The inducible heat shock protein 70 (Hsp70) is both cytoprotective and immunomodulatory, potentially accounting for its critical role in maintaining gastrointestinal homeostasis. When levels are reduced in conditions like inflammatory bowel diseases (IBD), loss of function contributes to the severity and chronicity of these diseases, although through which cell types and mechanisms remains unclear. Here, the role of Hsp70-mediated intestinal epithelial protection and immune regulation in experimental colitis was examined by using a villin-promoter driven Hsp70 transgene in the TNBS and DSS models and in IL10/Hsp70 double knockout (IL10-/-/Hsp70-/-) mice. In addition, Hsp70 mediated IL-10 production and immune protection were investigated by using CD45RBhigh transfer model, and measuring colonic and immune cell cytokine expression during colitis. We found that the epithelial-specific expression of Hsp70 transgene attenuated DSS-induced colitis in Hsp70-/- mice by protecting tight junctions (TJ) and their interaction with the TJ-associated protein, ZO-1. In the TNBS colitis and CD45RBhigh model, Hsp70 carried out its intracellular anti-inflammatory function by maintaining IL-10 production. Impaired ERK phosphorylation, but not p38 or JNK phosphorylation pathways, was associated with decreased IL-10 production in Hsp70 deficient cells. Together, these actions can be leveraged in the context of cellular specificity to develop complementary strategies that can lead to reduction in mucosal injury and immune activation in colonic colitis development.
In these studies, we developed a novel approach of in vivo magnetofection for localized delivery of nucleic acids such as micro-RNA-139-p (miR-139-5p, known to target Rho kinase2) to the circular smooth muscle layer of the internal anal sphincter (IAS). The IAS tone is known to play a major role in the rectoanal continence via activation of RhoA-associated kinase (RhoA/ROCK2). These studies established an optimized protocol for efficient gene delivery using an assembly of equal volumes of in vivo PolyMag and miR139-5p or anti-miR-139-5p (100 nM each) injected in the circular smooth muscle layer in the pin-pointed areas of the rat perianal region, and then incubated for 20 minutes under magnetic field. Magnetofection efficiency was confirmed and analyzed by confocal microscopy of FITC-tagged siRNA. Using physiological and biochemical approaches, we investigated the effects of miR-139-5p and anti-miR-139-5p on basal intraluminal IAS pressure (IASP), fecal pellet count, IAS tone, agonist-induced contraction, contraction-relaxation kinetics, and RhoA/ROCK2 signaling. Present studies demonstrate that magnetofection-mediated miR-139-5p delivery significantly decreased RhoA/ROCK2, p-MYPT1, and p-MLC20 signaling, leading to decreases in the lower basal IASP and IAS tone and in rates of contraction and relaxation, associated with increase in fecal pellet output. Interestingly, anti-miR-139-5p transfection had opposite effects on these parameters. Collectively, these data demonstrate that magnetofection is a promising novel method of in vivo gene delivery and of nucleotides to the internal anal sphincter for the site-directed and targeted therapy for rectoanal motility disorders.
Liver stiffness (LS) as measured by transient elastography is widely used to screen for liver fibrosis. However, LS also increases in response to pressure changes like congestion but no data on portal pressure are available. We here study the effect of rapid portal pressure changes on LS. Therefore, LS was assessed directly prior and after ligation of esophageal varices (n=11) as well as TIPS implantation in patients with established cirrhosis (n=14). Additionally, we retrospectively analyzed changes in LS and variceal size in patients with sequential gastroscopic monitoring and LS measurements (n=14). To study LS and portal pressure in healthy livers, LS (µFibroscan, Echosens, Paris) and invasive pressures (Powerlab, AD Instruments, New Zealand) were assessed in male Wistar rats after ligation of single liver lobes. Ligation of esophageal varices caused an immediate and significant increase of LS from 40.3±19.0 to 56.1±21.5 kPa. Likewise, LS decreased significantly from 53.1±16.6 to 43.8±17.3 kPa after TIPS placement which correlated significantly with portal pressure (r=0.558). In the retrospective cohort, the significant LS decrease from 54.9±23.5 to 47.9±23.8 kPa over a mean observation interval of 4.3±3 months was significantly correlated with a concomitant increase of variceal size (r=-0.605). In the animal model, LS and portal pressure increased significantly after single lobe ligation without changes of arterial or central venous pressure. In conclusion, rapid changes of portal pressure are a strong modulator of LS in healthy and cirrhotic organs. In patients with stable cirrhosis according to MELD, a decrease of LS may be indicative for enlarging varices.
Fatty liver disease (FLD), the most common chronic liver disease in the US, may be caused by alcohol or the metabolic syndrome. Alcohol is oxidized in the cytosol of hepatocytes by alcohol dehydrogenase (ADH), which generates NADH and increases cytosolic NADH/NAD+ ratio. The increased ratio may be important for development of FLD, but our ability to examine this question is hindered by methodological limitations. To address this, we used the genetically-encoded fluorescent sensor Peredox to obtain dynamic, real-time measurements of cytosolic NADH/NAD+ ratio in living hepatocytes. Peredox was expressed in dissociated rat hepatocytes and HepG2 cells by transfection, and in mouse liver slices by tail vein injection of adeno-associated virus (AAV)-encoded sensor. Under control conditions, hepatocytes and liver slices exhibit a relatively low (oxidized) cytosolic NADH/NAD+ ratio as reported by Peredox. The ratio responds rapidly and reversibly to substrates of lactate dehydrogenase (LDH) and sorbitol dehydrogenase (SDH). Ethanol causes a robust dose-dependent increase in cytosolic NADH/NAD+ ratio, and this increase is mitigated by the presence of NAD+-generating substrates of LDH or SDH. In contrast to hepatocytes and slices, HepG2 cells exhibit a relatively high (reduced) ratio, and show minimal responses to substrates of ADH and SDH. In slices, we show that comparable results are obtained with epifluorescence imaging and 2-photon fluorescence lifetime imaging (2p-FLIM). Live cell imaging with Peredox is a promising new approach to investigate cytosolic NADH/NAD+ ratio in hepatocytes. Imaging in liver slices is particularly attractive because it allows preservation of liver microanatomy and metabolic zonation of hepatocytes.
The expression of apelin and its receptors (APJ) in central autonomic networks suggests that apelin may regulate gastrointestinal motor functions. In rodents, central administration of apelin-13 has been shown to inhibit gastric emptying, however, the mechanisms involved remain to be determined. Using male adult Sprague-Dawley rats, the aims of the present study were i) to determine the expression of APJ receptor in DVC, ii) to assess the effects central application of apelin-13 into the dorsal vagal complex (DVC) on gastric tone and motility and iii) to investigate the neuronal pathways responsible for apelin-induced alterations. APJ receptor immunoreactivity was detected in gastric-projecting and choline acetyltransferase positive neurons of the DVC. Microinjection of apelin-13 into DVC decreased significantly gastric tone and motility in both corpus and antrum. The apelin-induced reduction in gastric tone and motility was prevented by surgical vagotomy or fourth ventricular application of the APJ receptor antagonist, F13A. Systemic administration of the muscarinic receptor antagonist atropine, but not the nitric oxide synthase inhibitor L-NAME, abolished the apelin-induced inhibitory responses. The present results indicate a central modulatory role of apelin in the vagal neuro-circuitry that controls gastric motor functions via withdrawal of the tonically active cholinergic pathway.
We examined the role of macrophages in inflammation associated colorectal cancer (CRC). Given the emerging evidence on immune-microbiota interactions in CRC, we also sought to examine the interaction between macrophages and gut microbiota. To induce CRC, male C57BL/6 mice (n=32) received a single injection of azoxymethane (AOM) followed by three cycles of dextran sodium sulfate (DSS) supplemented water at weeks 1, 4, and 7, respectively. Prior to the final DSS cycle (week 7) and twice weekly until sacrifice, mice (n=16/group) received either 200μl i.p. of clodronate filled liposomes (CLD) or phosphate buffered saline (PBS) encapsulated liposomes to deplete macrophages. Colon tissue was analyzed for polyp burden, macrophage markers, transcription factors, and inflammatory mediators. Stool samples were collected and DNA was isolated and subsequently sequenced for 16S rRNA. Clodronate liposomes decreased tumor number by ~36% and specifically large (≥1mm) tumors by ~36% (p<0.05). This was consistent with a decrease in gene expression of EMR1 in the colon tissue and polyp tissue as well as expression of select markers associated with M1 (IL-6) and M2 macrophages (IL-13, IL-10, TGFβ & CCL17) in the colon tissue (p<0.05). Similarly, there was a decrease in STAT3 and MAPK p38 and ERK signaling in colon tissue. Clodronate liposomes increased the relative abundance of the Firmicutes phylum (p<0.05) and specifically Lactobacillaceae and Clostridiaceae families, which have been associated with reduced CRC risk. Overall, these data support the development of therapeutic strategies to target macrophages in CRC and provide support for further evaluation of immune-microbiota interactions in CRC.
Previously we generated mouse models of Rack1 deficiency to identify key functions for Rack1 in regulating growth of intestinal epithelia: suppressing crypt cell proliferation and regeneration, promoting differentiation and apoptosis, and repressing development of neoplasia. However, other than low body weight, we did not detect an overt phenotype in mice constitutively deleted of Rack1 in intestinal epithelia (vil-Cre:Rack1fl/fl mice), presumably because Rack1 was deleted in < 10% of the total surface area of the epithelia. To assess the effect of Rack1 loss throughout the entire intestinal epithelia, we generated another mouse model of Rack1 deficiency, vil-Cre-ERT2:Rack1fl/fl. Within 5-10 days of the initial tamoxifen treatment, the mice lost over 20% of their body weight, developed severe diarrhea that for some was bloody, became critically ill and died, if not euthanized. Necropsies revealed mildly distended, fluid-, gas- and sometimes blood-filled loops of small and large bowel, inguinal lymphadenopathy and thrombocytosis. Rack1 was deleted in nearly 100% of the epithelia in both the small intestine and colon when assessed by immunofluorescent or immunoblot analyses. Rack1 expression in other tissues and organs was not different than in control mice, indicating tissue specificity of the recombination. Histopathology revealed a patchy, erosive, hemorrhagic, inflammatory enterocolitis with denuded, sloughed off surface epithelium and crypt hyperplasia. These results suggest a protective function for Rack1 in maintaining the integrity of intestinal epithelia and for survival.
