic ADN stimulation does not significantly modify the dynamic characteristics of the carotid sinus baroreflex.
ATP release from urinary bladder is vital for afferent signaling. The aims of this study were to localize CALHM1 and pannexin-1 expression and to determine their involvement in mediating ATP release in the bladder. Gene expression by PCR and immunohistochemistry were performed in the porcine bladder. CALHM1 and pannexin-1 mediated ATP release in response to hypotonic solution (0.45% NaCl) induced stretch and extracellular Ca2+ depletion ([Ca2+]0) was measured in isolated urothelial, suburothelial and detrusor muscle cells. CALHM1 and pannexin-1 mRNA and immunoreactivity were detected in urothelial, suburothelial and detrusor muscle layers with a highest expression on urothelium. Hypotonic stretch caused a 2.7-fold rise in ATP release from all three cell populations (P < 0.01), which was significantly attenuated by the pannexin-1 inhibitor, 10Panx1, and by CALHM1 antibody. Brefeldin A, a vesicular transport inhibitor, and ruthenium red, a non-selective CALHM1 channel blocker, also significantly inhibited stretch-mediated ATP release from urothelial cells. [Ca2+]0 caused a marked but transient elevation of extracellular ATP level in all three cell populations. CALHM1 antibody and ruthenium red inhibited [Ca2+]0-induced ATP release from urothelial cells, but their effects on suburothelial and detrusor cells was insignificant. 10Panx1 showed no significant inhibition of [Ca2+]0-induced ATP release in any types of cells. The results presented here provide compelling evidence that pannexin-1 and CALHM1 that are densely expressed in the porcine bladder function as ATP release channels in response to bladder distension. Modulation of extracellular Ca2+ may also regulate ATP release in the porcine bladder through voltage gated CALHM1 ion channels.
We recently showed the varying roles of Ca2+-activated (KCa), ATP-sensitive (KATP) and voltage-gated (KV) K+ channels in regulating cholinergic cutaneous vasodilation and sweating in normothermic conditions. However, it is unclear whether the respective contributions of these K+ channels remain intact during dynamic exercise in the heat. Eleven young (23±4 years) males completed a 30-min exercise bout at a fixed rate of metabolic heat production (400 W), followed by a 40-min recovery period in the heat (35°C, 20% relative humidity). Cutaneous vascular conductance (CVC) and local sweat rate were assessed at four forearm skin sites perfused via intradermal microdialysis with either: 1) lactated Ringer's solution (Control), 2) 50mM tetraethylammonium (non-specific KCa channel blocker), 3) 5mM glybenclamide (selective KATP channel blocker), or 4) 10mM 4-aminopyridine (non-specific KV channel blocker). Responses were compared at baseline and at 10-min intervals during and following exercise. KCa channel inhibition resulted in greater CVC versus Control at end-exercise (P=0.04) and 10- and 20-min into recovery (both P0.01). KATP channel blockade attenuated CVC compared to Control during baseline (P=0.04), exercise (all P≤0.04), and 10-min into recovery (P=0.02). No differences in CVC were observed with KV channel inhibition during baseline (P=0.15), exercise (all P≥0.06), or recovery (all P≥0.14). With the exception of KV channel inhibition augmenting sweating during baseline (P=0.04), responses were similar to Control with all K+ channel blockers during each time period (all P≥0.07). We demonstrated that KCa and KATP channels contribute to the regulation of cutaneous vasodilation during rest and/or exercise, and recovery in the heat.
The rostral raphe pallidus (rRPa) contains sympathetic premotor neurons controlling thermogenesis in brown adipose tissue (BAT). We sought to determine if a tonic activation of glycineA receptors (GlyAR) in the rRPa contributes to the inhibitory regulation of BAT SNA and of cardiovascular parameters in anesthetized rats. Nanoinjection of the GlyAR antagonist, strychnine (STR), into the rRPa of intact rats increased BAT sympathetic nerve activity (SNA, peak: +495%), BAT temperature (TBAT, +1.1 ºC), expired CO2, (+0.4 %), core body temperature (TCORE, +0.2 ºC), arterial pressure (MAP, +4 mmHg) and heart rate (HR, +57 bpm). STR into rRPa in rats with a post-dorsomedial hypothalamus transection produced similar increases in BAT thermogenic and cardiovascular parameters. Glycine (GLY) nanoinjection into the rRPa evoked a potent inhibition of the cooling-evoked increases in BAT SNA (nadir: -74 %), TBAT (-0.2 ºC), TCORE (-0.2 ºC), expired CO2 (-0.2 %), MAP (-8 mmHg), and HR (-22 bpm), but had no effect on the increases in these variables evoked by STR nanoinjection into rRPa. Nanoinjection of GABA into the rRPa inhibited the STR evoked BAT SNA (nadir: -86 %), and reduced the expired CO2 (-0.4 %). Blockade of glutamate receptors in rRPa reduced the STR-evoked increases in BAT SNA (nadir: -61 %), TBAT (-0.5 ºC), expired CO2 (-0.3 %), MAP (-9 mmHg) and HR (-33 bpm). We conclude that a tonically active glycinergic input to the rRPa contributes to the inhibitory regulation of the discharge of BAT sympathetic premotor neurons and of BAT thermogenesis and energy expenditure.
Swimming activity primarily accelerates growth in fish by increasing protein synthesis and energy efficiency. The role of muscle in this process is remarkable and especially important in teleosts, where muscle represents a high percentage of body weight, and by the continuous growth that many fish species present. The aim of this work was to characterize the effects of five weeks of moderate and sustained swimming in gene and protein expression of myogenic regulatory factors, proliferation markers and proteolytic molecules in two muscle regions (anterior and caudal) of gilthead sea bream fingerlings. Western blot results showed an increase in the proliferation marker PCNA, proteolytic systems' members CAPN1 and CTSD, as well as vascular-endothelial growth factor protein expression. Moreover, quantitative real-time PCR data showed that exercise increased the gene expression of proteases: calpains, cathepsins and members of the ubiquitin-proteasome system in the anterior muscle region; and the gene expression of the proliferation marker PCNA and the myogenic factor MyoD in the caudal area, compared to control fish. Overall, these data suggest a differential response of the two muscle regions during swimming adaptation, with tissue remodeling and new vessels formation occurring in the anterior muscle and enhanced cell proliferation and differentiation in the caudal area. In summary, the present study contributes to improve the knowledge on the role of proteolytic molecules and other myogenic factors in the adaptation of muscle to moderate sustained swimming in gilthead sea bream.
Caloric restriction decreases skeletal muscle mass in mammals, principally due to a reduction in fiber size. The effect of suboptimal nutrient intake on skeletal muscle metabolic properties in neonatal calves was examined. The longissimus muscle (LM) was collected after 8 wks consuming a control (CON) or caloric restricted (CR) diet and muscle fiber size, gene expression and metabolic signal transduction activity was measured. Results revealed that CR animals had smaller (P < 0.05) LM fiber cross-sectional area than CON, as expected. Western blot analysis detected equivalent amounts of PGC1α-1 but reduced (P < 0.05) amounts of the splice-variant, PGC1α-4 in CR LM. Expression of IGF-1, a PGC1α-4 target gene, was 40% less (P < 0.05) in CR than CON. Downstream mediators of autocrine IGF-1 signaling also are attenuated in CR by comparison to CON. The amount of phosphorylated AKT1 was less (P < 0.05) in CR than CON. The ratio of p4EBP1T37/46:total 4EBP1, a downstream mediator of AKT1, did not differ between CON and CR. By contrast, protein lysates from CR LM contained less (P < 0.05) total GSK3β and phosphorylated GSK3β than CON LM, suggesting blunted protein synthesis. Smaller CR LM fiber size associates with increased (P < 0.05) CAPN1 activity coupled with lower (P < 0.05) expression of calpastatin, the endogenous inhibitor of CAPN1. Atrogin-1 and MuRF expression and autophagy components were unaffected by CR. Thus, CR suppresses the hypertrophic PGC1α-4/IGF-1/AKT1 pathway while promoting activation of the calpain system.
This study was designed to determine the effect of active sensitization with ovalbumin (Ova) on cough responses to inhaled irritant gases in mice. Conscious mice moved freely in a recording chamber while the pressure change in the chamber, audio and video signals of the mouse movements were recorded simultaneously to measure the frequencies of cough reflex (CR) and expiration reflex (ER). To further verify the accuracy of cough analysis, the intra-pleural pressure was also recorded by a telemetry sensor surgically implanted in the intra-pleural space in a subgroup of mice. During the irritant gas inhalation challenge, sulfur dioxide (SO2; 200 & 400 ppm) or ammonia (NH3; 0.1% & 0.2%) was drawn into the chamber at a constant flowrate for 8 minutes. Ova sensitization and sham sensitization with vehicle (Veh) were performed over a 25-day period in separate groups of mice. Our results showed: 1) Both SO2 and NH3 inhalation challenges increased CR and ER frequencies in a concentration-dependent manner before Ova sensitization. 2) The baseline CR frequency was significantly elevated after Ova sensitization, accompanied by pronounced airway inflammation. 3) Ova sensitization also markedly augmented the responses of CR and ER to both SO2 and NH3 inhalation challenges; in sharp contrast, the cough responses did not change after sham sensitization in the Veh group. In conclusion, Ova sensitization caused distinct and lingering increases in baseline cough frequency, and also intensified both CR and ER responses to inhaled irritant gases, which probably resulted from an allergic inflammation-induced hypersensitivity of airway sensory nerves.
Perinatal hypoxia severely disrupts metabolic and somatotrophic development, as well as cerebral maturational programs. Hypoxia-inducible transcription factors (HIF) represent the most important endogenous adaptive mechanisms to hypoxia, activating a broad spectrum of growth factors that contribute to cell survival and energy homeostasis. To analyse effects of systemic hypoxia and growth hormone (GH) therapy (rhGH) on HIF-dependent growth factors during early postnatal development, we compared protein (ELISA) and mRNA levels (quantitative RT PCR) of growth factors in plasma and brain between normoxic and hypoxic mice (8% O2, 6 h; postnatal day 7, P7) at P14. Exposure to hypoxia led to reduced body weight (p<0.001) and length (p<0.04) compared to controls, and was associated with significantly reduced plasma levels of mouse GH (p<0.01) and IGF-1 (p<0.01). RhGH abrogated these hypoxia-induced changes of the GH/IGF-1 axis associated with normalization of weight and length gain until P14 compared to controls. In addition, rhGH treatment increased cerebral IGF-1, IGF-2, IGFBP-2, and EPO mRNA levels, resulting in significantly reduced apoptotic cell death in the hypoxic, developing mouse brain. These data indicate that rhGH may (i) functionally restore hypoxia-induced systemic dysregulation of the GH/IGF-1 axis, and (ii) induce up-regulation of neuroprotective, HIF-dependent growth factors in the hypoxic developing brain.
During heat stress, blunted increases in skin sympathetic nervous system activity (SSNA) and reductions in end-organ vascular responsiveness contribute to the age-related reduction in reflex cutaneous vasodilation. In older adults, folic acid supplementation improves the cutaneous vascular conductance (CVC) response to passive heating; however, the influence of folic acid supplementation on SSNA:CVC transduction is unknown. Fourteen older adults (66±1yrs, 8M/6F) ingested folic acid (5mg·day-1) or placebo for 6 weeks in a randomized, double-blind, crossover design. In protocol 1, esophageal temperature (Tes) was increased by 1.0ºC (water-perfused suit) while SSNA (peroneal microneurography) and red cell flux in the innervated dermatome (laser Doppler flowmetry; dorsum of the foot) were continuously measured. In protocol 2, two intradermal microdialysis fibers were placed in the skin of the lateral calf for graded infusions of acetylcholine (ACh; 10-10 to 10-1M) with and without nitric oxide synthase (NOS) blockade (20mM L-NAME). Folic acid improved reflex vasodilation (46±4% vs. 31±3 %CVCmax for placebo; P<0.001) without affecting the increase in SSNA (506±104% vs. 415±73% for placebo; NS). Folic acid increased the slope of the SSNA:CVC relation (0.08±0.02 vs. 0.05±0.01 for placebo; P<0.05) and extended the response range. Folic acid augmented ACh-induced vasodilation (83±3% vs. 66±4 %CVCmax for placebo; P=0.002); however there was no difference between treatments at the NOS-inhibited site (53±4% vs. 52±4% CVCmax for placebo; NS). These data demonstrate that folic acid supplementation enhances reflex vasodilation by increasing the sensitivity of skin arterioles to central sympathetic nerve outflow during hyperthermia in aged human subjects.
Accumulating evidence has shown that follicle stimulating hormone (FSH) and luteinizing hormone (LH) may influence the functions of non-gonadal tissues, in addition to their classic target gonads. Our previous studies revealed that the scented glands of male muskrats expressed prolactin receptor, steroidogenic enzymes and inhibin/activin subunits. To further seek the evidence of the activities of pituitary gonadotropins in scented glands, we investigated the seasonal expression patterns of FSH receptor (FSHR) and LH/choriogonadotropin receptor (LHCGR). The weight and size of scented glands in the breeding season were significantly higher than those in the non-breeding season. Immunohistochemical studies showed that FSHR was present in the serous cells of scented gland while LHCGR in the interstitial cells. The protein and mRNA expression levels of FSHR and LHCGR were significantly higher in the scented glands during the breeding season than those during the non-breeding season. Importantly, the levels of circulating FSH and LH were remarkably higher in the breeding season. Taken together, these results suggested that gonadotropins may affect the function of muskrat scented gland via the locally expressed receptors in a season-dependent manner.
μ-Opioid receptors are distributed widely in the brainstem respiratory network, and opioids with selectivity for μ-type receptors slow in-vivo respiratory rhythm in lowest effective doses. Several studies have reported μ-opioid receptor effects on the three-phase rhythm of respiratory neurons, but there are until now no reports of opioid effects on oscillatory activity within respiratory discharges. In this study, effects of the μ-opioid receptor agonist fentanyl on spike train discharge properties of several different types of rhythm-modulating medullary respiratory neuron discharges were analyzed. Doses of fentanyl that were just sufficient for prolongation of discharges and slowing of the three-phase respiratory rhythm also produced pronounced enhancement of spike train properties. Oscillation and burst patterns detected by autocorrelation measurements were greatly enhanced, and inter-spike intervals were prolonged. Spike train properties under control conditions and after fentanyl were uniform within each experiment, but varied considerably between experiments, which might be related to variability in acid-base balance in the brainstem extracellular fluid. Discharge threshold was shifted to more negative levels of membrane potential. The effects on threshold are postulated to result from opioid mediated disinhibition and postsynaptic enhancement of NMDA receptor current. Lowering of firing threshold, enhancement of spike train oscillations and bursts and prolongation of discharges by lowest effective doses of fentanyl could represent compensatory adjustments in the brainstem respiratory network to override opioid blunting of CO2/pH chemosensitivity.
Mutations in the brain-derived neurotrophic factor (BDNF) gene are associated with human obesity and BDNF has potent inhibitory effects on eating and body weight. Little is known about the effects of energy-balance manipulations on BDNF protein in the hypothalamus, though this brain region is critical for regulation of feeding and body weight and has high levels of BDNF. Here we investigated the effects of negative and positive energy status on BDNF protein levels in the arcuate (ARC), paraventricular (PVN), and ventromedial (VMH) hypothalamic nuclei, and the ectorhinal cortex. To achieve this, mice were food deprived for forty-eight hours or fed a western diet (WD), a restricted amount of WD, or chow for six hours, forty-eight hours, one week, or three weeks. BDNF protein levels were estimated as the number of neurons in each brain region that exhibited BDNF-like immunoreactivity (LIR). Food deprivation decreased BDNF protein (and mRNA) expression in the ARC compared with fed mice (32%). In contrast, one week of WD consumption increased BDNF protein expression in the VMH as compared with chow or restricted WD feeding (40%), and unexpectedly, increased BDNF protein in the ectorhinal cortex (20%). Furthermore, of the diet conditions and durations tested, only one week of WD consumption was associated with both hyperphagia and excess weight, suggesting effects of one or both contributed to the changes in BDNF levels. The decrease in ARC BDNF may support increased feeding in food-deprived mice, whereas, the increase in the VMH may moderate overeating in WD fed mice.
Roux-en-Y gastric bypass (RYGB) induces weight loss and improves insulin sensitivity when evaluated by the hyperinsulinemic-euglycemic clamp (HEC). Surrogate indices of insulin sensitivity calculated from insulin and glucose concentrations at fasting or after an oral glucose tolerance test (OGTT) are frequently used, but have not been validated after RYGB. Our aim was to evaluate whether surrogate indices reliably estimate changes in insulin sensitivity after RYGB. Four fasting (inverse-HOMA-IR, HOMA2-%S, QUICKI, revised-QUICKI) and three OGTT-derived surrogates (Matsuda, Gutt, OGIS) were compared with HEC-estimated peripheral insulin sensitivity (Rd or Rd/I, depending on how the index was originally validated) and the tracer-determined hepatic insulin sensitivity index (HISI) in patients with preoperative type 2 diabetes (n=10) and normal glucose tolerance (n=10) 1 week, 3 months and 1 year postoperatively. Post-RYGB changes in inverse-HOMA-IR and HOMA2-%S did not correlate with changes in Rd at any visit, but were comparable to changes in HISI at 1 week. Changes in QUICKI and revised-QUICKI correlated with Rd/I after surgery. Changes in Matsuda and Gutt did not correlate with changes in Rd/I and Rd, respectively, whereas OGIS-changes correlated with Rd-changes at 1 year post-RYGB. In conclusion, surrogate measures of insulin sensitivity may not reflect results obtained with gold standard methodology after RYGB, underscoring the importance of critical reflection when surrogate endpoints are used. Fasting surrogate indices may be particularly affected by post-RYGB changes in insulin clearance, whereas the validity of OGTT-derived surrogates may be compromised by the surgical rearrangements of the gut.
Hypohydration decreases cutaneous vasodilation and sweating during heat stress, but it is unknown if these decrements are from postsynaptic (i.e. sweat gland/blood vessel) alterations. Purpose: To determine if hypohydration affects postsynaptic cutaneous vasodilation and sweating responses. Methods: 12 healthy men participated in euhydrated (EU) and hypohydrated (HY) trials, with hypohydration induced via fluid restriction and passive heat-stress. Changes in cutaneous vascular conductance (CVC; %max) in response to incremental intradermal infusion of the endothelium-independent vasodilator sodium nitroprusside (SNP) and the endothelium-dependent vasodilator methacholine chloride (MCh) were assessed by laser Doppler flowmetry. Local sweat rate (LSR) was simultaneously assessed at the MCh site via ventilated capsule. At the end of the last dose, maximal CVC was elicited by delivering a maximal dose of SNP (5x10-2 M) for 30 min to both sites with simultaneous local heating (~44°C) at the SNP site. The concentration of drug needed to elicit 50% of the maximal response (Log EC50) was compared between hydration conditions. Results: %BM loss was greater when HY vs. EU (-2.2 ± 0.7 vs. -0.1 ± 0.7%, P < 0.001). Log EC50 of endothelium-dependent CVC was lower when EU (-3.62 ± 0.22) versus HY (-2.93 ± 0.08; P = 0.044). Hypohydration did not significantly alter endothelium-independent CVC or LSR (both P > 0.05). Conclusion: Hypohydration attenuated endothelium-dependent CVC but did not affect endothelium-independent CVC or LSR responses. These data suggest that reductions in skin blood flow accompanying hypohydration can be partially attributed to altered postsynaptic function.
Muscle weakness and exercise intolerance negatively affect the quality of life of mitochondrial myopathy patients. Short-term dietary nitrate supplementation has been shown to improve exercise performance and reduce oxygen cost of exercise in healthy humans and trained athletes. We investigated if 1 week of dietary inorganic nitrate supplementation decreases the oxygen cost of exercise and improves mitochondrial function in mitochondrial myopathy patients. Ten mitochondrial myopathy patients (40 ± 5 years, maximal whole-body oxygen uptake = 21.2 ± 3.2 mL/min/kg body weight, maximal workload = 122 ± 26 W) received 8.5 mg/kg body weight/day of inorganic nitrate (~7 mmol) for 8 days. Whole-body oxygen consumption at 50% of the maximal workload, in vivo skeletal muscle oxidative capacity (evaluated from post-exercise phosphocreatine recovery using 31P magnetic resonance spectroscopy) and ex vivo mitochondrial oxidative capacity in permeabilized skinned muscle fibers (measured with high-resolution respirometry) were determined before and after nitrate supplementation. Despite a 6-fold increase in plasma nitrate levels, nitrate supplementation did not affect whole-body oxygen cost during submaximal exercise. Additionally, no beneficial effects of nitrate were found on in vivo or ex vivo muscle mitochondrial oxidative capacity. This is the first time that the therapeutic potential of dietary nitrate for mitochondrial myopathy patients was evaluated. We conclude that 1 week of dietary nitrate supplementation does not reduce oxygen cost of exercise or improve mitochondrial function in the group of patients tested.
Acute amino acid (AA) infusion increases AA oxidation rates in normal late gestation fetal sheep. Because fetal oxygen consumption rate does not change with increased AA oxidation, we hypothesized that AA infusion would suppress glucose oxidation pathways and that the additional carbon supply from AA would activate hepatic glucose production. To test this, late gestation fetal sheep were infused intravenously for 3h with saline or exogenous AA (AA). Glucose tracer metabolic studies were performed and skeletal muscle and liver tissues samples were collected. AA infusion increased fetal arterial plasma branched chain AA, cortisol, and glucagon concentrations. Fetal glucose utilization rates were similar between basal and AA periods, yet the fraction of glucose oxidized and glucose oxidation rate were decreased by 40% in the AA period. AA infusion increased expression of PDK4, an inhibitor of glucose oxidation, nearly 2-fold in muscle and liver. In liver, AA infusion tended to increase PCK1 gluconeogenic gene and PCK1 correlated with plasma cortisol concentrations. AA infusion also increased liver mRNA expression of lactate transporter gene (MCT1), protein expression of GLUT2 and LDHA, and phosphorylation of AMPK, 4EBP1, and S6 proteins. In isolated fetal hepatocytes, AA supplementation increased glucose production and PCK1, LDHA, and MCT1 gene expression. These results demonstrate that AA infusion into fetal sheep competitively suppresses glucose oxidation and potentiates hepatic glucose production. These metabolic patterns support flexibility in fetal metabolism in response to increased nutrient substrate supply while maintaining a relatively stable rate of oxidative metabolism.
The taste of sugar elicits cephalic-phase insulin release (CPIR), which limits the rise in blood glucose associated with meals. Little is known, however, about the orosensory mechanisms that trigger CPIR. We asked whether oral stimulation with any of the following taste stimuli elicited CPIR in mice: glucose, sucrose, maltose, fructose, Polycose, saccharin, sucralose, AceK, SC45647 or a non-metabolizable sugar analog. The only taste stimuli that elicited CPIR were glucose and the glucose-containing saccharides (sucrose, maltose, Polycose). When we mixed an alpha-glucosidase inhibitor (acarbose) with the latter three saccharides, the mice no longer exhibited CPIR. This revealed that the carbohydrates were hydrolyzed in the mouth, and that the liberated glucose triggered CPIR. We also found that increasing the intensity or duration of oral glucose stimulation caused a corresponding increase in CPIR magnitude. To identify the components of the glucose-specific taste-signaling pathway, we examined the necessity of Calhm1, P2X2+P2X3, SGLT1 and Sur1. Among these proteins, only Sur1 was necessary for CPIR. Sur1 was not necessary, however, for taste-mediated attraction to sugars. Given that Sur1 is a subunit of the KATP channel, and that this channel functions as a part of a glucose-sensing pathway in pancreatic beta cells, we asked whether the KATP channel serves an analogous role in taste cells. We discovered that oral stimulation with drugs known to increase (glyburide) or decrease (diazoxide) KATP signaling produced corresponding changes in glucose-stimulated CPIR. We propose that the KATP channel is part of a novel signaling pathway in taste cells that mediates glucose-induced CPIR.
The removal of protons (H+) produced during intense exercise is important for skeletal muscle function, yet it remains unclear how best to structure exercise training to improve muscle pH regulation. We investigated whether 4 weeks of work-matched, sprint-interval training (SIT), performed 3 days per week, with either 1 min (Rest-1; n = 7) or 5 min (Rest-5; n = 7) of rest between sprints, influenced adaptations in acid/base transport protein content, non-bicarbonate muscle buffer capacity (βmin vitro), and exercise capacity in active women. Following one week of post-testing, comprising a biopsy, a repeated-sprint ability (RSA) test, and a graded-exercise test, maintenance of adaptations was then studied by reducing SIT volume to one day per week for a further 5 weeks. After 4 weeks of SIT, there was increased protein abundance of monocarboxylate transporter (MCT)1, sodium/hydrogen exchanger (NHE)1, and carbonic anhydrase (CA)XIV for both groups, but rest interval duration did not influence the adaptive response. In contrast, greater improvements in total work performed during the RSA test after 4 weeks of SIT was evident for Rest-5 compared to Rest-1 [effect size (ES): 0.51; 90% confidence limits ±0.37), whereas both groups had similarly modest improvements in VO2peak. When training volume was reduced to one day per week, enhanced acid/base transport protein abundance was maintained, although NHE1 content increased further for Rest-5 only. Finally, our data support intracellular lactate as a signaling molecule for inducing MCT1 expression, but neither lactate nor H+ accumulation appear to be important signaling factors in MCT4 regulation.
Cytochrome c oxidase (COX) subunit 4 has two paralogs in most vertebrates. The mammalian COX4-2 gene is hypoxia responsive and the protein has a disrupted ATP-binding site that confers kinetic properties on COX that distinguish it from COX4-1. The structure -function of COX4-2 orthologs in other vertebrates remains uncertain. Phylogenetic analyses suggest the two paralogs arose in basal vertebrates, but COX4-2 orthologs diverged faster than COX4-1 orthologs. COX4-1/4-2 protein levels in tilapia tracked mRNA levels across tissues, and did not change in hypoxia, arguing against a role for differential post-translational regulation of paralogs. Heart, and to a lesser extent brain, showed a size-dependent shift from COX4-1 to COX4-2 (transcript and protein). ATP allosterically inhibited both velocity and affinity for oxygen in COX assayed from both muscle (predominately COX4-2) and gill (predominately COX4-1). We saw some evidence of cellular and subcellular discrimination of COX4 paralogs in heart. In cardiac ventricle, some non-cardiomyocyte cells were COX positive but lacked detectible COX4-2. Within heart, the two proteins partitioned to different mitochondrial subpopulations. Cardiac subsarcolemmal mitochondria had mostly COX4-1 and intermyofibrillar mitochondria had mostly COX4-2. Collectively, these data argue that despite common evolutionary origins, COX4-2 orthologs of fish show unique patterns of subfunctionalization with respect to transcriptional and post-translation regulation, relative to the rodents and primates that have been studied to date.
Nerve damage can induce a heightened pain response to noxious stimulation, which is termed hyperalgesia. Pain itself acts as a stressor, initiating autonomic and sensory effects through the dorsal periaqueductal gray (dPAG) to induce both sympathoexcitation and analgesia, which prior studies have shown to be affected by endocannabinoid signaling. The present study addressed the hypothesis that neuropathic pain disrupts autonomic and analgesic regulation by endocannabinoid signaling in the dPAG. Endocannabinoid contents, transcript levels of endocannabinoid signaling components, and catabolic enzyme activity were analyzed in the dPAG of rats at 21 days after painful nerve injury. The responses to two nerve injury models were similar, with two-thirds of animals developing hyperalgesia that was maintained throughout the post injury period, while no sustained change in sensory function was observed in the remaining rats. Anandamide content was lower in the dPAG of rats that developed sustained hyperalgesia and activity of the catabolic enzyme fatty acid amide hydrolase (FAAH) was higher. Intensity of hyperalgesia was correlated to transcript levels of FAAH and negatively correlated to heart rate and sympatho-vagal balance. These data suggest that maladaptive endocannabinoid signaling in the dPAG after nerve injury could contribute to chronic neuropathic pain and associated autonomic dysregulation. This study demonstrates that reduced anandamide content and upregulation of FAAH in the dPAG are associated with hyperalgesia and reduced heart rate sustained weeks after nerve injury. These data provide support for the evaluation of FAAH inhibitors for the treatment of chronic neuropathic pain.
Recent evidence suggests that concussions may disrupt autonomic cardiovascular control. This study investigated the initial effects of concussion on cardiovascular function using three autonomic reflex tests. Twenty three recreational athletes (12 females, 11 males) were divided into concussed (n = 12) and control (n = 11) groups. Concussed participants performed forced breathing, standing and Valsalva autonomic tests four times: 1) within 48 hours of injury, 2) 24 hours later, 3) 1 week after injury and 4) 2 weeks after injury. The controls performed the same tests on the same schedule. Differences in heart rate (HR), systolic blood pressure (SBP) and diastolic blood pressure (DBP) responses to the tests were continuously measured using finger photoplethysmography and were analyzed using repeated measures MANOVAs and ANOVAs. Within 48 hours of injury, the concussed group had significantly greater resting SBP (t21 = 2.44, P = 0.02, d = 1.03), HR (t21 = 2.33, P = 0.03, d = 1.01) and SBP responses to standing (t21 = 2.98, P = 0.01, d = 1.24), and 90% SBP normalization times (t21 = 2.64, P = 0.02, d = 1.10) after the Valsalva, but those group differences subsided 24 hours later. There was also a significant interaction with the HR responses to forced breathing (F3,60 = 4.13, P = 0.01, p2 = 0.17), indicating the concussed responses declined relative to the control's over time. The results demonstrate that concussion disrupted autonomic cardiovascular control and that autonomic reflex tests are practical means by which to evaluate that dysfunction.
Preeclampsia affects 3-5% of pregnancies, contributes to 10-15% of maternal mortality, and is a leading cause of prematurity and increased long-term morbidity in those affected. Contemporary understanding of preeclampsia's maternal-fetal interface suggests a major role for placental oxidative stress resulting from ischemia-reperfusion injury. We hypothesized the pregnancy hormone relaxin would reduce cytotrophoblast apoptosis, necrosis (aponecrosis) and export of placental debris into maternal circulation. If so, relaxin might be employed as a therapeutic intervention to diminish activation of the maternal systemic inflammatory response central to clinical disease development. HTR-8/SVneo cells, a model for first trimester extravillous trophoblast, were subjected to serum deprivation and hypoxia or hypoxia-reoxygenation. The cells were treated with recombinant human relaxin or vehicle (VEH), and apoptosis and/or necrosis evaluated by terminal deoxynucleotidyl transferase mediated dUTP nick endlLabeling (TUNEL), CellEvent® Caspase-3/7 + SYTOX® AAdvance™, and propidium iodide staining determined by fluorescent microscopy or flow cytometry. To interrogate mechanisms of relaxin cytoprotection, HTR-8/SVneo cells were pre-treated with pharmacological inhibitors of PI3-kinase LY294004, Akt/PKB MK2206, or DMSO vehicle. Serum deprivation and hypoxia increased apoptotic cell death in HTR-8/SVneo cells, which was significantly ameliorated by concurrent relaxin treatment. Serum deprivation and hypoxia-reoxygenation increased necrotic cell death in HTR-8/SVneo cells, which was also significantly rescued by concurrent relaxin treatment. Pretreatment with LY294002 or MK2206 significantly blunted relaxin's cytoprotective effect. We demonstrated trophoblast cytoprotection by intervention with supraphysiologic relaxin concentrations, a process mediated through the PI3 kinase-AKT/PKB cell survival pathway. This provides further rationale for clinical investigation of relaxin as a potential preeclampsia therapeutic.
Type 2 diabetes (T2D) is characterized by chronic low-grade inflammation that contributes to disease pathophysiology. Exercise has anti-inflammatory effects but the impact of high-intensity interval training (HIIT) is not known. PURPOSE: To determine the impact of a single session of HIIT on cellular, molecular, and circulating markers of inflammation in individuals with T2D. METHODS: Participants with T2D (n=10) and healthy age-matched controls (HC; n=9) completed an acute bout of HIIT (7 X 1-min @ ~85% maximal aerobic power output, separated by 1-min recovery) on a cycle ergometer with blood samples obtained before (Pre), immediately after (Post), and at one-hour of recovery (1-h Post). Inflammatory markers on leukocytes were measured by flow cytometry, and tumor necrosis factor (TNF)-α assessed in both lipopolysaccharide (LPS)-stimulated whole blood cultures and plasma. RESULTS: A single session of HIIT had an overall anti-inflammatory effect, as evidenced by: i) significantly lower levels of toll-like receptor (TLR) 2 surface protein expression on both classical and CD16+ monocytes assessed at Post and 1-h Post compared with Pre (p<0.05 for all); ii) significantly lower LPS-stimulated TNF-α release in whole blood cultures at 1-h Post (p<0.05 vs. Pre); and iii) significantly lower levels of plasma TNF-α at 1-h Post (p<0.05 vs. Pre). There were no differences between T2D and HC except for a larger decrease in plasma TNF-α in HC vs. T2D (group x time interaction, p<0.05). CONCLUSIONS: One session of low-volume HIIT has immunomodulatory effects and provides potential anti-inflammatory benefits to people with, and without, T2D.
Aim: the liver is essential in maintaining and regulating glucose homeostasis during exercise. Interleukin 6(IL-6) has been shown to be secreted from skeletal muscle during exercise and has been suggested to signal to the liver. Therefore, the aim was to investigate the role of skeletal muscle IL-6 on hepatic glucose regulation and substrate choice during prolonged exercise. Methods: twelve weeks old skeletal muscle specific IL-6 knockout(IL-6 MKO) mice and littermate lox/lox(Control) mice were either rested(Rest) or completed a single bout of exercise for 10min, 60 min or 120 min and the liver was quickly obtained. Results: hepatic IL-6 mRNA was higher at 60 min of exercise and hepatic signal transducer and activator of transcription 3 was higher at 120 min of exercise than at Rest in both genotypes. Hepatic glycogen was higher in IL-6 MKO than Control at Rest, but decreased similarly during exercise in the two genotypes, and hepatic glucose content was lower in IL-6 MKO than Control at 120 min of exercise. Hepatic phosphoenolpyruvate carboxykinase mRNA and protein increased in both genotypes at 120 min of exercise, while hepatic glucose 6 phosphatase protein remained unchanged. Furthermore, IL-6 MKO mice had higher hepatic pyruvate dehydrogenase(PDH)Ser232 and PDHSer300 phosphorylation than Control at Rest. Conclusion: hepatic gluconeogenic capacity is increased during prolonged exercise independent of muscle IL-6. Skeletal muscle IL-6 influences hepatic substrate regulation at rest and hepatic glucose metabolism during prolonged exercise seemingly independent of IL-6 signaling in the liver.
The purpose of this study was to investigate the effect of chronic chlorella intake alone or in combination with high-intensity intermittent exercise (HIIE) training on exercise performance and muscle glycolytic and oxidative metabolism in rats. Forty male Sprague-Dawley rats were randomly assigned to the four groups: sedentary control, chlorella intake (0.5% chlorella powder in normal feed), HIIE training, and combination of HIIE training and chlorella intake for 6 weeks (n = 10 each group). HIIE training comprised 14 repeats of a 20-s swimming session with a 10-s pause between sessions, while bearing a weight equivalent to 16% of body weight, 4 days/week. Exercise performance was tested after the interventions by measuring the maximal number of HIIE sessions that could be completed. Chlorella intake and HIIE training significantly increased the maximal number of HIIE, and enhanced the expression of monocarboxylate transporter (MCT)1, MCT4, and peroxisome proliferator activated receptor coactivator-1α concomitantly with the activities of lactate dehydrogenase (LDH), phosphofructokinase, citrate synthase (CS), and cytochrome oxidase (COX) in the red region of the gastrocnemius muscle. Furthermore, the combination further augmented the increased exercise performance and the enhanced expressions and activities. By contrast, in the white region of the muscle, MCT1 expression and LDH, CS, and COX activities did not change. These results showed that compared to only chlorella intake and only HIIE training, chlorella intake combined with HIIE training has a more pronounced effect on exercise performance and muscle glycolytic and oxidative metabolism, in particular lactate metabolism.
Purpose: To examine the time-course of changes in the oxygen uptake (VO2) kinetics response subsequent to short-term exercise training (i.e., 24h, 48h, 72h, and 120h post-training) and examine the relationship with the time-course of changes in microvascular (deoxygenated hemoglobin concentration ([HHb])/VO2 ratio) and macrovascular (flow-mediated dilation (FMD)) O2 delivery to the active tissues/limbs. Methods: Seven healthy older (OA: 74±6yr) and young men (YA: 25±3yr) completed three endurance cycling exercise training sessions at 70% VO2max. Moderate-intensity exercise on-transient VO2 (measured breath-by-breath) and [HHb] (measured by near-infrared spectroscopy) were modeled with a mono-exponential and normalized (0-100% of response) and the [HHb]/VO2 ratio was calculated. Ultrasound-derived FMD of the popliteal artery was assessed following 5 minutes of cuff occlusion. %FMD was calculated as the greatest percent change in diameter from baseline. Results: Time constant of VO2 (VO2) was significantly reduced in both OA (~18%) and YA (~23%) 24h (P<0.001) post-training and remained decreased at 48h before returning toward PRE values. Both groups showed a significant decrease in the [HHb]/VO2 ratio at 24h, 48h and 72h (P=0.001, P=0.01 and P=0.03, respectively) post-training before returning toward PRE values at 120h. %FMD followed a similar time course as changes in the [HHb]/VO2 ratio; being significantly greater in both OA (by ~64%) and YA (by ~26%) at 24 h (P<0.001), remaining increased at 48h and 72h (P=0.02 and P=0.03, respectively) and returning toward PRE values at 120h. Conclusion: These data suggest the rate of adjustment of VO2 may be constrained by O2 availability in the active tissues.
Preeclampsia (PE) is a devastating disorder of pregnancy that classically presents with maternal hypertension and proteinuria after 20 weeks of gestation. In addition to being a leading cause of maternal and fetal morbidity/mortality, epidemiological and prospective studies have revealed long term consequences for both the mother and baby of preeclamptic pregnancies, including chronic hypertension as well as other cardiovascular diseases and metabolic derangements. To better understand the effect of in utero exposure of PE on offspring, we utilized the BPH/5 mouse, a spontaneous model of the maternal and fetal PE syndrome. We hypothesized that young BPH/5 offspring would have altered metabolic and cardiovascular phenotypes. Indeed, BPH/5 growth restricted offspring showed excess catch up growth by early adulthood due to hyperphagy and increased white adipose tissue (WAT) accumulation with inflammation markers isolated to the reproductive WAT depot only. Both excessive WAT accumulation and the inflammatory WAT phenotype was corrected by pair-feeding young BPH/5 female mice. We also found young BPH/5 female mice showed evidence of leptin resistance. Indeed, chronic hyperleptinemia has been shown to characterize other rodent models of PE; however, the maternal metabolic profile before pregnancy has not been fully understood. Furthermore, we found these mice show signs of cardiovascular anomalies (hypertension and cardiomegaly) and altered signaling within the reproductive axis in early life. Future studies will involve challenging the physiologic metabolic state of BPH/5 mice through pair-feeding to reduce WAT before pregnancy and determining its causal role in adverse pregnancy outcomes.
Muscle shortening and volume displacement are critical determinants of the pressure generating capacity of the diaphragm. This study was designed to test the hypothesis that diaphragm volume displacement is heterogeneous and distribution of volume displacement is dependent on regional muscle shortening, posture and level of muscle activation. Radio opaque markers were sutured along muscle bundles of the peritoneal surface of the crural muscle, dorsal costal, mid-costal, and ventral costal regions of the left hemi-diaphragm in four dogs. The markers were followed by bi-planar video fluoroscopy during quiet spontaneous breathing, passive inflation to total lung capacity (TLC), as well as inspiratory efforts against an occluded airway at three lung volumes spanning the vital capacity (functional residual capacity (FRC), FRC + 1/2 inspiratory capacity (IC), and TLC in both the prone and supine postures. Our data show the ventral costal diaphragm had the largest volume displacement (VD) and contributed nearly 2 times to the total diaphragm VD compared to the dorsal costal portion. In addition, the ventral costal diaphragm contributed nearly half of the total VD in the prone position while it only contributed a quarter of the total VD in the supine. During efforts against occluded airway and during passive inflation to TLC in the supine position, the crural diaphragm displaced volume equivalent to that of the mid-costal portion. We conclude that regional muscle shortening closely match regional volume displacement, and the primary force generator of the diaphragm is primarily dominated by contribution of ventral costal region to its volume displacement.
To explore the role of the testicular leptin and JAK-STAT(LEP-JAK-STAT) pathway in testosterone biosynthesis during juvenile stages and exercise for weight loss, male C57BL/6J mice were randomly divided into normal-diet (ND) and high-fat diet (HFD) groups. After 10 weeks, mice in the HFD group were further divided randomly into obese control (OC), obese moderate-volume exercise (OME), and obese high-volume exercise (OHE) groups. Mice in the OME group were provided with 2 h/day, 6 days/week swimming exercise for 8 weeks and mice in the OHE group underwent twice the amount of daily exercise intervention as the OME group. The results showed that high-fat diet causes obesity, leptin resistance, inhibition of the testicular LEP-JAK-STAT pathway, decreased mRNA and protein expression of SF-1, StAR, and P450scc, decrease in the serum T/E2 ratio, and decline in sperm quality parameters. Both moderate and high-volume exercise were able to reduce body fat and increase the mRNA and protein expression of LEP-JAK-STAT, but only moderate exercise significantly increased the mRNA and protein expression of SF-1, StAR, and P450scc, and significantly reversed the serum T/E2 ratio and sperm quality parameters. These findings suggest that by impairing the testicular LEP-JAK-STAT pathway, early-stage obesity inhibits the biosynthesis of testosterone and sexual development and reduces male reproductive potential. Long-term moderate and high-volume exercise can effectively reduce body fat and improve obesity-induced abnormalities in testicular leptin signal transduction, whereas only moderate-volume exercise can reverse the negative impacts of obesity on male reproductive function.
Fetal insulin secretion is inhibited by acute hypoxemia. The relationship between prolonged hypoxemia and insulin secretion, however, is less well defined. To test the hypothesis that prolonged fetal hypoxemia impairs insulin secretion, studies were performed in sheep fetuses that were bled to anemic conditions for 9 ± 0 days (anemic, n=19) and compared to control fetuses (n=15). Arterial hematocrit and oxygen content were 34% and 52% lower, respectively, in anemic vs. control fetuses (P<0.0001). Plasma glucose concentrations were 21% higher in the anemic group (P<0.05). Plasma norepinephrine and cortisol concentrations increased 70% in the anemic group (P<0.05). Glucose-, arginine-, and leucine-stimulated insulin secretion all were lower (P<0.05) in anemic fetuses. No differences in pancreatic islet size or β-cell mass were found. In vitro, isolated islets from anemic fetuses secreted insulin in response to glucose and leucine as well as control fetal islets. These findings indicate a functional islet defect in anemic fetuses which likely involves direct effects of low oxygen and/or increased norepinephrine on insulin release. In pregnancies complicated by chronic fetal hypoxemia, increasing fetal oxygen concentrations may improve insulin secretion.
Adipose tissue is an important energy depot and endocrine organ, and the degree of adiposity impacts the host response to infection. However, little is known regarding the mechanisms by which white adipose tissue (WAT) is lost acutely and then restored after resolution of sepsis. Therefore, signaling pathways governing protein synthesis, autophagy, apoptosis and the ubiquitin-proteasome were investigated to identify potential mechanisms mediating the acute (24 h) loss of WAT after cecal ligation and puncture (CLP) as well as the failure to replenish WAT during recovery (day 10). While whole-body fat mass was decreased equally in pair-fed control and septic mice at 5 days post-CLP, fat mass remained 35% lower in septic mice at day 10. During sepsis-recovery, protein synthesis in epididymal WAT (eWAT) was increased, compared to control values, and this increase was associated with an elevation in eIF2B but no change in mTORC1 activity (4E-BP1 or S6K1 phosphorylation). Protein breakdown was increased during sepsis-recovery as evidenced by the elevation in ubiquitin-proteasome activity. Moreover, indices of autophagy (LC3B-II, Atg5/12, beclin) were increased during sepsis-recovery and associated with increased AMPK-dependent S555-phosphorylated ULK1. Apoptosis was increased as suggested by increased cleavage of caspase-3 and PARP. These changes were associated with increased inflammasome activity (increased NLRP3, TMS1 and caspase-1 cleavage) and ER stress response (increased eIF2α and ATF4) and browning (UCP1) in eWAT. Our data suggest that WAT stores remain depleted during recovery from sepsis due to sustained inflammation and elevations in protein and cellular degradation, despite the increase in protein synthesis.
Adropin is a peptide hormone with cardiovascular and metabolic roles in the periphery, including effects on glucose and lipid homeostasis. Central administration of adropin has been shown to inhibit water intake in rats, however, the site at which central adropin acts has yet to be elucidated. The hypothalamic paraventricular nucleus (PVN), a critical autonomic control center, plays essential roles in the control of fluid balance, energy homeostasis and cardiovascular regulation, and is therefore a potential target for centrally acting adropin. In the present study, we used whole-cell patch-clamp techniques to examine the effects of adropin on the excitability of neurons within the PVN. All three neuronal subpopulations (magnocellular, preautonomic and neuroendocrine) in the PVN were found to be responsive to bath-application of 10 nM adropin, which elicited responses in 68% of cells tested (n = 57/84). The majority of cells (58%) depolarized (5.2 ± 0.3 mV; n = 49) in response to adropin, while the remaining responsive cells (10%) hyperpolarized (–3.4 ± 0.5 mV; n = 8), effects which were shown to be concentration-dependent. Additionally, responses were maintained in the presence of 1 μM TTX in 75% of cells tested (n = 9/12), and voltage-clamp analysis revealed that adropin had no effect on the amplitude or frequency of excitatory or inhibitory postsynaptic currents (EPSCs and IPSCs) in PVN neurons, suggesting the peptide exerts direct, postsynaptic actions on these neurons. Collectively, these findings suggest central adropin may exert its physiological effects through direct actions on neurons in the PVN.
The Na+-dependent taurocholate co-transporting polypeptide (NTCP/SLC10A1) is a hepatocyte specific solute carrier, which plays an important role in maintaining bile salt homeostasis in mammals. The absence of an hepatic Na+-dependent bile salt transport system in marine skate and rainbow trout raises a question regarding the function of the Slc10a1 gene in these species. Here we have characterized the Slc10a1 gene in the marine skate, Leucoraja erinacea. The transcript of skate Slc10a1 (skSlc10a1) encodes 319 amino acids and shares 46% identity to human NTCP (hNTCP) with similar topology to mammalian NTCP. SkSlc10a1 mRNA was mostly confined to the brain and testes with minimal expression in the liver. An FXR-bile salt reporter assay indicated that skSlc10a1 transported taurocholic acid (TCA) and scymnol sulfate, but not as effectively as hNTCP. A 3H-TCA uptake assay revealed that skSlc10a1 functioned as a Na+-dependent transporter but with low affinity for TCA (Km=92.4 µM) and scymnol sulfate (Ki=31 µM), compared to hNTCP (TCA, Km=5.4 µM; Scymnol sulfate, Ki=3.5 µM). In contrast, the bile salt concentration in skate plasma is 2 µM, similar to levels seen in mammals. Interestingly, skSlc10a1 demonstrated transport activity for the neurosteroids DHEAS and estrone-3-sulfate at physiological concentration, similar to hNTCP. Together, our findings indicate that skSlc10a1 is not a physiological bile salt transporter, providing a molecular explanation for the absence of a hepatic Na+-dependent bile salt uptake system in skate. We speculate that Slc10a1 is a neurosteroid transporter in skate that gained its substrate specificity for bile salts later in vertebrate evolution.
During hibernation, thirteen-lined ground squirrels (Ictidomys tridecemlineatus) regularly cycle between bouts of torpor and interbout arousal (IBA). Most of the brain is electrically quiescent during torpor, but regains activity quickly upon arousal to IBA resulting in extreme oscillations in energy demand during hibernation. We predicted increased functional capacity of brain mitochondria during hibernation compared to spring to accommodate the variable energy demands of hibernation. To address this hypothesis, we examined mitochondrial bioenergetics in the ground squirrel brain across three time points: spring (SP), torpor (TOR), and IBA. Respiration rates of isolated brain mitochondria through complex I of the electron transport chain were over twofold higher in TOR and IBA than in SP (P < 0.05). We also found a 10% increase in membrane potential between hibernation and spring (P < 0.05), and that proton leak was lower in TOR and IBA than in SP. Finally, there was a 30% increase in calcium loading in SP brain mitochondria compared to TOR and IBA (P < 0.01). To analyze brain mitochondrial abundance between spring and hibernation, we measured the ratio of copy number in a mitochondrial gene (ND1) versus a nuclear gene (B2M) in frozen cerebral cortex samples. No significant differences were observed in DNA copies between SP and IBA. These data show that brain mitochondrial bioenergetics are not static across the year, and suggest that brain mitochondria function more effectively during the hibernation season, allowing for rapid production of energy in order to meet demand when extreme physiological changes are occurring.
While seasonal modifications of brown adipose tissue (BAT) in hibernators are well documented, we know little about functional regulation of BAT in different phases of hibernation. In the 13-lined ground squirrel, liver mitochondrial respiration is suppressed by up to 70% during torpor. This suppression is reversed during arousal an°°d interbout euthermia (IBE), and corresponds with patterns of maximal activities of electron transport system (ETS) enzymes. Uncoupling of BAT mitochondria is controlled by free-fatty acid release stimulated by sympathetic activation of adipocytes, so we hypothesized that further regulation at the level of the ETS would be of little advantage. As predicted, maximal ETS enzyme activities of isolated BAT mitochondria did not differ between torpor and IBE. In contrast to this pattern, respiration rates of mitochondria isolated from torpid individuals were suppressed by ~60% compared with rates from IBE individuals when measured at 37°C. At 10°C, however, mitochondrial respiration rates tended to be greater in torpor than IBE. As a result, the temperature sensitivity (Q10) of mitochondrial respiration was significantly lower in torpor (~1.4) than IBE (~2.4), perhaps facilitating energy savings during entrance into torpor and thermogenesis at low body temperatures. Despite the observed differences in isolated mitochondria, norepinephrine-stimulated respiration rates of isolated BAT adipocytes did not differ between torpor and IBE, perhaps because the adipocyte isolation requires lengthy incubation at 37°C, potentially reversing any changes which occur in torpor. Such changes may include remodeling of BAT mitochondrial membrane phospholipids which could change in situ enzyme activities and temperature sensitivities.
The low oxygen experienced at high altitude is a significant challenge to effective aerobic locomotion, as it requires sustained tissue oxygen delivery in addition to the appropriate allocation of metabolic substrates. Here we test whether high- and low-altitude deer mice (Peromyscus maniculatus) have evolved different acclimation responses to hypoxia with respect to muscle metabolism and fuel use during submaximal exercise. Using F1-generation high- and low-altitude deer mice that were born and raised in common conditions, we assessed 1) fuel use during exercise, 2) metabolic enzyme activities, and 3) gene expression for key transporters and enzymes in the gastrocnemius. After hypoxia acclimation, highland mice showed a significant increase in carbohydrate oxidation and higher relative reliance on this fuel during exercise at 75% VO2max. Compared to the lowland mice, highlanders had consistently higher activities of oxidative and fatty acid oxidation enzymes in the gastrocnemius. In contrast, only after hypoxia acclimation did activities of hexokinase increase significantly in muscle of highland mice to levels greater than lowlanders. Highland mice also responded to acclimation with increases in muscle gene expression for genes Hk1 and Hk2, while both populations increase mRNA expression for glucose transporters. Changes in skeletal muscle with acclimation suggest highlanders had an increased capacity for uptake and oxidation of circulatory glucose. Our results demonstrate that highland mice have evolved a distinct mode of hypoxia acclimation that involves an increase in carbohydrate use during exercise.
The brain networks connected to the sympathetic motor and sensory innervations of brown (BAT) and white (WAT) adipose tissues were originally described using two transneuronally transported viruses: the retrogradely transported pseudorabies virus (PRV), and the anterogradely transported H129 strain of herpes simplex virus-1 (HSV-1 H129). Further complexity was added to this network organization when combined injections of PRV and HSV-1 H129 into either BAT or WAT of the same animal generated sets of co-infected neurons in the brain, spinal cord, sympathetic and dorsal root ganglia. These neurons are well positioned to act as sensorimotor links in the feedback circuits that control each fat pat. We have now determined the extent of sensorimotor crosstalk between inguinal (I)BAT and IWAT. PRV152 and HSV-1 H129 were each injected into IBAT or IWAT of the same animal: H129 into IBAT and PRV152 into IWAT. The reverse configuration was applied in a different set of animals. We found single labeled neurons together with H129+PRV152 co-infected neurons in multiple forebrain, midbrain, and hindbrain sites, with lesser numbers in the sympathetic and dorsal root ganglia that innervate IBAT and IWAT. We propose that these co-infected neurons mediate sensory-sympathetic motor crosstalk between IBAT and IWAT. Comparing the relative numbers of co-infected neurons between the two injection configurations showed a bias towards IBAT-sensory and IWAT-sympathetic motor feedback loops. These co-infected neurons provide a neuroanatomical framework for functional interactions between IBAT thermogenesis and IWAT lipolysis that occurs with cold exposure, food restriction/deprivation, exercise, and more generally with alterations in adiposity.
Heart failure is characterized by the loss of sympathetic innervation to the ventricles, contributing to impaired cardiac function and arrhythmogenesis. We hypothesized that renal denervation (RDx) would reverse this loss. Male Wistar rats underwent myocardial infarction (MI) or sham surgery and progressed into heart failure for four weeks before receiving bilateral RDx or sham RDx. After a further three weeks, left ventricular (LV) function was assessed and ventricular sympathetic nerve fibre density determined via histology. Post-MI heart failure rats displayed significant reductions in ventricular sympathetic innervation and tissue noradrenaline content (nerve fibre density in the LV of MI+sham RDx hearts was 0.31 ± 0.05 % vs. 1.00 ± 0.10 % in sham MI+sham RDx group, P < 0.05) and RDx significantly increased ventricular sympathetic innervation (0.76 ± 0.14 %, P < 0.05) and tissue noradrenaline content. MI was associated with an increase in fibrosis of the non-infarcted ventricular myocardium which was attenuated by RDx. RDx improved LV ejection fraction and end-systolic and -diastolic areas when compared to pre-RDx levels. This is the first study to show an interaction between renal nerve activity and cardiac sympathetic nerve innervation in heart failure. Our findings show denervating the renal nerves improves cardiac sympathetic innervation and function in the post-MI failing heart.
Sedentary lifestyle and lack of physical activity are well-established risk factors for chronic disease and adverse health outcomes. Thus there is enormous interest in measuring physical activity in biomedical research. Many consumer physical activity monitors, including Basis® Health Tracker, BodyMedia® Fit, DirectLife®, Fitbit® Flex, Fitbit One, Fitbit Zip, Garmin® Vivofit, Jawbone® UP, MisFit® Shine, Nike® FuelBand, Polar® Loop, Withings® Pulse O2, and others have accuracies similar to that of research-grade physical activity monitors for measuring steps. This review focuses on the unprecedented opportunities consumer physical activity monitors offer for human physiology and pathophysiology research because of their ability to measure activity continuously under real life conditions and because they are already widely used by consumers. We examine current and potential uses of consumer physical activity monitors as a measuring or monitoring device, or as an intervention in strategies to change behavior and predict health outcomes. The accuracy, reliability, reproducibility, and validity of consumer physical activity monitors is reviewed, as are limitations and challenges associated with using these devices in research. Other topics covered include how smartphone apps and platforms such as the Apple ResearchKit can be used in conjunction with consumer physical activity monitors for research. Lastly, the future of consumer physical activity monitors and related technology is considered: pattern recognition, integration of sleep monitors, and other biosensors in combination with new forms of information processing.
Physical exercise can improve brain function; however, the effects of exercise cessation are largely unknown. This study examined the time-course profile of hippocampal neurogenesis following exercise cessation. Male C57BL/6 mice were randomly assigned to either a control (Con) or an exercise cessation (ExC) group. ExC mice were reared in a cage with a running wheel for 8 weeks and subsequently placed in a standard cage to cease the exercise. Exercise significantly increased the density of doublecortin (DCX)-positive immature neurons in the dentate gyrus (at week 0). Following exercise cessation, the density of DCX+ neurons gradually decreased and was significantly lower than that in Con at 5 and 8 weeks after cessation, indicating that exercise cessation leads to a negative rebound in hippocampal neurogenesis. Immunohistochemistry analysis suggested the negative rebound in neurogenesis to be caused by diminished cell survival, not by suppression of cell proliferation and neural maturation. Neither elevated expression of FosB, a transcription factor involved in neurogenesis regulation, nor increased plasma corticosterone were involved in the negative neurogenesis rebound. Importantly, exercise cessation suppressed the ambulatory activity, and a significant correlation between change in activity and DCX+ neuron density suggested that the decrease in activity is involved in the impairment of neurogenesis. Forced treadmill running following exercise cessation failed to prevent the negative neurogenesis rebound. This study indicates that cessation of exercise or a decrease in physical activity is associated with an increased risk of impaired hippocampal function, which might increase vulnerability to stress-induced mood disorders.
Patients with heart failure and sleep apnea have greater chemoreflex sensitivity, presumably due to intermittent hypoxia (IH), and this is predictive of mortality. We hypothesized that endurance training would attenuate the effect of IH on peripheral chemoreflex sensitivity in healthy humans. Fifteen young healthy subjects (9 female, 26 ± 1 years) participated. Between visits, 11 subjects underwent eight weeks of endurance training that included running four times/week at 80% predicted max heart rate and interval training, and 4 control subjects did not change activity. Chemoreflex sensitivity (the slope of ventilation responses to serial oxygen desaturations), blood pressure, heart rate, and muscle sympathetic nerve activity (MSNA) were assessed before and after 30 minutes of IH. Endurance training decreased resting systolic blood pressure (119 ± 3 to 113 ± 3 mmHg, P = 0.027) and heart rate (67 ± 3 to 61 ± 2 beats/minute, P = 0.004), but did not alter respiratory parameters at rest (P > 0.2). Endurance training attenuated the IH-induced increase in chemoreflex sensitivity (pre-training: 0.045 ± 0.026 vs. post-training: - 0.028 ± 0.040 L/min/%O2 desaturation, P = 0.045). Furthermore, IH increased mean blood pressure and MSNA burst rate before training (P < 0.05), but IH did not alter these measures after training (P > 0.2). All measurements were similar in the control subjects at both visits (P > 0.05). Endurance training attenuates chemoreflex sensitization to IH, which may partially explain the beneficial effects of exercise training in patients with cardiovascular disease.
Despite decades of research, the magnitude and time course of hematologic and plasma volume (PV) changes following rapid ascent and acclimation to various altitudes are not precisely described. To develop a quantitative model, we utilized a comprehensive database and general linear mixed models to analyze 1055 hemoglobin ([Hb]) and hematocrit (Hct) measurements collected at sea level and repeated time points at various altitudes in 393 unacclimatized men (n=270) and women (n=123) who spent between 2 hours and 7 days at 2500-4500 m under well-controlled experimental conditions. The PV change (PV) was calculated from Hb and Hct measurements. The results are: 1) PV decreases rapidly (~6%) after the 1st day at 2500 m and [Hb] and Hct are increased by 0.5 g/dl and 1.5 points, respectively, 2) PV decreases an additional 1% and [Hb] and Hct increase an additional 0.1 g/dl and 0.2 points for every 500 m increase in elevation above 2500 m after the 1st day, 3) PV continues to decrease over time at altitude but the magnitude of this decrease and subsequent increase in [Hb] and Hct levels is dependent on elevation and sex, 4) individuals with high initial levels of [Hb] and Hct and older individuals hemoconcentrate less at higher elevations. This study provides the first quantitative delineation of PV and hematological responses during the first week of exposure over a wide range of altitudes and demonstrates that absolute altitude and time at altitude, as well as initial hematologic status, sex, and age impact the response.
Spontaneous physical activity (SPA) describes activity outside of formal exercise and shows large inter-individual variability. The hypothalamic orexin/hypocretin peptides are key regulators of SPA. Orexins drive SPA within multiple brain sites, including rostral lateral hypothalamus (LH) and nucleus accumbens shell (NAcSh). Rats with high basal SPA (high activity, HA) show higher orexin mRNA expression and SPA after injection of orexin-A in rostral LH compared to low activity (LA) rats. Here we explored the contribution of orexin signaling in rostral LH and NAcSh to the HA/LA phenotype. We found that HA rats have higher sensitivity to SPA after injection of orexin-A in rostral LH, but not in NAcSh. HA and LA rats showed similar levels of orexin receptor expression in rostral LH and activation of orexin producing neurons after orexin-A injection in rostral LH. Also, in HA and LA rats the co-injection of orexin-A in rostral LH and NAcSh failed to further increase SPA beyond the effects of orexin-A in rostral LH. Pre-treatment with muscimol, a GABA(A) receptor agonist, in NAcSh potentiated SPA produced by orexin-A injection in rostral LH in HA but not in LA rats. Our results suggest that a feedback loop from orexin responsive neurons in rostral LH to orexin neurons and a NAcSh - orexin neuron - rostral LH circuit regulate SPA. Overall, our data suggest that differences in orexin sensitivity in rostral LH and its modulation by GABA afferents from NAcSh contribute to individual SPA differences.
The purpose of this investigation was to examine the influence of short-term intense endurance training on cycling performance, along with the acute and chronic signaling responses of skeletal muscle stress and stability markers. Ten recreationally active subjects (25 ± 2 yr, 79 ± 3 kg, 47 ± 2 ml·kg·min-1) were studied before and after a 12-day cycling protocol to examine the effects of short-term intense (70-100% VO2max) exercise training on resting and exercise-induced regulation of molecular factors related to skeletal muscle cellular stress and protein stability. Skeletal muscle biopsies were taken at rest and 3h following a 20 km cycle time trial on days 1 and 12 to measure mRNA expression and protein content. Training improved (p<0.05) cycling performance by 5 ± 1%. Protein oxidation was unaltered on day 12, while resting SAPK/JNK phosphorylation was reduced (p<0.05), suggesting a reduction in cellular stress. The maintenance in the myocellular environment may be due to synthesis of cytoprotective markers along with enhanced degradation of damage proteins, as training tended (p<0.10) to increase resting protein content of MnSOD and HSP70, while mRNA expression of MuRF-1 was elevated (p<0.05). Following training (day 12), the acute exercise-induced transcriptional response of TNF-α, NFB, MuRF-1, and PGC1α was attenuated (p<0.05) compared to day 1. Collectively, these data suggest that short-term intense training enhances protein stability, creating a cellular environment capable of resistance to exercise-induced stress, which may be favorable for adaptation
The hypoxia inducible factor (HIF) family of transcription factors plays central roles in the development, physiology, pathology, and environmental adaptation of animals. Because many aquatic habitats are characterized by episodes of low dissolved oxygen, fish represent ideal models to study the roles of HIF in the response to aquatic hypoxia. The estuarine fish Fundulus heteroclitus occurs in habitats prone to hypoxia, it responds to low oxygen via behavioral, physiological, and molecular changes, and one member of the HIF family, HIF2α, has been previously described. Herein, cDNA sequencing, phylogenetic analyses, and genomic approaches were used to determine other members of the HIFα family from F. heteroclitus and their relationships to HIFα subunits from other vertebrates. In vitro and cellular approaches demonstrated that full-length forms of HIF1α, 2α, and 3α independently formed complexes with the β subunit (ARNT) to bind to hypoxia response elements and activate reporter gene expression. Quantitative PCR showed that HIFα mRNA abundance varied among organs of normoxic fish in an isoform-specific fashion. Analysis of the F. heteroclitus genome revealed a locus encoding a second HIF2α, HIF2αb, a predicted protein lacking oxygen sensing and transactivation domains. Finally, sequence analyses demonstrated polymorphism in the coding sequence of each F. heteroclitus HIFα subunit, suggesting that genetic variation in these transcription factors may play a role in the variation of hypoxia responses among individuals or populations.
Abstract Male muskrats have one pair of scented glands that grow and involute annually. To investigate the annual changes in the scented gland, we measured the expressions of aromatase cytochrome P450 (P450arom) and estrogen receptors (ERs) in the scented glands. P450arom was expressed in glandular cells and epithelial cells in the scented glands during the breeding season, and only in glandular cells during the non-breeding season. ERα and ERβ were also detected in different types of cells in the scented gland during the breeding and non-breeding seasons. Both mRNA and protein levels of P450arom, ERα and ERβ were higher in the scented glandular tissues during the breeding season than those during the non-breeding season. In addition, small RNAs sequencing showed that the predicted targets of the significantly changed microRNAs (miRNA) might be the genes encoding P450arom and ERs. In conclusion, the seasonal changes in the expression of P450arom and estrogen receptors may be involved in the regulation of scented gland functions.
Heat stress profoundly impairs tolerance to central hypovolemia in humans, via a number of mechanisms including heat-induced hypovolemia. However, heat stress also elevates plasma osmolality; the effects of which on tolerance to central hypovolemia remain unknown. This study examined the effect of plasma hyperosmolality on tolerance to central hypovolemia in heat stressed humans. Using a counterbalanced and cross-over design, 12 subjects (1 female) received intravenous infusion of either 0.9% (ISO) or 3.0% (HYPER) saline. Subjects were subsequently heated until core temperature increased ~1.4°C, following which all subjects underwent progressive lower-body negative pressure (LBNP) to pre-syncope. Plasma hyperosmolality improved LBNP tolerance (ISO: 288 ± 193 vs. HYPER: 382 ± 145 mmHg x min, P=0.04). However, no differences in mean arterial pressure (P=0.10), heart rate (P=0.09), or muscle sympathetic nerve activity (MSNA, P=0.60, n=6) were observed between conditions. When assessing individual data, LBNP tolerance improved ≥25% in 8 subjects, but remained unchanged in the remaining 4 subjects. In subjects who exhibited improved LBNP tolerance, plasma hyperosmolality resulted in elevated mean arterial pressure (ISO: 62 ± 10 vs. HYPER: 72 ± 9 mmHg, P<0.01) and a greater increase in heart rate (ISO: +12 ± 24 vs. HYPER: +23 ± 17 beats/min, P=0.05) prior to pre-syncope. No differences in these variables were observed between conditions in subjects that did not improve LBNP tolerance (all P≥0.55). These results suggest that plasma hyperosmolality improves tolerance to central hypovolemia during heat stress in most, but not all individuals.
Passive heat induces beneficial perfusion profiles, provides substantive cardiovascular strain and reduces blood pressure, thereby holding potential for healthy and cardiovascular disease populations. The aim of this study was to assess acute responses to passive heat via lower-limb hot-water immersion in patients with peripheral arterial disease (PAD) and healthy, elderly controls. Eleven patients with PAD (age 71±6 y, 7 male) and ten Controls (age 72±7 y, 8 male) underwent hot-water immersion (30 min waist-level immersion in 42.1±0.6°C water). Before, during and following immersion, brachial and popliteal artery diameter, blood flow and shear stress were assessed using duplex ultrasound. Lower-limb perfusion was measured also using venous occlusion plethysmography and near-infrared spectroscopy. During immersion, shear rate increased (p<0.0001) comparably between groups in the popliteal artery (Controls: +183±26%; PAD: +258±54%) and brachial artery (Controls: +117±24%; PAD: +107±32%). Lower-limb blood flow increased significantly in both groups, as measured from duplex ultrasound (>200%), plethysmography (>100%) and spectroscopy, while central and peripheral pulse wave velocity decreased in both groups. Mean arterial blood pressure was reduced by 22±9 mmHg (main effect p<0.0001, interaction p=0.60) during immersion, and remained 7±7 mmHg lower 3 h afterward. In PAD, popliteal shear profiles and claudication both compared favourably with those measured immediately following symptom-limited walking. A 30-min hot-water immersion is a practical means of delivering heat therapy to PAD patients and healthy, elderly individuals to induce appreciable systemic (chronotropic and blood pressure lowering) and hemodynamic (upper and lower-limb perfusion and shear rate increases) responses.
The subfornical organ (SFO) is a critical circumventricular organ involved in the control of cardiovascular and metabolic homeostasis. Despite the plethora of circulating signals continuously sensed by the SFO, studies investigating how these signals are integrated are lacking. In this study, we use patch clamp techniques to investigate how the traditionally classified 'cardiovascular' hormone angiotensin II (ANG), 'metabolic' hormone cholecystokinin (CCK) and 'metabolic' signal glucose interact and are integrated in the SFO. Sequential bath-application of CCK (10nM) and ANG (10nM) onto dissociated SFO neurons revealed that: 63% of responsive SFO neurons depolarized to both CCK & ANG; 25% depolarized to ANG only; and 12% hyperpolarized to CCK only. We next investigated the effects of glucose by incubating and recording neurons in either hypo-, normo- or hyperglycemic conditions and comparing the proportions of responses to ANG (n=55) or CCK (n=83) application in each condition. A hyperglycemic environment was associated with a larger proportion of depolarizing responses to ANG (X2, p<0.05), and a smaller proportion of depolarizing responses along with a larger proportion of hyperpolarizing responses to CCK (X2, p<0.01). These data demonstrate that SFO neurons excited by CCK are also excited by ANG, suggesting that CCK may influence fluid intake or blood pressure via the SFO. Additionally, the demonstration that glucose environment affects the responsiveness of neurons to both these hormones highlights the ability of SFO neurons to integrate multiple metabolic and cardiovascular signals, and has important implications for this structure's role in the control of various autonomic functions during hyperglycemia.
Marine teleosts can absorb imbibed seawater (SW) to maintain water balance, with esophageal desalination playing an essential role. NaCl absorption from luminal SW was enhanced 10-fold in the esophagus of SW-acclimated eels, and removal of Na+ or Cl- from luminal SW abolished the facilitated absorption, indicating coupled transport. Mucosal/serosal application of various blockers for Na+/Cl- transporters profoundly decreased the absorption. Among the transporter genes expressed in eel esophagus detected by RNA-seq, dimethyl amiloride-sensitive Na+/H+ exchanger (NHE3) and 4,4'-diisothiocyano-2,2'-disulfonic acid-sensitive Cl-/HCO3- exchanger (AE) coupled by the scaffolding protein on the apical membrane of epithelial cells, and ouabain-sensitive Na+/K+-ATPases (NKA1α1c and NKA3α) and diphenylamine-2-carboxylic acid-sensitive Cl- channel (CLCN2) on the basolateral membrane, may be responsible for enhanced transcellular NaCl transport because of their profound upregulation after SW acclimation. Upregulated carbonic anhydrase (CA2a) supplies H+ and HCO3- for activation of the coupled NHE and AE. Apical hydrochlorothiazide-sensitive Na+-Cl- cotransporters and basolateral Na+-HCO3- cotransporter (NBCe1) and AE1 are other possible candidates. Concerning the low water permeability that is typically seen in marine teleost esophagus, downregulated aquaporin genes (aqp1a and aqp3) and upregulated claudin gene (cldn15a) are candidates for transcellular/paracellular route. In situ hybridization showed that these upregulated transporters and tight-junction protein genes were expressed in the absorptive columnar epithelial cells of eel esophagus. These results allow us to depict a full picture of the molecular mechanism of active desalination and low water permeability that are characteristic to marine teleost esophagus and gain deeper insights into the role of gastrointestinal tracts in SW acclimation.
Sustained hypertension is an important consequence of obstructive sleep apnea. An animal model of the hypoxemia associated with sleep apnea, chronic intermittent hypoxia (CIH), produces increased sympathetic nerve activity (SNA) and sustained increases in blood pressure. Many mechanisms have been implicated in the hypertension associated with CIH including the role of FosB within the median preoptic nucleus (MnPO). Also, the renin-angiotensin system (RAS) has been associated with CIH hypertension. We conducted experiments in order to determine the possible association of FosB/FosB with a RAS component, angiotensin converting enzyme 1 (ACE1), within the MnPO following 7 days of CIH. Retrograde tract tracing from the paraventricular nucleus (PVN), a downstream region of the MnPO, was utilized in order to establish a potential pathway for FosB/FosB activation of MnPO ACE1 neurons. After CIH, ACE1 cells with FosB/FosB expression increased colocalization with a retrograde tracer that was injected unilaterally within the PVN. Also, Western blot examination showed ACE1 protein expression increasing within the MnPO following CIH. Chromatin immunoprecipitation (ChIP) assays demonstrated an increase in FosB/FosB association with the ACE1 gene within the MnPO following CIH. FosB/FosB may transcriptionally target ACE1 within the MnPO following CIH in order to affect the downstream PVN region, which may influence SNA and blood pressure.
Respiration varies from breath to breath. On the millisecond time scale of spiking, neuronal circuits exhibit variability due to the stochastic properties of ion channels and synapses. Does this fast, microscopic source of variability contribute to the slower, macroscopic variability of the respiratory period? To address this question, we modeled a stochastic oscillator with forcing; then, we tested its predictions experimentally for the respiratory rhythm generated by the in situ perfused preparation during vagal nerve stimulation (VNS). Our simulations identified a relationship among the gain of the input, entrainment strength and rhythm variability. Specifically, at high gain, the periodic input entrained the oscillator and reduced variability; whereas at low gain, the noise interacted with the input causing events known as "phase slips", which increased variability on a slow time scale. Experimentally, the in situ preparation behaved like the low-gain model: VNS entrained respiration, but exhibited phase slips that increased rhythm variability. Next, we used bilateral muscimol microinjections in discrete respiratory compartments to identify areas involved in VNS gain control. Suppression of activity in the nucleus tractus solitarii (nTS) occluded both entrainment and amplification of rhythm variability by VNS, confirming that these effects were due to the activation of the Hering-Breuer reflex. Suppressing activity of the Kölliker-Fuse nuclei (KFn) enhanced entrainment and reduced rhythm variability during VNS, consistent with the predictions of the high-gain model. Together, the model and experiments suggest that the KFn regulates respiratory rhythm variability via a gain control mechanism.
Mathematical modeling is an important tool for understanding quantitative relationships among components of complex physiological systems and for testing competing hypotheses. We used HumMod, a large physiological model, to test hypotheses of blood pressure (BP) salt sensitivity. Systemic hemodynamics, renal, and neurohormonal responses to chronic changes in salt intake were examined during normal renal function, fixed low or high plasma angiotensin II (Ang II) levels, bilateral renal artery stenosis, increased renal sympathetic nerve activity (RSNA), and decreased nephron numbers. Simulations were run for 4 weeks at salt intakes ranging from 30 to 1000 mmol/day. Reducing functional kidney mass or fixing Ang II increased salt sensitivity. Salt sensitivity, associated with inability of Ang II to respond to changes in salt intake, occurred with smaller changes in renal blood flow but greater changes in glomerular filtration rate, renal sodium reabsorption, and total peripheral resistance (TPR). However, clamping TPR at normal or high levels had no major effect on salt sensitivity. There were no clear relationships between BP salt sensitivity and renal vascular resistance or extracellular fluid volume. Our robust mathematical model of cardiovascular, renal, endocrine, and sympathetic nervous system physiology supports the hypothesis that specific types of kidney dysfunction, associated with impaired regulation of Ang II or increased tubular sodium reabsorption, contribute to BP salt sensitivity. However, increased preglomerular resistance, increased RSNA, or inability to decrease TPR does not appear to influence salt sensitivity. This model provides a platform for testing competing concepts of long-term BP control during changes in salt intake.
Effects of altered gastric emptying on glucose absorption and kinetics are not well understood in non-diabetic obese adults. The aim of this work was to develop a physiology-based model that can characterize and compare interactions among gastric emptying, glucose absorption and glycemic control in non-diabetic obese and lean healthy adults. Dynamic glucose, insulin and gastric emptying (measured with breath test) data from 12 non-diabetic obese and 12 lean healthy adults were available until 180 minutes after an oral glucose tolerance test (OGTT) with 10g, 25g and 75g of glucose. A physiology-based model was developed to characterize glucose kinetics applying nonlinear mixed-effects modelling with NONMEM7.3. Glucose kinetics after OGTT was described by a one-compartment model with an effect compartment to describe delayed insulin effects on glucose clearance. After accounting for interactions between individual gastric emptying and glucose absorption profiles the glucose absorption rate was found to be similar in non-diabetic obese and lean controls. Baseline glucose concentration was estimated to be only marginally higher in non-diabetic obese subjects (4.9 versus 5.2 mmol.L-1), whereas insulin-dependent glucose clearance in non-diabetic obese subjects was found to be cut in half compared to lean controls (0.052 versus 0.029 L.min-1) and insulin concentration associated with 50% of insulin-dependent glucose elimination rate was ~2-fold higher in non-diabetic obese subjects compared to lean controls (7.1 versus 15.3 μU.mL-1). Physiology-based models can characterize and compare the dynamic interaction among gastric emptying, glucose absorption and glycemic control in populations of interest such as lean healthy and non-diabetic obese adults.
This study investigated the role of -aminobutyric acid subtype B (GABAB) receptors in tibial and pudendal neuromodulation of bladder overactivity induced by intravesical administration of dilute (0.5%) acetic acid (AA) in α-chloralose anesthetized cats. To inhibit bladder overactivity, tibial or pudendal nerve stimulation (TNS or PNS) was applied at 5 Hz and 2 or 4 times threshold (T) intensity for inducing toe or anal sphincter twitch. TNS at 2T or 4T intensity significantly (p<0.05) increased the bladder capacity to 173.8±16.2% or 198.5±24.1%, respectively, of control capacity. Meanwhile, PNS at 2T or 4T intensity significantly (p<0.05) increased the bladder capacity to 217±18.8% and 221.3±22.3% of control capacity, respectively. CGP52432 (a GABAB receptor antagonist) at intravenous dosages of 0.1-1 mg/kg completely removed the TNS inhibition in female cats but had no effect in male cats. CGP52432 administered intravenously also had no effect on control bladder capacity or the pudendal inhibition of bladder overactivity. These results reveal a sex difference of GABAB receptors in tibial neuromodulation of bladder overactivity in cats and that GABAB receptors are not involved in either pudendal neuromodulation or irritation-induced bladder overactivity.
The ability of ectotherms to respond to changes in their thermal environment through plastic mechanisms is central to their adaptive capability. However, we still lack knowledge on physiological and functional responses by which ectotherms acclimate to temperatures during development, and in particular, how physiological stress at extreme temperatures may counteract beneficial acclimation responses at benign temperatures. We exposed Drosophila melanogaster to ten developmental temperatures covering their entire permissible temperature range. We obtained metabolic profiles and reaction norms for several functional traits: egg-to-adult viability, developmental time, and heat and cold tolerance. Females were more heat tolerant than males, whereas no sexual dimorphism was found in cold tolerance. A group of metabolites, mainly free amino acids, had linear reaction norms. Several energy carrying molecules, as well as some sugars, showed distinct inverted u-shaped norms of reaction across the thermal range, resulting in a positive correlation between metabolite intensities and egg-to-adult viability. At extreme temperatures, low levels of these metabolites were interpreted as a response characteristic of costs of homeostatic perturbations. Our results provide novel insights into a range of metabolites reported to be central for the acclimation response, and suggest several new candidate metabolites. Low and high temperatures result in different adaptive physiological responses, but they also have commonalities likely to be a result of the failure to compensate for the physiological stress. We suggest that the regulation of metabolites that are tightly connected to the performance curve is important for the ability of ectotherms to cope with variation in temperature.
Patients with ischemic heart failure (iHF) have a high risk of neurological complications such as cognitive impairment and stroke. We hypothesized that iHF patients have a higher incidence of impaired dynamic cerebral autoregulation (dCA). Adult patients with iHF and healthy volunteers were included. Cerebral blood flow velocity (CBFV, transcranial Doppler, middle cerebral artery), end-tidal CO2 (capnography), and arterial blood pressure (Finometer) were continuously recorded supine for five minutes at rest. Autoregulation index (ARI) was estimated from the CBFV step response derived by transfer function analysis using standard template curves. Fifty-two iHF patients and 54 age-, gender-, and BP-matched healthy volunteers were studied. Echocardiogram ejection fraction was 40 (20- 45) % in iHF group. iHF patients compared to control subjects had reduced EtCO2 (34.1 ± 3.7 vs. 38.3 ± 4.0 mmHg, p<0.001) and lower ARI values (5.1 ± 1.6 vs. 5.9 ± 1.0, p= 0.012). ARI < 4, suggestive of impaired CA, was more common in iHF patients (28.8% vs. 7.4%, p = 0.004). These results confirm that iHF patients are more likely to have impaired dCA in comparison with age-matched controls. The relationship between impaired dCA and neurological complications in iHF patients deserves further investigation.
Hypertensive pregnancy (HTNP) is a risk factor for future cardiovascular disease. Exaggerated cardiovascular responses to physical stress are also considered an independent marker of cardiovascular disease risk. However, there are limited data regarding the blood pressure (BP) responses to acute stress in women, who have a history of HTNP. Hence, the aim of the study is to compare BP responses to a physical stress in postmenopausal women with a history of HTNP to age- and parity-matched women with a history of normotensive pregnancy (NP). Beat-to-beat BP and heart rate was recorded in 64 postmenopausal women with [age = 58.5 (55.2, 62.2) yrs] and without [age = 59.4 (55.9, 62.4) yrs] a history of HTNP prior to and during isometric handgrip (IHG) exercise (30% of maximal voluntary contraction) to fatigue. Muscle metaboreflex was measured during post-exercise ischemia following IHG exercise. BP variables increased similarly in response to IHG exercise [systolic: NP=11.5 (8.9, 17.6) %, HTNP=11.3 (9.5, 15.9) %; diastolic NP=11.2 (7.9, 13.3) %, HTNP=9.5 (7.1, 14.3) %; mean blood pressure: NP= 9.8 (5.0, 13.6) %, HTNP=7.2 (4.4, 10.4) %)] and post-exercise ischemia [systolic: NP=14.1 (10.3, 23.0) %, HTNP=15.8 (10.6, 21.4) %; diastolic NP=12.2 (4.8, 17.0) %, HTNP=10.4 (5.3, 17.1) %; mean blood pressure: NP= 11.1 (6.1, 17.9) %, HTNP=9.4 (2.9, 14.8) %] in both groups. Although having a history of HTNP is associated with future cardiovascular disease risk, results from this study suggest that the risk may not be manifest through altered cardiovascular-metaboreflex response to physical stressors.
The purpose of this study was to evaluate the role of TGF-β1 in regulating tendon extracellular matrix (ECM) after acute exercise. Wistar rats exercised (n=15) on a treadmill for four consecutive days (60 min/day) or maintained normal cage activity. After each exercise bout, the peritendinous space of each Achilles tendon was injected with a TGF-β1 receptor inhibitor or sham. Independent of group, tendons injected with inhibitor exhibited ~50% lower Smad 3 (Ser423/425) (p<0.05) and 2.5-fold greater ERK1/2 phosphorylation (p<0.05) when compared to sham (p<0.05). Injection of the inhibitor did not alter collagen content in either group (p>0.05). In exercised rats, hydroxylyslpyridinoline (HP) content and collagen III expression were lower (p<0.05) in tendons injected with inhibitor when compared to sham. In non-exercised rats, collagen I and lysyl oxidase (LOX) expression were lower (p<0.05) in tendons injected with inhibitor when compared to sham. Decorin expression was not altered by inhibitor in either group (p>0.05). Based on evaluation of hematoxylin and eosin (H&E) stained cross-sections, cell numbers were not altered by inhibitor treatment in either group (p>0.05). Evaluation of H&E stained sections revealed no effect of inhibitor on collagen fibril morphology. In contrast, scores for regional variation in cellularity decreased in exercised rats (p<0.05). No differences in fiber arrangement, structure, and nuclei form were noted in either group (p>0.05). Our findings suggest that TGF-1 signaling is necessary for the regulation of tendon cross-link formation as well as collagen and LOX gene transcription in an exercise-dependent manner.