Dietary copper-fructose interactions contribute to the development of nonalcoholic fatty liver disease (NAFLD). Gut microbiota play critical roles in the pathogenesis of NAFLD. The aim of this study was to determine the effect of different dietary doses of copper and their interactions with high fructose on gut microbiome. Male weanling Sprague-Dawley rats were fed diets with adequate copper (6 ppm, CuA), marginal copper (1.5 ppm, CuM) (low-copper) or supplemented copper (20 ppm, CuS) (high-copper) for 4 weeks. Deionized water or deionized water containing 30% fructose (w/v) was given ad lib. Copper status, liver enzymes, gut barrier function and gut microbiome were evaluated. Both low- and high-copper diets led to liver injury in high fructose fed rats, and this was associated with gut barrier dysfunction, as shown by the markedly decreased tight junction proteins and increased gut permeability. 16S rDNA sequencing analysis revealed distinct alterations of the gut microbiome associated with dietary low- and high-copper/high-fructose feeding. The common features of the alterations of the gut microbiome were the increased abundance of Firmicutes and the depletion of Akkermansia. However, they differed mainly within the phylum, Firmicutes. Our data demonstrated that a complex interplay between host, microbes and dietary copper-fructose interaction regulates gut microbial metabolic activity, which may contribute to the development of liver injury and hepatic steatosis. The distinct alterations of gut microbial activity, which were associated with the different dietary doses of copper and fructose, imply that separate mechanism(s) may be involved.
S-allyl-glutathione (SAG) is one of the metabolites of diallyl sulfide (DAS), a component of garlic. DAS has shown preventative effects on carcinogenesis in animal models. However, whether synthetic SAG can improve liver fibrosis has not been investigated. We examined the potential preventive effects of SAG on acute and chronic models of liver fibrosis by chronic carbon tetrachloride (CCl4) administration. SAG inhibited liver fibrogenesis induced by CCl4 in a dose-dependent manner and reduced heat shock protein 47 (HSP47), a collagen-specific chaperone and other fibrosis markers. In fibrosis regression models, either after CCl4 administration for 9 weeks or dimethyl nitrosamine (DMN) for 6 weeks, SAG markedly accelerated fibrolysis in both models. In the regression stage of DMN-treated liver, SAG normalized the ratio of M2-phenotype (expression of mannose receptor) in Kupffer cells (KCs). Consistent with these results, the culture supernatants of SAG-treated M2-phenotype KCs inhibited COL1A1 mRNA expression in primary culture-activated rat hepatic stellate cells (HSCs). However, SAG did not directly inhibit HSCs activation. In acute model of CCl4 single injection, SAG inhibited hepatic injury dose-dependently in consistent with the inhibiting inflammatory KCs activation. These findings suggested that SAG could improve fibrogenic and fibrolysis cascade via the regulation of excess activated and polarized KCs. SAG may also serve as a preventive and therapeutic agent in fibrosis of other organs for which current clinical therapy is unavailable.
The role of Calcitonin Gene Related Peptide (CGRP) in visceral and somatic nociception is incompletely understood. CGRPα is highly expressed in sensory neurons of dorsal root ganglia (DRG) and particularly in neurons that also express the transient receptor potential cation channel subfamily V member 1 (Trpv1). Therefore, we investigated changes in visceral and somatic nociception following deletion of CGRPα from the Trpv1-Cre population using the Cre/lox system. In control mice, acetic acid injection (0.6%,i.p.) caused significant immobility (time stationary) - an established indicator of visceral pain. In CGRPα-mCherrylx/lx;Trpv1-Cre mice the duration of immobility was significantly less than controls and the distance CGRPα-mCherrylx/lx;Trpv1-Cre mice travelled over 20 minutes following acetic acid was significantly greater than controls. However, following acetic acid injection, there was no difference between genotypes in the writhing reflex, number of abdominal licks, or forepaw wipes of the cheek. CGRPα-mCherrylx/lx;Trpv1-Cre mice developed more pronounced inflammation-induced heat hypersensitivity above baseline values, compared to controls. However, analyses of noxious acute heat or cold transmission revealed no difference between genotypes. Also, odour avoidance test, odour preference test and buried food test for olfaction revealed no differences between genotypes. Our findings suggest that CGRPα-mediated transmission within the Trpv1-Cre population plays a significant role in visceral nociceptive pathways underlying voluntary movement. Monitoring changes in movement over time is a sensitive parameter to identify differences in visceral nociception, compared to writhing reflexes, abdominal licks, or forepaw wipes of the cheek that were unaffected by deletion of CGRPα- from Trpv1-Cre population and likely utilize different mechanisms.
Patients with type 2 diabetes (T2D) and patients with non-alcoholic fatty liver disease (NAFLD) frequently exhibit elevated plasma concentrations of glucagon (hyperglucagonemia). Hyperglucagonemia and alpha-cell hyperplasia may result from elevated levels of plasma amino acids when glucagon's action on hepatic amino acid metabolism is disrupted. We therefore measured plasma levels of glucagon and individual amino acids in patients with and without biopsy-verified NAFLD and with and without type T2D. Fasting levels of amino acids and glucagon in plasma were measured, using validated ELISAs and high-performance liquid chromatography, in obese, middle-aged individuals with I) normal glucose tolerance (NGT) and NAFLD, II) T2D and NAFLD, III) T2D without liver disease, and IV) NGT and no liver disease. Elevated levels of total amino acids were observed in participants with NAFLD and NGT compared to NGT controls (1,310±235 µM vs. 937±281 µM, p=0.03) and in T2D and NAFLD compared to T2D without liver disease (1,354±329 µM vs. 511±235 µM, p<0.0001). Particularly amino acids with known glucagonotropic effects (e.g. glutamine) were increased. Plasma levels of total amino acids correlated to plasma levels of glucagon also when adjusting for body mass index (BMI), glycated haemoglobin (HbA1c), and cholesterol levels (β=0.013±0.007, p=0.024). Elevated plasma levels of total amino acids associate with hyperglucagonemia in NAFLD patients independently of glycemic control, BMI or cholesterol - supporting the potential importance of a 'liver-alpha-cell axis' in which glucagon regulates hepatic amino acid metabolism. Fasting hyperglucagonemia as seen in T2D may therefore represent impaired hepatic glucagon action with increasing amino acids levels.
The Keap1-Nrf2 system has a wide variety of effects in addition to the oxidative stress response, such as growth promotion and chemoresistance of cancer cells. Nrf2 is constitutively activated in most cancer cells. However, the activation of Nrf2 together with oncogenic mutations does not always result in cancer promotion. K-rasLSL-G12D/+::p53LSL-R172H/+::Pdx-1-Cre (KPC) mice are an established model of pancreatic cancer, which specifically express mutants of both K-ras and p53 in the pancreas by using Pdx-1-Cre. We here generated KC::Keap1fl/fl (KC::Keap1) and KPC::Keap1fl/fl (KPC::Keap1) mice in which Nrf2 is constitutively activated by Keap1 deletion. KC::Keap1 and KPC::Keap1 mice started to die or showed obvious weakness at approximately around 40 days after birth. Histological examination revealed that KC::Keap1 and KPC::Keap1 mice did not develop pancreatic cancer, but, instead, progressive atrophy of the pancreatic parenchyma. In these mice, amylase-positive acinar cells as well as insulin- and glucagon-positive islet cells were decreased and surrounded by fibrotic tissues. KC::Keap1 and KPC::Keap1 mice presented lower body weight and glucose levels than C::Keap1 mice, presumably resulting from pancreatic exocrine insufficiency. Histological changes were not obvious in C::Keap1 and PC::Keap1 mice. The presence of the p53 mutation did not affect the phenotypes in KC::Keap1 mice. Heterologous or homologous Nrf2 deletion (Nrf2+/- or Nrf2-/-) rescued the pancreatic phenotypes, weight loss and hypoglycemia in KC::Keap1 mice, suggesting that Nrf2 is a major downstream target of Keap1. In conclusion, simultaneous K-ras activation and Keap1 deletion caused progressive atrophy of the pancreatic parenchyma in mice.
Eosinophilic pancreatitis (EP) is reported in human; however, the etiology and the role of eosinophils in EP pathogenesis is a poorly understood and not excusably explored. Therefore, it is interesting to examine the role of eosinophils in the initiation and progression of pancreatitis pathogenesis. Therefore, we performed real time PCR, ELISA, western blot, anti-MBP immunostaining, Chloroesterase and Massons' trichrome analysis were performed to examine transcript and protein levels of eosinophil active cytokines, chemokines, eosinophils, mast cells and collagen tissue accumulation. Pancreas is devoid of eosinophils in healthy individuals; however, eosinophils accumulation and degranulation in the tissue sections was observed in human pancreatitis. Herein, we first time located eosinophils accumulation and degranulation followed by induced mast cells and acinar cells atrophy in the pancreas of cerulein-induced murine model of pancreatitis. Additionally, induced transcript and protein levels of pro-inflammatory and pro-fibrotic cytokines, chemokines like IL-5, IL-18, eotaxin1 and eotaxin-2, TGF-β1, collagen-1, collagen-3, fibronectin and α-SMA were also observed in experimental pancreatitis in mice. Mechanistically, we report that eosinophil deficient GATA1 and endogenous IL-5 deficient mice were protected from the induction of proinflammatory and profibrotic cytokines, chemokines, tissue eosinophilia and mast cells, in cerulein-induced murine model of pancreatitis. These experimental data indicates that eosinophils accumulation and degranulation may be critical in promoting pancreatitis pathogenesis including fibrosis. Taken together, we report that eosinophil tissue accumulation has an important role in promoting pancreatitis pathogenesis including fibrosis that needs appropriate attention to understand and restrict disease progression and pathogenesis in human.
The gastrointestinal tract contains its own independent population of sensory neurons within the gut wall. These sensory neurons have been referred to as intrinsic primary afferent neurons (IPANs) and can be identified by immunoreactivity to calcitonin gene related peptide (CGRP) in mice. A common feature of IPANs is a paucity of fast synaptic inputs observed during sharp microelectrode recordings. Whether this is observed using different recording techniques is of particular interest for understanding the physiology of these neurons and neural circuit modelling. Here, we imaged spontaneous and evoked activation of myenteric neurons in isolated whole preparations of mouse colon and correlated recordings with CGRP and nitric oxide synthase (NOS) immunoreactivity, post hoc. Calcium indicator Fluo-4 was used for this purpose. Calcium responses were recorded in nerve cell bodies located 5-10mm oral to transmural electrical nerve stimuli. A total of 618 recorded neurons were classified for CGRP or NOS immunoreactivity. Aboral electrical stimulation evoked short-latency calcium transients in the majority of myenteric neurons, including ~90% of CGRP-immunoreactive Dogiel type II neurons. Activation of Dogiel type II neurons had a time course consistent with fast synaptic transmission and was always abolished by hexamethonium (300μM) and by low calcium Krebs solution. The nicotinic receptor agonist DMPP (during synaptic blockade) directly activated Dogiel type II neurons. The present study suggests that murine colonic Dogiel type II neurons receive prominent fast excitatory synaptic inputs from hexamethonium-sensitive neural pathways.
Previously we showed that Rack1 (Receptor for Activated C Kinase) regulates growth of colon cells in vitro, partly by suppressing Src kinase activity at key cell cycle checkpoints, in apoptotic and cell survival pathways and at cell-cell adhesions. Here, we generated mouse models of Rack1 deficiency to assess Rack1's function in intestinal epithelia in vivo. Intestinal Rack1 deficiency resulted in proliferation of crypt cells, diminished differentiation of crypt cells into enterocyte, goblet and enteroendocrine cell lineages and expansion of Paneth cell populations. Following radiation injury, the morphology of Rack1-deleted small bowel was strikingly abnormal with development of large polypoid structures that contained many partly formed villi, numerous back-to-back elongated and regenerating crypts, and high-grade dysplasia in surface epithelia. These abnormalities were not observed in Rack1-expressing areas of intestine or in control mice. Following irradiation, apoptosis of enterocytes was strikingly reduced in Rack1-deleted epithelia. These novel findings reveal key functions for Rack1 in regulating growth of intestinal epithelia: suppressing crypt cell proliferation and regeneration, promoting differentiation and apoptosis, and repressing development of neoplasia.
Riboflavin (RF) is essential for normal cellular functions and health. Humans obtain RF from exogenous sources via intestinal absorption that involves a highly specific carrier-mediated process. We have recently established that the riboflavin transporter-3 (RFVT3) is vital for the normal intestinal RF uptake process, and have characterized certain aspects of its transcriptional regulation. Little is known, however, about how this transporter is regulated at the post-transcriptional level. We address this issue focusing on the role of microRNAs. Using bioinformatics, we identified two potential interacting miRNAs with the human (h) RFVT3- 3'-UTR, and showed (using pmirGLO- hRFVT3-3'-UTR) that the hRFVT3-3'-UTR is indeed a target for miRNA effect. Of the two putative miRNAs identified, miR-423-5p was found to be highly expressed in intestinal epithelial cells, and that its mimic affected luciferase reporter activity of the pmirGLO-hRFVT3-3'-UTR construct, and also lead to inhibition in RF uptake by intestinal epithelial Caco-2 and HuTu-80 cells. Further cells transfected with mutated seed sequences for miR-423-5p showed an abrogation in inhibitory effect of the miR-423-5p mimic on luciferase activity. While miR-423-5p did not affect the level of expression of the hRFVT3 mRNA, it did lead to a significant inhibition in level of expression of its protein. Similarly, miR-423-5p was found to affect the level of expression of the mouse RFVT3 in cultured intestinal enteroids. These findings demonstrate, for the first time, that the RFVT3 is a target for post-transcriptional regulation by miRNAs in intestinal epithelial cells, and that this regulation has functional consequences on intestinal RF uptake.
The intestinal epithelial brush border Na+/H+ exchanger NHE3 accounts for a large component of intestinal Na absorption. NHE3 is regulated during digestion by signaling complexes on its C-terminus that include the four multi-PDZ domain containing NHERF family proteins. Because the roles of each NHERF appear to vary based on the cell model or intestinal segment studied and because of our recent finding that a NHERF3-NHERF2 heterodimer appears important for NHE3 regulation in Caco-2 cells, we examined the role of NHERF3 and NHERF2 in homozygous C57BL/6 NHERF2 and NHERF3 KO mouse jejunum. The jejunal apical membrane of NHERF3 null mice appeared similar to wild type (WT) in microvillus number and height, which is similar to results previously reported for NHERF2 null mice. NHE3 basal activity was not different from WT in either NHERF2 or NHERF3 null jejunum, while D-glucose stimulated NHE3 activity was reduced in NHERF2 but similar to WT in NHERF3 KO. LPA stimulation and UTP (elevated Ca2+) and cGMP inhibition of NHE3 were markedly reduced in both NHERF2 and NHERF3 null jejunum. Forskolin inhibited NHE3 in NHERF3 null jejunum, but the extent of inhibition was reduced compared to WT. The forskolin inhibition of NHE3 in NHERF2 null mice was inconsistent. These results demonstrate similar requirement for NHERF2 and NHERF3 in mouse jejunal NHE3 regulation by LPA, Ca2+, and cGMP. The explanation for the similarity is not known but is consistent with involvement of a brush border NHERF3-NHERF2 heterodimer or sequential NHERF dependent effects in these aspects of NHE3 regulation.
Enteric glia play an important neuroprotective role in the enteric nervous system (ENS) by producing neuroprotective compounds such as the antioxidant reduced glutathione (GSH). The specific cellular pathways that regulate glial production of GSH, and how these pathways are altered during, or contribute to, neuroinflammation in situ and in vivo are not fully understood. We investigated this issue using immunohistochemistry to localize GSH synthesis enzymes within the myenteric plexus and tested how the inhibition of GSH synthesis with the selective inhibitor L-Buthionine Sulfoximine (BSO) impacts neuronal survival and inflammation. Both enteric glia and neurons express the cellular machinery necessary for GSH synthesis. Further, glial GSH synthesis is necessary for neuronal survival in isolated preparations of myenteric plexus. In vivo depletion of GSH does not induce colitis but alters myenteric plexus neuronal phenotype and survival. Importantly, global depletion of glutathione is protective against some macro and microscopic measures of colonic inflammation. Together, our data highlights the heterogeneous roles of GSH in the myenteric plexus of the ENS and during GI inflammation.
It is generally accepted that the gastric mucus layer provides a protective barrier between the lumen and the mucosa, shielding the mucosa from acid and digestive enzymes and preventing auto-digestion of the stomach epithelium. However, the precise mechanisms that contribute to this protective function are still up for debate. In particular, it is not clear what physical processes are responsible for transporting hydrogen protons, secreted within the gastric pits, across the mucus layer to the lumen without acidifying the environment adjacent to the epithelium. One hypothesis is that hydrogen may be bound to the mucin polymers themselves as they are convected away from the mucosal surface and eventually degraded in the stomach lumen. It is also not clear what mechanisms prevent hydrogen from diffusing back toward the mucosal surface, thereby lowering the local pH. In this work we investigate a physics-based model of ion transport within the mucosal layer based on a Nernst-Planck-like equation. Analysis of this model shows that the mechanism of transporting protons bound to the mucus gel is capable of reproducing the trans-mucus pH gradients reported in the literature. Furthermore, when coupled with ion exchange at the epithelial surface, our analysis shows that bicarbonate secretion alone is capable of neutralizing the epithelial pH, even in the face of enormous diffusive gradients of hydrogen. Maintenance of the pH gradient is found to be robust to a wide array of perturbations in both physiological and phenomenological model parameters, suggesting a robust physiological control mechanism.
The Na+/H+ exchanger isoform-3 plays a key role in coupled electroneutral NaCl absorption in the mammalian intestine. Reduced NHE3 expression or function has been implicated in the pathogenesis of diarrhea associated with IBD or enteric infections. Our previous studies revealed transcriptional regulation of NHE3 by various agents such as TNF-, IFN- and butyrate involving the transcription factors SP1 and SP3. In silico analysis revealed that the NHE3 core promoter also contains a hepatocyte nuclear factor 4α (HNF4α) binding site that is evolutionarily conserved in several species suggesting that HNF4α has a role in NHE3 regulation. Nhe3 mRNA levels were reduced in intestine-specific Hnf4α-null mice. However, detailed mechanisms of NHE3 regulation by HNF4α are not known. We investigated the regulation of NHE3 gene expression by HNF4α in vitro in the human intestinal epithelial cell line C2BBe1, and in vivo in the intestine-specific Hnf4α-null (Hnf4αIEpC) and control (Hnf4αfl/fl) mice. HNF4α knockdown by siRNA in C2BBe1 cells significantly decreased NHE3 mRNA and NHE3 protein levels. Gel Mobility Shift and ChIP assays revealed that HNF4α directly interacts with the HNF4α motif in the NHE3 core promoter. Site-specific mutagenesis on the HNF4α motif decreased, whereas ectopic over expression of HNF4α increased NHE3 promoter activity. Further, loss of HNF4α in Hnf4αIEpC mice decreased colonic Nhe3 mRNA and NHE3 protein levels. Our results demonstrate a novel role for HNF4α in basal regulation of NHE3 expression. These studies represent an important and novel target for therapeutic intervention in IBD-associated diarrhea.
Background: Ultrasound Tissue Doppler imaging (US-TDI) has been used to diagnose regional wall motion (WM) abnormalities in coronary artery disease, but has not been applied to oropharyngeal diseases. This study aimed first to validate an US-TDI method to assess cervical esophageal (CE) WM, and secondly to use the method to evaluate CE WM in patients with oropharyngeal dysphagia (OD). Methods: First, we enrolled 22 healthy men (mean age: 59.7 years) who all underwent both US-TDI and videofluoroscopy (VF) and then esophageal high-resolution manometry (HRM) in the same week. We evaluated the reproducibility of the US-TDI, and the relationship between US-TDI and other modalities (VF and HRM). Second, we enrolled 56 mild OD patients (mean age: 58.0 years) and age- and sex-matched healthy controls. Difference in CE WM between these groups was evaluated by US-TDI. Results: All healthy subjects underwent US-TDI, VF, and HRM successfully, with a sufficiently high reproducibility coefficient for this method, and significant correlation between US-TDI and VF/HRM parameters. US-TDI showed mean time to open CE wall and mean velocity of CE wall opening significantly differed between patients and healthy controls (P<0.01). Conclusion: We have developed a US-TDI method for easily assessing CE WM in daily practice, and also found significant differences in CE WM between mild OD patients and healthy controls.