Autoantibodies to the angiotensin II (ANGII) type I receptor (AT1-AA) are associated with preeclampsia (PE). We found that Vitamin D supplementation reduced AT1-AA and blood pressure (MAP) in the RUPP rat model of PE. However, it was undetermined if the decrease in AT1-AA was the mechanism whereby Vitamin D lowered MAP or if it was through factors downstream of AT1-AA. Uterine artery resistance index, placental ET-1 and sFlt-1 are increased with AT1-AA induced hypertension and considered markers of PE in pregnant women. Therefore, we hypothesized that Vitamin D would reduce PE factors during AT1-AA induced hypertension and could lower blood pressure in a model of hypertension during pregnancy without PE features. Either ANGII (50ng/kg/day) or AT1-AA (1:40) was infused from gestational day (GD) 12-19. Vitamin D2 (VD2, 270 IU/day) or Vitamin D3 (VD3, 15 IU/day) was administered orally from GD14-18. MAP (mmHg) increased in AT1-AA (121±4) and ANGII (113±1) infused pregnant rats compared to normal pregnant rats (NP) (101±2) but was lower in AT1-AA+VD2 (105±2), AT1-AA+VD3 (109±2), ANGII+VD2 (104±4) and ANGII+VD3 (104±3). VD2 and/or VD3 improved PE features associated with AT1-AA during pregnancy, while ANGII did not induce such features, supporting the hypothesis that AT1-AA induces PE features during pregnancy and these are improved with Vit D. In this study we demonstrate that Vitamin D improved many factors associated with PE and reduced blood pressure in a hypertensive model without PE features, indicating that Vitamin D could be beneficial for various hypertensive disorders of pregnancy.
Substantial increases in cardiac output (CO), stroke volume (SV) and gastrointestinal blood flow are essential for euryhaline rainbow trout (Oncorhyncus mykiss) osmoregulating in seawater. However, the underlying hemodynamic mechanisms responsible for these changes are unknown. By examining a range of circulatory and cardiac morphological variables of seawater- and freshwater-acclimated rainbow trout, the present study revealed a significantly higher central venous pressure (CVP) in seawater-acclimated trout (~0.09 vs. -0.02 kPa). This serves to increase cardiac end-diastolic volume in seawater and explains the elevations in SV (~0.41 vs. 0.27 ml kg-1) and CO (~21.5 vs. 14.2 ml min-1 kg-1) when compared with trout in freshwater. Furthermore, these hemodynamic modifications coincided with a significant increase in the proportion of compact myocardium, which may be necessary to compensate for the increased wall tension associated with a larger stroke volume. Following a temperature increase from 10 to 16.5°C, both acclimation groups exhibited similar increases in heart rate (Q10 of ~2), but SV tended to decrease in seawater-acclimated trout despite the fact that CVP was maintained in both groups. This resulted in CO of seawater- and freshwater-acclimated trout stabilizing at a similar level after warming (~26 ml min-1 kg-1). The consistently higher CVP of seawater-acclimated trout suggests that factors other than compromised cardiac filling constrained the SV and CO of these individuals at high temperatures. The present study highlights, for the first time, the complex interacting effects of temperature and water salinity on cardiovascular responses in a euryhaline fish species.
Thyroid hormones (TH) regulate metabolism, but are typically suppressed during times of stressful physiological conditions, including fasting. Interestingly, prolonged fasting in northern elephant seal pups is associated with reliance on a lipid-based metabolism and increased levels of circulating thyroid hormones that are partially attributed to active secretion as opposed to reduced clearance. This apparent paradox is coupled with complementary increases in cellular TH-mediated activity suggesting that in mammals naturally adapted to prolonged fasting TH are necessary to support metabolism. However, the functional relevance of this physiological paradox has remained largely unexplored especially as it relates to the regulation of lipids. To address the hypothesis that thyroid stimulating hormone (TSH)-mediated increase in TH contributes to lipid metabolism, we infused early- and late-fasted pups with TSH and measured several key genes in adipose and muscle, and plasma hormones associated with the regulation of lipid metabolism. TSH infusion increased the mRNA expressions of peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) over 6.5-fold at 60 minutes in muscle, and the expression of uncoupling protein 2 (UCP2) over 27-fold during the early fast at 60 minutes, in adipose. Additionally, during the late fast the protein content of adipose CD36 increased 1.1-fold and plasma NEFA concentrations increased 25% at 120 mins, with NEFA levels returning to baseline after 24 hrs. Here we show that the TSH-induced increases in TH in fasting pups are functional and likely contribute to the maintenance of a lipid-based metabolism.
Temperature-induced changes in cardiac output (Q) in fish are largely dependent on thermal modulation of heart rate (fH) and at high temperatures Q collapses due to heat-dependent depression of fH. This study tests the hypothesis that firing rate of sinoatrial pacemaker cells sets the upper thermal limit of fH in vivo. To this end temperature-dependence of action potential (AP) frequency of enzymatically isolated pacemaker cells (pacemaker rate, fPM), spontaneous beating rate of isolated sinoatrial preparations (fSA) and in vivo heart rate (fH) of the cold-acclimated (4°C) brown trout (Salmo trutta fario) were compared under acute thermal challenges. With rising temperature, fPM steadily increased due to the acceleration of diastolic depolarization and shortening of AP duration up to the break point temperature (TBP) of 24.0±0.37°C where the electrical activity abruptly ceased. The maximum fPM at TBP was much higher (193±21.0 beats per minute (bpm)) than the peak fSA (94.3±6.0 bpm at 24.1°C) or peak fH (76.7±2.4 at 15.7±0.82°C) (P<0.05). These findings strongly suggest that the frequency generator of the sinoatrial pacemaker cells does not limit fH at high temperatures in the brown trout in vivo.
The aim of this study was to investigate the effect of repeated passive heat exposure (i.e. acclimation) on muscle contractility in humans. Fourteen non-heat acclimated males completed two trials including electrically evoked twitches and voluntary contractions in thermoneutral conditions (COOL: 24ºC, 40% RH) and hot ambient conditions in the hyperthermic state (HOT: 44-50ºC, 50% RH) on consecutive days in a counterbalanced order. Rectal temperature was ~36.5ºC in COOL and was maintained at ~39ºC throughout HOT. Both trials were repeated after 11 days of passive heat acclimation (1 h per day, 48-50ºC, 50% RH). Heat acclimation decreased core temperature in COOL (-0.2ºC, p<0.05), increased the time required to reach 39ºC in HOT (+9min, p<0.05) and increased sweat rate in HOT (+0.7 l.h-1, p<0.05). Moreover, passive heat acclimation improved skeletal muscle contractility as evidenced by an increase in evoked peak twitch amplitude both in COOL (20.5±3.6 vs. 22.0±4.0 N.m) and HOT (20.5±4.7 vs. 22.0±4.0 N.m) (+9%, p<0.05). Maximal voluntary torque production was also increased both in COOL (145±42 vs. 161±36 N.m) and HOT (125±36 vs. 145±30 N.m) (+17%, p<0.05), despite voluntary activation remaining unchanged. Furthermore, the slope of the relative torque/EMG linear relationship was improved post-acclimation (p<0.05). These adjustments demonstrate that passive heat acclimation improves skeletal muscle contractile function during electrically evoked and voluntary muscle contractions of different intensities both in COOL and HOT. These results suggest that repeated heat exposure may have important implications to passively maintain or even improve muscle function in a variety of performance and clinical settings.
White adipose tissue (WAT) and brown adipose tissue (BAT) are innervated and regulated by the sympathetic nervous system (SNS). It is not clear, however, whether there are shared or separate central SNS outflows to WAT and BAT that regulate their function. We injected two isogenic strains of pseudorabies virus, a retrograde transneuronal viral tract tracer, with unique fluorescent reporters into interscapular BAT (IBAT) and inguinal WAT (IWAT) of the same Siberian hamsters to define SNS pathways to both. To test the functional importance of SNS coordinated control of BAT and WAT, we exposed hamsters with denervated SNS nerves to IBAT to 4°C for 16-24 hours, and measured core and fat temperatures, and norepinephrine turnover (NETO) and uncoupling protein 1 (UCP1) expression in fat tissues. Overall, there were more SNS neurons innervating IBAT than IWAT across the neuroaxis. However, there was a greater percentage of singly labeled IWAT neurons in midbrain reticular nuclei than singly labeled IBAT neurons. The hindbrain had ~30-40% of doubly labeled neurons while the forebrain had ~25% suggesting shared SNS circuitry to BAT and WAT across the brain. The raphe nucleus, a key region in thermoregulation, had ~40% doubly labeled neurons. Hamsters with IBAT SNS denervation maintained core body temperature during acute cold challenge and had increased beige adipocyte formation in IWAT. They also had increased IWAT NETO, temperature, and UCP1 expression compared with intact hamsters. These data provide strong neuroanatomical and functional evidence of WAT and BAT SNS crosstalk for thermoregulation and beige adipocyte formation.
We recently showed that intradermal administration of endothelin-1 diminished endothelium-dependent and -independent cutaneous vasodilation. We evaluated the hypothesis that Rho kinase may be a mediator of this response. We also sought to evaluate if endothelin-1 increases sweating. In twelve adults (25±6 years), we measured cutaneous vascular conductance (CVC) and sweating during 1) endothelium-dependent vasodilation induced via administration of incremental doses of methacholine (0.25, 5, 100, 2000mM each for 25 min) and 2) endothelium-independent vasodilation induced via administration of 50mM sodium nitroprusside (20-25 min). Responses were evaluated at four skin sites treated with either 1) lactated Ringer solution (Control), 2) 400nM endothelin-1, 3) 3mM HA-1077 (Rho kinase inhibitor), or 4) endothelin-1+HA-1077. Pharmacological agents were intradermally administered via microdialysis. Relative to the Control site, endothelin-1 attenuated endothelium-dependent vasodilation (CVC at 2000mM methacholine, 80±10 vs. 56±15%max, P<0.01); however, this response was not detected when the Rho kinase inhibitor was simultaneously administered (CVC at 2000mM methacholine for Rho kinase inhibitor vs. endothelin-1 + Rho kinase inhibitor sites: 73±9 vs. 72±11%max, P>0.05). Endothelium-independent vasodilation was attenuated by endothelin-1 compared to the Control site (CVC, 92±13 vs. 70±14%max, P<0.01). However, in the presence of Rho kinase inhibition, endothelin-1 did not affect endothelium-independent vasodilation (CVC at Rho kinase inhibitor vs. endothelin-1+Rho kinase inhibitor sites: 81±9 vs. 86±10%max, P>0.05). There was no between-site difference in sweating throughout (P>0.05). We show that in young adults, Rho kinase is an important mediator of the endothelin-1 mediated attenuation of endothelium-dependent and -independent cutaneous vasodilation, and that endothelin-1 does not increase sweating.
We tested the hypothesis that female mice null for UCP1 would have increased susceptibility to Western diet-induced "whitening" of brown adipose tissue (AT) and glucose intolerance. Six-week old C57BL/6J wild-type (WT) and UCP1 knockout (UCP1-/-) mice, housed at 25°C, were randomized to either a control diet (10% kcal from fat) or Western diet (45% kcal from fat and 1% cholesterol) for 28 weeks. Loss of UCP1 had no effect on energy intake, energy expenditure, spontaneous physical activity, weight gain, or visceral white AT mass. Despite similar susceptibility to weight gain compared to WT, UCP1-/- exhibited whitening of brown AT evidenced by a striking ~500% increase in mass and appearance of large unilocular adipocytes, increased expression of genes related to inflammation, immune cell infiltration, and endoplasmic reticulum/oxidative stress (P<0.05), and decreased mitochondrial subunit protein (COX I, II, III, and IV, P<0.05), all of which were exacerbated by Western diet (P<0.05). UCP1-/- mice also developed liver steatosis and glucose intolerance, which was worsened by Western diet. Collectively, these findings demonstrate that loss of UCP1 exacerbates Western diet-induced whitening of brown AT, glucose intolerance, and induces liver steatosis. Notably, the adverse metabolic manifestations of UCP1-/- were independent of changes in body weight, visceral adiposity, and energy expenditure. These novel findings uncover a previously-unrecognized metabolic-protective role of UCP1 that is independent of its already established role in energy homeostasis.
In this review, we highlight that the relationship between muscle sympathetic nerve activity (MSNA) and mean arterial pressure is complex, differs by sex, and changes with age. In young men there is an inverse relationship between MSNA and cardiac output where high MSNA is compensated for by low cardiac output. This inverse relationship is not seen in older men. In young women sympathetic vasoconstriction is offset by β-adrenoreceptor mediated vasodilation, limiting the ability of young women to maintain blood pressure in response to orthostatic stress. However, β-mediated dilation in women is attenuated with age, leading to unopposed α-adrenergic vasoconstriction and a rise in the direct transduction of MSNA into increases in blood pressure. We propose that these changes with age and menopausal status are major contributing factors in the increased prevalence of hypertension in older women. In addition to aging, we highlight that changes in sex hormones in young women (across the menstrual cycle, with oral contraceptive use, or with pregnancy) influence MSNA and the transduction of MSNA into increases in blood pressure. It is likely that the β-adrenergic receptors and/or changes in baroreflex sensitivity play a large role in these sex differences and changes with alterations in sex hormones.
Deteriorated aerobic response to moderate exercise might precede the manifestation of clinical symptoms of non-communicable diseases. The purpose of the current study was to verify that the use of current wearable technologies for analysis of pulmonary oxygen uptake (VO2) dynamics during a pseudorandom ternary sequence (PRTS) over-ground walking protocol is a suitable procedure for the investigation of the aerobic response in more realistic settings. A wearable accelerometer assessed the input changes delivered to the aerobic system. Eight adults (23.5±3.7 years old, 174±7 cm and 71.4±7.4 kg) performed two identical PRTS over-ground walking protocols. In addition, they performed on the cycle ergometer two identical pseudo-random binary sequence (PRBS) protocols and one incremental protocol for maximal VO2 determination. In the frequency domain, mean normalized gain amplitude (MNG in %) quantified VO2 dynamics. The MNG during PRTS was correlated (r=-0.80, p=0.01) with the VO2 time constant obtain during cycling. The MNG estimated during PRBS was similar to the MNG estimated during PRTS (r=0.80, p=0.01). The maximal VO2 correlated with the MNG obtained during the PRBS (r=0.79, p=0.01) and PRTS (r=0.78, p=0.02) protocols. In conclusion, PRTS over-ground walking protocol can be used to evaluate the aerobic system dynamics by the simultaneous measurement of VO2 and hip acceleration. This study has shown that wearable technologies in combination with assessment of MNG, a novel indicator of system dynamics, open new possibilities to monitor cardiorespiratory health under conditions that better simulate activities of daily living than cardiopulmonary exercise testing performed in a medical environment.
High dose glucocorticoids (GC) induce skeletal muscle atrophy, insulin resistance and reduced muscle capillarization. Identification of treatments to prevent or reverse capillary rarefaction and metabolic deterioration caused by prolonged elevations in GCs would be therapeutically beneficial. Chronic administration of prazosin, α1-adrenergic antagonist, increases skeletal muscle capillarization, in healthy rodents and recently, a rodent model of elevated GCs and hyperglycemia. The purpose of this study was to determine whether prazosin administration would improve glucose tolerance and insulin sensitivity, through prazosin mediated sparing of capillary rarefaction, in this rodent model of increased GC exposure. Prazosin was provided in drinking water (50mg/L) to GC-treated or control rats (400 mg implants of either corticosterone or a wax pellet) for 7 or 14 days (n=5-14/group). Whole body measures of glucose metabolism were correlated with skeletal muscle capillarization (C:F) at 7 and 14 days in the 4 groups of rats. Individual C:F was found to be predictive of insulin sensitivity (r2=0.4781), but not of glucose tolerance (r2=0.1601) and compared with water only, prazosin treatment decreased insulin values during oral glucose challenge by~1/3rd in Cort-treated animals. Cort-treatment, regardless of duration, induced significant glycolytic skeletal muscle atrophy (P<0.05), decreased IRS-1 protein content (P<0.05), and caused elevations in FOXO1 protein expression (P<0.05), which were unaffected with prazosin administration. In summary, it appears that α1-adrenergic antagonism improves Cort-induced skeletal muscle vascular impairments and reduces insulin secretion during an OGTT, but is unable to improve the negative alterations directly affecting the myocyte, including muscle size and muscle signaling protein expression.
Skeletal muscle satellite cells (SC) play an important role in muscle adaptation. In untrained individuals, SC content and activation status has been observed to increase in response to a single bout of exercise. Muscle fiber characteristics change considerably when resistance exercise is performed chronically, but whether training status affects the activity of SC in response to a single bout of exercise remains unknown. We examined the changes in SC content and activation status following a single bout of resistance exercise, prior to and following a 16wk progressive resistance training (RT) program in fourteen young (25±3yr) men. Before and after RT, percutaneous biopsies from the vastus lateralis muscle were taken prior to a single bout of resistance exercise and after 24 and 72h of post-exercise recovery. Muscle fiber size, capillarization, and SC response were determined by immunohistochemistry. Following RT, there was a greater activation of SC after 24h in response to a single bout of resistance exercise (Pre:1.4±0.3,24h:3.1±0.3 Pax7+/MyoD+ cells/100 fibers) as compared to before RT (Pre:1.4±0.3,24h:2.2±0.3 Pax7+/MyoD+ cells/100 fibers, p<0.05); no difference was observed 72h post-exercise. Following 16wk of RT, MyoD mRNA expression increased from basal to 24h after the single bout of exercise (p<0.05); this change was not observed prior to training. Individual capillary-to-fiber ratio (C/Fi) increased in both type I (1.8±0.3 to 2.0±0.3 C/Fi, p<0.05) and type II (1.7±0.3 to 2.2±0.3 C/Fi, p<0.05) fibers in response to RT. Following RT, enhanced activation of SC in response to resistance exercise is accompanied by increases in muscle fiber capillarization.
The role of high fructose ingestion (HFI) in the development of conditions mimicking human metabolic syndrome has mostly been demonstrated in male animals; however, the extent of HFI-induced metabolic alterations in females remains unclear. The present study investigated whether HFI-induced metabolic perturbations differ between sexes and whether HFI aggravates the metabolic disturbances under ovarian hormone deprivation. Male, female and ovariectomized (OVX) Sprague-Dawley rats were given either water or liquid fructose (10% w/v) for 6 weeks. Blood pressure, glucose tolerance, insulin-stimulated glucose transport activity and signaling proteins, including insulin receptor (IR), insulin receptor substrate 1 (IRS-1), Akt, Akt substrate of 160 kDa (AS160), AMPKα, c-Jun NH2-terminal kinase (JNK), p38 MAPK, angiotensin converting enzyme (ACE), angiotensin II (AngII) type 1 receptor (AT1R), ACE2 and Mas receptor (MasR) in skeletal muscle, were evaluated. We found that HFI led to glucose intolerance and hypertension in male and OVX rats but not in female rats with intact ovaries. Moreover, HFI did not induce insulin resistance in the skeletal muscle of female and OVX rats but impaired the insulin-stimulated glucose transport activity in the skeletal muscle of male rats, which was accompanied by lower insulin-stimulated IRS-1 Tyr989 (44%), Akt Ser473 (30%) and AS160 Ser588 (43%) and increases in insulin-stimulated IRS-1 Ser307 (78%), JNK Thr183/Tyr185 (69%) and p38 MAPK Thr180/Tyr182 (81%). The results from the present study show sex differences in the development of metabolic syndrome-like conditions and indicate the protective role of female sex hormones against HFI-induced cardiometabolic abnormalities.
Cervical and high thoracic spinal cord injury (SCI) drastically impairs autonomic nervous system function. Individuals with SCI at thoracic spinal-level 5 (T5) or higher often present cardiovascular disorders that include resting systemic arterial hypotension. Gastrointestinal (GI) tissues are critically dependent upon adequate blood flow and even brief periods of visceral hypoxia triggers GI dysmotility. The aim of this study was to test the hypothesis that T3-SCI induces visceral hypoperfusion, diminished postprandial vascular reflexes and concomitant visceral inflammation. We measured in vivo systemic arterial blood pressure and superior mesenteric artery (SMA) and duodenal blood flow in anesthetized T3-SCI rats at 3 days and 3 weeks post-injury either fasted or following enteral feeding of a liquid mixed-nutrient meal (Ensure™). In separate cohorts of fasted T3-SCI rats, markers of intestinal inflammation were assayed by qRT-PCR. Our results show that T3-SCI rats displayed significantly reduced SMA blood flow under all experimental conditions (p<0.05). Specifically, the anticipated elevation of SMA blood flow in response to duodenal nutrient infusion (postprandial hyperemia) was either delayed or absent after T3-SCI. The dysregulated SMA blood flow in acutely-injured T3-SCI rats coincides with abnormal intestinal morphology and elevation of inflammatory markers, all of which resolve after 3 weeks. Specifically, Icam1, Ccl2 (MCP-1) and Ccl3 (MIP-1α) were acutely elevated following T3-SCI. Our data suggest that arterial hypotension diminishes mesenteric blood flow necessary to meet mucosal demands at rest and during digestion. The resulting GI ischemia and low-grade inflammation may be an underlying pathology leading to GI dysfunction seen following acute T3-SCI.
In supine humans the main drainage from the brain is through the internal jugular vein (IJV) but the vertebral veins (VV) become important during orthostatic stress because the IJV is partially collapsed. To identify the effect of this shift in venous drainage from the brain on the cerebral circulation, this study addressed both arterial and venous flow responses in the "anterior" and "posterior" parts of the brain when 9 healthy subjects (5 men) were seated and flow was manipulated by hyperventilation and inhalation of 6% carbon dioxide (CO2). From a supine to a seated position, both internal carotid artery (ICA) and IJV blood flow decreased (P=0.004 and P=0.002), while vertebral artery (VA) flow did not change (P=0.348) and VV flow increased (P=0.024). In both supine and seated positions the ICA response to manipulation of end-tidal CO2 tension was reflected in IJV (r=0.645 and r=0.790, P<0.001) and VV blood flow (r=0.771 and r=0.828, P<0.001). When seated the decrease in ICA blood flow did not affect venous outflow, but the decrease in IJV blood flow was associated with the increase in VV blood flow (r=0.479, P=0.044). In addition, the increase in VV blood flow when seated was reflected in VA blood flow (r=0.649, P=0.004) and the two flows were coupled during manipulation of the end-tidal CO2 tension (supine, r=0.551, P=0.004; seated, r=0.612, P<0001). These results support that VV compensates for the reduction in IJV blood flow when seated and that VV may influence VA blood flow.
Preeclampsia is a hypertensive disorder of pregnancy. Abnormal placentation associated with poor placental invasion of the uterine vasculature by trophoblast cells, leading to poor placental perfusion, oxidative stress and inflammation, is implicated in its pathogenesis. A dyslipidemia characterised by low plasma levels of high density lipoproteins (HDL) and elevated triglycerides has been described in preeclampsia. Apolipoprotein A-I (apoA-I), a constituent of HDL is an anti-inflammatory agent. This study investigated whether apoA-I protects against hypertension and adverse placental changes in a pro-inflammatory cytokine (TNF-α) induced model of preeclampsia. Further this study investigated whether apoA-I protects against the inhibitory effect of TNF-α in a human in vitro model of trophoblast invasion. Administration of apoA-I to pregnant mice prior to infusion with TNF-α resulted in a significant reduction in the cytokine induced increase in systolic BP. Magnetic resonance imaging (MRI) measurement of T2 relaxation, a parameter which is tissue specific and sensitive to physiological changes within tissues, showed a reversal of TNF-α induced placental changes. Pre-incubation of endothelial cells with apoA-I protected against the TNF-α induced inhibition of HTR-8/SVneo (trophoblast) cell integration into endothelial (UtMVEC) networks. This data suggests that a healthy lipid profile may affect pregnancy outcomes by priming endothelial cells in preparation for trophoblast invasion.
The cellular processes influenced by consuming polyunsaturated fatty acids remains poorly defined. Within skeletal muscle, a rate-limiting step in fatty acid oxidation is the of movement lipids across the sarcolemmal membrane, and therefore we aimed to determine the effects of consuming Flaxseed oil high in α-linolenic acid (ALA), on plasma membrane lipid composition and the capacity to transport palmitate. Rats fed a diet supplemented with ALA (10%) displayed marked increases in n-3 PUFAs within whole muscle and sarcolemmal membranes (~5-fold), at the apparent expense of arachidonic acid (-50%). These changes coincided with increased sarcolemmal palmitate transport rates (+20%), plasma membrane fatty acid translocase (FAT/CD36; +20%) abundance, skeletal muscle triacylglycerol content (~2-fold) and rates of whole body fat oxidation (~50%). The redistribution of FAT/CD36 to the plasma membrane could not be explained by increased phosphorylation of signaling pathways implicated in regulating FAT/CD36 trafficking events (i.e. phosphorylation of ERK1/2, CaMKII, AMPK, Akt), suggesting the increased n-3 PUFA composition of the plasma membrane influenced FAT/CD36 accumulation. Altogether, the present data provides evidence that a diet supplemented with ALA increases the transport of lipids into resting skeletal muscle in conjunction with increased sarcolemmal n-3 PUFA and FAT/CD36 contents.
While restoration of ACE2 activity in the pancreas leads to improvement of glycemia in experimental models of type 2 diabetes, global deficiency in ACE2 disrupts β-cell function and impairs glucose tolerance in mice, demonstrating the physiological role of ACE2 in glucose homeostasis. Although the contribution of pancreatic ACE2 to glucose regulation has been demonstrated in genetic models of diabetes and in models with overexpression of the renin-angiotensin system (RAS), it is unclear whether islet ACE2 is involved in glycemic control in common models of human type 2 diabetes. To determine whether diet-induced diabetes deregulates glucose homeostasis via reduction of ACE2 in the pancreatic islets, wildtype (WT) and ACE2 knockout (KO) male mice were fed a high-fat diet (HFD) for 16 weeks. ACE2 KO mice were more susceptible than WT mice to HFD-mediated glycemic dysregulation. Islet ACE2 activity and expression of various genes including Ang-II type 1a receptor (mAT1aR) were then assessed. Surprisingly, we observed no change in islet ACE2 activity and expression despite local RAS over-activity, indicated by up-regulation of mAT1aR expression. Despite a predominant expression in islet α-cells, further investigation highlighted a minor role for ACE2 on glucagon expression. Further, pancreatic ACE2 gene therapy improved glycemia in HFD-fed WT mice, leading to enhanced glucose-stimulated insulin secretion, reduced pancreatic Ang-II levels, fibrosis and ADAM17 activity. Altogether, our study demonstrates that HFD-feeding increases RAS activity and mediates glycemic dysregulation likely through loss of ACE2 present outside the islets but independently of changes in islet ACE2.
The epithelial Na+-coupled phosphate co-transporter family Slc34a (NaPi-II) is well conserved in vertebrates and plays an essential role in maintaining whole body levels of inorganic phosphate (Pi). A three dimensional model of the transport protein has recently been proposed with defined substrate coordination sites. Zebrafish express two NaPi-II isoforms with high sequence identity but a tenfold different apparent Km for Pi (K0.5Pi). We took advantage of the two zebrafish isoforms to investigate the contribution of specific amino acids to Pi coordination and transport. Mutations were introduced to gradually transform the low affinity isoform into a high affinity transporter. The constructs were expressed in Xenopus oocytes and functionally characterized. Since the co-transport of Pi and Na involves multiple steps that could all influence K0.5Pi we performed detailed functional analysis to characterize the impact of the mutations on particular steps of the transport cycle. We used varying concentrations of the substrates Pi and its slightly larger analogue arsenate, as well as the co-substrate Na+. Moreover, electrogenic kinetics were performed to assess intramolecular movements of the transporter. All the mutations were found to affect multiple transport steps which suggested that the altered amino acids induced subtle structural changes rather than coordinating Pi directly. The likely positions of the critical residues were mapped to the model of human Slc34a and their localization in relation to the proposed substrate binding pockets concurs well with the observed functional data.
Preeclampsia is associated with chronic inflammation and an imbalance among T-helper cell subtypes with an increase in T-helper 17 (TH17) cells. The objective of this study was to determine a role for TH17s from the RUPP rat model of preeclampsia to cause hypertension and chronic inflammation during pregnancy. CD4+/CD25- T cells were isolated from rat spleens, cultured in TH17 media, and were verified as TH17s via flow cytometry. On day 12 of gestation, 1x106 TH17 cells from RUPP rats were adoptively transferred into NP rats, carotid catheters inserted on day 18, and on day 19 mean arterial pressure (MAP) was recorded, serum and plasma were collected, and oxidative stress and AT1-AA production were analyzed. MAP increased from 100.3±1.7 mmHg in NP (n=17), to 124.8±2.1 mmHg in RUPP (n=22, P<0.0001) and to 110.8±2.8 mmHg in NP+RUPP TH17 (n=11). Pup weights in NP+RUPP TH17s were decreased to 1.92±0.09 g from 2.39±0.14 in NP rats (p<0.01). AT1-AA significantly increased from 0.1±0.2 beats/min in NP to 15.6±0.7 beats/min in NP+RUPP TH17s. IL-6 was 22.3±5.7 pg/mL in NP and increased to 60.45±13.8 pg/mL in RUPP (p<0.05) and 75.9 ±6.8 pg/mL in NP+RUPP TH17 rats (p<0.01). Placental and renal oxidative stress were 238±27.5 and 411±129.9 RLUs/min/mg in NP and 339±104.6 and 833±331.1 RLUs/min/mg in NP+RUPP TH17. In conclusion, RUPP TH17 cells induced intrauterine growth restriction and increased blood pressure, AT1-AA, IL-6, and tissue oxidative stress when transferred to NP rats, indicating a role for autoimmune associated TH17 cells, to cause much of the pathophysiology associated with preeclampsia.
Build-ups of ammonia can cause potentially fatal brain swelling in mammals, but such swelling is reversible in the anoxia- and ammonia-tolerant goldfish (Carassius auratus). We investigated brain swelling and its possible relationship to oxidative stress in the brain and liver of goldfish acutely exposed to high external ammonia (HEA; 5 mmol L-1 NH4Cl) at two different acclimation temperatures (14°C, 4°C). At 14ºC, HEA for 72 h increased internal ammonia and glutamine concentrations caused cellular oxidative damage in brain and liver. However, damage was most pronounced in brain, in which there was a 2-fold increase in thiobarbituric-acid reactive substances, a 3-fold increase in protein carbonylation, and a 20% increase in water volume (indicative of brain swelling). Increased activities of catalase, glutathione peroxidase, and glutathione reductase in the brain, suggested that goldfish upregulate their antioxidant capacity to partially offset oxidative stress during hyperammonemia at 14ºC. Notably, acclimation to colder (4°C) water completely attenuated the oxidative stress response to HEA in both tissues, and there was no change in brain water volume, despite similar increases in internal ammonia. We suggest that ammonia-induced oxidative stress may be responsible for the swelling of goldfish brain during HEA, but further studies are needed to establish a mechanistic link between ROS production and brain swelling. Nevertheless, a high capacity to withstand oxidative stress in response to variations in internal ammonia likely explains why goldfish are more resilient to this stressor than most other vertebrates.
Endothelial dysfunction and reduced nitric oxide (NO) signaling are key abnormalities leading to skeletal muscle oxygen delivery-utilization mismatch and poor physical capacity in patients with heart failure with reduced ejection fraction (HFrEF). Oral inorganic nitrate supplementation provides an exogenous source of NO that may enhance locomotor muscle function and oxygenation with consequent improvement in exercise tolerance in HFrEF. Thirteen patients (left ventricular ejection fraction ≤40%) were enrolled in a double-blind, randomized crossover study to receive concentrated nitrate-rich (nitrate) or nitrate-depleted (placebo) beetroot juice for 9 days. Low- and high-intensity constant-load cardiopulmonary exercise tests were performed with non-invasive measurements of central hemodynamics (stroke volume, heart rate and cardiac output via impedance cardiography), arterial blood pressure, pulmonary oxygen uptake, quadriceps muscle oxygenation (near-infrared spectroscopy) and blood lactate concentration. Ten patients completed the study with no adverse clinical effects. Nitrate-rich supplementation resulted in significantly higher plasma nitrite concentration compared to placebo (240±48 vs. 56±8 nM, respectively; P<0.05). There was no significant difference in the primary outcome of time to exercise intolerance between nitrate and placebo (495±53 vs. 489±58 s, respectively; P>0.05). Similarly, there were no significant differences in central hemodynamics, arterial blood pressure, pulmonary oxygen uptake kinetics, skeletal muscle oxygenation or blood lactate concentration from rest to low- or high-intensity exercise between conditions. Oral inorganic nitrate supplementation with concentrated beetroot juice did not present with beneficial effects on central or peripheral components of the oxygen transport pathway thereby failing to improve exercise tolerance in patients with moderate HFrEF.
Background: Exercise is beneficial in pulmonary arterial hypertension (PAH), although studies to date indicate little effect on the elevated pulmonary pressures or maladaptive RV hypertrophy associated with the disease. For chronic LV failure, high intensity interval training (HIIT) promotes greater endothelial stimulation and superior benefit than customary continuous exercise training (CExT); however, HIIT has not been tested for PAH. Therefore, here we investigated acute and chronic responses to HIIT vs. CExT in a rat model of monocrotaline- (MCT) induced mild PAH. Methods: Six weeks of treadmill training (5x/week) was performed, as either 30 min HIIT or 60 min low-intensity CExT. To characterize acute hemodynamic responses to the two approaches, novel recordings of simultaneous pulmonary and systemic pressures during running were obtained at pre- and 2, 4, 6, and 8 weeks post-MCT utilizing long-term implantable telemetry. Results: MCT-induced decrement in VO2max was ameliorated by both HIIT and CExT, with less pronounced pulmonary vascular remodeling and no increase in RV inflammation or apoptosis observed. Most importantly, only HIIT lowered RV systolic pressure, RV hypertrophy, and total pulmonary resistance, and prompted higher cardiac index; complemented by a RV increase in the positive inotrope apelin and reduced fibrosis. HIIT prompted a markedly pulsatile pulmonary pressure during running and was associated with greater lung endothelial nitric oxide synthase after 6 weeks. Conclusion: HIIT may be superior to CExT for improving hemodynamics and maladaptive RV hypertrophy in PAH. HIIT's superior outcomes may be explained by more favorable pulmonary vascular endothelial adaptation to the pulsatile HIIT stimulus.
Sympathetic outflow is modified during acute homeostatic stress through increased firing of low-threshold axons, recruitment of latent axons, and synaptic delay modifications. However, the role of central mechanisms versus peripheral-reflex control over sympathetic recruitment remains unknown. Here, we examined sympathetic discharge patterns during fatiguing static handgrip (SHG) exercise and post-exercise circulatory occlusion (PECO) to study the central versus peripheral-reflex elements of sympathetic neural coding. Muscle sympathetic nerve activity (MSNA; microneurography) was measured in six males (25±3 yrs) at baseline (3 minutes) and during 5 minutes of SHG exercise completed at 20% maximal voluntary contraction. Isolation of the peripheral metaboreflex component was achieved by PECO for 3 minutes. Action potential (AP) patterns were studied using wavelet-based methodology. Compared to baseline, total MSNA increased by minute three of SHG, remaining elevated throughout the duration of exercise and PECO (all P<0.05). The AP content per burst increased above baseline by minute four of SHG (4±2), remaining elevated at minute five (6±4) and PECO (4±4; all P<0.05). Similarly, total AP clusters increased by minute four of SHG (5±5), and remained elevated at minute five (6±3) and PECO (7±5; all P<0.01), indicating recruitment of latent sub-populations. Finally, the AP cluster size-latency profile was shifted downward during minute four (–32±22 ms) and five (–49±17 ms; both P<0.05) of SHG, but was not different than baseline during PECO (P>0.05). Our findings suggest that central perceptual factors play a specific role in the synaptic delay aspect of sympathetic discharge timing, whereas peripheral-reflex mechanisms affect recruitment of latent axons.
Intrauterine growth restriction (IUGR) is a common pregnancy complication and is a leading cause of fetal morbidity and mortality. Placental hypoxia contributes to adverse fetal consequences, including IUGR. Exposing pregnant rats to hypoxia can lead to IUGR; however, assessment of maternal vascular function in a rat model of hypoxia, and the mechanisms that may contribute to adverse pregnancy outcomes, has not been extensively studied. We hypothesized that exposing pregnant rats to hypoxia will affect maternal systemic vascular function and increase the uterine artery resistance index (RI), which will be associated with IUGR. To test this hypothesis, pregnant rats were kept in normoxia (21% O2) or hypoxia (11% O2) from gestational day (GD) 6 to 20. Maternal blood pressure, utero-placental resistance index (RI) (ultrasound biomicroscopy) and vascular function (wire myography) were assessed in uterine and mesenteric arteries. Fetal weight was significantly reduced (P< 0.001), while maternal blood pressure was increased (P<0.05) in rats exposed to hypoxia. Maternal vascular function was also affected after exposure to hypoxia, including impaired endothelium-dependent vasodilation responses to methacholine (MCh) in isolated uterine arteries (pEC50 normoxia: 6.55±0.23 vs. hypoxia: 5.02±0.35, P<0.01) and a reduced uterine artery RI in vivo (normoxia: 0.63±0.04 vs. hypoxia: 0.53±0.01, P<0.05); associated with an increase in umbilical vein RI (normoxia: 0.35±0.02 vs. hypoxia: 0.45±0.04, P<0.05). These data demonstrate maternal and fetal alterations in vascular function due to prenatal exposure to hypoxia. Further, although there was a compensatory reduction in uterine artery RI in the hypoxia groups, this was not sufficient to prevent IUGR.
Increasing evidence indicates a strong link between intestinal health and bone health. For example, inflammatory bowel disease can cause systemic inflammation, weight loss and extra-intestinal manifestations such as decreased bone growth and density. However, the effects of moderate intestinal inflammation without weight loss on bone health have never been directly examined; yet this condition is relevant not only to IBD but to conditions of increased intestinal permeability and inflammation as seen with ingestion of high fat diets, intestinal dysbiosis, irritable bowel syndrome, metabolic syndrome and food allergies. Here we induced moderate intestinal inflammation without weight loss in young male mice by treating with a low dose of DSS (1%) for 15 days. The mice displayed systemic changes marked by significant bone loss and a redistribution of fat from subcutaneous to visceral fat pad stores. Bone loss was caused by reduced osteoblast activity, characterized by decreased expression of osteoblast markers (runx 2, osteocalcin), histomorphometry and dynamic measures of bone formation. In addition, we observed a reduction in growth plate thickness and hypertrophic chondrocyte matrix components (collagen X). Correlation analyses indicate a link between gut inflammation and disease score, but more importantly we observed that bone density measures negatively correlated with intestinal disease score as well as colon and bone TNF levels. These studies demonstrate that colitis induced bone loss is not dependent upon weight loss and support a role for inflammation in the link between gut and bone health, an important area for future therapeutic development.
Maternal cigarette smoke (CS) exposure exhibits a strong epidemiological association with Sudden Infant Death Syndrome but other environmental stressors, including infection, hyperthermia and hypoxia have also been postulated as important risk factors. This study examines if maternal CS exposure causes maladaptations within homeostatic control networks by influencing the response to lipopolysaccharide, heat stress and/or hypoxia in neonatal rats. Pregnant dams were exposed to CS or parallel sham treatments daily for the length of gestation. Offspring were studied at postnatal day 6-8 at ambient temperatures (Ta) of 33°C or 38°C. Within each group, rats were allocated to control, saline or LPS (200µg/kg) treatments. Cardiorespiratory patterns were examined using head-out plethysmography and ECG surface electrodes during normoxia and hypoxia. Serum cytokine concentrations were quantified from samples taken at the end of each experiment. Our results suggest maternal CS exposure does not alter minute ventilation (VE) or heart rate (HR) response to infection or high temperature, but independently increases apnea frequency. CS also primes the inflammatory system to elicit a stronger cytokine response to bacterial insult. High Ta independently depresses VE but augments the hypoxia-induced increase in VE. Moreover, higher Ta increases HR during normoxia and hypoxia, and in the presence of an immune challenge, increases HR during normoxia and reduces the increase normally associated with hypoxia. Thus, while most environmental risk factors increase the burden on the cardiorespiratory system in early life, hyperthermia and infection blunt the normal HR response to hypoxia, and gestational CS independently destabilizes breathing by increasing apneas
We tested the hypothesis that sympathetic responses to baroreceptor unloading may be affected by circulating sex hormones. During lower body negative pressure at -30, -60, and -80 mmHg, muscle sympathetic nerve activity (MSNA), heart rate and blood pressure were recorded in women taking (n=8) and not taking (n=9) hormonal contraceptives. All women were tested twice, once during the low hormone phase (i.e. the early follicular phase of the menstrual cycle and the placebo phase of hormonal contraceptive use), and again during the high hormone phase (i.e. the midluteal phase of the menstrual cycle and active phase of contraceptive use). During baroreceptor unloading the reductions in stroke volume and resultant increases in MSNA and total peripheral resistance were greater in high hormone than low hormone phases in both groups. When normalized to the fall in stroke volume, increases in MSNA were no longer different between hormone phases. While stroke volume and sympathetic responses were similar between women taking and not taking hormonal contraceptives, mean arterial pressure was maintained during baroreceptor unloading in women not taking hormonal contraceptives but not in women using hormonal contraceptives. These data suggest that differences in sympathetic activation between hormone phases as elicited by lower body negative pressure are the result of hormonally-mediated changes in the hemodynamic consequences of negative pressure, rather than centrally-driven alterations to sympathetic regulation.
Diurnal or circadian rhythms are fundamentally important for healthy cardiovascular physiology, and play a role in timing of onset and tolerance to myocardial infarction (MI) in patients. Whether time of day of MI triggers different molecular and cellular responses that can influence myocardial remodelling is not known. This study was designed to test whether time of day of MI triggers different gene expression, humoral, and innate inflammatory responses that contribute to cardiac repair after MI. Mice were infarcted by left anterior descending coronary artery ligation (MI model) within a 2 hour time window either shortly after lights on or lights off, and the early remodelling responses at 8 hours post infarction were examined. We found that sleep-MI preferentially triggers early expression of genes associated with inflammatory responses, whereas wake-MI triggers more genes associated with metabolic pathways and transcription/translation, by microarray analyses. Homozygous clock mutant mice exhibit altered diurnal gene expression profiles, consistent with their cycling prior to onset of MI. In the first 8 hours, crucial for innate immune responses to MI, there are also significant differences in sleep-MI and wake-MI serum cytokine responses and in neutrophil infiltration to infarcted myocardium. By 1 week post-MI there are differences in survivorship between the sleep and wake MI mice that could be explained by the different molecular and cellular responses. Our whole-body physiology, tissues and cells exhibit endogenous daily rhythms and understanding their role in triggering effective responses after MI could lead to new strategies to benefit patients with cardiovascular disease.
Both cyclooxygenase (COX) and nitric oxide synthase (NOS) contribute to sweating, whereas NOS alone contributes to cutaneous vasodilation during exercise in the heat. Here, we evaluated if type 1 diabetes mellitus (T1DM) modulates these responses. Adults with (n=11, 25±5 years) and without (n=12, 24±4 years) T1DM performed two bouts of 30-min cycling at a fixed rate of heat production of 400W in the heat (35°C); each followed by a 20- and 40-min recovery period respectively. Sweat rate and cutaneous vascular conductance (CVC) were measured at four intradermal microdialysis sites treated with either 1) lactated Ringer (vehicle control site), 2) 10mM ketorolac (non-selective COX inhibitor), 3) 10mM NG-nitro-L-arginine methyl ester (non-selective NOS inhibitor), or 4) a combination of both inhibitors. In non-diabetic adults, separate and combined inhibition of COX and NOS reduced exercise sweat rate (P≤0.05) and the magnitude of reductions were similar across sites. In individuals with T1DM, inhibition of COX resulted in an increase in sweat rate of 0.10±0.09 and 0.09±0.08 mg.min-1.cm-2 for the first and second exercise bouts respectively relative to vehicle control site (P≤0.05), while NOS inhibition had no effect on sweating. In both groups, NOS inhibition reduced CVC during exercise (P≤0.05) although the magnitude of reduction did not differ between the non-diabetic and T1DM groups (Exercise 1: -28±10 vs. -23±8%max, P=0.51; Exercise 2: -31±12 vs. -24±10%max, P=0.38). We show that in individuals with T1DM performing moderate intensity exercise in the heat, NOS-dependent sweating but not cutaneous vasodilation is attenuated, whereas COX inhibition increases sweating.
Circadian clocks influence virtually all physiological processes, including lactation. Here we investigate the role of the CLOCK gene in regulation of mammary epithelial cell growth and differentiation. Comparison of mammary morphology in late pregnant wild-type and Clock19 mice, showed that gland development was negatively impacted by genetic loss of a functional timing system. To understand if these effects were due in part to loss of CLOCK function in the gland, the mouse mammary epithelial cell line, HC11, was transfected with shRNA that targeted Clock (shClock). Cells transfected with shClock expressed 70% less Clock mRNA than wild-type (WT) HC11 cultures, which resulted in significantly depressed levels of CLOCK protein (P< 0.05). HC11 lines carrying shClock had 4-fold higher growth rates (P< 0.05), and percent of cells in G1 phase was significantly higher (90.1± 1.1% of shClock versus 71.3 ± 3.6% of WT-HC11) following serum starvation. Q-PCR analysis showed shClock had significant effects (P< 0.0001) on relative expression levels of Ccnd1, Wee1 and Tp63. Q-PCR analysis of effect of shClock on Fasn and Cdh1 expression in undifferentiated cultures, and cultures treated 96 h with dexamethasone, insulin and prolactin (differentiated), found levels were reduced by 2-fold and 3-fold, respectively (P< 0.05), in shClock line relative to WT cultures. Abundance of CDH1 and TP63 proteins were significantly reduced in cultures transfected with shClock. These data support CLOCK plays a role in regulation of epithelial cell growth and differentiation in the mammary gland.
The effect of hypoxia on cellular metabolism is well-documented in adult vertebrates but information is entirely lacking for embryonic organisms. The effect of hypoxia on embryonic physiology is particularly interesting, as metabolic responses during development may have life-long consequences, due to developmental plasticity. To this end, we investigated the effects of chronic developmental hypoxia on cardiac mitochondrial function in embryonic and juvenile American alligators (Alligator mississippiensis). Alligator eggs were incubated in 21% or 10% oxygen from 20-90% of embryonic development. Embryos were either harvested at 90% development or allowed to hatch and then reared in 21% oxygen for 3 years. Ventricular mitochondria were isolated from embryonic/juvenile alligator hearts. Mitochondrial respiration and enzymatic activities of electron transport chain Complexes were measured with a microrespirometer and spectrophotometer, respectively. Developmental hypoxia induced growth restriction and increased relative heart mass, and this phenotype persisted into juvenile life. Embryonic mitochondrial function was not affected by developmental hypoxia, but at the juvenile life stage, animals from hypoxic incubations had lower levels of leak respiration and higher respiratory control ratios, which is indicative of enhanced mitochondrial efficiency. Our results suggest developmental hypoxia can have life-long consequences for alligator morphology and metabolic function. Further investigations are necessary to reveal the adaptive significance of the enhanced mitochondrial efficiency in the hypoxic phenotype.
The ionotropic purine type 2X7 receptor (P2X7R) is a non-specific cation channel implicated in sleep regulation and brain cytokine release. Many endogenous rhythms co-vary with sleep including locomotor activity and core body temperature. Further, brain-hypothalamic cytokines and purines play a role in the regulation of these physiological parameters as well as sleep. We hypothesized that these parameters are also affected by the absence of the P2X7 receptor. Herein we determine spontaneous expression of body temperature and locomotor activity in WT and P2X7R knockout (KO) mice and how they are affected by sleep deprivation (SD). We also compare hypothalamic, hippocampal and cortical cytokine and purine-related receptors and enzymes mRNA expressions before and after SD in WT and P2X7RKO mice. Next, in a hypothesis-generating survey of hypothalamic long non-coding (lnc) RNAs, we compare lncRNA expression levels between strains and after SD. During baseline conditions, P2X7RKO mice had attenuated temperature rhythms compared with WT mice, although locomotor activity patterns were similar in both strains. After 6h of SD, body temperature and locomotion were enhanced to a greater extent in P2X7RKO mice than in WT mice during the initial 2-3h after SD. Baseline mRNA levels of cortical TNFα and P2X4R were higher in the KO mice than WT mice. In response to SD, the KO mice failed to increase hypothalamic adenosine deaminase and P2X4R mRNAs. Further, hypothalamic lncRNA expressions varied by strain, and with SD. Current data are consistent with a role for the P2X7R in thermoregulation and lncRNA involvement in purinergic signaling.
This study explores the hypothesis that intracerebral hemorrhage (ICH) promotes release of diffusible factors that can significantly influence the structure and function of cerebral arteries remote from the site of injury, through action on platelet-derived growth factor (PDGF) receptors. Four groups of adult male Sprague Dawley rats were studied (n=8 each): 1) Sham; 2) Sham + 60 mg/kg IP imatinib; 3) ICH (collagenase method); and 4) ICH + 60 mg/kg IP imatinib given 60 min after injury. At 24 hours post-injury, sham artery passive diameters (+3 mM EGTA) averaged 244±7 µm (@60 mmHg). ICH significantly increased passive diameters up to 6.4% and decreased compliance up to 42.5%. For both pressure- and potassium-induced contractions, ICH decreased calcium mobilization up to 26.2% and increased myofilament calcium sensitivity up to 48.4%. ICH reduced confocal colocalization of Smooth Muscle α-Actin (SMαA) with Non-Muscle Myosin Heavy Chain (MHC) and increased its colocalization with Smooth Muscle MHC, suggesting that ICH promoted contractile differentiation. ICH also enhanced colocalization of MLCK with both SMαA and regulatory 20 kDa Myosin Light Chain. All effects of ICH on passive diameter, compliance, contractility, and contractile protein colocalization were significantly reduced or absent in arteries from animals treated with imatinib. These findings support the hypothesis that ICH promotes release into the CSF of vasoactive factors that can diffuse to and promote activation of cerebrovascular PDGF receptors, thereby altering the structure, contractile protein organization, contractility and smooth muscle phenotype of cerebral arteries remote from the site of hemorrhage.
Altricial bird species, like red-winged blackbirds, hatch at an immature state of functional maturity with limited aerobic capacity and no endothermic capacity. Over the next 10-12 days in the nest, red-winged blackbirds develop increased metabolic capacity before fledging. Although ontogeny of respiration has been described in precocial birds, ontogeny of ventilatory chemosensitivity is unknown in altricial species. Here we examined developmental changes in chemosensitivity of tidal volume (VT), breathing frequency (f), minute ventilation (VE), and whole-animal oxygen consumption (Vo2) from hatching to just before fledging in red-winged blackbirds on days 1, 3, 5, 7, and 9 post-hatching (dph) in response to hypercapnia (2 and 4% CO2) and hypoxia (15 and 10% O2). Under control conditions, there was a developmental increase in VE with age due to increased VT. Hypercapnic and hypoxic chemosensitivities were present as early as 1 dph. In response to hypoxia, 1, 3, and 9 dph nestlings increased VE at 10% O2, by increasing f with some change in VT in younger animals. In contrast to early neonatal altricial mammals, the hypoxic response of nestling red-winged blackbirds was not biphasic. In response to hypercapnia, 3 dph nestlings increased VE by increasing both f and VT. From 5 dph on, the hypercapnic increase in VE was accounted for by increased VT and not f. Chemosensitivity to O2 and CO2 matures early in nestling red-winged blackbird, well before the ability to increase Vo2 in response to cooling, and thus does not represent a limitation to the development of endothermy.
The aim of this study was to clarify the effect of vagal afferent activation on salivation and swallowing-like events. Salivation is part of a reflex induced by stimulation of the oral area during feeding or chewing. Recently, we reported that nausea induced by gastro-esophageal reflux (GER) activation produced salivation and swallowing in humans. Here, we investigated the ability of visceral sensation to enhance salivation and swallowing in rodents in order to inform the mechanism of GER-mediated stomatognathic activation. First, we administered LiCl to anesthetized male rats to induce nausea. LiCl significantly increased salivation and increased the activity of the vagal afferent nerve. Next, we simultaneously recorded salivation and swallowing using an electrode attached to the mylohyoid muscle during vagal afferent stimulation in a physiological range of frequencies. Vagal afferent stimulation significantly increased salivation and swallowing-like events in a frequency-dependent manner. A muscle relaxant, vecuronium bromide, diminished the swallowing-like response but did not affect salivation. These results indicate that visceral sensation induces salivation and swallowing-like events in anesthetized rodents through vagal afferent activation.
Ischemia-reperfusion (I/R) injury is a primary cause of poor outcomes following ischemic cardiovascular events. We tested whether acute hot water immersion protects against forearm vascular I/R. METHODS: Ten (5 male, 5 female) young (23±2 years), healthy subjects participated in two trials in random order 7-21 days apart, involving: 1) 60-min of seated rest (control), or 2) 60-min of immersion in 40.5°C water (peak rectal temperature: 38.9±0.2°C). I/R was achieved 70 min following each intervention by inflating an upper arm cuff to 250mmHg for 20-min followed by 20-min of reperfusion. Brachial artery flow-mediated dilation (FMD) and forearm post-occlusive reactive hyperemia (RH) were measured as markers of macro- and micro-vascular function at three time points: 1) pre-intervention, 2) 60-min post-intervention, and 3) post-I/R. RESULTS: Neither time control nor hot water immersion alone affected FMD (both p>0.99). I/R reduced FMD from 7.4±0.7 to 5.4±0.6% (p=0.03) and this reduction was prevented following hot water immersion (7.0±0.7 to 7.7±1.0%; p>0.99). I/R also impaired RH (peak vascular conductance: 2.6±0.5 to 2.0±4mL•min-1•mmHg-1, p=0.003), resulting in a reduced shear stimulus (SRAUC/10-3 : 22.5±2.4 to 16.9±2.4, p=0.04). The post-I/R reduction in peak RH was prevented by hot water immersion (2.5±0.4 to 2.3±0.4mL•min-1•mmHg-1; p=0.33). CONCLUSIONS: We observed a decline in brachial artery dilator function post-I/R, which may be (partly) related to damage incurred downstream in the microvasculature, as indicated by impaired RH and shear stimulus. Hot water immersion was protective against reductions in FMD and RH post-I/R, suggesting heat stress induces vascular changes consistent with reducing I/R injury following ischemic events.
Cardiac surgery triggers an inflammatory stress response leading to protein catabolism, a process which even high-dose insulin therapy alone cannot reverse. To determine whether hyperinsulinemic-normoglycemic clamp and perioperative amino acid supplementation improves whole-body protein balance, twenty patients scheduled for elective coronary artery bypass grafting surgery were randomly assigned to have intra and postoperative hyperinsulinemic-normoglycemic clamp with or without intravenous amino acid (AA) supplementation. Primed continuous infusions of [6,6-2H2]glucose and L-[1-13C]leucine were used to quantify whole-body protein and glucose metabolism before and after surgery. Adipose tissue and serum cytokines were also analyzed to measure their responsiveness to the anabolic effect of amino acid administration. During hyperinsulinemic-normoglycemic clamp, amino acid supplementation successfully stimulated whole-body protein synthesis, resulting in a positive whole-body protein balance after surgery (insulin: -13.6 +/- 4.5 vs insulin+AA: 2.1 +/- 5.4 micromol/kg/h, P<0.001). Endogenous glucose production was equally suppressed in both groups (insulin: 0.0 +/- 3.8 vs insulin+AA 1.6 +/- 1.6 micromol/kg/min, P=0.230). Amino acid supplementation led to significant changes in serum and tissue IL-6 (insulin: 246.6 +/- 111.2 vs insulin+AA: 124.5 +/- 79.3 pg/mL, P=0.011). In conclusion, hyperinsulinemic-normoglycemic clamp technique together with AA supplementation can induce an anabolic state after open-heart surgery as quantified by a positive whole body protein balance.
Shiga toxin 2 (Stx2)-producing enterohemorrhagic Escherichia Coli induced brain damage. Since a cerebroprotective action was reported for angiotensin (Ang) (1-7), our aim was to investigate whether Ang-(1-7) protects from brain damage induced by Stx2-producing enterohemorrhagic Escherichia Coli. The anterior hypothalamic area of adult male Wistar rats was injected with saline solution or Stx2 or Stx2 plus Ang-(1-7) or Stx2 plus Ang-(1-7) plus A779. Rats received a single injection of Stx2 at the beginning of the experiment and Ang-(1-7), A779 or saline was administered daily in a single injection for 8 days. Cellular ultrastructural changes were analyzed by transmission electron microscopy. Stx2 induced neurodegeneration, axonal demyelination, alterations in synapse and oligodendrocyte and astrocyte damage, accompanied by edema. Ang-(1-7) prevented neuronal damage triggered by the toxin in 55.6 ± 9.5% of the neurons and the Stx2-induced synapse dysfunction was reversed. In addition, Ang-(1-7) blocked Stx2-induced demyelination in 92 ± 4% of the axons. Oligodendrocyte damage caused by Stx2 was prevented by Ang-(1-7) but atrocytes were only partially protected by the peptide (38 ± 5% of astrocytes were preserved). Ang-(1-7) treatment resulted in 50% reduction in the number of activated microglial cells induced by Stx2, suggesting an anti-inflammatory action. All these beneficial effects elicited by Ang-(1-7) were blocked by the Mas receptor antagonist and thus it was concluded that Ang-(1-7) protects mainly neurons and oligodendrocytes, and partially astrocytes, in the central nervous system through Mas receptor stimulation.
Maternal overnutrition or associated complications putatively mediate the obesogenic effects of perinatal high-fat diet on developing offspring. Here, we tested the hypothesis that a Western diet developmental environment increases adiposity not only in male offspring from obesity-prone (DIO) mothers, but also in those from obesity-resistant (DR) dams, implicating a deleterious role for Western diet per se. Selectively bred DIO and DR female rats were fed chow (17% kcal fat) or Western diet (32%) for 54 days before mating and thereafter through weaning. As intended, despite chow-like caloric intake, Western diet increased pre-pregnancy weight gain and circulating leptin levels in DIO, but not DR, dams. Yet, in both genotypes, maternal Western diet increased the weight and adiposity of preweanlings, as early as day 1 in DR offspring, and increased plasma leptin, insulin and adiponectin of weanlings. Maternal Western diet disrupted the correlations otherwise seen in weanlings of GLP-1, amylin, leptin, insulin, and adiponectin with body fat. Though body weight normalized with chow feeding during adolescence, young adult Western diet offspring subsequently showed decreased energy expenditure and, in DR offspring, decreased lipid utilization as a fuel substrate. By mid-adulthood, maternal Western diet DR offspring ate more chow, weighed more and were fatter than controls. Thus, maternal Western diet covertly programmed increased adiposity in childhood and adulthood, disrupted relations of energy regulatory hormones with body fat, and decreased energy expenditure in offspring of lean, genetically obesity-resistant mothers. Maternal Western diet exposure alone, without maternal obesity or overnutrition, can promote offspring obesity.
To investigate time-dependent changes in sarcoplasmic reticulum (SR) Ca2+ release and myofibrillar (my-) Ca2+ sensitivity during recovery from prolonged low-frequency force depression (PLFFD), rat gastrocnemius muscles were electrically stimulated in situ. After 0 h (R0), 0.5 h (R0.5), 2 h (R2), 6 h (R6), or 12 h (R12) of recovery, the superficial gastrocnemius muscles were excised and used for biochemical and skinned fiber analyses. At R0, R0.5, R2, and R6, the ratio of force at 1 Hz to that at 50 Hz was decreased in the skinned fibers. The ratio of depolarization-induced force to the maximum Ca2+-activated force (depol/Ca2+ force ratio) was utilized as an indicator of SR Ca2+ release. At R0, both the depol/Ca2+ force ratio and my-Ca2+ sensitivity were decreased. At R0.5 and R2, my-Ca2+ sensitivity was recovered while the depol/Ca2+ force ratio remained depressed. At R6, my-Ca2+ sensitivity was decreased again whereas the depol/Ca2+ force ratio was nearly restored. Western blot analyses demonstrated that decreased my-Ca2+ sensitivity at R6 and reduced depol/Ca2+ force ratio at R0, R0.5, and R2 were accompanied by depressions in S-glutathionylated troponin I and increases in dephosphorylated ryanodine receptor 1, respectively. These results indicate that in the early stage of recovery, reduced SR Ca2+ release plays a primary role in the etiology of PLFFD, whereas decreased my-Ca2+ sensitivity is involved in the late stage and suggest that S-glutathionylation of troponin I and dephosphorylation of ryanodine receptor 1 contribute, at least partly, to fatiguing contraction-induced alterations in my-Ca2+ sensitivity and SR Ca2+ release, respectively.
Although vasovagal syncope (VVS) is a common clinical condition the underlying pathophysiology is not fully understood. A decrease in cardiac output has recently been suggested as a determinant factor for orthostatic VVS. The aim was to investigate compensatory mechanisms to maintain central blood volume and venous return during hypovolemic stress in women with VVS. 14 VVS women (25.7±5.0 years) and 15 matched controls (22.8±3.2 years) were investigated. Single step and graded lower body negative pressure (LBNP) to presyncope was used to create hypovolemic stress. Peripheral mobilization of venous blood from the arm (capacitance response and net capillary fluid absorption) and lower limb blood pooling (calf capacitance response) were evaluated with volumetric technique. Cardiovascular responses and plasma norepinephrine (P-NE) were measured. Resting P-NE was elevated in VVS (P<0.01). Despite similar hypovolemic stimulus, VVS displayed blunted increase in P-NE (P<0.01) and reduced maximal percentage increase in TPR (P<0.05) during graded LBNP. Arm capacitance response was slower (P<0.05) and reduced in VVS at higher levels of LBNP (P<0.05). Capillary fluid absorption from extra- to intravascular space was reduced by almost 40% in VVS (P<0.05). Accordingly, a more pronounced reduction in CO was found (P<0.05). In conclusion, women with VVS presented with decreased mobilization of peripheral venous blood and decreased net fluid absorption from tissue to blood during hypovolemic stress, partly explained by an attenuated vasoconstrictor response. This may seriously impede maintenance of cardiac output during hypovolemic stress and could contribute to the pathogenesis of VVS.
The role of cholinergic and β-adrenergic activity in mediating fetal cardiovascular recovery from brief repeated episodes of asphyxia consistent with established labor, remains unclear. In this study, we tested the effect of cholinergic and β-adrenergic blockade on the fetal chemoreflex and fetal heart rate (FHR) overshoot responses during brief repeated asphyxia at rates consistent with early or active labor. Chronically instrumented fetal sheep at 0.85 of gestation received either i.v. atropine sulfate (cholinergic blockade, n=8) or vehicle (n=7) followed by 3 x 1-minute umbilical cord occlusions repeated every 5 minutes (1:5; consistent with early labor), or i.v. propranolol hydrochloride (β-adrenergic blockade, n=6) or vehicle (n=6) followed by 3 x 2-minute occlusions repeated every 5 minutes (2:5; consistent with active labor). In vehicle-controls, 1:5 occlusions were associated with rapid and sustained FHR decelerations followed by rapid return of FHR to baseline values after release of the occlusion. Cholinergic blockade abolished FHR decelerations during occlusions and caused FHR overshoot after release of the occlusion (P<0.05 vs. control 1:5). In vehicle-controls, 2:5 occlusions caused rapid and sustained FHR decelerations followed by FHR overshoot after release of the occlusion. β-adrenergic blockade was associated with greater reduction in FHR during occlusions and attenuated FHR overshoot (P<0.05 vs. control 2:5). These data demonstrate that the FHR overshoot pattern after asphyxia is mediated by a combination of attenuated parasympathetic activity and increased β-adrenergic stimulation of the fetal heart.
The total baroreflex arc is the open-loop system relating carotid sinus pressure (CSP) to arterial pressure (AP). The nonlinear dynamics of this system were recently characterized. First, Gaussian white noise CSP stimulation was employed in open-loop conditions in normotensive and hypertensive rats with sectioned vagal and aortic depressor nerves. Then, nonparametric system identification was applied to measured CSP and AP to establish a second-order nonlinear Uryson model. The aim in this study was to assess the importance of higher-order nonlinear dynamics via development and evaluation of a third-order nonlinear model of the total arc using the same experimental data. Third-order Volterra and Uryson models were developed by employing nonparametric and parametric identification methods. The R2 values between the AP predicted by the best third-order Volterra model and measured AP in response to Gaussian white noise CSP not utilized in developing the model were 0.69±0.03 and 0.70±0.03 for normotensive and hypertensive rats, respectively. The analogous R2 values for the best third-order Uryson model were 0.71±0.03 and 0.73±0.03. These R2 values were not statistically different from the corresponding values for the previously established second-order Uryson model, which were both 0.71±0.03 (p > 0.1). Further, none of the third-order models predicted well-known nonlinear behaviors including thresholding and saturation better than the second-order Uryson model. Additional experiments suggested that the unexplained AP variance was partly due to higher brain center activity. In conclusion, the second-order Uryson model sufficed to represent the sympathetically-mediated total arc under the employed experimental conditions.
Preeclampsia is a hypertensive disorder of pregnancy with limited therapeutic options. In healthy pregnancy, relaxin plays an important vasodilatory role to maintain vascular compliance; however, there is currently no preclinical evidence to support the use of relaxin during preeclampsia. Therefore, the goal of this study was to test the hypothesis that recombinant human relaxin-2 (Serelaxin, Novartis, RLX) could reduce mean arterial pressure (MAP) and improve uterine artery resistance index (UARI) and nitric oxide bioavailability and/or decrease prepro-endothelin-1 (PPET-1), soluble fms-like tyrosine kinase-1 (sFlt-1) and tumor necrosis factor (TNF-alpha) in the Reduced Uterine Perfusion Pressure (RUPP) model of preeclampsia. On day 14 of gestation (GD14), pregnant rats were assigned to Normal pregnant (NP), RUPP, RUPP+RLX or NP+RLX groups. Treated rats received RLX at 0.4 ug/h or RLX2 4 ug/h RLX via minipump implanted on GD14. On GD18 carotid arterial catheters were inserted, and on GD19 MAP and tissues were collected. MAP was increased in RUPP rats compared to NP but was lowered with either dose of RLX. UARI and sFlt-1 were significantly improved in both treated RUPP groups. Total circulating nitrate-nitrite improved and placental PPET-1 and TNF-alpha were significantly decreased with the higher dose of RLX. Renal cortex PPET-1 was reduced with both doses of RLX. In conclusion, Serelaxin improved blood pressure, sFlt-1, TNF alpha, UARI and nitric oxide bioavailability and PPET-1 in a rat model of preeclampsia thereby suggesting a potential therapeutic role for RLX in maintaining maternal health and prolonging pregnancy in the face of placental ischemia.
β-carotene-15,15'-dioxygenase (BCO1) cleaves dietary carotenoids at the central 15,15' double bond, most notably acting on β-carotene to yield retinal. However, Bco1 disruption also impacts diverse physiologic endpoints independent of dietary carotenoid feeding, including expression of genes controlling androgen metabolism. Using the Bco1-/- mouse model, we sought to probe the effects of Bco1 disruption on testicular steroidogenesis, prostatic androgen signaling, and prostatic proliferation. Male wild-type (WT) and Bco1-/- mice were raised on carotenoid-free AIN-93G diets before euthanasia between 10-14 weeks of age. Weights of the prostate and seminal vesicles were significantly lower in Bco1-/- than in WT mice (-18% and -29%, respectively). Serum testosterone levels in Bco1-/- mice were significantly reduced by 73%. Bco1 disruption significantly reduced Leydig cell number and decreased testicular mRNA expression of Hsd17b3, suggesting inhibition of testicular testosterone synthesis. Immunofluorescent staining of the androgen receptor (AR) in the dorsolateral prostate lobes of Bco1-/- mice revealed a decrease in AR nuclear localization and analysis of prostatic morphology suggested decreases in gland size and secretion. These findings were supported by reduced expression of the proliferation marker Ki67 in Bco1-/- prostates. Expression analysis of 200 prostate cancer- and androgen-related genes suggested that Bco1 loss significantly disrupted prostatic androgen receptor signaling, cell cycle progression, and proliferation. This is the first demonstration that Bco1 disruption lowers murine circulating testosterone levels and thereby reduces prostatic androgen receptor signaling and prostatic cellular proliferation - further supporting the role of this protein in processes more diverse than carotenoid cleavage.
Selectively bred diet-induced obese (DIO) rats become obese on a high fat diet and are leptin resistant before becoming obese. As compared to diet-resistant (DR) neonates, DIO neonates have impaired leptin-dependent arcuate (ARC) neuropeptide Y/agouti-related peptide (NPY/AgRP) and α-melanocyte-stimulating hormone (αMSH; from pro-opiomelanocortin (POMC) neurons) axon outgrowth to the paraventricular nucleus (PVN). Using phosphorylation of STAT3 (pSTAT3) as a surrogate, we show that reduced DIO ARC leptin signaling develops by postnatal day 7 (P7) and is reduced within POMC, but not NPY/AgRP neurons. Since amylin increases leptin signaling in adult rats, we treated DIO neonates with amylin during the postnatal hypothalamic development and assessed leptin signaling, leptin-dependent ARC-PVN pathway development and metabolic changes. DIO neonates treated with amylin from P0-6 and from P0-16 increased ARC leptin signaling and both AgRP and αMSH ARC-PVN pathway development, but increased only POMC neuron number. Despite ARC-PVN pathway correction, P0-16 amylin-induced reductions in body weight did not persist beyond treatment cessation. Since amylin enhances adult DIO ARC signaling via an IL-6-dependent mechanism, we assessed ARC-PVN pathway competency in IL-6 knockout mice and found that the AgRP, but not αMSH, ARC-PVN pathway was reduced. These results suggest that both leptin and amylin are important neurotrophic factors for the postnatal development of the ARC-PVN pathway. Amylin might act as a direct neurotrophic factor in DIO rats to enhance both the number of POMC neurons and their αMSH ARC-PVN pathway development. This suggests important and selective roles for amylin during ARC hypothalamic development.
8-Prenylnaringenin (8-PN) is a prenylflavonoid that originates from hop extracts and is thought to help prevent disuse muscle atrophy. We hypothesized that 8-PN affects muscle plasticity by promoting muscle recovery under disuse muscle atrophy. To test the promoting effect of 8-PN on muscle recovery, we administered an 8-PN mixed diet to mice that had been immobilized with a cast to one leg for 14 days. Intake of the 8-PN mixed diet accelerated recovery from muscle atrophy, and prevented reductions in Akt phosphorylation. Studies on cell cultures of mouse myotubes in vitro demonstrated that 8-PN activated the PI3K/Akt/P70S6K1 pathway at physiologic concentrations. A cell-culture study using an inhibitor of estrogen receptors and an in vivo experiment with ovariectomized mice suggested that the estrogenic activity of 8-PN contributed to recovery from disuse muscle atrophy through activation of an Akt phosphorylation pathway. These data strongly suggest that 8-PN is a naturally occurring compound that could be used as a nutritional supplement to aid recovery from disuse muscle atrophy.
Purpose: Ideal therapy for lower urinary tract dysfunction in patients with spinal cord injury (SCI) should decrease detrusor overactivity, thereby promoting urine storage at low intravesical pressure and promoting efficient voiding at low pressure by decreasing detrusor-sphincter dyssynergia. Here we investigated blockade of various α1-adrenoceptors to determine the subtype that was principally responsible for improving the voiding dysfunction. Materials and Methods: The effects of the intravenous α1A/D-blocker naftopidil, the α1D-blocker BMY 7378, and the α1A-blocker silodosin were evaluated using cystometrography and external urethral sphincter-electromyography (EMG) in decerebrated, unanesthetized female Sprague-Dawley rats with chronic SCI following transection at Th8. Parameters measured included the voided volume, residual volume, voiding efficiency, and burst and silent periods on EMG. Results: Compared to values in decerebrated non-SCI rats, EMG of decerebrated SCI rats revealed more prominent tonic activity, significantly shorter periods of bursting activity, and a reduced ratio of the silent to active period during bursting compared to the values in decerebrated non-SCI rats. Compared to the value before drug administration (control), the voiding efficiency was significantly increased by naftopidil (1 and 3 mg/kg) (p < 0.05 each), and the burst (p < 0.01 and < 0.05, respectively) and silent periods (p < 0.01 each) of EMG were significantly lengthened. BMY 7378 (1 mg/kg) significantly increased voiding efficiency and lengthened the burst periods (each p < 0.05). Silodosin did not affect any parameters. Conclusions: These results suggest that α1D-blockade reduces the urethral resistance associated with detrusor-sphincter dyssynergia, thus improving voiding efficiency in SCI rats.
Alcohol use disorders (AUDs) frequently exist among persons living with HIV/AIDS (PLWHA). Chronic alcohol consumption, HIV infection, and anti-retroviral therapy (ART) are independently associated with impairments in glucose-insulin dynamics. Previous studies from our laboratory have shown that chronic binge alcohol (CBA) administration decreases body mass index, attenuates weight gain, and accentuates skeletal muscle wasting at end-stage disease in non-ART treated simian immunodeficiency virus (SIV)-infected male rhesus macaques. The aim of this study was to investigate whether CBA and ART alone or in combination alter body composition or glucose-insulin dynamics in SIV-infected male rhesus macaques during the asymptomatic phase of SIV infection. Daily CBA or sucrose (SUC) administration was initiated 3 mos. prior to intrarectal SIVmac251 inoculation and continued until the study end point at 11 mos. post-SIV infection. ART or placebo was initiated 2.5 months after SIV infection and continued until study end point. Four treatment groups (SUC/SIV±ART and CBA/SIV±ART) were studied. CBA/SIV macaques had a significantly decreased circulating adiponectin and resistin levels relative to SUC/SIV macaques and reduced disposition index (DI), acute insulin response to glucose (AIRg), insulin and C-peptide release during frequently sampled intravenous glucose tolerance test, irrespective of ART status. No statistically significant differences were observed in HOMA-insulin resistance (IR) values, body weight, total body fat, abdominal fat, or total lean mass or bone health among the four groups. These findings demonstrate CBA-mediated impairments in glucose-insulin dynamics and adipokine profile in asymptomatic SIV-infected macaques, irrespective of ART.
The impact of cerebral Epo in the regulation of the hypercapnic ventilatory response (HcVR) is controversial. While we reported that cerebral Epo does not affect the central chemosensitivity in C57Bl6 mice receiving an intracisternal injection of sEpoR (the endogenous antagonist of Epo), a recent study in transgenic mice with constitutive high levels of human Epo in brain and circulation (Tg6) and in brain only (Tg21), showed that Epo blunts the HcVR, maybe by interacting with central and peripheral chemoreceptors. High Epo serum levels in Tg6 mice lead to excessive erythrocytosis (hematocrit about 80-90%), the main symptom of chronic mountain sickness (CMS). These latter results support the hypothesis that reduced central chemosensitivity accounts for the hypoventilation observed in CMS patients. To solve this intriguing divergence, we re-evaluate HcVR in Tg6 and Tg21 mouse lines, by assessing the metabolic rate (O2 consumption; and CO2 production), a key factor modulating ventilation, which effect was not considered in the previous study. Our results showed that the decreased HcVR observed in Tg6 mice (~70% reduction; p<0.01) was due to a significant decrease in the metabolism (~40%; p<0.0001) rather than Epo's effect on CO2 chemosensitivity. Additional analysis in Tg21 mice did not revealed differences of HcVR or metabolism. We concluded that cerebral Epo does not modulate the central chemosensitivity system, and that a metabolic effect upon CO2 inhalation is responsible for decreased HcVR observed in Tg6 animals. As CMS patients also show decreased HcVR, our findings might help to better understand respiratory disorders at high altitude.
We aimed to determine whether oxidative pathways adapt to the overproduction of carbon skeletons resulting from the progressive activation of amino acid (AA) deamination and ureogenesis under a high protein (HP) diet. Ninety-four male Wistar rats, of which 54 were implanted with a permanent jugular catheter, were fed a normal protein diet for one week and were then switched to an HP diet for 1, 3, 6 or 14 days. On the experimental day, they were given their meal containing a mixture of 20 U-[15N]-[13C] AA, whose metabolic fate was followed for 4 h. Gastric emptying tended to be slower during the first 3 days of adaptation. 15N excretion in urine increased progressively during the first 6 days, reaching 29% of ingested protein. 13CO2 excretion was maximal as early as the first day, and represented only 16% of the ingested proteins. Consequently, the amount of carbon skeletons remaining in the metabolic pools 4 h after the meal ingestion progressively increased to 42% of the deaminated dietary AA after 6 days of HP diet. In contrast, 13C enrichment of plasma glucose tended to increase from 1 to 14 days of the HP diet. We conclude that there is no oxidative adaptation in the early postprandial period to an excess of carbon skeletons resulting from AA deamination in HP diets. This leads to an increase in the postprandial accumulation of carbon skeletons throughout the adaptation to an HP diet, which can contribute to the sustainable satiating effect of this diet.
Carbon monoxide (CO) is a gaseous signalling molecule and is produced in vivo from the intracellular breakdown of heme via the heme oxygenase (HO) family of enzymes. In this study we investigated the role of the HO-1/CO system in the control of ventilation in zebrafish, Danio rerio. Immunohistochemistry revealed the presence of HO-1 in the chemoreceptive neuroepithelial cells (NECs) of larvae (4 days post fertilization) and adults indicating the potential for endogenous CO production in the NECs. Hypoxia (20 min, water PO2 of 30 mm Hg) caused a significant increase in HO-1 activity in whole larvae and in the gills of adult fish. Zebrafish with reduced HO-1 activity (via HO-1 knockdown in larvae or zinc protoporphyrin IX (ZnPPIX) treatment in adults) exhibited increased ventilation frequency (Vf) under normoxic but not hypoxic conditions. The addition of exogenous CO restored resting Vf in fish with diminished CO production and in some cases (e.g. hypoxic sham larvae) CO modestly reduced Vf below resting levels. Larval fish were treated with phenylhydrazine (PHZ) to eliminate the potential confounding effects of CO-haemoglobin interactions which might influence ventilation. PHZ treatment did not cause changes in Vf of normoxic larvae and the addition of CO to PHZ-exposed larvae resulted in a significant decrease in sham and HO-1 deficient fish under normoxic conditions. This study demonstrates for the first time that CO plays an inhibitory role in the control of breathing in larval and adult zebrafish.
The mechanisms involved in the weight loss seen after Vertical Sleeve Gastrectomy (VSG) are not clear. The rat stomach has two morphologically and functionally distinct proximal and distal parts. The rat model for VSG involves complete removal of the proximal part and 80% removal of the distal part along the greater curvature. The purpose of this study was to understand the potential independent contributions of removal of these distinct gastric sections to VSG outcomes. We prepared four surgical groups of male Long Evans rats; VSG, sham-surgery (control), selective proximal section removal (PR) and selective distal section removal (DR). GER was highest after VSG compared to all other groups. However, PR, in turn, had significantly greater GER compared to both DR and sham groups. The surgery-induced weight loss followed the same pattern with VSG causing the greatest weight loss and PR having greater weight loss compared to DR and sham groups. The results were robust for rats fed regular chow or a high-fat diet. Body mass analysis revealed that the weight loss was due to loss of fat mass and there was no change in lean mass after the surgeries. In conclusion, removal of the proximal stomach contributes to most, but not all of the physiological impact of VSG.
Arterial baroreflex function is important for blood pressure control during exercise, but its contribution to cardiovascular adjustments at the onset of cycling exercise remains unclear. Fifteen healthy male subjects (24 ± 1 years) performed 45-s trials of low- and moderate-intensity cycling, with carotid baroreceptor stimulation by neck suction at -60 Torr applied 0-5, 10-15, and 30-35 s after the onset of exercise. Cardiovascular responses to neck suction during cycling were compared to those obtained at rest. An attenuated reflex decrease in heart rate following neck suction was detected during moderate-intensity exercise, as compared to the response at rest (P < 0.05). Furthermore, compared to the reflex decrease in blood pressure elicited at rest, neck suction elicited an augmented decrease in blood pressure at 0-5 and 10-15 s during low-intensity exercise and in all periods during moderate-intensity exercise (P < 0.05). The reflex depressor response at the onset of cycling was primarily mediated by increase in the total vascular conductance. These findings evidence altered carotid baroreflex function during the first 35 s of cycling as compared to rest, with attenuated bradycardic response, and augmented depressor response to carotid baroreceptor stimulation.
An animal-borne blood sampler with data logging functions was developed for phocid seals which collected two blood samples for the comparison of endocrinological/biochemical parameters under two different conditions. The sampler can be triggered by preset hydrostatic pressure, acceleration (descending or ascending), temperature and time, and also manually by light. The sampling was reliable with >78% successful attempts to collect blood samples. Contamination of fluids in the tubing to the next blood sample was <1%, following the prior clearance of the tubing to a waste syringe. In captive harbor seals (Phoca vitulina) the automated blood sampling method was less stressful than direct blood withdrawal as plasma levels of stress hormones were lower in the former (p<0.05 for ACTH and p=0.078 for cortisol). HPLC analyses showed that both cortisol and cortisone were circulating in seal blood. Using the sampler, plasma levels of cardiovascular hormones, atrial natriuretic peptide (ANP), arginine vasopressin (AVP), and angiotensin II (AngII), were compared in grey seals (Halichoerus grypus), between samples collected when the animals were on land and in the water. HPLC analyses determined that [Met12] ANP (1-28) and various forms of angiotensins (AngII, III and IV) were circulating in seal blood. Although water immersion profoundly changes the plasma levels of cardiovascular hormones in terrestrial mammals, there were only tendencies towards an increase in ANP (p=0.069) and a decrease in AVP (p=0.074) in the seals. These results suggest that cardiovascular regulation in phocid seals may have undergone adaptation during evolution of the carnivore to a semi-aquatic lifestyle.
Physical inactivity/disuse results in skeletal muscle metabolic disruption including insulin resistance and mitochondrial dysfunction. The role of the toll-like receptor 4 (TLR4) signaling pathway in contributing to metabolic decline with muscle disuse is unknown. Therefore, our goal was to determine if TLR4 is an underlying mechanism of insulin resistance, mitochondrial dysfunction, and skeletal muscle ceramide accumulation following muscle disuse in mice. To address this hypothesis, we subjected (n=6-8/group) male WT and TLR4-/- mice to 2-weeks of hindlimb unloading (HU) while a second group of mice served as ambulatory wild-type controls (WT CON, TLR4-/- CON). Mice were assessed for insulin resistance (HOMA-IR, glucose tolerance) and hindlimb muscles (soleus and gastrocnemius) were used to assess muscle sphingolipid abundance, mitochondrial respiration (respiratory control ratio (RCR)), and NFB signaling. The primary finding was that HU resulted in insulin resistance, increased total ceramides, specifically Cer18:0 and Cer20:0, and decreased skeletal muscle mitochondrial respiration. Importantly, TLR4-/- HU mice were protected from insulin resistance and altered NFB signaling and were partly resistant to muscle atrophy, ceramide accumulation and decreased RCR. Skeletal muscle ceramides and RCR were correlated with insulin resistance. We conclude that TLR4 is an upstream regulator of insulin sensitivity while partly upregulating muscle ceramides and worsening mitochondrial respiration during 2-weeks of HU.