The liver metabolizes alcohol using alcohol dehydrogenase (ADH) and cytochrome P450 2E1 (CYP2E1). Both enzymes metabolize ethanol into acetaldehyde, but CYP2E1 activity also results in the production of reactive oxygen species (ROS) that promote oxidative stress. We have previously shown that microtubules are hyperacetylated in ethanol-treated polarized, hepatic WIF-B cells and livers from ethanol fed rats. We have also shown that enhanced protein acetylation correlates with impaired clathrin-mediated endocytosis, constitutive secretion and nuclear translocation and that the defects are likely mediated by acetaldehyde. However, the roles of CYP2E1-generated metabolites and ROS in microtubule acetylation and these alcohol-induced impairments have not been examined. To determine if CYP2E1-mediated alcohol metabolism is required for enhanced acetylation and the trafficking defects, we co-incubated cells with ethanol and diallyl sulfide (a CYP2E1 inhibitor) or N-acetyl cysteine (an anti-oxidant). Both agents failed to prevent microtubule hyperacetylation in ethanol-treated cells and also failed to prevent impaired secretion or clathrin-mediated endocytosis. Somewhat surprisingly, both NAS and DAC prevented impaired STAT5B nuclear translocation. Further examination of microtubule-independent steps of the pathway revealed that Jak2/STAT5B activation by growth hormone (GH) was prevented by DAS and NAC. These results were confirmed in ethanol-exposed HepG2 cells expressing only ADH or CYP2E1. Using quantitative RT-PCR, we further determined that ethanol exposure led to blunted GH-mediated gene expression. In conclusion, we determined that alcohol-induced microtubule acetylation and associated defects in microtubule-dependent trafficking are mediated by ADH metabolism whereas impaired microtubule-independent Jak2/STAT5B activation is mediated by CYP2E1 activity.
Krüppel-like factor 5 (KLF5) is a member of the zinc finger family of transcription factors that regulates homeostasis of the intestinal epithelium. Previous studies suggested an indispensable role of KLF5 in maintaining intestinal barrier function. In the current study, we investigated the mechanisms by which KLF5 regulates colonic barrier function in vivo and in vitro. We used an inducible and a constitutive intestine-specific Klf5 knockout mouse model (Villin-CreERT2;Klf5fl/fl designated as Klf5IND and Villin-Cre;Klf5fl/fl as Klf5IS), and studied an inducible KLF5 knockdown in Caco-2 BBe cells using a lentiviral Tet-on system (Caco-2 BBe KLF5IND). Specific knockout of Klf5 in colonic tissues, either inducible or constitutive, resulted in increased intestinal permeability. The phenotype was accompanied by a significant reduction in Dsg2, which encodes desmoglein-2, a desmosomal cadherin, at both mRNA and protein levels. Transmission electron microscopy (TEM) showed alterations of desmosomal morphology in both KLF5 knockdown Caco-2 BBe cells and Klf5 knockout mouse colonic tissues. Inducible knockdown of KLF5 in Caco-2BBe cells grown on Transwell® plates led to impaired barrier function as evidenced by decreased transepithelial electrical resistance (TEER) and increased paracellular permeability to FITC-dextran 4kD. Furthermore, DSG2 was significantly decreased in KLF5 knockdown cells, and DSG2 overexpression partially rescued the impaired barrier function caused by KLF5 knockdown. Electron microscopy studies demonstrated altered desmosomal morphology after KLF5 knockdown. In combination with chromatin immunoprecipitation (ChIP) analysis and promoter study, our data show that KLF5 regulates intestinal barrier function by mediating the transcription of DSG2, a gene encoding a major component of desmosome structures.
During the post weaning period, piglets are prone to gastrointestinal infections. The resulting impairment of intestinal barrier function may cause diarrhea associated with growth retardation or even death of piglets. Orally applied zinc is commonly used to prevent and treat diarrhea but its mode of action still needs to be elucidated. In order to analyze the molecular mechanism whereby zinc acts on porcine intestinal barrier function, ex vivo studies on piglet jejunum and accompanying in vitro studies on a porcine jejunal epithelial cell line, IPEC-J2/PS, were performed with electrophysiological tools. Feeding pharmacological zinc doses exerted no significant electrophysiologically ascertainable short- and long-term effects on jejunal barrier function ex vivo. However, in IPEC-J2/PS, basolateral zinc was cytotoxic since its application caused a release of lactate dehydrogenase and an irreversible break-down of the epithelial barrier. In contrast, apical zinc application caused an immediate increase in paracellular resistance and a decrease in permeability to the paracellular marker fluorescein, reflecting overall barrier strengthening in vitro. Apical effects were fully reversible upon wash-out. This indicates that zinc supplemented to feed was completely washed out during ex vivo jejunum preparation. We conclude that there is no evidence for long-term barrier effects through prophylactic zinc supplementation and that extracellular zinc acts acutely and reversibly from the apical side via tightening the paracellular route, thus counteracting leak-flux diarrhea.
Clinical studies in burn patients demonstrate a close association between leaky guts and increased incidence or severity of sepsis and other complications. Severe thermal injury triggers intestinal inflammation that contributes to intestinal epithelial hyperpermeability, which exacerbates systemic response leading to multiple organ failure and sepsis. In this study, we identified a significant function of a particular palmitoyl acyltransferase (PAT), ZDHHC21, in mediating signaling events required for gut hyperpermeability induced by inflammation. Using qPCR, we show that ZDHHC21 mRNA, production was enhanced by two-fold when intestinal epithelial cells were treated with TNFα/IFN in vitro. In addition, pharmacological targeting of PATs with 2-bromopalmitate (2-BP) showed significant improvement in TNFα/IFN mediated epithelial barrier dysfunction by using electric cell-substrate impedance sensing (ECIS) assays, as well as FITC-dextran permeability assays. Using the ABE assay and click chemistry, we show that TNFα/IFN treatment of intestinal epithelial cells results in enhanced detection of total palmitoylated proteins, and this response is inhibited by 2-BP. Using ZDHHC21 deficient mice or wild-type mice treated with 2-BP, we showed that mice with impaired ZDHHC21 expression or pharmacological inhibition resulted in attenuated intestinal barrier dysfunction caused by thermal injury. Moreover, H&E staining of small intestine, as well as transmission electron microscopy (TEM), showed mice with genetic interruption of ZDHHC21 had attenuated villus structure disorganization associated with thermal injury induced intestinal barrier damage. Taken together, these results suggest an important role of ZDHHC21 in mediating gut hyperpermeability resulting from thermal injury.
Objective: Impaired gut motility may contribute, at least in part, to the development of systemic hyperammonemia and systemic neurological disorders in inherited metabolic disorders, or in severe liver and renal disease. It is not known whether enteric neurotransmission regulates intestinal luminal and hence systemic ammonia levels by induced changes in motility. Design: Here, we propose and test the hypothesis that ammonia acts through specific enteric circuits to influence gut motility. We tested our hypothesis by recording the effects of ammonia on neuromuscular transmission in tissue samples from mice, pigs and humans, and investigated specific mechanisms using novel mutant mice, selective drugs, cellular imaging and enzyme-linked immunosorbent assays. Results: Exogenous ammonia increased neurogenic contractions and decreased neurogenic relaxations in segments of mouse, pig and human intestine. Enteric glial cells responded to ammonia with intracellular Ca2+ responses. Inhibition of glial glutamine synthetase and the deletion of glial connexin-43 channels in hGFAP::CreERT2+/–/connexin43f/f mice potentiated the effects of ammonia on neuromuscular transmission. The effects of ammonia on neuromuscular transmission were blocked by GABAA receptor antagonists and ammonia drove substantive GABA release as did the selective pharmacological activation of enteric glia in GFAP::hM3Dq transgenic mice. Conclusion: We propose a novel mechanism whereby local ammonia is operational through GABAergic glial signaling to influence enteric neuromuscular circuits that regulate intestinal motility. Therapeutic manipulation of these mechanisms may benefit a number of neurological, hepatic and renal disorders manifesting hyperammonemia.
Studies show an age-related increase in the prevalence of anal incontinence and sphincter muscle atrophy. Wnt-β catenin signaling pathway has been recently recognized as the major molecular pathway involved in the age-related skeletal muscle atrophy and fibrosis. The goals of our study were to evaluate: (i) impact of normal aging on the EAS muscle length-tension (L-T) function & morphology; and (ii) specifically examine the role of Wnt signaling pathways in anal sphincter muscle fibrosis. New Zealand White female rabbits (six young 6 months old and six old 36 months of age were anesthetized anal canal pressure was measured to determine the L- T function of EAS. Animals were sacrificed at the end of study and anal canal was harvested and processed for histochemical studies (Masson trichrome stain for muscle /connective tissue) as well as for molecular markers known to induce fibrosis and atrophy (collagen-I, β-catenin, TGF- β, atrogin-1 and MURF-1). The L-T was significantly impaired in older animals compared to young animals. Anal canal sections stained with trichrome showed a significant decrease in the muscle content (52% in old compared to 70% in young) and an increase in the connective tissue/collagen content in the old animals. An increased protein and mRNA expression of all the fibrosis markers were seen in the older animals. Aging EAS muscle exhibits impairment of function and increase in connective tissue. Up regulation of atrophy and pro-fibrogenic proteins with aging may be the reason for the age-related decrease in anal sphincter muscle thickness and its function.
Aprepitant, an NK1 receptor antagonist, is approved for the treatment of chemotherapy-induced or postoperative emesis by blocking NK1 receptors in the brain stem vomiting center. Effects of NK1 receptors on gastric functions and postprandial symptoms in humans are unclear; a single, crossover study did not show a significant effect of aprepitant on gastrointestinal transit. Our aim was to compare, in a randomized, double-blind, placebo-controlled, parallel-group study (12 healthy volunteers per group), effects of aprepitant vs. placebo on gastric emptying (GE) of solids (by scintigraphy) with a 320kcal meal, gastric volumes (GV, fasting and accommodation by SPECT), satiation (maximum tolerated volume, MTV) and symptoms after a dyspeptogenic meal of Ensure®. Aprepitant (125mg on day 1, followed by 80mg on days 2-5) or placebo, one tablet daily, was administered for 5 consecutive days. Statistical analysis was by unpaired rank sum test, adjusted for gender and BMI. To assess treatment effects on symptoms, we incorporated MTV in the model. Aprepitant increased fasting, postprandial and accommodation GV, and tended to increase volume to fullness and MTV by ~200kcal. However, aprepitant increased aggregate symptom, nausea and pain scores after ingesting the MTV of Ensure®. There was no significant effect of aprepitant on GE T1/2 of solids. We conclude that NK1 receptors are involved in the control of GV and in determining postprandial satiation and symptoms. Further studies of the pharmacodynamics and therapeutic role of NK1 receptor antagonists in patients with gastroparesis and dyspepsia are warranted.