Recently, we used an ADP recycling approach to examine mouse skeletal muscle (SkM) mitochondrial function over respiratory states intermittent between state 3 and 4. We showed that respiration energized at complex II by succinate, in the presence of rotenone to block complex I, progressively increased with incremental additions of ADP. However, in the absence of rotenone, respiration peaked at low [ADP] but then dropped markedly as [ADP] was further increased. Here, we tested the hypothesis that these respiratory dynamics would differ between mitochondria of mice fed high-fat (HF) and treated with a low dose of streptozotocin to mimic type 2 diabetes and mitochondria from controls. We found that respiration and ATP production on succinate alone for both control and diabetic mice increased to a maximum at low [ADP] but dropped markedly as [ADP] was incrementally increased. However, peak respiration by the diabetic mitochondria required a higher [ADP] (right shift in the curve of O2 flux verses [ADP]). ATP production by diabetic mitochondria respiring on succinate alone was significantly less than controls whereas membrane potential trended higher, indicating that utilization of potential for oxidative phosphorylation was impaired. The rightward shift in the curve of O2 flux versus [ADP] is likely a consequence of these changes in ATP production and potential. In summary, using an ADP recycling approach, we demonstrated that ATP production by SkM mitochondria of HF/streptozotocin diabetic mice energized by succinate is impaired due to decreased utilization of and that more ADP is required for peak O2 flux.
Oxfenicine is a carnitine-palmitoyl transferase 1b (CPT-1b)-specific inhibitor that has been shown to improve whole-body insulin sensitivity while suppressing fatty acid (FA) oxidation and increasing circulating FA. Since the white adipose tissue (WAT) is an organ that stores and releases FAs, this study investigated whether oxfenicine-induced inhibition of FA oxidation affected adiposity and WAT metabolism in rats fed either low (LF) or high-fat (HF) diets. Following 8 weeks of dietary intervention, male Sprague-Dawley rats were given a daily i.p. injection of oxfenicine (150 mg/kg body weight) or vehicle (PBS) for 3 weeks. Oxfenicine treatment reduced whole-body fat oxidation, body weight, and adiposity and improved insulin sensitivity in HF-fed rats. All these effects occurred without alterations in food intake, energy expenditure, and ambulatory activity. In vivo oxfenicine treatment reduced FA oxidation and lipolysis in subcutaneous inguinal (SC Ing) adipocytes, whereas glucose incorporation into lipids (lipogenesis) was significantly reduced in both SC Ing and epididymal (Epid) adipocytes. In summary, our results show that oxfenicine-induced inhibition of CPT-1b markedly affects WAT metabolism, leading to reduced adiposity through a mechanism that involves reduced lipogenesis in the Sc Ing and Epid fat depots of rats.
Influenza is a significant health concern worldwide. Viral infection induces local and systemic activation of the immune system causing attendant changes in metabolism. High-resolution metabolomics (HRM) uses advanced mass spectrometry and computational methods to measure thousands of metabolites inclusive of most metabolic pathways. We used HRM to identify metabolic pathways and clusters of association related to inflammatory cytokines in lungs of mice with H1N1 influenza virus infection. Infected mice showed progressive weight loss, decreased lung function and severe lung inflammation with elevated cytokines [interleukin (IL)-1β, IL-6, IL-10, tumor necrosis factor (TNF)-α, and interferon (IFN)-] and increased oxidative stress via cysteine oxidation. HRM showed prominent effects of influenza virus infection on tryptophan and other amino acids, and widespread effects on pathways including purines, pyrimidines, fatty acids and glycerophospholipids. A metabolome-wide association study (MWAS) of the aforementioned inflammatory cytokines was used to determine the relationship of metabolic responses to inflammation during infection. This cytokine-MWAS (cMWAS) showed that metabolic associations consisted of distinct and shared clusters of 396 metabolites highly correlated with inflammatory cytokines. Strong negative associations of selected glycerosphingolipid, linoleate and tryptophan metabolites with IFN- contrasted strong positive associations of glycosphingolipid and bile acid metabolites with IL-1β, TNF-α and IL-10. Anti-inflammatory cytokine IL-10 had strong positive associations with vitamin D3, purine and vitamin E metabolism. The detailed metabolic interactions with cytokines indicate that targeted metabolic interventions may be useful during life-threatening crises related to severe acute infection and inflammation.
Increased NPY gene expression in the dorsomedial hypothalamus (DMH) has been shown to cause hyperphagia, but the pathway underlying this effect remains less clear. Hypothalamic neural systems play a key role in the control of food intake, in part, by modulating the effects of meal-related signals, such as cholecystokinin (CCK). An increase in DMH NPY gene expression decreases CCK-induced satiety. Since activation of catecholaminergic neurons within the nucleus of solitary tract (NTS) contributes to the feeding effects of CCK, we hypothesized that DMH NPY modulates NTS neural catecholaminergic signaling to affect food intake. We used an adeno-associated virus system to manipulate DMH NPY gene expression in rats to examine this pathway. Viral-mediated hrGFP anterograde tracing revealed that DMH NPY neurons project to the NTS; the projections were in close proximity to catecholaminergic neurons, and some contained NPY. Viral-mediated DMH NPY overexpression resulted in an increase in NPY content in the NTS, a decrease in NTS tyrosine hydroxylase (TH) expression and reduced exogenous CCK-induced satiety. Knockdown of DMH NPY produced the opposite effects. Direct NPY administration into the 4th ventricle of intact rats limited CCK-induced satiety and overall TH phosphorylation. Taken together, these results demonstrate that DMH NPY descending signals affect CCK-induced satiety, at least in part, via modulation of NTS catecholaminergic neuronal signaling.
Endothelial dysfunction and inflammation are characteristics of subclinical atherosclerosis and may increase through progressive menopausal stages. Evaluating endothelial responses to acute exercise can reveal underlying dysfunction not apparent in resting conditions. The purpose of this study was to investigate markers of endothelial function and inflammation before and after acute exercise in healthy low active perimenopausal (PERI) and late postmenopausal (POST) women. Flow mediated dilation (FMD), CD31+/CD42b- and CD62E+ endothelial microparticles (EMPs), and the circulating inflammatory factors monocyte chemoattractant protein 1 (MCP-1), interleukin 8 (IL-8), and tumor necrosis factor alpha (TNFα), were measured before and 30 minutes following acute exercise. Before exercise, FMD was not different between groups (PERI:6.4±0.9% vs. POST:6.5±0.8 %, p=0.97); however, following acute exercise PERI tended to improve FMD (8.5±0.9%, p=0.09) whereas POST did not (6.2±0.8%, p=0.77). Independent of exercise, we observed transient endothelial dysfunction in POST with repeated FMD measures. There was a group x exercise interaction for CD31+/CD42b- EMPs (p=0.04), where CD31+/CD42b- EMPs were similar before exercise (PERI:57.0±6.7 EMPs/μl vs. POST:58.5±5.3 EMPs/μl, p=0.86) but were higher in POST following exercise (PERI:48.2±6.7 EMPs/μl vs. POST:69.4±5.3 EMPs/μl, p=0.023). CD62E+ EMPs were lower in PERI compared with POST before exercise (p<0.001) and increased in PERI (p=0.04) but did not change in POST (p=0.68) in response to acute exercise. Following acute exercise, MCP-1 (p=0.055), TNFα (p= 0.02), and IL-8 (p<0.001) were lower in PERI but only IL-8 decreased in POST (p<0.001). Overall, these data suggest that perimenopausal and late postmenopausal women display different endothelial and inflammatory responses to acute exercise.
Humans and animals such as rats and mice tend to overconsume calorie-dense foods, a phenomenon that likely contributes to obesity. One often-advanced explanation for why we preferentially consume sweet and fatty foods is that they are more "rewarding" than low-calorie foods. "Reward" has been subdivided into three interdependent psychological processes: hedonia (liking a food), reinforcement (formation of associations among stimuli, actions and/or the food), and motivation (wanting the food). Research into these processes has focused on the mesolimbic system, which is comprised of both the dopamine neurons in the ventral tegmental area and the neurons in their major projection target, the nucleus accumbens. The mesolimbic system and closely connected structures are commonly referred to as the brain's "reward circuit." Implicit in this title is the assumption that "rewarding" experiences are generally the result of activity in this circuit. In this review, I argue that food intake and preference for calorie-dense foods can be explained without reference to subjective emotions. Furthermore, the contribution of mesolimbic dopamine to food intake and preference may not be a general one of promoting or coordinating behaviors that result in the most "reward" or caloric intake, but may instead be limited to facilitation of a specific form of neural computation that results in conditioned approach behavior. Studies on the neural mechanisms of caloric intake regulation must address how sensory information about calorie intake affects not just the mesolimbic system, but also many other forms of computation that govern other types of food-seeking and food-oriented behaviors.
Rats selectively bred to develop diet-induced obesity (DIO) have an early onset reduction in the sensitivity of their ventromedial hypothalamic nucleus (VMN) neurons to leptin as compared to diet-resistant (DR) rats. This reduced sensitivity includes decreased leptin receptor (Lepr-b) mRNA expression, leptin receptor binding, leptin-induced phosphorylation of STAT3 (pSTAT3) and impaired leptin excitation (LepE) of VMN neurons. When administered exogenously, the pancreatic peptide, amylin, acts synergistically to reduce food intake and body weight in obese, leptin resistant DIO rats by increasing VMN leptin signaling, likely by stimulation of microglia IL-6 which acts on its receptor to increase leptin-induced pSTAT3. Here we demonstrate that incubation of cultured VMN neurons of outbred rats with IL-6 increases their leptin sensitivity. Control, dissociated DIO VMN neurons express 66% less Lepr-b and 75% less Bardet Biedl Syndrome-6 (BBS6) mRNA and have reduced leptin-induced activation of LepE neurons compared to DR neurons. Incubation for 4 d with IL-6 increased DIO neuron Lepr-b expression by 77% and BBS6 by 290% and corrected their defective leptin activation of LepE neurons to DR levels. Since BBS6 enhances trafficking of Lepr-b to the cell membrane, the increases in Lepr-b and BBS6 expression appear to account for correction of the reduced leptin excitation of DIO LepE neurons to that of control DR rats. These data support prior findings suggesting that IL-6 mediates the leptin sensitizing effects of amylin on VMN neurons and that the inherent leptin resistance of DIO rats can be effectively reversed at a cellular level by IL-6.
Although obesity increases the risk for hypertension in pregnancy, the mechanisms responsible are unknown. Increased nitric oxide (NO) production results in vasodilation and reduced blood pressure during normal pregnancy in lean rats; however, the role of NO is less clear during obese pregnancies. We examined the impact of obesity on NO synthase (NOS)-mediated regulation of blood pressure during pregnancy by testing the hypothesis that NOS activity, expression and regulation of vascular tone and blood pressure are reduced in obese pregnant rats. At gestational day 19, melanocortin-4 receptor (MC4R)-deficient obese rats (MC4R+/-) had greater body weight and fat mass with elevated blood pressure and circulating sFlt-1 levels compared to MC4R+/+ pregnant rats. MC4R+/- pregnant rats also had less circulating cGMP levels and reduced total NOS enzymatic activity and expression in mesenteric arteries. Despite decreased biochemical measures of NO/NOS in MC4R+/- rats, NOS inhibition enhanced vasoconstriction only in mesenteric arteries from MC4R+/- rats, suggesting greater NOS-mediated tone. To examine the role of NOS on blood pressure regulation in obese pregnant rats, MC4R+/- and MC4R+/+ pregnant rats were administered the non-selective NOS inhibitor L-NG-Nitroarginine methyl ester (L-NAME, 100 mg/L) from gestational day 14 to 19 in drinking water. The degree by which L-NAME raised blood pressure was similar between obese and lean pregnant rats. Although MC4R+/- obese pregnant rats had elevated blood pressure associated with reduced total NOS activity and expression, they had enhanced NOS-mediated attenuation of vasoconstriction, with no evidence of alterations in NOS-mediated regulation of blood pressure.
The fetal cardiovascular responses to acute hypoxia includes a redistribution of the cardiac output towards the heart and the brain at the expense of other organs, such as the intestine. We hypothesized that hypoxia exerts a direct effect on the mesenteric artery (MA) that may contribute to this response. Using wire myography, we investigated the response to hypoxia (Po2 ~2.5 kPa for 20 min) of isolated MAs from 15- to 21-d chicken embryos (E15, E19 E21), and 1- to 45-d-old chickens (P1, P3, P14, P45). Agonist-induced pretone or an intact endothelium were not required to obtain a consistent and reproducible response to hypoxia, which showed a pattern of initial rapid phasic contraction followed by a sustained tonic contraction. Phasic contraction was reduced by elimination of extracellular Ca2+ or by presence of the neurotoxin tetrodotoxin, the α1-adrenoceptor antagonist prazosin, or inhibitors of L-type voltage-gated Ca2+ channels (nifedipine), mitochondrial electron transport chain (rotenone and antimycin A), and NADPH oxidase (VAS2870). The Rho-kinase inhibitor Y27632 impaired both phasic and tonic contraction and, when combined with elimination of extracellular Ca2+, hypoxia-induced contraction was virtually abolished. Hypoxic MA contraction was absent at E15 but present from E19 and increased towards the first days post-hatching. It then decreased during the first weeks of life and P45 MAs were unable to sustain hypoxia-induced contraction over time. In conclusion, the results of the present study demonstrate that hypoxic vasoconstriction is an intrinsic feature of chicken MA vascular smooth muscle cells during late embryogenesis and the perinatal period.
Recent fetal lamb data have suggested that the pulmonary trunk (PT) region displays a reservoir function, and that a pharmacologically-induced fall in pulmonary vascular resistance (PVR) increases and redistributes diastolic discharge from this central pulmonary reservoir towards the lungs, thereby producing a positive diastolic offset in the pulmonary arterial (PA) blood flow profile. As a similar offset in PA flow characteristically occurs after birth, this study tested the hypotheses that 1) central pulmonary reservoir discharge is both redistributed towards the lungs and increased in magnitude during the birth transition and 2) discharge from this reservoir constitutes a major component of increased PA diastolic blood flow after birth. Six anaesthetized near-term fetal lambs were instrumented with PT, ductal and left PA transit-time flow probes, and aortic, PT and left atrial catheters. Hemodynamic data were recorded in fetuses and at regular intervals during 2 hours' mechanical ventilation following cesarean section delivery. Diastolic PA blood flow rose from near-zero in fetuses to 468±188 ml/min by 15 min (P<0.001). Central pulmonary reservoir discharge in fetuses (99±44 ml/min) passed primarily right-to-left across the ductus. However, this reservoir discharge redistributed entirely to the lungs by 1 min after birth, and then doubled to a peak of 214±167 ml/min at 15 min (P<0.001). Reservoir discharge subsequently stabilized at 151±60 ml/min at 30-120 min, which comprised 50% of diastolic and 20% of mean PA blood flow. These findings suggest that enhanced diastolic central pulmonary reservoir discharge plays a major role in supporting an increased pulmonary perfusion after birth.
Several recent studies have shown that postoral sugar sensing rapidly stimulates ingestion. Here we explored the specificity with which early phase postoral sugar sensing influenced ingestive motivation. In Experiment 1, rats were trained to associate the consumption of 0.3M sucrose with injections of LiCl (3.0 mEq/kg IP, conditioned taste aversion) or given equivalent exposures to the stimuli, but in an unpaired fashion (UNPAIRED). Then, all rats were given two brief access tests to assess appetitive and consummatory responses to the taste properties of sucrose (0.01-1.0 M), 0.12M NaCl, and dH2O (in 10-s trials in randomized blocks). Intraduodenal (ID) infusions of either 0.3M sucrose or equiosmolar 0.15M NaCl (3.0 ml) were administered, beginning just before each test. For UNPAIRED rats, ID sucrose specifically enhanced licking for 0.03-1.0 M sucrose, with no effect on trial initiation, relative to ID NaCl. Rats with an aversion to sucrose suppressed licking responses to sucrose in a concentration-dependent manner, as expected, but the ID sucrose preload did not appear to further influence licking responses; instead, ID sucrose attenuated trial taking. Using a serial taste reactivity (TR) paradigm, however, Experiment 2 demonstrated that ID sucrose preloads suppressed ingestive oromotor responses to intraorally-delivered sucrose in rats with a sucrose aversion. Finally, Experiment 3 showed that ID sucrose preloads enhanced preferential licking to some representative tastants tested (sucrose, Polycose, and Intralipid), but not others (NaCl, quinine). Together, the results suggest the early phase reinforcing efficacy of postoral sugar is dependent on the sensory and motivational properties of the ingesta.
We tested the hypothesis that increases in blood pressure are sustained throughout 15 min of face cooling. Two independent trials were carried out. In the Face Cooling Trial, ten healthy adults underwent 15 min of face cooling where a 2.5 L bag of ice water (0 ± 0°C) was placed over their cheeks, eyes, and forehead. The Sham Trial was identical except that the temperature of the water was 34 ± 1°C. Primary dependent variables were forehead temperature, mean arterial pressure, and forearm vascular resistance. The square root of the mean of successive differences in R-R interval (RMSSD) provided an index of cardiac parasympathetic activity. In the Face Cooling Trial, forehead temperature fell from 34.1 ± 0.9°C at baseline to 12.9 ± 3.3°C at the end of face cooling (P<0.01). Mean arterial pressure increased from 83 ± 9 mmHg at baseline to 106 ± 13 mmHg at the end of face cooling (P<0.01). RMSSD increased from 61 ± 40 ms at baseline to 165 ± 97 ms during the first two min of face cooling (P≤0.05), but returned to baseline levels thereafter (65 ± 49 ms, P≥0.46). Forearm vascular resistance increased from 18.3 ± 4.4 mmHg/ml/100 g tissue/min at baseline to 26.6 ± 4.0 mmHg/ml/100 g tissue/min at the end of face cooling (P<0.01). There were no changes in the Sham Trial. These data indicated that increases in blood pressure are sustained throughout 15 min of face cooling and face cooling elicits differential time-dependent parasympathetic and likely sympathetic activation.
We examined whether older individuals with and without type 2 diabetes (T2D) experience differences in heart rate variability (HRV) during a 3-hour exposure to high heat stress compared to young adults. Young (YOUNG; n=22; 23±3 years) and older individuals with (T2D; n=11; 59±9 years) and without (OLDER; n=25; 63±5 years) T2D were exposed to heat stress (44°C, 30% relative humidity) for 3-hours. Fifty-five HRV measures were assessed for 15 minutes at baseline, and at minutes 82.5-97.5 (MID) and 165-180 (END) during heat stress. When compared to YOUNG, a similar number of HRV indices were significantly different (p<0.05) in OLDER (Baseline: 35; MID: 29; END: 32) and T2D (Baseline: 31; MID: 30; END: 27). In contrast, the number of HRV indices significantly different (p<0.05) between OLDER and T2D were far fewer (baseline: 13, MID: 1, END: 3). Within-group analyses demonstrated a greater change in the YOUNG group's HRV during heat stress, compared to OLDER and T2D; the number of significantly different (p<0.05) HRV indices between baseline and END were 42, 29, and 20, for YOUNG, OLDER, and T2D, respectively. Analysis of specific HRV domains suggest that YOUNG experienced greater sympathetic activity during heat stress compared to OLDER and T2D. In conclusion, compared to young, older individuals with and without type 2 diabetes demonstrate low HRV at baseline, and less change in HRV (including an attenuated sympathetic response) during 3-hour high heat stress, potentially contributing to impaired thermoregulatory function.
The purpose of this study was to determine whether chronic estrogen replacement in ovariectomized rats inhibits the pressor response to psychological stress by attenuating the activation of the renin-angiotensin system. Female Wistar rats aged 9 weeks were ovariectomized. After 4 weeks, the rats were randomly assigned to be implanted subcutaneously with pellets containing either 17β-estradiol (E2) or placebo (Pla). After 4 weeks of treatment, the rats underwent cage-switch stress and, in a separate experiment, a subset received an infusion of angiotensin II. The cage-switch stress rapidly elevated blood pressure (BP) and heart rate (HR) as measured by radiotelemetry in both groups. However, the BP and HR responses to the stress were significantly attenuated in the E2 group compared with the Pla group. An angiotensin II type 1 receptor blocker, losartan, given in drinking water, abolished the difference in the pressor response to stress between the two groups. Moreover, the stress-induced elevation in plasma renin activity and angiotensin II concentration was significant in the Pla group, but not in the E2 group. In addition, the expression of renin mRNA in the kidney was lower in the E2 group relative to the Pla group. Finally, we found that intravenous angiotensin II infusion increased BP and decreased HR to a similar degree in both groups. These results suggest that the inhibitory effects of estrogen on psychological stress-induced activation of the renin-angiotensin system could be at least partially responsible for the suppression of the pressor responses to psychological stress seen in estrogen-replaced ovariectomized rats.
Whole-body heat stress (WBH) results in numerous cardiovascular alterations that ultimately reduce orthostatic tolerance. While impaired carotid baroreflex (CBR) function during WBH has been reported as a potential reason for this decrement, study design considerations may limit interpretation of previous findings. We sought to test the hypothesis that CBR function is unaltered during WBH. CBR function was assessed in ten healthy male subjects (age, 26 ± 3; height, 185 ± 7 cm; weight, 82 ± 10 kg; BMI, 24 ± 3 kg/m2; mean ± sd) using 5 s trials of neck pressure (+45, +30 and +15 Torr) and neck suction (-20, -40, -60 and -80 Torr) during normothermia (NT) and passive WBH ( core temp ~1 °C). Analyses of stimulus response curves (four parameter logistic model) for CBR control of heart rate (CBR-HR) and mean arterial pressure (CBR-MAP), as well as separate 2-way ANOVA of the hypo- and hypertensive stimuli (factor 1: thermal condition, factor 2: chamber pressure) were performed. For CBR-HR, maximal gain was increased during WBH (-0.73±0.11) compared to NT (-0.39±0.04, mean±SE, p=0.03). In addition, the CBR-HR responding range was increased during WBH (33±5) compared to NT (19±2 bpm, p=0.03). Separate analysis of hypertensive stimulation revealed enhanced HR responses during WBH at -40, -60 and -80 Torr (condition*chamber pressure interaction, p=0.049) compared to NT. For CBR-MAP, both logistic analysis and separate 2-way ANOVA revealed no differences during WBH. Therefore, in response to passive WBH, CBR control of heart rate (enhanced) and arterial pressure (no change) is well-preserved.
It is currently unknown if sex differences exist in the cardiovascular consequences of the inspiratory muscle metaboreflex. We hypothesized that the activation of the inspiratory muscle metaboreflex will lead to less of an increase in mean arterial pressure (MAP) and limb vascular resistance (LVR) and less of a decrease in limb blood flow (QL) in women compared to men. Twenty healthy men (n=10, 23 ± 2 yrs) and women (n=10, 22 ± 3 yrs) were recruited for this study. Subjects performed inspiratory resistive breathing tasks (IRBTs) at 2% or 65% of their maximal inspiratory mouth pressure (PIMAX). During the IRBTs, the breathing frequency was 20 breaths min-1 with a 50% duty cycle. At rest and during the IRBTs, MAP was measured via automated oscillometry, QL was measured via Doppler ultrasound, and LVR was calculated. EMG was recorded on the leg to ensure no muscle contraction occurred. The 65% IRBT led to attenuated increases (p<0.01) from baseline in women compared to men for MAP (W: 7.3±2.0 mmHg; M: 11.1±5.0 mmHg) and LVR (W: 17.7%±14.0%; M: 47.9±21.0%) as well as less of a decrease (p<0.01) in QL (W: -7.5±9.9%; M: -23.3±10.2%). These sex differences in MAP, QL, and LVR were still present in a subset of subjects matched for PIMAX. The 2% IRBT resulted in no significant changes in MAP, QL, or LVR across time or between men and women. These data indicate pre-menopausal women exhibit an attenuated inspiratory muscle metaboreflex compared to age-matched men.
Thylakoids reduce body weight gain and body fat accumulation in rodents. This study investigated whether an enhanced oxidation of dietary fat-derived fatty acids in the intestine contributes to the thylakoid effects. Male Sprague-Dawley rats were fed a high-fat diet with (n = 8) or without thylakoids (n = 8) for two weeks. Body weight, food intake and body fat were measured, and intestinal mucosa was collected and analyzed. Quantitative real-time polymerase chain reaction (q-RT-PCR) was used to measure gene expression levels of key enzymes involved in fatty acid transport, fatty acid oxidation and ketogenesis. Another set of thylakoid-treated (n = 10) and control rats (n = 10) went through indirect calorimetry. In the first experiment, thylakoid-treated rats (n = 8) accumulated 25% less visceral fat than controls. Furthermore, fatty acid translocase (Fat/Cd36), carnitine palmitoyltransferase 1a (Cpt1a) and mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase 2 (Hmgcs2) genes were upregulated in the jejunum of the thylakoid-treated group. In the second experiment, thylakoid-treated rats (n = 10) gained 17.5% less weight compared to controls and their respiratory quotient was lower, 0.86 compared to 0.91. Thylakoid-intake resulted in decreased food intake and did not cause steatorrhea. These results suggest that thylakoids stimulated intestinal fatty acid oxidation and ketogenesis resulting in an increased ability of the intestine to handle dietary fat. The increased fatty acid oxidation and the resulting reduction in food intake may contribute to the reduced fat accumulation in thylakoid-treated animals.
The relationship between soluble epoxide hydrolase (sEH) and coronary reactive hyperemia (CRH) response to a brief ischemic insult is not known. Epoxyeicosatrienoic acids (EETs) exert cardioprotective effects in ischemia/reperfusion injury. sEH converts EETs into dihydroxyeicosatrienoic-acids (DHETs). Therefore, we hypothesized that knocking out sEH enhances CRH through modulation of oxylipin profiles including an increase in EETs/DHETs ratio. Compared to sEH+/+, sEH-/- mice showed enhanced CRH, including greater repayment volume (RV; 28% higher, p<0.001) and repayment/debt ratio (32% higher, p<0.001). Oxylipins from the heart perfusates were analyzed by LC-MS/MS. The 14,15-EET/14,15-DHET ratio was 3.7 fold higher at baseline (p<0.001) and 5.6 fold higher post-ischemia (p<0.001) in sEH-/- compared to sEH+/+ mice. Likewise, the baseline 9,10- and 12,13-EpOME/DiHOME ratios were 3.2 (p<0.01) and 3.7 (p<0.001) fold higher, respectively in sEH-/- compared to sEH+/+ mice. 13-HODE was also significantly increased at baseline by 71% (p<0.01) in sEH-/- vs. sEH+/+ mice. Levels of 5-, 11-, 12-, and 15-HETEs were not significantly different between the two strains (p>0.05), but were decreased post-ischemia in both groups (p=0.02, p=0.04, p=0.05, p=0.03 respectively). Modulation of CRH by PPAR was demonstrated using a PPAR-antagonist (T0070907), which reduced repayment volume by 25% in sEH+/+ (p<0.001) and 33% in sEH-/- mice (p<0.01), and a PPAR-agonist (rosiglitazone), which increased repayment volume by 37% in both sEH+/+ (p=0.04) and sEH-/- mice (p=0.04). L-NAME attenuated CRH in both sEH-/- and sEH+/+. These data demonstrate that genetic deletion of sEH resulted in an altered oxylipin profile which may have led to an enhanced CRH response.
Countercurrent systems have evolved in a variety of biological systems that allow transfer of heat, gases and solutes. For example, in the renal medulla, the countercurrent arrangement of vascular and tubular elements facilitates the trapping of urea and other solutes in the inner medulla, which in turn enables the formation of concentrated urine. Arteries and veins in the cortex are also arranged in a countercurrent fashion, as are descending and ascending vasa recta in the medulla. For countercurrent diffusion to occur, barriers to diffusion must be small. This appears to be characteristic of larger vessels in the renal cortex. There must also be gradients in the concentration of molecules between afferent and efferent vessels, with the transport of molecules possible in either direction. Such gradients exist for oxygen in both the cortex and medulla, but there is little evidence that large gradients exist for other molecules such as carbon dioxide, nitric oxide, superoxide, hydrogen sulfide, and ammonia. There is some experimental evidence for arterial-to-venous (AV) oxygen shunting. Mathematical models also provide evidence for oxygen shunting in both the cortex and medulla. However, the quantitative significance of AV oxygen shunting remains a matter of controversy. Thus, while the countercurrent arrangement of vasa recta in the medulla appears to have evolved as a consequence of the evolution of Henle's loop, the evolutionary significance of the intimate countercurrent arrangement of blood vessels in the renal cortex remains an enigma.
The physiological transition to aerial breathing in larval air-breathing fishes is poorly understood. We investigated gill ventilation frequency (fG), heart rate (fH) and air breathing frequency (fAB) as a function of development, activity, hypoxia and temperature in embryos/larvae from Day (D)2.5 to D30 post hatch of the tropical gar Atractosteus tropicus, an obligate air breather. Gill ventilation at 28°C began at ~D2, peaking at ~75 beats/min on D5, before declining to ~55 beats/min at D30. Heart beat began ~36-48 h post-fertilization and ~1 day before hatching. fH peaked between D3 and D10 at ~140 beats/min, remaining at this level through D30. Air breathing started very early at D2.5 to D3.5 at 1-2 breaths/h, increasing to ~30 breaths/h at D15 and D30. Forced activity at all stages resulted in a rapid but brief increase in both fG and fH, (but not fAB), indicating that even in these early larval stages reflex control existed over both ventilation and circulation prior to its increasing importance in older fishes. Acute progressive hypoxia increased fG in D2.5-D10 larvae, but decreased fG in older larvae (>D15), possibly to prevent branchial O2 loss into surrounding water. Temperature sensitivity of fG and fH measured at 20, 25, 28 and 38°C was largely independent of development, with a Q10 between 20°C and 38°C of ~2.4 and ~1.5 for fG and fH, respectively. The rapid onset of air breathing, coupled with both respiratory and cardiovascular reflexes as early as D2.5 indicates that larval A. tropicus develops "in the fast lane".
In humans numbers of circulating T cells show a circadian rhythm with peak counts during the night and a steep decline in the morning. Sleep per se appears to counter this rhythm by acutely reducing the number of total T cells. The T-cell population, however, is rather heterogeneous comprising various subpopulations with different features and functions and also different circadian rhythms. Therefore, here, we examined whether sleep likewise differentially affects these subsets. We measured eight different T-cell subsets (naïve, central memory, effector memory, and effector CD4+ and CD8+ T cells) over a 24-hour period under conditions of sustained wakefulness compared to a regular sleep-wake cycle in 14 healthy young men. Sleep reduced the number of all T-cell subsets during nighttime with this effect reaching the p < 0.05 level of significance in all but one subpopulation, i.e., effector CD4+ T cells, where it only approached significance. Sleep was furthermore associated with an increase in growth hormone, prolactin, and aldosterone levels, whereas concentrations of catecholamines tended to be lower than during nocturnal wakefulness. The effect of sleep uniformly decreasing the different T-cell subsets is surprising considering their differential function and circadian rhythms, and even more so, since the sleep-induced decreases in these subsets are probably conveyed by different hormonal mediators. Although the reductions in cell numbers are rather small, they are comparable to changes seen for example after vaccination and are therefore likely to be of physiological relevance.
The present study aimed to investigate the effects of aerobic exercise on human somatosensory processing recorded by somatosensory-evoked potentials (SEPs) under temperate (TEMP, 20 °C and 40% relative humidity; RH) and hot (HOT, 35 °C and 30% RH) environments. Fifteen healthy subjects performed 4 x 15-min bouts of a moderate cycling exercise (mean power output: 156.5 ± 7.7 (SE) W), with a 10-min rest period, and received a posterior tibial nerve stimulation at the left ankle before and after each exercise bout; SEPs were recorded in five sessions; 1st (pre), 2nd (post 1st exercise bout), 3rd (post 2nd exercise bout), 4th (post 3rd exercise bout), and 5th (post 4th exercise bout). The peak latencies and amplitudes of the P37, N50, P60, and N70 components at Cz were evaluated. The latencies of P37, N50, P60, and N70 were significantly shorter with the repetition of aerobic exercise, and these shortened latencies were significantly greater in the HOT condition than in the TEMP condition (P37: 3rd, p < 0.05, and 5th, p < 0.01; P60: 4th, p < 0.05, and 5th, p < 0.01; N70: 4th, p < 0.05, and 5th, p < 0.001). No significant differences were observed in the amplitudes of any SEP component under either thermal condition. These results suggest that the conduction velocity of the ascending somatosensory input was accelerated by increases in body temperature, and aerobic exercise did not alter the strength of neural activity in cortical somatosensory processing.
The overload-induced increase in muscle mass is accompanied by protein accretion, however the initiating events are poorly understood. Regulated in Development and DNA Damage 1 (REDD1), a repressor of the mechanistic target of rapamycin in complex 1 (mTORC1), blunts the elevation in protein synthesis induced by acute muscle contractions. Therefore, this study was designed to determine whether REDD1 alters the rate of the overload-induced increase in muscle mass. Wild type (WT) and REDD1 null mice underwent unilateral functional overload (OV) of the plantaris, while the contralateral sham leg served as a control. After 3 and 5 days of OV, puromycin incorporation was used as measurement of protein synthesis. The percent increase in plantaris wet weight and protein content was greater in REDD1 null mice after 3, 5, and 10 days OV. The overload-stimulated rate of protein synthesis in the plantaris was similar between genotypes after 3 days OV, but translational capacity was lower in REDD1 null mice indicating elevated translational efficiency. This was likely due to elevated absolute mTORC1 signaling (phosphorylation of p70S6K1 (Thr389) and 4E-BP1 (Ser65)). By 5 days of OV, the rate of protein synthesis in REDD1 null mice was lower than WT mice with no difference in absolute mTORC1 signaling. Additionally, markers of autophagy (LC3II/I ratio and p62 protein) were decreased to a greater absolute extent after 3 days OV in REDD1 null mice. These data suggest that loss of REDD1 augments the rate of the overload-induced increase in muscle mass by altering multiple protein balance pathways.
13% of the world's population suffers from obesity and 39% from being overweight, which correlates with an increase in numerous secondary metabolic complications including type 2 diabetes mellitus. Bariatric surgery is the most effective treatment for severe obesity and results in significant weight loss and the amelioration of obesity-related comorbidities through changes in enteroendocrine activity, caloric intake, and alterations in gut microbiota composition. The circadian system has recently been found to be a critical regulatory component in the control of metabolism and thus may potentially play an important role in inappropriate weight gain. Indeed, some behaviors and lifestyle factors associated with an increased risk of obesity are also risk factors for misalignment in the circadian clock system and for the metabolic syndrome. It is thus possible that alterations in peripheral circadian clocks in metabolically relevant tissues are a contributor to the current obesity epidemic. As such, it is plausible that post-surgical alterations in central circadian alignment as well as peripheral gene expression in metabolic tissues may represent another mechanism for the beneficial effects of bariatric surgery. Bariatric surgery may represent an opportunity to identify changes in the circadian expression of clock genes that have been altered by environmental factors, allowing for a better understanding of the mechanism of action of surgery. These studies could also reveal an overlooked target for behavioral intervention to improve metabolic outcomes following bariatric surgery.
The family Channichthyidae or "icefishes" (suborder Notothenioidei) represents the only vertebrates lacking hemoglobin (Hb) as adults. Several icefish species also do not express cardiac myoglobin (Mb). We address how levels of proteins involved in iron (Fe) processing (transport, sequestration, and export) vary among white- and red-blooded notothenioids, and whether absence of Hb and/or Mb in channichthyids is accompanied by expansion of contents of Fe-binding proteins to protect against unchaperoned Fe. Levels of transferrin (Tf), ferritin (Ft), ceruloplasmin (Cp), and non-heme Fe were quantified in plasma, serum, and/or non-hematopoietic tissues (cardiac ventricle, skeletal muscle, and liver) from species of white-blooded (Chaenocephalus aceratus, Champsocephalus gunnari, Chionodraco rastrospinosus, Pseudochaenichthys georgianus) (the first two species not expressing Mb) and red-blooded (Notothenia coriiceps, Gobionotothen gibberifrons) notothenioids. We also measured levels of ascorbate (Asc), a mediator of Fe uptake. While plasma concentrations of Tf, and tissue levels of Asc are similar among species, concentrations of plasma Asc are lower in white-blooded species. Concentrations of Ft and non-heme Fe and activities of Cp are also generally reduced in icefishes compared with red-blooded notothenioids. The presence of cardiac Mb in some icefish species does not appear to influence levels of proteins involved in Fe processing. To address further the question of Fe sequestration within a physiological context, we account for well-characterized differences in blood volume and heart mass among white- and red-blooded notothenioids. We report that total contents of plasma Tf are greater, while ventricle non-heme Fe is at least at parity in white- versus red-blooded species.
We hypothesize that ceramides are involved in the regulation of food intake in fish. Therefore, we assessed in rainbow trout (Oncorhynchus mykiss) the effects of intracerebroventricular (ICV) treatment with C6:0 ceramide on food intake. In a second experiment, we assessed the effects in brain areas of ceramide treatment on neuropeptide expression, fatty acid sensing systems, and cellular signaling pathways. Ceramide treatment induced a decrease in food intake, a response opposed to the orexigenic effect described in mammals, which can be related to enhanced mRNA abundance of CART and POMC and decreased mRNA abundance of AgRP and NPY. Fatty acid sensing systems appear to be inactivated by ceramide treatment. The mRNA abundance of integrative sensors AMPK and SIRT-1, and the phosphorylation status of cellular signaling pathways dependent on Akt, AMPK, mTOR, and Fox01 are generally activated by ceramide treatment. However, Tthere are differences between hypothalamus and hindbrain in the response phosphorylation status of AMPK (decreased in hypothalamus and increased in hindbrain), mTOR (decreased in hypothalamus and increased in hindbrain), and Fox01 (increased in hypothalamus and decreased in hindbrain) to ceramide treatment. The results suggest that ceramides are involved in the regulation of food intake in rainbow trout through mechanisms comparable to those characterized previously in mammals in some cases but not in others.
In lung epithelial cells, hypoxia decreases the expression and activity of sodium transporting molecules, thereby reducing the rate of transepithelial sodium absorption. The mechanisms underlying the sensing of hypoxia and subsequent coupling to sodium transporting molecules remain unclear. Hydrogen sulfide (H2S) has recently been recognized as a cellular signaling molecule whose intracellular concentrations critically depend on oxygen levels. Therefore it was questioned whether endogenously produced H2S contributes to hypoxic inhibition of sodium transport. In electrophysiological Ussing chamber experiments, hypoxia was established by decreasing oxygen concentrations in the chambers. Hypoxia concentration-dependently and reversibly decreased amiloride-sensitive sodium absorption by cultured H441 monolayers and freshly dissected porcine tracheal epithelia due to inhibition of basolateral Na+/K+-ATPase. Exogenous application of H2S by the sulfur salt Na2S mimicked the effect of hypoxia and inhibited amiloride-sensitive sodium absorption by both tissues in an oxygen-dependent manner. Hypoxia increased intracellular concentrations of H2S and decreased the concentration of polysulfides. Pre-treatment with the cystathionine--lyase inhibitor D/L-propargylglycine (PAG) decreased hypoxic inhibition of sodium transport by H441 monolayers, whereas inhibition of cystathionine-β-synthase (with aminooxy-acetic acid; AOAA) or 3-mercaptopyruvate sulfurtransferase (with aspartate) had no effect. Inhibition of all of these H2S-generating enzymes with a combination of AOAA, PAG and aspartate decreased the hypoxic inhibition of sodium transport by H441 cells and pig tracheae and decreased H2S production by tracheae. These data suggest that airway epithelial cells endogenously produce H2S during hypoxia and this contributes to hypoxic inhibition of transepithelial sodium absorption.
Sexual maturation and maintenance of reproductive function are regulated by neurohormonal communication between the hypothalamus, pituitary, and gonads (referred to as the HPG axis). Phoenixin (PNX) is a newly identified, endogenous peptide abundantly produced in the hypothalamus and shown to be an important mediator of ovarian cyclicity. However, the underlying mechanisms by which phoenixin functions within the HPG axis are unknown. Previous in vitro studies demonstrated a direct action of PNX on gonadotrophs to potentiate gonadotrophin releasing hormone (GnRH) induced luteinizing hormone (LH) secretion. Therefore, we hypothesized that centrally-derived phoenixin regulates the pre-ovulatory LH surge required for ovarian cyclicity. We observed a significant dose-related increase in the level of plasma LH in diestrous, female rats that were given an intracerebroventricular injection of PNX compared to vehicle-treated controls. While this suggests that even under low estrogen conditions PNX acts centrally to stimulate the HPG-axis, further characterization is contingent on the elucidation of its cognate receptor. Using the "Deductive Ligand Receptor Matching Strategy," we identified the orphan G protein-coupled receptor, Gpr173, as our top candidate. In cultured pituitary cells, siRNA-targeted compromise of Gpr173 abrogated PNX's action to potentiate GnRH-stimulated LH secretion. In addition, siRNA-mediated knockdown of endogenous Gpr173, which localized to several hypothalamic sites related to reproductive function, not only significantly extended the estrous cycle but also prevented the PNX-induced LH secretion in diestrous, female rats. These studies are the first to demonstrate a functional relationship between PNX and Gpr173 in reproductive physiology and identify a potential therapeutic target for ovulatory dysfunction.
Acetylcholine released from cholinergic nerves is involved in heat loss responses of cutaneous vasodilation and sweating. K+ channels are thought to play a role in regulating cholinergic cutaneous vasodilation and sweating, though which K+ channels are involved in their regulation remains unclear. We evaluated the hypotheses that 1) Ca2+-activated K+ (KCa), ATP-sensitive K+ (KATP), and voltage-gated K+ (KV) channels all contribute to cholinergic cutaneous vasodilation; and 2) KV channels, but not KCa and KATP channels, contribute to cholinergic sweating. In 13 young adults (24±5 years), cutaneous vascular conductance (CVC) and sweat rate were evaluated at intradermal microdialysis sites that were continuously perfused with: 1) lactated Ringer (Control), 2) 50mM tetraethylammonium (KCa channel blocker), 3) 5mM glybenclamide (KATP channel blocker), and 4) 10mM 4-aminopyridine (KV channel blocker). At all sites, cholinergic cutaneous vasodilation and sweating were induced by co-administration of methacholine (0.0125, 0.25, 5, 100, and 2000mM, each for 25 min). The methacholine-induced increase in CVC was lower with the KCa channel blocker relative to Control at 0.0125 (1±1 vs. 9±6%max) and 5 (2±5 vs. 17±14%max) mM methacholine, whereas it was lower in the presence of KATP (69±7%max) and KV (57±14%max) channel blocker in comparison to Control (79±6%max) at 100mM methacholine. Further, methacholine-induced sweating was lower at the KV channel blocker site (0.42±0.17mg•min-1•cm-2) compared to Control (0.58±0.15mg•min-1•cm-2) at 2000mM methacholine. In conclusion, we show that KCa, KATP, and KV channels play a role in cholinergic cutaneous vasodilation, whereas only KV channels contribute to cholinergic sweating in normothermic resting humans.
Hypothalamic orexin neurons project to numerous brain areas, including the ventral tegmental area (VTA), which is involved in motivation and food seeking behavior. Here we address how exogenously administered orexin-A, and endogenous orexin 1 receptor (OX1R) activation in the VTA affects feeding behavior. We hypothesized that orexin-A and OX1R antagonist SB334867 delivered to the VTA, at doses that were subthreshold for effect when injected into the ventricle, would affect intake of palatable foods in multiple test situations. We first used a hedonic feeding model in which satiated rats selectively consume high-fat diet (HFD). Intra-VTA orexin-A stimulated additional consumption of chow and increased HFD intake in this model. In ad lib-fed rats given daily 30-min test sessions, intra-VTA orexin-A also increased intake of HFD and 0.1M sucrose. Further analysis of licking patterns revealed that that VTA orexin-A increased meal size and licking burst size only toward the end of the meal. Consistent with this finding, a subthreshold dose of VTA orexin-A prevented intake suppression induced by gastrointestinal nutrient infusion. Surprisingly, intra-VTA orexin-A had no effect on operant responding for sucrose pellets on a progressive ratio schedule of reinforcement. A role for endogenous VTA OX1R stimulation is supported by our finding that bilateral VTA injection of the selective OX1R antagonist SB334867 suppressed 0.1M sucrose intake. Together, our data suggest that OX1R activity in the VTA facilitates food intake, potentially by counteracting post-ingestive negative feedback that would normally suppress feeding later in a meal.
Central pathways regulate metabolic responses to cold in endotherms to maintain relatively stable internal core body temperatures. However, peripheral muscles routinely experience temperatures lower than core body temperature, so that it would be advantageous for peripheral tissues to respond to temperature changes independently from core body temperature regulation. Early developmental conditions can influence offspring phenotypes, and here we tested whether developing muscle can compensate locally for the effects of cold exposure independently from central regulation. Muscle myotubes originate from undifferentiated myoblasts that are laid down during embryogenesis. We show that in a murine myoblast cell line (C2C12), cold exposure (32°C) increased myoblast metabolic flux compared to 37°C control conditions. Importantly, myotubes that differentiated at 32°C compensated for the thermodynamic effects of low temperature by increasing metabolic rates, ATP production, and glycolytic flux. Myotube responses were also modulated by the temperatures experienced by "parent" myoblasts. Myotubes that differentiated under cold exposure increased activity of the AMP-stimulated protein kinase (AMPK), which may mediate metabolic changes in response cold exposure. Moreover, cold exposure shifted myosin heavy chains from slow to fast, presumably to overcome slower contractile speeds resulting from low temperatures. Adjusting thermal sensitivities locally in peripheral tissues complements central thermoregulation, and permits animals to maintain function in cold environments. Muscle also plays a major metabolic role in adults, so that developmental responses to cold are likely to influence energy expenditure later in life.
Oxygen tension (PO2) of urine in the bladder could be used to monitor risk of acute kidney injury if it varies with medullary PO2. Therefore, we examined this relationship, and characterized oxygen diffusion across walls of the ureter and bladder, in anesthetized rabbits. A computational model was then developed to predict medullary PO2 from bladder urine PO2. Both intravenous infusion of [Phe2,Ile3,Orn8]-vasopressin and infusion of NG-nitro-L-arginine reduced urinary PO2 and medullary PO2 (8-17%), yet had opposite effects on renal blood flow and urine flow. Changes in bladder urine PO2 during these stimuli correlated strongly with changes in medullary PO2 (within-rabbit r2 = 0.87-0.90). Differences in the PO2 of saline infused into the ureter close to the kidney could be detected in the bladder, although this was diminished at lesser ureteric flow. Diffusion of oxygen across the wall of the bladder was very slow, so was not considered in the computational model. The model predicts PO2 in the pelvic ureter (presumed to reflect medullary PO2) from known values of bladder urine PO2, urine flow and arterial PO2. Simulations suggest that, across a physiological range of (single kidney) urine flow in anesthetized rabbits (0.1 - 0.5 ml/min), a change in bladder urine PO2 reflects 10 - 50% of the change in pelvic urine/medullary PO2. Thus, it is possible to infer changes in medullary PO2 from changes in urinary PO2, so urinary PO2 may have utility as a biomarker of risk of acute kidney injury.
Fish oil is commonly taken by pregnant women, and supplements sold at retail are often oxidised. We aimed to assess the effects of supplementation with oxidised fish oil during pregnancy in mothers and offspring, using a rat model, focussing on newborn viability and maternal insulin sensitivity. Female rats were allocated to a control or high-fat diet and then mated. They were subsequently randomised to receive a daily treatment of 1 ml of unoxidised fish oil, a highly oxidised fish oil, or control (water) throughout pregnancy by gavage. At birth, the gavage treatment was stopped, but the same maternal diets were fed ad libitum throughout lactation. Supplementation with oxidised fish oil during pregnancy had a marked adverse effect on newborn survival at day 2, leading to much greater odds of mortality than in the control (odds ratio 8.26) and unoxidised fish oil (odds ratio 13.70) groups. In addition, maternal intake of oxidised fish oil during pregnancy led to increased insulin resistance at the time of weaning (three weeks after exposure) compared to control dams (HOMA-IR 2.64 vs 1.42; p=0.044). These data show that the consumption of oxidised fish oil is harmful in rat pregnancy, with deleterious effects in both mothers and offspring.
The anti-hyperglycemic agent linagliptin, a dipeptidyl peptidase-4 (DPP-IV) inhibitor, has been shown to reduce inflammation and improve endothelial cell function. In this study, we hypothesized that DPP-IV inhibition with linagliptin would improve impaired cerebral perfusion in diabetic rats as well as improve insulin-induced cerebrovascular relaxation and reverse pathological cerebrovascular remodeling. We further postulated that these changes would lead to a subsequent improvement of cognitive function. Male type-2 diabetic and nondiabetic Goto-Kakizaki (GK) rats were treated with linagliptin for four weeks, and blood glucose and DPP-IV plasma levels were assessed. Cerebral perfusion was assessed post-treatment using laser Doppler imaging, and dose response to insulin (10-13- 10-6 M) in middle cerebral arteries was tested on a pressurized arteriograph. The impact of DPP-IV inhibition on diabetic cerebrovascular remodeling was assessed over a physiologically relevant pressure range, and changes in short-term hippocampal-dependent learning were observed using a novel object recognition test. Linagliptin lowered DPP-IV activity but did not change blood glucose or insulin levels in diabetes. Insulin-mediated vascular relaxation and cerebral perfusion were improved in the diabetic rats with linagliptin treatment. Indices of diabetic vascular remodeling such increased cross-sectional area, media thickness, and wall to lumen ratio were also ameliorated, however improvements in short-term hippocampal-dependent learning were not observed. The present study provides evidence that linagliptin treatment improves cerebrovascular dysfunction and remodeling in a type-2 model of diabetes independent of glycemic control. This has important implications in diabetic patients who are predisposed to the development of cerebrovascular complications such as stroke and cognitive impairment.
Heat therapy has been shown to promote capillary growth in skeletal muscle and in the heart in several animal models, but the effects of this therapy on angiogenic signaling in humans in unknown. We evaluated the acute effect of lower body heating (LBH) and unilateral thigh heating (TH) on the expression of angiogenic regulators and heat shock proteins (HSPs) in healthy young individuals. Exposure to LBH (n=18) increased core temperature (Tc) from 36.9±0.1 to 37.4±0.1ºC (p<0.01) and average leg skin temperature (Tleg) from 33.1±0.1 to 39.6±0.1ºC (p<0.01), but did not alter the levels of circulating angiogenic cytokines and bone marrow-derived pro-angiogenic cells (CD34+CD133+). In skeletal muscle, the change in mRNA expression from baseline of vascular endothelial growth factor (VEGF), angiopoietin 2 (ANGPT2), chemokines CCL2 and CX3CL1, platelet factor-4 (PF4) and several members of the HSP family was higher 30 min after the intervention in the individuals exposed to LBH (n=11) as compared to the control group (n=12). LBH also reduced the expression of transcription factor FOXO1 (p=0.03). Exposure to TH (n=14) increased Tleg from 32.8±0.2 to 40.3±0.1ºC (p<0.05) but Tc remained unaltered (36.8±0.09ºC at baseline and 36.9±0.07ºC at 90 min). This protocol upregulated the expression of VEGF, ANGPT1, ANGPT2, CCL2 and HSPs in skeletal muscle but did not affect the levels of CX3CL1, FOXO-1 and PF4. These findings suggest that both LBH and TH increase the expression of factors associated with capillary growth in human skeletal muscle.
Limited nutrient availability is a cause of intrauterine growth restriction (IUGR), a condition that has important implications for the wellbeing of the offspring. Using the established IUGR model of maternal fasting in the rat, we investigated mechanisms that control gene expression and mRNA translation in late gestation fetal liver. Maternal fasting for 48 hr during the last third of gestation was associated with a 10-15% reduction in fetal body weight and a disproportionate one third reduction in total fetal liver protein. The fetal liver transcriptome showed only subtle changes consistent with reduced cell proliferation and enhanced differentiation in IUGR. Effects on the transcriptome could not be attributed to specific transcription factors. We purified translating polysomes to profile the population of mRNAs undergoing active translation. Microarray analysis of the fetal liver translatome indicated a global reduction of translation. The only targeted effect was enhanced translation of mitochondrial ribosomal proteins in IUGR, consistent with enhanced mitochondrial biogenesis. There was no evidence for attenuated signaling through the mammalian target of rapamycin (mTOR). Western blotting showed no changes in fetal liver mTOR signaling. However, eukaryotic initiation factor 2α (eIF2α) phosphorylation was increased in livers from IUGR fetuses, consistent with a role in global translation control. Our data indicate that IUGR-associated changes in hepatic gene expression and mRNA translation likely involve a network of complex regulatory mechanisms, some of which are novel and distinct from those that mediate the response of the liver to nutrient restriction in the adult rat.
We investigated the effects different diets on adipose tissue, liver and serum morphology and biomarkers in rats that voluntarily exercised. Male Sprague-Dawley rats (~9-10 weeks of age) exercised with resistance-loaded voluntary running wheels (EX; wheels loaded with 20-60% body mass) or remained sedentary (SED) over 6 weeks. EX and SED rats were provided isocaloric amounts of either a ketogenic diet (KD; 20.2%-10.3%-69.5% protein-carbohydrate-fat), a Western diet (WD; 15.2%-42.7-42.0%), or standard chow (SC; 24.0%-58.0%-18.0%); n=8-10 in each diet for SED and EX rats. Following the intervention, body mass and feed efficiency was lowest in KD rats independent of exercise (p<0.05). Absolute and relative (body mass-adjusted) omental adipose tissue (OMAT) masses were greatest in WD rats (p<0.05) and OMAT adipocyte diameters were lowest in KD-fed rats (p<0.05). None of the assayed OMAT or subcutaneous (SQ) protein markers were affected by the diets [total acetyl coA carboxylase (ACC), CD36 and CEBPα or phosphorylated NF-B/p65, AMPKα and hormone-sensitive lipase (HSL)], although EX unexpectedly altered some OMAT markers (i.e., higher ACC and phosphorylated NF-B/p65, and lower phosphorylated AMPKα and phosphorylated HSL). Liver triglycerides were greatest in WD rats (p<0.05) and liver phosphorylated NF-B/p65 was lowest in KD rats (p<0.05). Serum insulin, glucose, triglycerides and total cholesterol were greater in WD and/or SC rats compared to KD rats (p<0.05), and serum β-hydroxybutyrate was greater in KD versus SC rats (p<0.05). In conclusion, KD rats presented a healthier metabolic profile, albeit the employed exercise protocol minimally impacts any potentiating effects that KD has on fat loss.
Leg thermotherapy (TT) application reduces blood pressure (BP) and increases both limb blood flow and circulating levels of anti-inflammatory mediators in healthy, young humans and animals. The purpose of the present study was to determine the impact of TT application using a water-circulating garment on leg and systemic hemodynamics and on the concentrations of circulating cytokines and vasoactive mediators in patients with symptomatic peripheral artery disease (PAD). Sixteen patients with PAD and intermittent claudication (age: 63±9 yrs) completed three experimental sessions in a randomized order: TT, control intervention and one exercise testing session. The garment was perfused with 48ºC water for 90 min in the TT session and with 33ºC water in the control intervention. A subset of 10 patients also underwent a protocol for the measurement of blood flow in the popliteal artery during 90 min of TT using phase-contrast MRI. Compared to the control intervention, TT promoted a significant reduction in systolic (~11 mmHg) and diastolic (~6 mmHg) BP (p<0.05) that persisted for nearly 2 hrs after the end of the treatment. The serum concentration of endothelin-1 (ET-1) was significantly lower 30 min after exposure to TT (Control: 2.3±0.1 vs. TT: 1.9±0.09 pg/mL, p=0.026). In addition, TT induced a marked increase in peak blood flow velocity (~68%), average velocity (~76%), and average blood flow (~102%) in the popliteal artery (p<0.01). These findings indicate that TT is a practical and effective strategy to reduce BP and circulating ET-1 concentration and enhance leg blood flow in patients with PAD.
Changes in osmolality or extracellular NaCl concentrations are detected by specialized neurons in the hypothalamus to increase vasopressin (VP) and stimulate thirst. Recent in vitro evidence suggests this process is mediated by an N-terminal variant of the transient receptor potential vanilloid type 1 (TRPV1) channel expressed by osmosensitive neurons of the lamina terminalis and vasopressinergic neurons of the supraoptic nucleus. The present study tested this hypothesis in vivo by analysis of plasma VP levels during acute hypernatremia in awake control and TRPV1-/- rats. TRPV1-/- rats were produced by a Zinc-finger-nuclease 2-bp deletion in exon 13. Intravenous injection of the TRPV1 agonist capsaicin produced hypotension and bradycardia in control rats, but this response was absent in TRPV1-/- rats. Infusion of 2M NaCl (1 mL per hr, IV) increased plasma osmolality, electrolytes, and VP levels in both control and TRPV1-/- rats. However, plasma VP levels did not differ between strains at any time. Furthermore, a linear regression between plasma VP versus osmolality revealed a significant correlation in both control and TRPV1-/- rats, but the slope of the regression lines was not attenuated in TRPV1-/- versus control rats. Hypotension produced by intravenous injection of minoxidil decreased blood pressure and increased plasma VP levels similarly in both groups. Finally, both treatments stimulated thirst; however, cumulative water intakes in response to hypernatremia or hypotension were not different between control and TRPV1-/- rats. These findings suggest that TRPV1 channels are not necessary for VP secretion and thirst stimulated by hypernatremia.
We investigated whether heat-induced hyperventilation during exercise is affected by time-of-day. Nineteen male subjects were divided into two experiments (protocol 1, n=10 and protocol 2, n=9). In protocol 1, subjects performed cycle exercise at 50% peak oxygen uptake in the heat (37°C and 50% RH) in the morning (06:00) and evening (18:00). Results showed that baseline resting and exercising esophageal temperature (Tes) were significantly (0.5°C) higher in the evening than morning. Minute ventilation (VE) increased from 54.3±7.9 and 54.9±6.8 l•min-1 at 10 min to 71.4±8.1 and 76.5±11.8 l•min-1 at 48.5 min in the morning and evening, respectively (both P<0.01). Time-of-day had no effect on VE (P=0.44). When VE as the output response was plotted against Tes as thermal input, the Tes threshold for increases in VE was higher in the evening than morning (37.2±0.7 vs. 36.6±0.6°C, P=0.009), indicating the ventilatory response to the same core temperature is smaller in the evening. In protocol 2, the circadian rhythm-related higher resting Tes seen in the evening was adjusted down to the same temperature seen in the morning by immersing the subject in cold-water. Importantly, time-course of changes in VE during exercise were smaller in the evening, but the threshold for VE remained higher in the evening than morning (P<0.001). Collectively, those results suggest that time-of-day has no effect on time-course of hyperventilation during exercise in the heat, despite higher core temperatures in the evening. This is likely due to diurnal variation in the control of ventilation in response to rising core temperature.
Aortic pulse pressure arises from the interaction of the heart, the systemic arterial system and peripheral microcirculations. The complex interaction between hemodynamics and arterial remodeling precludes the ability to experimentally ascribe changes in aortic pulse pressure to particular adaptive responses. Therefore, the purpose of the present work was to use a human systemic arterial system model to test the hypothesis that pulse pressure homeostasis can emerge from physiologic adaptation of systemic arteries to local mechanical stresses. First we assumed a systemic arterial system that had a realistic topology consisting of 121 arterial segments. Then the relationships of pulsatile blood pressures and flows in arterial segments were characterized by standard pulse transmission equations. Finally, each arterial segment was assumed to remodel to local stresses following three simple rules: 1) increases in endothelial shear stress increases radius, 2) increases in wall circumferential stress increases wall thickness, and 3) increases in wall circumferential stress decreases wall stiffness. Simulation of adaptation by iteratively calculating pulsatile hemodynamics, mechanical stresses and vascular remodeling led to a general behavior in response to mechanical perturbations: initial increases in pulse pressure led to increased arterial compliances, and decreases in pulse pressure led to decreased compliances. Consequently, vascular adaptation returned pulse pressures back towards baseline conditions. This behavior manifested when modeling physiologic adaptive responses to changes in cardiac output, changes in peripheral resistances, and changes in local arterial radii. The present work thus revealed that pulse pressure homeostasis emerges from physiologic adaptation of systemic arteries to local mechanical stresses.
Collecting duct endothelin-1 (ET-1), endothelin B receptor (ETB) and nitric oxide synthase-1 (NOS1) pathway is critical for regulation of fluid-electrolyte balance and blood pressure control during high salt feeding. ET-1, ETB receptor, and NOS1 are highly expressed in the IMCD and vasa recta, suggesting that there may be cross talk or paracrine signaling between the vasa recta and IMCD. The purpose of this study was to test the hypothesis that endothelial cell-derived ET-1 (paracrine) and collecting duct-derived ET-1 (autocrine) promote IMCD NO production through activation of the ETB receptor during high salt feeding. We determined that after 7 days of high salt diet (HS7) there was a shift to 100% ETB expression in IMCDs as well as a 2-fold increase in nitrite production (a metabolite of NO) and this increase could be prevented by acute inhibition of the ETB receptor. ETB receptor blockade or NOS1 inhibition also prevented the ET-1 dependent decrease in ion transport from primary IMCDs as determined by transepithelial resistance. IMCD were also isolated from vascular endothelial ET-1 knockout mice (VEETKO), collecting duct ET-1 KO (CDET-1KO) and flox controls. Nitrite production by IMCD from VEETKO and flox mice was similarly increased 2-fold with HS7. However, IMCD NO production from CDET-1KO mice was significantly blunted with HS7 compared to flox control. Taken together these data indicate that during high salt feeding it is the autocrine actions of ET-1 via up-regulation of the ETB receptor that are critical for IMCD NO production facilitating inhibition of ion reabsorption.
Obesity leads to altered autonomic reflexes that reduce stability of mean arterial pressure (MAP). Sympathoinhibitory reflexes such as baroreflexes are impaired, but reflexes that raise MAP appear to be augmented. In obese Zucker rats (OZR) sciatic nerve stimulation evokes larger increases in MAP by unknown mechanisms. We sought to determine the autonomic underpinnings of this enhanced somatic pressor reflex, and whether other sympathoexcitatory reflexes are augmented. We also determined whether their final common pathway, glutamatergic activation of the rostral ventrolateral medulla (RVLM), was enhanced in male OZR compared to lean Zucker rats (LZR). Sciatic nerve stimulation or activation of the nasopharyngeal reflex evoked larger rises in splanchnic SNA (79% and 45% larger in OZR respectively, P<0.05) and MAP in urethane-anesthetized, ventilated, paralyzed adult OZR compared to LZR. After elimination of baroreflex feedback by pharmacological prevention of changes in MAP and heart rate, these two sympathoexcitatory reflexes were still exaggerated in OZR (167% and 69% larger respectively, P<0.05). In adult OZR microinjections of glutamate, AMPA, or NMDA into the RVLM produced larger rises in SNA (~61% larger in OZR, P<0.05 for each drug) and MAP, but stimulation of axonal fibers in the upper thoracic spinal cord yielded equivalent responses in OZR and LZR. In juvenile OZR and LZR, sympathoexcitatory reflexes and physiological responses to RVLM activation were comparable. These data suggest the ability of glutamate to activate the RVLM becomes enhanced in adult OZR and may contribute to the development of exaggerated sympathoexcitatory responses independent of impaired baroreflexes.
Preeclampsia (PE) is a pregnancy-related hypertensive disorder (HTN-Preg) with unclear mechanism. Imbalance between anti-angiogenic soluble fms-like tyrosine kinase-1 (sFlt-1) and angiogenic placental growth factor (PlGF) has been observed in PE, but the vascular targets and signaling pathways involved are unclear. We tested if inducing sFlt-1/PlGF imbalance by infusing sFlt-1 (10 µg/kg/day) in pregnant (Preg) rats increases BP and vascular reactivity; and if restoring sFlt-1/PlGF balance by infusing PIGF (20 µg/kg/day) in a rat model of reduced uterine perfusion pressure (RUPP) improves BP and vascular function. On gestation day 19, BP was in Preg+sFlt-1 and RUPP>Preg and in RUPP+PlGF<RUPP. Plasma sFlt-1/PlGF ratio was increased in Preg+sFlt-1 and RUPP, and was reduced in RUPP+PlGF rats. In endothelium-intact aorta, carotid, mesenteric and renal artery, pheylephrine (Phe) and high KCl-induced contraction was in Preg+sFlt-1 and RUPP>Preg and RUPP+PlGF. The differences in vascular reactivity between groups were less apparent in vessels treated with NOS inhibitor L-NAME or guanylate cyclase inhibitor ODQ or endothelium-denuded, suggesting changes in endothelial NO-cGMP. Acetylcholine (ACh)-induced relaxation was in Preg+sFlt-1 and RUPP<Preg and RUPP+PlGF, and was blocked by L-NAME, ODQ or endothelium-removal. Aortic total eNOS and phospho-eNOS were in Preg+sFlt-1 and RUPP<Preg and RUPP+PlGF. ACh-induced vascular nitrate/nitrite production was in Preg+sFlt-1 and RUPP<Preg and RUPP+PlGF. Vascular relaxation to sodium nitroprusside was not different among groups. Thus, a tilt in angiogenic/anti-angiogenic balance towards sFlt-1 is associated with decreased vascular relaxation via NO-cGMP and increased vasoconstriction and BP. Restoring angiogenic/anti-angiogenic balance using PlGF enhances vascular relaxation and decreases vasoconstriction and BP in HTN-Preg.
The present study was undertaken to examine whether variations in endocannabinoid signaling in the dorsal periaqueductal gray (dPAG) are associated with baseline autonomic nerve activity, heart rate and blood pressure. Blood pressure was recorded telemetrically in rats, and heart rate and power spectral analysis of heart rate variability were determined. Natural variations from animal to animal provided a range of baseline values for analysis. Transcript levels of endocannabinoid signaling components in the dPAG was analyzed and endocannabinoid content and catabolic enzyme activity were measured. Higher baseline heart rate was associated with increased anandamide content and with decreased activity of the anandamide hydrolyzing enzyme, fatty acid amide hydrolase (FAAH), and was negatively correlated with transcript levels of both FAAH and monoacylglycerol lipase (MAGL), a catabolic enzyme for 2-arachidonoylglycerol (2-AG). Autonomic tone and heart rate, but not blood pressure, were correlated to levels of FAAH mRNA. In accord with these data, exogenous anandamide in the dPAG of anesthetized rats increased heart rate. These data indicate that in the dPAG, anandamide, a FAAH-regulated lipid, contributes to regulation of baseline heart rate through influences on autonomic outflow.
McArdle disease (muscle glycogenosis type V) is a disease caused by myophosphorylase deficiency leading to 'blocked' glycogen breakdown. A significant but varying glycogen accumulation in especially distal hind limb muscles of mice affected by McArdle disease has recently been demonstrated. In this study, we investigated how myophosphorylase deficiency affects glucose metabolism in hind limb muscle of 20-week old McArdle mice and vastus lateralis muscles from patients with McArdle disease. Western blotting and activity assay demonstrated that glycogen synthase was inhibited in glycolytic muscle from McArdle mice. The level and activation of proteins involved in contraction-induced glucose transport (AMPK, GLUT4) and glycogen synthase inhibition were increased in quadriceps muscle of McArdle mice. In addition, pCaMKII in quadriceps was reduced, suggesting lower insulin-induced glucose uptake, which could lead to lower glycogen accumulation. In comparison, tibialis anterior, extensor digitorum longus and soleus had massive glycogen accumulation, but few, if any, changes or adaptations in glucose metabolism compared to wild-type. The findings suggest plasticity in glycogen metabolism in the McArdle mouse that is related to myosin heavy chain type IIB content in muscles. In patients, the level of GLUT4 was vastly increased as were hexokinase II and phosphofructokinase, and glycogen synthase was more inhibited, suggesting that patients adapt by increasing capture of glucose for direct metabolism, thereby significantly reducing glycogen buildup compared to the mouse model. Hence, the McArdle mouse may be a useful tool for further comparative studies of disease mechanism caused by myophosphorylase deficiency and basic studies of metabolic adaptation in muscle.
Early-life experience (ELE) can significantly affect life-long health and disease, including cardiovascular function. Specific dimensions of emotionality also modify risk of disease, and aggressive traits along with social inhibition have been established as independent vulnerability factors for the progression of cardiovascular disease. Yet, the biological mechanisms mediating these associations remain poorly understood. The present study utilized the inherently stress-susceptible and socially inhibited Wistar-Kyoto rats to determine the potential influences of ELE and trait aggression (TA) on cardiovascular parameters throughout the lifespan. Pups were exposed to maternal separation (MS), consisting of daily three-hour separations of the entire litter from postnatal day (P)1 to P14. The rats were weaned at P21, and as adults were instrumented for chronic radiotelemetry recordings of blood pressure and heart rate (HR). Adult aggressive behavior was assessed using the resident-intruder test, which demonstrated that TA was independent of MS exposure. MS-exposed animals (irrespective of TA) had significantly lower resting HR accompanied by increases in HR variability. No effects of MS on resting blood pressure were detected. In contrast, TA correlated with increased resting mean, systolic, and diastolic arterial pressures, but had no effect on HR. TA rats (relative to non-aggressive animals) also manifested: increased wall-to-lumen ratio in the thoracic aorta, increased sensitivity to phenylephrine-induced vascular contractility and increased norepinephrine content in the heart. Together these data suggest that ELE and TA are independent factors that impact baseline cardiovascular function.
Oxygen supply to the heart has been hypothesized to limit cardiac performance and whole animal acute thermal tolerance (CTmax) in fish. We tested these hypotheses by continuously measuring venous oxygen tension (PVO2) and cardiovascular variables in vivo during acute warming in European perch (Perca fluviatilis) from a reference area during summer (18°C) and a chronically heated area (Biotest enclosure) that receives warm effluent water from a nuclear power plant and is normally 5-10°C above ambient (24°C at the time of experiments). While CTmax was 2.2°C higher in Biotest compared to reference perch, the peaks in cardiac output and heart rate prior to CTmax occurred at statistically similar PVO2 values (2.3 - 4.0 kPa), suggesting that cardiac failure occurred at a common critical PVO2 threshold. Environmental hyperoxia (200% air saturation) increased PVO2 across temperatures in reference fish, but heart rate still declined at a similar temperature. CTmax of reference fish increased slightly (by 0.9°C) in hyperoxia, but remained significantly lower than in Biotest fish despite an improved cardiac output due to an elevated stroke volume. Thus, while cardiac oxygen supply appears critical to elevate stroke volume at high temperatures, oxygen limitation may not explain the bradycardia and arrhythmia that occur prior to CTmax. Acute thermal tolerance and its thermal plasticity can therefore only be partially attributed to cardiac failure from myocardial oxygen limitations, and likely involves limiting factors on multiple organizational levels.
A single MIPS gene (Isyna1/Ino1) exists in eel and tilapia genomes with a single MIPS transcript identified in all eel tissues, although two MIPS spliced variants (termed MIPS(s) and MIPS(l)) are found in all tilapia tissues. The larger tilapia transcript (MIPS(l)) results from the inclusion of the 87-nucleotide intron between exons 5 and 6 in the genomic sequence. In most tilapia tissues the MIPS(s) transcript exhibits much higher abundance (generally >10-fold) with the exception of white skeletal muscle and oocytes where the MIPS(l) transcript predominates. SW-acclimation resulted in large (6- to 32-fold) increases in mRNA expression for both MIPS(s) and MIPS(l) in all tilapia tissues tested whereas in the eel changes in expression were limited to a more modest 2.5-fold increase and only in the kidney. Western blots identified a number of species- and tissue-specific immunoreactive MIPS proteins ranging from 40 to 67 kDa molecular weight. SW-acclimation failed to affect the abundance of any immunoreactive protein in any tissue tested from the eel. However, a major 67 kDa immunoreactive protein (presumed to be MIPS) found in tilapia tissues exhibited 11- and 54-fold increases in expression in gill and fin samples from SW-acclimated fish. Immunohistochemical investigations revealed specific immunoreactivity in the gill, fin, skin and intestine taken from only SW-acclimated tilapia. Immunofluorescence indicated that MIPS was expressed within gill chondrocytes and epithelial cells of the primary filaments, basal epithelial cell layers of the skin and fin, the cytosol of columnar intestinal epithelial and mucous cells as well as unknown entero-endocrine-like cells.
Chronic intermittent hypoxia (IH) induces oxidative stress and inflammation, which impair vascular endothelial function. Long-term insulin resistance also leads to endothelial dysfunction. We determined, in vivo, whether the effects of chronic IH and insulin resistance on endothelial function augment each other. Male 12 week old Goto-Kakizaki (GK) and Wistar control rats were subjected to normoxia or chronic IH (90-s N2, 5% O2 at nadir, 90-s air, 20 cycles/h, 8 h per day) for 4 weeks. Coronary endothelial function was assessed using microangiography with synchrotron radiation. Imaging was performed at baseline, during infusion of acetylcholine (ACh, 5 μg/kg/min) and then sodium nitroprusside (SNP, 5 μg/kg/min), after blockade of both nitric oxide (NO) synthase (NOS) with N-nitro-L-arginine methyl ester (L-NAME, 50 mg/kg) and cyclooxygenase (COX, meclofenamate, 3 mg/kg), and during subsequent ACh. In GK rats, coronary vasodilatation in response to ACh and SNP was blunted compared to Wistar rats, and responses to ACh were abolished after blockade. In Wistar rats, IH blunted the ability of ACh or SNP to increase the number of visible vessels. In GK rats exposed to IH, neither ACh nor SNP were able to increase visible vessel number or caliber, and blockade resulted in marked vasoconstriction. Our findings indicate that IH augments the deleterious effects of insulin resistance on coronary endothelial function. They appear to increase the dependence of the coronary microcirculation on NO and/or vasodilator prostanoids, and greatly blunt the residual vasodilation in response to ACh after blockade of NOS/COX, presumably mediated by endothelium derived hyperpolarizing factors.
Water-intake behavior is under the control of brain systems that sense body-fluid conditions at sensory circumventricular organs (sCVOs); however, the underlying mechanisms have not yet been elucidated in detail. Nax is a sodium (Na+) level sensor in the brain, and the transient receptor potential vanilloid (TRPV) channels, TRPV1 and TRPV4, have been proposed to function as osmosensors. We herein investigated voluntary water intake immediately induced after an intracerebroventricular (icv) administration of a hypertonic NaCl solution in TRPV1-, TRPV4-, Nax-, and their double-gene knockout (KO) mice. The induction of water intake by TRPV1-KO mice was normal, whereas that by TRPV4-KO and Nax-KO mice was significantly less than that by WT mice. Water intake by Nax/TRPV4-double KO mice was similar to that by the respective single KO mice. When TRPV4 activity was blocked with a specific antagonist HC-067047, water intake by WT mice was significantly reduced, whereas that by TRPV4-KO and Nax-KO mice was not. Similar results were obtained with the administration of miconazole, which inhibits the biosynthesis of epoxyeicosatrienoic acids (EETs), endogenous agonists for TRPV4, from arachidonic acid (AA). Icv injection of hypertonic NaCl with AA or 5,6-EET restored water intake by Nax-KO mice to the WT level, but not that by TRPV4-KO mice. These results suggest that the Na+ signal generated in Nax-positive glial cells leads to the activation of TRPV4-positive neurons in sCVOs in order to stimulate water intake by using EETs as gliotransmitters.
The oxidative phosphorylation (OxPhos) pathway is responsible for most aerobic ATP production and is the only metabolic pathway with proteins encoded by both nuclear and mitochondrial genomes. In studies examining mito-nuclear interactions among distant populations within a species or across species, the interactions between these two genomes can affect metabolism, growth, and fitness, depending on the environment. However, there is little data on whether these interactions impact natural populations within a single species. In an admixed Fundulus heteroclitus population with northern and southern mitochondrial haplotypes, there are significant differences in allele frequencies associated with mitochondrial haplotype. In this study, we investigate how mitochondrial haplotype and any associated nuclear differences affect six OxPhos parameters within a population. The data demonstrate significant OxPhos functional differences between the two mitochondrial genotypes. These differences are most apparent when individuals are acclimated to high temperatures with the southern mitochondrial genotype having a large acute response and the northern mitochondrial genotype having little, if any acute response. Furthermore, acute temperature effects and the relative contribution of Complex I and II depend on acclimation temperature: when individuals are acclimated to 12°C, the relative contribution of Complex I increases with higher acute temperatures, whereas at 28°C acclimation the relative contribution of Complex I is unaffected by acute temperature change. These data demonstrate a complex gene by environmental interaction affecting the OxPhos pathway.
Repeated social defeat in the rat induces long-lasting cardiovascular changes associated with anxiety. In this study, we investigated the effects of repeated social defeat on breathing. Respiratory rate was extracted from the respiratory sinus arrhythmia (RSA) peak frequency of the ECG in rats subjected to social defeat for four consecutive days. Respiratory rate was recorded under anesthesia six days (D+10) or 26 days (D+30) after social defeat. At D+10, defeated (D) rats spent less time in the open arms of the elevated plus maze test, had heavier adrenal glands, and displayed bradypnea, unlike non-defeated (ND) animals. At D+30, all signs of anxiety had disappeared. However, half of the rats still displayed bradypnea (DL rats, for low respiratory rate indicated by a lower RSA frequency), while those with higher respiratory rate (DH rats) had recovered. Acute blockade of the dorsomedial hypothalamus (DMH) or nucleus tractus solitarii (NTS) 5-HT3 receptors reversed bradypnea in all D rats at D+10 and in DL rats at D+30. Respiratory rate was also recorded in conscious animals implanted with radiotelemetric ECG probes. DH rats recovered between D+10 and D+18, while DL rats remained bradypneic until D+30. In conclusion, social stress induces sustained chronic bradypnea mediated by DMH neurons and NTS 5-HT3 receptors. These changes are associated with an anxiety-like state that persists until D+10, followed by recovery. However, bradypnea may persist in half of the population up until D+30 despite apparent recovery of the anxiety-like state.
Pro-inflammatory cytokines play a critical role in the pathophysiology of muscle atrophy. We hypothesized that glycine exerted an anti-inflammatory effect and alleviated lipopolysaccharide (LPS)-induced muscle atrophy in piglets. Pigs were assigned to four treatments including: (1) non-challenged control; (2) LPS-challenged control; (3) LPS+1.0% glycine; (4) LPS+2.0% glycine. After receiving the control, 1.0% or 2.0% glycine supplemented diets, piglets were treated with either saline or LPS. At 4 h after treatment with saline or LPS, blood and muscle samples were harvested. We found that 1.0% or 2.0% glycine increased protein/DNA ratio, protein content and RNA/DNA ratio in gastrocnemius or longissimus dorsi (LD) muscles. Glycine also resulted in decreased mRNA expression of muscle atrophy F-box (MAFbx) and muscle RING finger 1 (MuRF1) in gastrocnemius muscle. In addition, glycine restored the phosphorylation of Akt, mammalian target of rapamycin (mTOR), eukaryotic initiation factor 4E binding protein 1 (4E-BP1) and Forkhead Box O 1 (FOXO1) in gastrocnemius or LD muscles. Furthermore, glycine resulted in decreased plasma tumor necrosis factor-α (TNF-α) concentration and muscle TNF-α mRNA abundance. Moreover, glycine resulted in decreased mRNA expresson of toll-like receptor 4 (TLR4), nucleotide-binding oligomerization domain protein 2 (NOD2) and their respective downstream molecules in gastrocnemius or LD muscles. These results indicate glycine enhances muscle protein mass under an inflammatory condition. The beneficial roles of glycine on the muscle are closely associated with maintaining Akt-mTOR-FOXO1 signaling, and suppressing the activation of TLR4 and/or NOD2 signaling pathways.
The analysis of heart rate variability (HRV) by nonlinear methods has been gaining increasing interest due to their ability to quantify the complexity of cardiovascular regulation. In this study, multiscale entropy (MSE) and refined MSE (RMSE) were applied to track the complexity of HRV as a function of time scale in three pathological conscious animal models: rat with heart failure (HF), spontaneously hypertensive rat (SHR) and rat with sinoaortic-denervation (SAD). Results showed that HF did not change HRV complexity, although there was a tendency to decrease the entropy in HF animals. On the other hand, SHR group was characterized by reduced complexity at long time scales, whereas SAD animals exhibited a smaller short- and long-term irregularity. We propose that short time scales (1 to 4), accounting for fast oscillations, are more related to vagal and respiratory control, whereas long time scales (5 to 20), accounting for slow oscillations, are more related to sympathetic control. The increased sympathetic modulation is probably the main reason for the lower entropy observed at high scales for both SHR and SAD groups, acting as a negative factor for the cardiovascular complexity. This study highlights the contribution of the multiscale complexity analysis of HRV for understanding the physiological mechanisms involved in cardiovascular regulation
In healthy young women, basal cerebral blood flow (CBF) and cerebrovascular reactivity may change across the menstrual cycle, but mechanisms remain untested. Compared to the early follicular phase of the menstrual cycle, we hypothesized women in late follicular phase would exhibit: 1) greater basal CBF, 2) greater hypercapnic increases in CBF, 3) greater hypoxic increases in CBF, and 4) increased cyclooxygenase (COX) signaling. We measured middle cerebral artery velocity (MCAv, transcranial Doppler ultrasound) in 11 healthy women (23±1 yrs) during rest, hypoxia, and hypercapnia. Subjects completed four visits; two during the early follicular (~day 3) and two during the late follicular (~day 14) phases of the menstrual cycle, with and without COX inhibition (oral indomethacin). Isocapnic hypoxia elicited an SPO2=90% and SPO2=80% for 5-minutes each. Separately, hypercapnia increased end-tidal carbon dioxide 10 mmHg above baseline. Cerebral vascular conductance index (CVCi=MCAv/MABP *100) was calculated and a positive change reflected vasodilation (CVCi). Basal CVCi was greater in the late follicular phase (P<0.001). Indomethacin decreased basal CVCi (~37%) and abolished the phase difference (P<0.001). Hypoxic CVCi was similar between phases and unaffected by indomethacin. Hypercapnic CVCi was similar between phases, and indomethacin decreased hypercapnic CVCi (~68%; P<0.001) similarly between phases. In summary, while neither hypercapnic nor hypoxic vasodilation is altered by menstrual phase, increased basal CBF in the late follicular phase is fully explained by a greater contribution of COX. These data provide new mechanistic insight into anterior CBF regulation across menstrual phases and contribute to our understanding of CBF regulation in women.