Iron homeostasis is tightly regulated and the peptide hormone hepcidin is considered to be a principal regulator of iron metabolism. Previous studies in a limited number of mouse strains found equivocal sex and strain dependent differences in mRNA and serum levels of hepcidin. Similarly, conflicting data on the relationship between hepcidin (Hamp1) mRNA levels and iron status as assessed by transferrin saturation and tissue iron concentrations were reported. Our aim was to clarify the relationships between strain, sex and hepcidin expression by examining multiple tissues and the effects of different dietary conditions in multiple inbred strains. Two studies were done: first, Hamp1 mRNA, liver iron and plasma diferric transferrin levels were measured in fourteen inbred strains on a control iron diet; and second, Hamp1 mRNA and plasma hepcidin levels in both sexes, as well as iron levels in the heart, kidneys, liver, pancreas and spleen in males, were measured in nine inbred/recombinant inbred strains raised on an iron sufficient or high iron diet. Both sex and strain have a significant effect on both hepcidin mRNA (primarily a sex effect) and plasma hepcidin levels (primarily strain effect). Hepatic hepcidin mRNA level is a highly significant predictor of plasma hepcidin levels. However, liver iron or diferric transferrin levels are not a predictor of Hamp1 mRNA levels in iron sufficient or high iron diet, nor are the Hamp1 mRNA and plasma hepcidin levels good predictors of tissue iron levels, at least in males.
Background and aims: Rising evidence points to endothelial-to-mesenchymal transition (EndMT) as a significant source of the mesenchymal cell population in fibrotic diseases. In this context, we hypothesized that liver endothelial cells undergo EndMT during fibrosis progression. Methods: Cirrhosis in mice was induced by CCl4. A transgenic mouse expressing a red fluorescent protein reporter under the control of Tie2 promoter (Tie2-tdTomato) was used to trace the acquisition of EndMT. Sinusoidal vascular connectivity was evaluated by intravital microscopy and high resolution 3D confocal microscopy. Results: A modest but significant fraction of liver endothelial cells from both cirrhotic patients and CCl4-treated Tie2-tdTomato mice acquired an EndMT phenotype characterized by the co-expression of CD31 and α-SMA, compared with non-cirrhotic livers. BMP-7 inhibited the acquisition of EndMT induced by TGF-β1 treatment in cultured MLiECs from control mice. EndMT was also reduced significantly in vivo in cirrhotic Tie2-tdTomato mice treated intraperitoneally with BMP-7 compared with untreated mice (1.9±0.2 vs. 3.8±0.3 %, respectively; p<0.05). The decrease of EndMT in cirrhotic livers correlated with a significant decrease in liver fibrosis (p<0.05) and an improvement in the vascular disorganization rate (p<0.05). Conclusions: We demonstrated the acquisition of the EndMT phenotype by a subpopulation of endothelial cells from cirrhotic livers in both animal models and patients. BMP-7 treatment decreases the occurrence of the EndMT phenotype and has a positive impact on the severity of disease by reducing fibrosis and sinusoidal vascular disorganization.
G protein-coupled receptors (GPCRs) make up the largest transmembrane receptor superfamily in the human genome and are expressed in nearly all gastrointestinal (GI) cell types. Coupling of GPCRs and their respective ligands activates various phosphotransferase in the cytoplasm, and thus, activation of GPCR signaling in intestine regulates many cellular and physiological processes. Studies in microRNAs (miRNAs) demonstrate that they represent critical epigenetic regulators of different pathophysiologic responses in different organs and cell types in humans and animals. Here, we reviewed recent research on GPCR/miRNA interactions related to GI pathophysiology, such as, Inflammatory Bowel Diseases (IBD), Irritable Bowel Syndrome (IBS) and GI cancers. Given that the presence of different types of cells in the GI tract suggest the importance of cell-cell interactions in maintaining GI homeostasis, we also discuss how GPCR/miRNAs interactions regulate gene expression at the cellular level and subsequently modulate GI pathophysiology through molecular regulatory circuits and cell-cell interactions. These studies helped identified novel molecular pathways, leading to the discovery of potential biomarkers for GI diseases.
Nonalcoholic fatty liver disease (NAFLD) is characterized by lipid accumulation in the liver that may progress to hepatic fibrosis and nonalcoholic steatohepatitis (NASH). Mechanisms underlying NAFLD and NASH are not yet fully understood. Dietary cholesterol was recently shown to be a risk factor for the development of NASH suggesting a role for intestinal handling of cholesterol. One important regulator of cholesterol homeostasis is the Sterol Response Element Binding Protein SREBP2 transcription factor. We tested the hypothesis that the overactivation of intestinal SREBP2 increases the susceptibility to diet-induced NASH. A transgenic mouse model with intestine specific overexpression of active SREBP2 (ISR2 mice) driven by villin promoter was used. ISR2 mice and wild type littermates were fed a regular chow diet or a high fat high cholesterol diet (HFHC: 15 % fat, 1 % cholesterol) for 15 weeks. Results showed that HFHC feeding to ISR2 mice caused hepatic inflammation with increased levels of proinflammatory cytokines. Histological examination demonstrated extensive fibrosis after a HFHC diet associated with a perivascular as well as pericellular collagen deposits in ISR2 mice as compared to wild type littermates. The severe hepatic inflammation and advanced fibrosis in ISR2 mice was not associated with a difference in lipid accumulation in ISR2 mice as compared to wild type littermates after HFHC feeding. These data indicate that overactivation of intestinal SREBP2 promotes diet-induced hepatic inflammation with features of human NASH resulting in rapid severe fibrosis and provide a novel link between regulatory processes of intestinal cholesterol and progression of fatty liver.
The contributions of gastric emptying (GE) and gastric accommodation (GA) to satiation, satiety, and postprandial symptoms remain unclear. We aimed to evaluate the relationships between GA or GE with satiation, satiety, and postprandial symptoms in healthy overweight or obese volunteers (total n=285, 73% females, mean BMI 33.5kg/m2): 26 prospectively studied obese, otherwise healthy participants and 259 healthy subjects with previous similar GI testing. We assessed GE of solids, gastric volumes, calorie intake at buffet meal, satiation by measuring volume to comfortable fullness (VTF) and maximum tolerated volume (MTV) using Ensure® nutrient drink test (30mL/min), and symptoms 30 minutes after MTV. Relationships between GE or GA with satiety, satiation, and symptoms were analyzed using Spearman's rank (Rs) and Pearson (R) linear correlation coefficients. We found a higher volume to comfortable fullness during satiation test correlated with a higher calorie intake at ad libitum buffet meal (Rs=0.535; p<0.001). There was a significant inverse correlation between GE T1/2 and volume to fullness (Rs= -0.317; p<0.001) and the calorie intake at buffet meal (Rs= -0.329; p<0.001), and an inverse correlation between GE Tlag and GE25% emptied with volume to fullness (Rs= -0.273, p<0.001and Rs= -0.248, p<0.001, respectively). GE T1/2 was significantly associated with satiation (MTV; R= -0.234; p<0.0001), nausea (R=0.145; p=0.023), pain (R=0.149; p=0.012), and higher aggregate symptom score (R=0.132; p=0.026). There was no significant correlation between GA and satiation, satiety, postprandial symptoms or GE. We concluded that GE of solids, rather than GA, is associated with postprandial symptoms, satiation and satiety in healthy participants.
Necrotizing enterocolitis (NEC), a gastrointestinal inflammatory disease of unknown etiology, causes a great deal of morbidity and mortality in premature infants. The liver as well as the gut may be affected in NEC. We tested the hypothesis that signaling molecules, which are endogenous to the bowel, regulate the severity of experimental NEC in mice. Specifically, we postulated that mucosal serotonin (5-HT), which is proinflammatory, would increase the severity of NEC and that oxytocin (OT), which is present in enteric neurons and is anti-inflammatory, would oppose NEC. Genetic deletion of the 5-HT transporter (SERT), which increases and prolongs the effects of 5-HT, was found to increase the severity of the systemic manifestations of NEC as well as intestinal inflammation and associated hepatotoxicity. In contrast, genetic deletion of tryptophan hydroxylase 1 (TPH1), which is responsible for 5-HT biosynthesis in enterochromaffin (EC) cells of the intestinal mucosa, and TPH inhibition with LP-920540 both decrease the severity of NEC in intestine and liver. These observations suggest that 5-HT from EC cells helps to drive both the enteric and hepatic manifestations of NEC. Administration of OT decreased NEC-induced intestinal inflammation, while that of atosiban, an OT receptor antagonist, caused NEC-induced intestinal inflammation to become more severe. Data from the current investigation are consistent with the tested hypotheses, that the enteric signaling molecules, 5-HT (positively) and OT (negatively) regulate NEC severity; moreover, we suggest that mucosally restricted inhibition of 5-HT biosynthesis and/or administration of OT may be useful in the treatment of NEC.
Melatonin is a hormone produced by the pineal gland with increased circulating levels shown to inhibit biliary hyperplasia and fibrosis during cholestasis. Melatonin also has the capability to suppress the release of hypothalamic gonadotropin releasing hormone (GnRH), a hormone that promotes cholangiocyte proliferation when serum levels are elevated. However, the interplay and contribution of neural melatonin and GnRH to cholangiocyte proliferation and fibrosis in bile duct-ligated (BDL) rats has not been investigated. In order to test this, cranial levels of melatonin were increased by implanting osmotic minipumps that performed an intracerebroventricular (ICV) infusion of melatonin or saline for 7 days starting at the time of BDL. Hypothalamic GnRH mRNA and cholangiocyte secretion of GnRH and melatonin was assessed. Cholangiocyte proliferation and fibrosis was measured. Primary human hepatic stellate cells (HSCs) were treated with cholangiocyte supernatants, GnRH, or the GnRH receptor 1 (GnRHR1) antagonist, cetrorelix acetate and cell proliferation and fibrosis gene expression were assessed. Melatonin infusion reduced hypothalamic GnRH mRNA expression and led to decreased GnRH and increased melatonin secretion from cholangiocytes. Infusion of melatonin was found to reduce hepatic injury, cholangiocyte proliferation and fibrosis during BDL-induced liver injury. HSCs supplemented with BDL cholangiocyte supernatant had increased proliferation and this increase was reversed when HSCs were supplemented with supernatants from melatonin-infused rats. GnRH stimulated fibrosis gene expression in HSCs and this was reversed by cetrorelix acetate co-treatment. Increasing bioavailability of melatonin in the brain may improve outcomes during cholestatic liver disease.
Excessive or persistent inflammation and hepatocyte death are the key triggers of liver diseases. The poly(ADP-ribose) polymerase (PARP) proteins induce cell death and inflammation. Chemical inhibition of PARP activity protects against liver injury during Concanavalin A (ConA)-induced hepatitis. In this mice model, ConA activates immune cells which promote inflammation and induce hepatocyte death, mediated by the activated invariant NKT-(iNKT) lymphocyte population. We analyzed immune cell populations in the liver and several lymphoid organs such as spleen, thymus, and bone marrow in Parp2 deficient mice to better define the role of PARP proteins in liver immunity and inflammation at steady state and during ConA-induced hepatitis. We show that i) the genetic inactivation of Parp2, but not Parp1, protected mice from ConA hepatitis without deregulating cytokine expression and leucocyte recruitment; ii) cellularity was lower in the thymus, but not in spleen, liver, or bone marrow of Parp2-/- mice; iii) spleen and liver iNKT lymphocytes, as well as thymic T and NKT lymphocytes were reduced in Parp2 knockout mice. In conclusion, our results suggest that the defect of T-lymphocyte maturation in Parp2 knockout mice leads to a systemic reduction of iNKT cells, reducing hepatocyte death during ConA-mediated liver damage, thus protecting the mice from hepatitis.
A deeper understanding of the radiation-induced pathophysiological processes that develop in the gut is imperative in order to prevent, alleviate or eliminate cancer survivorship diseases after radiotherapy to the pelvic area. Most rodent models of high-dose gastrointestinal radiation injury are limited by high mortality. We therefore established a model that allows for the delivering of radiation in fractions at high doses, while maintaining long-term survival. Adult male C57/BL6 mice were exposed to small-field irradiation, restricted to 1,5 cm of the colorectum using a linear accelerator. Each mouse received 6 or 8 Gy, twice daily in 12 hours intervals, in 2, 3 or 4 fractions. Acute cell death was examined at 4.5 hours post-irradiation, and histological changes at six weeks post-irradiation. Another group was given 4 fractions of 8 Gy and followed over time for development of visible symptoms. Irradiation caused immediate cell death, mainly limited to the colorectum. At six weeks post-irradiation, several crypts displayed signs of radiation-induced degeneration. The degenerating crypts were seen alongside crypts that appeared perfectly healthy. Crypt survival was reduced after the fourth fraction regardless of dose, while the number of macrophages increased. Angiogenesis was induced, likely as a compensatory mechanism for hypoxia. Four months post-irradiation, mice began to show radiation-induced symptoms, and histological examination revealed an extensive crypt loss and fibrosis. Our model is uniquely suitable for studying the long-term trajectory and underlying mechanisms of radiation-induced gastrointestinal injury.
Dopamine (DA) containing fibers and neurons are embedded within the brainstem dorsal vagal complex (DVC); we have shown previously that DA modulates the membrane properties of neurons of the dorsal motor nucleus of the vagus (DMV) via DA1 and DA2 receptors. The vagally-dependent modulation of gastric tone and phasic contractions, i.e. motility, by DA, however, has not been characterized. Using microinjections of DA in the DVC while recording gastric tone and motility, the aims of the present study were: i) assess the gastric effects of brainstem DA application, ii) identify the DA receptor subtype, and, iii) identify the postganglionic pathway(s) activated. Dopamine microinjection in the DVC decreased gastric tone and motility in both corpus and antrum in 29 of 34 rats, the effects were abolished by ipsilateral vagotomy and fourth ventricular treatment with the selective DA2 receptor antagonist L741,626, but not by application of the selective DA1 receptor antagonist SCH 23390. Systemic administration of the cholinergic antagonist atropine attenuated the inhibition of corpus and antrum tone in response to DA microinjection in the DVC. Conversely, systemic administration of the nitric oxide synthase inhibitor L-NAME did not alter the DA-induced decrease in gastric tone and motility. Our data provide evidence of a dopaminergic modulation of a brainstem vagal neurocircuit that controls gastric tone and motility.
The essentiality of thiamin stems from its roles as a co-factor (mainly in the form of thiamin pyrophosphate, TPP) in critical metabolic reactions including oxidative energy metabolism and reduction of cellular oxidative stress. Like other mammalian cells, pancreatic acinar cells (PAC) obtain thiamin from their surroundings and convert it to TPP; mitochondria then take up TPP by a carrier-mediated process that involves the mitochondrial TPP (MTPP) transporter (MTPPT; product of SLC25A19 gene). Previous studies have characterized different physiological/biological aspects of the MTPP uptake process, but little is known about its possible adaptive-regulation. We addressed this issue using pancreatic acinar 266-6 cells (PAC 266-6) maintained under thiamin-deficient (DEF) and over-supplemented (OS) conditions, as well as thiamin-DEF and -OS transgenic mice carrying the SLC25A19 promoter. We found that maintaining PAC 266-6 under thiamin-DEF condition leads to a significant induction in mitochondrial [3H]-TPP uptake, as well as in level of expression of the MTPPT protein and mRNA compared to -OS cells. Similar findings were observed in mitochondria from thiamin-DEF mice compared to -OS. Subsequently, we demonstrated that adaptive-regulation of MTTP protein was partly mediated via transcriptional mechanism(s) via studies with PAC 266-6 transfected with the SLC25A19 promoter, and transgenic mice carrying the SLC25A19 promoter. This transcriptional regulation appeared to be, at least in part, mediated via epigenetic mechanism(s) involving histone modifications. These studies report, for the first time, that the PAC mitochondrial TPP uptake process is adaptively-regulated by the prevailing thiamin level and that this regulation is transcriptionally mediated and involves epigenetic mechanism(s).
Regulation of colonic motility depends upon the integrity of enteric inhibitory neurotransmission mediated by nitric oxide (NO), purine neurotransmitters, and neuropeptides. Intramuscular interstitial cells of Cajal (ICC-IM) and platelet-derived growth factor receptor α-positive (PDGFRα+) cells are involved in generating responses to NO and purine neurotransmitters, respectively. Previous studies have suggested a decreased nitrergic and increased purinergic neurotransmission in KitW/KitW-v (W/Wv) mice that display lesions in ICC-IM along the gastrointestinal tract. However, contributions of NO to these phenotypes have not been evaluated. We utilized small-chamber superfusion assays and HPLC to measure the spontaneous and electrical field stimulation (EFS)-evoked release of nicotinamide adenine dinucleotide (NAD+)/ADP-ribose, uridine adenosine tetraphosphate (Up4A), adenosine 5'-triphosphate (ATP) and metabolites from the tunica muscularis of human, monkey and murine colons and circular muscle of monkey colon, and we tested drugs that modulate NO levels or blocked NO receptors. NO inhibited EFS-evoked release of purines in the colon via presynaptic neuromodulation. Colons from W/Wv, Nos1-/- and Prkg1-/- mice displayed augmented neural release of purines that was likely due to altered nitrergic neuromodulation. Colons from W/Wv mice demonstrated decreased nitrergic and increased purinergic relaxations in response to nerve stimulation. W/Wv mouse colons demonstrated reduced Nos1 expression and reduced NO release. Our results suggest that enhanced purinergic neurotransmission may compensate for the loss of nitrergic neurotransmission in muscles with partial loss of ICC. The interactions between nitrergic and purinergic neurotransmission in the colon provides novel insight into the role of neurotransmitters and effector cells in the neural regulation of gastrointestinal motility.
There is a lack of tools that selectively target vagal afferent neurons (VAN) innervating the gut. We use saporin (SAP), a potent neurotoxin, conjugated to the GI hormone cholecystokinin (CCK-SAP) injected into the nodose ganglia (NG) of male Wistar rats to specifically ablate GI-VAN. We report that CCK-SAP ablates a sub-population of VAN in culture. In vivo, CCK-SAP injection into the NG reduces VAN innervating the mucosal and muscular layers of the stomach and small intestine, but not the colon, while leaving vagal efferent neurons intact. CCK-SAP abolishes feeding induced c-Fos in the NTS, as well as satiation by CCK or glucagon like peptide -1 (GLP-1). CCK-SAP in the NG of mice also abolishes CCK-induced satiation. Therefore we provide multiple lines of evidence that injection of CCK-SAP in NG is a novel selective vagal deafferentation technique of the upper gastrointestinal tract that works in multiple vertebrate models. This method provides improved tissue specificity and superior separation of afferent and efferent signaling compared to vagotomy, capsaicin, and subdiaphragmatic deafferentation.
Pancreatic ductal adenocarcinoma (PDAC) displays extensive and poorly vascularized desmoplastic stromal reaction, and therefore pancreatic cancer (PaCa) cells are confronted with nutrient deprivation and hypoxia. Here, we investigate the roles of autophagy and metabolism in PaCa cell adaptation to environmental stresses, amino acid (AA) depletion and hypoxia. It is known that in healthy cells, basal autophagy is at a low level, but it is greatly activated by environmental stresses. By contrast, we find that in PaCa cells basal autophagic activity is relatively high, but AA depletion and hypoxia activate autophagy only weakly or not at all, due to their failure to inhibit mTOR. Basal, but not stress-induced, autophagy is necessary for PaCa cell proliferation; and AA supply is even more critical to maintain PaCa cell growth. To gain insight into the underlying mechanisms, we analyzed the effects of autophagy inhibition and AA depletion on PaCa cell metabolism. PaCa cells display mixed oxidative/glycolytic metabolism, with oxidative phosphorylation (OXPHOS) predominant. Both autophagy inhibition and AA depletion dramatically decreased OXPHOS; furthermore, pharmacologic inhibitors of OXPHOS suppressed PaCa cell proliferation. The data indicate that maintaining OXPHOS is a key mechanism through which autophagy and AA supply support PaCa cell growth. We find that the expression of oncogenic Kras markedly promotes basal autophagy and stimulates OXPHOS through autophagy-dependent mechanism. The results suggest that approaches aimed to suppress OXPHOS, particularly through limiting AA supply, could be beneficial in treating PDAC.