External lateral parabrachial nucleus (elPBN) within the pons and rostral ventrolateral medulla (rVLM) contribute to central processing of excitatory cardiovascular reflexes during stimulation of cardiac sympathetic afferent nerves (CSAN). However, the importance of elPBN cardiovascular neurons in regulation of the rVLM activity during CSAN activation remains unclear. We hypothesized that CSAN stimulation excites the elPBN cardiovascular neurons and, in turn, increases the rVLM activity through elPBN-rVLM projections. Compared to controls, rats subjected to microinjection of retrograde tracer into the rVLM, the numbers of elPBN neurons double-labeled with c-Fos (an immediate early gene) and the tracer were increased after CSAN stimulation (P<0.05). Majority of these elPBN neurons contain vesicular glutamate transporter 3. In felines, epicardial bradykinin (BK) and electrical stimulation of CSAN increased the activity of elPBN cardiovascular neurons, which was reversed (n=6, P<0.05) after blockade of glutamate receptors with iontophoresis of kynurenic acid (Kyn; 25 mM). In separate cats, microinjection of Kyn (1.25 nmol/50 nl) into the elPBN reduced the rVLM activity evoked by both BK and electrical stimulation (n=5, P<0.05). Furthermore, excitation of elPBN with microinjection of dl-homocysteic acid (DLH; 2 nmol/50 nl) significantly increased basal and CSAN-evoked rVLM activity. In contrast, the DLH-enhanced rVLM activity was reversed following iontophoresis of Kyn into the rVLM (n=7, P<0.05). These data suggest that cardiac sympathetic afferent stimulation activates cardiovascular neurons in the elPBN and rVLM sequentially through monosynaptic excitatory elPBN-rVLM pathways and glutamate mechanism important in elPBN processing of cardiac-cardiovascular sympathoexcitatory responses.
Brown adipose tissue (BAT) is a thermogenic organ that is vital for hibernation in mammals. Throughout the hibernation season BAT mitochondrial uncoupling protein 1 (UCP1) enables rapid rewarming from hypothermic torpor to periodic interbout arousals (IBAs) as energy is dissipated as heat. However, BAT's unique ability to rewarm the body via non-shivering thermogenesis is not necessary outside the hibernation season, suggesting a potential seasonal change in the regulation of BAT function. Here, we examined the BAT mitochondrial proteome and mitochondrial bioenergetics in the thirteen-lined ground squirrel (Ictidomys tridecemlineatus) across four time points: spring, fall, torpor and IBA. Relative mitochondrial content of BAT was estimated by measuring BAT pad mass, UCP1 protein content, and mitochondrial DNA (mtDNA) copy number. BAT mitochondrial DNA content was significantly lower in spring compared to torpor and IBA (P < 0.05). UCP1 mRNA and protein levels were highest during torpor and IBA. Respiration rates of isolated BAT mitochondria were interrogated at each complex of the electron transport chain. Respiration at complex II was significantly higher in torpor and IBA compared to spring (P < 0.05), suggesting an enhancement in mitochondrial respiratory capacity during hibernation. Additionally, proteomic iTRAQ® labeling identified 778 BAT mitochondrial proteins. Proteins required for mitochondrial lipid translocation and β-oxidation were up-regulated during torpor and IBA, and down-regulated in spring. These data imply that BAT bioenergetics and mitochondrial content are not static across the year, despite the year-round presence of UCP1.
A single bout of exhaustive exercise signals expression of vascular endothelial growth factor (VEGF) in muscle. Previous studies have reported that mice with life-long deletion of skeletal myofiber VEGF have fewer capillaries and a severe reduction in endurance exercise. However, in adult mice VEGF gene deletion conditionally targeted to skeletal myofibers limits exercise capacity without evidence of capillary regression. To explain this, we hypothesized that adult skeletal myofiber VEGF acutely regulates skeletal muscle perfusion during muscle contraction. A tamoxifen-inducible skeletal myofiber-specific VEGF gene deletion mouse (skmVEGF-/-) was used to reduce skeletal muscle VEGF protein by 90% in adult mice. Three weeks after inducing deletion of the skeletal myofiber VEGF gene, skmVEGF-/- mice exhibited diminished maximum running speed (-10%, P < 0.05) and endurance capacity (-47%; P < 0.05) that did not persist after eight weeks. In skmVEGF-/- mice gastrocnemius complex time to fatigue measured in situ was 71% lower than control mice. Contraction-induced perfusion measured by optical imaging during a period of electrically stimulated muscle contraction was 85% lower in skmVEGF-/- than control mice. No evidence of capillary rarefication was detected in the soleus, gastrocnemius and EDL up to 8 weeks after tamoxifen-induced VEGF ablation, and contractility and fatigue resistance of the soleus measured ex vivo were also unchanged. The force-frequency of the EDL showed a small right shift, but fatigue resistance did not differ between EDL from control and skmVEGF-/- mice. These data suggest myofiber VEGF is required for regulating perfusion during periods of contraction and may in this manner affect endurance capacity.
Prostanoids generated by the cyclooxygenase (COX) pathway appear to contribute to the neurogenic hypertension (HTN) in rats. The first goal of this study was to establish the time frame during which prostanoids participate in AngII-salt HTN. We induced HTN using AngII (150 ng/kg/min, sc) infusion for 14 days in rats on a high salt (2% NaCl) diet. When ketoprofen pretreatment was combined with treatment during the first 7 days of AngII infusion, development of HTN and increased neurogenic pressor activity (indexed by the depressor response to ganglion blockade) were significantly attenuated for the entire AngII infusion period. This suggest that prostanoid generation caused by administration of AngII and salt leads to an increase in neurogenic pressor activity and blood pressure (BP) via a mechanism that persists without the need for continuing prostanoid input. Second goal of this study was to determine if prostanoid products specifically in the brain contribute to HTN development. Expression of prostanoid pathway genes was measured in brain regions known to affect neurogenic BP regulation. AngII treated rats exhibited changes in gene expression of phospholipase A2 (upregulated in organum vasculosum of the lamina terminalis, paraventricular nucleus, nucleus of the solitary tract, and middle cerebral artery) and lipocalin type prostaglandin D synthase (upregulated in the organum vasculosum of the lamina terminalis). Based on our results we propose that activation of the brain prostanoid synthesis pathway both upstream and downstream from COX at early stages plays an important role in the development of the neurogenic component of AngII-salt HTN.
Hindbrain glucagon-like peptide 1 (GLP-1) neurons project to numerous forebrain areas, including the lateral septum (LS). Using a fluorescently labeled GLP-1R agonist, Exendin 4 (Ex4) we demonstrated GLP-1 receptor binding throughout the rat LS. We examined the feeding effects of Ex4 and the GLP-1R antagonist, Exendin (9-39) (Ex9), at doses subthreshold for effect when delivered to the lateral ventricle. Intra-LS Ex4 suppressed overnight chow and high-fat diet (HFD) intake, and Ex9 increased chow and HFD intake relative to vehicle. During 2-h tests, intra-LS Ex9 significantly increased 0.25M sucrose and 4% corn oil. Ex4 can cause nausea, but intra-LS administration of Ex4 did not induce pica. Furthermore, intra-LS Ex4 had no effect on anxiety-like behavior in the elevated plus maze. We investigated the role of LS GLP-1R in motivation for food by examining operant responding for sucrose on a progressive ratio (PR) schedule, with and without a nutrient preload to maximize GLP-1 neuron activation. The preload strongly suppressed PR responding, but blockade of GLP-1R in the intermediate subdivision of the LS did not affect motivation for sucrose under either load condition. The ability of the nutrient load to suppress subsequent chow intake was significantly attenuated by iLS Ex9 treatment. By contrast, blockade of GLP-1R in the dorsal subdivision of the LS increased both PR responding and overnight chow intake. Together, these studies suggest that endogenous activity of GLP-1R in the LS influence feeding, and dLS GLP-1R, in particular, play a role in motivation.
Hepatic glucose production (HGP) normally begins just prior to birth. Prolonged fetal hypoglycemia, intrauterine growth restriction, and acute hypoxemia produce an early activation of fetal HGP. To test the hypothesis that prolonged hypoxemia increases factors which regulate HGP, studies were performed in fetuses that were bled to anemic conditions (anemic, n=11) for 8.9 ± 0.4 days and compared to control fetuses (n=7). Fetal arterial hematocrit and oxygen content were 32% and 50% lower, respectively, in anemic vs. controls (p<0.005). Arterial plasma glucose was 15% higher in the anemic group (p<0.05). Hepatic mRNA expression of phosphonenolpyruvate carboxykinase (PCK1) was 2-fold higher in the anemic group (p<0.05). Arterial plasma glucagon concentrations were 70% higher in anemic fetuses compared to controls (p<0.05) and they were positively associated with hepatic PCK1 mRNA expression (p<0.05). Arterial plasma cortisol concentrations increased 90% in the anemic fetuses (p<0.05), but fetal cortisol concentrations were not correlated with hepatic PCK1 mRNA expression. Hepatic glycogen content was 30% lower in anemic vs. control fetuses (p<0.05) and was inversely correlated with fetal arterial plasma glucagon concentrations. In isolated primary fetal sheep hepatocytes, incubation in low oxygen (3%) increased PCK1 mRNA 3-fold compared to incubation in normal oxygen (21%). Together, these results demonstrate that glucagon and PCK1 may potentiate fetal HGP during chronic fetal anemic hypoxemia.
The mounting of appropriate emotional and neuroendocrine responses to environmental stressors critically depend on the hypothalamic pituitary adrenal (HPA) axis and associated limbic circuitry. Although its function is currently unknown, the highly evolutionarily conserved Transmembrane Protein 35 (TMEM35) is prominently expressed in HPA circuitry and limbic areas including the hippocampus and amygdala. To investigate the possible involvement of this protein in neuroendocrine function, we generated tmem35 knockout (KO) mice to characterize the endocrine, behavioral, electrophysiological, and proteomic alterations caused by deletion of the tmem35 gene. While capable of mounting a normal corticosterone response to restraint stress, KO mice showed elevated basal corticosterone accompanied by increased anxiety-like behavior. The KO mice also displayed impairment of hippocampus-dependent fear and spatial memories. Given the intact memory acquisition but a deficit in memory retention in the KO mice, TMEM35 is likely required for long-term memory consolidation. This conclusion is further supported by a loss of long-term potentiation in the Schaffer collateral-CA1 pathway in the KO mice. To identify putative molecular pathways underlying alterations in plasticity, proteomic analysis of synaptosomal proteins revealed lower levels of postsynaptic molecules important for synaptic plasticity in the KO hippocampus, including PSD95 and NMDA receptors. Pathway analysis (Ingenuity Pathway Analysis®) of differentially expressed synaptic proteins in tmem35 KO hippocampus implicated molecular networks associated with specific cellular and behavioral functions, including decreased LTP, and increased startle reactivity and locomotion. Collectively, these data suggest that TMEM35 is a novel factor required for normal activity of the HPA axis and limbic circuitry.
An increasing number of studies have linked high dietary phosphate (Pi) intake to hypertension. It is well established that the rise in sympathetic nerve activity (SNA) and blood pressure (BP) during physical exertion is exaggerated in many forms of hypertension, which are primarily mediated by an overactive skeletal muscle exercise pressor reflex (EPR). However, it remains unknown whether high dietary Pi intake potentiates the EPR-mediated SNA and BP response to exercise. Accordingly, we measured renal SNA (RSNA) and mean BP (MBP) in normotensive Sprague-Dawley rats fed a normal Pi diet (0.6%, n=13) or high Pi diet (1.2%, n=13) for 3 months. As previously reported, we found that resting BP was significantly increased by 1.2% Pi diet in both conscious and anesthetized animals. Activation of the EPR by electrically-induced hindlimb contraction triggered greater increases in RSNA and MBP in the 1.2% as compared to 0.6% Pi group (126 ±25 vs. 42±9 %; 44±5 vs. 14±2 mmHg, respectively, P<0.01). Activation of the muscle mechanoreflex, a component of the EPR, by passively stretching hindlimb muscle also evoked greater increases in RSNA and MBP in the 1.2% as compared to 0.6% Pi group (109±27vs. 24±7%, 38±7 vs. 8±2 mmHg, respectively, P<0.01). A similar response was produced by hindlimb intra-arterial capsaicin administration to stimulate the metaboreflex arm of the EPR. Thus, our data demonstrate a novel action of dietary Pi loading in augmenting EPR function through overactivation of both the muscle mechanoreflex and metaboreflex function.
The gastric hormone ghrelin positively affects energy balance by increasing food intake and reducing energy expenditure. Ghrelin mimetics are a possible treatment against cancer anorexia-cachexia syndrome (CACS). This study aimed to characterize the action of the non-peptidergic ghrelin receptor agonist HM01 on neuronal function, energy homeostasis and muscle mass in healthy rats and to evaluate its possible usefulness for the treatment of CACS in a rat tumor model. Using extracellular single-unit recordings, we tested if HM01 mimics the effects of ghrelin on neuronal activity in the arcuate nucleus (Arc). Furthermore, we assessed the effect of chronic HM01 treatment on food intake (FI), body weight (BW), lean and fat volumes and muscle mass in healthy rats. Using a hepatoma model, we investigated the possible beneficial effects of HM01 on tumor-induced anorexia, BW loss, muscle wasting and metabolic rate. HM01 (10-7-10-6M) mimicked the effect of ghrelin (10-8M) by increasing the firing rate in 76% of Arc neurons. HM01 delivered chronically for 12 days via osmotic minipumps (50 μg/h) increased FI in healthy rats by 24%, paralleled by increased BW, higher fat and lean volumes and higher muscle mass. Tumor-bearing rats treated with HM01 had 30% higher FI than tumor-bearing controls and were protected against BW loss. HM01 treatment resulted in higher muscle and fat mass. Moreover, tumor-bearing rats reduced their metabolic rate following HM01 treatment. Our studies substantiate the possible therapeutic usefulness of ghrelin receptor agonists like HM01 for the treatment of CACS and possibly other forms of disease-related anorexia and cachexia.
The aim was to investigate the effect of the long-acting β2-adrenergic agonist formoterol on muscle strength and power output, muscle metabolism and phosphorylation of CaMKII Thr287 and FXYD1 during maximal sprinting. In a double-blind crossover study, thirteen males (VO2max: 45.0±0.2 (mean±SE) mL min-1 kg-1) performed a 30-s cycle ergometer sprint after inhalation of either 54 µg formoterol (FOR) or placebo (PLA). Before and after the sprint, muscle biopsies were collected from vastus lateralis and maximal voluntary contraction (MVC) and contractile properties of quadriceps were measured. Oxygen uptake was measured during the sprint. During the sprint, peak, mean and end power were 4.6±0.8, 3.9±1.1 and 9.5±3.2% higher (P<0.05) in FOR than in PLA, respectively. Net rates of glycogenolysis and glycolysis were 45.7±21.0 and 28.5±13.4% higher (P<0.05) in FOR than in PLA, respectively, and the decrease in ATP content was lower (P<0.05) in FOR than in PLA (3.7±1.5 vs. 8.0±1.6 mmol kg dw-1). There was no difference in breakdown of phosphocreatine and oxygen uptake between treatments. Before and after the sprint, MVC and peak twitch force were higher (P<0.05) in FOR than in PLA. No differences were observed in phosphorylation of CaMKII Thr287 and FXYD1 between treatments before the sprint, whereas phosphorylation of CaMKII Thr287 and FXYD1 was greater (P<0.05) in FOR than in PLA after the sprint. In conclusion, formoterol-induced enhancement in power output during maximal sprinting is associated with increased rates of glycogenolysis and glycolysis that may counteract development of fatigue.
The role of vagal function in cardiovascular risk in older women remains unclear. Autonomic modulation following carbohydrate ingestion (CI), and postural stress (PS) was investigated in 14 healthy men and 21 age-matched post-menopausal women (age: 65.0±2.1 vs 64.1±1.6 years), with normal and comparable insulin sensitivity. Continuous non-invasive finger arterial pressure and ECG were recorded in the lying and the standing positions before and after ingestion of a carbohydrate-rich meal (600kcal, carbohydrate 78%, protein 13%, and fat 8%). Low frequency (LF, 0.04Hz - 0.15Hz) and high- frequency (HF, 0.15Hz - 0.4Hz) components (ms2) of heart rate variability (HRV), low frequency power (mmHg2) of systolic blood pressure variability (SBP LF power),and the sequence method for spontaneous baroreflex sensitivity (BRS, ms/mmHg), were used to quantify autonomic modulation. In response to CI and PS, MAP maintained stable and HR increased in women and men in the lying and standing positions. Following CI (60, 90, and 120 minutes post-prandially) in the standing position, SBP LF power increased by 40% in men (P=0.02), with unchanged HRV parameters; in contrast, in women, HRV HF power halved (P=0.02), with unaltered SBP LF power. During PS before and after CI, similar magnitude of SBP LF power, HRV and BRS changes was observed in men and women. In conclusion, CI induces sex-specific vascular sympathetic activation in healthy older men, and cardiac vagal inhibition in healthy older women; this CI mediated efferent vagal inhibition may suggest differential cardiovascular risk factors in women, irrespective of insulin resistance, and impairment of autonomic control.
Following binding to receptors in the arcuate nucleus (ArcN), insulin increases sympathetic nerve activity (SNA) and baroreflex control of SNA via a pathway that includes the paraventricular nucleus of the hypothalamus (PVN). Previous studies in males indicate that the sympathoexcitatory response is mediated by α-melanocyte stimulating hormone (α-MSH), which binds to PVN melanocortin type 3/4 receptors (MC3/4R). The present study was conducted in α-chloralose-anesthetized female rats to test the hypothesis that suppression of inhibitory Neuropeptide Y (NPY) inputs to the PVN is also involved. In support, blockade of PVN NPY Y1 receptors with BIBO 3304 (NPY1x), ArcN insulin nanoinjections, and PVN NPY1x followed by ArcN insulin each increased lumbar SNA (LSNA) and its baroreflex regulation similarly. Moreover, prior PVN injections of NPY blocked the sympathoexcitatory effects of ArcN insulin. Finally, PVN nanoinjections of the MC3/4R inhibitor SHU9119 prevented both the acute (15 min) and longer, more slowly developing (60 min), increases in LSNA in response to ArcN insulin. In conclusion, in females, ArcN insulin increases LSNA in part by suppressing tonic PVN NPY inhibition, which unmasks excitatory α-MSH drive of LSNA. Moreover, the steadily increasing rise in LSNA induced by ArcN insulin is also dependent on PVN MC3/4R.
The pressure-volume relationship in the ascending aorta ("windkessel") of the hooded seal was determined and the morphology of its vasa vasorum described in some detail. We found that the ascending aorta has a high compliance and can easily accommodate the entire stroke volume when the peripheral vascular resistance becomes much increased and maintain perfusion pressure during the much extended diastole and thereby reduce cardiac stroke work during diving. We also found that the 3-5 mm thick wall of the ascending aorta had a very elaborate vasa vasorum interna with a hitherto undescribed vascular structure that penetrates the entire vascular wall. If similar structures with similar importance for the nutrition of the wall of the vessel are found in man, important implications for the understanding of pathological conditions, such as aneurisms, may be indicated.
Estradiol (E2) decreases both water and saline intakes by female rats. The ERα and ERβ subtypes are expressed in areas of the brain that control fluid intake; however, the role these receptors play in E2's anti-dipsogenic and anti-natriorexigenic effects have not been examined. Accordingly, we tested the hypothesis that activation of ERα and ERβ decrease water and saline intakes by female rats. We found a divergence in E2's inhibitory effect on intake: activation of ERα decreased water intake, whereas activation of ERβ decreased saline intake. E2 decreases expression of the angiotensin II type 1 receptor (AT1R), a receptor with known relevance to water and salt intakes, in multiple areas of the brain where ERα and ERβ are differentially expressed. We, therefore, tested for agonist-induced changes in AT1R mRNA expression by RT-PCR and protein expression by analyzing receptor binding to test the hypothesis that the divergent effects of these ER subtypes are mediated by region specific changes in AT1R expression. Although we found no changes in AT1R mRNA or binding in areas of the brain known to control fluid intake associated with agonist treatment, the experimental results replicate and extend previous findings that body weight changes mediate alterations in AT1R expression in distinct brain regions. Together, the results reveal selective effects of ER subtypes on ingestive behaviors, advancing our understanding of E2's inhibitory role in the controls of fluid intake by female rats.
Obesity and type 2 diabetes are major worldwide public health issues where there appears to be a relationship between total fat intake and obesity. In addition, the mechanisms of long-term and excessive high-fat diet (HFD) intake in the development of obesity still need to be elucidated. The ventromedial hypothalamus (VMH) is a major site involved in the regulation of glucose and energy homeostasis where "metabolic sensing neurons" integrate metabolic signals from the periphery. Among these signals, fatty acids (FA) modulate the activity of VMH neurons utilizing the FA translocator/CD36 which plays a critical role in the regulation of energy and glucose homeostasis. During low fat diet (LFD) intake, FA are oxidized by VMH astrocytes to fuel their on-going metabolic needs. However, HFD intake causes VMH astrocytes to utilize FA to generate ketones bodies. We postulate that these astrocyte-derived ketone bodies are exported to neurons where they produce excess ATP and ROS which override CD36-mediated FA sensing and act as a signal to decrease short-term food intake. On HFD, VMH astrocyte-produced ketones reduce elevated caloric intake to LFD levels after 3 d in rats genetically-predisposed to resist (DR) diet-induced obesity (DIO), but not leptin resistant DIO rats. This suggests that, while VMH ketone production on HFD can contribute to protection from obesity, the inherent leptin resistance overrides this inhibitory action of ketone bodies on food intake. Thus, astrocytes and neurons form a tight metabolic unit that is able to monitor circulating nutrients in order to alter food intake and energy homeostasis.
Most vertebrates, including cartilaginous fishes, maintain their plasma SO42- concentration ([SO42-]) within a narrow range of 0.2-1 mM. As seawater has a [SO42-] about 40 times higher than that of the plasma, SO42- excretion is major role of the kidneys in marine teleost fishes. It has been considered that cartilaginous fishes also excrete excess SO42- via the kidney. However, little is known about the underlying mechanisms for SO42- transport in cartilaginous fish, largely due to the extraordinarily elaborate four-loop configuration of the nephron, which consists of at least 10 morphologically distinguishable segments. In the present study, we determined cDNA sequences from the kidney of holocephalan elephant fish (Callorhinchus milii) that encoded solute carrier family 26 member 1 (Slc26a1) and member 6 (Slc26a6), which are SO42- transporters that are expressed in mammalian and teleost kidneys. Elephant fish Slc26a1 (cmSlc26a1) and cmSlc26a6 mRNAs were co-expressed in the proximal II (PII) segment of the nephron, which comprises the second loop in the sinus zone. Functional analyses using Xenopus oocytes and the results of immunohistochemistry revealed that cmSlc26a1 is a basolaterally-located electroneutral SO42- transporter, while cmSlc26a6 is an apically-located, electrogenic Cl-/SO42- exchanger. In addition, we found that both cmSlc26a1 and cmSlc26a6 were abundantly expressed in the kidney of embryos; SO42- was concentrated in a bladder-like structure of elephant fish embryos. Our results demonstrated that the PII segment of the nephron contributes to the secretion of excess SO42- by the kidney of elephant fish. Possible mechanisms for SO42- secretion in the PII segment are discussed.
Cardiovagal baroreflex sensitivity (cvBRS) measures the efficiency of the cardiovagal baroreflex to modulate heart rate in response to rises or falls in systolic blood pressure (SBP). Given that baroreceptors are located in the walls of the carotid sinuses (CS) and aortic arch (AA), the arterial mechanics of these sites are important contributors to cvBRS. However, the relative contribution of CS and AA mechanics to cvBRS remains unclear. This study employed sex differences as a model to test the hypothesis that differences in cvBRS between groups would be explained by the vascular mechanics of the AA but not the CS. Thirty-six young, normotensive individuals (18 female; 24 ±2 years) were recruited. cvBRS was measured using transfer function analysis of the low-frequency region (0.04-0.15 Hz). Ultrasonography was performed at the CS and AA to obtain arterial diameters for the measurement of distensibility. Local pulse pressure (PP) was taken at the CS using a hand held tonometer while AA PP was estimated using a transfer function of brachial PP. Both cvBRS (25 ±11 vs. 19 ±7 ms/mmHg, p = 0.04) and AA distensibility (16.5 ±6.0 vs. 10.5 ±3.8 mmHg-1 x10-3, p = 0.02) were greater in females than males. Sex differences in cvBRS were eliminated after controlling for AA distensibility (p = 0.19). There were no sex differences in CS distensibility (5.32 ±2.3 vs. 4.63 ±1.3 mmHg-1 x10-3, p = 0.32). The present data demonstrate that AA mechanics are an important contributor to differences in cvBRS.
Preeclampsia (PE) is a pregnancy associated disorder that affects 5-8% of pregnancies and is a major cause of maternal, fetal, and neonatal morbidity and mortality. Hallmark characteristics of preeclampsia are new onset hypertension after 20 weeks gestation with or without proteinuria, chronic immune activation, fetal growth restriction, and maternal endothelial dysfunction. However, the pathophysiologic mechanisms that lead to the development of preeclampsia are poorly understood. Recent data from studies of both clinical and animal models demonstrate an imbalance in the subpopulations of CD4+ T cells and a role for these cells as mediators of inflammation and hypertension during pregnancy. Specifically, it has been proposed that the imbalance between two CD4+ T cell subtypes, regulatory T cells (TRegs) and T-helper 17 cells (Th17s), is involved in the pathophysiology of preeclampsia. Studies from our laboratory highlighting how this imbalance contributes to vasoactive factors, endothelial dysfunction and hypertension during pregnancy will be discussed in this review (Fig 1). Therefore, the purpose of this review is to highlight hypertensive mechanisms stimulated by inflammatory factors in response to placental ischemia, thereby elucidating a role for inflammation in the pathophysiology of PE.
Adipose tissue PKA, has roles in adipogenesis, lipolysis and mitochondrial function. PKA transduces the cAMP signal downstream of G protein-coupled receptors, which are being explored for therapeutic manipulation to reduce obesity and improve metabolic health. This study aimed to determine the overall physiological consequences of PKA activation in adipose tissue. Mice expressing an activated PKA catalytic subunit in adipose tissue (Adipoq-caPKA mice) showed increased PKA activity in subcutaneous, epididymal and mesenteric white adipose tissue (WAT) depots and brown adipose tissue (BAT) compared to controls. Adipoq-caPKA mice weaned onto a high fat diet (HFD) or switched to the HFD at 26 weeks of age were protected from diet-induced weight gain. Metabolic health was improved, with enhanced insulin sensitivity, glucose tolerance, and β-cell function. Adipose tissue health was improved, with smaller adipocyte size and reduced macrophage engulfment of adipocytes. Using metabolic cages, Adipoq-caPKA mice were shown to have increased energy expenditure, but no difference to littermate controls in physical activity or food consumption. Immunoblotting of adipose tissue showed increased expression of uncoupling protein-1 (UCP1) in BAT and dramatic UCP1 induction in subcutaneous WAT, but no induction in the visceral depots. Feeding a HFD increased PKA activity in epididymal WAT of wildtype mice compared to chow, but did not change PKA activity in subcutaneous WAT or BAT. This was associated with changes in PKA regulatory subunit expression. This study shows that adipose tissue PKA activity is sufficient to increase energy expenditure and indicates that PKA is a beneficial target in metabolic health.
Neuronal circuits in the hypothalamus and hindbrain are of importance for control of food intake, energy expenditure and fat mass. We have recently shown that treatment with exendin-4 (Ex-4), an analogue of the pro-glucagon derived molecule glucagon-like peptide 1 (GLP-1), markedly increases mRNA-expression of the cytokine interleukin-6 (IL-6) in hypothalamus and hindbrain, and that this increase partly mediates the suppression of food intake and body weight by Ex-4. Endogenous GLP-1 in the central nervous system (CNS) is produced by preproglucagon (PPG) neurons of the nucleus of the solitary tract (NTS) in the hindbrain. These neurons project to various parts of the brain, including the hypothalamus. Outside the brain, IL-6 stimulates GLP-1 secretion from the gut and pancreas. In this study, we aim to investigate whether IL-6 can affect GLP-1 producing PPG neurons in the NTS in mouse hindbrain via the ligand binding part of the IL-6 receptor, IL-6 Receptor-α (IL-6Rα). Using immunohistochemistry, we found that IL-6Rα was localized on PPG neurons of the NTS. Recordings of these neurons in GCaMP3/GLP-1 reporter mice showed that IL-6 enhances cytosolic Ca2+ concentration in neurons capable of expressing PPG. We also show that the Ca2+ increase originates from the extracellular space. Furthermore, we found that IL-6Rα was localized on cells in the caudal hindbrain expressing immunoreactive NeuN (a neuronal marker) or CNPase (an oligodendrocyte marker). In summary, IL-6Rα is present on PPG neurons in the NTS, and IL-6 can stimulate these cells by increasing influx of Ca2+ to the cytosol from the extracellular space.
We have recently demonstrated that specific overexpression of DEP-domain containing mTOR-interacting protein (DEPTOR) in the mediobasal hypothalamus (MBH) protects mice against high-fat diet-induced obesity, revealing DEPTOR as a significant contributor to energy balance regulation. Based on evidence that DEPTOR is expressed in the pro-opiomelanocortin (POMC) neurons of the MBH, the present study aimed at investigating whether these neurons mediate the metabolic effects of DEPTOR. Here, we report that specific DEPTOR overexpression in POMC neurons does not recapitulate any of the phenotypes observed when the protein was overexpressed in the MBH. Unlike the previous model, mice overexpressing DEPTOR only in POMC neurons i) did not show differences in feeding behavior, ii) did not exhibit changes in locomotion activity and oxygen consumption, iii) did not show an improvement in systemic glucose metabolism and iv) were not resistant to high-fat diet-induced obesity. These results support the idea that other neuronal populations are responsible for these phenotypes. We nonetheless observed a mild elevation in fasting blood glucose, insulin resistance, and alterations in liver glucose and lipid homeostasis in mice overexpressing DEPTOR in POMC neurons. Taken together, these results show that DEPTOR overexpression in POMC neurons does not affect energy balance regulation but could modulate metabolism through a brain-liver connection.
Recent reports suggest that aerobic exercise may boost the hypertrophic response to short-term resistance training. This study explored the effects of an acute aerobic exercise bout on the transcriptional response to subsequent resistance exercise. Ten moderately trained men performed ~45 min cycling on one leg followed by 4x7 maximal knee extensions for each leg, 15 min later. Thus, one limb performed aerobic and resistance exercise (AE+RE), while the opposing leg did resistance exercise only (RE). Biopsies were obtained from m. vastus lateralis of each leg 3-h after the resistance exercise bout. Using DNA microarray, we analyzed differences (≥1.5-fold, FDR ≤10%) in gene expression profiles for the two modes of exercise. There were 176 genes up- (127) or down-regulated (49) by AE+RE compared with RE. Among the most significant differentially expressed genes were established markers for muscle growth and oxidative capacity, novel cytokines, transcription factors and microRNAs. The most enriched functional categories were those linked to carbohydrate metabolism and transcriptional regulation. Upstream analysis revealed that VEGF, CREB, TET2 and mTOR were regulators highly activated by AE+RE, whereas JnK, Nfβ, MAPK and several miRNA's were inhibited. Thus, aerobic exercise alters the skeletal muscle transcriptional signature of resistance exercise to initiate important gene programs promoting both myofiber growth and improved oxidative capacity. These results provide novel insight into human muscle adaptations to diverse exercise modes and offer the very first genomic basis explaining how aerobic exercise may augment, rather than compromise muscle growth induced by resistance exercise.
The health benefits of garlic and other organosulfur-containing foods are well recognized and have been attributed to both pro-oxidant and antioxidant activities. The effects of garlic are surprisingly similar to those of hydrogen sulfide (H2S) which is also known to be released from garlic under certain conditions. However, recent evidence suggests that polysulfides, not H2S, may be the actual mediator of physiological signaling. In this study we monitored formation of H2S and polysulfides from garlic oil in buffer and in HEK293 cells with fluorescent dyes, AzMC and SSP4, respectively and redox activity with two redox indicators roGFP and DCF. Our results show that H2S release from garlic oil in buffer requires other low molecular weight thiols such as cysteine (Cys) or glutathione (GSH), whereas polysulfides are readily detected in garlic oil alone. Administration of garlic oil to cells rapidly increases intracellular polysulfide but has minimal effects on H2S unless Cys or GSH are also present in the extracellular medium. We also observed that garlic oil and diallyltrisulfide (DATS) potently oxidized roGFP in buffer but did not affect DCF. This appears to be a direct polysulfide-mediated oxidation that does not require a reactive oxygen species intermediate. Conversely, when applied to cells, garlic oil became a significant intracellular reductant independent of extracellular Cys or GSH. This suggests that intracellular metabolism and further processing of the sulfur moieties are necessary to confer anti-oxidant properties to garlic oil in vivo.
High fat diets rapidly cause weight gain and glucose intolerance. We sought to determine whether these changes could be mitigated with prior exercise training. Male C57BL/6J mice were exercise trained by treadmill running (1 hr/day, 5 days/week) for four weeks. Twenty-four hours after the final bout of exercise, mice were provided with a high fat diet (60% kcal from lard) for four days, with no further exercise. In mice fed the high fat diet prior exercise training resulted in blunted weight gain, reduced fat mass and a slight attenuation in glucose intolerance that was mirrored by greater insulin-induced Akt phosphorylation in skeletal muscle compared to sedentary mice fed the high fat diet. When ad libitum fed sedentary mice were compared to sedentary high fat fed mice that were calorie restricted (-30%) to match the weight gain of the previously trained high fat fed mice, the same attenuated impairments in glucose tolerance were found. Blunted weight gain was associated with a greater capacity to increase energy expenditure in trained compared to sedentary mice when challenged with a high fat diet. Although mitochondrial enzymes in white adipose tissue and UCP-1 protein content in brown adipose tissue were increased in previously exercised compared to sedentary mice fed a HFD, ex vivo mitochondrial respiration was not increased in either tissue. Our data suggest that prior exercise training attenuates high fat diet-induced weight gain and glucose intolerance and is associated with a greater ability to increase energy expenditure in response to a high fat diet.
The affect of hyperthermia on cognitive function remains equivocal, perhaps because of methodological discrepancy. Using electroencephalographic event-related potentials (ERPs), we tested the hypothesis that a passive heat stress impairs cognitive processing. Thirteen volunteers performed repeated auditory oddball paradigms under two thermal conditions, normothermic Time control and Heat stress, on different days. For the Heat stress trial, these paradigms were performed at pre-heat stress (i.e., normothermic) baseline, when esophageal temperature (Tes) had increased by ~0.8°C, when Tes had increased by ~2.0°C, and during cooling following the heat stress. The reaction time and ERPs were recorded in each session. For the Time control trial, subjects performed the auditory oddball paradigms at approximately the same time interval as they did in the Heat stress trial. The peak latency and amplitude of an indicator of auditory processing (N100) were not altered regardless of thermal conditions. An indicator of stimulus classification/evaluation time (latency of P300) and the reaction time were shortened during heat stress, moreover an indicator of cognitive processing (the amplitude of P300) was significantly reduced during severe heat stress (8.3±1.3μV) relative to the baseline (12.2±1.0μV, p<0.01). No changes in these indices occurred during the Time control trial. During subsequent whole body cooling, the amplitude of P300 remained reduced, and the reaction time and latency of P300 remained shortened. These results suggest that excessive elevations in internal temperature reduce cognitive processing but promote classification time.
During embryonic development, environmental perturbations can affect organisms' developing phenotype, a process known as developmental plasticity. Resulting phenotypic changes can occur during discrete, critical windows of development. Critical windows are periods when developing embryos are most susceptible to these perturbations. We have previously documented that hypoxia reduces embryo size and increases relative heart mass in American alligator, and this study identified critical windows when hypoxia altered morphological, cardiovascular function and cardiac gene expression of alligator embryos. We hypothesized that incubation in hypoxia (10% O2) would increase relative cardiac size due to cardiac enlargement rather than suppression of somatic growth. We exposed alligator embryos to hypoxia during discrete incubation periods to target windows where the embryonic phenotype is altered. Hypoxia affected heart growth between 20-40% of embryonic incubation, whereas somatic growth was affected between 70-90% of incubation. Arterial pressure was depressed by hypoxic exposure during 50-70% of incubation, whereas heart rate was depressed in embryos exposed to hypoxia during a period spanning 70-90% of incubation. Expression of Vegf and PdgfB was increased in certain hypoxia-exposed embryo treatment groups, and hypoxia towards the end of incubation altered β-adrenergic tone for arterial pressure and heart rate. It is well known that hypoxia exposure can alter embryonic development, and in the present study we have identified brief, discrete windows that alter the morphology, cardiovascular physiology, and gene expression in embryonic American alligator.
We compared the effects of concurrent exercise, incorporating either high-intensity interval training (HIT) or moderate-intensity continuous training (MICT), on mTORC1 signalling and microRNA expression in skeletal muscle, relative to resistance exercise (RE) alone. Eight males (mean ± SD: age, 27 ± 4 y; "V" O2peak, 45.7 ± 9 ml•kg-1•min-1) performed three experimental trials in a randomised order: i) RE (8 x 5 leg press repetitions at 80% 1RM) performed alone, and RE preceded by either ii) HIT cycling (10 x 2 min at 120% lactate threshold [LT]; HIT+RE) or iii) work-matched MICT cycling (30 min at 80% LT; MICT+RE). Vastus lateralis muscle biopsies were obtained immediately before RE, either without (REST) or with (POST) preceding endurance exercise, +1 h (RE+1 h) and +3 h (RE+3 h) after RE. Prior HIT and MICT similarly reduced muscle glycogen content and increased ACCSer79 and p70S6KThr389 phosphorylation before subsequent RE (i.e., at POST). Compared with MICT, HIT induced greater mTORSer2448 and rps6Ser235/236 phosphorylation at POST. RE-induced increases in p70S6K and rps6 phosphorylation were not influenced by prior HIT or MICT; however, mTOR phosphorylation was reduced at RE+1 h for MICT+RE vs. both HIT+RE and RE. Expression of miR-133a, miR-378 and miR-486 was reduced at RE+1 h for HIT+RE vs. both MICT+RE and RE. Post-exercise mTORC1 signalling following RE is therefore not compromised by prior HIT or MICT, and concurrent exercise incorporating HIT, but not MICT, reduces post-exercise expression of miRNAs implicated in skeletal muscle adaptation to RE.
Cardiac natriuretic peptides (NP) are involved in cardio-renal regulation and in lipolysis. The NP activity is largely dependent on the ratio between the signaling receptor NPRA and the clearance receptor NPRC. Lipolysis increases when NPRC is reduced by starving or very-low calorie diet. On the contrary, insulin is an anti-lipolytic hormone that increases sodium-retention, suggesting a possible functional link with NP. We examined the insulin-mediated regulation of NP receptors in differentiated human adipocytes and tested the association of NP receptors expression in visceral adipose tissue (VAT) with metabolic profiles of patients undergoing renal surgery. Differentiated human adipocytes from VAT and Simpson-Golabi-Behmel Syndrome (SGBS) adipocyte cell line were treated with insulin in the presence of high glucose or low-glucose media to study NP receptors and insulin/glucose regulated pathways. Fasting blood samples and VAT samples were taken from patients in the day of renal surgery. We observed a potent insulin-mediated and glucose-dependent up-regulation of NPRC, through the PI3K pathway, associated with lower lipolysis in differentiated adipocytes. No effect was observed on NPRA. Low-glucose medium, utilized to simulate in vivo starving conditions, hampered the insulin effect on NPRC through modulation of insulin/glucose regulated pathways, allowing ANP to induced lipolysis and thermogenic genes. An expression ratio in favor of NPRC in adipose tissue was associated with higher fasting insulinemia, HOMA-IR and atherogenic lipid levels. Insulin /glucose dependent NPRC induction in adipocytes might be a key factor linking hyperinsulinemia, metabolic syndrome and higher blood pressure by reducing NP effects on adipocytes.
Low temperature directly alters turtle cardiovascular physiology, causing bradycardia, arterial hypotension and a reduction in systemic blood pressure. Additionally, blood viscosity, systemic resistance and sensitivity to cardiac preload increase. However, the long-term effects of these seasonal responses are unclear. We acclimated red-eared sliders to a control temperature (25 °C) or chronic cold (5 °C). To differentiate the direct effects of temperature from a cold-induced remodeling response, all measurements were conducted at the control temperature. In anesthetized turtles, cold acclimation reduced systemic resistance by 1.8-fold and increased systemic blood flow by 1.4-fold, resulting in a 2.3-fold higher right to left (R-L) cardiac shunt flow. Following a volume load by bolus injection of saline (calculated to increase stroke volume by 5-fold, ~2.2 % of total blood volume), systemic resistance was reduced while pulmonary blood flow and systemic pressure increased. An increased systemic blood flow meant the R-L cardiac shunt was further pronounced. In the isolated ventricle, passive stiffness increased following cold acclimation with 4.2-fold greater collagen deposition in the myocardium. Histological sections of the major outflow arteries revealed a 1.4-fold higher elastin content in cold-acclimated animals. These results suggest that cold acclimation alters cardiac shunting patterns with an increased R-L shunt flow, achieved through reducing systemic resistance and increasing systemic blood flow. Furthermore, our data suggests the that cold-induced cardiac remodeling may reduce the stress of high cardiac preload by increasing compliance of the vasculature and decreasing ventricular compliance. Together, these responses could compensate for reduced systolic function at low temperatures.
Leptin signals energy sufficiency to the reproductive hypothalamic-pituitary-gonadal (HPG) axis. Studies using genetic models have demonstrated that hypothalamic neurons are major players mediating these effects. Leptin receptor (LepR) is also expressed in the pituitary gland and in the gonads, but the physiological effects of leptin in these sites are still unclear. Female mice with selective deletion of LepR in a subset of gonadotropes show normal pubertal development but impaired fertility. Conditional deletion approaches however often result in redundancy or developmental adaptations which may compromise the assessment of leptin's action in gonadotropes for pubertal maturation. To circumvent these issues, we adopted a complementary genetic approach and assessed if selective re-expression of LepR only in gonadotropes is sufficient to enable puberty and improve fertility of LepR null female mice. We initially assessed the co-localization of GnRHR and LepR in the HPG axis using GnRH Receptor-IRES-Cre (GRIC) and LepR-Cre reporter (tdTomato or eGFP) mice. We found that GRIC and leptin-induced pSTAT3 are expressed in distinct hypothalamic neurons. Whereas LepR-Cre was observed in theca cells, GRIC expression was rarely found in the ovarian parenchyma. In contrast, a subpopulation of gonadotropes expressed the LepR-Cre reporter gene (tdTomato). We then crossed the GRIC mice with the LepR null reactivable (LepRloxTB) mice. These mice showed an increase in FSH levels, but they remained in a prepubertal state. Together with previous findings, our data indicate that leptin-selective action in gonadotropes serves a role in adult reproductive physiology but is not sufficient to allow pubertal maturation in mice.
Life cycle delays are beneficial for opportunistic species encountering suboptimal environments. Many animals display a programmed arrest of development (diapause) at some stage(s) of their development, and the diapause state may or may not be associated with some degree of metabolic depression. In this review, we will evaluate current advancements in our understanding of the mechanisms responsible for the remarkable phenotype, as well as environmental cues that signal entry and termination of the state. The developmental stage at which diapause occurs dictates and constrains the mechanisms governing diapause. Considerable progress has been made in clarifying proximal mechanisms of metabolic arrest and the signaling pathways like insulin/Foxo that control gene expression patterns. Overlapping themes are also seen in mechanisms that control cell cycle arrest. Evidence is emerging for epigenetic contributions to diapause regulation via small RNAs in nematodes, crustaceans, insects, and fish. Knockdown of circadian clock genes in selected insect species supports the importance of clock genes in the photoperiodic response that cues diapause. A large suite of chaperone-like proteins, expressed during diapause, protects biological structures during long periods of energy-limited stasis. More information is needed to paint a complete picture of how environmental cues are coupled to the signal transduction that initiates the complex diapause phenotype, as well as molecular explanations for how the state is terminated. Excellent examples of molecular memory in post-dauer animals have been documented in Caenorhabditis elegans. It is clear that a single suite of mechanisms does not regulate diapause across all species and developmental stages.
Recurrent dehydration, such as commonly occurs with manual labor in tropical environments, has been recently shown to result in chronic kidney injury, likely through the effects of hyperosmolarity to activate both vasopressin and aldose reductase-fructokinase pathways. The observation that the latter pathway can be directly engaged by simple sugars (glucose and fructose) leads to the hypothesis that soft drinks (which contain these sugars) might worsen rather than benefit dehydration associated kidney disease. Recurrent dehydration was induced in rats by exposure to heat (36°C) for one hour/ 24 h followed by access for 2 hours to plain water (W), a 11% fructose-glucose solution (FG, same composition as typical soft drinks), or water sweetened with non-caloric stevia (ST). After 4 weeks plasma and urine samples were collected, and kidneys examined for oxidative stress, inflammation and injury. Recurrent heat-induced dehydration with ad libitum water repletion resulted in plasma and urinary hyperosmolarity with stimulation of the vasopressin (copeptin) levels and resulted in mild tubular injury and renal oxidative stress. Rehydration with 11% FG solution, despite larger total fluid intake, resulted in greater dehydration (higher osmolarity and copeptin levels), and worse renal injury, with activation of aldose reductase and fructokinase, whereas rehydration with stevia water had opposite effects. Similar findings were also shown in a second model of dehydration. In animals that are dehydrated, rehydration acutely with soft drinks worsens dehydration and exacerbates dehydration associated renal damage. These studies emphasize the danger of drinking soft drink-like beverages as an attempt to rehydrate following dehydration.
We investigated whether corticotropin-releasing factor receptor 2 (CRF2) and its high affinity agonist urocortin 1 (Ucn1) mediate sex-specific signaling and immune responses. Intrarectal trinitrobenzene sulfonic acid (TNBS) was used to induce experimental colitis in wildtype (WT), CRF2 knockout (CRF2KO), and heterozygous (CRF2Ht) mice of both sexes. Changes in plasma extravasation, organ weight, survival, immune cell numbers, inflammatory cytokines, and the MAPK signaling pathway were assessed. Stored intestinal biopsies from patients with Crohn's disease (CD) and age- and sex-matched individuals without inflammatory bowel disease (IBD) were examined by immunofluorescence and confocal microscopy to characterize Ucn1 and CRF receptor expression. CRF2Ht mice of both sexes showed decreased survival during colitis compared to other genotypes. Ucn1 improved survival in male mice alone. Ucn1 restored colon length, spleen and adrenal weight, and decreased colonic TNF-α, IL-6, and IL-1β levels in male CRF2Ht mice alone. CRF2Ht mice of both sexes showed decreased phosphorylation of MAPK p38 and heat shock protein 27 (Hsp27) levels. Ucn1 restored p-Hsp27 levels in male CRF2Ht mice alone. Expression of the chaperone protein Hsp90 decreased during colitis, except in male CRF2Ht mice. Taken together, our data indicate that sex shows significant interaction with genotype and Ucn1 during colitis. Human duodenal and colonic biopsies revealed that sex-specific differences exist in levels of CRF receptors and Ucn1 expression in patients with CD compared with the matched non-IBD subjects. To conclude, Ucn1 mediates sex-specific immune and cellular signaling responses via CRF2, emphasizing the need for inclusion of females in preclinical studies.
We examined whether the sustained activation of metaboreceptor in forearm during cycling exercise can modulate sweating and cutaneous vasodilation. On separate days, twelve young participants performed a 1.5-min isometric handgrip exercise at 40% maximal voluntary contraction followed by 1) 9-min forearm ischemia (Occlusion, to activate metaboreceptor) or 2) no ischemia (Control) in thermoneutral conditions (27°C, 50%) with mean skin temperature clamped at 34°C. Thirty seconds after the handgrip exercise, participants cycled for 13.5 min at 40% VO2max. For Occlusion, forearm ischemia was maintained for 9 min followed by no ischemia thereafter. Local sweat rate (SR, ventilated capsule) and cutaneous vascular conductance (CVC, laser-Doppler perfusion units/mean arterial pressure) on the contralateral non-ischemic arm as well as esophageal and skin temperatures were measured continuously. The period of ischemia in the early stages of exercise increased SR (+0.03 mg/cm2/min, P<0.05) but not CVC (P>0.05) above Control levels. No differences were measured in the esophageal temperature at which onset of sweating (Control: 37.19±0.09 vs. Occlusion: 37.07±0.09°C) or CVC (Control: 37.21±0.08 vs. Occlusion: 37.08±0.10°C) as well as slopes for these responses (all P>0.05). However, a greater elevation in SR occurred thereafter such that SR was significantly elevated at the end of the ischemic period relative to Control (0.37±0.05 vs. 0.23±0.05 mg/cm2/min respectively, P<0.05) despite no differences in esophageal temperature. We conclude that the activation of forearm muscle metaboreceptor can modulate sweating, but not CVC, during cycling exercise without affecting the core temperature-SR relationship.
Teleosts living in seawater continually absorb water across the intestine to compensate for branchial water loss to the environment. The present study reveals that the Gulf toadfish (Opsanus beta) rectum plays a comparable role to the posterior intestine in ion and water absorption. However, the posterior intestine appears to rely more on SLC26a6 (a HCO3-/Cl- antiporter) and the rectum on NKCC2 (SLC12a1) for the purposes of solute-coupled water absorption. The present study also demonstrates that the rectum responds to renoguanylin (RGN), a member of the guanylin family of peptides that alters the normal osmoregulatory processes of the distal intestine, by inhibited water absorption. RGN decreases rectal water absorption more greatly than in the posterior intestine, and leads to net Na+ and Cl- secretion, and a reversal of the absorptive short-circuit current (ISC). It is hypothesized that maintaining a larger fluid volume within the distal segments of intestinal tract facilitates the removal of CaCO3 precipitates and other solids from the intestine. Indeed, the expression of the components of the Cl--secretory response, apical CFTR and basolateral NKCC1 (SLC12a2), are upregulated in the rectum of the Gulf toadfish after 96 h in 60 ppt, an exposure that increases CaCO3 precipitate formation relative to 35 ppt. Moreover, the downstream intracellular effects of RGN appear to directly inhibit ion absorption by NKCC2 and anion exchange by SLC26a6. Overall, the present findings elucidate key electrophysiological differences between the posterior intestine and rectum of Gulf toadfish and the potent regulatory role renoguanylin plays in osmoregulation.
Heat stress causes morbidity and mortality in humans and animals and threatens food security by limiting livestock productivity. Inflammatory signaling may contribute to heat stress-mediated skeletal muscle dysfunction. Previously we discovered increased circulating endotoxin and intramuscular oxidative stress and TNFα protein abundance but not inflammatory signaling following 24 and 72 hours of heat stress. Thus, the purpose of this investigation was to clarify the role of inflammatory signaling in heat stressed skeletal muscle. Crossbred gilts (n=8/group) were assigned to either thermal neutral (24° C), heat stress (37° C), or pair-fed thermal neutral (24° C) conditions for 12 hours. Following treatment, animals were euthanized and the semitendinosus red (STR) and white (STW) were recovered. Heat stress did not alter inflammatory signaling in STW. In STR, relative heat shock protein abundance was similar between groups as was nuclear content of HSF1. In whole homogenate, relative abundance of the NF-B activator IKKα was increased by heat stress though abundance of NF-B was similar between groups. Relative abundance of phosphorylated NF-B was increased by heat stress in nuclear fractions. AP-1 signaling was similar between groups. While there were few differences in transcript expression between thermal neutral and heat stress, 80 and 56% of measured transcripts driven by NF-B or AP-1, respectively, were increased by heat stress compared to pair-fed thermal neutral. Heat stress also caused a reduction in IL-6 transcript and relative protein abundance. These data demonstrate that short-term heat stress causes inflammatory signaling through NF-B in oxidative, but not glycolytic, skeletal muscle.
Intrauterine growth restriction (IUGR) induced by placental restriction (PR) in sheep leads to chronic hypoxemia and reduced surfactant maturation. The underlying molecular mechanism involves altered regulation of hypoxia signaling by increased prolyl hydroxylase domain (PHD) expression. Here, we evaluated the effect of intratracheal administration of the PHD inhibitor, dimethyloxalylglycine (DMOG), on functional, molecular and structural determinants of lung maturation in the Control and PR sheep fetus. There was no effect of DMOG on fetal blood pressure or fetal breathing movements. DMOG reduced lung expression of genes regulating hypoxia signaling (HIF-3α, ACE1), anti-oxidant defence (CAT), lung liquid reabsorption (SCNN1-A, ATP1-A1, AQP-1, AQP-5) and surfactant maturation (SFTP-A, SFTP-B, SFTP-C, PCYT1A, LPCAT, ABCA3, LAMP3) in Control fetuses. There were very few effects of DMOG on gene expression in the PR fetal lung (reduced lung expression of angiogenic factor ADM, water channel AQP-5 and increased expression of glucose transporter SLC2A1). DMOG administration in Controls reduced total lung lavage phosphatidylcholine to the same degree as in PR fetuses. These changes appear to be regulated at the molecular level as there was no effect of DMOG on the percent tissue, air space or numerical density of SFTP-B positive cells in the Control and PR lung. Hence, DMOG administration mimics the effects of PR in reducing surfactant maturation in the lung of Control fetuses. The limited responsiveness of the PR fetal lung suggests a potential biochemical limit or reduced plasticity to respond to changes in regulation of hypoxia signaling following exposure to chronic hypoxemia in utero.
Despite significant clinical interest in renal denervation as a therapy, the role of the renal nerves in the physiological regulation of renal blood flow (RBF) remains debated. We hypothesized that the renal nerves physiologically regulate beat-to-beat RBF variability (RBFV). This was tested in chronically instrumented, healthy rabbits that underwent either bilateral surgical renal denervation (DDNx) or a sham denervation procedure (INV). Artifact-free segments of RBF and arterial pressure (AP) from calmly resting, conscious rabbits were used to extract RBFV and AP variability for time-domain, frequency-domain, and nonlinear analysis. While steady-state measures of RBF, AP, and heart rate did not statistically differ between groups, DDNx rabbits had greater RBFV than INV rabbits. AP-RBF transfer function analysis showed greater admittance gain in DDNx rabbits than in INV rabbits, particularly in the low-frequency (LF) range where systemic sympathetic vasomotion gives rise to AP oscillations. In the LF range, INV rabbits exhibited a negative AP-RBF phase shift and low coherence, consistent with the presence of an active control system. Neither of these features were present in the LF range of DDNx rabbits, which showed no phase shift and high coherence, consistent with a passive, Ohm's law pressure-flow relationship. Renal denervation did not significantly affect nonlinear RBFV measures of chaos, self-affinity, or complexity, nor did it significantly affect glomerular filtration rate or extracellular fluid volume. Cumulatively, these data suggest that the renal nerves mediate LF renal sympathetic vasomotion which buffers RBF from LF AP oscillations in conscious, healthy rabbits.
During lactation, highly-specialized secretory mammary epithelial cells (MECs) produce and secrete huge quantities of nutrients and non-nutritive factors into breast milk. The zinc (Zn) transporter ZnT4 (SLC30A4) transports Zn into the trans-Golgi apparatus for lactose synthesis, and across the apical cell membrane for efflux from MECs into milk. This is consistent with observations in "lethal milk" (lm/lm) mice, which have a truncation mutation in SLC30A4, and present with not only low milk Zn concentration, but also smaller mammary glands, decreased milk volume and lactation failure by lactation day 2. However, the molecular underpinnings of these defects are not understood. Here, we used lactating C57bl/6Jlm/lm (ZnT4-null) mice to explore the consequences of a ZnT4-null phenotype on mammary gland function during early lactation. Lactating C57bl/6Jlm/lm mice had significantly fewer, smaller and collapsed alveoli comprised of swollen, lipid-filled MECs during early lactation. These defects were associated with decreased Akt expression and STAT5 activation, indicative of defects in MEC secretion. In addition, increased expression of ZnT2, TNFα and cleaved e-cadherin concomitant with increased activation of STAT3 implicated the loss of ZnT4 in precocious activation of involution. Collectively, our study indicates that loss of ZnT4 has profound consequences on MEC secretion and may promote tissue remodeling in the mammary gland during early lactation.
De novo brown adipogenesis involves the proliferation and differentiation of progenitors, yet the mechanisms that guide these events in vivo are poorly understood. We previously demonstrated that treatment with a β3-adrenergic receptor (ADRB3) agonist triggers brown/beige adipogenesis in gonadal white adipose tissue following adipocyte death and clearance by tissue macrophages. The close physical relationship between adipocyte progenitors and tissue macrophages suggested that the macrophages that clear dying adipocytes might generate proadipogenic factors. Flow cytometric analysis of macrophages from mice treated with CL 316,243 identified a subpopulation that contained elevated lipid and expressed CD44. Lipidomic analysis of FACS-isolated macrophages demonstrated that CD44+ macrophages contained 4-5 fold higher levels of the endogenous PPAR ligands 9-HODE and 13-HODE compared to CD44- macrophages. Gene expression profiling and immunohistochemistry demonstrated that ADRB3 agonist treatment upregulated expression of Alox15, the lipoxygenase responsible for generating 9- and 13-HODE. Using an in vitro model of adipocyte efferocytosis, we found that IL-4 primed tissue macrophages accumulated lipid from dying fat cells and upregulated expression of Alox15. Furthermore, treatment of differentiating adipocytes with 9-and 13-HODE potentiated brown/beige adipogenesis. Collectively, these data indicate that non-inflammatory removal of adipocyte remnants and coordinated generation of PPAR ligands by M2 macrophages provides localized adipogenic signals to support de novo brown/beige adipogenesis.
Androgens are essential for the development and maintenance of male reproductive tissues and sexual function and for overall health and well-being. Testosterone, the predominant and most important androgen, not only affects the male reproductive system, but also influences the activity of many other organs. In the cardiovascular system, the actions of testosterone are still controversial, ranging from protective to deleterious effects. While early studies showed that testosterone-replacement therapy exerted beneficial effects on cardiovascular disease, some recent safety studies point to a positive association between endogenous and supraphysiological levels of androgens/testosterone and cardiovascular disease risk. Among the possible mechanisms involved in the actions of testosterone on the cardiovascular system, indirect actions (changes in the lipid profile, insulin sensitivity, and hemostatic mechanisms, modulation of the sympathetic nervous system and renin-angiotensin-aldosterone system) as well as direct actions (modulatory effects on pro-inflammatory enzymes, on the generation of reactive oxygen species, nitric oxide bioavailability, and on vasoconstrictor signaling pathways) have been reported. This mini-review focuses on evidence indicating that testosterone has pro-oxidative actions that may contribute to its deleterious actions in the cardiovascular system. The controversial effects of testosterone on ROS generation/oxidant status, both pro-oxidant and antioxidant, in the cardiovascular system and in cells/tissues of other systems are reviewed.
Sodium intake occurs either as a spontaneous or induced behavior, which is enhanced, i.e. sensitized, by repeated episodes of water deprivation followed by subsequent partial rehydration (WD-PR). In the present work, we examined if repeated WD-PR alters hypothalamic transcripts related to the brain renin-angiotensin system (RAS) and apelin system in male normotensive Holtzman rats (HTZ). We also examined if the sodium intake of a strain with genetically inherited high expression of the brain RAS, the spontaneously hypertensive rat (SHR), responds differently than HTZ to repeated WD-PR. We found that repeated WD-PR, besides enhancing spontaneous and induced 0.3 M NaCl intake, increased the hypothalamic expression of angiotensinogen, aminopeptidase N, and apelin receptor transcripts (43%, 60%, and 159%, respectively) in HTZ at the end of the 3rd WD-PR. Repeated WD-PR did not change the daily spontaneous 0.3 M NaCl intake and barely changed the need-induced 0.3 M NaCl intake of SHR. The same treatment consistently enhanced spontaneous daily 0.3 M NaCl intake in the normotensive Wistar-Kyoto rats. The results show that repeated WD-PR produces alterations in hypothalamic transcripts in addition to sensitize sodium appetite in HTZ. They suggest an association between the components of hypothalamic RAS and apelin system with neural and behavioral plasticity produced by repeated episodes of WD-PR in a normotensive strain. The results also indicate that the inherited hyperactive brain RAS is not a guarantee for sensitization of sodium intake in the male adult SHR exposed to repeated WD-PR.
Adipose triglyceride lipase (ATGL) catalyzes the rate-limiting removal of the first fatty acid from a triglyceride. ATGL is activated by comparative gene identification-58 (CGI-58) and inhibited by G(0)/G(1) switch gene-2 protein (G0S2). Research in other tissues and cell culture indicates that inhibition is dependent on relative G0S2-to-ATGL protein content. G0S2 may also have several roles within mitochondria, however this has yet to be observed in skeletal muscle. The purpose of this study was to determine if muscle G0S2 relative to ATGL content would decrease to facilitate intramuscular lipolysis following endurance training. Male Sprague-Dawley rats (n = 10, starting age 51-53 days) were progressively treadmill trained at a 10% incline for 8-weeks ending with 25m/min for 1 hour, compared to control. Sciatic nerve stimulation for hind limb muscle contraction (and lipolysis) was administered for 30 minutes to one leg, leaving the opposing leg as a resting control. Soleus (SOL), red (RG) and white gastrocnemius (WG) were excised from both legs following stimulation or control. ATGL protein increased in all trained muscles. Unexpectedly, G0S2 protein was greater in the trained SOL and RG. In RG isolated mitochondria, G0S2 also increased with training, yet mitochondrial G0S2 content was unaltered with acute contraction, therefore any role of G0S2 in the mitochondria does not appear to be acutely mediated by content alone. In summary, G0S2 increased with training in oxidative muscles and mitochondria but not following acute contraction, suggesting that inhibition is not through relative G0S2-to-ATGL content, but through more complicated intracellular mechanisms.
While the neural control of glucoregulatory responses to insulin-induced hypoglycemia is beginning to be elucidated, brain sites responsible for behavioral responses to hypoglycemia are relatively poorly understood. To help elucidate central control mechanisms associated with hypoglycemia unawareness, we first developed an animal model of hypoglycemia-induced behavioral arousal, as a surrogate for "awareness", using a conditioned place preference (CPP) model. We found that hypoglycemia reversed previously acquired CPP in rats and that recurrent hypoglycemia prevented this reversal. Pre-treatment with a brain-penetrant selective orexin receptor-1 antagonist, SB334867A, blocked hypoglycemia-induced reversal of CPP. Recurrently hypoglycemic rats also showed decreased prepro-orexin mRNA expression in the perifornical hypothalamus by 50% but adjacent lateral hypothalamic expression was unaffected. Pre-treatment with sertraline, previously shown to prevent hypoglycemia-associated glucoregulatory failure, did not prevent attenuation of hypoglycemia-induced CPP reversal by recurrent hypoglycemia. This work describes the first behavioral model of hypoglycemia unawareness and suggests a role for orexin neurons in mediating behavioral responses to hypoglycemia.
The parabrachial nucleus is important for thermoregulation because it relays skin temperature information from the spinal cord to the hypothalamus. Prior work in rats localized thermosensory relay neurons to its lateral subdivision (LPB), but the genetic and neurochemical identity of these neurons remains unknown. To determine the identity of LPB thermosensory neurons, we exposed mice to a warm (36 °C) or cool (4 °C) ambient temperature. Each condition activated neurons in distinct LPB subregions that receive input from the spinal cord. Most Fos+ neurons in these LPB subregions expressed the transcription factor marker, FoxP2. Consistent with prior evidence that LPB thermosensory relay neurons are glutamatergic, all FoxP2+ neurons in these subregions co-localized with GFP in reporter mice for vglut2, but not for vgat. Prodynorphin (pdyn)-expressing neurons were identified using a GFP reporter mouse and formed a caudal subset of LPB FoxP2+ neurons, primarily in the dorsal lateral subnucleus (PBdL). Warm exposure activated many FoxP2+ neurons within PBdL. Half of the Fos+ neurons in PBdL were pdyn+, and most of these project to the preoptic area. Cool exposure activated a separate FoxP2+ cluster of neurons in the far-rostral LPB, which we named the rostral-to-external lateral subnucleus (PBreL). These findings improve our understanding of LPB organization and reveal that pdyn-cre mice provide genetic access to warm-activated, FoxP2+ glutamatergic neurons in PBdL, many of which project to the hypothalamus.
Premenopausal females are resistant to the genesis development of hypertension, and this protection is lost following the onset of menopause, resulting in a sharp increase in disease onset and severity. However, it is unknown how a fluctuating ovarian hormone environment during the transition from perimenopause to menopause impacts the onset of hypertension, and whether interventions during perimenopause prevent disease onset after menopause. A gradual transition to menopause was induced by repeated daily injections of 4-vinylcyclohexene diepoxide (VCD). Ang II (800 ng/kg/min) was infused into peri- and menopausal female mice for 14 days. A separate cohort of mice received 17-β estradiol replacement during perimenopause. Ang II-infusion produced significantly higher systolic blood mean arterial pressures (MAP) (SBP) in menopausal vs. cycling females and 17-β estradiol replacement prevented this increase. In contrast, MAP was not significantly different when Ang II was infused into perimenopausal and cycling females, suggesting that female resistance to Ang II-induced hypertension is intact during perimenopause. Similar to studies in male mice, Ang II-infusion caused a significant glomerular hypertrophy and hypertrophy was not impacted by hormonal status. Expression levels of aquaporin-2, a collecting duct protein have been suggested to reflect blood pressure. AQP2 protein expression was significantly down regulated in the renal cortex of the Ang II-infused menopause group, where blood pressure was increased. AQP2 expression levels were restored to control levels with 17-β estradiol replacement. This study indicates that the changing hormonal environment in the VCD model of menopause impacts the severity of Ang II-induced hypertension. These data highlight the utility of the ovary-intact VCD model of menopause as a clinically relevant model to investigate the physiological mechanisms of hypertension that occur in women during the transition into menopause.
Metabolic disease, specifically obesity, has now become the greatest challenge to improving cardiovascular health. The renin-angiotensin system (RAS) exists as both a circulating hormone system and as a local paracrine signaling mechanism within various tissues including the brain, kidney, and adipose, and this system is strongly implicated in cardiovascular health and disease. Growing evidence also implicates the RAS in the control of energy balance, supporting the concept that the RAS may be mechanistically involved in the pathogenesis of obesity and obesity-hypertension. Here, we review the involvement of the RAS in the entire spectrum of whole-organism energy balance mechanisms, including behaviors (food ingestion and spontaneous physical activity) and biological processes (digestive efficiency and both aerobic and non-aerobic resting metabolic rates). We hypothesize that opposing, tissue-specific effects of the RAS to modulate these various components of energy balance can explain the apparently paradoxical results reported by energy-balance studies that involve stimulating, versus disrupting, the RAS. We propose a model in which such opposing and tissue-specific effects of the RAS can explain the failure of simple, global RAS blockade to result in weight loss in humans, and hypothesize that obesity-mediated uncoupling of endogenous metabolic rate control mechanisms can explain the phenomenon of obesity-hypertension.
Candida glabrata (CG) is an opportunistic fungal pathogen that initiates infection by binding to host cells via specific lectin-like adhesins proteins. We have previously shown the importance of lectin-oligosaccharide binding in cardiac responses to flow and agonists. Due to the lectinic-oligosaccharide nature of CG binding, we tested the ability of CG to alter the agonist- and flow-induced changes in cardiac function in isolated perfused guinea pig hearts. Both transmission and scanning electron microscopy showed strong attachment of CG to the coronary endothelium, even after extensive washing. CG shifted the coronary flow versus AV-delay relationship upward, indicating that greater flow was required to achieve the same AV delay. This effect was completely reversed with mannose, partially reversed with galactose and N- acetylgalactosamine, but hyaluronan had no effect. Western blot was used to determine binding of CG to isolated coronary endothelial luminal membrane (CELM) receptors and the results indicate that flow sensitive CELM receptors, Angiotensin II type I, α- adrenergic 1A, endothelin-2 and VCAM-1 bind to CG. In addition, CG inhibited agonist-induced effects of bradykinin, angiotensin and phenylephrine on AV delay, coronary perfusion pressure and left ventricular pressure. Mannose reversed the inhibitory effects of CG on the agonist responses. These results suggest that CG directly binds to flow sensitive CELM receptors via lectinic-oligosaccharide interactions with mannose and disrupts the lectin-oligosaccharide binding necessary for flow-induced cardiac responses.
The link between dietary carbohydrate/protein and de novo lipogenesis (DNL) remains debatable in carnivorous fish. We evaluate the response of hepatic lipogenic gene expression to dietary carbohydrate intake/glucose and dietary protein intake/amino acids (AAs) during acute stimulations in trout. For the in vivo trial, three different diets and a controlled-feeding method were employed to supply fixed amount of dietary protein or carbohydrate in a single meal; for the in vitro trial, primary hepatocytes were stimulated with low/high level of glucose (3 mM or 20 mM) and low/high level of AAs (one-fold or four-fold concentrated AAs). In vitro data showed that high level of AAs up-regulated the expression of enzymes involved in DNL (FAS and ACLY), lipid bioconversion (Elovl5, Elovl2, D6D and SCD1), NADPH production (G6PDH and ME), and transcriptional factor SREBP1c, while high level of glucose only elevated the expression of ME. Data in trout liver also showed that high dietary protein intake induced higher lipogenic gene expression (FAS, ACLY and Elovl2) regardless of dietary carbohydrate intake, while high carbohydrate intake markedly suppressed the expression of ACC and Elovl5. Overall, we conclude that, unlike rodents or humans, DNL gene expression in rainbow trout is more responsive to dietary protein intake/AAs than dietary carbohydrate intake/glucose during acute stimulations. This discrepancy probably represents one important physiological and metabolic difference between carnivores and omnivores.
Objective: 1) To assess hemodynamic responses and baroreflex sensitivity (BRS) indices during Valsalva maneuver (VM) and head-up tilt (HUT) testing in orthostatic intolerance (OI). Methods: Patients with neurogenic orthostatic hypotension (NOH, n=26), postural tachycardia syndrome (POTS, n=26) and symptomatic orthostatic intolerance (SOI, n=14) were compared to healthy population (Control, n=107) and inappropriate sinus tachycardia (IST, n=7). Hemodynamic assessment included patterning and quantification with vagal and adrenergic BRS (BRSv and BRSa/BRSa1). Results: In NOH, cardiovagal SBP decrements in VM and HUT were correlated (r=0.660, p<0.001); a "V" pattern of VM indicated alpha BRSa failure. Yet, BRSa1 did not reveal changes vs. Control (p>0.05) or was not applicable in 60% of NOH. In SOI, compared to Control cardiovagal SBP decrements were larger (p<0.05); higher BRSa1 contradicted higher adrenergic index (CASS). Overshoot in phase IV dipped below baseline or dropped ≥ 10 mmHg over 8 s in POTS ("N" pattern), but by 3 s in IST ("M" pattern"). Conclusions: Visualization of distinct VM patterns allows primary evaluation of autonomic dysfunction and differentiation of the various forms of OI. BRSa1 evaluation is compromised by pathological SBP patterns. Significance: VM patterning is a valuable non-postural supplement to HUT capable of detecting and differentiating OI.
Fetal overgrowth is common in obese women and is associated with perinatal complications and increased risk for the child to develop metabolic syndrome later in life. Placental nutrient transport capacity has been reported to be increased in obese women giving birth to large infants, however the underlying mechanisms are not well established. Obesity in pregnancy is characterized by elevated maternal serum insulin and leptin, hormones that stimulate placental amino acid transporters in vitro. We hypothesized that maternal obesity activates placental insulin/IGF-I/mTOR and leptin signaling pathways. We tested this hypothesis in a mouse model of obesity in pregnancy that is associated with fetal overgrowth. C57BL/6J female mice were fed a control (C) or a high fat/high sugar (HF/HS) pelleted diet supplemented by ad libitum access to sucrose (20%) solution. Placentas were collected at embryonic day 18.5. Using Western blot analysis, placental mTOR activity was determined along with energy, inflammatory, leptin and insulin signaling pathways (upstream modulators of mTOR). Phosphorylation of S6 ribosomal protein (S-235/236), 4E-BP1 (T-37/46), IRS-1 (Y-608), Akt (T-308) and STAT-3 (Y-705) was increased in obese dams. In contrast, expression of placental caspase-1, IBα, IL-1β and phosphorylated-JNK p46/54 T183/Y185 was unaltered. Fetal amino acid availability is a key determinant of fetal growth. We propose that activation of placental insulin/IGF-I/mTOR and leptin signaling pathways in obese mice stimulates placental amino acid transport and contributes to increased fetal growth.
Obesity in pregnancy is associated with increased fetal growth and adiposity, which, in part, is determined by transplacental nutrient supply. Trophoblast uptake and intracellular trafficking of lipids are dependent on placental fatty acid transport proteins (FATP), translocase (FAT/CD36) and fatty acid binding proteins (FABP). We hypothesized that maternal obesity in mice leads to increased placental expression of FAT/CD36, FATPs and FABPs, and lipid accumulation in the fetal liver. C57/BL6J female mice were fed either a control (C; n=10) or an obesogenic (OB; n=10) high fat-high sugar diet before mating and throughout pregnancy. At E18.5 placentas and fetal livers were collected. Trophoblast plasma membranes (TPM) were isolated from placental homogenates. Expression of FAT/CD36 and FATP (TPM) and FABP (homogenates) was determined by immunoblotting. Gene expression was assessed by RT-qPCR. Sections of fetal livers were stained for Red Oil O and lipid droplets were quantified. TPM protein expression of FAT/CD36, FATP 2 and 4 was comparable between C and OB groups. Conversely, TPM FATP 6 expression was increased by 35% in OB compared to C placentas without changes in mRNA expression. FABP 1, 3-5 and PPAR were expressed in homogenates and FABP 3 expression increased 27% in OB compared to C placentas; however no changes were observed in mRNA expression. Lipid droplet accumulation was 10-fold higher in the livers of fetuses from OB compared to C group. We propose that increased lipid transport capacity in obese mice promotes transplacental fatty acid transport and contributes to excess lipid accumulation in the fetal liver.
Heart Failure (HF) is associated with increased sympathetic nerve activity to the heart (CSNA), which is directly linked to mortality in HF patients. Previous studies indicate that HF is associated with high levels of plasma endothelin-1 (ET-1), which correlates with the severity of the disease. We hypothesized that blockade of endothelin receptors would decrease CSNA. The effects of intravenous tezosentan ((a non-selective ETA and ETB receptor antagonist) (8 mg/kg/h) on resting levels of CSNA, arterial pressure and heart rate were determined in conscious normal sheep (n= 6) and sheep with pacing induced HF (n= 7). HF was associated with a significant decrease in ejection fraction (from 74 ± 2% to 38 ± 1%, P < 0.001) and a significant increase in resting levels of CSNA burst incidence (from 56 ± 11 to 87 ± 2 bursts / 100 heart beats, P < 0.01). Infusion of tezosentan for 60 minutes significantly decreased resting MAP in both normal and HF sheep (-8 ± 4 mmHg and -4 ± 3 mmHg respectively; p<0.05). This was associated with a significant decrease in CSNA (by 25 ± 26% of control) in normal sheep, but there was no change in CSNA in HF sheep. Calculation of spontaneous baroreflex gain indicated significant impairment of the baroreflex control of HR after intravenous tezosentan infusion in normal animals, but no change in HF animals. These data suggest that endogenous levels of ET-1 contribute to the baseline levels of CSNA in normal animals, but this effect is absent in HF.
We herein investigated the effects of passive heat stress on human somatosensory processing recorded by somatosensory-evoked potentials (SEPs). Fifteen healthy subjects received a median nerve stimulation at the left wrist under two thermal conditions: Heat stress and normothermic Time Control. The latencies and amplitudes of P14, N20, P25, N35, P45, and N60 at C4' and P14, N18, P22, and N30 at Fz were evaluated. Under the Heat stress condition, SEPs were recorded at normothermic baseline (1st), early in heat stress (2nd), when esophageal temperature had increased by ~1.0 °C (3rd) and ~2.0 °C (4th), and after heat stress (5th). In the Time control condition, SEPs were measured at the same time intervals as those in the Heat stress condition. The peak latencies and amplitudes of SEPs did not change early in heat stress. However, the latencies of P14, N20, and N60 at C4' and P14, N18, and P22 at Fz were significantly shorter in the 4th session than in the 1st session. Furthermore, the peak amplitudes of P25 and N60 at C4', and P22 and N30 at Fz decreased with increases in body temperature. On the other hand, under the Time control condition, no significant differences were observed in the amplitudes or latencies of any component of SEPs. These results suggested that the conduction velocity of the ascending somatosensory input was accelerated by increases in body temperature, and hyperthermia impaired the neural activity of cortical somatosensory processing.
The ability of the human body to maintain arterial blood pressure (BP) during orthostatic stress is determined by several reflex neural mechanisms. Renal vasoconstriction progressively increases during graded elevations in lower body negative pressure (LBNP). This sympathetically mediated response redistributes blood flow to the systemic circulation to maintain BP. However, how healthy aging affects the renal vasoconstrictor response to LBNP is unknown. Therefore, ten young (25 ± 1 years; mean ± SE) and ten older (66 ± 2 years) subjects underwent graded LBNP (-15 and -30 mmHg) while beat-to-beat renal blood flow velocity (RBFV; Doppler ultrasound), arterial blood pressures (BP; Finometer), and heart rate (HR; electrocardiogram) were recorded. Renal vascular resistance (RVR), an index of renal vasoconstriction, was calculated as mean BP/RBFV. All baseline cardiovascular variables were similar between groups, except diastolic BP was higher in older subjects (P < 0.05). Increases in RVR during LBNP were greater in the older group compared to the young group (older: -15 mmHg 10 ± 3%, -30 mmHg 20 ± 5%; young: -15 mmHg 2 ± 2%, -30 mmHg 6 ± 2%; P < 0.05). RBFV tended to decrease more (P = 0.10) and mean BP tended to decrease less (P = 0.09) during LBNP in the older group compared to the young group. Systolic and diastolic BP, pulse pressure, and HR responses to LBNP were similar between groups. These findings suggest that aging augments the renal vasoconstrictor response to orthostatic stress in humans.
We evaluated postural effects on intracranial pressure (ICP) and cerebral perfusion pressure (CPP: mean arterial pressure (MAP) - ICP) in neurosurgical patients undergoing 24-hour ICP monitoring as part of their diagnostic workup. We identified 9 patients (5 women, age 44±20 yrs.; mean±SD) who were "as normal as possible" i.e. without indication for neurosurgical intervention (e.g. focal lesions, global edema, abnormalities in ICP-profile or cerebrospinal fluid dynamics). ICP (tip-transducer probe, Raumedic) in the brain parenchyma (N=7) or in the lateral ventricles (N=2) and cardiovascular variables (Nexfin) were determined from 20° head-down tilt to standing up. Compared to the supine position, ICP increased during 10° and 20° of head-down tilt (from 9.4±3.8 to 14.3±4.7 and 19±4.7 mmHg, P<0.001). Conversely, 10° and 20° head-up tilt reduced ICP to 4.8±3.6 and 1.3±3.6 mmHg and ICP reached -2.4±4.2 mmHg when standing up (P<0.05). Concordant changes in MAP maintained CPP at 77±7 mmHg regardless of body position (P=0.95). During head-down tilt, the increase in ICP corresponded to a hydrostatic pressure gradient with reference just below the heart, likely reflecting the venous hydrostatic indifference point. When upright, the decrease in ICP was attenuated, corresponding to formation of a separate hydrostatic gradient with reference to the base of the skull, likely reflecting the site of venous collapse. ICP therefore seems to be governed by pressure in the draining veins and collapse of neck veins may protect the brain from being exposed to a large negative pressure when upright. Despite positional changes in ICP, MAP keeps CPP tightly regulated.
Fat transplants increase body fat mass without changing the energy status of an animal and provide a tool for investigating control of total body fat. Early transplant studies found that small pieces of transplanted fat took on the morphology of the transplant recipient. Experiments described here tested whether this response was dependent upon expression of leptin receptors in either transplanted fat or the recipient mouse. Fat from leptin receptor deficient db/db mice or wild type mice was placed subcutaneously in db/db mice. After 12 weeks cell size distribution in the transplant was the same as in endogenous fat of the recipient. Thus wild type fat cells, which express leptin receptors, were enlarged in a hyperleptinemic environment indicating that leptin does not directly control adipocyte size. By contrast, db/db or wild type fat transplanted into wild type mice decreased in size, suggesting that a functional leptin system in the recipient is required for body fat mass to be controlled. In the final experiment wild type fat was transplanted into a db/db mouse parabiosed to either another db/db mouse to an ob/ob mouse or in control pairs in which both parabionts were ob/ob mice. Transplants increased in size in db/db-db/db pairs, decreased in db/db-ob/ob pairs and did not change in ob/ob-ob/ob pairs. We propose that leptin from db/db parabionts activated leptin receptors in their ob/ob partners. This in turn stimulated release of unidentified circulating factors which travelled back to the db/db partner and acted on the transplant to reduce fat cell size.
Although the associations between chronic levels of arterial stiffness and blood pressure (BP) have been fairly well studied, it is not clear if and how much arterial stiffness is influenced by acute perturbations in BP. The primary aim of this study was to determine magnitudes of BP-dependence of various measures of arterial stiffness during acute BP perturbation maneuvers. Fifty apparently healthy subjects, including 25 young (20-40 years) and 25 older adults (60-80 years), were studied. A variety of BP perturbations, including head-up tilt, head-down tilt, mental stress, isometric handgrip exercise, and cold pressor test, were used in order to encompass BP changes induced by physical, mental, and/or physiological stimuli. When each index of arterial stiffness was plotted with mean BP, all arterial stiffness indices, including cardio-ankle vascular index or CAVI (r=0.50), carotid-femoral pulse wave velocity or cfPWV (r=0.51), brachial-ankle pulse wave velocity or baPWV (r=0.61), arterial compliance (r=-0.42), elastic modulus (r=0.52), arterial distensibility (r=-0.32), β-stiffness index (r=0.19), and Young's modulus (r=0.35) were related to mean BP (all P<0.01). Changes in CAVI, cfPWV, baPWV, and elastic modulus were significantly associated with changes in mean BP in the pooled conditions while changes in arterial compliance, arterial distensibility, β-stiffness index, and Young's modulus were not. In conclusion, this study demonstrated that BP changes in response to various forms of pressor stimuli were associated with the corresponding changes in arterial stiffness indices and that the strengths of associations with BP varied widely depending on what arterial stiffness indices were examined.
Hyperbaric oxygen (HBO) is a major therapeutic treatment for ischemic ulcerations that perforate skin and underlying muscle in diabetic patients. These lesions do not heal effectively, in part, because of the hypoxic microvascular O2 partial pressures (PmvO2) resulting from diabetes-induced cardiovascular dysfunction which alters the dynamic balance between O2 delivery and utilization rates. We tested the hypothesis that HBO in diabetic muscle would exacerbate the hyperoxic PmvO2 dynamics due, in part, to a reduction or slowing of the cardiovascular, sympathetic nervous and respiratory system responses to acute HBO exposure. Adult male Wistar rats were divided randomly into diabetic (DIA: Streptozotocin i.p.) and healthy (CONT) groups. A small animal hyperbaric chamber was pressurized with oxygen (100% O2) to 3.0 ATA at 0.2 ATA/min. Phosphorescence quenching techniques were used to measure PmvO2 in tibialis anterior muscle of anesthetized rats during HBO. Lumbar sympathetic nerve activity (LSNA), heart rate (HR) and respiratory rate (RR) were measured electrophysiologically. During the normobaric hyperoxia and HBO, DIA tibialis anterior PmvO2 increased faster than CONT. Subsequently, PmvO2 remained elevated at similar levels in CONT. Sympathetic nervous system, cardiac and respiratory responses to HBO were slower in DIA versus CONT. HBO treatment increases tibialis anterior muscle PmvO2 more rapidly and for a longer duration in DIA than CONT, but not to a greater level. Whereas, respiratory, cardiovascular and LSNA responses to HBO are profoundly slowed in DIA only the cardiovascular arm (via HR) may contribute to the muscle vascular incompetence and these faster PmvO2 kinetics.
We assessed the presence of fatty acid (FA) sensing mechanisms in hypothalamus of Senegalese sole (Solea senegalensis) and investigated their sensitivity to FA chain length and/or level of unsaturation. Stearate (SA, saturated FA), oleate (OA, monounsaturated FA of the same chain length), α-linolenate (ALA, a n-3 PUFA of the same chain length), and eicosapentanoate (EPA, a n-3 PUFA of a larger chain length) were injected intraperitoneally. Parameters related to FA sensing and neuropeptide expression in the hypothalamus were assessed after 3h and changes in accumulated food intake after 4, 24, and 48 h. Three FA sensing systems characterized in rainbow trout were also found in Senegalese sole, and were activated by OA in a way similar to that previously characterized in rainbow trout and mammals. These hypothalamic FA sensing systems were also activated by ALA, differing from mammals, where n-3 PUFA do not seem to activate FA sensors. This might suggest additional roles and highlights the importance of n-3 PUFA in fish diets, especially in marine species. The activation of FA sensing seems to be partially dependent on acyl chain length and degree of saturation, as no major changes were observed after treating fish with SA or EPA. The activation of FA sensing systems by OA and ALA, but not SA or EPA, is further reflected in the expression of hypothalamic neuropeptides involved in the control of food intake. Both OA and ALA enhanced anorexigenic capacity compatible with the activation of FA sensing systems.
The present study investigated the type 1 cannabinoid receptor (CB1R) as a potential candidate to mediate the homeostatic responses triggered by 24 hours of water deprivation (WD), which constitutes primarily a hydroelectrolytic challenge and also significantly impacts energy homeostasis. The present results demonstrated for the first time that CB1R mRNA expression is increased in the hypothalamus of WD rats. Furthermore, the administration of ACEA, a CB1R selective agonist, potentiated WD-induced dipsogenic effect, whereas AM251, a CB1R antagonist, attenuated not only water but also salt intake in response to WD. In parallel with the modulation of thirst and salt appetite, we confirmed that CB1Rs are essential for the development of appropriated neuroendocrine responses. Although the administration of ACEA or AM251 did not produce any effects on WD-induced vasopressin (AVP) secretion, oxytocin (OXT) plasma concentrations were significantly decreased in WD rats treated with ACEA. At the genomic level, ACEA significantly decreased AVP and OXT mRNA expression in the hypothalamus of WD rats, whereas AM251 potentiated both basal and WD-induced stimulatory effects on the transcription of AVP and OXT genes. In addition, we showed that WD alone upregulates proopiomelanocortin, Agouti-related protein, melanin-concentrating hormone and Orexin A mRNA levels in the hypothalamus, and that CB1Rs regulate main central peptidergic pathways controlling food intake, being most of these effects also significantly influenced by the hydration status. In conclusion, the present study demonstrated that CB1Rs participate in the homeostatic responses regulating fluid balance and energy homeostasis during WD.
The electrogenic sodium bicarbonate cotransporter (NBCe2) is encoded by SLC4A5, variants of which have been associated with salt sensitivity of blood pressure, which affects 25% of the adult population. NBCe2 is thought to mediate sodium bicarbonate cotransport primarily in the renal collecting duct, but NBCe2 mRNA is also found in the rodent renal proximal tubule (RPT). The protein expression or function of NBCe2 has not been demonstrated in the human RPT. We validated an NBCe2 antibody by shRNA and western blot, as well as overexpression of an epitope-tagged NBCe2 construct in both RPT cells (RPTCs) and HEK293 cells. Using this validated NBCe2 antibody, we found NBCe2 protein expression in the RPT of fresh and frozen human kidney slices, RPTCs isolated from human urine, and isolated RPTC apical membrane. Under basal conditions, NBCe2 was primarily found in the Golgi while NBCe1 was primarily found at the basolateral membrane. Following an acute short-term increase in intracellular sodium, NBCe2 expression was increased at the apical membrane in cultured slices of human kidney and polarized, immortalized RPTCs. Sodium bicarbonate transport was increased by monensin and overexpression of NBCe2, decreased by NBCe2 shRNA but not by NBCe1 shRNA, and blocked by 2,2'-(1,2-ethenediyl)bis[5-isothiocyanato-benzenesulfonic acid. NBCe2 appears to be important in apical sodium and bicarbonate cotransport under high salt conditions. Future studies will examine the role of NBCe2 in mediating increased renal sodium transport in humans whose blood pressures are elevated by an increase in sodium intake.
Preeclampsia (PE) is a pregnancy-specific disorder typically presenting as new-onset hypertension and proteinuria. While numerous epidemiological studies have demonstrated that obesity increases the risk of PE, the mechanisms have yet to be fully elucidated. Growing evidence from animal and human studies implicate placental ischemia in the etiology of this maternal syndrome. It is thought that placental ischemia is brought about by dysfunctional cytotrophoblast migration and invasion into the uterus and subsequent lack of spiral arteriole widening and placental perfusion. Placental ischemia/hypoxia stimulates the release of soluble placental factors into the maternal circulation where they cause endothelial dysfunction, particularly in the kidney, to elicit the clinical manifestations of PE. The most recognized of these factors are the anti-angiogenic sFlt-1 and pro-inflammatory TNFα and AT1-AA, which promote endothelial dysfunction by reducing levels of the pro-vasodilator nitric oxide and stimulating production of the potent vasoconstrictor endothelin-1 and reactive oxygen species. We hypothesize that obesity-related metabolic factors increase the risk for developing PE by impacting various stages in the pathogenesis of PE, namely, 1) cytotrophoblast migration and placental ischemia; 2) release of soluble placental factors into the maternal circulation; and 3) maternal endothelial and vascular dysfunction. This review will summarize the current experimental evidence supporting the concept that obesity and metabolic factors like lipids, insulin, glucose and leptin affect placental function and increase the risk for developing hypertension in pregnancy by reducing placental perfusion; enhancing placental release of soluble factors; and by increasing the sensitivity of the maternal vasculature to placental ischemia-induced soluble factors.
Background: It is now well-established that after menopause cardiometabolic disorders become more common. Recently, resistance exercise has been recommended as a complement to aerobic (combined training, CT) for the treatment of cardiometabolic diseases. Objective: To evaluate the effects of CT in hypertensive ovariectomized rats undergoing fructose overloadon blood pressure variability (BPV), inflammation and oxidative stress parameters. Methods:Wistar and spontaneously hypertensive (SHR) rats were divided into (n=8/group): Control (C), hypertensive ovariectomized undergoingfructose overload, sedentary (HOF) and trained (HOFT). CT was performed on a treadmill and ladder adapted to rats in alternate days (8 weeks; 40-60% maximal capacity). Arterial pressure (AP) signals were directly recorded. Oxidative stress and inflammation were measured on cardiac and renal tissues. Results: The association of risk factors (hypertension+ovariectomy+fructose) promoted an increase in mean AP (FHO: 174±4 vs. C: 108±1 mmHg) and heart rate (FHO: 403±12 vs. C: 352±11 bpm), induced impairment in insulin sensitivity, BVP, cardiac and renal oxidative stress and cardiac inflammation (TNF-α- FHO: 65.8±9.9 vs. C: 23.3±4.3pg/mg protein). However, CT was able to reduce MAP (FHOT: 158±4 mmHg) and heart rate (FHOT: 303±5 bpm), normalize insulin sensitivity and sympathetic modulation. Moreover, the trained rats presented reduced lipoperoxidation and increased antioxidant defenses in cardiac and renal tissues; reduced TNF- α (FHOT: 33.1±4.9 pg/mg protein) and increased IL-10 and nitric oxide bioavailability. Conclusion: The association of risk factors promoted an additional impairment in metabolic, cardiovascular, autonomic, inflammatory and oxidative stress parameters and combined exercise training was able to attenuate these dysfunctions.
Fetal growth restriction programs an increased risk of cardiovascular disease in adulthood but the actual mechanisms of this developmental programming are not fully understood. Previous studies in mammalian models suggest that hearts of growth-restricted fetuses have reduced cardiomyocyte number due to reduced proliferation and premature cardiomyocyte maturation. Chicken embryos incubated under chronic hypoxia are also growth-restricted, have smaller hearts and show signs of cardiac insufficiency post-hatching. The aim of the present study was to investigate how chronic hypoxia (14% O2) during development affects cardiomyocyte mass and how myocardial structure is altered. Hypoxic incubation reproduced the well-characterized embryonic growth restriction and an increased ventricle-to-body mass ratio (at E11, E15, E17 and E19) with reduced absolute heart mass only at E19. Cell density, apoptosis and cardiomyocyte size were insensitive to hypoxia at E15 and E19 and no signs of ventricular wall remodeling or myocardial fibrosis were detected. Bayesian modeling provided strong support for hypoxia affecting absolute mass and proliferation rates at E15, indicating that the growth impairment, at least partly, occurs earlier in development. Neither E15 nor E19 hearts contained binucleated cardiomyocytes, indicating that fetal hypoxia does not trigger early maturation of cardiomyocytes in the chicken, which contrasts with previous results from hypoxic rat pups. In conclusion, prenatal hypoxia in the chick embryo results in a reduction in the number of cardiomyocytes without inducing ventricular remodeling, cell hypertrophy or premature cardiomyocyte maturation.
Background: Angiotensin-II (Ang-II) interacts with the sympathetic nervous system at central nervous blood pressure regulating structures including the baroreflex. It is unknown whether prolonged BP elevation mediated by high Ang-II plasma levels could induce a persistent shift of the central nervous baroreflex setpoint, lasting beyond the short Ang-II plasmatic half time of few seconds, thereby consolidating elevated BP and / or increased SNA in healthy humans. Methods: In a blinded cross-over design Ang-II or placebo (saline) was infused for a 6-hour period in 12 resting normotensive students (6 males) raising BP to borderline hypertensive levels. Between 60-120 minutes after the infusion period muscle sympathetic nerve activity (MSNA) was assessed microneurographically, and correlated with oscillometric BP measurements and heart rate at supine rest (baseline) and during pharmacologic baroreceptor challenge. Results: Infusion of Ang-II increased BP to borderline-hypertensive levels, as intended whereas heart rate remained unaltered. At baroreflex assessment (i.e. 60-120 minutes after end of infusion period), systolic BP was significantly higher compared to placebo (8.4±3.1 mmHg; p<0.05), whereas diastolic values were near equal between conditions. Baseline MSNA was neither decreased nor increased and baroreflex sensitivity to vasoactive drug challenge was not altered. Conclusion: Our results show that elevation of Ang-II plasma levels over 6-hours was able to increase systolic but not diastolic BP far beyond blood-mediated Ang-II effects. MSNA or heart rate did not counter-regulate this BP elevation, indicating that Ang-II had sustainably reset the central nervous BP threshold of sympathetic baroreflex function to accept elevated BP input-signals without counter-regulatory response.
Pregnancy is characterized by increased blood volume and baseline sympathetic nerve activity (SNA), vasodilation, and tachycardia. Relaxin (RLX), an ovarian hormone elevated in pregnancy, activates forebrain sites involved in control of blood volume and SNA through angiotensin (Ang II) dependent mechanisms, and contributes to adaptations during pregnancy. In anesthetized arterial baroreceptor denervated nonpregnant (NP) rats, RLX microinjected into the subfornical organ (SFO, 0.77 pmol in 50 nl) produced sustained increases in lumbar SNA (8 ± 3%) and mean arterial pressure (MAP, 26 ± 4 mmHg). Low dose intra-carotid artery (ica) infusion of RLX (155 pmol/ml/hr; 1.5 hr) had minor transient effects on arterial pressure and activated neurons (increased Fos-IR) in the SFO and in spinally-projecting (19 ± 2%) and AVP-IR (21 ± 5%) cells in the paraventricular nucleus of the hypothalamus (PVN) of NP, but not pregnant (P) rats. However, mRNA for RLX and Ang II AT1a receptors in the SFO were preserved in pregnancy. RLX receptor-IR is present in the region of the SFO in NP and P rats and is localized in astrocytes, the major source of angiotensinogen in the SFO. These data provide an anatomical substrate for a role of RLX in resetting of AVP secretion and increased baseline SNA in pregnancy. Since RLX and Ang II receptor expression was preserved in the SFO of P rats, we speculate that the lack of response to exogenous RLX may be due to maximal activation by elevated endogenous levels of RLX in near-term pregnancy.
The AMP-activated protein kinase (AMPK) is an endogenous energy sensor that regulates lipid and carbohydrate metabolism. Non-alcoholic fatty liver disease (NAFLD) is regarded as a hepatic manifestation of metabolic syndrome with impaired lipid and glucose metabolism, and increased oxidative stress. Our recent study showed that folic acid supplementation attenuated hepatic oxidative stress and lipid accumulation in high fat diet fed mice. The aim of the present study was to investigate the effect of folic acid on hepatic AMPK during high fat diet feeding and the mechanisms involved. Male C57BL/6J mice were fed a control diet (10% kcals fat), a high fat diet (60% kcals fat) or a high fat diet supplemented with folic acid (26mg/kg diet) for 5 weeks. Mice fed a high fat diet exhibited hyperglycemia, hepatic cholesterol accumulation and reduced hepatic AMPK phosphorylation. Folic acid supplementation restored AMPK phosphorylation (activation), and reduced blood glucose and hepatic cholesterol levels. Activation of AMPK by folic acid was mediated through an elevation of its allosteric activator AMP and activation of its upstream kinase, namely, liver kinase B1 (LKB1) in the liver. Consistent with in vivo findings, 5-methyltetrahydrofolate (bioactive form of folate) restored phosphorylation (activation) of both AMPK and LKB1 in palmitic acid-treated HepG2 cells. Activation of AMPK by folic acid might be responsible for AMPK-dependent phosphorylation of HMG-CoA reductase, leading to reduced hepatic cholesterol synthesis during high fat diet feeding. These results suggest that folic acid supplementation may improve cholesterol and glucose metabolism by restoration of AMPK activation in the liver.
Mental stress consistently increases arterial blood pressure, but this reliable pressor response is often associated with highly variable muscle sympathetic nerve activity (MSNA) responsiveness between individuals. Although MSNA has been shown to be reproducible within individuals at rest and during cold pressor test (CPT), intra-individual reproducibility of MSNA responsiveness to mental stress has not been adequately explored. The purpose of this study was to examine MSNA reactivity to mental stress across three experimental sessions. Sixteen men and women (age 21±1 years) performed two experimental sessions within a single laboratory visit and a third experimental session one month later. Each experimental session consisted of a mental stress trial via mental arithmetic and a CPT trial. Blood pressure, heart rate (HR) and MSNA were measured, and the consistencies of these variables were determined using intraclass correlation (Cronbach's α coefficient). MSNA, mean arterial pressure (MAP), and HR were highly reproducible across the baselines preceding mental stress (Cronbach's α ≥0.816, p≤0.001) and CPT (Cronbach's α ≥0.782, p≤0.001). Across the three mental stress trials, changes in MSNA (Cronbach's α = .875; p=.001), MAP (Cronbach's α =.749; p<.001) and HR (Cronbach's α =.9919; p<.001) were reproducible . During CPT, changes in MSNA (Cronbach's α = .805; p=.008), MAP (Cronbach's α =.878; p<.001), and HR (Cronbach's α =.927; p<.001) remained consistent across the three sessions. In conclusion, our findings demonstrate that MSNA reactivity to mental stress is consistent within a single laboratory visit and across laboratory sessions conducted on separate days.
Cerebrovascular complications and increased risk of encephalopathies are characteristic of preeclampsia and contribute to 40% of preeclampsia/ eclampsia related deaths. Circulating tumor necrosis factor α (TNFα) is elevated in preeclamptic women and infusion of TNFα into pregnant rats mimics characteristics of preeclampsia. While this suggests that TNFα has a mechanistic role to promote preeclampsia, the impact of TNFα on the cerebral vasculature during pregnancy remains unclear. We tested the hypothesis that TNFα contributes to cerebrovascular abnormalities during placental ischemia by first infusing TNFα in pregnant rats (200 ng/day i.p, from gestational day 14 to 19), at levels to mimic those reported in preeclamptic women. TNFα increased mean arterial pressure (MAP, p<0.05) and brain water content in the anterior cerebrum (p<0.05), however, TNFα infusion had no effect on blood-brain barrier (BBB) permeability in the anterior cerebrum or posterior cerebrum. We then assessed the role of endogenous TNFα in mediating these abnormalities in a model of placental ischemia induced by reducing uterine perfusion pressure followed by treatment with the soluble TNFα receptor (etanercept, 0.8 mg/kg, sc.) on gestational day 18. Etanercept reduced placental ischemia-mediated increases in MAP, anterior brain water content (p<0.05), and BBB permeability (202±44% in placental ischemic rats to 101±28% of normal pregnant rats). Our results indicate that TNFα mechanistically contributes to cerebral edema by increasing BBB permeability and is an underlying factor in the development of cerebrovascular abnormalities associated with preeclampsia complicated by placental ischemia.
Reducing blood flow to working muscles during dynamic exercise causes metabolites to accumulate within the active muscles and evokes systemic pressor responses. Whether a similar cardiovascular response is elicited with normal blood flow to exercising muscles during dynamic exercise remains unknown, however. To address that issue, we tested whether cardiovascular responses are affected by increases in blood flow to active muscles. Thirteen healthy subjects performed dynamic plantarflexion exercise for 12 minutes at 20%, 40% and 60% of peak workload (EX20, EX40 and EX60) with their lower thigh enclosed in a negative pressure box. Under control conditions, the box pressure was the same as the ambient air pressure. Under negative pressure conditions, beginning 3 min after the start of the exercise, the box pressure was decreased by 20, 45 and then 70 mmHg in stepwise fashion with 3-min step durations. During EX20, the negative pressure had no effect on blood flow or the cardiovascular responses measured. However, application of negative pressure increased blood flow to the exercising leg during EX40 and EX60. This increase in blood flow had no significant effect on systemic cardiovascular responses during EX40, but it markedly attenuated the pressor responses otherwise seen during EX60. These results demonstrate that during mild exercise, normal blood flow to exercising muscle is not a factor eliciting cardiovascular responses, whereas it elicits an important pressor effect during moderate exercise. This suggests blood flow to exercising muscle is a major determinant of cardiovascular responses during dynamic exercise at higher than moderate intensity.
An adequate supply of oxygen is important for the survival of all tissues, but is especially critical for tissues with high energy demands such as the heart. Insufficient tissue oxygenation occurs under a variety of conditions, including high altitude, embryonic and fetal development, inflammation, and thrombotic diseases, often affecting multiple organ systems. Responses and adaptations of the heart to hypoxia are of particular relevance in human cardiovascular and pulmonary diseases, where the effects of hypoxic exposure can range in severity from transient to long-lasting. This study uses the genetic model system Drosophila to investigate cardiac responses to acute (30 minutes), sustained (18 hours), and chronic (3 weeks) hypoxia with reoxygenation. Whereas hearts from wild type flies recovered quickly after acute hypoxia, exposure to sustained or chronic hypoxia significantly compromised heart function upon reoxygenation. Hearts from flies with mutations in sima, Drosophila homolog of the Hypoxia Inducible Factor alpha subunit (HIFα), exhibited exaggerated reductions in cardiac output in response to hypoxia. Heart function in hypoxia-selected flies, selected over many generations for survival in a low oxygen environment, revealed reduced cardiac output in terms of decreased heart rate and fractional shortening compared to their normoxia controls. Hypoxia-selected flies also had smaller hearts, myofibrillar disorganization and increased extracellular collagen deposition, consistent with the observed reductions in contractility. This study indicates that longer duration hypoxic insults exert deleterious effects on heart function that are mediated in part by sima and advances Drosophila models for the genetic analysis of cardiac-specific responses to hypoxia and reoxygenation.
This study characterized the local effects of extracellular osmolality and prolactin (PRL) on branchial ionoregulatory function of a euryhaline teleost, Mozambique tilapia (Oreochromis mossambicus). First, gill filaments were dissected from freshwater (FW)-acclimated tilapia and incubated in four different osmolalities, 280, 330, 380 and 450 mOsm/kg. The mRNA expression of Na+/K+-ATPase a1a (NKA a1a) and Na+/Cl- cotransporter (NCC) showed higher expression with decreasing media osmolalities, while Na+/K+/2Cl- cotransporter 1a (NKCC1a) and PRL receptor 2 (PRLR2) mRNA levels were upregulated by increases in media osmolality. We then incubated gill filaments in media containing ovine PRL (oPRL) and native tilapia PRLs (tPRL177 and tPRL188). oPRL and the two native tPRLs showed concentration-dependent effects on NCC, NKA a1a and PRLR1 expression; Na+/H+ exchanger 3 (NHE3) expression was increased by 24 h of incubation with tPRLs. Immunohistochemical observation showed that oPRL and both tPRLs maintained a high density of NCC- and NKA-immunoreactive ionocytes in cultured filaments. Furthermore, we found that tPRL177 and tPRL188 differentially induce expression of these ion transporters according to incubation time. Together, these results provide evidence that ionocytes of Mozambique tilapia may function as osmoreceptors as well as directly respond to PRL to modulate branchial ionoregulatory functions.
Background &Aims: The pregnant uterus is a smooth muscle organ whose pattern of contraction is dictated by the propagation of electrical impulses. Such electrical activity may originate from one or more pacemakers but the location of these sites have not yet been determined. Methods: To detect the location of the pacemaker in the gravid uterus, two approaches were used: A) determine the site from where the contraction started using isolated uteri from the pregnant guinea pig and video-taping their contractions. B) Recording in isolated uteri from pregnant term rats with 240 extracellular electrodes simultaneously and determine where the electrical bursts started. Results: In both the contractile and electrophysiological experiments, there was not a single specific pacemaker area. However, most contractions (guinea pig 87%) and bursts (rat 76%) started close to the mesometrial border (mean 2.7±4.0 mm SD in guinea pigs and 1.3±1.4 mm in the rats). In addition, in the rat, most of the sites of initiations were located closer to the ovarial end of the horn (mean distance from the ovarial end 6.0±6.2 mm SD) whereas such an orientation was not seen in the guinea pig. Conclusions: In both guinea pig and rat uteri at term, there is not one specific pacemaker area. Rather, contractile and electrical activity may arise from any site with the majority starting close to the mesometrial border. Furthermore, in the rat, most activities started at the ovarial end of the horn. This may suggest slightly different pattern of contraction in both species.
We examined whether sustained changes in baroreceptor loading status during prolonged postexercise recovery can alter the metaboreceptors' influence on heat loss. Thirteen young males performed a 1-min isometric handgrip exercise (IHG) at 60% maximal voluntary contraction followed by 2-min of forearm ischemia (to activate metaboreceptors) before and 15, 30, 45 and 60-min after a 15-min intense treadmill running exercise (>90% maximal heart rate) in the heat (35°C). This was repeated on three separate days with continuous lower-body positive (LBPP, +40 mmHg), negative (LBNP, -20 mmHg), or no pressure (Control) from 13- to 65-min postexercise. Sweat rate (ventilated capsule; forearm, chest, upper back) and cutaneous vascular conductance (CVC; forearm, upper back) were measured. Relative to pre-IHG levels, sweating at all sites increased during IHG and remained elevated during ischemia at baseline and similarly at 30, 45, and 60-min postexercise (site average sweat rate increase during ischemia: Control, 0.13±0.02; LBPP, 0.12±0.02; LBNP, 0.15±0.02 mg·min-1·cm-2; all P<0.01), but not at 15-min (all P>0.10). LBPP and LBNP did not modulate the pattern of sweating to IHG and ischemia (all P>0.05). At 15-min postexercise, forearm CVC was reduced from pre-IHG levels during both IHG and ischemia under LBNP only (ischemia: 3.9±0.8 %CVCmax; P<0.02). Therefore, we show metaboreceptors increase postexercise sweating in the mid-to-late stages of recovery (30-60 min), independent of baroreceptor loading status and similarly between skin sites. In contrast, metaboreflex modulation of forearm but not upper back CVC occurs only in the early stages of recovery (15-min) and is dependent upon baroreceptor unloading.
The aim of the study was to identify the contribution of myelinated (A-fiber) and unmyelinated (C-fiber) baroreceptor central pathways to the baroreflex control of sympathetic nerve activity (SNA) and arterial pressure (AP), in anesthetized Wistar-Kyoto (WKY, n = 8) and spontaneously hypertensive rats (SHR, n = 8). The left aortic depressor nerve (ADN) was electrically stimulated with two types of binary white noise signals designed to preferentially activate A-fibers (A-BRx protocol) or C-fibers (C-BRx protocol). In WKY, the central arc transfer function from ADN stimulation to SNA estimated by A-BRx showed strong derivative characteristics with the slope of dynamic gain between 0.1 and 1 Hz (Gslope) of 14.63±0.89 dB/decade. In contrast, the central arc transfer function estimated by C-BRx exhibited non-derivative characteristics with Gslope of 0.64±1.13 dB/decade. This indicates that A-fibers are important for rapid baroreflex regulation, whereas, C-fibers are likely important for more sustained regulation of SNA and AP. In SHR, the central arc transfer function estimated by A-BRx showed higher Gslope (18.46±0.75 dB.decade, P < 0.01), and that estimated by C-BRx showed higher Gslope (8.62±0.64 dB/decade, P<0.001) with significantly lower dynamic gain at 0.01 Hz (6.29±0.48 vs. 2.80±0.36 %/Hz, p<0.001) compared with WKY. In conclusion, the dynamic characteristics of the A-fiber central pathway are enhanced in the high modulation frequency range (0.1-1 Hz) and those of the C-fiber central pathway are attenuated in the low modulation frequency range (0.01-0.1 Hz) in SHR.
While abnormal hemodynamic forces alter fetal myocardial growth, little is known about whether such insults dysregulate fetal cardiac valve development. We hypothesized that chronically elevated systolic load would detrimentally alter fetal valve growth. Sixteen chronically instrumented fetal sheep received either a continuous infusion of adult sheep plasma to increase fetal blood pressure (n=8), or a Lactated Ringers infusion as a volume control (n=8) beginning on day 126±4 of gestation. After 8 days, mean arterial pressure was higher in the plasma infusion group (63.0 mm Hg vs. 41.8 mm Hg, P < 0.01). Mitral annular septal-lateral diameter (11.9 mm vs. 9.1 mm, P < 0.05), anterior leaflet length (7.7mm vs. 6.4 mm, P < 0.05), and posterior leaflet length (4.0 mm vs. 3.0 mm, P < 0.05) were greater in the elevated load group. mRNA levels of Notch-1, TGF-β2, Wnt-2b, BMP-1, and versican were suppressed in aortic and mitral valve leaflets; elastin and alpha-1 type I collagen mRNA levels were suppressed in the aortic valves only. We conclude that sustained elevated pressure load on the fetal heart valve leads to anatomic remodeling and, surprisingly, suppression of signaling and extracellular matrix genes that are important to valve development. These novel findings have important implications on the developmental origins of valve disease and may have long term consequences on valve function and durability.
Vasoactive agents are used in critical care to optimize circulatory function, but their effects on renal tissue oxygenation in the absence of anesthesia remain largely unknown. Therefore, we assessed the effects of multiple vasoactive agents on regional kidney oxygenation in awake sheep. Sheep were surgically instrumented with pulmonary and renal artery flow probes, and combination fiber-optic probes, in the renal cortex and medulla, comprising a fluorescence optode to measure tissue PO2 and a laser-Doppler probe to assess tissue perfusion. Carotid arterial and renal venous cannulae enabled measurement of arterial pressure and total renal oxygen delivery and consumption. Norepinephrine (0.1 or 0.8 µg/kg/min) dose-dependently reduced cortical and medullary laser Doppler flux (LDF) and PO2 without significantly altering renal blood flow (RBF), or renal oxygen delivery or consumption. Angiotensin II (9.8±2.1 µg/h) reduced RBF by 21%, renal oxygen delivery by 28%, oxygen consumption by 18% and medullary PO2 by 38%, but did not significantly alter cortical PO2 or cortical or medullary LDF. Arginine vasopressin (3.3±0.5 µg/h) caused similar decreases in RBF and renal oxygen delivery, but did not significantly alter renal oxygen consumption or cortical or medullary LDF or PO2. Captopril had no observable effects on cortical or medullary LDF or PO2, at a dose that increased renal oxygen delivery by 24%, but did not significantly alter renal oxygen consumption. We conclude that vasoactive agents have diverse effects on regional kidney oxygenation in awake sheep that are not predictable from their effects on LDF, RBF or total renal oxygen delivery and consumption.
The role of serotonin (5-HT) neurons in cardiovascular responses to acute intermittent hypoxia (AIH) has not been studied in the neonatal period. We hypothesized that a partial loss of 5-HT neurons would reduce arterial blood pressure (BP) at rest, increase the fall in BP during hypoxia, and reduce the long term facilitation of breathing (vLTF) and BP following AIH. We exposed 2 week-old, 5,7-dihydroxytryptamine (5,7-DHT)-treated and controls to AIH (10% O2; n=13 control, 14 treated), acute intermittent hypercapnia (AIHC; 5% CO2; n=12 and 11), or acute intermittent hypercapnic hypoxia (AIHH; 10% O2, 5% CO2; n=15 and 17). We gave five 5-min challenges of AIH and AIHC, and twenty ~20 sec challenges of AIHH to mimic sleep apnea. Systolic (sBP), diastolic (dBP), mean arterial pressure (MAP),heart rate(HR),and metabolic rate (VO2) were continuously monitored. 5,7-DHT induced an ~35% loss of 5-HT neurons from the medullary raphe. Compared to controls, pups deficient in 5-HT neurons had reduced resting sBP (~6 mmHg), MAP (~5mmHg), HR (56 beats/min),and experienced a reduced drop in BP during hypoxia. AIHH induced vLTF in both groups, reflected in increased VE and VE/VO2,and decreased arterial PaCO2. The sBP of pups deficient in 5-HT neurons, but not controls, was increased 1 hr following AIHH. Our data suggest that a relatively small loss of 5-HT neurons compromises resting BP and HR, but has no influence on ventilatory plasticity induced by AIHH. AIHH may be useful for reversing cardio-respiratory defects related to partial 5-HT system dysfunction.
The total baroreflex arc (the open-loop system relating carotid sinus pressure (CSP) to arterial pressure (AP)) is known to exhibit nonlinear behaviors. However, few studies have quantitatively characterized its nonlinear dynamics. The aim of this study was to develop a nonlinear model of the sympathetically-mediated total arc without assuming any model form. Normal rats were studied under anesthesia. The vagal and aortic depressor nerves were sectioned, the carotid sinus regions were isolated and attached to a servo-controlled piston pump, and AP and sympathetic nerve activity (SNA) were measured. CSP was perturbed using a Gaussian white noise signal. A second-order Volterra model was developed by applying nonparametric identification to the measurements. The second-order kernel was mainly diagonal, but the diagonal differed in shape from the first-order kernel. Hence, a reduced second-order model was similarly developed comprising a linear dynamic system in parallel with a squaring system in cascade with a slower linear dynamic system. This "Uryson" model predicted AP changes 12% better (p < 0.01) than a linear model in response to new Gaussian white noise CSP. The model also predicted nonlinear behaviors including thresholding and mean responses to CSP changes about the mean. Models of the neural arc (the system relating CSP to SNA) and peripheral arc (the system relating SNA to AP) were likewise developed and tested. However, these models of sub-systems of the total arc showed approximately linear behaviors. In conclusion, the validated nonlinear model of the total arc revealed that the system takes on an Uryson structure.
The brain of mammalian hibernators is naturally protected. Hibernating ground squirrels undergo rapid and extreme changes in body temperature and brain perfusion as they cycle between lengthy torpor bouts and brief periods of euthermia called interbout arousals (IBAs). Arousal from torpor to IBA occurs rapidly, but there is no evidence of brain injury accompanying this extreme physiological transition. Production of the hormone melatonin accompanies arousal, suggesting that it plays a protective role at this time. Here, we investigated mechanisms of melatonin receptor-mediated protection in the brain of the hibernating ground squirrel. We administered the competitive melatonin receptor antagonist luzindole (30 mg/kg, i.p.) to ground squirrels at the predicted end of a torpor bout, triggering an arousal. We found that luzindole treated animals exhibited caspase-3 activity two times higher than vehicle treated animals in the hypothalamus at mid-arousal (P=0.01), suggesting that melatonin receptor signaling is important for protection in this brain region. We also found a 30% decline in succinate-fueled mitochondrial respiration in luzindole-treated animals compared to vehicle treated animals (P=0.019), suggesting that melatonin receptor signaling is important for optimal mitochondrial function during arousal from torpor. The mitochondrial effects of luzindole treatment were seen only during the hibernation season, indicating that this effect is specifically important for arousal from torpor. These data provide evidence for the protective role of melatonin receptor signaling during the extreme physiological transition that occurs when a hibernating mammal arouses from torpor and provide further evidence for regional and seasonal changes in the hibernator brain.
We examined blood flow in the submandibular gland (SMGBF) and sublingual gland (SLGBF) during electrical stimulation of the central cut end of the lingual nerve (LN) in the urethane-anesthetized rats using a laser speckle imaging flow meter. LN stimulation elicited intensity- and frequency-dependent SMGBF and SLGBF increases, and the magnitude of the SMGBF increase was higher than that of the SLGBF increase. The increase in both glands was significantly inhibited by intravenous administration of the autonomic cholinergic ganglion blocker hexamethonium. The antimuscarinic agent atropine markedly inhibited the SMGBF increase and partly inhibited the SLGBF increase. The atropine resistant SLGBF increase was significantly inhibited by infusion of vasoactive intestinal peptide (VIP) receptor antagonist, although administration of VIP receptor antagonist alone had no effect. The recovery time to the basal blood flow level was shorter after LN stimulation than after administration of VIP. However, the recovery time after LN stimulation was significantly delayed by administration of atropine in a dose-dependent manner to the same level as after administration of VIP. Our results indicate that 1) LN stimulation elicits a parasympathetic SMGBF increase mainly evoked by cholinergic fibers, and a parasympathetic SLGBF increase evoked by cholinergic and non-cholinergic fibers, and 2) VIP-ergic mechanisms are involved in the non-cholinergic SLGBF increase, and are activated when muscarinic mechanisms are deactivated.
A high salt diet can lead to hydromineral imbalance and increases in plasma sodium and osmolality. It is recognized as one of the major contributing factors for cardiovascular diseases such as hypertension. The PVN plays a pivotal role in osmotically-driven sympathoexcitation and high blood pressure, which precise mechanisms are not fully understood. Recent evidence indicates that vasopressin (AVP) released from magnocellular neurons might be involved in this process. Using a combination of in vivo and in situ studies, we sought to investigate whether AVP, acting on PVN neurons, can change mean arterial pressure (MAP) and sympathetic nerve activity (SNA) in euhydrated male rats. Furthermore, we wanted to determine whether V1a receptors on PVN neurons would be involved in salt-induced sympathoexcitation and hypertension. In rats, 4 days of salt loading (NaCl 2%) elicited a significant increase in plasma osmolality (39±7 mOsm/L), increase in MAP (26±2 mmHg), and sympathoexcitation when compared to euhydrated rats. Microinjection of AVP into the PVN of conscious euhydrated animals (100 nL, 3 µM) elicited a pressor response (14±2 mmHg) and a significant increase in lumbar SNA (100 nL, 1mM) (19±5%). Pre-treatment with a V1a receptor antagonist, microinjected into the PVN of salt-loaded animals, elicited a decrease in lumbar SNA (-14±5%) and MAP (-19±5 mmHg), when compared with the euhydrated group. Our findings show that AVP plays an important role in modulating the salt-induced sympathoexcitation and high blood pressure, via V1a receptors, within the PVN, which might contribute to neurogenic hypertension in individuals consuming a high salt diet.
Our recent study has shown that hyperventilation of humidified warm air (HWA) triggered cough and reflex bronchoconstriction in patients with mild asthma. We suggested that a sensitizing effect on bronchopulmonary C-fibers by increasing airway temperature was involved, but direct evidence was lacking. This study was carried out to test the hypothesis that HWA enhances the pulmonary C-fiber sensitivity in Brown-Norway rats sensitized with ovalbumin (Ova). In anesthetized rats, isocapnic hyperventilation of HWA for 3 min rapidly elevated airway temperature to a steady state of 41.7°C. Immediately after the HWA challenge, the baseline fiber activity (FA) of pulmonary C-fibers was markedly elevated in sensitized rats, but not in control rats. Furthermore, the response of pulmonary C-fibers to right atrial injection of capsaicin in sensitized rats was significantly higher than control rats before the HWA challenge, and the response to capsaicin was further amplified after HWA in sensitized rats (FA = 4.51 ± 1.02 imp/s before, and 9.26 ± 1.74 imp/s after the HWA challenge). A similar pattern of the HWA-induced potentiation of the FA response to phenylbiguanide, another chemical stimulant of C-fibers, was also found in sensitized rats. These results clearly demonstrated that increasing airway temperature significantly elevated both the baseline activity and responses to chemical stimuli of pulmonary C-fibers in Ova-sensitized rats. In conclusion, this study supports the hypothesis that the increased excitability of these afferents may have contributed to the cough and reflex bronchoconstriction evoked by hyperventilation of HWA in patients with asthma.
Voltage gated sodium channels (NaV) 1.7 are highly expressed on the axons of somatic afferent neurons and are thought to play an important role in the signaling of inflammatory pain. NaV 1.7 channels are classified as Tetrodotoxin (TTX) sensitive, meaning that they are blocked by TTX concentrations of less than 300 nM. These findings prompted us to determine in decerebrated, unanesthetized rats, the role played by NaV 1.7 channels in the transmission of muscle afferent input evoking the exercise pressor reflex. We first showed that the exercise pressor reflex, which was evoked by static contraction of the triceps surae muscles, was reversibly attenuated by application of 50 nM TTX, but not 5 nM TTX, to the L4-L5 dorsal roots (control: 21±1 mmHg, TTX: 8±2 mmHg, restored: 21±3 mmHg; n=6; p<0.01). We next found that the peak pressor responses to contraction were significantly attenuated by dorsal root application of 100 nM Ssm6a, a compound that is a selective NaV 1.7 channel inhibitor. Removal of Ssm6a restored the reflex to its control level (control: 19±3 mmHg, Ssm6a: 10±1 mmHg, recovery: 19±4 mmHg; n=6; p<0.05). Compound action potentials recorded from the L4 and L5 dorsal roots and evoked by single pulse stimulation of the sciatic nerve showed that both TTX and Ssm6a attenuated input from group III as well as group IV afferents. We conclude that NaV 1.7 channels play a role in the thin fiber muscle afferent pathway evoking the exercise pressor reflex.
The hormonal family of vasoinhibins, which derive from the anterior pituitary hormone prolactin, are known for their inhibiting effects on blood vessel growth, vasopermeability, and vasodilation. As pleiotropic hormones, vasoinhibins act in multiple target organs and tissues. The generation, secretion, and regulation of vasoinhibins are embedded into the organizational principle of an axis, which integrates the hypothalamus, the pituitary, and the target tissue microenvironment. This axis is designated as the prolactin/vasoinhibin axis. Disturbances of the prolactin/vasoinhibin axis are associated with the pathogenesis of retinal and cardiac diseases, and with diseases occurring during pregnancy. New phylogenetical, physiological, and clinical implications are discussed.
Preeclampsia (PE) is associated with altered immune activation during pregnancy. We have previously shown that adoptive transfer of CD4+ T cells from the reduced uterine perfusion pressure (RUPP) rat model of PE increases blood pressure, oxidative stress (ROS), and inflammation in normal pregnant recipient rats. The objective of this study was to determine if blockade of communication via the CD40-CD40 ligand interaction between placental ischemia-induced CD4+ T cells with endogenous normal pregnant (NP) cells would improve pathophysiology that was previously observed in NP recipient rats of RUPP CD4+ T cells. Splenic CD4+ T lymphocytes were magnetically separated, incubated with 2.5 µg/mL anti-CD40 ligand (αCD40L) overnight and transferred into NP rats on day 12 of gestation (NP+RUPP CD4+T cells+CD40L). On day 19 of gestation, blood pressure (MAP), blood, and tissues were collected. MAP was 99±2 in NP (n=13), 116±4 in NP+RUPP CD4+ T cells (n=7; p<0.01); MAP only increased to 104 ±2 in NP+RUPP CD4+Tcells+CD40L (n=24) (p<0.05 vs NP+RUPP CD4+T cells). Mechanisms of hypertension in response to RUPP CD4+ T cells include ET-1, ROS, and AT1-AA were analyzed. Inhibition of CD40 ligand binding reduced placental ET-1 to 2.3 fold above NP rats, and normalized placental ROS from 318.6 ±89 in NP+RUPP CD4+T cells (p<0.05) to 118.7 ±24 in NP+RUPP CD4+T cells+CD40L (p<0.05). AT1-AA was also normalized with inhibition of CD40 ligand. This data suggests that placental ischemia-induced T cell communication via the CD40 ligand is one important mechanism leading to much of the pathophysiology of preeclampsia.
After decades of investigation, the causes of essential hypertension remain obscure. The contribution of the nervous system has been excluded by some on the basis that baroreceptor mechanisms maintain blood pressure only over the short term. However, this point of view ignores one of the most powerful contributions of the brain in maintaining biological fitness-specifically; the capacity to promote adaptation of behavioral and physiological responses to cope with new challenges and maintain this new level of activity through processes involving neuroplasticity. This review presents a body of recent findings demonstrating that experiences encountered earlier in life can produce persistent conditions resulting in an enhanced blood pressure response to hypertension-eliciting stimuli. This sensitized hypertensinogenic state is maintained in the absence of the inducing stimuli, and it is accompanied by sustained up-regulation of components of the brain renin-angiotensin-aldosterone system and other molecular changes recognized to be associated with central nervous system neuroplasticity. While the heritability of hypertension is high, it is becoming increasingly clear that factors beyond just genes contribute to the etiology of this disease. Life experiences and attendant changes in molecular components in the neural network controlling sympathetic tone can enhance the hypertensive response to recurrent, sustained or new stressors. Although the epigenetic mechanisms that allow the brain to be reprogrammed in the face of challenges to cardiovascular homeostasis can be adaptive, this capacity can also be maladaptive under conditions present in different evolutionary eras or ontogenetic periods.
Heat stroke (HS) remains a significant public health concern. Despite the substantial threat posed by HS, there is still no field or clinical test of HS severity. We suggested previously that circulating cardiac troponin (cTnI) could serve as a robust biomarker of HS severity after heating. In the current study, we hypothesized that cTnI band intensity on a portable point-of-care test (ctPOC) could be used to predict severity and organ damage at the onset of HS. Conscious male Fischer 344 rats (N=16) continuously monitored for HR, BP, and core temperature (Tc) (radiotelemetry) were heated to maximum Tc (Tc,Max) of 41.9 ± 0.1°C and recovered undisturbed for 24h at an ambient temperature of 20°C. Blood samples were taken at Tc,Max and 24hrs-post heat via submandibular bleed and analyzed on ctPOC test. POC cTnI band intensity was ranked using a simple 4 point scale via 2 blinded observers, and compared to cTnI levels measured by a clinical blood analyzer. Blood was also analyzed for biomarkers of systemic organ damage. HS severity, as previously defined using HR, BP, and recovery Tc profile during heat exposure, correlated strongly with cTnI (R2 = 0.69) at Tc,Max. POC cTnI band intensity ranking accurately predicted cTnI levels (R2 = 0.64) and HS severity (R2 = 0.83). Five markers of systemic organ damage also correlated with ctPOC score (ALB, ALT, BUN, CHOL, TBIL; R2 > 0.4). This suggests that cTnI POC tests can accurately determine HS severity and could serve as simple, portable, cost effective HS field tests.
The relevance of functional brown adipose tissue (BAT) depots in human adults was undisputedly proven approximately seven years ago. Here we give an overview of all dedicated studies that were published on cold-induced BAT activity in adult humans that appeared since then. Different cooling protocols and imaging techniques to determine BAT activity are reviewed. BAT activation can be achieved by means of air- or water-cooling protocols. The most promising approach is individualized cooling, during which subjects are studied at the lowest temperature for nonshivering condition, probably revealing maximal nonshivering thermogenesis. The highest BAT prevalence (i.e. close to 100%) is observed using the individualized cooling protocol. Currently, the most widely used technique to study the metabolic activity of BAT is [18F]FDG-PET/CT-imaging. Dynamic imaging provides quantitative information about glucose uptake rates, while static imaging reflects overall BAT glucose uptake, localization and distribution. In general, standardized uptake values (SUV) are used to quantify BAT activity. An accurate determination of total BAT volume is hampered by the limited spatial resolution of the PET-image, leading to spill over. Different research groups use different SUV threshold values, which make it difficult to directly compare BAT activity levels between studies. Another issue is the comparison of [18F]FDG uptake in BAT with respect to other tissues or upon with baseline values. This comparison can be performed by using the 'fixed volume' methodology. Finally, the potential use of other relatively noninvasive methods to quantify BAT, like MRI or thermography, is discussed.
Heart failure (HF) is associated with increased cardiac and renal sympathetic drive, which are both independent predictors of poor prognosis. A candidate mechanism for the centrally mediated sympatho-excitation in HF is reduced synthesis of the inhibitory neuromodulator nitric oxide (NO), resulting from down-regulation of neuronal NO synthase (nNOS). We therefore investigated the effects of increasing the levels of NO in the brain, or selectively in the PVN, on cardiac sympathetic nerve activity (CSNA) and baroreflex control of CSNA and heart rate in ovine pacing-induced HF. The resting level of CSNA was significantly higher in the HF than the normal group, but the resting level of RSNA was unchanged. Intracerebroventricular (ICV) infusion of the NO donor sodium nitroprusside (SNP; 500 μg/mL/hr) in conscious normal sheep and HF sheep inhibited CSNA and restored baroreflex control of heart rate, but there was no change in RSNA. Microinjection of SNP into the PVN did not cause a similar cardiac sympatho-inhibition in either group, although the number of nNOS positive cells was decreased in the PVN of sheep in HF. Reduction of endogenous NO with ICV infusion of N-nitro-L-arginine methyl ester decreased CSNA in normal but not HF sheep, and caused no change in RSNA in either group. These findings indicate that endogenous NO in the brain provides tonic excitatory drive to increase resting CSNA in the normal state, but not in HF. In contrast, exogenously administered NO inhibited CSNA in both normal and HF groups via an action on sites other than the PVN.
Insulin administration during insulin-modified intravenous glucose tolerance test (IM-IVGTT) can induce transient hypoglycemia in healthy insulin-sensitive subjects. This triggers counterregulatory responses (CRR), which influence the kinetics of glucose and non-esterified fatty acids (NEFA) and undermines the accuracy of mathematical modeling methods that do not explicitly account for CRR. The aim of this study is to evaluate mathematical models of glucose and NEFA kinetics against experimental data in presence or absence of CRR. Thirteen healthy non-diabetic subjects underwent a standard IM-IVGTT and a modified test (GC-IM-IVGTT) with a variable glucose infusion preventing hypoglycemia. While model predictions fit very well glucose and NEFA data from GC-IM-IVGTT, they lagged behind observations from IM-IVGTT during recovery from hypoglycemia, independently of insulinemia, which did not differ significantly between protocols. A modification to the glucose minimal model, using glucose concentration below a threshold as signal for CRR, improves model predictions for both glucose and NEFA. The associated increase in endogenous glucose production correlates, among various CRR hormones, mainly with the dynamics of glucagon concentration. The modified minimal models introduce new parameters that quantify strength and duration of CRR following hypoglycemia. Although CRR represents an unwanted side effect in IM-IVGTT occurring only in insulin-sensitive subjects, this study provides new insights leading to improved procedures for estimating insulin sensitivity from IM-IVGTT, which may also allow for assessing the individual capacity of recovery from hypoglycemic events in patients treated with insulin or insulin-releasing drugs.
Ischemic heart disease (IHD) is the single most common cause of death. New approaches to enhance myocardial perfusion are needed to improve outcomes for patients with IHD. Thyroid hormones (TH) are known to increase blood flow; however, their usefulness for increasing perfusion in IHD is limited because TH accelerates heart rate which can be detrimental. Therefore, selective activation of TH effects is desirable. We hypothesized that cell-type-specific TH receptor (TR) expression can increase TH action in the heart while avoiding the negative consequences of TH treatment. We generated a binary transgenic mouse (BTG) that selectively expresses TRα1 in endothelial cells in a tetracycline-inducible fashion. In BTG mice endothelial TRα1 protein expression was increased by twofold, which in turn increased coronary blood flow by 77%, coronary conductance by 60%, and coronary reserve by 47% when compared to wild type mice. Systemic blood pressure was decreased by 20% in BTG mice after TRα1 expression. No effects on heart rate were observed. Endothelial TRα1 expression activated AKT/eNOS pathway and increased A2aR adenosine receptor. Furthermore, hearts from BTG mice overexpressing TRα1 submitted to 20 min. ischemia and 20 min. reperfusion showed a 20% decline in left ventricular pressure (LVP) compared with control mice where LVP was decreased by 42%. Studies using an infarction mouse model demonstrated that endothelial overexpression of TRα1 decreased infarct size by 45%. In conclusion, selective expression of TRα1 in endothelial cells protects the heart against injury after an ischemic insult and does not result in adverse cardiac or systemic effects.
The aim of this study was to investigate effects and mechanisms of electroacupuncture (EA) on blood glucose and insulin sensitivity in mice fed with a high fat diet. Methods: Both wild type (WT) and adipose Ecto-nucleotide pyrophosphate phosphodiesterase (ENPP1) transgenic (TG) mice were fed with high-fed diet for 12 weeks; each mouse was studied intra-peritoneal glucose tolerance test (IPGTT) and insulin tolerance test (ITT) with or without EA at abdomen or auricular areas. Results: 1). High fat diet induced insulin resistance in both WT and TG mice. 2). In the WT mice, EA at 3Hz and 15Hz but not at 1Hz or 100Hz via CV4+CV12 significantly reduced postprandial glucose levels; EA at 3Hz was most potent. The glucose level was reduced by 61.7% at 60 min and 74.5% at 120 min with EA at 3 Hz (all P<0.001 vs. control). 2) Similar hypoglycemic effect was noted in the TG mice; 3) on contrary, EA at auricular points increased postprandial glucose level (P<0.03). 4). EA at 3Hz via CV4+CV12 significantly enhanced the decrease of blood glucose after insulin injection, suggesting improvement of insulin sensitivity. 4). Plasma free fatty acid was significantly suppressed by 42.5% at 15 min and 50.8% at 30 min with EA (P < 0.01) in both WT and TG mice. Conclusions: EA improves glucose tolerance in both WT and TG mice fed with high fat diet, and the effect is associated with stimulation parameters and acupoints, and is probably attributed to the reduction of free fatty acid.
Initial studies found that female Dahl salt-sensitive (DS) rats exhibit greater blood pressure (BP) salt sensitivity than female spontaneously hypertensive rats (SHR). Based on the central role played by NO in sodium excretion and BP control, we further tested the hypothesis that blunted increases in BP in female SHR will be accompanied by greater increases in renal inner medullary NOS activity and expression in response to a HS diet compared to DS rats. Gonad-intact and ovariectomized (OVX) female SHR and DS rats were placed on normal salt (NS; 0.4% salt) or HS (4% salt) diet for 2 weeks. OVX did not alter BP in SHR and HS diet produced a modest increase in BP. OVX significantly increased BP in DS rats on NS; HS further increased BP in all DS rats although OVX had a greater increase in BP. Renal inner medullary NOS activity, total NOS3 protein and NOS3 phosphorylated on serine residue 1177 were not altered by salt or OVX in either strain. NOS1 protein expression, however, significantly increased with HS only in SHR and this corresponded to an increase in urinary nitrate/nitrite excretion. SHR also exhibit greater NOS1 and NOS3 protein expression than DS rats. These data indicate that female sex hormones offer protection against HS mediated elevations in BP in DS rats but not SHR. We propose that the relative resistance to HS-mediated increases in BP in SHR is related to greater NOS expression and the ability to increase NOS1 protein expression compared to DS rats.
The endogenous lipid messenger OEA inhibits eating and modulates fat metabolism supposedly through the activation of PPAR-α and vagal sensory fibers. We tested in adult male rats whether OEA stimulates fatty acid oxidation (FAO) and ketogenesis and whether it increases plasma levels of the satiating gut peptides glucagon-like peptide-1 (GLP-1) and peptide tyrosine-tyrosine (PYY). We also explored whether OEA still inhibits eating after subdiaphragmatic vagal deafferentation (SDA). We found that intraperitoneally (IP) injected OEA (10 mg/kg body weight = BW) reduced (P < 0.05) food intake mainly by increasing meal latency and that this effect was stronger in rats fed a 60% high-fat diet (HFD) than in chow-fed rats. OEA increased (P < 0.05) postprandial plasma non-esterified fatty acids and β-hydroxybutyrate (BHB) in the hepatic portal vein (HPV) and vena cava (VC) 30 min after injection, which was more pronounced in HFD- than in chow-fed rats. OEA also increased the protein expression of the key-ketogenetic enzyme, mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase, in the jejunum of HFD-fed rats, but not in the liver or duodenum of either diet group. Furthermore, OEA decreased GLP-1 and PYY concentrations (Ps < 0.05) in the HPV and VC 30 min after administration. Finally, OEA reduced food intake in SDA and sham-operated rats similarly. Our findings indicate that neither intact abdominal vagal afferents nor prandial increases in GLP-1 or PYY are necessary for the satiety effect of OEA. The enhanced FAO and ketogenesis raise the possibility of an involvement of intestinal-derived BHB in OEA's satiety effect under certain conditions.
Rhythms in glucocorticoids are the product of interactions between the hypothalamic-pituitary-adrenal (HPA) axis and the mammalian clock gene system. The adrenal clock drives the glucocorticoid rhythm that synchronizes other peripheral clocks to maintain homeostasis. Stress acutely activates and chronically upregulates the HPA axis, suggesting that the adrenal clock could be modulated by stress. However, there is no direct evidence that stress affects the adrenal clock rhythm. We tested the hypothesis that a model of chronic subordination stress (CSS) that has a major impact on HPA axis regulation, metabolism and emotional behavior, alters adrenal and pituitary clock gene rhythms. Clock gene rhythms were assessed using mPER2::Luciferase (PER2Luc) knockin mice in which in vitro bioluminescence rhythms reflect the Per2 clock gene expression. PER2Luc mice that experienced CSS for 2 weeks showed positive energy balance reflected by increased body weight and food intake. Additionally, CSS phase-advanced the adrenal (~2h) and the pituitary (~1h) PER2Luc rhythm compared to control mice. The activity rhythm was not affected. The adrenal clock phase shift was associated with increased feed conversion efficiency, suggesting that the metabolic phenotype in CSS mice may be related to altered adrenal clock rhythmicity. Interestingly, a single subordination experience followed by 8h sensory housing also phase advanced the adrenal, but not the pituitary, PER2Luc rhythm. Overall, these data demonstrate a stress-induced phase shift in a peripheral clock gene rhythm and differential stress-sensitivity of two peripheral clocks within the HPA axis, suggesting a link between clock desynchrony and individual vulnerability to stress.
We determined the contribution of vascular BK and L-type Ca2+ channel dysregulation to exaggerated mortality in cecal ligation/puncture (CLP)-induced septic BK channel β1-subunit knockout (BK β1-KO, smooth muscle specific) mice. CLP-induced hemodynamic changes and mortality were assessed over 7 days in wild-type (WT) and BK β1-KO mice that were either untreated, given volume resuscitation (saline) or saline + calcium channel blocker nicardipine. Some mice were sacrificed 24 hours post-CLP to measure tissue injury, vascular and immune responses. CLP-induced hypotension was similar in untreated WT and BK β1-KO mice, but BK β1-KO mice died sooner. At 24 hours post-CLP (mortality latency in BK β1-KO mice), untreated CLP-BK β1-KO mice showed more severe hypothermia, lower tissue perfusion, polymorphonuclear neutrophil infiltration-independent severe intestinal necrosis, and higher serum cytokine levels than CLP-WT mice. Saline resuscitation improved survival in CLP-WT, but not CLP-BK β1-KO mice. Saline + nicardipine treated CLP-BK β1-KO mice exhibited longer survival times, higher tissue perfusion, less intestinal injury and lower cytokines vs. untreated CLP-BK β1-KO mice. These improvements were absent in treated CLP-WT mice, although saline + nicardipine improved blood pressure similarly in both septic mice. At 24 hours post-CLP, BK and L-type Ca2+ channel functions in vitro were maintained in mesenteric arteries from WT mice. Mesenteric arteries from BK β1-KO mice had blunted BK/enhanced L-type Ca2+ channel function. We conclude that vascular BK channel deficiency exaggerates mortality in septic BK β1-KO mice by activating L-type Ca2+ channels leading to blood pressure-independent tissue ischemia.
Repeated stress and chronically elevated glucocorticoids cause exaggerated cardiovascular responses to novel stress, elevations in baseline blood pressure and increased risk for cardiovascular disease. We hypothesized that elevated corticosterone (Cort) within the dorsal hindbrain (DHB): (1) enhances arterial pressure and neuroendocrine responses to novel and repeated restraint stress, (2) increases c-Fos expression in regions of the brain involved in sympathetic stimulation during stress and, (3) recruits a vasopressin-mediated blood pressure response to acute stress. Small pellets made of 10% Cort were implanted on the surface of the DHB in male Sprague-Dawley rats. Blood pressure was measured by radiotelemetry. Cort concentration was increased in the DHB in Cort-treated compared with Sham-treated rats (60±15 vs. 14±2 ng Cort/g of tissue, P<0.05). DHB Cort significantly increased the integrated arterial pressure response to 60 min of restraint stress on days 6, 13 and 14 following pellet implantation (e.g. 731±170 vs. 1204±68 mmHg/60 min in Sham- vs. Cort-treated rats, day 6, P<0.05). Cort also increased baseline blood pressure by day 15 (99±2 vs. 108±3 mmHg for Sham- vs. Cort-treated rats, P<0.05) and elevated baseline plasma norepinephrine and neuropeptide Y concentrations. Cort significantly enhanced stress-induced c-Fos expression in vasopressin-expressing neurons in the paraventricular nucleus of the hypothalamus, and blockade of peripheral vasopressin V1 receptors attenuated the effect of DHB Cort to enhance the blood pressure response to restraint. These data indicate that glucocorticoids act within the DHB to produce some of the adverse cardiovascular consequences of chronic stress in part by a peripheral vasopressin-dependent mechanism.
Nesfatin-1/NucB2, an anorexigenic molecule, is expressed mainly in the hypothalamus, particularly in the supraoptic nucleus (SON) and the paraventricular nucleus (PVN). Nesfatin-1/NucB2 is also expressed in the subfornical organ (SFO). Because the SON and PVN are involved in body fluid regulation, nesfatin-1/NucB2 may be involved in dehydration-induced anorexia. To clarify the effects of endogenous nesfatin-1/NucB2, we studied changes in nesfatin-1/NucB2 mRNA levels in the SFO, SON, and PVN in adult male Wistar rats after exposure to osmotic stimuli by using in situ hybridization histochemistry. Significant increases in nesfatin-1/NucB2 mRNA levels, about 2- to 3-folds compared to control, were observed in the SFO, SON, and PVN following water deprivation for 48 h, consumption of 2% NaCl hypertonic saline in drinking water for 5 days, and polyethylene glycol-induced hypovolemia. In addition, nesfatin-1/NucB2 expression was increased in response to water deprivation in a time-dependent manner. These changes in nesfatin-1/NucB2 mRNA expression were positively correlated with plasma sodium concentration, plasma osmolality, and total protein levels in all the examined nuclei. Immunohistochemistry for nesfatin-1/NucB2 revealed that nesfatin-1/NucB2 protein levels were also increased after 48 h of dehydration and attenuated by 24 h of rehydration. Moreover, intracerebroventricular administration of nesfatin-1/NucB2 neutralizing antibody after 48 h of water deprivation resulted in a significant increase in food intake compared to administration of vehicle alone. These results suggested that nesfatin-1/NucB2 is a crucial peptide in dehydration-induced anorexia.
Mindfulness meditation (MM) is a stress-reduction technique that may have real biological effects on hemodynamics, but has never previously been tested in chronic kidney disease (CKD). In addition, the mechanisms underlying the potential BP-lowering effects of MM are unknown. We sought to determine if MM acutely lowers BP in CKD patients, and if these hemodynamic changes are mediated by a reduction in sympathetic nerve activity. In 15 hypertensive African-American (AA) males with CKD, we conducted a randomized, crossover study in which participants underwent 14 minutes of MM, or 14 minutes of BP education (control intervention) during 2 separate random-order study visits. Muscle sympathetic nerve activity (MSNA), beat-to-beat arterial BP, heart rate (HR), and respiratory rate (RR) were continuously measured at baseline, and during each intervention. A subset had a third study visit to undergo controlled breathing (CB), to determine if a reduction in RR alone was sufficient in exacting hemodynamic changes. We observed a significantly greater reduction in systolic BP, diastolic BP, mean arterial pressure, HR, as well as MSNA, during MM compared to the control intervention. Participants had a significantly lower RR during MM; however, in contrast to MM, CB alone did not reduce BP, HR, or MSNA. MM acutely lowers BP and HR in AA males with hypertensive CKD, and these hemodynamic effects may be mediated by a reduction in sympathetic nerve activity. RR is significantly lower during MM, but CB alone without concomitant meditation does not acutely alter hemodynamics or sympathetic activity in CKD.
Epidemiological and experimental evidence suggests that a sub-optimal environment during perinatal life programs offspring susceptibility to the development of metabolic syndrome and Type 2 diabetes. We hypothesized that the lasting impact of perinatal protein deprivation on mitochondrial fuel oxidation and insulin sensitivity would depend on the time-window of exposure. To improve our understanding of underlying mechanisms, an integrative approach was used, combining the assessment of insulin sensitivity and untargeted mass spectrometric-based metabolomics in the offspring. A hyperinsulinemic-euglycemic clamp was performed in adult male rats born from dams fed a low protein diet during gestation and/or lactation, and subsequently exposed to a Western diet (WD) for 10 weeks. Metabolomics was combined with targeted acylcarnitine profiling and analysis of liver gene expression to identify markers of adaptation to WD that influence the phenotype outcome evaluated by body composition analysis. At adulthood offspring of protein-restricted dams had impaired insulin secretion when kept under standard diet. Moreover, rats who demonstrated catch-up growth at weaning displayed higher gluconeogenesis and branched-chain amino acid catabolism, and lower fatty acid β-oxidation compared to control rats. Post-weaning exposure of IUGR-born rats to a WD exacerbated incomplete fatty acid β-oxidation and excess fat deposition. Control offspring nursed by protein-restricted mothers showed peculiar low fat accretion through adulthood and preserved insulin sensitivity even after WD-exposure. Altogether our findings suggest testable hypothesis about how maternal diet might influence metabolic outcomes (insulin sensitivity) in the next generation such as mitochondrial overload and/or substrate oxidation inflexibility dependent on the time-window of perinatal dietary manipulation.
The present study was designed to investigate the role of the medial preoptic nucleus (MPO) as a site of the thermogenic and metabolic effects of the αMSH analog Melanotan II (MTII). We also assessed the involvement of the dorsomedial hypothalamic nucleus (DMH) by investigating the effects of the MPO infusion of MTII in rats with DMH lesions produced by kainic acid. Infusion of MTII in the MPO led to increases in interscapular BAT (iBAT) temperature and iBAT uptake of 14C-bromopalmitate. Both increases were blocked by DMH lesions. iBAT temperature increase (area under curve) and 14C-bromopalmitate uptake emerged as two correlated variables (r = 0.63, P < 0.001). DMH lesions also blocked MTII-induced expression of mRNAs coding for proteins involved in (i) thermogenesis [type II iodothyronine deiodinase (Dio2) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (Pgc1α)], (ii) lipolysis [hormone sensitive lipase (Hsl)] and (iii) lipogenesis [diacylglycerol O-acyltransferase 2 (Dgat2), fatty acid synthase (Fas)], in iBAT of rats killed one hour after MPO infusion of MTII. MTII also stimulated expression of genes in iWAT but only in rats with DMH lesions. These genes included glucose transporter member 4 (Glut4), glycerol-3-phosphate acyltransferase 3 (Gpat3), Dgat1, Dgat2, triglyceride lipase (Atgl), Hsl and carnitine palmitoyltransferase 1β (Cpt1β). Altogether, the present results reveal the MPO as a site of the thermogenic and metabolic actions of MTII. They also contribute to establish the MPO-DMH duet as a significant target for melanocortins to modulate energy homeostasis.
Parameters of glucose dynamics recorded by the continuous glucose monitoring system (CGMS) could help the controlling of glycemic fluctuations that is important in diabetes management. Multiscale entropy (MSE) analysis has recently been developed to measure the complexity of physical and physiological time sequences. A reduced MSE complexity index indicates the increased repetition patterns of the time sequence, and thus a decreased complexity in this system. No study investigated the MSE analysis of glucose dynamics in diabetes. This study was designed to compare the complexity of glucose dynamics between the diabetic patients (n = 17) and the control subjects (n = 13) matched for sex, age and body mass index via MSE analysis using CGMS data. Compared with the control subjects, the diabetic patients revealed a significant increase (P < 0.001) in the mean (diabetic patients 166.0 ± 10.4 versus control subjects 93.3 ± 1.5 mg/dL), standard deviation (51.7 ± 4.3 versus 11.1 ± 0.5 mg/dL) and mean amplitude of glycemic excursions (127.0 ± 9.2 versus 27.7 ± 1.3 mg/dL) of the glucose levels; and a significant decrease (P < 0.001) in the MSE complexity index (5.09 ± 0.23 versus 7.38 ± 0.28). In conclusion, the complexity of glucose dynamics is decreased in diabetes. This finding implies the reactivity of glucoregualtion is impaired in the diabetic patients. Such impairment presenting as an increased regularity of glycemic fluctuating pattern could be detected by MSE analysis. Thus, the MSE complexity index could potentially be used as a biomarker in the monitoring of diabetes.
The reported effects of atrial natriuretic peptide (ANP) on sympathetic nerve activity (SNA) are variable, dependent on concomitant hemodynamic actions and likely to be regionally differentiated. There are few reports of the effect of B-type natriuretic peptide (BNP) on SNA and none have measured cardiac SNA (CSNA) by direct microneurography. We measured the effects of low dose ANP and BNP (2.4pmol/kg/min infused for 120 min) on CSNA and hemodynamics in conscious sheep (n=8). Whilst there was a trend for mean arterial pressure and cardiac output to fall with both ANP and BNP, changes were not significant compared with vehicle control. However, BNP did significantly reduce systolic arterial (97±4.2 vs 107±6.8mmHg during control; p=0.043) and pulse pressures (0.047) and increase heart rate (110±6.7 vs 96±7.3bpm; p=0.044). Trends for these hemodynamic parameters to change with ANP did not achieve statistical significance. ANP also had no significant effect on any CSNA parameters measured. In contrast, BNP induced a rise in both CSNA burst frequency (~20 bursts/min higher than control, p=0.011) and burst area (~40% higher than control, p=0.013). BNP-induced rises in burst incidence (bursts/100 beats) and burst area/100 beats, however, were not significant. In conclusion, BNP infused at low doses that only had subtle effects on hemodynamics increased CSNA burst frequency and burst area/min. This increase in CSNA may in large part be secondary to an increase in heart rate as CSNA burst incidence and burst area/100beats were not significantly increased. This study provides no evidence for inhibition of CSNA by natriuretic peptides.
The stress response in Drosophila melanogaster reveals sex differences in behavior, similar to what has been observed in mammals. However, unlike mammals, the sex determination pathway in Drosophila is well established, making this an ideal system to identify factors involved in the modulation of sex-specific responses to stress. In this study, we show that the Drosophila fat body, which has been shown to be important for energy homeostasis and sex determination, is a dynamic tissue that is altered in response to stress in a sex and time dependent manner. We manipulated the sex determination pathway in the fat body via targeted expression of transformer and transformer-2, and analyzed these animals for changes in their response to stress. In the majority of cases, manipulation of transformer or transformer-2 was able to change the physiological output in response to starvation and oxidative stress to that of the opposite sex. Our data also uncover the possibility of additional downstream targets for transformer and transformer-2 that are separate from the sex determination pathway and can influence behavioral and physiological responses.
Circadian rhythms are essential to cardiovascular health and disease. Temporal coordination of cardiac structure and function has focussed primarily at the physiologic and gene expression levels, but these analyses are invariably incomplete not the least because proteins underlie many biological processes. The purpose of this study was to reveal the diurnal cardiac proteome and important contributions to cardiac function. The 24 hour (h) day/night murine cardiac proteome was assessed by 2-dimensional difference in gel electrophoresis (2D-DIGE) and liquid chromatography-mass spectrometry. Daily variation was considerable, as ~7.8% (90/1147) of spots exhibited statistical changes at paired times across the 24h light (L) dark (D) cycle. JTK_CYCLE was used to investigate underlying diurnal rhythms in corresponding mRNA. We next revealed that disruption of the L:D cycle altered protein profiles, and diurnal variation in cardiac function in Langendorff-perfused hearts, relative to the L:D cycle. To investigate the role of the circadian clock mechanism, we used cardiomyocyte clock mutant (CCM) mice. CCM myofilaments exhibited a loss of time-of-day-dependent maximal calcium-dependent ATP consumption, and altered phosphorylation rhythms. Moreover, the cardiac proteome was significantly altered in CCM hearts, especially enzymes regulating vital metabolic pathways. Lastly, we used a model of pressure overload cardiac hypertrophy to demonstrate the temporal proteome during heart disease. Our studies demonstrate that time of day plays a direct role in cardiac protein abundance, and indicate a novel mechanistic contribution of circadian biology to cardiovascular structure and function.
Changes in the maternal nutritional environment during fetal development can influence offspring metabolic risk in later life. Animal models have demonstrated that offspring of diet-induced obese dams develop metabolic complications, including non-alcoholic fatty liver disease. In this study we investigated the mechanisms in young offspring that lead to the development of NAFLD. Female offspring of C57BL/6J dams fed either a control or obesogenic diet were studied at 8 weeks of age. We investigated the roles of oxidative stress and lipid metabolism in contributing to offspring fatty liver. There were no differences in body weight or adiposity at 8 weeks of age; however offspring of obese dams were hyperinsulinemic. Oxidative damage markers were significantly increased in their livers, with reduced levels of the antioxidant enzyme glutathione peroxidase-1. Mitochondrial complex I and II activities were elevated, while levels of mitochondrial cytochrome c were significantly reduced and glutamate dehydrogenase was significantly increased, suggesting mitochondrial dysfunction. Offspring of obese dams also had significantly greater hepatic lipid content, associated with increased levels of PPAR and reduced triglyceride lipase. Liver glycogen and protein content were concomitantly reduced in offspring of obese dams. In conclusion, offspring of diet-induced obese dams have disrupted liver metabolism and develop NAFLD prior to any differences in body weight or body composition. Oxidative stress may play a mechanistic role in the progression of fatty liver in these offspring.
Effective treatment of sepsis remains a significant challenge in intensive care units. During sepsis there is widespread activation of the sympathetic nervous system, which is thought to have both beneficial and detrimental effects. The sympathoexcitation is thought to be partly due to the developing hypotension, but may also be a response to the inflammatory mediators released. Thus, we investigated whether intracarotid infusion of prostaglandin E2 (PGE2) induced similar cardiovascular changes to those caused by intravenous infusion of E. coli in sheep, and whether inhibition of prostaglandin synthesis, with the non-selective cyclooxygenase (COX) inhibitor indomethacin, administered at two and eight hours after the onset of sepsis, reduced sympathetic nerve activity (SNA) and heart rate (HR). Studies were performed in conscious sheep instrumented to measure mean arterial pressure (MAP), HR, cardiac SNA (CSNA) and renal SNA (RSNA). Intracarotid infusion of PGE2 (50 ng/kg/min) increased temperature, CSNA and HR, but not MAP or RSNA. Sepsis, induced by infusion of E. coli, increased CSNA, but caused an initial, transient inhibition of RSNA. At two hours of sepsis, indomethacin (1.25 mg/kg bolus) increased MAP and caused reflex decreases in HR and CSNA. After eight hours of sepsis, indomethacin did not alter MAP, but reduced CSNA and HR, without altering baroreflex control. These findings indicate an important role for prostaglandins in mediating the increase in CSNA and HR during the development of hyperdynamic sepsis, whereas prostaglandins do not have a major role in determining the early changes in RSNA.
The Kölliker-Fuse region (KF) and the lateral parabrachial nucleus (LPBN) have been implicated in the maintenance of cardiorespiratory control. Here, we evaluated the involvement of the KF region and the LPBN in cardiorespiratory responses elicited by chemoreceptor activation in unanesthetized rats. Male Wistar rats (280-330g, n=5-9/group) with bilateral stainless steel guide-cannulas implanted in the KF region or the LPBN were used. Injection of muscimol (100 and 200 pmol/100 nl) in the KF decreased resting ventilation (1140 ± 68 and 978 ± 100, vs. saline: 1436 ± 155 ml/kg/min), without changing mean arterial pressure (MAP) and heart rate (HR). Bilateral injection of the GABA-A antagonist bicuculline (1 nmol/100 nl) in the KF blocked the inhibitory effect on ventilation (1418 ± 138, vs. muscimol: 978 ± 100 ml/kg/min) elicited by muscimol. Muscimol injection in the KF reduced the increase in ventilation produced by hypoxia (8% O2) (1827 ± 61, vs. saline: 3179 ± 325 ml/kg/min) or hypercapnia (7% CO2) (1488 ± 277, vs. saline: 3539 ± 374 ml/kg/min) in unanesthetized rats. Bilateral injection of bicuculline in the KF blocked the decrease in ventilation produced by muscimol in the KF during peripheral or central chemoreflex activation. Bilateral injection of muscimol in the LPBN did not change resting ventilation or the increase in ventilation elicited by hypoxia or hypercapnia. The results of the present study suggest that the KF region, but not LPBN, have mechanisms to control ventilation in resting, hypoxic or hypercapnic conditions in unanesthetized rats.
Urinary bladder dysfunction presents a major problem in the clinical management of patients suffering from pathological conditions and neurological injuries or disorders. Currently, the etiology underlying altered visceral sensations from the urinary bladder that accompany the chronic pain syndrome, bladder pain syndrome (BPS)/interstitial cystitis (IC), is not known. Bladder irritation and inflammation are histopathological features that may underlie BPS/IC that can change the properties of lower urinary tract sensory pathways (e.g., peripheral and central sensitization, neurochemical plasticity) and contribute to exaggerated responses of peripheral bladder sensory pathways. Among the potential mediators of peripheral nociceptor sensitization and urinary bladder dysfunction are neuroactive compounds (e.g., purinergic and neuropeptide/receptor pathways), sensory transducers (e.g., transient receptor potential channels) and target-derived growth factors (e.g., nerve growth factor). We review studies related to the organization of the afferent limb of the micturition reflex and discuss neuroplasticity in an animal model of urinary bladder inflammation to increase the understanding of functional bladder disorders and to identify potential novel targets for development of therapeutic interventions. Given the heterogeneity of BPS/IC and the lack of consistent treatment benefits, it is unlikely that a single treatment directed at a single target in micturition reflex pathways will have a mass benefit. Thus, identification of multiple targets is a prudent approach and use of cocktail treatments directed at multiple targets should be considered.
Bioactive oxidized linoleic acid metabolites (OXLAMs) include 13- and 9-hydroxy-octadecadienoic acid (13-HODE + 9-HODE), and have been linked to oxidative stress, inflammation, and pathological and physiological states. The purpose of this study was to measure changes in plasma 13-HODE + 9-HODE following a 75-km cycling bout and identify potential linkages to linoleate metabolism and established biomarkers of oxidative stress (F2-isoprostanes) and inflammation (cytokines) using a metabolomics approach. Trained male cyclists (N=19, age 38.0±1.6 y, wattsmax 304±10.5) engaged in a 75-km cycling time trial on their own bicycles using electromagnetically-braked cycling ergometers (2.71±0.07 h). Blood samples were collected pre-exercise, and immediately post-, 1.5-h post-, and 21-h post-exercise, and analyzed for plasma cytokines (IL-6, IL-8, IL-10, TNFα, MCP-1, GCSF), F2-isoprostanes, and shifts in metabolites using global metabolomics procedures with GC–MS and LC–MS. 13-HODE + 9-HODE increased 3.1-fold and 1.7-fold immediately post- and 1.5-h post-exercise (both p<0.001), and returned to pre-exercise levels by 21-h post-exercise. Post-75-km cycling plasma levels of 13-HODE + 9-HODE were not significantly correlated with increases in plasma cytokines, but were positively correlated with post-exercise F2-isoprostanes (r=0.75, p<0.001), linoleate (r=0.54, P=0.016), arachidate (r=0.77, p<0.001), 12,13-dihydroxy-9Z-octadecenoate (12,13-DiHOME) (r=0.60, p=0.006), dihomo-linolenate (r=0.57, p=0.011), and adrenate (r=0.56, p=0.013). These findings indicate that prolonged and intensive exercise caused a transient, 3.1-fold increase in the stable linoleic acid oxidation product 13-HODE + 9-HODE, and was related to increases in F2-isoprostanes, linoleate, and fatty acids in the linoleate conversion pathway. These data support the use of 13-HODE + 9-HODE as an oxidative stress biomarker in acute exercise investigations.
Insulin controls hyperglycemia after severe burns, and its use opposes the hypermetabolic response. Underlying molecular mechanisms are poorly understood and previous research in this area has been limited due to the inadequacy of animal models to mimic the physiologic effects seen in humans with burns. Using a recently published rat model the combines both burn and disuse components, we compare the effects of insulin treatment versus vehicle on glucose tolerance, hypermetabolic response, muscle loss, and circadian-metabolic protein expression after burns. Male Sprague Dawley rats were assigned to three groups: cage-controls (n=6); vehicle-treated (VBH; n=11) and insulin-treated (IBH; n=9). With the exception of cage-controls, rats underwent a 40% TBSA burn with hindlimb unloading, then IBH rats received 12 days of subcutaneous insulin injections (5 units/kg/day), and VBH rats received an equivalent dose of vehicle. Glucose tolerance testing was performed on day 14, after which blood and tissues were collected for analysis. Body mass loss was attenuated by insulin treatment (VBH=265±17 g versus IBH=283±14 g, p=0.016) and glucose clearance capacity was increased. Soleus and gastrocnemius muscle loss was decreased in the IBH group. IRS-1, AKT, FOXO-1, Caspase-3, and PER1 phosphorylation was altered by injury and disuse, with levels restored by insulin treatment in almost all cases. Insulin treatment after burn and disuse attenuated the hypermetabolic response, increased glucose clearance , and normalized circadian-metabolic protein expression patterns. Therapies aimed at targeting downstream effectors may provide the beneficial effects of insulin without hypoglycemic risk.
Tissue hypoxia likely contributes to anemia-induced organ injury and mortality. Severe anemia activates hypoxia-inducible factor (HIF) signaling by hypoxic- and neuronal nitric oxide synthase- (nNOS) dependent mechanisms. However, organ-specific hemoglobin (Hb) thresholds for increased HIF expression have not been defined. To assess organ specific Hb thresholds for tissue hypoxia, HIF-α (ODD) luciferase mice were hemodiluted to mild, moderate, or severe anemia corresponding to Hb levels of 90, 70, and 50g/L, respectively. HIF luciferase reporter activity, HIF protein, and HIF-dependent RNA levels were assessed. In the brain, HIF-1α was paradoxically decreased at mild anemia, returned to baseline at moderate anemia, and then increased at severe anemia. Brain HIF-2α remained unchanged at all Hb levels. Both kidney HIF-1α and -2α increased earlier (Hb~70-90g/L) in response to anemia. Liver also exhibited an early HIF-1α response. Carotid blood flow was increased early (Hb~70g/L) but renal blood flow remained relatively constant, only increased at Hb of 50g/L. Anemia increased nNOS (brain and kidney) and eNOS (kidney) levels. Whereas anemia-induced increases in brain HIFα were nNOS-dependent, our current data demonstrate that increased renal HIFα was nNOS-independent. HIF-dependent RNA levels increased linearly (~10 fold) in the brain. However, renal HIF-RNA responses (MCT4, EPO) increased exponentially (~100 fold). Plasma EPO levels increased near Hb threshold of 90g/L, suggesting that the EPO response is sensitive. Collectively, these observations suggest that each organ expresses a different threshold for cellular HIF/NOS hypoxia responses. This knowledge may help define the mechanism(s) by which the brain and kidney maintain oxygen homeostasis during anemia.
Beta-mercaptoacetate (MA) is a drug known to block mitochondrial oxidation of medium and long chain fatty acids (FAs) and to stimulate feeding. Because MA-induced feeding is vagally-dependent, it has been assumed that the feeding response is mediated by MA's antimetabolic action at a peripheral, vagally-innervated site(s). However, MA's site of action has not yet been identified. Therefore, we used fluorescent calcium measurements in isolated neurons from rat nodose ganglia to determine whether MA has direct effects on vagal sensory neurons. We found that MA alone did not alter cytosolic calcium concentrations in nodose neurons. However, MA (60 µM to 6 mM) significantly decreased calcium responses to both linoleic acid (LA, 10 µM) and caprylic acid (C8, 10 μM) in all neurons responsive to LA and C8. GW9508 (40 μM), an agonist of the FA receptor, G-protein coupled receptor 40 (GPR40), also increased calcium levels almost exclusively in FA-responsive neurons. MA significantly inhibited this response to GW9508. MA did not inhibit calcium responses to serotonin, high K+, or capsaicin, which do not utilize GPRs, or to cholecystokinin, which acts on a different GPR. GPR 40 was detected in nodose ganglia by RT-PCR. Results suggest that FAs directly activate vagal sensory neurons via GPR40, and that MA antagonizes this effect. Thus, we propose that MA's nonmetabolic actions on GPR40 membrane receptors, expressed by multiple peripheral tissues in addition to the vagus nerve, may contribute to or mediate MA-induced feeding.
Increasing evidence suggests that TRB3, a mammalian homolog of Drosophila tribbles, plays an important role in cell growth, differentiation and metabolism. In the liver, TRB3 binds and inhibits Akt activity, whereas in adipocytes, TRB3 upregulates fatty acid oxidation. In cultured muscle cells, TRB3 has been identified as a potential regulator of insulin signaling. However, little is known about the function and regulation of TRB3 in skeletal muscle in vivo. In the current study, we found that four weeks of voluntary wheel running (6.6±0.4 km/day) increased TRB3 mRNA by 1.6-fold and protein by 2.5-fold in triceps muscle. Consistent with this finding, muscle-specific transgenic mice that overexpress TRB3 (TG) had a pronounced increase in exercise capacity compared to wild type (WT) littermates (TG:1535 ± 283; WT:644 ± 67 joules). The increase in exercise capacity in TRB3 TG mice was not associated with changes in glucose uptake or glycogen levels; however, these mice displayed a dramatic shift toward a more oxidative/fatigue-resistant (Type I/ IIA) muscle fiber type, including 3-fold more type I fibers in soleus muscles. Skeletal muscle from TRB3 TG mice had significantly decreased PPARα expression, 2-fold higher levels of miR208b and miR499, and corresponding increases in the myosin heavy chain isoforms Myh7 and Myb7b which encode these microRNAs. These findings suggest that TRB3 regulates muscle fiber type via a PPARα-regulated miR499/miR208b pathway, revealing a novel function for TRB3 in the regulation of skeletal muscle fiber type and exercise capacity.