Hydrogen sulfide (H2S), like nitric oxide (NO), causes smooth muscle relaxation, but unlike NO, does not stimulate soluble guanylyl cyclase (sGC) activity and generate cGMP. The aim of this study was to investigate the interplay between NO and H2S in colonic smooth muscle. In colonic smooth muscle from rabbit, mouse and human, L-cysteine, substrate of cystathionine--lyase (CSE), or NaHS, an H2S donor, inhibited phosphodiesterase 5 (PDE5) activity and augmented the increase in cGMP levels, IP3 receptor phosphorylation at Ser1756 (measured as a proxy for PKG activation), and muscle relaxation in response to NO donor, s-nitrosoglutathione (GSNO), suggesting augmentation of cGMP/PKG pathway by H2S. The inhibitory effect of L-cysteine, but not NaHS, on PDE5 activity, was blocked in cells transfected with CSE siRNA or treated with CSE inhibitor, DL-propargylglycine (DL-PPG), suggesting activation of CSE and generation of H2S in response to L-cysteine. H2S levels were increased in response to L-cysteine and the effect of L-cysteine was augmented by GSNO in a cGMP-dependent protein kinase-sensitive manner, suggesting augmentation of CSE/H2S by cGMP/PKG pathway. As a result, GSNO-induced relaxation was inhibited by DL-PPG. In flat-sheet preparation of colon, L-cysteine augmented calcitonin-gene-related peptide release in response to mucosal stimulation and in intact segments, L-cysteine increased the velocity of pellet propulsion. These results demonstrate that in colonic smooth muscle, there is a novel interplay between NO and H2S. NO generates H2S via cGMP/PKG pathway and H2S, in turn, inhibits PDE5 activity and augments NO-induced cGMP levels. In the intact colon, H2S promotes colonic transit.
Dclk1-expressing tuft cells constitute a unique intestinal epithelial lineage that is distinct from enterocytes, Paneth cells, goblet cells, and enteroendocrine cells. Tuft cells express taste-related receptors and distinct transcription factors, and interact closely with the enteric nervous system, suggesting a chemosensory cell lineage. In addition, recent work has shown that tuft cells interact closely with cells of the immune system, with a critical role in the cellular regulatory network governing responses to luminal parasites. Importantly, ablation of tuft cells severely impairs epithelial proliferation and tissue regeneration after injury, implicating tuft cells in the modulation of epithelial stem/progenitor function. Finally, tuft cells expand during chronic inflammation and in preneoplastic tissues, suggesting a possible early role in inflammation-associated tumorigenesis. Hence, we outline and discuss emerging evidence that strongly supports tuft cells as key regulatory cells in the complex network of the intestinal microenvironment.
Inflammation plays a role in abdominal surgery (AS)-induced intestinal ileus that is alleviated by electrical vagal stimulation. The stable thyrotropin-releasing hormone agonist, RX77368 injected intracisternally (ic) activates dorsal motor nucleus neurons and gastric vagal efferent discharges. We investigated the gastric inflammation induced by AS and the modulation by ic RX-77368 in rats. RX77368 (50 ng/rat) or saline was injected ic followed, 1 h later, by laparotomy and small intestinal/cecal manipulation. Sham group had anesthesia alone. After 6-h, gastric emptying (GE) and the inflammation in gastric corpus were determined. AS inhibited GE by 72% vs control. In whole mount preparation of myenteric plexus, AS doubled the number of M1-like macrophage immunoreactive for MHCII (M1 marker) but not for CD206 (M2 marker) (MHCII+/CD206-) while there was no change in M2-like macrophages (MHCII-/CD206+). AS increased mRNA levels of interleukin-1β (IL-1β) and tumor necrosis factor α (TNF-α) in the gastric submucosa plus muscle layers by 1.7- and 1.5-fold respectively and the infiltration of neutrophils labeled by myeloperoxidase in the muscularis externa by 9.5-fold. RX77368 inhibited AS-related gastric changes while not altering these parameters in sham group. There was a significant negative correlation between GE and IL-1β (r=-0.46), TNF-α (r=-0.44), M1 macrophage (r=-0.82) and neutrophils (r=-0.91). The M2 like macrophages and IL-10 expression were unchanged after AS with ic saline or RX77368. These data indicate that AS activates gastric M1 macrophages and increases proinflammatory cytokines expression that are prevented by central vagal activation and may contribute to the correlated prevention of postoperative gastric ileus.
Bile acids (BA) are signaling molecules with a wide range of biological effects, also identified amongst the most responsive plasma metabolites in the post-prandial state. We here describe this response to different dietary challenges and report on key determinants linked to its inter-individual variability. Healthy men and women (N=72, 62 ± 8 years) were enrolled into a 12-week weight loss intervention. All subjects underwent an oral glucose tolerance test (OGTT) and a mixed meal tolerance test (MMTT) before and after the intervention. BA were quantified in plasma by LC-MS/MS combined with whole genome exome sequencing and fecal microbiota profiling. Considering the average response of all 72 subjects, no effect of the successful weight loss intervention was found on plasma BA profiles. Fasting and post-prandial BA profiles revealed high inter-individual variability and 3 main patterns in post-prandial BA response were identified using multivariate analysis. Although the women enrolled were postmenopausal, gender effects in BA response were evident. Exome data revealed the contribution of preselected genes to the observed inter-individual variability. In particular, a variant in the SLCO1A2 gene, encoding the small intestinal BA transporter OATP1A2 was associated with delayed post-prandial BA increases. Fecal microbiota analysis did not reveal evidence for a significant influence of bacterial diversity and/or composition on plasma BA profiles. The analysis of plasma BA profiles in response to two different dietary challenges revealed a high inter-individual variability, which was mainly determined by genetics and gender of host with minimal effects of the microbiota.
Small for size and flow syndrome (SFSF) is one of the most challenging complications following extended hepatectomy (EH). After EH, hepatic artery flow decreases and portal vein flow increases per 100 gram remnant liver volume (RLV). This causes hypoxia followed by metabolic acidosis. A correlation between acidosis and post-hepatectomy liver failure has been postulated, but not studied systematically in a large animal model or clinical setting. In our study, we performed stepwise liver resections on nine pigs to defined SFSF limits as follows: step 1: segment II/III resection, step 2: segment IV resection, step 3: segment V/VIII resection (RLV: 75%, 50% and 25% respectively). Blood gas values were measured before and after each step using four catheters inserted into the carotid artery, internal jugular vein, hepatic artery, and portal vein. The pH, HCO3- and base excess (BE) decreased but HCO3- values increased after 75% resection in the portal and jugular veins. EH correlated with reduced BE in the hepatic artery. PCO2 values increased after 75% resection in the jugular vein. In contrast, arterial pO2 increased after every resection while the venous pO2 decreased slightly. There were differences in venous HCO3-, BE in the hepatic artery, and PCO2 in the jugular vein after 75% liver resection. Since 75% resection is the limit for SFSF, these noninvasive blood evaluations may be used to predict SFSF. Further studies with long-term follow-up are required to validate this correlation.
Background and Aims: Patients with alcohol-related cirrhosis (ALD) are prone to infection. Circulating neutrophils in ALD are dysfunctional and predict development of sepsis, organ dysfunction and survival. Neutrophil granules are important effector organelles containing a toxic array of microbicidal proteins, whose controlled release is required to kill micro-organisms whilst minimising inflammation and damage to host tissue. We investigated the role of these granular responses in contributing to immune disarray in ALD. Methods: Neutrophil granular content and mobilisation was measured by flow cytometric quantitation of cell-surface/intracellular markers, [secretory vesicles (CD11b), secondary granules (CD66b) and primary granules (CD63; myeloperoxidase)] before and after bacterial stimulation in 29 patients with ALD cirrhosis compared to healthy controls (HC). ImageStream Flow Cytometry characterised localisation of granule subsets within the intracellular and cell-surface compartments. The plasma cytokine environment was analysed using ELISA/Cytokine Bead Array. Results: Circulating neutrophils were primed in the resting state with upregulated surface expression of CD11b (p=0.0001) in a cytokine milieu rich in IL-8 (p<0.001) and lactoferrin (p=0.035). Neutrophils showed exaggerated mobilisation to the cell surface of primary granules at baseline (p=0.001) and in response to fMLP (p=0.009) and E. coli (p=0.0003). There was no deficit in granule content or mobilisation to the cell membrane in any granule subset observed. Paradoxically, active alcohol consumption abrogated the hyper-responsive neutrophil granular responses compared to their abstinent counterparts. Conclusions: Neutrophils are pre-primed at baseline with augmented effector organelle mobilisation in response to bacterial stimulation; neutrophil degranulation is not a mechanism leading to innate immunoparesis in ALD.
Na+/H+ exchanger NHE3 mediates the majority of intestinal and renal electroneutral sodium absorption. Dysfunction of NHE3 is associated with a variety of diarrheal diseases. We previously reported that the NHE3 gene (SLC9A3) has more than 400 single nucleotide polymorphisms (SNPs) but few non-synonymous polymorphisms. Among the latter, one polymorphism (rs2247114-G>A), which causes a substitution from arginine to cysteine at amino acid position 799 (p.R799C), is common in Asian populations. To improve our understanding of the population distribution and potential clinical significance of the NHE3-799C variant, we investigated the frequency of this polymorphism in different ethnic groups using bioinformatics analyses, and in a cohort of Japanese patients with cardiovascular or renal disease. We also characterized the function of human NHE3-799C and its sensitivity to regulatory ligands in an in vitro model. NHE3-799C had an allele frequency of 29.5-57.6% in Asian populations, 11.1-23.6% in European populations, and 10.2-22.7% in African populations. PS120/FLAG-NHERF2 fibroblasts stably expressing NHE3-799C had lower total protein expression but higher percentage of surface expression than those expressing NHE3-799R. NHE3-799C had similar basal activity to NHE3-799R and was similarly stimulated or inhibited, by serum or forskolin, respectively. Tenapanor, a small-molecule NHE3 inhibitor, dose-dependently inhibited NHE3-799R and NHE3-799C activities. The IC50 values of tenapanor for NHE3-799C and NHE3-799R were significantly different, but both were in the nanomolar range. These results suggest that NHE3-799C is a common variant enriched in Asian populations, is not associated with compromised function or abnormal regulation, and is unlikely to contribute to clinical disease.
Notch signaling is thought to act to drive cell versification in the lining of the small intestine. The purpose of the present study was to evaluate the role of Notch signaling pathway in stem cell differentiationin the late stages of intestinal adaptation after massive small bowel resection in a rat. Male Sprague-Dawley rats were randomly assigned to one of two experimental groups of 8 rats each: Sham rats underwent bowel transection and re-anastomosis, SBS- rats underwent 75% small bowel resection. Rats were sacrificed on day 14. Illumina's Digital Gene Expression (DGE) analysis was used to determine Notch signaling gene expression profiling. Notch-related gene and protein expression were determined using Real Time PCR, Western blotting and immunohistochemistry. From 7 investigated Notch-related (by DGE analysis) genes 6 genes were up-regulated in SBS vs control animals with a relative change in gene expression level of 20% or more. A significant up-regulation of Notch signaling related genes in resected animals was accompanied by a significant increase in Notch-1 protein levels (Western Blot) and a significant increase in the number of NOTCH-1 and Hes -1 (target gene) positive cells (immunohistochemistry) compared to sham animals. Evaluation of cell differentiation has shown a strong increase in total number of absorptive cells (unchanged secretory cells) compared to control rats. In conclusion, two weeks after bowel resection in rats, stimulated Notch signaling directs crypt cells population toward absorptive progenitors.