Greater peripheral quadriceps fatigue at the voluntary termination of single-leg knee-extensor exercise (KE), in comparison to whole-body cycling, has been attributed to confining group III and IV skeletal muscle afferent feedback to a small muscle mass, enabling the central nervous system (CNS) to tolerate greater peripheral fatigue. However, as task specificity and vastly differing systemic challenges may have complicated this interpretation, 8 males were studied during constant workload trials to exhaustion at 85% of peak workload during single- and double-leg KE. It was hypothesized that due to the smaller muscle mass engaged during single-leg KE, a greater magnitude of peripheral quadriceps fatigue would be present at exhaustion. Vastus lateralis integrated electromyogram (iEMG) signal relative to the first minute of exercise, pre- to post-exercise maximal voluntary contractions (MVCs) of the quadriceps, and twitch-force evoked by supramaximal magnetic femoral nerve stimulation (Qtw,pot) quantified peripheral quadriceps fatigue. Trials performed with single-leg KE (8.1±1.2 min; 45±4 W) resulted in significantly greater peripheral quadriceps fatigue than double-leg KE (10±1.3 min; 83±7 W), as documented by changes in the iEMG signal (147±24 vs. 85±13%), MVC (-25±3 vs. -12±3%), and Qtw,pot (-44±6 vs. -33±7%), for single- and double-leg KE, respectively. Therefore, avoiding concerns over task specificity and cardiorespiratory limitations, this study reveals that a reduction in muscle mass permits the development of greater peripheral muscle fatigue and supports the concept that the CNS tolerates a greater magnitude of peripheral fatigue when the source of group III/IV afferent feedback is limited to a small muscle mass.
Insulin stimulates nerve arterial vasodilation through a nitric-oxide synthase (NOS) mechanism. Experimental diabetes reduces vasa nervorum NO-reactivity. Studies investigating hyperglycemia and nerve arterial vasodilation typically omit insulin-treatment and use sedentary rats resulting in severe hyperglycemia. We tested the hypotheses that i) insulin-treated experimental diabetes and inactivity (DS) will attenuate insulin-mediated nerve arterial vasodilation and ii) deficits in vasodilation in DS rats will be overcome by concurrent exercise training (DX; 75-85% VO2max, 1 h/d, 5 d/week, for 10 weeks). The baseline index of vascular conductance values (iVC = nerve blood flow velocity/mean arterial blood pressure) were similar (P≥0.68), but peak iVC and the area under the curve (AUCi) for the iVC during a euglycemic hyperinsulinemic clamp (EHC; 10 mU/kg/min), were lower in DS rats vs. control sedentary (CS) rats and DX rats (P≤0.01). Motor nerve conduction velocity (MNCV) was lower in DS rats vs. CS rats and DX rats (P≤0.01). Compared with DS rats, DX rats expressed greater nerve endothelial NOS (eNOS) protein content (P=0.04). In a separate analysis, we examined the impact of diabetes in exercise trained rats alone. Compared with exercise trained control rats (CX), DX rats had a lower AUCi during the EHC, lower MNCV values and lower sciatic nerve eNOS protein content (P≤0.03). Therefore, vasa nervorum and motor nerve function are impaired in DS rats. Such deficits in rats with diabetes can be overcome by concurrent exercise training. However, in exercise trained rats (CX and DX groups), poorly controlled blood glucose lowers vasa nervorum and nerve function.
Both human and animal studies have demonstrated that respiratory parameters change in response to presentation of alerting stimuli as well as during stress; yet central neuronal pathways that mediate such responses remain unknown. The aim of our study was to investigate the involvement of the amygdala in mediating respiratory responses to stressors of various intensities and duration. Adult male Wistar rats (n=8) received microinjections of GABA-A agonist muscimol or saline into the amygdala bilaterally and were subjected to a respiratory recording using whole-body plethysmography. Presentation of acoustic stimuli (500-ms white noise, 40-90 dB) caused transient responses in respiratory rate and in tidal volume that were proportional to the stimulus intensity, ranging from +13 ± 9 cpm to +276 ± 67 cpm for 40 and 90 dB stimuli, respectively. Inhibition of the amygdala significantly suppressed respiratory rate responses to the high-intensity stimuli (70-90dB). Submitting rats to the restraint stress significantly elevated the mean respiratory rate (+72 ± 8 cpm) and the dominant respiratory rate (+51 ± 12 cpm) as well as the fraction of high-frequency respiratory rate (+10 ± 3 %). Inhibition of the amygdala by muscimol significantly suppressed these responses. We conclude that the amygdala is one of the key structures that are essential for the expression of respiratory responses to stressful or alerting stimuli in rats.
We previously demonstrated white adipose tissue (WAT) innervation using the established WAT retrograde sympathetic nervous system (SNS)-specific transneuronal viral tract tracer, pseudorabies virus (PRV152) and showed its role in the control of lipolysis. Conversely, we demonstrated WAT sensory innervation using the established anterograde sensory system (SS)-specific transneuronal viral tracer, the H129 strain of herpes simplex virus-1, with sensory nerves showing responsiveness with increases in WAT SNS drive. Several brain areas were part of the SNS outflow to and SS inflow from WAT between these studies suggesting SNS-SS feedback loops. Therefore, we injected both PRV152 and H129 into inguinal WAT (IWAT) of Siberian hamsters. Animals were perfused on Days 5 and 6 post-inoculation after H129 and PRV152 injections, respectively, and brains, spinal cords, sympathetic and dorsal root ganglia (DRG) were processed for immunohistochemical detection of each virus across the neuroaxis. The presence of H129+PRV152 colocalized neurons (~50%) in the spinal segments innervating IWAT suggested short SNS-SS loops with significant co-infections (>60%) in discrete brain regions, signifying long SNS-SS loops. Notably, the most highly populated sites with the doubly-infected neurons were the medial part of medial preoptic nucleus, medial preoptic area, hypothalamic paraventricular nucleus, lateral hypothalamus, periaqueductal gray, oral part of the pontine reticular nucleus and the nucleus of the solitary tract. Collectively, these results strongly indicate the neuroanatomical reality of the central SNS-SS feedback loops with short loops in the spinal cord and long loops in the brain, both likely involved in the control of lipolysis or other WAT pad-specific functions.
Two types of AQP5 genes (aqp-xt5a and aqp-xt5b) were identified in the genome of Xenopus tropicalis by synteny comparison and molecular phylogenetic analysis. When the frogs were in water, AQP-xt5a mRNA was expressed in the skin and urinary bladder. The expression of AQP-xt5a mRNA was significantly increased in dehydrated frogs. AQP-xt5b mRNA was also detected in the skin and showed an increase in its expression in response to dehydration. Additionally, AQP-xt5b mRNA began to be slightly expressed in the lung and stomach under dehydration. For the pelvic skin, immunofluorescence staining localized AQP-xt5a and AQP-xt5b to the cytoplasm of secretory cells of the granular glands and the apical plasma membrane of secretory cells of the small granular glands, respectively, in normally hydrated frogs. Under dehydration, the locations of both AQPs in their respective glands did not change, but AQP-xt5a appeared in the cytoplasm of secretory cells of the small granular glands. For the urinary bladder, AQP-xt5a was observed in the apical plasma membrane and cytoplasm of a number of granular cells under normal hydration. In contrast, AQP-xt5a was found in the apical membrane and cytoplasm of most granular cells after dehydration. Injection of vasotocin into hydrated frogs did not induce these changes in the localization of AQP-xt5a in the small granular glands and urinary bladder, however. The results suggest that AQP-xt5a might be involved in water reabsorption from the urinary bladder under dehydration, whereas AQP-xt5b might play a role in water secretion from the small granular gland.
Marinobufagenin (MBG) is a cardiotonic steroid that increases in the circulation in preeclampsia. Preeclampsia and eclampsia are associated with cerebral edema. Therefore, we examined the effects of MBG on human brain microvascular endothelial cells (HBMEC) in vitro. MBG enhanced the permeability of HBMEC monolayers at 1, 10 and 100 nM doses, but had no effect at 0.1 nM. Agilent Human Gene Expression microarrays were utilized in these studies. MBG treatment (10 nM for 12 h) down-regulated concentrations of the soluble VEGFR transcript sFLT by 59%, but did not alter those of FLTv3 mRNA (determined by quantitative PCR). When treated and control HBMEC transcriptomes were interrogated on microarrays, 1069 genes appeared to be regulated by MBG. Quantitative RT-PCR confirmed that MBG treatment up-regulated ENKUR mRNA concentrations by 57%. Its protein product interacts with calmodulin and calcium channel proteins. MBG treatment down-regulated several genes whose protein products are involved in cell adhesion (ITGA2B, FERMT1, CLDN16 and TMEM207) and cell signaling (GRIN2C, SLC8A1 and ESR1). The level of down-regulation ranged from 22 to 66%. Altogether, MBG actively enhanced the permeability of HBMEC monolayers while down-regulating genes involved in adhesion. MBG treatment had variable effects on ENKUR, GRIN2C and SLC8A1 genes, all associated with calcium transport. These studies provide the basis for future investigations of MBG actions in normal physiology and disease.
Although sensitivity to high dietary NaCl is regarded to be a risk factor for cardiovascular disease, the causes of salt-sensitive hypertension remain elusive. Previously we have shown that rats pretreated with sub-pressor doses of either angiotensin II (ANG II) or aldosterone (Aldo) show sensitized hypertensive responses to a mildly pressor dose of ANG II when tested after an intervening delay. The current studies investigated whether such treatments will induce salt-sensitivity. In studies employing an Induction-Delay-Expression experimental design, male rats were instrumented for chronic blood pressure (BP) recording. In separate experiments, ANG II, Aldo or vehicle were delivered either subcutaneously or intracerebroventricularly during Induction. There were no sustained differences in BP during Delay prior to being given 2% saline. While consuming 2% saline during Expression, both ANG II- and Aldo-pretreated rats showed significantly greater hypertension. When hexamethonium was used to assess autonomic control of BP, no differences in the decrease of BP in response ganglionic blockade were detected during Induction. However, during Expression the fall was greater in sensitized rats. In separate experiments, brain tissue collected at the end of Delay showed increases in message or activation of putative markers of neuroplasticity (i.e., brain-derived neurotrophic factor, p38 mitogen-activated protein kinase, cAMP response element-binding protein). These experiments demonstrate that prior administration of non-pressor doses of either ANG II or Aldo will induce salt-sensitivity. Collectively our findings indicate that treatment with sub-pressor doses of ANG II and Aldo initiate central neuroplastic changes that are involved in hypertension of different etiologies.
Lingual lipase generates non esterified fatty acids (NEFA) from dietary fats during oral processing by lipolysis. Lingual lipase in rodents has strong lipolytic activity and plays a critical role in oral detection of fats. The functional activity of lingual lipase during oral processing of high fat foods in humans remains poorly characterized. Five commonly consumed high fat foods varying in physical states and fatty acid composition -almond, almond butter, olive oil, walnut and coconut were masticated by 15 healthy human subjects at the rate of one chew/second with and without a lipase inhibitor, Orlistat. Salivary NEFA concentrations were measured. To determine the role of lingual lipase in oral fat detection, sensory ratings were obtained from the same 15 human subjects for almond butter with and without Orlistat. Lingual lipase was active during oral processing of almond and coconut. No activity of lingual lipase was detected during processing of almond butter. There was only weak evidence lingual lipase is a determinant of oral fat detection. Lingual lipase may only contribute to NEFA generation and oral fat detection of fatty foods that require stronger oral processing effort.
The mechanisms of autonomic imbalance and subsequent cardiovascular manifestations in HIV-1-infected patients are poorly understood. We report here that HIV-1 trans-activator of transcription (Tat, fragment 1-86) produced a concentration-dependent increase in cytosolic Ca2+ in cardiac-projecting parasympathetic neurons of nucleus ambiguus retrogradely labeled with rhodamine. Using stores-specific pharmacological agents, we identified several mechanisms of the Tat-induced Ca2+ elevation: (1) lysosomal Ca2+ mobilization; (2) Ca2+ release via inositol 1,4,5-trisphosphate-sensitive endoplasmic reticulum pools; and (3) Ca2+ influx via transient receptor potential vanilloid type 2 (TRPV2) of nonselective cation channels. Activation of TRPV2, nonselective cation channels, induced a robust and prolonged neuronal membrane depolarization, thus triggering an additional P/Q-mediated Ca2+ entry (4). In vivo microinjection studies indicate a dose-dependent, prolonged bradycardic effect of Tat administration into the nucleus ambiguus of conscious rats, in which neuronal TRPV2 played a major role. Our results support previous studies indicating that Tat promotes bradycardia and consequently may be involved in the QT interval prolongation reported in HIV-infected patients. In the context of an overall HIV-dependent autonomic dysfunction, these Tat-mediated mechanisms may account for the higher prevalence of sudden cardiac death in HIV-1-infected patients as compared with general population with similar risk factors. Our results may be particularly relevant in view of the recent findings that significant Tat levels can still be identified in the cerebrospinal fluid of HIV-infected patients with viral load suppression due to efficient antiretroviral therapy.
Contracting muscle releases interleukin-6 (IL-6) enabling the metabolic switch from carbohydrate to fat utilization. Similarly, metabolism is switched during transition from fed to fasting state. Herein, we examined a putative role for IL-6 in the metabolic adaptation to normal fasting. In lean C57BL/6J mice, 6 hours of food withdrawal increased gene transcription levels of IL-6 in skeletal muscle but not in white adipose tissue. Concomitantly, circulating IL-6 and free fatty acid (FFA) levels were significantly increased, whereas respiratory quotient (RQ) was reduced in 6-hour fasted mice. In white adipose tissue, phosphorylation of hormone-sensitive lipase (HSL) was increased upon fasting, indicating increased lipolysis. Intriguingly, fasting-induced increase in circulating IL-6 levels and parallel rise in FFA concentration were absent in obese and glucose intolerant mice. A causative role for IL-6 in the physiological adaptation to fasting was further supported by the fact that fasting-induced increase in circulating FFA levels was significantly blunted in lean IL-6 knockout (KO) and lean C57BL/6J mice treated with neutralizing IL-6 antibody. Consistently, phosphorylation of HSL was significantly reduced in adipose tissue of IL-6 depleted mice. Hence, our findings suggest a novel role for IL-6 in energy supply during early fasting.
The zebrafish (ZF) has emerged as an important model for developmental cardiovascular (CV) biology, however, much less is known about the cardiac function of the adult zebrafish that can be used as a model of teleost CV biology. Here, we describe electrophysiological parameters, such as HR, AP duration (APD) and atrio-ventricular (AV) delay, in the ZF heart over a range of physiological temperatures (18-28 °C). Using the potentiometric dye RH-237 the electrical activity in several distinct regions of the heart were assessed simultaneously. While intrinsic HR varied considerably between fish, the ex vivo preparation exhibited impressively stable HRs and sinus rhythm for >5 hours, with a mean HR of 158 ± 9 bpm (mean ± SEM) (n = 20) at 28 °C. Atrial and ventricular APDs at 50% repolarization (APD50) were 33 ± 1 ms and 98 ± 2 ms, respectively. The AV delay was 61 ± 3 ms at 28 oC. APD and AV delay did not appear to be statistically dependent on intrinsic basal HR, likely due to the innate beat-to-beat variability within each heart. Cooling to 18 °C decreased HR by ~40% and atrial and ventricular APD50 increased by ~3x and 2x, respectively. The acute effect of temperature was most apparent on the atrial APD. In conclusion, optical mapping studies utilizing zebrafish heart can yield insightful information into cardiac arrhythmias as well as serving as a model for other teleosts.
Myosin heavy chain (MHC) isoform complement is intimately related to a muscle's contractile properties, yet relatively little is known about avian MHC isoforms or how they may vary with fiber type and/or the contractile properties of a muscle. The rapid shortening of muscles necessary to power flight at the high wingbeat frequencies of ruby-throated hummingbirds and zebra finches (25-60 Hz), along with the varied morphology and use of the hummingbird hindlimb, provides a unique opportunity to understand how contractile and morphological properties of avian muscle may be reflected in MHC expression. Isoforms of the hummingbird and zebra finch flight and hindlimb muscles were electrophoretically separated and compared to those of other avian species representing different contractile properties and fiber types. The flight muscles of the study species operate at drastically different contraction rates and are composed of different histochemically-defined fiber types, yet each exhibited the same, single MHC isoform corresponding to the chicken adult fast isoform. Thus, despite quantitative differences in the contractile demands of flight muscles across species, this isoform appears necessary for meeting the performance demands of avian powered flight. Variation in flight muscle contractile performance across species may be due to differences in the structural composition of this conserved isoform and/or variation within other mechanically-linked proteins. The leg muscles were more varied in their MHC isoform composition across both muscles and species. The disparity in hindlimb MHC expression between hummingbirds and the other species highlights previously observed differences in fiber type composition and thrust production during take-off.
Autonomic and endocrine profiles of chronic hypertension and heart failure resemble those of acute dehydration. Importantly, all of these conditions are associated with exaggerated sympathetic nerve activity (SNA) driven by glutamatergic activation of the hypothalamic paraventricular nucleus (PVN). Here, studies sought to gain insight into mechanisms of disease by determining the role of PVN ionotropic glutamate receptors in supporting SNA and mean arterial pressure (MAP) during dehydration and by elucidating mechanisms regulating receptor activity. Blockade of PVN NMDA receptors reduced (P<0.01) renal SNA and MAP in urethane-chloralose anesthetized dehydrated (DH) (48 h water deprivation) rats, but had no effect in euhydrated (EH) controls. Blockade of PVN AMPA receptors had no effect in either group. NMDA in PVN caused dose-dependent increases of renal SNA and MAP in both groups, but the Emax renal SNA response was greater (P<0.05) in DH rats. The latter was not explained by increased PVN expression of NMDA receptor NR1 subunit protein, increased PVN neuronal excitability, or decreased brain water content. Interestingly, PVN injection of the pan-specific excitatory amino acid transporter (EAAT) inhibitor TBOA produced smaller sympathoexcitatory and pressor responses in DH rats, which was associated with reduced glial expression of EAAT2 in PVN. Like chronic hypertension and heart failure, dehydration increases excitatory NMDA receptor tone in PVN. Reduced glial-mediated glutamate uptake was identified as a key contributing factor. Defective glutamate uptake in PVN could therefore be an important, but as yet unexplored, mechanism driving sympathetic hyperactivity in chronic cardiovascular diseases.
Lymph flow is the primary mechanism for returning interstitial fluid to the blood circulation. Currently, the adaptive response of lymphatic vessels to mesenteric venous hypertension is not known. This study sought to determine the functional responses of post-nodal mesenteric lymphatic vessels. We surgically occluded bovine mesenteric veins to create mesenteric venous hypertension to elevate mesenteric lymph flow. Three days after surgery, post-nodal mesenteric lymphatic vessels from mesenteric venous hypertension (MVH; n=7) and sham surgery (SHAM; n=6) group animals were evaluated and compared. Contraction frequency (MVH: 2.98±0.75 min-1; SHAM: 5.42±0.81 min-1) and fractional pump flow (MVH: 1.14±0.30 min-1; SHAM: 2.39±0.32 min-1) were significantly lower in the venous occlusion group. These results indicate that post-nodal mesenteric lymphatic vessels adapt to mesenteric venous hypertension by reducing intrinsic contractile activity.
Chronic alcohol abuse is associated with skeletal muscle myopathy. Previously we demonstrated that chronic binge alcohol (CBA) consumption by rhesus macaques accentuates skeletal muscle wasting at end-stage of simian immunodeficiency virus (SIV) infection. A pro-inflammatory, pro-oxidative milieu and enhanced ubiquitin proteasome activity were identified as possible mechanisms leading to loss of skeletal muscle. The possibility that impaired regenerative capacity, as reflected by the ability of myoblasts derived from satellite cell (SCs) to differentiate into myotubes has not been examined. We hypothesized that the inflammation and oxidative stress in skeletal muscle from CBA animals, impairs the differentiation capacity of myoblasts to form new myofibers in in vitro assays. We isolated primary myoblasts from the quadriceps femoris of rhesus macaques that were administered CBA or isocaloric sucrose (SUC) for 19 months. Proliferation and differentiation potential of cultured myoblasts was examined in vitro. Myoblasts from the CBA group had significantly reduced PAX7 MYOD1, myogenin, MYF5 and MEF2C expression. This was associated with decreased myotube formation as evidenced by Jenner-Giemsa staining and myonuclei fusion index. No significant difference in the proliferative ability, cell cycle distribution or autophagy was detected between myoblasts isolated from CBA and SUC groups. Together, these results reflect marked dysregulation of myoblast myogenic gene expression and myotube formation that we interpret as evidence of impaired skeletal muscle regenerative capacity in CBA administered macaques. The contribution of this mechanism to alcoholic myopathy warrants further investigation.
Continuous subcutaneous administration of leptin normalizes blood glucose levels in rodent models of type 1 and type 2 diabetes independent of changes in food intake, body weight and plasma insulin. We tested whether an acute intravenous leptin infusion changed blood glucose in normal and diet-induced leptin resistant rats to determine if this measure could be used as a marker of leptin sensitivity. Leptin responsive chow fed rats and diet induced leptin resistant Male Sprague Dawley rats were fitted with thoracic jugular vein catheters. Four days after surgery, conscious rats were infused intravenously (i.v) with either saline for 32 minutes, low dose (LD) leptin (1.9µg/kg/min) followed by high dose (HD) leptin (3.8µg/kg/min) for 16 minutes each, or only HD leptin for 16 minutes. There was no change in blood glucose after an acute i.v infusion of either LD leptin or HD leptin alone for 16 minutes. An i.v. infusion of LD followed by HD leptin for 16 minutes each, significantly decreased serum glucose in leptin responsive rats but not in leptin resistant rats. Leptin infusions increased serum leptin in all rat groups, but had no effect on plasma glucagon or 12 hour weight gain and energy intake in any group of rats. These results show that leptin has an acute glucose lowering effect that reflects the leptin responsiveness of the rat. This effect is consistent across controls and different leptin resistant rat models and the acute, non-lethal test provides a novel method of testing leptin responsiveness in rats.
Sympathetic nervous system (SNS) mediated peripheral vasoconstriction plays a key role in initial maintenance of blood pressure during rapid onset asphyxia in the mammalian fetus, but is attenuated after the first few minutes. It is unclear whether the SNS response is sustained during the brief but frequently repeated episodes of asphyxia characteristic of labor. In the present study, 14 fetal sheep at 0.85 of gestation received either chemical sympathectomy with 6-hydroxydopamine (6-OHDA, n = 7) or sham injection (control, n = 7), followed 4-5 days later by repeated 2-minute episodes of complete umbilical cord occlusion every 5 minutes for up to 4 hours or until mean arterial blood pressure (MAP) fell to < 20 mmHg for 2 successive occlusions. In controls, umbilical cord occlusions were associated with a rapid initial fall in fetal heart rate (FHR) and femoral blood flow (FBF), with initial hypertension, followed by progressive development of hypotension during ongoing occlusions. Sympathectomy was associated with attenuation of the initial rise in MAP during umbilical cord occlusion, and after the onset of hypotension, markedly more rapid fall of MAP to the nadir, with a correspondingly slower fall in FBF (P < 0.05). In contrast, MAP and FHR between successive occlusions were higher after sympathectomy (P < 0.05), and there was no significant difference in the number of occlusions before terminal hypotension (16.1 ± 2.2 vs. control; 18.7 ± 2.3). These data show that SNS activity provides ongoing support for fetal MAP during prolonged exposure to brief repeated asphyxia.
Cerebrovascular lesions, mainly germinal matrix hemorrhage and ischemic injury to the periventricular white matter, are major causes of adverse neurodevelopmental outcome in preterm infants. Cerebrovascular lesions and neuromorbidity increase with decreasing gestational age, with the white matter predominantly affected. Developmental immaturity in the cerebral circulation, including ongoing angiogenesis and vasoregulatory immaturity, play a major role in the severity and pattern of preterm brain injury. Prevention of this injury requires insight into pathogenesis. Cerebral blood flow (CBF) is low in the preterm white matter, which also has blunted vasoreactivity compared to other brain regions. Vasoreactivity in the preterm brain to cerebral perfusion pressure, oxygen, carbon dioxide and neuronal metabolism is also immature. This could be related to immaturity of both the vasculature and vasoactive signaling. Other pathologies arising from preterm birth and the neonatal intensive care environment itself may contribute to impaired vasoreactivity and ineffective CBF regulation, resulting in the marked variations in cerebral hemodynamics reported both within and between infants depending on their clinical condition. Many gaps exist in our understanding of how neonatal treatment procedures and medications impact upon cerebral hemodynamics and preterm brain injury. Future research directions for neuroprotective strategies include establishing cotside, real-time clinical reference values for cerebral hemodynamics and vasoregulatory capacity, and to demonstrate that these thresholds improve long-term outcomes for the preterm infant. In addition, stimulation of vascular development and repair with growth factor and cell-based therapies also hold promise.
With this study we tested the hypothesis that six weeks of endurance training increases maximal cardiac output (Qmax) relatively more by elevating blood volume (BV) than by inducing structural and functional changes within the heart. Nine healthy but untrained volunteers (VO2max 47 ± 5 ml.min-1.kg-1) underwent supervised training (60 min; 4 times weekly at 65% VO2max for six weeks) and Qmax was determined by inert gas re-breathing during cycle ergometer exercise before and after the training period. After the training period, blood volume (determined in duplicates by CO re-breathing) was re-established to pre-training values by phlebotomy and Qmax was quantified again. Resting echography revealed no structural heart adaptations as a consequence of the training intervention. Following the training period, plasma volume (PV), red blood cell volume (RBCV) and BV increased (p<0.05) by 147 ± 168 (5 ± 5 %), 235 ± 64 (10 ± 3 %) and 382 ± 204 ml (7 ± 4 %), respectively. VO2max was augmented (p<0.05) by 10 ± 7 % following the training period and decreased (p<0.05) by 8 ± 7 % with phlebotomy. Concomitantly, Qmax was increased (p<0.05) from 18.9 ± 2.1 to 20.4 ± 2.3 l.min-1 (9 ± 6 %) as a consequence of the training intervention, and following normalization of BV by phlebotomy Qmax returned to pre training values (18.1 ± 2.5 l.min-1; 12 ± 5 % reversal). Thus, the exercise training induced increase in BV is the main mechanism increasing Qmax following six weeks of endurance training in previously untrained subjects.
Hydrogen sulfide (H2S) is a toxic gas getting recognized as an endogenous signaling molecule in multiple organ systems, in particular cardiovascular system. H2S is known to regulate cardiac function and protect against ischemic injury. However, little information is available regarding the effect of H2S on cardiac function in insulin resistance. This study was designed to examine the impact of H2S supplementation on cardiac function using an Akt2 knockout model of insulin resistance. Wild-type and Akt2 knockout mice were treated with NaHS (50 micromole/kg/d, i.p. for 10 days) prior to evaluation of echocardiographic, cardiomyocyte contractile and intracellular Ca2+ properties, apoptosis and mitochondrial damage. Our results revealed that Akt2 ablation led to overtly enlarged ventricular end systolic diameter, reduced myocardial and cardiomyocyte contractile function, disrupted intracellular Ca2+ homeostasis and apoptosis, the effects of which were ameliorated by H2S. Furthermore, Akt2 knockout displayed upregulated apoptotic protein markers (Bax, Caspase-3, -9 and -12) and mitochondrial damage (reduced aconitase activity and NAD+, elevated cytochrome C release from mitochondria) along with reduced phosphorylation of PTEN, Akt and GSK3β in the absence of changes in pan protein expression, the effects of which were abolished or significantly ameliorated by H2S treatment. In vitro data revealed that H2S-induced beneficial effect against Akt2 ablation was obliterated by mitochondrial uncoupling. Taken together, our findings suggest the H2S may reconcile Akt2 knockout-induced myocardial contractile defect and intracellular Ca2+ mishandling, possibly via attenuation of mitochondrial injury and apoptosis.
Background: Free vascular endothelial growth factor (VEGF) is undetectable in plasma during human pregnancy. However, studies examining pregnant rats have reported both low (8-29 pg/mL) and high (527-1030 pg/mL) free VEGF. These discrepancies cast uncertainty over the use of rat models to study angiogenic factors in pregnancy and preeclampsia. This study investigates methodological factors that may explain these discrepancies. Methods: Plasma VEGF in non-pregnant, day 7 pregnant and day 19 pregnant rats was measured using rat and mouse ELISAs. Results: The rat ELISA detected VEGF in plasma from non-pregnant rats, but not in plasma from day 19 pregnant rats. The mouse ELISA detected higher VEGF concentrations than the rat ELISA in every sample tested. This discrepancy was greater in day 19 pregnant rats (Median: 2,273 vs. 0 pg/mL) than in non-pregnant (97 vs. 20 pg/mL) and day 7 pregnant (66 vs. 2 pg/mL) rats. Recovery of recombinant rat VEGF (rrVEGF) spiked into plasma from non-pregnant and day 7 pregnant rats was high for the rat ELISA (82-105%), but low for the mouse ELISA (17-22%). The rat ELISA did not recover rrVEGF in plasma from day 19 pregnant rats, suggesting that this ELISA measures free VEGF. Conclusions: The use of the rat vs. mouse ELISA likely explains the differences in reported VEGF concentrations in pregnant rats. While the rat ELISA appears to measure free VEGF, plasma concentrations are below the assay sensitivity limit. As most previous studies of pregnant rats used the mouse VEGF ELISA, this data should be interpreted cautiously.
Nesfatin-1 is produced in the periphery and in brain where it has been demonstrated to regulate appetite, stress hormone secretion and cardiovascular function. The anorexigenic action of central nesfatin-1 requires recruitment of neurons producing the melanocortins and centrally-projecting oxytocin (OT) and corticotropin releasing hormone (CRH) neurons. We previously have shown that two components of this pathway, the central melanocortin and oxytocin systems, contribute to the hypertensive action of nesfatin-1 as well. We hypothesized that the cardiovascular effect of nesfatin-1 also was dependent upon activation of neurons expressing CRH receptors, and that the order of activation of the melanocortin-CRH-oxytocin circuit was preserved for both the anorexigenic and hypertensive actions of the peptide. Pretreatment of male rats with the CRH-2 receptor antagonist, astressin2B, abrogated nesfatin-1-induced increases in mean arterial pressure (MAP). Furthermore, the hypertensive action of CRH was blocked by pretreatment with an oxytocin receptor antagonist, OVT, indicating that the hypertensive effect of nesfatin-1 may require activation of OTergic neurons in addition to recruitment of CRH neurons. Interestingly, we found that the hypertensive effect of alpha-melanocyte stimulating hormone (alpha-MSH) itself was not blocked by either astressin2B or OVT. These data suggest that while alpha-MSH-producing neurons are part of a core melanocortin-CRH-oxytocin circuit regulating food intake, and a subpopulation of melanocortin neurons activated by nesfatin-1 do mediate the hypertensive action of the peptide, alpha-MSH can signal independently from this circuit to increase MAP.
Activation of muscle metaboreceptors and mechanoreceptors has been shown to independently influence the sweating response, while their integrative control effects remain unclear. We examined the sweating response when the two muscle receptors are concurrently activated in different limbs as well as the blood pressure response. In total, 27 young males performed passive calf muscle stretches (muscle mechanoreceptor activation) for 30 s in a semi-supine position with and without post-isometric handgrip exercise muscle ischemia (PEMI, muscle metaboreceptor activation) at exercise intensities of 35 and 50% of maximum voluntary contraction (MVC) under hot conditions (ambient temperature, 35°C, relative humidity, 50%). Passive calf muscle stretching alone increased the mean sweating rate significantly on the forehead, chest, and thigh (SRmean) and mean arterial blood pressure (MAP), but not the heart rate (HR), from pre-stretching levels by 0.04±0.01 mg/cm2/min, 4.0±1.3 mmHg (P < 0.05), and -1.0±0.5 beats/min (P > 0.05), respectively. The SRmean and MAP during PEMI were significantly higher than those at rest. The passive calf muscle stretch during PEMI increased MAP significantly, by 3.4±1.0 and 2.0±0.7 mmHg for 35 and 50% of MVC, respectively (P < 0.05), but not that of SRmean or HR at either exercise intensity. These results suggest that sweating and blood pressure respond to concurrent activation of the two muscle receptors in different limbs differ, and that the influence of calf muscle mechanoreceptor activation alone on the sweating response disappears during forearm muscle metaboreceptor activation.
The present study examined to what extent an acute bout of hypotension influences blood flow in the external carotid artery (ECA) and the corresponding implications for blood flow regulation in the internal carotid artery (ICA). Nine healthy male participants were subject to an abrupt decrease in arterial pressure via the thigh-cuff inflation-deflation technique. Duplex ultrasound was employed to measure beat-to-beat ECA and ICA blood flow. Compared to the baseline normotensive control, acute hypotension resulted in a heterogeneous blood flow response. ICA blood flow initially decreased following cuff-release and then returned quickly to baseline levels. In contrast, the reduction in ECA blood flow persisted for 30 sec following cuff-release. Thus, the contribution of common carotid artery blood flow to the ECA circulation decreased during acute hypotension (-10 ± 4 %, P<0.001). This finding suggests that a preserved reduction in ECA blood flow as well as dynamic cerebral autoregulation likely prevent a further decrease in intracranial blood flow during acute hypotension. The peripheral vasculature of the ECA may thus be considered an important vascular bed for intracranial cerebral blood flow regulation.
Our laboratory has suggested that central command provides selective inhibition of the cardiomotor component of aortic baroreflex at the start of exercise, preserving carotid sinus baroreflex. It is postulated that central command may modify the signal transduction of aortic baroreceptors, so as to decrease aortic baroreceptor input to the cardiovascular centers, and thereby can cause the selective inhibition of aortic baroreflex. To test the hypothesis, we directly analyzed the responses in multifiber aortic nerve activity (AoNA) and carotid sinus nerve activity (CsNA) during spontaneous motor activity in decerebrate, paralyzed cats. The increases of 62-104% in mean AoNA and CsNA were found during spontaneous motor activity, in proportion to a rise of 35 ± 3 mmHg (mean ± SE) in mean arterial blood pressure (MAP), and had an attenuating tendency by restraining heart rate (HR) at the lower intrinsic frequency of 154 ± 6 beats/min. Brief occlusion of the abdominal aorta was conducted before and during spontaneous motor activity to produce a mechanically-evoked increase in MAP and thereby to examine the stimulus-response relationship of arterial baroreceptors. Although the sensitivity of the MAP-HR baroreflex curve was markedly blunted during spontaneous motor activity, the stimulus-response relationships of AoNA and CsNA were not influenced by spontaneous motor activity, irrespective of the absence or presence of the HR restraint. Thus it is concluded that aortic and carotid sinus baroreceptors can code beat-by-beat blood pressure during spontaneous motor activity in decerebrate cats and that central command is unlikely to modulate the signal transduction of arterial baroreceptors.
Serotonergic (5-hydroxytryptamine, 5-HT) neurons of the area postrema (AP) represent one neuronal phenotype implicated in the regulation of salt appetite. Tryptophan hydroxylase (Tryp-OH, synthetic enzyme producing 5-HT) immunoreactive neurons in the AP of rats become c-Fos activated following conditions in which plasma sodium levels are elevated; these include intraperitoneal injections of hypertonic saline and sodium-repletion. Non Tryp-OH neurons also became c-Fos activated. Sodium depletion, which induced an increase in plasma osmolality but caused no significant change in the plasma sodium concentration, had no effect on the c-Fos activity in the AP. Epithelial sodium channels (ENaCs) are expressed in the Tryp-OH immunoreactive AP neurons, possibly functioning in the detection of changes in plasma sodium levels. Since little is known about the neural circuitry of these neurons, we tested one hypothesis and that was whether the AP contributes to a central pathway that innervates the reward center of the brain. Stereotaxic injections of pseudorabies virus were made in the nucleus accumbens (NAc), and after 4 days, this viral tracer produced retrograde transneuronal labeling in the Tryp-OH and non-Tryp-OH AP neurons. Both sets of neurons innervate the NAc via a multisynaptic pathway. Besides sensory information regarding plasma sodium levels, the AP->NAc pathway may also transmit other types of chemosensory information, such as those related to metabolic functions, food intake, and immune system, to the subcortical structures of the reward system. Since these subcortical regions ultimately project to the medial prefrontal cortex, different types of chemical signals from visceral systems may influence affective functions.
Recent work has shown that the carotid chemoreceptor (CC) contributes to sympathetic control of cardiovascular function during exercise, despite no evidence of increased circulating CC stimuli, suggesting enhanced CC activity/sensitivity. As interactions between metaboreceptors and chemoreceptors have been previously observed, the purpose of this study was to isolate the metaboreflex while acutely stimulating or inhibiting the CC to determine if the metaboreflex increased CC activity/sensitivity. Fourteen young healthy men (Height: 177.0±2.1 cm, Weight: 85.8±5.5 kg, Age: 24.6±1.1) performed three trials of 40 % maximal voluntary contraction handgrip for 2min, followed by 3min of post-exercise circulatory occlusion (PECO) to stimulate the metaboreflex. In random order, subjects either breathed room air, hypoxia (target SpO2=85 %), or hyperoxia (FIO2=1.0) during the PECO in order to modulate the chemoreflex. Following these trials, a resting hypoxia trial was conducted without handgrip or PECO. Ventilation (VE), heart rate (HR), blood pressure and muscle sympathetic nervous activity (MSNA) data were continuously obtained. Relative to normoxic PECO, inhibition of the CC during hyperoxic PECO resulted in lower MSNA (p=0.038) and HR (p=0.021). Relative to normoxic PECO, stimulation of the CC during hypoxic PECO resulted in higher HR (p<0.001) and VE (p<0.001). The ventilatory and MSNA responses to hypoxic PECO were not greater than the sum of the responses to hypoxia and PECO individually, indicating that the CC are not sensitized during metaboreflex activation. These results demonstrate that stimulation of the metaboreflex activates, but does not sensitize the CC, and help explain the enhanced CC activity with exercise.
A wide variety of species, including vertebrate and invertebrates, consume food in bouts (i.e., meals). Decades of research suggest that different mechanisms regulate meal initiation (when to start eating) versus meal termination (how much to eat in a meal, also known as satiety). There is a very limited understanding of the mechanisms that regulate meal onset and the duration of the postprandial intermeal interval (ppIMI). In the present review, we examine issues involved in measuring meal onset and some of the limited available evidence regarding how it is regulated. Then, we describe our recent work indicating dorsal hippocampal neurons inhibit meal onset during the ppIMI and describe the processes that may be involved in this. We also synthesize recent evidence, including evidence from our laboratory, suggesting that overeating impairs hippocampal functioning and that impaired hippocampal functioning, in turn contributes to the development and/or maintenance of diet-induced obesity. Finally, we identify critical questions and challenges for future research investigating neural controls of meal onset.
Adult obese Zucker rats (OZR; >12 wks) develop elevated sympathetic nerve activity (SNA) and mean arterial pressure (MAP) with impaired baroreflexes compared to adult lean Zucker rats (LZR) and juvenile OZR (6-7 wks). In adult OZR, baroreceptor afferent nerves respond normally to changes in MAP, whereas electrical stimulation of baroreceptor afferent fibers produces smaller reductions in SNA and MAP compared to LZR. We hypothesized that impaired baroreflexes in OZR are linked to reduced activation of brainstem sites that mediate baroreflexes. In conscious adult rats a hydralazine (HDZ)-induced reduction in MAP evoked tachycardia that was initially blunted in OZR, but equivalent to LZR within 5 minutes. In agreement, HDZ-induced expression of c-Fos in the rostral ventrolateral medulla (RVLM) was comparable between groups. In contrast, phenylephrine (PE)-induced rise in MAP evoked markedly attenuated bradycardia with dramatically reduced c-Fos expression in nucleus tractus solitarius (NTS) of adult OZR compared to LZR. However, in juvenile rats PE-induced hypertension evoked comparable bradycardia in OZR and LZR with similar or augmented c-Fos expression in NTS of the OZR. In urethane-anesthetized rats, microinjections of glutamate into NTS evoked equivalent decreases in SNA, HR, and MAP in juvenile OZR and LZR, but attenuated decreases in SNA and MAP in adult OZR. In contrast, microinjections of glutamate into the caudal ventrolateral medulla, a target of barosensitive NTS neurons, evoked comparable decreases in SNA, HR, and MAP in adult OZR and LZR. These data suggest OZR develop impaired glutamatergic activation of the NTS that likely contributes to attenuated baroreflexes in adult OZR.
How sex steroids modulate glucocorticoid feedback on the hypothalamic-pituitary-corticotrope (HPC) unit is controversial in humans. We postulated that testosterone (T) in men and estradiol (E2) in women govern unstressed cortisol-mediated negative feedback on ACTH secretion. To test this hypothesis, 24 men and 24 women age 58 ± 2.4 yr were pre-treated with leuprolide and either sex steroid (E2 in women, T in men) or placebo addback. Placebo or ketoconazole (KTCZ) was administered overnight to inhibit adrenal steroidogenesis during overnight 14-h i.v. infusions of saline or cortisol in a continuous vs pulsatile manner to test for feedback differences. ACTH was measured every 10 min during the last 8 hr of the infusions. The main outcome measures were mean ACTH concentrations, pulsatile ACTH secretion and ACTH ApEn (approximate entropy). ACTH concentrations were lower in women than men (P<0.01), and in women in the E2 + compared with E2 - group under both continuous (P=0.01) and pulsatile (P=0.006) cortisol feedback, despite higher CBG and lower free cortisol levels in women than men (P<0.01). In the combined groups, under both modes of cortisol addback, ACTH concentrations, pulsatile ACTH secretion and ACTH secretory-burst mass correlated negatively and univariately with E2 levels (each P<0.005). E2 also suppressed ACTH ApEn (process randomness) during continuous cortisol feedback (P=0.004). T had no univariate effect but was a positive correlate of ACTH when assessed jointly with E2 (negative) under cortisol pulses. In conclusion, sex steroids modulate selective gender-related HPA-axis adaptations to cortisol feedback in unstressed humans.
Episodic increases in cerebrovascular perfusion and shear stress may have beneficial impacts on endothelial function that improve brain health. We hypothesised that water immersion to the level of the right atrium in humans would increase cerebral perfusion. We continuously measured, in 9 young (mean±SD, 24.6 ± 2.0 yrs) healthy men, systemic hemodynamic variables along with blood flows in the common carotid and middle and posterior cerebral arteries during controlled filling and emptying of a water tank to the level of the right atrium. Mean arterial pressure (80 ± 9 vs 91 ± 12 mmHg, P<0.05), cardiac output (4.8 ± 0.7 vs 5.1±0.6 L/min, P<0.05) and end-tidal carbon dioxide (PetCO2, 39.5 ± 2.0 vs 44.4 ± 3.5 mmHg, P<0.05) increased with water immersion, along with middle (59 ± 6 vs 64 ± 6 cm/s, P<0.05) and posterior cerebral artery blood flow velocities (41 ± 9 vs 44 ± 10 cm/s, P<0.05). These changes were reversed when the tank was emptied. Water immersion is associated with haemodynamic and PetCO2 changes, which increase cerebral blood velocities in humans. This study provides an evidence base for future studies to examine the potential addictive effect of exercise in water on improving cerebrovascular health.
ABSTRACT Since the discovery of the rectal gland of the dogfish shark 50 years ago, experiments with this tissue have greatly aided our understanding of secondary active chloride secretion and the secretagogues responsible for this function. In contrast, very little is known about the rectal gland of skates. In the present experiments, we performed the first studies in the perfused rectal gland of the little skate (Leucoraja erinacea), an organ weighing less than one tenth of the shark rectal gland. Our results indicate that the skate gland can be studied by modified perfusion techniques and in primary culture monolayers and that secretion is blocked by the inhibitors of membrane proteins required for secondary active chloride secretion. Our major finding is that three G protein coupled receptor agonists, the incretin gastric inhibitory polypeptide (GIP), also known as glucose-dependent insulinotropic peptide, as well as glucagon and serotonin, are unexpected potent chloride secretagogues in the skate but not the shark. Glucagon stimulated chloride secretion to a mean value of 1661 ± 587 µEq/h/g and serotonin stimulated to 2893 ± 699 µEq/h/g. GIP stimulated chloride secretion to 3733 ± 679 µEq/h/g and significantly increased tissue cyclic AMP content compared to basal conditions. This is the first report of GIP functioning as a chloride secretagogue in any species or tissue.
Preeclampsia is thought to arise from inadequate cytotrophoblast migration and invasion of the maternal spiral arteries, resulting in placental ischemia and hypertension. Evidence suggests that altered expression of Epithelial Na+ Channel (ENaC) proteins may be a contributing mechanism for impaired cytotrophoblast migration. ENaC activity is required for normal cytotrophoblast migration. Moreover, βENaC, the most robustly expressed placental ENaC message, is reduced in placentas from preeclamptic women. We recently demonstrated that heme oxygenase-1 (HO-1) protects against hypertension in a rat model of placental ischemia; however, whether HO-1 regulation of βENaC contributes to the beneficial effects of HO-1 is unknown. The purpose of this study was to determine whether βENaC mediates cytotrophoblast migration and if HO-1 enhances ENaC-mediated migration. We showed that placental ischemia, induced by reducing uterine perfusion, suppressed, and HO-1 induction restored βENaC expression in ischemic placentas. Using an in vitro model, we found that HO-1 induction, using cobalt protoporphyrin, stimulates cytotrophoblast βENaC expression by 1.5 and 1.8-fold (10 and 50 µM). We then showed that silencing of βENaC in cultured cytotrophoblasts (BeWo cells), by expression of dominant-negative constructs, reduced migration to 56 ± 13% (p<0.05) of control. Importantly, HO-1 induction enhanced migration (43 ± 5% of control, p<0.05), but the enhanced migratory response was entirely blocked by ENaC inhibition with amiloride (10 µM). Taken together, our results suggest that βENaC mediates cytotrophoblast migration and increasing βENaC expression by HO-1 induction enhances migration. HO-1 regulation of cytotrophoblast βENaC expression and migration may be a potential therapeutic target in preeclamptic patients.
Leptin released peripherally acts within the central nervous system (CNS) to modulate numerous physiological and behavioral functions. Histochemical identification of leptin-responsive CNS cells can reveal the specific cellular phenotypes and neural circuits through which leptin signaling modulates these functions. Leptin signaling elicits phosphorylation of signal transducer and activator of transcription 3 (pSTAT3), making pSTAT3-immunoreactivity (ir) a useful proxy for identifying leptin-responsive cells. Relatively low systemic doses of leptin (i.e., 10-130 μg/kg BW) are sufficient to decrease food intake, inhibit gastric emptying, and increase sympathetic activity, but there are no histological reports of central pSTAT3-ir following leptin doses within this range. Considering this, we quantified central pSTAT3-ir in rats after i.p. injections of leptin at doses ranging from 50-800 μg/kg BW. Tissue sections were processed to identify pSTAT3-ir alone or in combination with immunolabeling for cocaine- and amphetamine-regulated transcript (CART), glucagon-like peptide-1 (GLP-1), prolactin-releasing peptide (PrRP), or dopamine-β-hydroxylase (DβH). Leptin doses as low as 50, 100, and 200 μg/kg BW significantly increased the number of pSTAT3-ir cells in the arcuate nucleus of the hypothalamus (ARC), nucleus of the solitary tract (NTS), and ventromedial nucleus of the hypothalamus, respectively, and also led to robust pSTAT3 labeling in neural processes. The differential dose-dependent increases in pSTAT3-ir across brain regions provides new information regarding central leptin sensitivity. Within the ARC, CART- and pSTAT3-ir were often colocalized, consistent with evidence of leptin sensitivity in this neural population. Conversely, within the NTS, pSTAT3 only rarely colocalized with PrRP and/or DβH, and never with GLP-1.
Diets high in sugar and saturated fat (Western diet) contribute to obesity and pathophysiology of metabolic syndrome. A common physiological response to obesity is hypertension, which induces cardiac remodeling and hypertrophy. Hypertrophy is regulated at chromatin by repressor element 1-silencing transcription factor (REST), and pathological hypertrophy is associated with reexpression of a fetal cardiac gene program. Reactivation of fetal genes is commonly observed in hypertension-induced hypertrophy; however, this response is blunted in diabetic hearts, partially due to upregulation of the post-translational modification O-linked-β-N-acetylglucosamine (O-GlcNAc) to proteins by O-GlcNAc transferase (OGT). OGT and O-GlcNAc are found in chromatin-modifying complexes, but it is unknown if they play a role in Western diet-induced hypertrophic remodeling. Therefore, we investigated the interactions between O-GlcNAc, OGT, and the fetal gene-regulating transcription factor complex REST/mSin3A/histone deacetylase (HDAC). Five-week-old male C57BL/6 mice were fed a Western (n=12) or control diet (n=12) for two weeks to examine the early hypertrophic response. Western diet fed mice exhibited fasting hyperglycemia and increased body weight (P<0.05). As expected for this short duration of feeding, cardiac hypertrophy was not yet evident. We found that REST is O-GlcNAcylated and physically interacts with OGT in mouse hearts. Western blotting showed that HDAC protein levels were not different between groups; however, relative to controls, Western diet hearts showed increased REST and decreased ANP and skeletal α-actin. Transcript levels of HDAC2 and cardiac α-actin were decreased in Western diet hearts. These data suggest that REST coordinates regulation of diet-induced hypertrophy at the level of chromatin.
This study investigated the effects of angiotensin II (Ang II) on slow and rapid baroreflex responses of barosensitive bulbospinal neurons in the rostral ventrolateral medulla (RVLM) in urethane-anesthetized rabbits to determine whether the sympathetic baroreflex modulation induced by application of Ang II into the RVLM can be explained by the total action of Ang II on individual RVLM neurons. In response to pharmacologically induced slow ramp changes in mean arterial pressure (MAP), individual RVLM neurons exhibited a unit activity-MAP relationship that was fitted by a straight line with upper and lower plateaus. Iontophoretically applied Ang II raised the upper plateau without changing the slope, and thereby increased the working range of the baroreflex response. An asymmetric sigmoid curve that was determined by averaging individual unit activity-MAP relationship lines became more symmetric with Ang II application. The characteristics of the average curves, both before and during Ang II application, were consistent with the renal sympathetic nerve activity-MAP relationship curves obtained under the same experimental conditions. Ang II also affected rapid baroreflex responses of RVLM neurons that were induced by cardiac beats, as application of Ang II predominantly raised the average unit activities in the downstroke phase of arterial pulse waves. The present study provides a possible explanation for the Ang II-induced sympathetic baroreflex modulation based on the action of Ang II on barosensitive bulbospinal RVLM neurons. Our results also suggest that Ang II changes both static and dynamic characteristics of baroreflex responses of RVLM neurons.
We tested the hypothesis that excess saturated fat consumption during pregnancy, lactation and/or post-weaning alters the expression of genes mediating hippocampal synaptic efficacy and impairs spatial learning and memory in adulthood. Dams were fed control chow or a diet high in saturated fat before mating, during pregnancy and into lactation. Offspring were weaned to either standard chow or a diet high in saturated fat. Morris Water Maze was used to evaluate spatial learning and memory. Open field testing was used to evaluate motor activity. Hippocampal gene expression in adult males was measured using RT-PCR and ELISA. Offspring from high-fat fed dams took longer, swam farther and faster to try and find the hidden platform during the five day learning period. Control offspring consuming standard chow spent the most time in memory quadrant during the probe test. Offspring from high-fat fed dams consuming excess saturated fat spent the least. The levels of mRNA and protein for brain-derived neurotrophic factor and activity-regulated cytoskeletal associated protein were significantly decreased by maternal diet effects. Nerve growth factor mRNA and protein levels were significantly reduced in response to both maternal and postweaning high-fat diets. Expression levels for the NMDA receptor subunit, NR2B, as well as synaptophysin were significantly decreased in response to both maternal and post-weaning diets. Synaptotagmin was significantly increased in offspring from high-fat fed dams. These data support the hypothesis that exposure to excess saturated fat during hippocampal development is associated with complex patterns of gene expression and deficits in learning and memory.
Hypertonic NaCl infused into the carotid arteries increases arterial blood pressure (AP) and changes sympathetic nerve activity (SNA) via cerebral mechanisms. We hypothesized that elevated sodium levels in the blood supply to the brain would induce differential responses in renal and cardiac SNA via sensors located outside the blood-brain barrier. To investigate this hypothesis we measured renal and cardiac SNA simultaneously in conscious sheep during intracarotid (IC) infusions of NaCl (1.2 M), sorbitol (2.4 M) or urea (2.4 M) at 1 mL/min up each carotid. IC NaCl significantly increased AP (91±2 to 97±3 mmHg, p<0.05) without changing heart rate (HR). IC NaCl was associated with no change in cardiac SNA (11±5.0%), but a significant inhibition of renal SNA (-32.5±6.4%, p<0.05). Neither IC sorbitol nor urea changed AP, HR, CVP or cardiac and renal SNA. The changes in AP and renal SNA were completely abolished by microinjection of the GABA agonist muscimol (5mM, 500nL) into the paraventricular nucleus of the hypothalamus (PVN). Infusion of IC NaCl for 20 min stimulated a larger increase in water intake (1100±75 mL) than IC sorbitol (683±125 mL) or IC urea (0 mL). These results demonstrate that acute increases in blood sodium levels cause a decrease in renal but no change in cardiac SNA in conscious sheep. These effects are mediated by cerebral sensors located outside the blood-brain barrier that are more responsive to changes in sodium concentration than osmolality. The renal sympatho-inhibitory effects of sodium are mediated via a pathway that synapses in the PVN.
Adipose tissue (AT)-derived cytokines are proposed to contribute to obesity-associated vascular insulin resistance. We tested the hypothesis that voluntary physical activity and diet restriction-induced maintenance of body weight would both result in decreased AT inflammation and concomitant improvements in insulin-stimulated vascular relaxation in the hyperphagic, obese OLETF rat. Rats (age 12 wk) were randomly assigned to sedentary (SED, n=10), wheel running (WR, n=10), and diet restriction (DR, n=10; fed 70% of SED) for 8 weeks. WR and DR rats exhibited markedly lower adiposity (7.1±0.4 and 15.7±1.1% body fat, respectively) relative to SED (27±1.2% body fat), as well as improved blood lipid profiles and systemic markers of insulin resistance. Reduced adiposity in both WR and DR was associated with decreased AT mRNA expression of inflammatory genes (e.g., MCP-1, TNF-α, IL-6) and markers of immune cell infiltration (e.g., CD8, CD11c, F4/80). The extent of these effects were most pronounced in visceral AT compared to subcutaneous and periaortic AT. Markers of inflammation in brown AT were upregulated with WR but not DR. In periaortic AT, WR and DR-induced reductions in expression and secretion of cytokines were accompanied with a more athero-protective gene expression profile in the adjacent aortic wall. WR, but not DR, resulted in greater insulin-stimulated relaxation in the aorta; an effect that was in part mediated by a decrease in insulin-induced endothelin-1 activation in WR aorta. Collectively, we show in OLETF rats that lower adiposity leads to less AT and aortic inflammation as well as an exercise-specific improvement in insulin-stimulated vasorelaxation.
Vascular endothelial growth factor (VEGF) is exercise-responsive, pro-angiogenic, and expressed in several muscle cell types. We hypothesized that in adult mice, VEGF generated within skeletal myofibers (and not other cells within muscle) is necessary for the angiogenic response to exercise training. This was tested in adult conditional, skeletal myofiber-specific VEGF gene-deleted mice (skmVEGF-/-), with VEGF levels reduced by >80%. After 8 weeks of daily treadmill training, speed and endurance were unaltered in skmVEGF-/- mice, but increased by 18% and 99% (p < 0.01), respectively, in controls trained at identical absolute speed, incline, and duration. In vitro, isolated soleus and EDL contractile function was not impaired in skmVEGF-/- mice. However, training-induced angiogenesis was inhibited in plantaris (WT, 38%, skmVEGF-/- 18%, p < 0.01), and gastrocnemius (WT, 43%, p < 0.01; skmVEGF-/-, 7%, n.s.). Capillarity was maintained (different from VEGF gene deletion targeted to multiple cell types) in untrained skmVEGF-/- mice. Arteriogenesis (smooth muscle actin+, artery number and diameter) and remodeling (vimentin+, BrDU+ and F4/80+ cells) occurred in skmVEGF-/- mice, even in the absence of training. SkmVEGF-/- mice also displayed a limited oxidative enzyme (citrate synthase and β -HAD) training response; β-HAD activity levels were elevated in the untrained state. These data suggest that myofiber expressed VEGF is necessary for training responses in capillarity and oxidative capacity, and for improved running speed and endurance.
Adverse conditions in utero can have transgenerational effects, in the absence of a subsequent insult. We aimed to investigate the contribution of the maternal pregnancy environment vs. germ-line effects in mediating alterations to cardio-renal and metabolic physiology in offspring from mothers born small. Uteroplacental insufficiency was induced by bilateral uterine artery and vein ligation (Restricted) or sham surgery (Control) in Wistar-Kyoto rats. Restricted and Control female offspring (F1) were mated with either breeder males (embryo donor) or vasectomised males (embryo recipient). Embryo transfer was performed at embryonic day (E) 1 whereby second generation (F2) embryos gestated (donor-in-recipient) in either a Control (Cont-in-Cont, Rest-in-Cont) or Restricted (Cont-in-Rest, Rest-in-Rest) mother. In male and female offspring, glomerular number and size were measured at postnatal day (PN) 35 and systolic blood pressure, glucose control, insulin sensitivity and pancreatic β-cell mass were measured in separate sibling cohorts at 6 months. Rest-in-Rest offspring were hypothesised to have similar characteristics (reduced growth, altered metabolic control and hypertension) to non embryo-transferred Rest, such that embryo transfer would not be a confounding experimental influence. However, embryo-transferred Rest-in-Rest offspring underwent accelerated growth during the peri-pubertal phase, followed by slowed growth between 2-3 months of age compared with non embryo-transferred Rest groups. Furthermore, renal function and insulin response to a glucose load were different to respective non embryo-transferred groups. Our data demonstrate the long-term effects of in vitro embryo manipulation, which confounded the utility of this approach in delineating between the maternal pregnancy environment and germ-line effects that drive transgenerational outcomes.
A seven amino acid peptide (PEP7) is encoded within a short open reading frame (sORF) within exon 2 (E2) in the 5'-leader sequence (5'LS) upstream of the rat angiotensin 1a-receptor (rAT1aR) mRNA. A chemically synthesized PEP7 markedly inhibited angiotensin II (Ang II)-induced Erk1/2 activation in cells by 62% vs. a scrambled PEP7 (sPEP7) [pErk1/2/Erk1/2 (AU): Ang II, 1.000±0.0, Ang II+PEP7, 0.3812±0.086, Ang II+sPEP7, 1.069±0.18; n=3]. Under these conditions, PEP7 had no effect on Ang II-stimulated inositol-trisphosphate production. PEP7 also had no effect on EGF- and PMA-induced Erk1/2 activation, suggesting PEP7 selectively inhibits AT1aR-mediated Erk1/2 signaling. PEP7 i.c.v. inhibited Ang II-induced saline intake but had no effect on water intake in male and female rats indicating PEP7 also selectively inhibits the Ang II-Erk1/2 pathway in vivo since saline drinking is Erk1/2-mediated while water drinking is not. PEP7 inhibition of Ang II-induced saline ingestion was rapidly reversed by a subsequent i.c.v. injection of an oxytocin antagonist suggesting when PEP7 blocks Ang II-stimulated Erk1/2 activation, animals no longer ingest saline to balance the continued water intake, due to release of oxytocin and its subsequent inhibitory effects on saline drinking. PEP7 also attenuated Ang II-induced increases in arterial pressure by 35% when compared to sPEP7. Thus, we have identified a novel peptide encoded within the rAT1aR E2 that selectively inhibits Erk1/2 activation resulting in physiological consequences for sodium ingestion and arterial pressure that has implications for treating sodium-sensitive diseases like hypertension and kidney disease.
Reproducibly differential responses to different classes of antihypertensive agents are observed among hypertensive patients, and may be due to interindividual differences in hypertension pathology. Computational models provide a tool for investigating the impact of underlying disease mechanisms on the response to antihypertensive therapies with different mechanisms of action. Here we present the development, calibration, validation, and application of an extension of the Guyton/Karaaslan model of blood pressure regulation. The model incorporates a detailed submodel of the Renin Angiotensin Aldosterone System (RAAS), allowing therapies that target different parts of this pathway to be distinguished. Literature data on RAAS biomarker and blood pressure responses to different classes of therapies were used to refine the physiological actions of Angiotensin II and aldosterone on renin secretion, renal vascular resistance, and sodium reabsorption. The calibrated model was able to accurately reproduce the RAAS biomarker and blood pressure responses to combinations of dual-RAAS agents, as well as RAAS therapies in combination with diuretics or calcium channel blockers. The final model was used to explore the impact of underlying mechanisms of hypertension on blood pressure response to different classes of antihypertensive agents. Simulations indicate that the underlying etiology of hypertension can impact the magnitude of response to a given class of therapy, making a patient more sensitive to one class and less sensitive others. Given that hypertension is usually the result of multiple mechanisms, rather than a single factor, these findings yield insight into why combination therapy is often required in order to adequately control blood pressure.
Methamphetamine (Meth) can evoke extreme hyperthermia, which correlates with both neurotoxicity and death in laboratory animals and humans. The objective of this study was to uncover the mechanisms of a complex dose-dependence of temperature responses to Meth by mathematical modeling the neural circuitry involved. Based on previous studies, we composed an artificial neural network with the core comprised of three sequentially connected nodes: Excitatory, Medullary and sympathetic preganglionic SPN. Meth directly stimulated the Excitatory node, an inhibitory drive targeting the Medullary node, and in high doses - additional excitatory drive affecting the SPN. All model parameters (weights of connections, sensitivities, time constants) were subject to fitting experimental time series of temperature responses to 1, 3, 5, and 10 mg/kg of Meth. Modeling suggested that the temperature response to the lowest dose of Meth which caused an immediate and short hyperthermia, involves neuronal excitation at a supramedullary level. The delay in response seen after intermediate doses of Meth is a result of neuronal inhibition at the medullary level. Finally, the rapid and robust increase in body temperature induced by the highest dose of Meth involves activation of high-dose excitatory drive. The impairment in the inhibitory mechanism can provoke life-threatening temperature rise, and makes it a plausible cause of fatal hyperthermia in Meth users. We expect that studying putative neuronal sites of Meth action and involved neuromediators resulting in a detailed model of this system may lead to more effective strategies of prevention and treatment of hyperthermia induced by amphetamine-like stimulants.
Maternal high-fat diet appears to disrupt several energy balance mechanisms in offspring. Here, female offspring from dams fed high fat (HF) did not significantly differ in body weight compared to those fed chow (CHOW), when weaned onto chow diet. Yet when presented with both chow and high fat diet, high fat intake was significantly higher in HF compared to CHOW offspring. To assess taste-based responsiveness, offspring (12 wks old) were tested in 30-min sessions (10-s trials) to a sucrose concentration series in a brief-access taste test. Compared to CHOW, the HF offspring initiated significantly fewer trials but did not significantly differ in the amount of concentration-dependent licking. Thus, rather than affect lick response (consummatory), maternal diet affects spout approach (appetitive) which may be attributed to motivation-related mechanisms. Consistent with this possibility, naltrexone, an opioid receptor antagonist, further reduced trial initiation, but not licking in both groups. With naltrexone administration, the group difference in trial initiation was no longer evident suggesting differences in endogenous opioid activity between the two groups. Relative expression of mu-opioid receptor in the ventral tegmental area was significantly lower in HF rats. When trial initiation was not required in one-bottle intake tests, no main effect of maternal diet on the intake of sucrose and corn oil emulsions was observed. Thus the maternal high fat diet induced difference in diet preference is not likely due to changes in the sensory orosensory component of the taste stimulus but may depend on alterations in satiety signals or absorptive mechanisms.
Prenatal and early childhood exposures are implicated as causes of allergy, but the effects of intrauterine growth restriction on immune function and allergy are poorly defined. We therefore evaluated effects of experimental restriction of fetal growth on immune function and allergic sensitization in adolescent sheep. Immune function (circulating total red and white blood cells, neutrophils, lymphocytes, monocytes, eosinophils and basophils, and the antibody response to Clostridial vaccination) and responses to house dust mite (HDM) allergen and ovalbumin (OVA) antigen sensitization (specific total Ig, IgG1 and IgE antibodies, and cutaneous hypersensitivity) were investigated in adolescent sheep from placentally-restricted (PR, n = 23) and control (n = 40) pregnancies. Increases in circulating HDM-specific IgE (P = 0.007) and OVA-specific IgE (P = 0.038) were greater in PR than control progeny. PR did not alter total Ig, IgG1 or IgM responses to either antigen. PR increased OVA-specific but not HDM-specific IgA responses in females only (P = 0.023). Multiple birth increased Ig responses to OVA in a sex-specific manner. PR decreased the proportion of positive cutaneous hypersensitivity responders to OVA at 24 h (P = 0.030), but had no effect on cutaneous responses to HDM. Acute wheal responses to intradermal histamine correlated positively with birth weight in singletons (P = 0.023). Intrauterine growth restriction may suppress inflammatory responses in skin downstream of IgE induction, without impairment in antibody responses to a non-polysaccharide vaccine. Discord between cutaneous and IgE responses following sensitization suggests new mechanisms for prenatal allergy programming.
The inflammatory response to muscle damaging exercise requires monocyte mobilization and adhesion. Complement receptor type 3 (CR3) and macrophage inflammatory protein (MIP)-1β enables monocyte recruitment, adhesion, and subsequent infiltration into damaged muscle tissue. The purpose of this study was to examine the effects of cold water immersion (CWI) and/or β-hydroxy-β-methylbutyrate free acid (HMB-FA) on CR3 expression and MIP-1β concentration following four sets of up to ten repetitions of squat, dead lift, and split squat exercises at 70-80% 1-repetition maximum. Thirty-nine resistance-trained men (22.2 ± 2.5 y) were randomly divided into four groups: 1)Placebo (PL); 2)HMB-FA; 3)HMB-FA-CWI; 4)PL-CWI. The HMB-FA groups ingested 3 g•d-1 and CWI groups submersed into 10-12°C water for 10 minutes following exercise. Blood was sampled at baseline (PRE), immediately post (IP), 30 minutes post (30P), 24 hours post (24P), and 48 hours post (48P) exercise. Circulating MIP-1β was assayed and CR3 expression on CD14+ monocytes was measured by flow cytometry. Without treatment, CR3 expression significantly elevated at 30P when compared to other time points (p=0.030-0.047). HMB-FA significantly elevated the percent of monocytes expressing CR3 between IP and 24P (p=0.046) and between IP and 48P (p=0.046). No time effect was observed for MIP-1β concentration. The recovery modalities showed to attenuate the rise in CR3 following exercise. Additionally, supplementation with HMB-FA significantly elevated the percent of monocytes expressing CR3 during recovery. Although the time course which inflammatory responses are most beneficial remains to be determined, recovery modalities may alter immune cell mobilization and adhesion mechanisms during tissue recovery.
Reduced mechanical loading during bedrest, spaceflight, casting, etc. causes rapid morphological changes in skeletal muscle: fiber atrophy and reduction of slow-twitch fibers. An emerging signaling event in response to unloading is the translocation of neuronal nitric oxide synthase (nNOSµ) from the sarcolemma to the cytosol. We used EUK-134, a cell-permeable mimetic of superoxide dismutase and catalase, to test the role of redox signaling in nNOSµ translocation and muscle fiber atrophy as a result of short-term (54 hours) hindlimb unloading. Fischer-344 rats were divided into ambulatory controls (CON), hindlimb unloaded (HU), and hindlimb unloaded + EUK-134 (HU-EUK) groups. EUK-134 mitigated the unloading-induced phenotype, including muscle fiber atrophy and muscle fiber-type shift from slow to fast. nNOSµ immunolocalization at the sarcolemma of the soleus was reduced with HU, while nNOSµ protein content in the cytosol increased with unloading. Translocation of nNOS from the sarcolemma to cytosol was virtually abolished by EUK-134. EUK-134 also mitigated dephosphorylation at Thr32 of FoxO3a during HU. Hindlimb unloading elevated oxidative stress (4-hydroxynonenal) and increased sarcolemmal localization of Nox2 subunits gp91phox (Nox2) and p47phox, effects normalized by EUK-134. Thus, our findings are consistent with the hypothesis that oxidative stress triggers nNOSµ translocation from the sarcolemma and FoxO3a dephosphorylation as an early event during mechanical unloading. Thus redox signaling may serve as a biological switch for nNOS to initiate morphological changes in skeletal muscle fibers.
Aims: It is unknown whether cardiomyocyte hypertrophy and the transition to fatty acid oxidation as the main source of energy after birth is dependent on the maturation of the cardiomyocytes' metabolic system, or on the limitation of substrate availability before birth. This study aimed to investigate whether intrafetal administration of a PPAR agonist, rosiglitazone, during late gestation can stimulate the expression of factors regulating cardiac growth and metabolism in preparation for birth, and the consequences on cardiac contractility in the fetal sheep at ~140d gestation. Methods: The mRNA expression and protein abundance of key factors regulating growth and metabolism were quantified using qRT-PCR and Western blotting, respectively. Cardiac contractility was determined by measuring the Ca2+ sensitivity and maximum Ca2+ activated force of skinned cardiomyocyte bundles. Results: Rosiglitazone treated fetuses had a lower cardiac abundance of insulin signaling molecules, including IRβ, IRS-1, phospho-IRS-1(Tyr895), PI3K regulatory subunit p85, PI3K catalytic subunit p110α, phospho-PDPK-1(Ser241), Akt-1, phospho-Akt(ser273), PKC, phospho-PKC(Thr410), AS160, phospho-AS160(Thr642) and GLUT-4. Additionally, cardiac abundance of regulators of fatty acid β-oxidation, including AdipoR1, AMPKα, phospho-AMPKα(Thr172), phospho-ACC(Ser79), CPT-1 and PGC-1α was lower in the rosiglitazone treated group. Rosiglitazone administration also resulted in a decrease in cardiomyocyte size. Conclusions: Rosiglitazone administration in the late gestation sheep fetus resulted in a decreased abundance of factors regulating cardiac glucose uptake, fatty acid β-oxidation and cardiomyocyte size. These findings suggest that activation of PPAR using rosiglitazone does not promote the maturation of cardiomyocyte, rather, it may decrease cardiac metabolism and compromise cardiac health later in life.
Although the effects of prenatal under-nutrition on adult cardiovascular health have been well studied, its effects on the cerebrovascular structure and function remain unknown. We used a pair-fed rat model of 50% caloric restriction from day 11 of gestation to term, with ad libitum feeding after birth. We validated that maternal food restriction (MFR) stress is mediated by glucocorticoids by administering Metyrapone, a corticosterone synthesis inhibitor, to MFR mothers at day 11 of gestation. At age 8-months, Control, MFR, and MFR+Metyrapone offspring were sacrificed and middle cerebral artery (MCA) segments were studied using vessel-bath myography and confocal microscopy. Colocalization of smooth muscle α-actin (SMαA) with non-muscle (NM), SM1 and SM2 myosin heavy chain (MHC) isoforms was used to assess smooth muscle phenotype. Our results indicate that artery stiffness and wall thickness were increased, pressure-evoked myogenic reactivity was depressed, and myofilament Ca++ sensitivity was decreased in MFR compared to Control offspring. MCA from MFR offspring exhibited a significantly greater SMαA/NM colocalization, suggesting that the SMC's had been altered toward a non-contractile phenotype. MET significantly reversed the effects of MFR on stiffness but not myogenic reactivity, lowered SMαA/NM colocalization, and increased SMαA/SM2 colocalization. Together, our data suggest that MFR alters cerebrovascular contractility via both glucocorticoid-dependent and glucocorticoid-independent mechanisms.
Glucagon-like peptide 1 receptors (GLP-1R) are expressed in multiple tissues and activation results in metabolic benefits including enhanced insulin secretion, slowed gastric emptying, suppressed food intake, and improved hepatic steatosis. Limited and inconclusive knowledge exists regarding whether the effects of chronic exposure to a GLP-1R agonist are solely mediated via this receptor. Therefore, we examined 3-month dosing of exenatide in mice lacking a functional GLP-1R (Glp1r-/-). Exenatide (30 nmol/kg/d) was infused subcutaneously for 12 wk in Glp1r-/- and wild-type (Glp1r+/+) control mice fed a high-fat diet. HbA1c, plasma glucose, insulin, amylase, lipase, ALT, AST, body weight, food intake, terminal hepatic lipid content (HLC) and plasma exenatide levels were measured. At study end, oral glucose tolerance (OGTT) and rate of gastric emptying was assessed. Exenatide produced no significant changes in Glp1r-/- mice at study end. In contrast, exenatide decreased body weight, food intake, and glucose in Glp1r+/+ mice. Compared to vehicle, exenatide reduced insulin, OGTT glucose AUC0-2h, ALT, and HLC in Glp1r+/+ mice. Exenatide had no effect on plasma amylase or lipase levels. Exenatide concentrations were approximately 8-fold higher in Glp1r-/- vs. Glp1r+/+ mice after 12 wk infusion whereas renal function was similar. These data support the concept that exenatide requires a functional GLP-1R to exert chronic metabolic effects in mice, and that novel "GLP-1" receptors may not substantially contribute to these changes. Differential exenatide plasma levels in Glp1r+/+ vs. Glp1r-/- mice suggest that GLP-1R may play an important role in plasma clearance of exenatide, and potentially other GLP-1-related peptides.
The adrenal cortex has a molecular clock that generates circadian rhythms in glucocorticoids, yet how the clock is synchronized to the external environment is unknown. Using mPER2::Luciferase (mPER2Luc) knockin mice, in which luciferase is rhythmically expressed under the control of the mouse Per2 clock gene, we hypothesized that ACTH transmits entrainment signals to the adrenal. Adrenal explants were administered ACTH at different phases of the mPER2Luc rhythm. Treatment with ACTH 1-39 produced a phase delay that was phase-dependent, with a maximum at circadian time (CT)18; ACTH did not alter the period of the rhythm. Forskolin produced a parallel response, suggesting that the phase delay was cyclic AMP-mediated. The response to ACTH was concentration-dependent and peptide-specific. Pulse administration (60 min) of ACTH 1-39 also produced phase delays restricted to late CTs. In contrast to ACTH 1-39, other ACTH fragments including alpha melanocyte-stimulating hormone that do not activate the melanocortin 2 (MC2/ACTH) receptor had no effect. The finding that ACTH in vitro phase delays the adrenal mPER2luc rhythm in a monophasic fashion argues for ACTH as a key resetter, but not the sole entrainer, of the adrenal clock.
Neurons in the supraoptic nuclei (SON) produce oxytocin and vasopressin and express insulin receptors (InsR) and glucokinase. Since oxytocin is an anorexigenic agent and glucokinase and InsR are hallmarks of cells that function as glucose and/or metabolic sensors, we evaluated the effect of glucose, insulin, and their downstream effector, KATP channels on calcium signaling in SON neurons and on oxytocin and vasopressin release from explants of the rat hypothalamo-neurohypophyseal system. We also evaluated the effect of blocking glucokinase and phosphatidylinositol 3 kinase (PI3K; mediates insulin-induced mobilization of glucose transporter, GLUT4) on responses to glucose and insulin. Glucose and insulin increased intracellular calcium ([Ca++]i ). The responses were glucokinase and PI3K dependent, respectively. Insulin and glucose alone increased vasopressin release (p<0.002). Oxytocin release was increased by glucose in the presence of insulin. The oxytocin and vasopressin responses to insulin+glucose were blocked by the glucokinase inhibitor, alloxan (4mM; p≤0.002), and the PI3K inhibitor, wortmannin (50nM; OT: p=0.03; VP: p≤0.002). Inactivating KATP channels with 200nM glibenclamide increased oxytocin and vasopressin release (OT: p<0.003; VP: p<0.05). These results suggest that insulin activation of PI3K increases glucokinase-mediated ATP production inducing closure of KATP channels, opening of voltage sensitive calcium channels, and stimulation of oxytocin and vasopressin release. The findings are consistent with SON oxytocin and vasopressin neurons functioning as glucose and 'metabolic' sensors to participate in appetite regulation.
Menopause is associated with an accelerated decline in vascular function, however, whether this is an effect of age and/or menopause and how exercise training may affect this decline remains unclear. We examined a range of molecular measures related to vascular function in matched pre- and post-menopausal women before and after 12 weeks of exercise training. Thirteen pre-menopausal and ten recently post-menopausal (1.6±0.3 (mean±SEM) years after final menstrual period) women only separated by three years (48±1 vs. 51±1 years) were included. Before training, diastolic blood pressure, soluble intercellular adhesion molecule-1 (sICAM-1) and skeletal muscle expression of thromboxane A synthase were higher in the post-menopausal women compared to the pre-menopausal women, all indicative of impaired vascular function. In both groups, exercise training lowered diastolic blood pressure, the levels of sICAM-1, soluble vascular adhesion molecule-1 (sVCAM-1), as well as plasma and skeletal muscle endothelin-1. The vasodilator prostacyclin tended (P=0.061) to be higher in plasma with training in the post-menopausal women only. These findings demonstrate that already within the first years after menopause, several biomarkers of vascular function are adversely altered, indicating that these biomarker changes are more related to hormonal changes than aging. Exercise training appears to have a positive impact on vascular function, as indicated by a marked improvement in the biomarker profile, in both pre- and post-menopausal women.
This study examined the effects of two weeks of high NaCl diet on kidney function and dynamic renal blood flow autoregulation (RBFA) in rats with adenine-induced chronic renal failure (ACRF). Male Sprague-Dawley rats received either chow containing adenine or were pair-fed an identical diet without adenine (controls, C). After 10 weeks rats were randomized to either remain on the same diet (0.6% NaCl) or to be switched to high 4% NaCl chow. Two weeks after randomization renal clearance experiments were performed under isoflurane anesthesia and dynamic RBFA, baroreflex sensitivity (BRS), systolic arterial pressure variability (SAPV) and heart rate variability (HRV) were assessed by spectral analytical techniques. Rats with ACRF showed marked reductions in GFR and renal blood flow (RBF) whereas mean arterial pressure and SAPV were significantly elevated. In addition, spontaneous BRS was reduced by approximately 50 % in ACRF animals. High NaCl diet significantly increased transfer function fractional gain values between arterial pressure and RBF in the frequency range of the myogenic response (0.06-0.09 Hz) only in ACRF animals (0.3±4.0 vs. -4.4±3.8 dB, P<0.05) . Similarly, high NaCl diet significantly increased SAPV in the low frequency range only in ACRF animals. To conclude, a two week period of high NaCl diet in ACRF rats significantly impaired dynamic RBFA in the frequency range of the myogenic response and increased SAPV in the low frequency range. These abnormalities may increase the susceptibility to hypertensive end-organ injury and progressive renal failure by facilitating pressure transmission to the microvasculature.
Intestinal nutrient infusions result in variable decreases in food intake and body weight based on the nutrient type and the specific intestinal infusion site. We previously found that intrajejunal infusions of a fatty acid and glucose, but not casein hydrolysate, decreases food intake and body weight in lean chow-fed laboratory rats. To test whether obese, high fat-fed animals would show similar decreases in food intake and body weight in response to intrajejunal infusions of the same nutrients, equal kilocalorie loads of these nutrients (11.4 kcal) or vehicle were infused into the jejunum of obese, high fat-fed male Sprague-Dawley rats over 7 h/day for 5 consecutive days. Food intake was continuously monitored and body weight was measured daily. After the infusion on the final day, rats were sacrificed and plasma collected. Similar to lean chow-fed rats, intrajejunal infusions of LA and Glu, but not Cas, suppressed food intake with no compensatory increase in food intake after the infusion period. In contrast to lean chow-fed rat, only the LA, and not the Glu or Cas, produced decreases in body weight in the obese high fat fed rat. There also were no differences in plasma GLP-1 levels in any of the nutrient infusion groups compared to saline infusion. These results suggest that there is a differential response to the same nutrients in lean versus obese animals.
Body temperature increases when individuals experience salient, emotionally significant events. There is controversy concerning the contribution of non-shivering thermogenesis in brown adipose tissue (BAT) to emotional hyperthermia. In the present study we compared BAT, core body, and brain temperature, and tail blood flow, simultaneously measured, to determine whether BAT thermogenesis contributes to emotional hyperthermia in a resident Sprague-Dawley rat when an intruder rat, either freely-moving or confined to a small cage, is suddenly introduced into the cage of the resident rat for 30 min. Introduction of the intruder rat promptly increased BAT, body and brain temperatures in the resident rat. For the caged intruder these temperature increases were 1.4±0.2, 0.8±0.1, 1.0±0.1C° respectively, with the increase in BAT temperature being significantly greater (p<0.01) than the increases in body and brain. The initial 5 min slope of the BAT temperature record (0.18±0.02C°/min) was significantly greater (p<0.01) than the corresponding value for body (0.10±0.01C°/min) and brain (0.09±0.02C°/min). Tail artery pulse amplitude fell acutely when the intruder rat was introduced, possibly contributing to the increases in body and brain temperature. Prior blockade of beta-3 adrenoceptors (SR59230A 10 mg/kg i.p.) significantly reduced the amplitude of each temperature increase. Intruder-evoked increases in BAT temperature were similar in resident rats maintained at 11°C for 3 days. In the caged intruder situation there is no bodily contact between the rats, so the stimulus is psychological rather than physical. Our study thus demonstrates that BAT thermogenesis contributes to increases in body and brain temperature occurring during emotional hyperthermia.
There is a link between visceral white adipose tissue (WAT) and the metabolic syndrome in humans, with health improvements produced with small visceral WAT reduction. By contrast, subcutaneous WAT provides a site for lipid storage that is rather innocuous relative to ectopic lipid storage in muscle or liver. The sympathetic nervous system (SNS) is the principal initiator for lipolysis in WAT by mammals. Nothing is known, however, about the central origins of the SNS circuitry innervating the only true visceral WAT in rodents - mesenteric WAT (MWAT) which drains into the hepatic portal vein. We tested whether the central sympathetic circuits to subcutaneous [inguinal WAT (IWAT)] and visceral WAT (MWAT) are separate or shared and whether they possess differential sympathetic drives with food deprivation in Siberian hamsters. Using two isogenic strains of pseudorabies virus, a retrograde transneuronal viral tract tracer within the same hamsters we found overlap (~20-55% doubly infected neurons) between the two circuitries across the neural axis with lesser overlap proximal to the depots (spinal cord and sympathetic chain) and with more neurons involved in the innervation of IWAT than MWAT in some brain regions. Food deprivation triggered a greater sympathetic drive to subcutaneous (IWAT) than visceral (MWAT) depots. Collectively, we demonstrated both shared and separate populations of brain, spinal cord and sympathetic chain neurons ultimately project to a subcutaneous WAT depot (IWAT) and the only visceral WAT depot in rodents (MWAT). In addition, the lipolytic stimulus of food deprivation only increased SNS drive to subcutaneous fat (IWAT).
Signals from the vestibular system, area postrema, and forebrain elicit nausea and vomiting, but gastrointestinal (GI) vagal afferent input arguably plays the most prominent role in defense against food poisoning. It is difficult to determine the contribution of GI vagal afferent input on emesis because various agents (e.g., chemotherapy) often act on multiple sensory pathways. Intragastric copper sulfate (CuSO4) potentially provides a specific vagal emetic stimulus but its actions are not well defined in musk shrews, a primary small animal model used to study emesis. The aims of the current study were (1) to investigate the effects of subdiaphragmatic vagotomy on CuSO4-induced emesis and (2) to conduct preliminary transneuronal tracing of the GI-brain pathways in musk shrews. Vagotomy failed to inhibit the number of emetic episodes produced by optimal emetic doses of CuSO4 (60 and 120 mg/kg, ig), but the effects of lower doses were dependent on an intact vagus (20 and 40 mg/kg). Vagotomy also failed to affect emesis produced by motion (1 Hz, 10 min) or nicotine administration (5 mg/kg, sc). Anterograde transport of the H129 strain of herpes simplex virus-1 from the ventral stomach wall identified the following brain regions as receiving inputs from vagal afferents: the nucleus of the solitary tract, area postrema, and lateral parabrachial nucleus. These data indicate that the contribution of vagal pathways to intragastric CuSO4-induced emesis is dose dependent in musk shrews. Furthermore, the current neural tracing data suggests brainstem anatomical circuits that are activated by GI signaling in the musk shrew.
The subfornical organ (SFO) is an important sensory circumventricular organ implicated in the regulation of fluid homeostasis and energy balance. We investigated whether the SFO is activated by the hormone cholecystokinin (CCK). CCK1 and CCK2 receptors were identified in the SFO by RT-PCR. Dissociated SFO neurons that responded to CCK (40/77), were mostly depolarized (9.2±0.9mV, 30/77); but some were hyperpolarized (-7.3±1.1 mV, 10/77). We next examined the responses of SFO neurons in vivo to CCK (16µg/kg, i.p.), in the presence and absence of CCK1 or CCK2 receptor antagonists (Devazepide; 600 µg/kg and L-365,260; 100 µg/kg, respectively), using the functional activation markers c-Fos and p-ERK. The nucleus of the solitary tract (NTS) served as a control for CCK-induced activity. There was a significant increase in c-Fos expression in the NTS (259.2±20.8 neurons) compared to vehicle (47.5±2.5). Similarly, in the SFO, c-Fos was expressed in 40.5±10.6 neurons in CCK-treated compared to 6.6 ±2.7 in vehicle-treated rats (P<0.01). Devazepide significantly reduced the effects of CCK in the NTS, but not in SFO. L-365,260 blocked the effects of CCK in both brain regions. CCK increased the number of p-ERK neurons in NTS (27.0±4.0) as well as SFO (18.0±4.0), compared to vehicle (8.0 ±2.6 and 4.3±0.6, respectively; P<0.05). Both devazepide and L-365,260 reduced CCK induced p-ERK in NTS, but only L-365,260 reduced it in the SFO. In conclusion, the SFO represents a novel brain region at which circulating CCK may act via CCK2 receptors to influence central autonomic control.
Exaggerated GLP-1 and PYY secretion is thought to be a major mechanism in the reduced food intake and body weight after Roux-en-Y gastric bypass surgery. Here we use complementary pharmacological and genetic loss-of-function approaches to test the role of increased signaling by these gut hormones in high-fat diet-induced obese rodents. Chronic brain infusion of a supra-maximal dose of the selective GLP-1 receptor antagonist exendin-3-39 into the lateral cerebral ventricle significantly increased food intake and body weight in both RYGB and sham-operated rats, suggesting that, while contributing to the physiological control of food intake and body weight, central GLP-1 receptor signaling tone is not the critical mechanism uniquely responsible for the body weight lowering effects of RYGB. Central infusion of the selective Y2R-antagonist BIIE0246 had no effect in either group, suggesting that it is not critical for the effects of RYGB on body weight under the conditions tested. In a recently established mouse model of RYGB that closely mimics surgery and weight loss dynamics in humans, obese GLP-1R-deficient mice lost the same amount of body weight and fat mass and maintained similarly lower body weight compared with wildtype mice. Together, the results surprisingly provide no support for important individual roles of either gut hormone in the specific mechanisms by which RYGB rats settle at a lower body weight. It is likely that the beneficial effects of bariatric surgeries are expressed through complex mechanisms that require combination approaches for their identification.
The choroid plexus epithelium forms the blood-cerebrospinal fluid barrier and accumulates essential minerals and heavy metals. Choroid plexus is cited as being a 'sink' for heavy metals and excess minerals, serving to minimize accumulation of these potentially toxic agents in the brain. Understanding of how low doses of contaminant metals might alter transport of other solutes in choroid plexus is limited. Using primary cultures of epithelial cells isolated from neonatal rat choroid plexus, our objective was to characterize modulation of apical uptake of the model organic cation choline elicited by low concentrations of the contaminant metal cadmium (CdCl2). At 50-1000 nM, cadmium did not directly decrease or increase 30-min apical uptake of 10 µM [3H]choline. However, extended exposure to 250-500 nM cadmium increased [3H]choline uptake by as much as 75% without marked cytotoxicity. In addition, cadmium induced heat shock protein 70 and heme oxygenase-1 protein expression and markedly induced metallothionein (Mt-1) gene expression. The antioxidant N-acetylcysteine attenuated stimulation of choline uptake and induction of stress proteins. Conversely, an inhibitor of glutathione synthesis L-buthionine-sulfoximine (BSO) enhanced stimulation of choline uptake and induction of stress proteins. Cadmium also activated ERK1/2 MAP kinase. The MEK1 inhibitor PD98059 diminished ERK1/2 activation and attenuated stimulation of choline uptake. Furthermore, inhibition of ERK1/2 activation abated stimulation of choline uptake in cells exposed to cadmium with BSO. These data indicate in choroid plexus exposure to low concentrations of cadmium may induce oxidative stress and consequently stimulate apical choline transport through activation of ERK1/2 MAP kinase.
Zebrafish Na+/H+ exchanger 3b (zNHE3b) is highly expressed in the apical membrane of ionocytes where Na+ is absorbed from ion-poor fresh water against a concentration gradient. Much in vivo data indicated that zNHE3b is involved in Na+ absorption but not leakage. However, zNHE3b-mediated Na+ absorption has not been thermodynamically explained, and zNHE3b activity has not been measured. To address this issue, we overexpressed zNHE3b in Xenopus oocytes and characterized its activity by electrophysiology. Exposure of zNHE3b oocytes to Na+-free media resulted in significant decrease in intracellular pH (pHi) and intracellular Na+ activity (aNai). aNai increased significantly when the cytoplasm was acidified by media containing CO2/HCO3- or butyrate. Activity of zNHE3b was inhibited by amiloride or 5-ethylisopropyl amiloride (EIPA). Although the activity was accompanied by a large hyperpolarization of ~50 mV, voltage clamp experiments showed that Na+/H+-exchange activity of zNHE3b is electroneutral. Exposure of zNHE3b oocytes to medium containing NH3/NH4+ resulted in significant decreases in pHi and aNai and significant increase in intracellular NH4+ activity, indicating that zNHE3b mediates the Na+/NH4+ exchange. In low-Na+ (0.5 mM) media, zNHE3b oocytes maintained intracellular Na+ activity of 1.3 mM, and Na+-influx was observed when intracellular pH was decreased by media containing CO2/HCO3- or butyrate. These results provide thermodynamic evidence that zNHE3b mediates Na+ absorption from ion-poor fresh water by its Na+/H+ and Na+/NH4+-exchange activities.
Nerve cell metabolic activity is monitored in multiple brain regions, including the hypothalamus and hindbrain dorsal vagal complex (DVC), but it is unclear if individual metabolo-sensory loci operate autonomously or interact to coordinate CNS reactivity to energy imbalance. This research addressed the hypothesis that hypoglycemia-associated DVC lactoprivation stimulates hypothalamic AMPK activity and metabolic neurotransmitter expression. As DVC catecholaminergic neurons express biomarkers for metabolic monitoring, we investigated whether these cells are a source of lactate deficit signaling to the hypothalamus. Caudal fourth ventricle (CV4) infusion of the glucose metabolite, L-lactate, during insulin-induced hypoglycemia reversed changes in DVC A2 noradrenergic, arcuate neuropeptide Y (NPY) and pro-opiomelanocortin (POMC), and lateral hypothalamic orexin-A (ORX) neuronal phosphoAMPK (pAMPK) expression, coincident with hypoglycemic intensification. Hindbrain lactate repletion also blunted hypoglycemic up-regulation of arcuate NPY mRNA and protein. This treatment did not alter hypoglycemic paraventricular oxytocin (OT) and lateral hypothalamic ORX mRNA profiles, but exacerbated or reversed adjustments in OT and ORX neuropeptide synthesis, respectively. CV4 delivery of the monocarboxylate transporter inhibitor, 4-CIN, increased A2 pAMPK levels, elevated circulating glucose, and stimulated feeding, responses that were attenuated by 6-hydroxydopamine pretreatment. 4-CIN-infused rats exhibited increased (NPY, ORX neurons) or decreased (POMC neurons) pAMPK expression concurrent with hyperglycemia. These data show that hindbrain lactoprivic signaling regulates hypothalamic AMPK and key effector neurotransmitter responses to hypoglycemia. Evidence that A2 AMPK activity is lactate-dependent, and that DVC catecholamine cells are critical for lactoprivic control of glucose, feeding, and hypothalamic AMPK, implies A2 derivation of this metabolic regulatory stimulus
Activation of mineralocorticoid receptors (MR) of the hypothalamic paraventricular nucleus (PVN) increases sympathetic excitation. To determine whether MR and glucocorticoid receptors (GR) are expressed in pre-autonomic neurons of the PVN and how they relate to endogenous aldosterone levels in healthy normotensive rats, retrograde tracer was injected into the intermediolateral cell column at T4 to identify pre-autonomic neurons in the PVN. Expression of MR, GR, 11-β hydroxysteroid dehydrogenase1 & 2 (11βHSD1&2), and hexose-6-phosphate dehydrogenase (H6PD) required for 11βHSD1 reductase activity by immunohistochemistry. RT-PCR and western blot analysis was used to determine MR gene and protein expression. Most pre-autonomic neurons were in the caudal mediocellular region of PVN and most expressed MR; none expressed GR. 11βHSD1, but not 11βHSD2 or H6PD immunoreactivity, was detected in the PVN. In rats receiving chronic low or high sodium intakes, the low sodium diet was associated with significantly higher plasma aldosterone, MR mRNA and protein expression, and c-fos immunoreactivity within pre-autonomic neurons. Plasma corticosterone and sodium and expression of tonicity-responsive enhancer binding protein in the PVN did not differ between groups, suggesting osmotic adaptation to the altered sodium intake. These results suggest that MR within pre-autonomic neurons in the PVN directly participate in the regulation of sympathetic nervous system drive, aldosterone may be a relevant ligand for MR in pre-autonomic neurons of the PVN under physiological conditions due to the dehydrogenase activity of 11βHSD1 in the absence of H6PD that regenerates NADP+ from NADPH and may increase MR gene expression under physiological conditions.
Transient receptor potential canonical subtype 4 (TRPC4) is expressed in the magnocellular paraventricular nucleus (PVN) and supraoptic nucleus (SON) of the hypothalamus. In this study, the regulation of TRPC4 expression was investigated in water deprivation and hepatic cirrhosis. We utilized laser capture microdissection technique for precise dissection of pure arginine-vasopressin (AVP) cell population in the PVN and SON followed by quantitative real time RT-PCR, and immunodetection techniques by western blot analysis and immunofluorescence. Bile duct ligation elevated TRPC4 transcripts in the SON but not PVN with correlated changes in the protein expression in these regions as well as increased colocalization with AVP in the SON with no changes in the PVN. Water deprivation resulted in increased TRPC4 mRNA expression in the PVN while it decreased channel expression levels in the SON. In both these regions, protein expression measured from tissue punches were unaltered following water deprivation with no changes in the number of TRPC4 positive cells. Thus, TRPC4 expression is differentially regulated in physiological and pathophysiological models of vasopressin release.
Hypophagia induced by inflammation is associated with JAK2-STAT3 signaling and leptin-mediated hypophagia is also mediated by JAK2-STAT3 pathway. We have previously reported that lipopolysaccharide (LPS) did not reduce food intake in leptin-resistant high-fat diet (HFD) rats, but maintained body weight loss. We investigated if changes in p-STAT3 expression in the hypothalamus and brainstem could account for the desensitization of hypophagia in HFD animals after a low LPS dose (100μg/Kg). Wistar rats fed standard diet (3.95Kcal/g) or HFD (6.3Kcal/g) for 8 weeks were assigned into control diet-saline, control diet-LPS, HFD-saline and HFD-LPS groups. LPS reduced feeding in the control diet, but not HFD. This group showed no p-STAT3 expression in the PVN and VMH, but sustained, though lower than control, p-STAT3 in the NTS and RPA. LPS decreased body weight in HFD rats, and increased Fos expression in the NTS. LPS increased body temperature, oxygen consumption and energy expenditure in both control diet and HFD rats, and this response was more pronounced in HFD-LPS group. BAT thermogenesis and increased energy expenditure seem to contribute to body weight loss in HFD-LPS. This response might be related with increased brainstem activation. In conclusion, LPS activates STAT3-mediated pathway in the hypothalamus and brainstem, leading to hypophagia, however LPS effects on food intake, but not body weight loss, are abolished by leptin resistance induced by HFD. The preserved STAT3 phosphorylation in the brainstem suggests that unresponsiveness to LPS on STAT3 activation under HFD might be selective to the hypothalamus.
Metaboreceptor activation during passive heating is known to influence cutaneous vascular conductance (CVC) and sweat rate (SR). However, whether metaboreceptors modulate the suppression of heat loss following dynamic exercise remains unclear. On separate days, before and after 15-min of high intensity treadmill running in the heat (35°C), eight males performed either: i) no isometric handgrip exercise (IHG) or ischemia (CON); ii) 1-min IHG (60% of maximum, IHG); iii) 1-min IHG followed by 2-min of ischemia (IHG+OCC); iv) 2-min of ischemia (OCC); or v) 1-min IHG followed by 2-min of ischemia with application of lower body negative pressure (IHG+LBNP). SR (ventilated capsule), cutaneous blood flow (Laser-Doppler) and mean arterial pressure (Finometer) were measured continuously before and after dynamic exercise. Following dynamic exercise, CVC was reduced with IHG exercise (P<0.05) and remained attenuated with post-IHG ischemia during IHG+OCC relative to CON (39±2 vs 47±6%, P<0.05). Furthermore, the reduction in CVC was exacerbated by application of LBNP during post-IHG ischemia (35±3%, P<0.05) relative to IHG+OCC. SR increased during IHG exercise (P<0.05) and remained elevated during post-IHG ischemia relative to CON following dynamic exercise (0.94±0.15 vs. 0.53±0.09 mg•min-1•cm-2, P<0.05). In contrast, application of LBNP during post-IHG ischemia had no effect on SR (0.93±0.09 mg•min-1•cm-2, P>0.05) relative to post-IHG ischemia during IHG+OCC. We show that CVC is reduced and that sweat rate is increased by metaboreceptor activation following dynamic exercise. In addition, we show that the metaboreflex-induced loading of the baroreceptors can influence the CVC response, but not the sweating response.
The purpose of this study was to assess whether the deleterious effect of chronic alcohol consumption differs in adult and aged female rats. To address this aim, adult (4 m) and aged (18 m) F344 rats were fed a nutritionally complete liquid diet containing alcohol (36% total calories) or an isocaloric isonitrogenous control diet for 20 wks. Cardiac structure and function, assessed by echocardiography, as well as myocardial protein synthesis and proteolysis did not differ in either alcohol- vs control-fed adult rats or in adult vs aged control-fed rats. In contrast, cardiac function was impaired in alcohol-fed aged rats, compared to age-matched control rats. Additionally, alcohol feeding decreased cardiac protein synthesis which was associated with decreased phosphorylation of 4E-BP1 and S6K1. This reduction in mTOR kinase activity was associated with reduced eIF3f and binding of both Raptor and eIF4G to eIF3. Proteasome activity was increased in alcohol-fed aged rats with a coordinate elevation in the E3 ligases atrogin-1 and MuRF1. These changes were associated with increased REDD1 and phosphorylation of AMPK, but no increase in AKT or FOXO3 phosphorylation. Finally, markers of autophagy (e.g., LC3B, Atg7, Atg12) and TNFα were increased to a greater extent in alcohol-fed aged rats. These data demonstrate that aged female rats exhibit an enhanced sensitivity to alcohol, compared to adult animals. Our data are consistent with a model whereby alcohol increases proteolysis via FOXO-independent increase in atrogin-1 which degrades eIF3f and therefore impairs formation of a functional pre-initiation complex and protein synthesis.
Chronic obstructive pulmonary disease (COPD) often results in increased levels of tumor necrosis factor-alpha (TNF-α), a pro-inflammatory cytokine, which circulates in the blood. However, it is not clear whether pulmonary TNF-α overexpression (a COPD mimic) induces excessive reactive oxygen species (ROS) formation in skeletal muscle, and thereby may contribute to the muscle impairment often seen in COPD. We hypothesized that ROS generation in contracting skeletal muscle is elevated when there is TNF-α overproduction in the lung, and that this can induce muscle dysfunction. Cytochrome c (cyt c) in the perfusate was used to assay superoxide (O2•-) release from isolated contracting soleus muscles from transgenic mice of pulmonary TNF-α overexpression (Tg+) and wild type (WT) mice. Our results showed that Tg+ muscle released significantly higher levels of O2•- than WT during a period of intense contractile activity (in nmol/mg wt.; 17.5 ± 2.3 vs. 4.4 ± 1.3 respectively; n = 5; P < 0.05). In addition, the soleus muscle demonstrated a significantly reduced fatigue resistance in Tg+ mice compared to WT mice. Perfusion of the contracting soleus muscle with superoxide dismutase, which specifically scavenges O2•- in the perfusate, resulted in significantly less cyt c reduction, thereby indicating that the type of ROS released from the Tg+ muscles is O2•-. Our results demonstrate that pulmonary TNF-α overexpression leads to a greater O2•- release from contracting soleus muscle in Tg+ compared to WT, and that the excessive formation of O2•- in the contracting muscle of Tg+ mice leads to earlier fatigue.
The remarkable development and refinement of the Cre-loxP system coupled with the non-stop production of new mouse models and virus vectors have impelled the growth of various fields of investigation. In this article, I will discuss the data collected using these genetic tools in our area of interest, giving specific emphasis to the identification of the neuronal population(s) that relay leptin action in reproductive physiology. A series of mouse models that allow manipulation of the leptin receptor gene have been generated. Of those, I will discuss the use of two models of leptin receptor gene re-expression (LepRneo/neo and LepRloxTB/loxTB) and one model of leptin signaling blockade (LepRflox/flox). I will also highlight the differences of using stereotaxic delivery of virus vectors expressing DNA-recombinases (Flp and Cre) and mouse models expressing Cre-recombinase. Our findings indicate that leptin action in the ventral premammillary nucleus is sufficient but not required for leptin action in reproduction and that leptin action in Kiss1 neurons arises after pubertal maturation; therefore, direct leptin signaling in Kiss1 neurons is neither required nor sufficient for the permissive action of leptin in pubertal development. It also became evident that the full action of leptin in the reproductive neuroendocrine axis requires the engagement of an integrated circuitry, yet to be fully unveiled.
In addition to orosensory signals, post-oral actions of fat stimulate appetite and condition flavor preferences but the gut sensors mediating these responses are unknown. Here we investigated the role of the fatty acid sensors GPR40 and GPR120 in post-oral and oral preferences for a soybean oil emulsion (Intralipid). Mice were trained to drink a flavored solution (CS+) paired with intragastric (IG) oil infusions and another flavored solution (CS-) paired with water infusions. Knockout (KO) mice missing GPR40 or GPR120 sensors increased their CS+ intake in one-bottle tests (1 h/day) but less so than wildtype (WT) mice. The KO mice also preferred the CS+ to CS- in a two-bottle test but the preference was attenuated in GPR40 KO mice. Double knockout (DoKO) mice missing both GPR40 and GPR120 displayed attenuated stimulation of CS+ intake and only a marginal CS+ preference. The DoKO mice developed a more substantial CS+ preference when tested 24 h/day, although weaker than that of WT mice. The DoKO mice also consumed less of the CS+ paired with IG Intralipid as well as less Intralipid in oral tests. However, DoKO mice, like GPR40 KO and GPR120 KO mice did not differ from WT mice in their preference for Intralipid over water at 0.001% - 20% concentrations. In contrast to prior results obtained with mice missing the CD36 fatty acid sensor, these findings indicate that together GPR40 and GPR120 play a critical role in the post-oral stimulation of appetite by fat but are not essential for oral fat preferences.
Differential sensing of dietary fat and fatty acids by the oral cavity is proposed to regulate the susceptibility to obesity. In the current experiments, animals which differ in their susceptibility to obesity were used to investigate the influence of the oral cavity on the preference for the polyunsaturated fatty acid, linoleic acid. In Experiment 1, the preference for differing concentrations of linoleic acid was determined in obesity-prone, Osborne-Mendel (OM) and obesity-resistant S5B/Pl (S5B) rats. The preference threshold for linoleic acid was lower in S5B rats, compared to OM rats. To determine if differences in linoleic acid preference threshold were related to innate strain differences fatty acid receptors on the tongue, the expression of GPR120, GPR40 and CD36 on the circumvallate papillae were assessed in OM and S5B rats. Results indicated that the expression of CD36, GPR40 and GPR120 did not differ between these two strains. Numerous studies have examined the role of CD36 on fat intake, therefore in Experiment 3, RNA interference was used to decrease the expression of CD36 on the tongues of OM and S5B rats and the effect of decreased CD36 expression on linoleic acid preference was determined. CD36 siRNA attenuated linoleic acid preference for the most preferred concentration in both OM and S5B rats. Overall these data indicate that there are innate differences in the preference threshold for linoleic acid in obesity-prone and obesity-resistant rats. Experimentally reducing the expression of CD36 on the circumvallate papillae attenuated the preference for linoleic acid in both strains.
To explore developmental changes in circadian organization of central and peripheral oscillators, circadian rhythms in clock gene expression were examined in 12 organs in transgenic rats carrying a bioluminescence reporter for Per2. Organ slices were obtained from different developmental stages starting at postnatal day 5 and tissue was cultured for more than 6 days. In addition, four organs were examined from embryonic day 20. Robust circadian rhythms in bioluminescence were detected in all organs examined. The circadian period in vitro was specific to each organ and remained essentially the same during development. The circadian peak phase on the first day of culture was significantly different not only among organs but also in the same organ. Three patterns in circadian phase were detected during development. Thus, during development, circadian phase did not change in the suprachiasmatic nucleus, adrenal gland and liver, whereas delay-shifts were seen in the pineal, lung, heart, kidney, spleen, thymus and testis. Finally, circadian phase advanced at postnatal day 10-15, and subsequently phase-delayed in skeletal muscle and stomach. Circadian amplitude also showed developmental changes in several organs. These findings indicate that the temporal orders of physiological functions of various organs change in the course of development. Such age dependent and organ specific changes in the phase relationship among circadian clocks most likely reflect entrainment to organ specific time cues at different developmental stages.
Tumor necrosis factor (TNF) is considered an adverse mediator of heat stroke (HS) based on clinical studies showing high serum levels. However, soluble TNF receptors (sTNFR; TNF antagonists) were higher in survivors than nonsurvivors and TNFR KO mice showed a trend towards increased mortality suggesting TNF has protective actions for recovery. We delineated TNF actions in HS by comparing thermoregulatory, metabolic and inflammatory responses between B6129F2 (WT) and TNFR KO mice. Prior to heat exposure, TNFR KO mice showed ~0.4°C lower core temperature (Tc; radiotelemetry), ~10% lower metabolic rate (Mr; indirect calorimetry) and reduced plasma IL-1α and sIL-1RI than WT mice. KO mice selected warmer temperatures than WT mice in a gradient, but remained hypothermic. In the calorimeter, both genotypes showed a similar heating rate, but TNFR KO maintained lower Tc and Mr than WT mice for a given heat exposure duration and required ~30 min longer to reach maximum Tc (42.4°C). Plasma IL-6 increased at ~3h of recovery in both genotypes, but KO mice showed a more robust sIL-6R response. Higher sIL-6R in the KO mice was associated with delayed liver p-STAT3 protein expression and attenuated serum amyloid A3 (SAA3) gene expression suggesting the acute phase response (APR) was attenuated in these mice. Our data suggest that the absence of TNF signaling induced a regulated hypothermic state in the KO mice, TNF-IL-1 interactions may modulate Tc and Mr during homeostatic conditions, and TNF modulates the APR during HS recovery through interactions with the liver IL-6-STAT3 pathway of SAA3 regulation.
This work examined the effects of age on salt appetite measured in the form of daily saline drinking in response to administration of deoxycorticosterone acetate (DOCA; 5 mg/ kg BW) using young (4 mo), "middle-aged" adult (12 mo) and old (30 mo) male Brown Norway rats. Water and sodium intakes, excretions and balances were determined daily. The salt appetite response was age-dependent with "middle-aged" rats ingesting the most sodium saline solution followed in order by young and then old rats. While old rats drank the least saline solution, the amounts of saline ingested still were copious, and comprise an unambiguous demonstration of salt appetite in old rats. Middle-aged rats had the highest saline preference ratios of the groups under baseline conditions and throughout testing consistent with an increased avidity for sodium taste. There were age differences in renal handling of water and sodium that were consistent with a renal contribution to the greater saline intakes by middle-aged rats. There was evidence of impaired renal function in old rats, but this did not account for the reduced saline intakes of the oldest rats.
Heat dissipation from the rat's tail is reduced in response to cold and during fever. The sympathetic premotor neurons for this mechanism, located in the medullary raphé, are under tonic inhibitory control from the preoptic area. In parallel with the inhibitory pathway, an excitatory pathway from the rostromedial preoptic region (RMPO) to the medullary raphé mediates the vasoconstrictor response to cold skin. Whether this applies also to the tail vasoconstrictor response in fever is unknown. Single- or a few-unit tail sympathetic nerve activity (SNA) was recorded in urethane-anaesthetized, artificially ventilated rats. Experimental fever was induced by prostaglandin E2 (PGE2) injected into the lateral cerebral ventricle (icv, 50ng in 1.5µl) or into the RMPO (0.2ng in 60nl); in both cases there was a robust increase in tail SNA and a delayed rise in core temperature. Microinjection of glutamate receptor antagonist kynurenate (50mM, 120nl) into the medullary raphé completely reversed the tail SNA response to icv or RMPO PGE2 injection. Inhibiting RMPO neurons by microinjecting glycine (0.5M, 60nl) or the GABAA receptor agonist, muscimol (2mM, 30-60nl), reduced the tail SNA response to PGE2 injected into the same site by approximately half. Vehicle injections into the medullary raphé or RMPO were without effect. These results suggest that the tail vasoconstrictor response during experimental fever depends on a glutamatergic excitatory synaptic relay in the medullary raphé, and that an excitatory output signal from the RMPO contributes to the tail vasoconstrictor response during fever.
The locus coeruleus (LC) is a chemoreceptive brainstem region in anuran amphibians and contains neurons sensitive to physiological changes in CO2/pH. The ventilatory and central sensitivity to CO2/pH is proportional to the temperature in amphibians, i.e., sensitivity increases with increasing temperature. We hypothesized that LC neurons from bullfrogs, Lithobates catesbeianus, would increase CO2/pH sensitivity with increasing temperature and decrease CO2/pH sensitivity with decreasing temperature. Further, we hypothesized that cooling would decrease, while warming would increase, normocapnic firing rates of LC neurons. To test these hypotheses, we used whole-cell patch clamp electrophysiology to measure firing rate, membrane potential (Vm), and input resistance (Rin) in LC neurons in brainstem slices from adult bullfrogs over a physiological range of temperatures during normocapnia and hypercapnia. We found that cooling reduced chemosensitive responses of LC neurons as temperature decreased until elimination of CO2/pH sensitivity at 10°C. Chemosensitive responses increased at elevated temperatures. Surprisingly, chemosensitive LC neurons increased normocapnic firing rate and underwent membrane depolarization when cooled and decreased normocapnic firing rate and underwent membrane hyperpolarization when warmed. These responses to temperature were not observed in non-chemosensitive LC neurons or neurons in a brainstem slice 500μm rostral to the LC. Our results indicate that modulation of cellular chemosensitivity within the LC during temperature changes may influence temperature-dependent respiratory drive during acid-base disturbances in amphibians. Additionally, cold-activated/ warm-inhibited LC neurons introduce paradoxical temperature sensitivity in respiratory control neurons of amphibians.
Entrainment of circadian behavior rhythms by daily exposure to running-wheel was examined in mice under constant darkness. Spontaneous movement was individually monitored for more than 6 months by thermal sensor. After establishment of steady state free-running, mice were placed in a different cage equipped with a running-wheel for 3 h once per day at 6:00 am. The daily exchange was continued for 80 days. The number of wheel revolutions during exposure to running-wheel was also measured simultaneously with spontaneous movement. In 13 out of 17 mice, circadian behavior rhythm was entrained by daily wheel exposure, showing a period indistinguishable from 24 h. The entrainment occurred in parallel with an increase in spontaneous movement immediately prior to the daily wheel exposure. A similar pre-exposure increase was observed in only one out of 4 un-entrained mice. The pre-exposure increase appeared in 19.5 days on average after the start of daily wheel exposure and persisted for 36 days on average after the termination of the exposure schedule. The pre-exposure increase was detected only when daily wheel exposure came into the activity phase of the circadian behavior rhythm, which was accompanied by an increase in the number of wheel-revolutions. These findings indicate that a novel oscillation with a circadian period is induced in mice by daily exposure to running-wheel at a fixed time of day and suggest that the oscillation is involved in the non-photic entrainment of circadian rhythms in spontaneous movement.
This review describes the changes that occur in circulating renin-angiotensin-aldosterone (RAAS) system components in human pregnancy. These changes depend on endocrine secretions from the ovary and possibly the placenta and decidua. Not only do these hormonal secretions directly contribute to the increase in RAAS levels, they also cause physiological changes within the cardiovascular system and the kidney, which in turn induce reflex release of renal renin. High levels of Ang II play a critical role in maintaining circulating blood volume, blood pressure and uteroplacental blood flow through interactions with the Ang II type I receptor, and through increased production of downstream peptides acting on a changing Ang receptor phenotype. The increase in Ang II early in gestation is driven by estrogen-induced increments in angiotensinogen (AGT) levels, so there cannot be negative feedback leading to reduced Ang II production. AGT can exist in various forms in terms of redox state or complexed with other proteins as polymers; these affect the ability of renin to cleave Ang I from AGT. Thus during pregnancy the rate of Ang I production varies not only because levels of renin change in response to homeostatic demand but also because AGT changes not only in concentration but in form. Activation of the circulating and intrarenal RAASs is essential for normal pregnancy outcome subserving the increased demand for salt and hence water during pregnancy. Thus the complex integration of the secretions and actions of the circulating maternal renin-angiotensin system in pregnancy plays a key role in pregnancy outcome.
Classical pro-inflammatory, and more recently discovered lipid mediators with pro-resolving bioactivity, exert a complex role in the initiation, control, and resolution of inflammation. We investigated lipid mediator responses to resistance exercise and treatment with the non-steroidal anti-inflammatory drug (NSAID) ibuprofen. Human subjects undertook a single bout of resistance exercise (80% 1RM) following oral ingestion of ibuprofen (400 mg) or placebo control. Blood was collected during early recovery (0-3 h and 24 h post-exercise) and serum lipid mediator composition analyzed by LC-MS based targeted lipidomics. Post-exercise recovery was characterized by elevated levels of cyclooxygenase (COX-1 and 2) derived prostanoids (TXB2, PGE2, PGD2, PGF2α, PGI2), lipooxygenase (5-, 12-, and 15-LOX) derived hydroxyeicosatetraenoic acids (HETEs) and leukotrienes (e.g. LTB4), and epoxygenase (CYP) derived epoxy/dihydroxyeicosatrienoic acids (EpETrEs/DiHETrEs). Additionally, we detected elevated levels of lipid mediators with anti-inflammatory and pro-resolving properties including AA derived lipoxins (LXA4 & LXB4), and the EPA (E-series) and DHA (D-series) derived resolvins (RvD1 & RvE1), and protectins (PD1 isomer 10S, 17S-diHDoHE). Ibuprofen blocked increases in COX-1 and 2 derived prostanoids, but also resulted in off target reductions in leukotriene biosynthesis, and a diminished pro-resolving lipid mediator response. CYP pathway product metabolism was also altered by ibuprofen treatment, as indicated by elevated post-exercise 5,6- and 8,9- DiHETrE only in those receiving ibuprofen. These findings characterize the lipid mediator response to resistance exercise in humans and show that pro-inflammatory signals are mechanistically linked to the induction of a biological active inflammatory resolution program, regulated by pro-resolving lipid mediators during post-exercise recovery.
The cessation of physical activity in rodents and humans initiates obesogenic mechanisms. The overall purpose of the current study was to determine how the cessation of daily physical activity in rats at 49-56 days of age and at 70-77 days of age via wheel lock (WL) affects adipose tissue characteristics. Male Wistar rats began voluntary running at 28 days old and were either sacrificed at 49-56 days old or at 70-77 days old. Two cohorts of rats always had wheel access (RUN), a second two cohorts of rats had wheel access restricted during the last seven days (7d-WL), and a third two cohorts of rats did not have access to a voluntary running wheel after the first 6 days of (SED). We observed more robust changes with WL in the 70-77 day-old rats. Compared to RUN rats, 7d-WL rats exhibited greater rates of gain in fat mass and % body fat, increased adipocyte number, higher percentage of small adipocytes, and greater cyclin A1 mRNA in epididymal and perirenal adipose tissue. In contrast, 49-56 day-old rats had no change in most of the same characteristics. There was no increase in inflammatory mRNA expression in either cohort with WL. These findings suggest that adipose tissue in 70-77 day-old rats is more protected from WL than 49-56 day-old rats and responds by expansion via hyperplasia.
Maternal obesity increases the risk of obesity in offspring, and obesity is accompanied by an increase in blood leptin levels. The "yellow" mutation at the mouse agouti locus (Ay) increases blood leptin levels in C57Bl pre-obese pregnant mice without affecting other metabolic characteristics. We investigated the influence of the Ay mutation or leptin injection at the end of pregnancy in C57Bl mice on metabolic phenotypes and the susceptibility to diet-induced obesity (DIO) in offspring. In both C57Bl-Ay and leptin-treated mice, the maternal effect was more pronounced in male offspring. Compared to males born to control mothers, males born to Ay mothers displayed equal food intake (FI) but decreased body weight (BW) gain after weaning, equal glucose tolerance, enhanced FI/BW ratios on the standard diet but the same FI and BW on the high-fat diet. Males born to Ay mothers were less responsive to the anorectic effect of exogenous leptin and less resistant to fasting (were not hyperphagic and gained less weight during re-feeding after food deprivation) compared to males born to control mothers. However, all progeny displayed equal hypothalamic expression of AgRP, NPY and POMC and equal plasma leptin and glucose levels after food deprivation. Leptin injections to C57Bl mice on day 17 of pregnancy decreased BW in both male and female offspring but inhibited the FI and DIO only in male offspring. Our results show that hyperleptinemia during pregnancy has gender-specific long-term effects on energy balance regulation in progeny and does not predispose offspring to developing obesity.
Orexin/hypocretin terminals innervate noradrenergic locus coeruleus (LC) neurons that project to the prefrontral cortex, which may influence spontaneous physical activity (SPA) and energy balance. Obesity resistant (OR) rats have higher orexin receptors (OXR) mRNA in the LC and other brain regions, as well as lower adiposity compared to obese rats. These findings led us to hypothesize that orexin activity in the LC is relevant for the OR phenotype. We compared OR rats to Sprague-Dawley rats. We predicted that: 1) brain OXR expression pattern is sufficient to differentiate OR from non-bred Sprague-Dawley rats; 2) non-resting energy expenditure (NREE) and orexin A (OXA) stimulated SPA after injection in LC would be greater in OR rats; and 3) the effect of OXA on SPA would be greater than its effect on feeding. OXR mRNA from 11 brain sites, and the SPA and feeding responses to OXA in the LC were determined. Body composition, basal SPA and EE were determined. Principal component analysis of the OXR expression pattern differentiates OR and Sprague-Dawley rats and suggests OXR mRNA in the LC is important in defining the OR phenotype. In comparison to Sprague-Dawley rats, OR rats had greater SPA and NREE, lower resting EE and adiposity. SPA responsivity to OXA in the LC was greater in OR rats compared to Sprague-Dawley rats. OXA in the LC did not stimulate feeding in OR or Sprague-Dawley rats. These data suggest that the LC is a prominent site modulating OXA-stimulated SPA, which promotes lower adiposity and higher non-resting EE.
Introduction Ischemia reperfusion injury (IRI) contributes to partial flap and solid organ transplant failure. Heme-oxygenase 1 (HO-1) is an inducible, cytoprotective enzyme which protects against IRI in solid organ transplant models. Heme arginate (HA), a HO-1 inducer, is a promising, translatable, preconditioning agent. This study investigated the effects of preconditioning with HA on the clinical outcome of a myocutaneous IRI model. Methodology Forty, male, Lewis rats were randomized to receive intravenous: (1) Control- NaCl; (2) HA; (3) HA and tin mesoporphyrin (SnMP), a HO-1 inhibitor; (4) SnMP alone. 24 h later an in situ transverse rectus abdominis myocutaneous (TRAM) flap was performed under isofluorane anaesthesia. Viability of flaps was measured clinically and by laser Doppler perfusion scanning. In vitro work on human epidermal keratinocytes (HEKa) assessed the effects of: HA; SnMP; the iron chelator desferrioxamine (DF) on: (1) cytotoxicity; (2) intracellular reactive oxygen species (ROS) concentration; and (3) ROS-mediated DNA damage. Results In contrast to our hypothesis, HA preconditioning produced over 30% more flap necrosis at 48 h compared with controls (p = 0.02). HA containing treatments produced significantly worse flap perfusion at all postoperative time points. In vitro work showed that HA is cytotoxic to keratinocytes. This cytotoxicity was independent of HO-1 and was mediated by the generation of reactive oxygen species by free heme. Conclusion In contrast to solid organ data, pharmacological preconditioning with HA significantly worsened clinical outcome thus indicating that this is not a viable approach in free flap research.
The classical renin-angiotensin system (RAS) pathway has been recently updated with the identification of additional molecules (such as ACE2, Ang-(1-7) and Mas receptor) that might improve some pathophysiological processes in chronic inflammatory diseases. In the present study, we focused on the potential protective role of Mas receptor activation on mouse lipid profile, liver steatosis and atherogenesis. Mas/ApoE-Double-Knockout (DKO) mice (based on C57BL/6 strain of 20 weeks of age) were fed under normal diet and compared with aged-matched Mas and ApoE single KO as well as wild type mice. Mas/ApoE double deficiency was associated with increased serum levels of atherogenic fractions of cholesterol, triglycerides and fasting glucose when compared to wild type or single KO. Serum levels of HDL or leptin in DKO were lower than other groups. Hepatic lipid content as well as ALT serum levels were increased in DKO as compared to wild type or single KO animals. Accordingly, the hepatic protein content of mediators related to atherosclerotic inflammation, such as PPARα and LXR, was altered in an adverse way in DKO as compared to ApoE KO. On the other hand, DKO mice did not display increased atherogenesis and intraplaque inflammation as compared to ApoE KO group. In conclusion, Mas deletion in ApoE knockout mice was associated with development of severe liver steatosis and dyslipidemia without affecting concomitant atherosclerosis. Mas receptor activation might represent a promising strategies for future treatments targeting both hepatic and metabolic alterations in chronic conditions clustering these disorders.
Feeding a diet high in fat and sucrose (HFS) during pregnancy and lactation is known to increase susceptibility to develop metabolic derangements later in life. A trait for increased behavioral activity may oppose these effects, since this would drain energy from milk produced to be made available to the offspring. To investigate these interactions, we assessed several components of behavioral energetics during lactation in control mice (C) and in mice of two lines selectively bred for high wheel-running activity (S1, S2) subjected to a HFS diet or a low-fat (LF) diet. Energy intake, litter growth, and milk energy output at peak lactation (MEO; assessed by subtracting maternal metabolic rate from energy intake) were elevated in HFS-feeding dams across all lines compared to the LF condition, an effect that was particularly evident in the S dams. This effect was not preceded by improved lactation behaviors assessed between PND1-7. In fact, S1 dams had less high-quality nursing and S2 dams showed poorer pup-retrieval than C dams during PND1-7, and S dams had generally higher levels of physical activity at peak lactation. These data demonstrate that HFS-feeding increases MEO underlying increased litter and pup growth, particularly in mice with a trait for increased behavioral physical activity.
Nitric oxide (NO) is metabolized in plasma, partly by the ferroxidase ceruloplasmin (Cp), to form nitrite and nitrosothiols (SNOs), which are proposed to mediate protective responses to hypoxia and ischemia. We hypothesized that NO metabolism would be attenuated in fetal plasma due to low Cp activity. We measured Cp concentrations and activity in plasma samples collected from adults and fetuses of humans and sheep. We then added NO ([NO]: 1.5 µM or 100 µM) to plasma and aqueous buffer and measured rates of NO disappearance and the production of nitrite and SNO. Cp concentrations in fetal plasma were <15 % of adult levels. In aqueous buffer, 1.5 µM NO disappeared with a half-life of 347 ± 64 s (mean±SE) but in plasma of humans the half-life was 19 ± 2 s (adult) and 11 ± 1 s (fetus, p=0.004), and in sheep it was 31 ± 3 s (adult) and 43 ± 5 s (fetus, p=0.04). Cp activity was not correlated with the overall elimination half-life of NO nor with the amount of SNO ([NO]: 100 µM) or nitrite ([NO]: 1.5 µM, 100 µM) produced, but correlated with SNO yields at 1.5 μM [NO] (r=0.92, p=0.04). Our data demonstrate that Cp is not essential to the increased rate of metabolism of NO in plasma relative to aqueous buffers, and that it is not essential to the production of nitrite from NO. Cp may be involved in the conversion of NO to SNO in plasma under near-physiological concentrations of NO.
Fetal and neonatal iron deficiency results in cognitive impairments in adulthood despite prompt postnatal iron replenishment. To systematically determine whether abnormal expression and localization of proteins that regulate adult synaptic efficacy are involved, we used a quantitative proteomic approach (iTRAQ) and pathway analysis to identify dysregulated proteins in hippocampal synapses of fetal iron deficiency model. Rat pups were made iron-deficient (ID) from gestational day 2 through postnatal day (P) 7 by providing pregnant and nursing dams an ID diet (4 ppm Fe) after which they were rescued with an iron-sufficient diet (200 ppm Fe). This paradigm resulted in a 40% loss of brain iron at P15 with complete recovery by P56. Synaptosomes were prepared from hippocampi of the formerly iron deficient (FID) and always iron-sufficient controls rats at P65 using a sucrose gradient method. Six replicates per group that underwent iTRAQ labeling and LC-MS/MS analysis for protein identification and comparison elucidated 331 differentially expressed proteins. Western analysis was used to confirm findings for selected proteins in the glutamate receptor signaling pathway, which regulates hippocampal synaptic plasticity, a cellular process critical for learning and memory. Bioinformatics were performed using knowledge-based Interactive Pathway Analysis®. FID synaptosomes show altered expression of synaptic proteins mediated cellular signalings, supporting persistent impacts of fetal iron deficiency on synaptic efficacy, which likely cause the cognitive dysfunction and neurobehavioral abnormalities. Importantly, the findings uncover previously unsuspected pathways, including nNOS signaling, identifying additional mechanisms that may contribute to the long-term biobehavioral deficits.
Severe-intensity exercise initiated from an elevated metabolic rate would be expected to enhance the proportional activation of higher-order (type II) muscle fibers. The purpose of this study was therefore to test the hypothesis that, compared to placebo (PL), NO3--rich beetroot juice (BR) supplementation would speed the phase II vo2 kinetics (p) and enhance exercise tolerance during severe-intensity exercise initiated from a baseline of moderate-intensity exercise. Nine healthy, physically-active subjects were assigned in a randomized, double-blind, crossover design to receive BR (140 mL/day, containing ~8 mmol of NO3-) and PL (140 mL/day, containing ~0.003 mmol of NO3-) for 6 days. On days 4, 5 and 6 of the supplementation periods, subjects completed a double-step exercise protocol that included transitions from unloaded-to-moderate intensity exercise (U->M) followed immediately by moderate-to-severe-intensity exercise (M->S). Compared to PL, BR elevated resting plasma nitrite concentration (PL: 65 ± 32 vs. BR: 348 ± 170 nM, P<0.01) and reduced the vo2 p in M->S (PL: 46 ± 13 vs. BR: 36 ± 10 s, P<0.05) but not U->M (PL: 25 ± 4 vs. BR: 27 ± 6 s, P>0.05). During M->S exercise, the faster vo2 kinetics coincided with faster NIRS-derived muscle [deoxyhemoglobin] kinetics (; PL: 20 ± 9 vs. BR: 10 ± 3 s, P<0.05) and a 22% greater time-to-task failure (PL: 521 ± 158 vs. BR: 635 ± 258 s, P<0.05). Dietary supplementation with NO3--rich BR juice speeds vo2 kinetics and enhances exercise tolerance during severe-intensity exercise when initiated from an elevated metabolic rate.
Prenatal hypoxia leads to an increased risk of adult cardiovascular disease. We have previously demonstrated a programming effect of prenatal hypoxia on the cardiac beta-adrenergic (βAR) response. The aim of this study was to determine 1) if the decrease in βAR sensitivity in prenatally hypoxic 5 week chicken hearts is linked to changes in β1AR/β2ARs, Gαi expression and cAMP accumulation and 2) if prenatal hypoxia has an effect on heart function in vivo. We incubated eggs in normoxia (N, 21% O2) or hypoxia from day 0 (H, 14% O2) and raised the post-hatchlings to 5 weeks of age. Cardiac β1AR/β2ARs were assessed through competitive binding of [3H]CGP-12177 with specific β1AR or β2AR blockers. Gαs and Gαi proteins were assessed by Western blot and cAMP accumulation by ELISA. Echocardiograms were recorded in anesthetized birds to evaluate diastolic/systolic diameter and heart rate and tissue sections were stained for collagen. We found an increase in relative heart mass, β1ARs and Gαs in prenatally hypoxic hearts. cAMP levels after isoproterenol stimulation and collagen content was not changed in H compared to N but in vivo echocardiograms showed systolic contractile dysfunction. The changes in βAR and G-protein subtypes may be indicative of an early compensatory stage in the progression of cardiac dysfunction, further supported by the cardiac hypertrophy and systolic contractile dysfunction. We suggest that it is not the changes in the proximal part of the βAR system that causes the decreased cardiac contractility, but Ca2+ handling mechanisms further downstream in the βAR signaling cascade.
Erythrocytes participate in the matching of oxygen (O2) delivery with local need in skeletal muscle via the release of O2 and the vasodilator, ATP. It was reported that a concentration of insulin found in humans with insulin resistance inhibits low O2-induced ATP release. However, in vivo, insulin is co-released with connecting peptide (C-peptide) at equimolar concentrations, but due to shorter insulin half-life, the peptides circulate at ratios of C-peptide to insulin ranging from 1:1 to 6:1. Here we investigate the hypothesis that C-peptide and insulin work synergistically to maintain low O2-induced ATP release from human erythrocytes. Using a thin-film tonometer to alter O2 tension we determined that either C-peptide or insulin alone inhibits low O2-induced ATP release in a concentration-dependent manner; however, co-administration of the peptides at a 1:1 ratio does not (n=5, p<0.05). Since this ratio of C-peptide to insulin is not present in vivo for extended periods, we also investigated the effect of additional physiological ratios on ATP release. In the presence of insulin concentrations that would be found in fasting humans (0.05 nM), C-peptide to insulin ratios of 4:1 and 6:1 did not adversely affect low O2-induced ATP release. However, at a concentration of insulin found in the peripheral circulation of humans under post-prandial conditions (0.5 nM), a ratio of C-peptide to insulin of 6:1 inhibited low O2-induced ATP release (n=5). These findings demonstrate a heretofore unrecognized synergism between C-peptide and insulin that could have physiological importance in the regulation of perfusion distribution in skeletal muscle.
Exercise-induced changes in -aminobutyric acid (GABA) or nitric oxide signaling within paraventricular nucleus (PVN) have not been studied in renovascular hypertension. We tested whether exercise training decreases mean arterial pressure (MAP) and renal sympathetic nerve activity (RSNA) in two-kidney one-clip hypertensive rats due to enhanced nitric oxide or GABA signaling within PVN. Conscious, unrestrained male Sprague Dawley rats with either sham (Sham) or right renal artery clipping (2K-1C) were assigned to sedentary (SED) or voluntary wheel running (ExT) for 6 or 12 weeks. MAP and angiotensin II (Ang II) were elevated in 2K-1C SED rats. The 2K-1C ExT rats displayed lower MAP at 6 weeks that did not decline further by 12 weeks. Ang II was lower in 2K-1C ExT rats. Increases in MAP, heart rate, and RSNA to blockade of PVN nitric oxide in 2K-1C SED rats were attenuated compared with either Sham group. Exercise training restored the responses in 2K-1C ExT rats. The increase in MAP in response to bicuculline was inversely correlated with baseline MAP. The rise in MAP was lower in 2K-1C SED vs either Sham group, and was normalized in the 2K-1C ExT rats. Paradoxically, heart rate and RSNA responses were not diminished in 2K-1C SED rats but were significantly lower in the 2K-1C ExT rats. Thus, the decrease in arterial pressure in 2K-1C hypertension associated with exercise training is likely due to diminished excitatory inputs to PVN because of lower Ang II and higher nitritergic tone rather than enhanced GABA inhibition of sympathetic output.
We have previously observed that many of the renal and hemodynamic adaptations seen in normal pregnancy can be induced in virgin female rats by chronic systemic vasodilation. Fourteen-day vasodilation with sodium nitrite (NaNO2) or nifedipine (NIF) produced plasma volume expansion (PVE), hemodilution and increased renal medullary phosphodiesterase 5 A (PDE5A) protein. The present study examined the role of the RAAS in this mechanism. Virgin females were treated for 14 days with NIF; 10 mg/kg/day, NIF with spironolactone (SPR; mineralocorticoid receptor (MR) blocker, 200-300 mg/kg/day), NIF with losartan (LOS; AT1 receptor blocker, 20 mg/kg/day), enalapril (ENAL; ACE inhibitor, 62.5 mg/L), or vehicle (CON). Mean arterial pressure (MAP) was reduced 7.4±0.5% with NIF, 6.33±0.5% with NIF+SPR, 13.3±0.9% with NIF+LOS, and 12.0±0.4% with ENAL vs. baseline MAP. Compared to CON (3.6±0.3%), plasma volume factored for body weight was increased by NIF (5.2±0.4%) treatment but not by NIF+SPR (4.3±0.3%), NIF+LOS (3.6±0.1%), or ENAL (4.0±0.3%). NIF increased PDE5A protein abundance in the renal inner medulla and SPR did not prevent this increase (188±16% and 204±22% of CON respectively). NIF increased the α-subunit of the epithelial sodium channel (ENaC) protein in renal outer (365±44%) and inner medulla (526±83%), and SPR prevented these changes. There was no change in either PDE5A or ENaC abundance vs. CON in rats treated with NIF+LOS or ENAL. These data indicate that the PVE and renal medullary adaptations in response to chronic vasodilation result from RAAS signaling, with increases in PDE5A mediated through AT1R and ENaC through the MR.
We previously described synaptic currents between baroreceptor fibers and second-order neurons in the nucleus tractus solitarius (NTS) that were larger in Syrian hamsters than in rats. This suggested that although electrical activity throughout the hamster brain decreased as brain temperature declined, the greater synaptic input to its NTS would support continued operation of cardiorespiratory reflexes at low body temperatures. Here we focused on properties that would protect these neurons against potential damage from the larger synaptic inputs, testing the hypotheses that hamster NTS neurons exhibit: 1) intrinsic NMDAR properties that limit Ca2+ influx to a greater degree than do rat NTS neurons; and 2) properties that reduce gating signals to NMDARs to a greater degree than in rat NTS neurons. Whole-cell patch-clamp recordings on anatomically identified second-order NTS baroreceptive neurons showed that NMDAR-mediated synaptic currents between sensory fibers and second-order NTS neurons were larger in hamsters than in rats at 33°C and 15°C, with no difference in their permeability to Ca2+. However, at 15°C, but not at 33°C, non-NMDAR currents evoked by glutamate released from baroreceptor fibers had significantly shorter durations in hamsters than in rats. Thus, hamster NMDARs did not exhibit lower Ca2+ influx than did rats (negating hypotheses 1), but they did exhibit significant differences in non-NMDAR neuronal properties at low temperature (consistent with hypothesis 2). The latter (shorter duration of non-NMDAR currents) would likely limit NMDAR coincidence gating and may help protect hamster NTS neurons, enabling them to contribute to signal processing at low body temperatures.
For terrestrial vertebrates, water economy is a prerequisite for survival, and the kidney is their major osmoregulatory organ. Birds are the only vertebrates other than mammals that can concentrate urine in adaptation to terrestrial environments. Aquaporin (AQP) and glyceroporin (GLP) are phylogenetically old molecules and have been found in plants, microbial organisms, invertebrates, and vertebrates. Currently, 13 AQPs/aquaGLP, and isoforms are known to be present in mammals. AQPs 1, 2, 3, 4, 6, 7, 8, and 11 are expressed in the kidney; of these, AQPs 1, 2, 3, 4, and 7 are shown to be involved in fluid homeostasis. In avian kidneys, AQPs 1, 2, 3, and 4 have been identified and characterized. Also, gene and/or amino acid sequences of AQP5, AQP7, AQP8, AQP9, AQP11, and AQP12 have been reported in birds. AQPs 2 and 3 are expressed along cortical and medullary collecting ducts (CDs) and are responsible, respectively, for the water inflow and outflow of CD epithelial cells. While AQP4 plays important role in water exit in the CD of mammalian kidneys, it is unlikely to participate in water outflow in avian CDs. This review summarizes current knowledge on structure and function of avian AQPs and compares them to those in mammalian and nonmammalian vertebrates. Also, we aim to provide input into, and perspectives on, the role of renal AQPs in body water homeostasis during ontogenic and phylogenetic advancement.
The immature heart is known to be resistant to ischemia-reperfusion (IR) injury, however key proteins engaged in phospho-dependent signaling pathways crucial to cell survival are not yet defined. Our goal was to determine the post-natal changes in myocardial tolerance to IR, including baseline expression of key proteins governing IR tolerance and their phosphorylation during IR. Hearts from male C57Bl/6 mice (neonates, 2, 4, 8, and 12 weeks of age, n=6/group) were assayed for survival signaling/effectors (Akt, p38MAPK, GSK3β, HSP27, connexin-43, HIF1α, caveolin-3), regulators of apoptosis (Bax, Bcl-2) and autophagy (LC3B, Parkin, Beclin1). The effect of IR on ventricular function was measured in isolated perfused hearts from immature (4 week) and adult (12 week) mice. Neonatal myocardium exhibits a large pool of inactive Akt; high phospho-activation of p38MAPK, HSP27 and connexin-43; phospho-inhibition of GSK3β; and high expression of caveolin-3, HIF1α, LC3B, Beclin1, Bax and Bcl-2. Immature hearts sustained less dysfunction and infarction following IR than adults. Emergence of IR intolerance in adult vs. immature hearts was associated with complex proteomic changes: decreased expression of Akt, Bax and Bcl-2; increased GSK3β, connexin-43, HIF1α, LC3B and Bax:Bcl-2; enhanced post-ischemic HIF1α, caveolin-3, Bax and Bcl-2; and greater post-ischemic GSK3β and HSP27 phosphorylation. Neonatal myocardial stress-resistance reflects high expression of pro-survival and autophagy proteins, and apoptotic regulators. Notably, there is high phosphorylation of GSK3β, p38MAPK, HSP27, and low phosphorylation of Akt (high Akt 'reserve'). Subsequent maturational reductions in IR tolerance are associated with reductions in Akt, Bcl-2, LC3B and Beclin1.
A large proportion of vagal afferents are dependent on neurotrophin-3 (NT-3) for survival. NT-3 is expressed in developing gastrointestinal (GI) smooth muscle, a tissue densely innervated by vagal mechanoreceptors, and thus could regulate their survival. We genetically ablated NT-3 from developing GI smooth muscle and examined the pattern of loss of NT-3 expression in the GI tract and whether this loss altered vagal afferent signaling or feeding behavior. Meal-induced c-Fos activation was reduced in the solitary tract nucleus and area postrema in mice with a smooth muscle-specific NT-3 knockout (SM-NT-3KO) compared to controls, suggesting a decrease in vagal afferent signaling. Daily food intake and body weight of SM-NT-3KO mice and controls were similar. Meal pattern analysis revealed that mutants, however, had increases in average and total daily meal duration compared to controls. Mutants maintained normal meal size by decreasing eating rate compared to controls. Although microstructural analysis did not reveal a decrease in the rate of decay of eating in SM-NT-3KO mice, they ate continuously during the 30-minute meal, whereas controls terminated feeding after 22 minutes. This led to a 74% increase in first daily meal size of SM-NT-3KO mice compared to controls. The increases in meal duration and first meal size of SM-NT-3KO mice are consistent with reduced satiation signaling by vagal afferents. This is the first demonstration of a role for GI NT-3 in short-term controls of feeding, most likely involving effects on development of vagal GI afferents that regulate satiation.
Many diseases associated with sympathoexcitation also exhibit elevated reactive oxygen species (ROS). A recent animal study indicated that exogenous administration of the sympathetic neurotransmitter, norepinephrine (NE), increased systemic ROS via circulating leukocytes. The mechanisms contributing to this effect of NE and whether these findings can be translated to humans is unknown. Thus, we tested the hypothesis that NE increases superoxide production in human peripheral blood mononuclear cells (PBMCs) via NADPH oxidase. Primary human PBMCs were freshly isolated from healthy young men and placed in culture. Following NE (50pg/ml, 50ng/ml and 50µg/ml concentrations) or control treatments, NADPH oxidase mRNA expression (gp91phox, p22phox and p67phox) was assessed using real time RT-PCR and intracellular superoxide production was measured using dihydroethidium fluorescence. PBMCs were also treated with selective adrenergic agonists-antagonists to determine the receptor population involved. In addition, CD14+ monocyte-endothelial cell adhesion was determined using a fluorescent-based assay. NE significantly increased NADPH oxidase gene expression and intracellular superoxide production in a time dependent manner (superoxide: 0.9±0.2 fold 6 hours vs. 3.0±0.3 fold 36 hours (NE, 50μg/ml); P < 0.05). The sustained increase in NE-induced superoxide production was primarily mediated via α-adrenergic receptors, preferentially α2-receptors. The NADPH oxidase blocker diphenylene iodonium and protein kinase C inhibitor staurosporine significantly attenuated NE-induced increases in superoxide production. Importantly, NE treatment increased CD14+ monocyte-endothelial cell adhesion. These findings indicate for the first time that NE increases superoxide production in freshly isolated primary human PBMCs via NADPH oxidase through α-adrenergic receptors; an effect facilitating monocyte adhesion to the endothelium.
Chronic obstructive pulmonary disease (COPD) is associated with systemic oxidative stress and skeletal muscle dysfunction. The purpose of this study was to examine the impact of intravenous ascorbate administration (AO) on biological markers of antioxidant capacity and oxidative stress, and subsequently skeletal muscle function during dynamic, small muscle mass exercise in patients with COPD. Ten patients with spirometric evidence of COPD performed single-leg knee extensor (KE) trials matched for intensity and time (isotime) following intravenous ascorbate (2g) or saline infusion (PL). Quadriceps fatigue was quantified by changes in force elicited by maximal voluntary contraction (MVC) and magnetic femoral nerve stimulation (Qtw,pot). AO administration significantly increased antioxidant capacity, as measured by the ferric reducing ability of plasma (PL: 1±0.1 vs AO: 5±0.2 mM), and significantly reduced malondialdehyde levels (PL: 1.16±0.1 vs AO: 0.97±0.1 mmol). Additionally, resting blood pressure was significantly reduced (PL: 104±4 vs AO: 93±6 mmHg) and resting femoral vascular conductance was significantly elevated after AO (PL: 2.4±0.2 vs AO: 3.6±0.4 ml/min/mmHg). During isotime exercise, the AO significantly attenuated both the ventilatory and metabolic responses, and patients accumulated significantly less peripheral quadriceps fatigue, as illustrated by less of a fall in MVC (PL: -11±2% vs AO: -5±1%) and Qtw,pot (PL: -37±1% vs AO: -30±2%). These data demonstrate a beneficial role of AO administration on skeletal muscle fatigue in patients with COPD and further implicate systemic oxidative stress as a causative factor in the skeletal muscle dysfunction observed in this population.
We investigated the responses of intramuscular phosphate-linked metabolites and pH (as assessed by 31P-MRS) during intermittent high-intensity exercise protocols performed with different recovery-interval durations. Following estimation of the parameters of the power-duration relationship (i.e., CP and W') for severe-intensity constant-power exercise, eight male subjects completed three intermittent exercise protocols to exhaustion where periods of high-intensity exercise (60-s) were separated by different durations of passive recovery (18-s, 30-s and 48-s). The tolerable duration of exercise was 304 ± 68 s, 516 ± 142 s and 847 ± 240 s for the 18-s, 30-s and 48-s recovery protocols, respectively (P<0.05). The work done >CP (W>CP) was significantly greater for all intermittent protocols compared to the subjects' W' and this difference became progressively greater as recovery-interval duration was increased. Similarly, the degree of intramuscular phosphocreatine restoration during recovery was greatest, intermediate and least for 48-s, 30-s and 18-s of recovery, respectively (P<0.05). The W>CP in excess of W' increased with greater durations of recovery and this was correlated with the mean magnitude of muscle phosphocreatine reconstitution between work intervals (r = 0.61; P<0.01). During intermittent high-intensity exercise, recovery intervals allow intramuscular homeostasis to be restored, with the degree of restoration being related to the duration of the recovery interval. Consequently, the ability to perform W>CP during intermittent high-intensity exercise and, therefore, exercise tolerance, increases in a predictable manner when recovery-interval duration is extended.
Recently, it has been recognized that a single airway sensory unit may contain multiple receptive fields, and that each field houses at least one encoder. Since some units respond to both lung inflation and deflation, we hypothesized that these units contain heterogeneous encoders for sensing inflation and deflation, respectively. Single unit activities were recorded from the cervical vagus nerve in anesthetized, open chest, and mechanically ventilated rabbits. Fifty two airway sensory units with multiple receptive fields and responding to both lung inflation and deflation were identified. Among them, 13 units had separate receptive fields for inflation and deflation, where one of the fields could be blocked by local injection of 2% lidocaine (10 μl). In 8 of the 13 units, the deflation response was blocked without affecting the unit's response to inflation, whereas in the remaining 5 units, the inflation response was blocked without affecting the deflation response. Our results support the hypothesis that a single mechanosensory unit may contain heterogeneous encoders that can respond to either inflation or deflation.
The myocardial Na+/H+ exchanger-1 (NHE1) plays a major role in regulation of intracellular pH and its upregulation has been implicated in increased ischemia-reperfusion injury and other pathologies. Hydrogen peroxide (H2O2) increases NHE1 activity acutely via ERK1/2 signaling. Chronic strenuous exercise upregulates NHE1 in skeletal muscle, but we hypothesize this will not occur in the heart because exercise creates a cardioprotective phenotype. NHE1 activity and its regulation by H2O2 were examined at physiological pH using isolated cardiomyocytes from female Sprague-Dawley rats exercised on a treadmill for 5 wks (E, n=11). Compared to sedentary (S, n=15), E displayed increases (P<0.05) in heart:body weight (6.8%) and plantaris mitochondria content (89%). NHE1 activity (acid efflux rate following an acid load) was 209% greater in E (0.65±0.12 v 2.01±0.29 fmol/min). The difference was attributed primarily to greater cell volume (22.2±0.6 v 34.3±1.1 pL) and pHi-buffering capacity (33.94±1.59 v 65.82±5.20 mM/pH unit) of E myocytes. H2O2 stimulation (100 µM) raised NHE1 activity significantly less in E (45%) than S (167%); however, activity remained 185% greater in E. ERK1/2 inhibition abrogated the increases. H2O2-stimulated ERK1/2 phosphorylation levels normalized to total ERK1/2 were similar between groups. Content of NHE1 and activities of H2O2 scavengers were also similar. We observed that pHi-buffering capacity differences between groups became progressively less with declining pH, which may be an exercise-induced cardioprotective adaptation to lower NHE1 activity during certain pathological situations. We conclude that strenuous endurance exercise increases myocardial NHE1 activity at physiological pH, which would likely enhance cardiac performance under physiological conditions.
The sensory circumventricular organs (CVOs) are specialized collections of neurons and glia that lie in the midline of the IIIrd and IVth ventricles of the brain, lack a blood-brain barrier, and function as chemosensors, sampling both the cerebrospinal fluid and plasma. These structures, which include the organum vasculosum of the lamina terminalis (OVLT), subfornical organ (SFO), and area postrema (AP), are sensitive to changes in sodium concentration but the cellular mechanisms involved remain unknown. Epithelial sodium channel (ENaC)-expressing neurons of the CVOs may be involved in this process. Here we demonstrate with immunohistochemical and in situ hybridization methods that ENaC-expressing neurons are densely concentrated in the sensory CVOs. These neurons become c-Fos activated, a marker for neuronal activity, after various manipulations of peripheral levels of sodium including systemic injections with hypertonic saline, dietary sodium deprivation, and sodium repletion after prolonged sodium deprivation. The increases seen c-Fos activity in the CVOs were correlated with parallel increases in plasma sodium levels. Since ENaCs play a central role in sodium reabsorption in kidney and other epithelia, we present a hypothesis here suggesting that these channels may also serve a related function in the CVOs. ENaCs could be a significant factor in modulating CVO neuronal activity by controlling the magnitude of sodium permeability in neurons. Hence, some of the same circulating hormones controlling ENaC expression in kidney, such as angiotensin II and atrial natriuretic peptide, may coordinate ENaC expression in sensory CVO neurons, and could potentially orchestrate sodium appetite, osmoregulation, and vasomotor sympathetic drive.
Noradrenergic A2 neurons in NTS respond to stressors such as hypoxia. We hypothesize that tyrosine hydroxylase (TH) knockdown in NTS reduces cardiovascular responses to chronic intermittent hypoxia (CIH), a model of the arterial hypoxemia observed during sleep apnea in humans. Adult male Sprague-Dawley rats were implanted with radio telemetry transmitters and adeno-associated viral constructs with a GFP reporter having either short hairpin RNA (shRNA) for TH or scrambled virus (scRNA) were injected into caudal NTS. Virus injected rats were exposed to 7 days of CIH (alternating 6 min periods of 10% O2 and 4 min of 21% O2 from 8am-4pm; from 4pm-8am rats were exposed to 21% O2). CIH increased MAP and HR during the day in both the scRNA (n= 14, P <.001 MAP and HR) and shRNA (n=13, P <.001 MAP and HR) groups. During the night MAP and HR remained elevated in the scRNA rats (P <0.001 MAP and HR) but not in the shRNA group. TH immunoreactivity and protein were reduced in shRNA group. FosB/FosB immunoreactivity was decreased in paraventricular nucleus (PVN) of shRNA group (P < 0.001). However, shRNA group did not show any change in the FosB/FosB immunoreactivity in the rostral ventro-lateral medulla. Exposure to CIH increased MAP which persisted beyond the period of exposure to CIH. Knockdown of TH in the NTS reduced this CIH-induced persistent increase in MAP and reduced the transcriptional activation of PVN. This indicates that NTS A2 neurons play a role in the cardiovascular responses to CIH.
Use of protease inhibitors (PI) in HIV patients is associated with hyperlipidemia and increased risk of CHD. Chronic systemic and cardiac effects of ritonavir (RTV), a universal PI booster, and Mg-supplementation were examined. RTV was administered (75 mg/kg/day p.o.) to LewisXBrown-Norway hybrid (LBNF1) rats for up to 8 weeks; significant increases in plasma triglyceride and cholesterol occurred from 8 days to 8 weeks. At 5 weeks, the expression of selected hepatic genes (CYP7A1, CITED2, G6PC, and ME-1), which are key to lipid catabolism/synthesis, were altered towards lipogenesis. Dietary Mg-supplementation (6-fold higher) completely reversed the altered expression of these genes, and attenuated both hypertriglyceridemia and hypercholesterolemia. Neutrophils isolated from the RTV treated rats displayed a 3-fold higher basal and a 2-fold higher stimulated superoxide production; plasma isoprostane and RBC GSSG levels were elevated 2-3-fold. All oxidative indices were normalized by Mg-supplementation. After 5 weeks, RTV caused significant decreases in cardiac left ventricular (LV) shortening fraction and LV ejection fraction; mitral valve E/A ratio was reduced accompanied by LV posterior wall thinning. Immunohistochemical staining revealed significant WBC infiltration (5-wks) and prominent fibrosis (8-wks) in the RTV hearts. Mg-supplementation attenuated RTV-induced declines in systolic and diastolic function (>70%), improved mitral E/A ratio and lessened LV posterior wall thinning (by 75%), and substantially decreased the pathological markers. Conclusions: The known clinical hyperlipidemia effects of RTV could be mimicked in the LBNF1 rats; in association, systemic oxidative stress and progressive cardiac dysfunction occurred. Remarkably, Mg-supplementation alone suppressed RTV-mediated hyperlipidemia, oxidative stress and cardiac dysfunction.
Hypoxia activates catecholamine neurons in the caudal ventrolateral medulla (CVLM). The hypothalamic paraventricular nucleus (PVN) modulates arterial chemoreflex responses and receives catecholaminergic projections from the CVLM, but it is not known whether the CVLM-PVN projection is activated by chemoreflex stimulation. We hypothesized that acute hypoxia (AH) activates PVN-projecting catecholaminergic neurons in the CVLM. Fluorogold (FG, 2%, 60-90nL) was microinjected into the PVN to retrogradely label CVLM neurons. After recovery, conscious rats underwent three hours of normoxia (21% O2, n=4) or AH (12, 10, or 8% O2; n=5 each group). We used Fos-immunoreactivity (IR) as an index of CVLM neuronal activation and tyrosine hydroxylase (TH)-IR to identify catecholaminergic neurons. Positively labeled neurons were counted in six caudal-rostral sections containing CVLM. Hypoxia progressively increased the number of Fos-IR CVLM cells (21%: 19 ± 6; 12%: 49 ± 2; 10%: 117 ± 8; 8%: 179 ± 7; p<0.001). Catecholaminergic cells co-labeled with Fos-IR in the CVLM were observed following 12% O2 and further increases in severity of hypoxia caused markedly more activation. PVN-projecting CVLM cells were activated following more severe hypoxia (10% and 8% O2). A large proportion (89 ± 3%) of all activated PVN-projecting CVLM neurons were catecholaminergic, regardless of hypoxia intensity. Data suggest that catecholaminergic, PVN-projecting CVLM neurons are particularly hypoxia-sensitive and these neurons may be important in the cardiorespiratory or neuroendocrine responses elicited by the chemoreflex.
Ca2+-activated Cl- channels (CaCCs) are critical to processes such epithelial transport, membrane excitability, and signal transduction. Anoctamin, or TMEM16, is a family of ten mammalian transmembrane proteins, two of which were recently shown to function as CaCCs. The functions of other family members have not been firmly established and almost nothing is known about anoctamins in invertebrates. Therefore, we performed a phylogenetic analysis of anoctamins across the animal kingdom and examined the expression and function of anoctamins in the genetically tractable nematode Caenorhabditis elegans. Phylogenetic analyses support five anoctamin clades that are at least as old as the deuterostome/protosome ancestor. This includes a branch containing two Drosophila paralogs that group with mammalian ANO1 and ANO2, the two best characterized CaCCs. We identify two anoctamins in C. elegans (ANOH-1 and ANOH-2) that are also present in basal metazoans. The anoh-1 promoter is active in amphid sensory neurons that detect external chemical and nociceptive cues. Within amphid neurons, ANOH-1::GFP fusion protein is enriched within sensory cilia. RNAi silencing of anoh-1 reduced avoidance of steep osmotic gradients without disrupting amphid cilia development, chemotaxis, or withdrawal from noxious stimuli suggesting that ANOH-1 functions in a sensory mode-specific manner. The anoh-2 promoter is active in mechanoreceptive neurons and the spermatheca, but loss of anoh-2 had no effect on motility or brood size. Our study indicates that at least five anoctamin duplicates are evolutionarily ancient and suggest that sensory signaling may be a basal function of the anoctamin protein family.
Estuarine species frequently encounter areas of simultaneously low dissolved O2 (hypoxia) and high CO2 (hypercapnia). Organisms exposed to hypoxia experience a metabolic depression, which serves to decrease ATP utilization and O2 demand during stress. This down-regulation is typically facilitated by a reduction in protein synthesis, a process that can be responsible for up to 60% of basal metabolism. The added effects of hypercapnia, however, are unclear. Certain decapods also exhibit a metabolic depression in response to bacterial challenges, leading us to hypothesize that protein synthesis may also be reduced during infection. In the present study, we examined the effects of hypoxia (H) and hypercapnic hypoxia (HH), as well as bacterial infection (Vibrio campbellii), on tissue-specific (muscle and hepatopancreas) fractional protein synthesis rates (ks) in Litopenaeus vannamei. We observed a significant decrease in ks in muscle after 24h exposure to both H and HH, and in hepatopancreas after 24h exposure to HH. Thus, ks is responsive to changes in O2, and the combined effect of hypercapnic hypoxia on ks is more severe than hypoxia alone. These reductions in ks appear to be driven by changes in RNA translational efficiency (kRNA), and not RNA capacity (Cs). Bacterial infection, however, had no significant effect on ks in either tissue. These results suggest that crustaceans reduce metabolic demand during environmental hypoxia by reducing global protein synthesis, and that this effect is magnified when hypercapnia is concomitantly present. Conversely, an immune-mediated metabolic depression is not associated with a decrease in overall protein production.
Heat stroke (HS) is characterized by a systemic inflammatory response syndrome (SIRS) consisting of profound core temperature (Tc) changes in mice. Encephalopathy is common at HS collapse, but inflammatory changes occurring in the brain during the SIRS remain unidentified. We determined the association between inflammatory gene expression changes in the brain with Tc disturbances during HS recovery in mice. Gene expression changes of heat shock protein (HSP)72, heme-oxygenase (hmox1), cytokines (IL-1β, IL-6, TNFα), cyclooxygenase enzymes (COX-1, COX-2), chemokines (MCP-1, MIP-1α, MIP-1β, CX3CR1), and glia activation markers (CD14, aif1, vimentin) were examined in the hypothalamus (HY) and hippocampus (HC) of control (Tc~36.0ºC) and HS mice at Tc,Max (42.7°C), hypothermia depth (HD; 29.3±0.4°C) and fever (37.8±0.3°C). HSP72 (HY<HC) and IL-1β (HY only) were the only genes that showed increased expression at Tc,Max. HSP72 (HY<HC), hmox1 (HY<HC), cytokine (HY=HC), and chemokine (HY=HC) expression was highest at HD and similar to controls during fever. COX-1 expression was unaffected by HS, whereas HD was associated with ~3-fold increase in COX-2 expression (HY only). COX-2 expression was not increased during fever and indomethacin (COX inhibitor) had no effect on this Tc response indicating fever is regulated by other inflammatory pathways. CD14, aif1, and vimentin activation at HD coincided with maximal cytokine and chemokine expression suggesting glia cells are a possible source of brain cytokines and chemokines during HS recovery. The inflammatory gene expression changes during HS recovery suggest cytokines and/or chemokines may be initiating development or re-warming from hypothermia whereas fever pathway(s) remain to be elucidated.
Roux-en-Y gastric bypass (RYGB) surgery leads to bone loss in humans, which may be caused by vitamin D and calcium malabsorption and subsequent secondary hyperparathyroidism. However, because these conditions occur frequently in obese people, it is unclear if they are the primary causes of bone loss after RYGB. To determine the contribution of calcium and vitamin D malabsorption to bone loss in a rat RYGB model, adult male Wistar rats were randomized for RYGB surgery, sham-operation ad libitum fed or sham-operation body weight-matched. Bone mineral density, calcium and phosphorus balance, acid-base status and markers of bone turnover were assessed at different time points for 14 weeks after surgery. Bone mineral density decreased for several weeks after RYGB. Intestinal calcium absorption was reduced early after surgery, but plasma calcium and parathyroid hormone levels were normal. 25-hydroxyvitamin D levels decreased, while levels of active 1,25-dihydroxyvitamin D increased after surgery. RYGB rats displayed metabolic acidosis due to increased plasma lactate levels and increased urinary calcium loss throughout the study. These results suggest that initial calcium malabsorption may play a key role in bone loss early after RYGB in rats, but other factors, including chronic metabolic acidosis, contribute to insufficient bone restoration after normalization of intestinal calcium absorption. Secondary hyperparathyroidism is not involved in postoperative bone loss. Upregulated vitamin D activation may compensate for any vitamin D malabsorption.
Two human hemoglobin (Hb) variants, Hb C and Hb S, are known to protect against Plasmodium falciparum malaria and have evolved repeatedly in malaria endemic areas. Both aggregate to insoluble crystals (Hb C) or polymers (Hb S) under certain physiological conditions, impair parasite growth and may facilitate retention of infected red blood cells (RBCs) in the spleen. Given the profound effects of parasites on host evolution in general, and RBC Hb concentrations close to the solubility limit throughout vertebrates, similar mechanisms may operate in non-human vertebrates. Here we show exercise-induced, profound in vivo Hb polymerization in RBCs of the Gulf toadfish. Hb aggregation was closely associated with the extent of plasma acidosis, fully reversible and without any signs of haemolysis or anemia. Our literature analysis suggests that aggregation prone Hbs may be relatively old, evolved multiple times in non-human vertebrates, show enhanced aggregation during hemoparasite infections and can be uncovered in vivo by splenectomy. We suggest that they are the result of on-going selection pressure against RBC parasites. Comparative studies of these long established systems may provide valuable insights into hemoparasite susceptibility and reservoir potential of livestock and companion animals, but also into human malaria and sickle cell disease.
Vitamin D receptors (VDR) are found in cells throughout the cardiovascular system. A variety of experimental studies indicate that the liganded VDR may play an important role in controlling cardiac hypertrophy and fibrosis, regulating blood pressure and suppressing the development of atherosclerosis. Some, but not all, observational studies in humans provide support for these experimental findings, raising the possibility that vitamin D or its homologues might prove useful therapeutically in the prevention or treatment of cardiovascular disease.
Chronic intermittent hypoxia (CIH) increases mean arterial pressure (MAP) and FosB/FosB staining in central autonomic nuclei. To test the role of the brain renin-angiotensin system (RAS) in CIH hypertension, rats were implanted with intracerebroventricular (ICV) cannulae delivering losartan (1ug/h) or vehicle (VEH) via mini-osmotic pumps and telemetry devices for arterial pressure recording. A third group was given the same dose of losartan subcutaneously (SC). Two groups of losartan treated rats served as normoxic controls. Rats were exposed to CIH or normoxia for 7 days then sacrificed for immunohistochemistry. ICV losartan attenuated CIH-induced increases in arterial pressure during CIH exposure (0800-1600 during the light phase) on days 1, 6, and 7 and each day during the normoxic dark phase. FosB/FosB staining in the organum vasculosum of the lamina terminalis (OVLT), median preoptic nucleus (MnPO), paraventricular nucleus of the hypothalamus (PVN) the rostral ventrolateral medulla (RVLM) and the nucleus of the solitary tract (NTS) was decreased in ICV losartan treated rats. SC losartan also reduced CIH hypertension during the last two days of CIH and produced bradycardia prior to the effect on blood pressure. Following SC losartan, FosB/FosB staining was reduced only in the OVLT, MnPO, PVN, and NTS. These data indicate that the central and peripheral RAS contribute to CIH-induced hypertension and transcriptional activation of autonomic nuclei and that the contribution of the central RAS is greater during the normoxic dark phase of CIH hypertension.
Maternal high-fat (HF) diet has long term consequences on the metabolic phenotype of the offspring. Here, we determined the effects of postweaning exercise in offspring of rat dams fed HF diet during gestation and lactation. Pregnant Sprague-Dawley rats were maintained on chow or HF diet throughout gestation and lactation. All pups were weaned onto chow diet on postnatal day (PND) 21. At 4 weeks of age, male pups were given free access to running wheels (RW) or remained sedentary (SED) for three weeks after which all rats remained sedentary, resulting in four groups: CHOW-SED, CHOW-RW, HF-SED, HF-RW. Male HF offspring gained more body weight by PND7 compared to CHOW pups and maintained this weight difference through the entire experiment. Three weeks of postweaning exercise did not affect body weight gain in either CHOW or HF offspring, but reduced adiposity in HF offspring. Plasma leptin was decreased at the end of the 3 week running period in HF-RW rats, but was not different from HF-SED nine weeks after the exercise period ended. At 14 weeks of age, intracerebroventricular injection of leptin suppressed food intake in CHOW-SED, CHOW-RW and HF-RW, while it did not affect food intake in HF-SED group. At sacrifice, HF-RW rats also had higher leptin-induced phospho-STAT3 level in the arcuate nucleus than HF-SED rats. Both maternal HF diet and postweaning exercise had effects on hypothalamic neuropeptide and receptor mRNA expression in adult offspring. Our data suggest that postweaning exercise improves central leptin sensitivity and signaling in this model.
The high pressure in giraffe leg arteries renders them vulnerable to edema. We investigated whether large and small arteries in the legs and the tight fascia protect leg capillaries. Ultrasound imaging of foreleg arteries in anesthetized giraffes and ex vivo examination revealed abrupt thickening of the arterial wall and a reduction of its internal diameter just below the elbow. At and distal to this narrowing, the artery constricted spontaneously and in response to norepinephrine and intravascular pressure recordings revealed a dynamic, viscous pressure drop along the artery. Histology of the isolated median artery confirmed dense sympathetic innervation at the narrowing. Structure and contractility of small arteries from muscular beds in the leg and neck were compared. The arteries from the legs demonstrated an increased media thickness to lumen diameter ratio, increased media volume, and increased numbers of smooth muscle cells per segment length and they furthermore contracted more strongly than arteries from the neck (500 ± 49 vs. 318 ± 43 mmHg, n=6 legs and neck, respectively). Finally the transient increase in interstitial pressure following injection of saline was 5.5 ± 1.7 times larger (n=8) in the leg than in the neck. We conclude that 1) tissue compliance in the legs is low; 2) large arteries of the legs function as resistance arteries; and 3) structural adaptation of small muscle arteries allow them to develop an extraordinary tension. All three findings can contribute to protection of the capillaries in giraffe legs from a high arterial pressure.
High levels of sympathetic drive in several cardiovascular diseases including post myocardial infarction, chronic congestive heart failure and hypertension are reinforced through dysregulation of afferent input and central integration of autonomic balance. However, recent evidence suggests that a significant component of sympathetic hyperactivity may also reside peripherally at the level of the post-ganglionic neuron. This has been studied in depth using the animal model of genetic essential hypertension, the spontaneously hypertensive rat, where larger neuronal calcium transients, increased release and impaired re-uptake of norepinephrine in neurons of the stellate ganglia lead to a significant tachycardia even before hypertension has developed. The release of additional sympathetic co-transmitters during high levels of sympathetic drive can also have deleterious consequences for peripheral cardiac parasympathetic neurotransmission even in the presence of beta-adrenergic blockade. Stimulation of the cardiac vagus reduces heart rate, lowers myocardial oxygen demand, improves coronary blood flow and independently raises ventricular fibrillation threshold. Recent data demonstrates a direct action of the sympathetic co-transmitters neuropeptide-Y (NPY) and galanin on the ability of the vagus to release acetylcholine and control heart rate. Moreover, there is as a strong correlation between plasma NPY levels and coronary microvascular function in patients with ST-elevation myocardial infarction being treated with primary percutaneous coronary intervention. Antagonists of the NPY receptors Y1 and Y2 may be therapeutically beneficial both acutely during myocardial infarction, and also during chronic heart failure and hypertension. Such medications would be expected to act synergistically with beta-blockers and implantable vagus nerve stimulators to improve patient outcome.
The ontogeny of pectoralis muscle bioenergetics was studied in growing Adélie penguin chicks during the first month after hatching and compared with adults using permeabilized fibers and isolated mitochondria. With pyruvate/malate/succinate or palmitoyl-carnitine as substrates, permeabilized fiber respiration markedly increased during chick growth (3-fold) and further rose in adults (1.4-fold). Several markers of muscle fiber oxidative activity (cytochrome oxidase, citrate synthase, hydroxyl-acyl-CoA dehydrogenase) increased 6-19 fold with age together with large rises in intermyofibrillar (IMF) and subsarcolemmal (SS) mitochondrial content (3-5 fold) and oxidative activities (1.5-2.4 fold). The proportion of IMF relative to SS mitochondria increased with chick age but markedly dropped in adults. Differences in oxidative activity between mitochondrial fractions were reduced in adults as compared with hatched chicks. Extrapolation of mitochondrial to muscle respirations revealed similar figures with isolated mitochondria and permeabilized fibers with carbohydrate-derived but not with lipid-derived substrates suggesting diffusion limitations of lipid substrates with permeabilized fibers. Two immunoreactive fusion proteins, Mitofusin 2 (Mfn2) and Optic Atrophy 1 (OPA1), were detected by western blots on mitochondrial extracts and their relative abundance increased with age. Muscle fiber respiration was positively related with Mfn2 and OPA1 relative abundance. Present data showed by two complementary techniques large ontogenic increases in muscle oxidative activity that may enable birds to face thermal emancipation and growth in childhood and marine life in adulthood. The concomitant rise in mitochondrial fusion protein abundance suggests a role of mitochondrial networks in the skeletal muscle processes of bioenergetics that enable penguins to overcome harsh environmental constraints.
Little is known about the mechanisms underpinning thermal plasticity of vertebrate hearts. Bluefin tuna hearts offer a unique model to investigate processes underlying thermal acclimation. Their hearts, while supporting an endothermic physiology, operate at ambient temperature and are presented with a thermal challenge when migrating to different thermal regimes. Here, we examined the molecular responses in atrial and ventricular tissues of pacific bluefin tuna acclimated to 14oC, 20oC and 25oC. qPCR studies showed an increase in sarcoplasmic reticulum Ca2+ATPase gene expression with cold acclimation and an induction of Na+/Ca2+-exchanger gene at both cold and warm temperatures. This data provide evidence for thermal plasticity of excitation-contraction coupling gene expression in bluefin tunas and indicate an increased capacity for internal Ca2+ storage in cardiac myocytes at 14oC. Transcriptomic analysis showed profound changes in cardiac tissues with acclimation. A principal component analysis revealed that temperature effect was greatest on gene expression in warm-acclimated atrium. Overall data showed an increase in cardiac energy metabolism at 14oC, potentially compensating for cold temperature to optimize bluefin tuna performance in colder oceans. In contrast, metabolic enzyme activity and gene expression data suggest a decrease in ATP production at 25oC. Expression of genes involved in protein turnover and molecular chaperons was also decreased at 25oC. Expression of genes involved in oxidative stress response and programmed cell-death suggest an increase in oxidative damage and apoptosis at 25oC, particularly in the atrium. These findings provide insights into molecular processes that may characterize cardiac phenotypes at upper thermal limits of teleosts.
A hallmark of chronic heart failure (CHF) is an increased sympathetic tone resulting in autonomic imbalance. Renal denervation (DNx) in CHF patients has resulted in symptomatic improvement, but the protective mechanisms remain unclear. We hypothesized in CHF, unilateral renal DNx would improve cardiac autonomic balance. The present study utilized conscious, chronically instrumented New Zealand White rabbits undergoing renal DNx prior to pacing-induced CHF. Four treatment groups were used: non-pace non-DNx (Sham-INV), non pace DNx (sham-DNx), pace non-DNx (CHF-INV) or pace DNx (CHF-DNx). We examined several markers indicative of autonomic balance. Baroreflex sensitivity and time domain heart rate variability (HRV) were both decreased in the CHF-INV group compared to sham-INV and restored to sham levels by renal DNx. Power spectral analysis indicated an increase in LF/HF ratio in the CHF-INV as compared to the sham-INV, which was normalized to sham levels by DNx. In order to assess if this was due to a withdrawal of sympathetic tone or an increase in parasympathetic tone, the heart rate response was measured after an IV bolus of metoprolol or atropine. Bradycardia induced by IV metoprolol (indicative of cardiac sympathetic tone) was exacerbated in CHF-INV rabbits compared to sham-INV but was normalized in CHF-DNx. Conversely, the tachycardia in response to IV atropine (indicative of cardiac vagal tone) was not improved in CHF-DNx vs CHF-INV animals. Renal DNx also prevented the increase in circulating plasma NE seen in CHF-INV rabbits. These results suggest renal DNx improves cardiac autonomic balance in CHF by a reduction sympathetic tone.
Background: Mild decrease of core temperature (32-34°C), also known as therapeutic hypothermia, is a highly effective strategy of neuroprotection from ischemia and holds significant promise in the treatment of stroke. However, induction of hypothermia in conscious stroke patients is complicated by cold-defensive responses, such as shivering and tachycardia. Modulators of thermosensitive ion channels TRPM8 and TRPV1 alter multiple thermoregulatory responses. However, the regulation of shivering by TRPM8 and TRPV1 is not fully understood. Objective: To determine the effects of TRPM8 inhibition and TRPV1 activation on the shivering and tachycardic responses to external cooling. Methods: Conscious mice were treated with TRPM8 inhibitor "compound 5" or TRPV1 agonist dihydrocapsaicin and exposed to cooling at 10°C. Shivering was measured by electromyography using implanted electrodes in back muscles, tachycardic response by electrocardiography and core temperature by wireless transmitters in abdominal cavity. The role of TRPM8 was further determined using TRPM8 KO mice. Results: TRPM8 ablation had no effect on total EMG muscle activity (vehicle: 24.0±1.8; "compound 5": 23.8±2.0; TRPM8 KO: 19.7±1.9 V*s/min), tachycardia (HR=124±31; 121±13; 121±31 beats/min) and drop in core temperature (-3.6±0.1; -3.4±0.4; -3.6±0.5°C) during cold exposure. TRPV1 activation substantially suppressed EMG muscle activity (vehicle: 25.6±3.0 vs. DHC: 5.1±2.0 V*s/min), tachycardia (HR=204±25 vs. 3±35 beats/min) and produced a profound drop in core temperature (-2.2±0.6 vs. -8.9±0.6°C). Conclusions: External cooling-induced shivering and tachycardia are suppressed by TRPV1 activation, but not by TRPM8 inhibition. This suggests that TRPV1 agonists may be combined with external physical cooling to achieve more rapid and effective hypothermia.
Angiotensin II (ANG II) alters the short-term blood pressure buffering capacity of the baroreflex in many adult animals. In embryonic chickens, high plasma ANG II levels contribute to baseline mean arterial pressure (MAP, kPa) without changing heart rate (fH, beats min-1). Based on these features we hypothesized an ANG II-induced reduction in baroreflex sensitivity is present in embryonic chickens as in adults. We examined baroreflex function in day 19 embryonic chickens (Gallus gallus domesticus) after chronic depletion of endogenous ANG II via angiotensin-converting enzyme (ACE) inhibition with captopril (5 mg kg-1) from day 5-18 of incubation. The correlation between MAP and fH was assessed using increasing doses of sodium nitroprusside, a vasodilator, and phenylephrine, a vasoconstrictor. We used two analytical methods to evaluate baroreflex function: a conventional 'static' method in which maximal MAP and fH responses were examined, and a 'dynamic' method that assessed beat-to-beat changes during the response to pharmacological manipulation. Captopril-treated embryos were hypotensive by 19% with baroreflex slopes ~40% steeper and normalized gains ~50% higher than controls, and differences across treatments were similar using either analytical method. Furthermore, reintroduction of ANG II via infusion raised MAP back to control levels and decreased the baroreflex gain in captopril-treated embryos. Therefore, during typical chicken development, ANG II dampens the baroreflex regulatory capacity and chicken embryos can be used as a natural model of elevated ANG II for studying developmental cardiovascular function. This study is the first to demonstrate that reduction of embryonic ANG II alters normal baroreflex function.
Previously we investigated the role of NPY and leptin sensitive networks in the mediobasal hypothalamus (MBH) in sleep and feeding and found profound homeostatic and circadian deficits with an intact suprachiasmatic nucleus (SCN). We propose that the arcuate nuclei (Arc) are required for the integration of homeostatic circadian systems including temperature and activity. We tested this hypothesis using saporin toxin conjugated to leptin (Lep-SAP) injected into Arc in rats. Lep-SAP rats became obese and hyperphagic and progressed through a dynamic phase to a static phase of growth.. Circadian rhythms were examined over 49 days during the static phase. Rats were maintained on a 12:12 light:dark (LD) schedule for 13 days and thereafter maintained in continuous dark (DD). After the first thirteen days of DD, food was restricted to four hours per day for ten days. We found that the activity of Lep-SAP rats was arrhythmic in DD, but that food anticipatory activity was nevertheless entrainable to the restricted feeding schedule and the entrained rhythm persisted during the subsequent 3-day fast in DD. Thus, for activity, the light-entrainable oscillator, but not the food entrainable oscillator, was disabled by the Arc lesion. In contrast, temperature remained rhythmic in DD in the Lep-SAP rats and did not entrain to restricted feeding. We conclude that the leptin-sensitive network that includes the Arc is required for entrainment of activity by photic cues and entrainment of temperature by food, but is not required for entrainment of activity by food or temperature by photic cues.
End- tidal breath carbon monoxide (CO) is abnormally low in women with preeclampsia (PE), while women smoking during pregnancy have shown an increase in CO levels and a 33% lower incidence of PE. This effect may be in part due to lowered sFLT1 plasma levels in smokers, and perhaps low- level CO inhalation can attenuate the development of PE in high risk women. Our previous work showed maternal chronic CO exposure (< 300ppm) throughout gestation had no maternal or fetal deleterious effects in mice. Our current study evaluated the uteroplacental vascular effects in CD-1 maternal mice who inhaled CO (250ppm) both chronically, gestation day (GD) 0.5 to 18.5, and acutely, 2.5hrs on each of GD 10.5 and 14.5. We demonstrated, using micro-ultrasound measurements of blood velocity and micro-computed tomography imaging of the uteroplacental vasculature, that chronic maternal exposure to CO doubled uterine artery blood flow and augmented uteroplacental vascular diameters and branching. This finding may be of benefit to women with PE, as they exhibit uteroplacental vascular compromise. The ratio of VEGF protein to its FLT1 receptor was increased in the placenta suggesting a shift to a more angiogenic state, however maternal circulating levels of VEGF, sFLT1, and their ratio were not significantly changed. Doppler blood velocities in the maternal uterine artery and fetal umbilical artery and vein were unaltered. This study provides in vivo evidence that chronic inhalation of 250ppm CO throughout gestation augments uterine blood flow and uteroplacental vascular growth, changes that may protect against the subsequent development of preeclampsia.
When freshwater turtles acclimatize to winter hibernation, there is a gradual transition from aerobic to anaerobic metabolism, which may require adjustments of blood O2 transport before turtles become anoxic. Here we report the effects of protons, anionic cofactors and temperature on the O2-binding properties of isolated hemoglobin (Hb) isoforms, HbA and HbD, in the turtle Trachemys scripta. We determined the primary structures of the constituent subunits of the two Hb isoforms, and we related the measured functional properties to differences in O2 affinity between untreated hemolysates from turtles that were acclimated to normoxia and anoxia. Our data show that HbD has a consistently higher O2 affinity compared to HbA, whereas Bohr and temperature effects as well as thiol reactivity are similar. Although sequence data show amino acid substitutions at two known β-chain ATP-binding site positions, we find high ATP affinities for both Hb isoforms, suggesting an alternative and stronger binding site for ATP. The high ATP affinities indicate that, although ATP levels decrease in red blood cells of turtles acclimating to anoxia, the O2 affinity would remain largely unchanged, as confirmed by O2-binding measurements of untreated hemolysates from normoxic and anoxic turtles. Thus, the increase in blood-O2 affinity that accompanies winter acclimation is mainly attributable to a decrease in temperature rather than in concentrations of organic phosphates. This is the first extensive study on freshwater turtle Hb isoforms providing molecular evidence for adaptive changes in O2 transport associated with acclimation to severe hypoxia.
Over 26,000 manuscripts have been published dealing with endothelins since their discovery 25 years ago. These peptides, and particularly endothelin-1 (ET-1), are expressed by, bind to, and act upon virtually every cell type in the body, influencing multiple biologic functions. Among these actions, the effects of ET-1 on arterial pressure and volume homeostasis have been most extensively studied. While ET-1 modulates arterial pressure through regulation of multiple organ systems, the peptide's actions in the kidney in general, and the collecting duct in particular, are of unique importance. The collecting duct produces large amounts of ET-1 which bind in an autocrine manner to endothelin A and B receptors, causing inhibition of Na+ and water reabsorption; absence of collecting duct ET-1 or its receptors is associated with marked salt-sensitive hypertension. Collecting duct ET-1 production is stimulated by Na+ and water loading through local mechanisms that include sensing of salt and other solute delivery as well as shear stress. Thus, the collecting duct ET-1 system exists, at least in part, to detect alterations in, and maintain homeostasis for, extracellular fluid volume. Derangements in collecting duct ET-1 production may contribute to the pathogenesis of genetic hypertension. Blockade of endothelin receptors causes fluid retention due, in large part, to inhibition of the action of ET-1 in the collecting duct; this side effect has substantially limited the clinical utility of this class of drugs. Herein, the biology of the collecting duct ET-1 system is reviewed, with particular emphasis on key issues and questions that need addressing.
Endothelium-dependent vasodilation is mediated by nitric oxide (NO), prostaglandins (PG), and endothelium-derived hyperpolarizing factor (EDHF). We studied the contributions and temporal characteristics of these components in the renal vasodilator responses to acetylcholine (ACh) and bradykinin (BK) and in the buffering of vasoconstrictor responses to norepinephrine (NE) and angiotensin II (Ang II). Renal blood flow (RBF) and vascular conductance (RVC) were studied in anesthetized rats in response to renal arterial bolus injections before and after inhibition of NO-synthase (LNAME), cyclooxygenase (indomethacin, INDO), or both. ACh increased RVC peaking at tmax=29s. LNAME (n=8) diminished the integrated response, and made it substantially faster (tmax=18s). The point-by-point difference caused by LNAME (=NO component) integrated to 74% of control and was much slower (tmax=38s). INDO (n=9) reduced the response without affecting tmax (36 vs. 30s). The difference (=PG) reached 21% of the control with tmax=25s. LNAME+INDO (n=17) reduced the response to 18% and markedly accelerated tmax to 16s (=EDHF). Results were similar for BK with slightly more PG- and less NO-contribution than for ACh. Constrictor responses to NE and AngII were augmented and decelerated by LNAME and LNAME+INDO. The calculated difference (=buffering by NO or NO+PG) was slower than the constriction. It is concluded that NO, PG, and EDHF contribute >50%, 20-40%, and <20% to the renal vasodilator effect of ACh and BK. EDHF acts substantially faster and less sustained (tmax=16s) than NO and PG (tmax=30s). Constrictor buffering by NO and PG is not constant over time, but renders the constriction less sustained.
Heart failure (HF) is characterized by elevated sympathetic activity and reduced parasympathetic control of the heart. Experimental evidence suggests that the increase in parasympathetic function can be a therapeutic alternative to slow HF evolution. The parasympathetic neurotransmission can be improved by acetylcholinesterase inhibition. We investigated the long term (four weeks) effects of the acetylcholinesterase inhibitor pyridostigmine on sympathovagal balance, cardiac remodeling and cardiac function in the onset of HF following myocardial infarction. Myocardial infarction was elicited in adult male Wistar rats. After four weeks of pyridostigmine administration, per os, methyl-atropine and propranolol were used to evaluate the cardiac sympathovagal balance. The tachycardic response caused by methyl-atropine was considered to be the vagal tone, while the bradycardic response caused by propranolol was considered to be the sympathetic tone. In conscious HF rats, pyridostigmine reduced the basal heart rate, increased vagal and reduced sympathetic control of heart rate. Pyridostigmine reduced the myocyte diameter and collagen density of the surviving left ventricle. Pyridostigmine also increased vascular endothelial growth factor protein in the left ventricle, suggesting myocardial angiogenesis. Cardiac function was assessed by means of the pressure-volume conductance catheter system. HF rats treated with pyridostigmine exhibited a higher stroke volume, ejection fraction, cardiac output and contractility of the left ventricle. It was demonstrated that the long term administration of pyridostigmine started right after coronary artery ligation augmented cardiac vagal and reduced sympathetic tone, attenuating cardiac remodeling and left ventricular dysfunction during the progression of HF in rats.
The effect of hypercapnia on outwardly-rectifying currents was examined in locus coeruleus (LC) neurons in slices from neonatal rats (P3-P15). Two outwardly-rectifying currents (4-aminopyridine [4-AP]-sensitive transient current and tetraethyl ammonium [TEA]-sensitive sustained current) were found in LC neurons. 4-AP induced a membrane depolarization of 3.6 ± 0.6mV (n = 4), while TEA induced a smaller membrane depolarization of 1.2 ± 0.3 mV (n = 4). Hypercapnic acidosis (HA) inhibited both currents. The maximal amplitude of the TEA-sensitive current was reduced by 52.1 ± 4.5% (n=5) in 15% CO2 (pHo 7.00, pHi 6.96). The maximal amplitude of the 4-AP-sensitive current was reduced by 34.5 ± 3.0% (n = 6) in 15% CO2 (pHo 7.00, pHi 6.96), by 29.4 ± 6.8% (n = 6) in 10% CO2 (pHo 7.15, pHi 7.14), and increased by 29.0 ± 6.4% (n = 6) in 2.5% CO2 (pHo 7.75, pHi 7.35). 4-AP completely blocked hypercapnia-induced increased firing rate but TEA did not affect it. When LC neurons were exposed to HA with either pHo or pHi constant, the 4-AP-sensitive current was inhibited. The data show that the 4-AP-sensitive current (likely an A current) is inhibited by decreases in either pHo or pHi. The inhibition of the A current by various levels of CO2 is correlated with the change in firing rate induced by CO2, implicating the 4-AP-sensitive current in chemosensitive signaling in LC neurons.
Anaphylactic shock is sometimes life-threatening, and is accompanied by hepatic venoconstriction in animals, which in part accounts for anaphylactic hypotension. Roles of norepinephrine and α-adrenoceptor in anaphylaxis-induced hypotension and portal hypertension were investigated in anesthetized ovalbumin-sensitized Sprague-Dawley rats. The sensitized rats were randomly allocated to the following pretreatment groups (n=6/group): (1) control (non-pretreatment), (2) α1-adrenoceptor antagonist prazosin, (3) non-selective α-adrenoceptor antagonist phentolamine, (4) 6-hydroxydopamine-induced chemical sympathetectomy, and (5) surgical hepatic sympathetectomy. Anaphylactic shock was induced by an intravenous injection of the antigen. The systemic arterial pressure (SAP), central venous pressure (CVP), portal venous pressure (PVP) and portal venous blood flow (PBF) were measured, and splanchnic (Rspl: (SAP-PVP)/PBF) and portal venous (Rpv: (PVP-CVP)/PBF) resistances were determined. Separately, we measured efferent hepatic sympathetic nerve activity during anaphylaxis. In the control group, SAP markedly decreased, followed by a gradual recovery toward baseline. PVP and Rpv increased 3.2- and 23.3-fold, respectively, after antigen. Rspl decreased immediately but only transiently after antigen, and then increased 1.5-fold later than 10 min. The α-adrenoceptor antagonist pretreatment or chemical sympathetectomy inhibited the late increase in Rspl and the SAP recovery. Pretreatment with α-adrenoceptor antagonists, or either chemical or surgical hepatic sympathetectomy did not affect the antigen-induced increase in Rpv. Hepatic sympathetic nerve activity did not significantly change after antigen. In conclusion, α-adrenoceptor antagonists and chemical sympathetectomy exacerbate anaphylaxis-induced hypotension, but not portal hypertension, in anesthetized rats. Hepatic sympathetic nerves are not involved in anaphylactic portal hypertension.
In pregnancy, α-adrenoceptor mediated vasoconstriction is augmented in uterine radial arteries, and is accompanied by underlying changes in smooth muscle (SM) Ca2+ activity. This study aims to determine the Ca2+ entry channels associated with altered vasoconstriction in pregnancy, with the hypothesis that augmented vasoconstriction involves transient receptor potential canonical type-3 (TRPC3) and L- and T-type voltage-dependent Ca2+ channels. Immunohistochemistry showed TRPC3, L-type Cav1.2 (as the α1C sub-unit), and T-type Cav3.1 (α1G) and Cav3.2 (α1H) localization to the uterine radial artery SM. Fluorescence intensity of TRPC3, Cav1.2 and Cav3.2 was increased, and Cav3.1 decreased in radial artery SM from pregnant rats. Western blotting confirmed increased TRPC3 protein expression in the radial artery from pregnant rats. Pressure myography incorporating pharmacological intervention to examine the role of these channels in uterine radial arteries showed an attenuation of phenylephrine (PE)-induced constriction with Pyr3-mediated TRPC3 inhibition, or with nifedipine-mediated L-type channel block alone in vessels from pregnant rats; both effects of which were diminished in radial arteries from non-pregnant rats. Combined TRPC3 and L-type inhibition attenuated PE-induced constriction in radial arteries; and the residual vasoconstriction was reduced, and abolished, with T-type channel block with NNC 55-0396 in arteries from non-pregnant and pregnant rats, respectively. With SM Ca2+ stores depleted, and in the presence of PE, nifedipine and NNC 55-0396, blockade of TRPC3 reversed PE-induced constriction. These data suggest that TRPC3 channels act synergistically with L-and T-type channels to modulate radial artery vasoconstriction, with the mechanism being augmented in pregnancy.
Prolactin (PRL) is a potent liver mitogen and proangiogenic hormone. Here, we used hyperprolactinemic rats and PRL receptor null mice (PRLR-/-) to study the effect of PRL on liver growth and angiogenesis before and after partial hepatectomy (PH). Liver to body weight ratio (LBW), hepatocyte and sinusoidal endothelial cell (SEC) proliferation, and hepatic expression of VEGF were measured before and after PH in hyperprolactinemic rats, generated by placing two anterior pituitary glands (AP) under the kidney capsule. Also, LBW and hepatic expression of IL-6, and suppressor of cytokine signaling-3 (SOCS-3) were evaluated in wild type and PRLR-/- mice before and after PH. Hyperprolactinemia increased the LBW, the proliferation of hepatocytes and SEC, and VEGF hepatic expression. Also, liver regeneration was increased in AP-grafted rats and was accompanied by elevated hepatocyte and SEC proliferation, and VEGF expression compared to non-grafted controls. Lowering circulating PRL levels with CB-154, an inhibitor of AP PRL secretion, prevented AP-induced stimulation of liver growth. Relative to wild type animals, PRLR-/- mice had smaller livers, and soon after PH they displayed a ~2-fold increased mortality, and elevated and reduced hepatic IL-6 and SOCS-3 expression, respectively. However, liver regeneration was improved in surviving PRLR-/- mice. PRL stimulates normal liver growth, promotes survival, and regulates liver regeneration by mechanisms that may include hepatic downregulation of IL-6 and upregulation of SOCS-3, increased hepatocyte proliferation, and angiogenesis. PRL contributes to physiological liver growth and has potential clinical utility for ensuring survival and regulating liver mass in diseases, injuries, or liver surgery.
Skeletal muscles can be injured by lengthening contractions, when the muscles are stretched while activated. Lengthening contractions produce structural damage that leads to the degeneration and regeneration of damaged muscle fibers by mechanisms that have not been fully elucidated. Reactive oxygen species (ROS) generated at the time of injury may initiate degenerative or regenerative processes. In the present study we hypothesized that lengthening contractions that damage the muscle would generate more ROS than isometric contractions that do not cause damage. To test our hypothesis, we subjected muscles of mice to lengthening contractions or isometric contractions and simultaneously monitored intracellular ROS generation with the fluorescent indicator 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein (CM-DCFH), which is oxidized by ROS to form the fluorescent product CM-DCF. We found that CM-DCF fluorescence was not different during or shortly after lengthening contractions compared with isometric controls, regardless of the amount of stretch and damage that occurred during the lengthening contractions. The only exception was that after severe stretches, the increase in CM-DCF fluorescence was impaired. We conclude that lengthening contractions that damage the muscle do not generate more ROS than isometric contractions that do not cause damage. The implication is that ROS generated at the time of injury are not the initiating signals for subsequent degenerative or regenerative processes.
The reduction in nitric oxide (NO)-mediated vascular function with age has largely been determined by flow mediated dilation (FMD). However, in light of recent uncertainty surrounding the NO-dependency of FMD and the recognition that brachial artery (BA) vasodilation during handgrip exercise is predominantly NO-mediated in the young, we sought to determine the contribution of NO to BA vasodilation in the elderly using the handgrip paradigm. BA vasodilation during progressive dynamic (1 Hz) handgrip exercise performed at 3, 6, 9, and 12 kg was assessed with and without NO synthase (NOS) inhibition [intra-arterial NG-monomethyl-L-arginine (L-NMMA)] in 7 healthy older subjects (69±2 yr). Handgrip exercise in the control condition evoked significant BA vasodilation at 6 (4.7±1.4%), 9 (6.5±2.2%), and 12 kg (9.5±2.7%). NOS inhibition attenuated BA vasodilation, as the first measurable increase in BA diameter did not occur until 9 kg (4.0±1.8%) and the change in BA diameter at 12 kg was reduced by ~30% (5.1 ± 2.2%), with unaltered shear rate (CONTROL: 407±57, L-NMMA: 427±67 sec-1). Although shifted downwards, the slope of the relationship between BA diameter and shear rate during handgrip exercise was unchanged (CONTROL: 0.0013±0.0004, L-NMMA: 0.0011±0.007, p = 0.6) as a consequence of NOS inhibition. Thus, progressive handgrip exercise in the elderly evokes a robust BA vasodilation, the magnitude of which was only minimally attenuated following NOS inhibition. This, somewhat anticipated, modest contribution of NO to BA vasodilation in the elderly supports the use of the handgrip exercise paradigm to assess NO-dependent vasodilation across the lifespan.
Antenatal betamethasone (BM) therapy accelerates lung development in preterm infants but may induce early programming events with long-term cardiovascular consequences. To elucidate these events, we developed a model of programming whereby pregnant ewes are administered BM (2 doses of 0.17 mg/kg) or vehicle at the 80th day of gestation and offspring are delivered at term. The BM-exposed (BMX) offspring develop elevated blood pressure, decreased baroreflex sensitivity, and alterations in the circulating, renal, and brain renin-angiotensin systems (RAS) by 6-months of age. We compared components of the choroid plexus 4th ventricle (ChP4) and CSF RAS between control and BMX male offspring at 6-months of age. In the ChP, high molecular weight renin protein and Ang I-intact angiotensinogen were unchanged between BMX and control animals. ACE2 activity was 3-fold higher than either NEP or ACE in control and BMX animals. Moreover, all three enzymes were equally enriched ~2.5 fold in ChP4 brush border membrane preparations. CSF Ang-(1-7) levels were significantly lower in BMX animals (351.8 ± 76.8 vs. 77.5 ± 29.7 fmol/mg; p<0.05) and ACE activity was significantly higher (6.6 ± 0.5 vs. 8.9 ± 0.5 fmol/min/ml; P<0.05) while ACE2 and NEP activities were below measurable limits. A thiol-sensitive peptidase contributed to the majority of Ang-(1-7) metabolism in the CSF, with higher activity in the BMX animals. We conclude that in utero BM exposure alters CSF but not ChP RAS components resulting in lower Ang-(1-7) levels in exposed animals.
Neuropeptide W (NPW), an endogenous ligand for G protein-coupled receptors NPBWR1 (GPR7) and NPBWR2 (GPR8), has been detected in neurons in limbic and reticular activating system areas known to be important in arousal, as well as hypothalamic nuclei known to be important in food and water intake and the neuroendocrine response to stress. In rat, central administration of NPW increased mean arterial pressure (MAP) and behaviors associated with locomotion and grooming. We hypothesized that the NPW-induced increase in MAP was secondary to those increases in physical activity. Since peptides that stimulate arousal have been shown to increase sympathetic activity (e.g. orexin), we tested the ability of the mixed α1- and α2-adrenergic antagonist, phentolamine, to block the NPW-23-induced rise in MAP. Phentolamine pretreatment abrogated the NPW-induced MAP increase. However, we noticed the animals no longer exhibited NPW-associated behavioral arousal when pretreated with phentolamine. Anesthesia also blocked the NPW-induced increase in MAP although the animals still were able to respond with an increase in MAP to centrally administered angiotensin II. Additionally, pretreatment with an orexin type 1 receptor antagonist significantly reduced the behavioral action of NPW-23 and completely blocked the peptide's action to increase MAP, suggesting that orexin neurons are downstream targets of NPW. Our results suggest that NPW increased MAP secondary to increased behavioral arousal.
Aging is associated with attenuated thermoregulatory function which varies regionally over the body. Decrements in vasodilation and sweating are well documented with age yet limited data are available concerning the regional relation between these responses. We aimed to examine age-related alterations in the relation between regional sweating (RSR) and skin blood flow (SkBF) to thermal and pharmacological stimuli. Four microdialysis fibers were inserted in the ventral forearm, abdomen, thigh, and lower back of eight healthy aged subjects (64±7 years) and nine young (23±3 years) during 1) acetylcholine dose response (ACh 1x10-7 - 0.1 M, mean skin temperature 34°C) and 2) passive whole body heating to 1°C rise in oral temperature (Tor). RSR and SkBF were measured over each microdialysis membrane using ventilated capsules and laser-Doppler flowmetry. Maximal SkBF was measured at the end of both protocols (50mM SNP). Regional sweating thresholds and RSR were attenuated in aged versus young at all sites (p<0.0001) during whole body heating. Vasodilation thresholds were similar between groups (p>0.05). Attenuated SkBF were observed at the arm and back in the aged, representing 56% and 82% of those in the young at these sites, respectively (0.5 Tor). During ACh perfusion SkBF (p=0.137) and RSR were similar between groups (p= 0.326). Together these findings suggest regional age-related decrements in heat-activated sweat gland function but not cholinergic sensitivity. Functional consequences of such thermoregulatory impairment include the compromised ability of older individuals to defend core temperature during heat exposure, and a subsequently greater susceptibility to heat-related illness and injury.
Post-oral sugar actions enhance the intake of and preference for sugar-rich foods, a process referred to as appetition. Here we investigated the role of intestinal sodium glucose co-transporters (SGLTs) in sugar appetition in C57BL/6J mice using sugars and non-metabolizable sugar analogs that differ in their affinity for SGLT1 and SGLT3. In Experiments 1 and 2 food-restricted mice were trained (1 h/day) to consume a flavored saccharin solution (CS-) paired with intragastric (IG) self-infusions of water and a different flavored solution (CS+) paired with infusions of 8 or 12% sugars (glucose; fructose; galactose) or sugar analogs (α-methyl-D-glucopyranoside, MDG; 3-O-methyl-D-glucopyranoside: OMG). Subsequent two-bottle CS+ vs. CS- choice tests were conducted without co-infusions. Infusions of the SGLT1 ligands glucose, galactose, MDG and OMG stimulated CS+ licking above CS- levels. However, only glucose, MDG and galactose conditioned significant CS+ preferences, with the SGLT3 ligands (glucose, MDG) producing the strongest preferences. Fructose, which is not a ligand for SGLTs, failed to stimulate CS+ intake or preference. Experiment 3 revealed that IG infusion of MDG + phloridzin (an SGLT1/3 antagonist) blocked MDG appetition, whereas phloridzin had minimal effects on glucose-induced appetition. However, adding phloretin (a GLUT2 antagonist) to the glucose + phloridzin infusion blocked glucose appetition. Taken together, these findings suggest that humoral signals generated by intestinal SGLT1 and SGLT3, and to a lesser degree GLUT2, mediate post-oral sugar appetition in mice. The MDG results indicate that sugar metabolism is not essential for the post-oral intake stimulating and preference conditioning actions of sugars in mice.
The present study compared the progression of renal injury in Sprague Dawley (SD) and Dahl salt-sensitive (SS) treated with streptozotocin (STZ). The rats received an injection of STZ (50 mg/kg, i.p.) and an insulin pellet (2 U/day, s.c.) to maintain the blood glucose levels between 400-600 mg/dL. Twelve weeks later, arterial pressure (143±6 vs. 107±8 mmHg) and proteinuria (557±85 vs. 81±6 mg/day) were significantly elevated in STZ-SS rats compared to the values observed in STZ-SD rats, respectively. The kidneys from STZ-SS rats exhibited thickening of glomerular basement membrane, mesangial expansion, severe glomerulosclerosis, and renal interstitial fibrosis occasional glomerular nodule formation. In additional studies, treatment with a therapeutic dose of insulin (4 U/day, s.c.) attenuated the development of proteinuria (212±32 mg/day) and renal injury independent of changes in arterial pressure in STZ-SS rats. Since STZ-SS rats developed severe renal injury, we characterized the time course changes in renal hemodynamics during the progression of renal injury. After 9 weeks of diabetes, there was a 42% increase in GFR in STZ-SS rats versus time-control SS rats with reduced RBF. These results indicate that SS rats treated with STZ develop hyperfiltration and progressive proteinuria and display renal histological lesions characteristic to those seen in patients with diabetic nephropathy. Overall, this model may be useful to study signaling pathways and mechanisms that play role in the progression of diabetes-induced renal disease and the development of new therapies to slow the progression of diabetic nephropathy.
Systemic lupus erythematosus (SLE) is a chronic inflammatory disorder with prevalent hypertension and renal injury. In this study, we tested whether the renal nerves contribute to the development of hypertension in an established mouse model of SLE (NZBWF1). Female SLE and control (NZW/LacJ) mice were subjected to either bilateral renal denervation or a sham procedure at 32 weeks of age. Two weeks later, blood pressure was assessed in conscious mice using carotid artery catheters. Blood pressure was higher in SLE mice compared to controls as previously reported; however, blood pressure was not altered in the denervated SLE or control mice. The development of albuminuria was markedly blunted in denervated SLE mice; however, glomerulosclerosis was increased. Renal denervation reduced renal cortical expression of monocyte-chemoattractant protein in SLE mice but did not significantly alter renal monocyte/macrophage infiltration. Renal cortical TNF-α expression was also increased in sham SLE mice but this was not impacted by denervation. This study suggests that the renal nerves do not have a significant role in the pathogenesis of hypertension, but have a complex effect on the associated renal inflammation and renal injury.
Costameres are mechano-sensory sites of focal adhesion in the sarcolemma that reinforce the muscle-fiber composite and provide an anchor for myofibrillogenesis. We hypothesized that elevated content of the integrin-associated regulator of costamere turnover in culture, focal adhesion kinase (FAK), drives changes in costamere component content in anti-gravity muscle in a load-dependent way in correspondence with altered muscle weight. The content of FAK in soleus muscle being phosphorylated at auto-regulatory tyrosine 397 (FAK-pY397) was increased after 20 seconds of stretch. FAK-pY397 content remained elevated after 24 hours of stretch-overload due to up-regulated FAK content. Overexpression of FAK in soleus muscle fibers by means of gene electro-transfer increased the beta 1 integrin (+56%) and meta-vinculin (+88%) content. Alpha 7 integrin (p=0.46) and gamma-vinculin (p=0.18) content was not altered after FAK overexpression. Co-overexpression of the FAK inhibitor FRNK reduced FAK-pY397 content by 33% and increased the percentage of fast type fibers that arose in connection with hybrid fibers with gene transfer. Transplantation experiments confirmed the association of FRNK expression with slow-to-fast fiber transformation. 7 days of unloading blunted the elevation of FAK-pY397, beta 1 integrin and meta-vinculin content with FAK-overexpression and this was reversed by one day of reloading. The results highlight that the expression of components for costameric attachment sites of myofibrils is under load- and fiber type-related control via FAK and its inhibitor FRNK.
Our aim was to establish in spontaneously-breathing urethane-anesthetized rats, the relationship between the concentrations of H2S transported in the blood and the corresponding clinical manifestations, i.e. breathing stimulation followed by terminal apnea, during and following infusion of NaHS at increasing rates. The gaseous concentration of H2S (CgH2S, 1/3 of the total soluble form) was computed from the continuous determination of H2S partial pressure in the alveolar gas, while H2S, both dissolved and combined to hemoglobin, was measured at specific time points by sulfide complexation with monobromobimane (CMBBH2S). We found that, using a potent reducing agent in-vitro, H2S added to the whole blood had very little interaction with the plasma proteins, as sulfide appeared to be primarily combined then oxidized by hemoglobin. In vivo, H2S was undetectable in the blood in its soluble form in baseline conditions, while CMBBH2S averaged 0.7±0.5 µM. During NaHS infusion, H2S was primarily present in non-soluble form in the arterial blood; e.g. CMBBH2S was about 50 times higher than CgH2S at the lowest levels of exposure and 5-6 times at the levels wherein fatal apnea occurred. CgH2S averaged only 1.1±0.7 µM when breathing increased, corresponding to a CMBBH2S of 11.1±5.4 µM. Apnea occurred at CgH2S above 5.1 µM and CMBBH2S aove 25.4 µM. At the cessation of exposure, CMBBH2S remained elevated, at about 3 times above baseline for at least 15 min. These data provide a frame of reference for studying the putative effects of endogenous H2S and for testing antidotes against its deadly effects.
Both innate and adaptive immunity in birds are different from their mammalian counterparts. Understanding bird immunity is important because of the enormous potential impact of avian infectious diseases, both in their role as food animals and as potential carriers of zoonotic diseases in man. The anti-inflammatory protein tristetraprolin (TTP) is an important component of the mammalian innate immune response, in that it binds to and destabilizes key cytokine mRNAs. TTP KO mice exhibit a severe systemic inflammatory syndrome, and are abnormally sensitive to innate immune stimuli such as lipopolysaccharide (LPS). TTP orthologues have been found in most vertebrates studied, including frogs. Here we attempted to identify TTP orthologues in chicken and other birds, using database searches and deep mRNA sequencing. Although sequences encoding the two other widely expressed TTP family members, ZFP36L1 and ZFP36L2, were identified, we did not find sequences corresponding to TTP in any bird species. Sequences corresponding to TTP were identified in both lizards and alligators, close evolutionary relatives of birds. The induction kinetics of Zfp36l1 and Zfp36l2 mRNAs in LPS-stimulated chicken macrophages or serum-stimulated chick embryo fibroblasts did not resemble the normal mammalian TTP response to these stimuli, suggesting that the other two family members might not compensate for the TTP deficiency in regulating rapidly induced mRNA targets. Several mammalian TTP target transcripts have chicken counterparts that contain one or more potential TTP binding sites, raising the possibility that birds express other proteins that subsume TTP's function as a rapidly inducible regulator of AU-rich mRNA turnover.
Hypothalamic-pituitary-gonadal (HPG) axis function fundamentally affects the physiology of eating. We review sex differences in the physiological and pathophysiological controls of amount eaten in rats, mice, monkeys and humans. These controls result from interactions among genetic effects, organizational effects of reproductive hormones (i.e., permanent early developmental effects), and activational effects of these hormones (i.e., effects dependent on hormone levels). Male-female sex differences in the physiology of eating involve both organizational and activational effects of androgens and estrogens. An activational effect of estrogens decreases eating (1) during the periovulatory period of the ovarian cycle in rats, mice, monkeys and women and (2) tonically between puberty and reproductive senescence or ovariectomy in rats and monkeys, sometimes in mice, and possibly in women. Estrogens acting on estrogen receptor-α (ERα) in the caudal medial nucleus of the solitary tract appear to mediate these effects in rats. Androgens, prolactin, and other reproductive hormones also affect eating in rats. Sex differences in eating are mediated by alterations in orosensory capacity and hedonics, gastric mechanoreception, ghrelin, CCK, GLP-1, glucagon, insulin, amylin, apolipoprotein A-IV, fatty-acid oxidation and leptin. The control of eating by central neurochemical signaling via serotonin, MSH, NPY, AgRP, MCH and dopamine are modulated by HPG function. Finally, sex differences in the physiology of eating may contribute to human obesity, anorexia nervosa, and binge eating. The variety and physiological importance of what has been learned so far warrant intensifying basic, translational and clinical research on sex differences in eating.
Hibernating mammals undergo torpor during which blood pressure (BP), heart rate (HR), metabolic rate, and core temperature (TC) dramatically decrease, conserving energy. While the cardiovascular system remains functional, temporal changes of BP, HR, and baroreceptor-HR reflex sensitivity (BRS) over complete hibernation bouts and their relation to TC are unknown. We implanted BP/temperature telemetry transmitters into Syrian hamsters to test three hypotheses: (H-1) BP, HR, and BRS decrease concurrently during entry into hibernation and increase concurrently during arousal; (H-2) these changes occur before changes in TC; (H-3) the pattern of changes is consistent over successive bouts. We found: (a) upon entry, BP and HR declined before TC and BRS, suggesting baroreflex control of HR continues to regulate BP as the BP set point decreases; (b) during the later phase of entry, BRS decreased rapidly while BP and TC fell gradually, suggesting the importance of TC in further BP declines; (c) during torpor, BP slowly increased (but remained relatively low) without changes in HR or BRS or increased TC, suggesting minimal baroreflex or temperature influence; (d) during arousal, increased TC and BRS significantly lagged increases in BP and HR, consistent with establishment of tissue perfusion before increased TC /metabolism; (e) the temporal pattern of these changes was similar over successive bouts in all hamsters. These results negate H-1, support H-2 with respect to BP and HR, support H-3, and indicate that the baroreflex contributes to cardiovascular regulation over a hibernation bout, albeit operating in a fundamentally different manner during entry versus arousal.
Mitochondria are dynamic organelles forming a tubular network that is continuously fusing and dividing to control their morphology and functions. Recent literature has shed new light on a potential link between the dynamic behavior of mitochondria and muscle development. In this study, we investigate the role of mitochondrial fission factor, dynamin-related protein 1 (Drp1), in myogenic differentiation. We found that differentiation of C2C12 myoblasts induced by serum starvation was accompanied by a gradual increase in Drp1 protein expression (to ~350% up to 3 days) and a fast reduction of Drp1 phosphorylation at Ser-637 (to ~30%) resulting in translocation of Drp1 protein from the cytosol to mitochondria. During differentiation, treatment of myoblasts with mdivi-1, a specific inhibitor of Drp1 GTPase activity, caused extensive formation of elongated mitochondria, which coincided with increased apoptosis evidenced by both enhanced caspase-3 activity and increased number of TUNEL-positive cells. Furthermore, the mdivi-1 treated myotubes (day 3 in differentiation media) showed a reduction in mitochondrial DNA content, mitochondrial mass and membrane potential in a dose-dependent manner indicating defects in mitochondrial biogenesis during myogenic differentiation. Most interestingly, mdivi-1 treatment significantly suppressed myotube formation in both C2C12 cells and primary myoblasts. Likewise, stable overexpression of a dominant negative mutant Drp1 (K38A) dramatically reduced myogenic differentiation. These data suggest that Drp-1-dependent mitochondrial division is a necessary step for successful myogenic differentiation, and perturbation of mitochondrial dynamics hinders normal mitochondrial adaptations during muscle development. Therefore, in the present study, we report a novel physiological role of mitochondrial dynamics in myogenic differentiation.
While a paucity of information exists regarding post-transcriptional mechanisms influencing mitochondrial biogenesis, in resting muscle the stability of peroxisome proliferator-activated receptor co-activator 1α (PGC-1α) mRNA has been linked to mitochondrial content. Therefore, in the current study we have examined if exercise promotes mRNA accumulation through the induction of proteins affiliated with mRNA stabilization (HuR) or conversely by decreasing the expression of mRNA destabilizing proteins (AUF1 and CUG-BP1). A single bout of exercise increased (P<0.05) the mRNA content of the transcriptional co-activator PGC-1α ~3.5 fold without affecting mRNA content for HuR, CUG-BP1 or AUF1. One week of treadmill exercise training did not alter markers of mitochondrial content, the mRNA stabilizing protein HuR, or the mRNA destabilizing protein AUF1. In contrast, the mRNA destabilizing protein CUG-BP1 increased ~40%. Four weeks of treadmill training increased the content of subunits of the electron transport chain ~50%, suggesting induction of mitochondrial biogenesis. Expression levels for HuR and CUG-BP1 were not altered with chronic training; however AUF1 expression was increased post-training. Specifically, training increased (P<0.05) total muscle expression of two of the four AUF1 isoforms ~50% (AUF1p37, AUF1p40). Interestingly, these two isoforms were not detected in isolated nuclei; however, a large band representing the other two isoforms (AUF1p42, AUF1p45) was present in nuclei and increased ~35% following chronic training. Altogether the current data provides evidence that mitochondrial biogenesis occurs in the presence of increased CUG-BP1 and AUF1 following, suggesting that reductions in known mRNA destabilizing proteins likely does not contribute to exercise-induced mitochondrial biogenesis.
The effects of muscle contractions on the profile of post-contraction resting intracellular Ca2+ ([Ca2+]i) accumulation in Type I diabetes are unclear. We tested the hypothesis that, following repeated muscle contractions, the rise in resting [Ca2+]i evident in healthy rats would be increased in diabetes rats and that these changes would be associated with a decreased cytoplasmic Ca2+ buffering capacity. Adult male Wistar rats were divided into diabetic (DIA: Streptozocin i.p.) and healthy (CONT) groups. Four weeks later animals were anesthetized and spinotrapezius muscle contractions (10 sets of 50 contractions) were elicited by electrical stimulaton (100 Hz). Ca2+ imaging was achieved using Fura2-AM in the spinotrapezius muscle in vivo (i.e., circulation intact). The ratio (340/380 nm) was determined from fluorescence images following each contractions set, for estimation of [Ca2+]i. Also, muscle Ca2+ buffering was studied in individual myocytes microinjected with 2 mM Ca2+ solution. After muscle contractions, resting [Ca2+]i in DIA increased earlier and more rapidly than in CONT (P < 0.05). Peak [Ca2+]i in response to the [Ca2+]i injection was significantly higher in CONT (25.8%) than DIA (10.2%). Subsequently, CONT decreased rapidly to plateau 9-10% above baseline whereas DIA remained throughout the 60 s measurement window. No differences in SERCA (Ca2+ uptake) levels were evident between CONT and DIA, whereas RyR (Ca2+ release) level and mitochondrial enzyme activity was decreased in DIA (P < 0.05). In conclusion, diabetes impairs resting [Ca2+]i homeostasis following muscle contractions. Markedly different responses to Ca2+ injection in DIA versus CONT suggest fundamentally deranged Ca2+ handling.
We employed the work-loop method to study the ability of ventricular and atrial trabeculae from Atlantic cod to sustain power production during repeated contractions at acclimation temperatures (10°C) and when acutely warmed (20°C). Oxygen tension (PO2) was lowered from 450 to 34% air saturation to augment the thermal stress. Preparations worked under conditions simulating either a large stroke volume (35 min-1 contraction rate, 8-12% muscle strain) or a high heart rate (70 min-1 rate, 2-4% strain), with power initially equal under both conditions. The effect of declining PO2 on power was similar under both conditions, but was temperature and tissue dependent. In ventricular trabeculae at 10°C (and atria at 20°C), shortening power declined across the full range of PO2 studied whereas the power required to lengthen the muscle was unaffected. Conversely, in ventricular trabeculae at 20°C, there was no decline in shortening power but an increase in lengthening power when PO2 fell below 100% air saturation. Finally, when ventricular trabeculae were paced at rates up to 115 min-1 at 20°C (vs. the maximum of 70 min-1 in vivo), they showed marked increases in both shortening and lengthening power. Our results suggest that while elevated heart rates may not impair ventricular power as they commonly do isometric force, limited atrial power and the increased work required to expand the ventricle during diastole may compromise ventricular filling, and hence stroke volume, in Atlantic cod at warm temperatures. Neither large strains nor high contraction rates convey an apparent advantage in circumventing this.
Lifestyle intervention programs currently emphasize weight loss secondary to obesity as the primary determinant of phenotypic changes. We examined whether the effects of a short-term lifestyle intervention program differs in normal weight vs. overweight/obese children. 19 overweight/obese (O, BMI=33.6±1.9 kg/m2) and 14 normal weight (N, BMI=19.9±1.5 kg/m2) children participated in a 2-week program consisting of an ad libitum high-fiber, low-fat diet and daily exercise (2-2.5 hr). Fasting serum samples were taken pre- and post-intervention for determination of lipids, glucose homeostasis, inflammatory cytokines, and adipokines. Only O lost weight (3.9%), but remained overweight/obese (32.3±1.9 kg/m2). Both groups exhibited significant intervention-induced decreases (p<0.05) in serum insulin (N: 52.5% vs. O: 28.1%, between-group p=0.38), homeostatic model assessment for insulin resistance (N: 53.1% vs. O: 28.4%, p=0.43), leptin (N: 69.3% vs. O: 44.1%, p=0.10), amylin (N: 28.7% vs. O: 26.1%, p=0.80), resistin (N: 40.0% vs. O: 35.1%, p=0.99), plasminogen activator-inhibitor-1 (N: 30.8% vs. O: 25.6%, p=0.59), interleukin (IL)-6 (N: 58.8% vs. O: 48.5%, p=0.78), IL-8 (N: 46.0% vs. O: 42.2%, p=0.49), and tumor necrosis factor-α (N: 45.8% vs. O: 40.8%, p=0.99). No associations between indices of weight change and phenotypic changes were noted. A short-term, intensive lifestyle modification program is effective in ameliorating metabolic risk factors in normal weight and overweight/obese children. These results suggest that obesity per se was not the primary driver of the phenotypes noted and dietary intake and physical inactivity induce the phenotypic abnormalities. These data may have implications for the weight-loss independent management of cardiometabolic risk in pediatric populations.
Methods to predict onset of cardio-pulmonary (CP) decompression sickness (DCS) would be of great benefit to clinicians caring for stricken divers. Principal dynamic mode (PDM) analysis of the electrocardiogram has been shown to provide accurate separation of the sympathetic and parasympathetic tone dynamics. Nine swine (Sus scrofa) underwent a 15 hour saturation dive at 184 kPa (60 ft. of saltwater) in a hyperbaric chamber followed by dropout decompression, while six swine, used as a control, underwent a 15 hour saturation dive at 15 kPa (5 ft. of saltwater). Non-invasive electrocardiograms were recorded throughout the experiment and autonomic nervous system dynamics evaluated by heart rate series analysis using power spectral density (PSD) and PDM methods. We observed a significant increase in the sympathetic and parasympathetic tones using the PDM method on average 20 minutes prior to DCS onset following a sudden induction of decompression. Parasympathetic activities remained elevated but the sympathetic modulation was significantly reduced at onset of cutis and CP DCS signs, as reported by a trained observer. Similar non-significant observations occurred during PSD analysis. PDM observations contrast with previous work showing neurological DCS resulted in >50% reduction in both sympathetic and parasympathetic tone. Therefore, tracking dynamics of the parasympathetic tones via the PDM method may allow discrimination between CP DCS and neurological DCS and this significant increase in parasympathetic tone has potential use as a marker for early diagnosis of CP DCS.
Studies in young adults show that a greater proportion of heat is gained shortly following the start of exercise and that temporal changes in whole-body heat loss during intermittent exercise have a pronounced effect on body heat storage. The consequences of short duration intermittent exercise on heat storage with aging are unclear. We compared evaporative heat loss (HE) and changes in body heat content (Hb) between young (20-30 years), middle-age (40-45 years) and older males (60-70 years) of similar body mass and surface area, during successive exercise (4x15-min) and recovery periods (4x15-min) at a fixed rate of heat production (400W) and under fixed environmental conditions (35°C/20% relative humidity). HE was lower in older males versus young males during each exercise (Ex1: 283±10 vs. 332±11 kJ, Ex2: 334±10 vs. 379±5 kJ, Ex3: 347±11 vs. 392±5 kJ, Ex4: 347±10 vs. 387±5 kJ, all P<0.02), whereas HE in middle-age males was intermediate to that measured in young and older adults (Ex1: 314±13, Ex2: 355±13, Ex3:371±13, Ex4:365±8 kJ). HE was not significantly different between groups during the recovery periods. The net effect over 2-hours was a greater Hb in older (267±33 kJ, P=0.016) and middle-age adults (245±16 kJ, P=0.073) relative to younger counterparts (164±20 kJ). As a result of a reduced capacity to dissipate heat during exercise, which was not compensated by a sufficiently greater rate of heat loss during recovery, both older and middle-age males had a progressively greater rate of heat storage in comparison to young males over 2-hours of intermittent exercise.
In addition to effects on appetite and metabolism, leptin influences many neuroendocrine and physiologic systems including the sympathetic nervous system. Building on my Carl Ludwig Lecture of the American Physiological Society, I review the sympathetic and cardiovascular actions of leptin. The review focuses on a critical analysis of the concept of selective leptin resistance (SLR) and the role of leptin in the pathogenesis of obesity-induced hypertension in both experimental animals and humans. We introduced the concept of SLR in 2002 to explain how leptin might increase blood pressure (BP) in obese states, such as diet-induced obesity (DIO), that are accompanied by partial leptin resistance. This concept, analogous to selective insulin resistance in the metabolic syndrome, holds that in several genetic and acquired models of obesity, there is preservation of the renal sympathetic and pressor actions of leptin despite attenuation of the appetite and weight reducing actions. Two potential overlapping mechanisms of SLR are reviewed: 1. Differential leptin molecular signaling pathways that mediate selective as opposed to universal leptin action, and 2. Brain site-specific leptin action and resistance. Although the phenomenon of SLR in DIO has so far focused on preservation of sympathetic and BP actions of leptin, consideration should be given to the possibility that this concept may extend to preservation of other actions of leptin. Finally, I review perplexing data on the effects of leptin on sympathetic activity and BP in humans and its role in human obesity-induced hypertension.
Premenopausal women have lower blood pressure and a reduced incidence of cardiovascular disease compared to age-matched men. Similar sex differences have been seen across species and in multiple animal models of hypertension. While important progress over the last decade has been made in elucidating some of the mechanisms underlying these differences, there are still significant gaps in our knowledge. Understanding the cellular and molecular mechanisms responsible for sex differences in hypertension will be important for developing sex specific therapies targeted toward the prevention and treatment of hypertension. Female sex hormones, especially estrogen, have been demonstrated to modulate the renin-angiotensin-aldosterone system (RAAS) and to have beneficial effects on cardiovascular function through actions not only on the kidney, heart and vasculature, but also on the central nervous system (CNS). This review primarily focuses on the central regulatory actions of estrogen on brain nuclei involved in blood pressure regulation and the interactions between estrogen and the RAAS in the CNS by which estrogen plays an important protective role against the development of hypertension.
Nitric oxide is a critical regulator of blood pressure (BP) and inflammation and female spontaneously hypertensive rats (SHR) have higher renal nitric oxide bioavailability than males. We hypothesize that female SHR will have a greater rise in BP and renal T cell infiltration in response to nitric oxide synthase (NOS) inhibition than males. Both male and female SHR displayed a dose-dependent increase in BP to the non-specific NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME: 2, 5, and 7 mg/kg/day for 4 days each); however, females exhibited a greater increase in BP than males. Treatment of male and female SHR with 7 mg/kg/day L-NAME for 2 weeks significantly increased BP in both sexes; however, prior exposure to L-NAME only increased BP sensitivity to chronic NOS inhibition in females. L-NAME-induced hypertension increased renal T cell infiltration and indices of renal injury in both sexes, yet female SHR exhibited greater increases in Th17 cells and greater decreases in regulatory T cells than males. Chronic L-NAME was also associated with larger increases in renal cortical adhesion molecule expression in female SHR. The use of triple therapy to block L-NAME-mediated increases in BP attenuated L-NAME-induced increases in renal T cell counts and normalized adhesion molecule expression in SHR, suggesting that L-NAME-induced increases in renal T cells were dependent on both increases in BP and NOS inhibition. Our data suggest that NOS is critical in the ability of SHR, females in particular, to maintain BP and limit a pro-inflammatory renal T cell profile.
Maintenance of body water homeostasis is critical for preventing hyperthermia, because evaporative cooling is the most efficient means of dissipating excess body heat. Water homeostasis is achieved by regulation of water intake and water loss by the kidneys. The former is achieved by sensations of thirst that motivate water acquisition. While the latter is regulated by the antidiuretic action of vasopressin. Vasopressin secretion and thirst are stimulated by increases in the osmolality of the extracellular fluid as well as decreases in blood pressure and/or blood volume, signals that are precipitated by water depletion associated with the excess evaporative water loss required to prevent hyperthermia. In addition, they are stimulated by increases in body temperature. The sites and molecular mechanisms involved in integrating thermal and osmotic regulation of thirst and vasopressin secretion are reviewed here with a focus on the role of the thermal and mechanosensitive TRPV family of ion channels.
The effects of intensified training in combination with a reduced training volume on muscle ion kinetics, transporters and work capacity were examined. Eight well-trained cyclists replaced their regular training with speed-endurance training (12x30-s sprints) 2-3 times per wk and aerobic high-intensity training (4-5x3-4min at 90-100% of maximal heart rate) 1-2 times per wk for 7 wks, and reduced training volume by 70% (intervention period; IP). The duration of an intense exhaustive cycling bout (EX2; 356±6 W), performed 2.5min after a 2-min intense cycle bout (EX1), was longer (p<0.05) after than before IP (4:16±0:34 vs. 3:37±0:28 min:s), and mean and peak power during a repeated sprint test improved (p<0.05) by 4 and 3%, respectively. Femoral venous K+ concentration in recovery from EX1 and EX2 was lowered (p<0.05) after compared to before IP, whereas muscle interstitial K+ concentration and net muscle K+ release during exercise was unaltered. No changes in muscle lactate and H+ release during and after EX1 and EX2 were observed, but the in vivo buffer capacity was higher (p<0.05) after IP. Expression of the K+ATP channel 2 (Kir6.2) decreased by IP, with no change in the strong inward rectifying K+ channel (Kir2.1), muscle Na+-K+ pump subunits, monocarboxylate transporters 1 and 4 (MCT1 and MCT4) and Na+-H+ exchanger 1 (NHE1). In conclusion, 7wks of intensified training with a reduced training volume improved performance during repeated intense exercise, which was associated with a greater muscle re-uptake of K+ and muscle buffer capacity but not with the amount of muscle ion transporters.
Emerging evidence supports a potential therapeutic role of relaxin in fibrotic diseases including chronic kidney disease. Relaxin is a pleiotropic hormone, best characterized for its role in the reproductive system; however, recent studies have demonstrated a role of relaxin in the cardio-renal system. Both relaxin and its receptor, RXFP1, are expressed in the kidney, and relaxin has been shown to play a role in renal vasodilation, in sodium excretion, and as an anti-fibrotic agent. Together, these findings suggest that the kidney is a target organ of relaxin. Therefore, the purpose of this review is to describe the functional and structural impacts of relaxin treatment on the kidney and to discuss evidence that relaxin prevents disease progression in several experimental models of kidney disease. In addition, this review will present potential mechanisms that are involved in the therapeutic actions of relaxin.
Obesity is an increasing health problem. Since drug treatments are limited, diets remain popular. High-protein diets (HPD) reduce body weight (BW) although the mechanisms are unclear. We investigated physiological mechanisms altered by switching rats from Western-type diet (WTD)-induced obesity (DIO) to HPD. Male rats were fed standard (SD) or WTD (45% calories from fat). After developing DIO (50% of rats), they were switched to SD or HPD (52% calories from protein) for 14 days. Food intake (FI), BW, body composition, glucose tolerance, insulin sensitivity and intestinal hormone plasma levels were monitored. Rats fed WTD showed an increased FI starting at 3 weeks and had a 25% greater BW gain after 9 weeks compared to SD (P<0.05). DIO rats switched from WTD to HPD reduced daily FI by 30% on day 1 which lasted to day 9 (-9%) and decreased BW during the 2-week period compared to SD/SD (P<0.05). During these 2 weeks, WTD/HPD rats lost 72% more fat mass than WTD/SD (P<0.05), whereas lean mass was unaltered. WTD/HPD rats had lower blood glucose than WTD/SD at 30 min post glucose gavage (P<0.05). The increase of gastric inhibitory polypeptide and pancreatic polypeptide during the 2-h dark phase feeding was higher and that of peptide YY lower in WTD/HPD compared to WTD/SD (P<0.05). These data indicate that HPD reduces BW in WTD rats, which may be related to decreased FI and the selective reduction of fat mass accompanied by improved glucose tolerance suggesting relevant benefits for HPD in the treatment of obesity.
Heat-related decreases in cerebral perfusion are partly the result of ventilatory related reductions in arterial carbon dioxide (CO2) tension. Cerebral perfusion likely contributes to an individual's tolerance to a challenge like lower body negative pressure (LBNP). Thus, increasing cerebral perfusion may prolong LBNP tolerance. This study tested the hypothesis that a hypercapnia-induced increase in cerebral perfusion improves LBNP tolerance in hyperthermic individuals. Eleven individuals (31±7 y; 75±12 kg) underwent passive heat stress (increased intestinal temperature ~1.5°C) followed by a progressive LBNP challenge to tolerance on two separate days (randomized). From 30 mm Hg LBNP, subjects inhaled either (blinded) a hypercapnic gas mixture (5% CO2, 21% oxygen, balanced nitrogen) or room air (SHAM). LBNP tolerance was quantified via the cumulative stress index (CSI). Mean middle cerebral artery blood velocity (MCAvmean,) and end-tidal CO2 (PETCO2,) were also measured. 5% CO2 inhalation increased PETCO2 at ~40 mm Hg LBNP (by 16±4 mmHg) and at LBNP tolerance (by 18±5 mmHg), compared to SHAM (P<0.01). Subsequently, MCAvmean was higher in the 5% CO2 trial during ~40 mm Hg LBNP (by 21±12 cm.s-1, ~31%) and at LBNP tolerance (by 18±10 cm.s-1, ~25%) relative to the SHAM (P<0.01). However, hypercapnia-induced increases in MCAvmean did not alter LBNP tolerance (5% CO2 CSI: 339±155 mm Hg x min; SHAM CSI: 273±158 mm Hg x min; P=0.26). These data indicate that inhaling a hypercapnic gas mixture increases cerebral perfusion during LBNP but does not improve LBNP tolerance when hyperthermic.
During drought periods camels are watered at long intervals, but effects on body fluid homeostasis of lactating camels are not known. It was hypothesized that camels store water after drinking and minimize water losses by diurnal variation in body temperature, changes in behavior, and release of vasopressin. The aim was to find a sustainable watering interval for lactating camels. Seven lactating camels were studied in a cross-over trial in which they were watered once daily (W1), every fourth day (W4), every eighth day (W8), or after 16 days (W16) with a five day interval between treatments. When offered water every fourth or eighth days, the camels drank sufficient amounts to cover their needs for subsequent days, but after 16 days of dehydration they did not drink enough to compensate the body weight loss. Rectal temperature fell at night and the camels searched shade during daytime minimizing evaporative fluid losses. Plasma osmolality and sodium concentration were elevated after four days of water deprivation, and plasma protein and vasopressin concentrations after eight days. Milk production decreased during the last week of W16. Plasma aldosterone concentration was elevated upon rehydration after W16, indicating sodium deficiency. In conclusion, lactating camels stored water after drinking and reduced water losses by staying in shade, keeping body temperature low and releasing plasma vasopressin. However, serious dehydration was observed during W8, and after 16 days of water deprivation recovery took a long time. A watering interval between four and seven days seems advisable under similar environmental conditions.
CTRP9 is a secreted multimeric protein of the C1q family and the closest paralog of the insulin-sensitizing adipokine, adiponectin. The metabolic function of this adipose tissue-derived plasma protein remains largely unknown. Here, we show that the circulating levels of CTRP9 are downregulated in diet-induced obese mice and upregulated upon refeeding. Over-expressing CTRP9 resulted in lean mice that dramatically resisted weight gain induced by a high-fat diet, largely through decreased food intake and increased basal metabolism. Enhanced fat oxidation in CTRP9 transgenic mice resulted from increases in skeletal muscle mitochondrial content, expression of enzymes involved in fatty acid oxidation (LCAD and MCAD), and chronic AMPK activation. Hepatic and skeletal muscle triglyceride levels were substantially decreased in transgenic mice. Consequently, CTRP9 transgenic mice had a greatly improved metabolic profile with markedly reduced fasting insulin and glucose levels. The high-fat diet-induced obesity, insulin resistance, and hepatic steatosis observed in wild-type mice were prevented in transgenic mice. Consistent with the in vivo data, recombinant protein significantly enhanced fat oxidation in L6 myotubes via AMPK activation and reduced lipid accumulation in H4IIE hepatocytes. Collectively, these data establish CTRP9 as a novel metabolic regulator and a new component of the metabolic network that links adipose tissue to lipid metabolism in skeletal muscle and liver.
The sensitization of capsaicin-sensitive lung vagal (CSLV) afferents by inflammatory mediators is important in the development of airway hypersensitivity. Hydrogen sulfide (H2S) is an endogenous mediator inducing hyperalgesia through transient receptor potential ankyrin 1 (TRPA1) receptors located on nociceptors. We conducted this study to determine whether H2S elevates the sensitivity of rat CSLV afferents. In anesthetized, artificially ventilated rats, the inhalation of aerosolized sodium hydrosulfide (NaHS, a H2S donor) caused no significant changes in the baseline activity of CSLV afferents. However, the afferent responses to right atrial injection of capsaicin or phenylbiguanide and to lung inflation were all markedly potentiated after NaHS inhalation. By contrast, the inhalation of its vehicle or NaOH (with a similar pH to NaHS) failed to enhance the afferent responses. Additionally, the potentiating effect on the afferent responses was found in rats inhaling L-cysteine (a substrate of H2S synthase) that slowly releases H2S. The potentiating effect of NaHS on the sensitivity of CSLV afferents was completely blocked by pretreatment of HC-030031 (a TRPA1 receptor antagonist), but was unaffected by its vehicle. In isolated rat CSLV neurons, the perfusion of NaHS alone did not influence the intracellular Ca2+ concentration but markedly potentiated the Ca2+ transients evoked by capsaicin. The NaHS-caused effect was totally abolished by HC-030031 pretreatment. These results suggest that H2S induces a nonspecific sensitizing effect on CSLV fibers to both chemical and mechanical stimulation in rat lungs, which appears mediated through an action on the TRPA1 receptors expressed on the nerve endings of CSLV afferents.
Mechanical ventilation (MV) is used clinically to maintain gas exchange in patients that require assistance in maintaining adequate alveolar ventilation. Common indications for MV include respiratory failure, heart failure, drug overdose, and surgery. Although MV can be a life-saving intervention for patients suffering from respiratory failure, prolonged MV can promote diaphragmatic atrophy and contractile dysfunction which is referred to as ventilator-induced diaphragm dysfunction (VIDD). This is significant because VIDD is thought to contribute to problems in weaning patients from the ventilator. Extended time on the ventilator increases health care costs and greatly increases patient morbidity and mortality. Research reveals that only 18-24 hours of MV is required to develop VIDD in both laboratory animals and humans. Studies using animal models reveal that MV-induced diaphragmatic atrophy occurs due to increased diaphragmatic protein breakdown and decreased protein synthesis. Recent investigations have identified calpain, caspase-3, autophagy, and the ubiquitin-proteasome system as key proteases that participate in MV-induced diaphragmatic proteolysis. The challenge for the future is to define the MV-induced signaling pathways that promote the loss of diaphragm protein and depress diaphragm contractility. Indeed, forthcoming studies that delineate the signaling mechanisms responsible for VIDD will provide the knowledge necessary for the development of a pharmacological approach that can prevent VIDD and reduce the incidence of weaning problems.
Obese individuals are characterized by low circulating adiponectin concentrations and an increased number of macrophages in adipose tissue, which is believed to be causally associated with chronic low-grade inflammation and insulin resistance. Regular physical exercise decreases overall morbidity in obese subjects which may be due to modulations of inflammatory pathways. In this randomized clinical trial we investigated the separate effects of endurance training-induced weight loss, diet-induced weight loss and endurance training per se (without weight loss) on plasma adiponectin multimer composition (Western blotting) and adipose tissue macrophage content (immunohistochemistry) in young, moderately overweight men. Weight loss and endurance training per se decreased whole body fat percentage in an additive manner. No intervention-induced changes were observed for plasma total adiponectin. Surprisingly, endurance training, irrespectively of any associated weight loss, shifted the adiponectin multimer distribution towards a lower molecular weight (21% decrease in HMW/LMW, P=0.015) whereas diet-induced weight loss shifted the distribution towards a higher molecular weight (42% increase in HMW/MMW, P<0.001). Furthermore, endurance training per se increased the number of anti-inflammatory CD163+ macrophages (from 12.7 [2.1] (mean [SE]) to 16.1 [3.1] CD163+ cells/100 adipocytes, P=0.013), whereas diet-induced weight loss tended to decrease CD68+ macrophages in subcutaneous abdominal adipose tissue. Thus, regular physical exercise influences systemic and adipose tissue inflammatory pathways differently than diet-induced weight loss in younger, moderately overweight men. Our data suggest that some of the health benefits of a physically active lifestyle may occur through modulations of anti- rather than pro-inflammatory pathways in young, overweight men.
Administration of metformin is known to reduce both body weight and food intake. While the hypothalamus is recognized as a critical regulator of energy balance and body weight, there is currently no evidence for an effect of metformin in the hypothalamus. Therefore, we sought to determine the central action of metformin on energy balance and body weight as well as its potential involvement with key hypothalamic energy sensors, including AMP-activated protein kinase (AMPK) and S6 kinase (S6K). We used meal pattern analysis and a conditioned taste aversion (CTA) test, and measured energy expenditure in C56BL/6 mice administered metformin (0, 7.5, 15, or 30 μg) into the third ventricle (I3V). Furthermore, we I3V-administered either control or metformin (30 μg) and compared the phosphorylation of AMPK and S6K in the mouse mediobasal hypothalamus. Compared to the control, I3V administration of metformin decreased body weight and food intake in a dose-dependent manner and did not result in CTA. Furthermore, the reduction in food intake induced by I3V administration of metformin was accomplished by decreases in both nocturnal meal size and number. Compared to the control, I3V administration of metformin significantly increased phosphorylation of S6K at Thr389 and AMPK at Ser485/491 in the mediobasal hypothalamus, while AMPK phosphorylation at Thr172 was not significantly altered. Moreover, I3V rapamycin pretreatment restored the metformin-induced anorexia and weight loss. These results suggest that the reduction in food intake induced by the central administration of metformin in the mice may be mediated by activation of S6K pathway.
The present paper provides an overview on adaptive changes in brain structure and learning abilities during hibernation as a behavioral strategy used by several mammalian species to minimize energy expenditure under current or anticipated inhospitable environmental conditions. One cellular mechanism that contributes to the regulated suppression of metabolism and thermogenesis during hibernation is reversible phosphorylation of enzymes and proteins that limits rates of flux through metabolic pathways. Reversible phosphorylation during hibernation also affects synaptic membrane proteins, a process known to be involved in synaptic plasticity. This mechanism of reversible protein phosphorylation also affects the microtubule-associated protein tau, thereby generating a condition that in the adult human brain is associated with aggregation of tau protein to paired helical filements (PHFs) as observed in Alzheimer's disease. Here, we put forward the concept that phosphorylation of tau is a neuroprotective mechanism to escape NMDA-mediated hyperexcitability of neurons that would otherwise occur during slow gradual cooling of the brain. Phosphorylation of tau and its subsequent targeting to subsynaptic sites might, thus work as a kind of "master switch", regulating NMDA receptor mediated synaptic gain in a wide array of neuronal networks, thereby enabling entry into torpor. If this condition lasts to long, however, it may eventually turn into a pathological trigger driving a cascade of events leading to neurodegeneration as in Alzheimer's disease or other "tauopathies".
Our objective was to test the hypothesis that fetal urine contains a substance(s) that regulates amniotic fluid volume by altering the rate of intramembranous absorption of amniotic fluid. In late gestation ovine fetuses, amniotic fluid volumes, urine and lung liquid production rates, swallowed volumes and intramembranous volume and solute absorption rates were measured over 2 day periods under control conditions and when urine was removed and continuously replaced at an equal rate with exogenous fluid. Intramembranous volume absorption rate decreased by 40% when urine was replaced with lactated Ringer's solution or lactated Ringer's solution diluted 50% with water. Amniotic fluid volume doubled under both conditions. Analysis of the intramembranous sodium and chloride fluxes suggests that the active but not passive component of intramembranous volume absorption was altered by urine replacement whereas both active and passive components of solute fluxes were altered. We conclude that fetal urine contains an unidentified substance(s) that stimulates active intramembranous transport of amniotic fluid across the amnion into the underlying fetal vasculature and thereby functions as a regulator of amniotic fluid volume.
Mounting evidence suggests that the circadian clock plays an integral role in the regulation of many physiological processes including blood pressure, renal function, and metabolism. The canonical molecular clock functions via activation of circadian target genes by Clock/Bmal1 and repression of Clock/Bmal1 activity by Per1-3 and Cry1/2. However, we have previously shown that Per1 activates genes important for renal sodium reabsorption, which contradicts the canonical role of Per1 as a repressor. Moreover, Per1 KO mice exhibit a lowered blood pressure and heavier body weight phenotype similar to Clock KO mice, and opposite that of Cry1/2 KO mice. Recent work has highlighted the potential role of Per1 in repression of Cry2. Therefore, we postulated that Per1 potentially activates target genes through a Cry2-Clock/Bmal1dependent mechanism, in which Per1 antagonizes Cry2, preventing its repression of Clock/Bmal1. This hypothesis was tested in vitro and in vivo. The Per1 target genes αENaC and Fxyd5 were identified as Clock targets in mpkCCDc14 cells, a model of the renal cortical collecting duct. We identified PPARα and DEC1 as novel Per1 targets in the mouse hepatocyte cell line, AML12 and in the liver in vivo. Per1 knockdown resulted in upregulation of Cry2 in vitro and this result was confirmed in vivo in mice with reduced expression of Per1. Importantly, siRNA-mediated knockdown of Cry2 and Per1 demonstrated opposing actions for Cry2 and Per1 on Per1 target genes, supporting the potential Cry2-Clock/Bmal1 dependent mechanism underlying Per1 action in the liver and kidney.
Resveratrol (RSV) is a polyphenolic compound suggested to have anti-diabetic properties. Surprisingly, little is known regarding the effects of RSV supplementation on adipose tissue metabolism in vivo. The purpose of this study was to assess the effects of RSV on mitochondrial content and respiration, glyceroneogenesis (GNG), and adiponectin secretion in adipose tissue from Zucker Diabetic Fatty (ZDF) rats. Five-week old ZDF rats were fed a chow diet with (ZDF RSV) or without (ZDF chow) RSV (200 mg/kg body weight) for 6 weeks. Changes in adipose tissue metabolism were assessed in subcutaneous (scAT) and intra-abdominal (retroperitoneal [rpWAT], epididymal [eWAT]) adipose tissue depots. ZDF RSV rats showed lower fasting glucose and higher circulating adiponectin, as well as lower glucose area under the curve during i.p. glucose and insulin tolerance tests than ZDF chow. 14C-pyruvate incorporation into triglycerides and adiponectin secretion were higher in scAT from ZDF RSV rats, concurrent with increases in adipose tissue triglyceride lipase (ATGL), hormone sensitive lipase (HSL), and the phosphorylation of pyruvate dehydrogenase-E1α (PDH) (ser293) protein content in this depot. Moreover, uncoupled mitochondrial respiration and complex I and II-supported respiration were increased in both scAT and rpWAT, which correlated with increases in cytochrome C oxidase subunit IV (COX4) protein content. In vitro treatment of scAT with RSV (50 μmol/L; 24h) induced pyruvate dehydrogenase kinase 4 (PDK4) and PPAR gamma co-activator-1α (PGC-1α) mRNA expression. Collectively, these data demonstrate that RSV can induce adipose tissue mitochondrial biogenesis in parallel with increases in GNG and adiponectin secretion.
H2S derived from thiol metabolism has been proposed serve as an oxygen sensor in many systems due to its susceptibility for oxidation and ability to mimic hypoxic responses in oxygen sensing tissues. Thiosulfate, an intermediate in oxidative H2S metabolism can be reduced and regenerate H2S. We propose this contributes to the H2S-mediated oxygen sensing mechanism. H2S formation from thiosulfate in buffers and variety of mammalian tissues and lamprey aorta was examined in real-time using a polarographic H2S sensor. Inferences of intracellular H2S production were made by examining hypoxic pulmonary vasoconstriction (HPV) in bovine pulmonary arteries under conditions expected to increase H2S production and in mouse and rat aortas where reducing conditions mediate vasorelaxation. In mammalian and lamprey buffers, H2S was generated from thiosulfate in the presence of the exogenous reductant, dithiothreitol (DTT), or the endogenous reductant dihydrolipoic acid (DHLA). Both magnitude and rate of H2S production was greatly increased by these reductants in the presence of tissue with the most notable effects occurring in the liver. H2S production was only observed when tissues were hypoxic; exposure to room air, or injecting oxygen inhibited H2S production and resulted in net H2S consumption. DTT and DHLA augmented HPV and DHLA dose-dependently relaxed precontracted mouse and rat aortas. These results indicate that thiosulfate can contribute to H2S signaling under hypoxic conditions and that this is a ready source of H2S production and it serves as a means of recycling sulfur thereby conserving biologically relevant thiols.
Exposure to hypobaric hypoxia is sufficient to decrease cardiac PCr/ATP, and alters skeletal muscle energetic in humans. Cellular mechanisms underlying the different metabolic responses of these tissues, and the time-dependent nature of these changes, are currently unknown but altered substrate utilization and mitochondrial function may be a contributory factor. We therefore sought to investigate the effects of acute (1 day) and more sustained (7 days) hypoxia (13% O2) on the transcription factor, peroxisome proliferator-activated receptor alpha (PPARα) and its targets in cardiac and skeletal muscle. In heart, PPARα expression was 40% higher than in normoxia after 1 and 7 days of hypoxia. Activities of carnitine palmitoyltransferase (CPT) I and β-hydroxyacyl-CoA dehydrogenase (HOAD) were 75% and 35% lower, respectively, after 1 day of hypoxia, returning to normoxic levels after 7 days. Oxidative phosphorylation respiration rates using palmitoyl-carnitine followed a similar pattern, whilst respiration using pyruvate decreased. In skeletal muscle, PPARα expression and CPT I activity were 20% and 65% lower, respectively, after 1 day of hypoxia, remaining at this level after 7 days with no change in HOAD activity. Oxidative phosphorylation respiration rates using palmitoyl-carnitine were lower in skeletal muscle throughout hypoxia, whilst respiration using pyruvate remained unchanged. The rate of CO2 production from palmitate oxidation was significantly lower in both tissues throughout hypoxia. Thus, cardiac muscle may remain reliant on fatty acids during sustained hypoxia, whilst skeletal muscle decreases fatty acid oxidation and maintains pyruvate oxidation.
With current techniques, experimental measurements alone cannot characterize the effects of oxygen blood-tissue diffusion on muscle oxygen uptake (VO2) kinetics in contracting skeletal muscle. To complement experimental studies, a computational model is used to quantitatively distinguish the contributions of convective oxygen delivery, diffusion into cells, and oxygen utilization to VO2 kinetics. The model is validated using previously published experimental VO2 kinetics in response to slowed blood flow (Q) on-kinetics in canine muscle (Q=20s, 46s, and 64s) (Goodwin ML, Hernández A, Lai N, Cabrera ME, Gladden LB. J Appl Physiol. 2012 112(1):9-19). Distinctive effects of permeability-surface area or diffusive conductance (PS) and Q on VO2 kinetics are investigated. Model simulations quantify the relationship between PS and Q, as well as the effects of diffusion associated with PS and Q dynamics on the mean response time of VO2. The model indicates that PS and Q are linearly related and that PS increases more with Q when convective delivery is limited by slower Q dynamics. Simulations predict that neither oxygen convective nor diffusive delivery are limiting VO2 kinetics in the isolated canine gastrocnemius preparation under normal spontaneous conditions during transitions from rest to moderate (submaximal) energy demand, although both operate close to the tipping point.
High strength static magnetic fields ( >7T) perturb the vestibular system causing dizziness, nystagmus, and nausea in humans, and head motion, locomotor circling, conditioned taste aversion, and c-Fos induction in brainstem vestibular nuclei in rodents. To determine the role of head orientation, mice were exposed for 15 min within a 14.1 T magnet at 6 different angles (mice oriented parallel to the field with the head towards B+ at 0°, or pitched rostrally down at 45°, 90°, 90° "sideways", 135°, and 180°), followed by a 2-min swimming test. Additional mice were exposed at 0°, 90°, and 180° and processed for c-Fos immunohistochemistry. Magnetic field exposure induced circular swimming that was maximal at 0° and 180° but attenuated at 45° and 135°. Mice exposed at 0° and 45° swam counterclockwise, while mice exposed at 135° and 180° swam clockwise. Mice exposed at 90° (with their rostral-caudal axis perpendicular to the magnetic field) did not swim differently than controls. In parallel, exposure at 0° and 180° induced c-Fos in vestibular nuclei with left-right asymmetries that were reversed at 0° vs. 180°. No significant c-Fos was induced after 90° exposure. Thus, the optimal orientation for magnetic field effects is the rostral-caudal axis parallel to the field, such that the horizontal canal and utricle are also parallel to the field. These results have mechanistic implications for modeling magnetic field interactions with the vestibular apparatus of the inner ear, e.g. the model of Roberts et al. of an induced Lorenz force causing horizontal canal cupula deflection.