A response, i.e., the supragastric belch (SGB), in which a belch occurs without gastric involvement has been characterized in humans. The aims of this study were to determine whether animals have an SGB and if so to determine its mechanisms. Studies were conducted in decerebrate cats (N=30) with EMG electrodes on hyoid, pharyngeal, esophageal, and diaphragm muscles. The effects of distending different regions of the esophagus in different manners using a balloon were quantified to determine the most appropriate stimulus for activating the cat SGB. The effects of esophageal perfusion of lidocaine (N=3), vagus nerve transection (N=3), or esophageal acidification (N=5) on activation of the SGB were determined. Rapid large distensions of the thoracic esophagus best activated responses similar to the human SGB, i.e., rapid inhalation followed by a belch. The rapid inhalation was associated with activation of hiatal fibers and the belch with activation of dome fibers of the diaphragm. The rapid inhalation response was independent of the belch response. Lidocaine perfusion of the esophagus blocked the belch response without blocking the rapid inhalation, HCl perfusion sensitized the esophagus to activation of both the rapid inhalation and the belch response, and vagotomy blocked both responses. We conclude that the cat has an SGB which is composed of two independent reflex responses, i.e., rapid inhalation and belch, that are mediated by the vagus nerves and tension/mucosal receptors of the esophagus and sensitized by esophageal acid exposure. We hypothesize that the SGB is a learned voluntarily activated reflex response.
High-iron feeding of rodents has been commonly used to model human iron-overload disorders. We recently noted that high-iron consumption impaired growth and caused severe systemic copper deficiency in growing rats, but the mechanism by which this occurred could not be determined due to technical limitations. In the current investigation, we thus utilized mice; first to determine if the same phenomenon occurred in another mammalian species, and secondly since we could assess in vivo copper absorption in mice. We hypothesized that excessive dietary iron impaired intestinal copper absorption. Weanling, male mice were thus fed AIN-93G-based diets containing high (HFe) (~8800 ppm) or adequate (AdFe) (~80 ppm) iron in combination with low (~0.9 ppm), adequate (~9 ppm) or high (~180 ppm) copper for several weeks. Iron and copper homeostasis was subsequently assessed. Mice consuming the HFe diets grew slower, were anemic, and had lower hepatic copper levels and serum ceruloplasmin activity. These physiologic perturbations were all prevented by higher dietary copper, demonstrating that copper depletion was the underlying cause. Furthermore, homeostatic regulation of copper absorption was noted in the mice consuming the AdFe diets, with absorption increasing as dietary copper decreased. HFe-fed mice did not have impaired copper absorption (disproving our hypothesis), but homeostatic control of absorption was disrupted. There were also noted perturbations in the tissue distribution of copper in the HFe-fed mice, suggesting that altered storage and thus bioavailability contributed to the noted copper deficiency. Dietary iron loading thus antagonizes copper homeostasis leading to pathological symptoms of severe copper depletion.
The liver is one of the largest and most functionally diverse organs in the human body. In addition to roles in detoxification of xenobiotics, digestion, synthesis of important plasma proteins, gluconeogenesis, lipid metabolism and storage, the liver also plays a significant role in iron homeostasis. Apart from being the storage site for excess body iron, it also plays a vital role in regulating the amount of iron released into the blood by enterocytes and macrophages. Since iron is essential for many important physiological and molecular processes, it increases the importance of liver in the proper functioning of the body's metabolism. This hepatic iron-regulatory function can be attributed to the expression of many liver-specific or liver-enriched proteins, all of which play an important role in the regulation of iron homeostasis. This review focuses on these proteins and their known roles in the regulation of body iron metabolism.
Scope: The aim of this study was to determine the extent to which oat particle size in a porridge could alter glucose absorption, gastric emptying, gastrointestinal hormone response and subjective feelings of appetite and satiety. Method and results: Porridge was prepared from either oat flakes or oat flour with the same protein, fat, carbohydrate and mass. These were fed to eight volunteers on separate days in a crossover study and subjective appetite ratings, gastric contents and plasma glucose, insulin, and gastrointestinal hormones were determined over a period of three hours. The flake porridge gave a lower glucose response than the flour porridge and there were apparent differences in gastric emptying in both the early and late post prandial phases. The appetite ratings showed similar differences between early and late phase behavior. Conclusions: The structure of the oat flakes remained sufficiently intact to delay their gastric emptying leading to a lower glycemic response, even though initial gastric emptying rates were similar for the flake and flour porridge. This highlights the need to take food structure into account when considering relatively simple physiological measures and offering nutritional guidance.
Esophageal injury is a risk factor for diseases such as Barrett's Esophagus (BE) and Esophageal Adenocarcinoma. In order to improve understanding of signaling pathways associated with both normal and abnormal repair, animal models are needed. Traditional rodent models of esophageal repair are limited by the absence of esophageal submucosal glands (ESMGs), which are present in human esophagus. Previously, we identified acinar ductal metaplasia in human ESMGs in association with both esophageal injury and cancer. In addition, the SOX9 transcription factor has been associated with generation of columnar epithelium and the pathogenesis of BE and is present in ESMGs. In order to test our hypothesis that ESMGs activate after esophageal injury with an increase in proliferation, generation of a ductal phenotype and expression of SOX9, we developed a porcine model of esophageal injury and repair using radiofrequency ablation (RFA). The porcine esophagus contains ESMGs and RFA produces a consistent and reproducible mucosal injury in the esophagus. Here we present a temporal assessment of this model of esophageal repair. Porcine esophagus was evaluated at 0, 6, 18, 24, 48, 72 hours, 5 and 7 days following RFA and compared to control uninjured esophagus. Following RFA, ESMGs demonstrated an increase in ductal phenotype, echoing our prior studies in humans. Proliferation increased in both squamous epithelium and ESMGs post injury with a prominent population of SOX9 positive cells in ESMGs post-injury. This model promises to be useful in future experiments evaluating mechanisms of esophageal repair.
SLC26A3 (Down Regulated in Adenoma, DRA) plays a key role in mammalian intestinal NaCl absorption via mediating apical membrane Cl-/HCO3- exchange. DRA function and expression are significantly decreased in diarrhea associated with inflammatory bowel diseases (IBD). DRA is also considered to be a marker of cellular differentiation, and is predominantly expressed in differentiated epithelial cells. Caudal type homeobox protein-2 (CDX2) is known to regulate genes involved in intestinal epithelial differentiation and proliferation. Reduced expression of both DRA and CDX2 in intestinal inflammation prompted us to study whether DRA gene is directly regulated by CDX2. Our initial studies utilizing CDX2 knockout (CDX2fV/fV; Cre+) mice showed marked reduction in DRA mRNA and protein levels in the proximal and distal colon. In silico analysis of DRA promoter showed 2 consensus sites for CDX2 binding. Therefore, we utilized Caco-2 cells as an in vitro model to examine if DRA is a direct target of CDX2 regulation. siRNA-mediated silencing of CDX2 in Caco2 cells resulted in a marked (~ 50%) decrease in DRA mRNA and protein levels, whereas ectopic over-expression of CDX2 upregulated DRA expression and also stimulated DRA promoter activity suggesting transcriptional regulation. EMSA and ChIP assays demonstrated direct binding of CDX2 to one of the two putative CDX2 binding sites in the DRA promoter (+645/+663). In summary, our studies, for the first time, demonstrated transcriptional regulation of DRA expression by CDX2 implicating that reduced expression of DRA in IBD-associated diarrhea may partly be due to downregulation of CDX2 in the inflamed mucosa.
The lower esophageal sphincter (LES) and crural diaphragm (CD) provide the closure mechanism at esophago-gastric junction (EGJ). We used CT imaging in combination with 3D EGJ pressure recordings to determine the functional morphology of the LES and CD and its relationship to the EGJ anatomy. A 3D-HRM catheter recorded the EGJ pressure in 10 healthy subjects. A 0.5 mm diameter metal ball (BB) was taped to the catheter, adjacent to transducer #1. The EGJ pressure was recorded under following conditions; 1) end-expiration (LES pressure) a) before swallow, b) after swallow, and 2) peak inspiration (CD contraction) c) tidal inspiration and d) forced inspiration. A CT scan was performed to localize the circumferential orientation of the BB. The LES pressure varied under different conditions but the shape of pressure profile was not different with the LES length longer towards the lesser as compared to greater curvature of stomach. The LES pressure profile revealed circular pressure asymmetry, with greatest pressure and shortest cranio-caudal length on the left. The CD contraction with inspiration increased pressure in the cranial half of the LES pressure profile and it was placed horizontally. The CT images revealed that the hiatus is placed obliquely across the esophagus, however, due to the bend of the esophagus to the left the two are placed at right angle to each other. Our observations suggest a unique shape of the LOS, CD and the anatomical relationship between the two, which provides explanation as to why the LOS pressure is circumferentially asymmetric.
Background: In eosinophilic esophagitis (EoE) the esophageal barrier integrity is impaired. Integrity can be assessed with different techniques. Aims: To assess the correlations between esophageal eosinophilia and various measures of mucosal integrity, and to evaluate whether endoscopic impedance measurements can predict disease activity. Methods: Endoscopies and mucosal integrity measurements were performed in adult EoE patients with active disease (≥15 eosinophils/HPF) at baseline (n=32), after fluticasone (n=15) and elemental dietary treatment (n=14) and in controls (n=19). Mucosal integrity was evaluated during endoscopy using electrical tissue spectroscopy (ETIS) measuring mucosal impedance, and transepithelial electrical resistance (TER) and transepithelial molecule-flux through biopsy specimens in Ussing chambers. Results: We included 61 measurements; 32 of patients at baseline and 29 after treatment, 3 patients dropped-out. After treatment 20 patients were in remission (≤15 eosinophils/HPF) and these measurements were compared with 41 measurements of patients with active disease (at baseline or after failed treatment). All 4 mucosal integrity measures showed significant impairment in active EoE compared with remission. Eosinophilia was negatively correlated with ETIS and TER and positively with transepithelial molecule-flux (p≤0.001). The optimal ETIS cutoff to predict disease activity was 6000 (•m) with a sensitivity of 79% (95% CI 54 - 94%), specificity of 84% (95% CI 69 - 94%), positive predictive values of 89% (95% CI 77- 95%) and negative predictive values of 71% (95% CI 54 - 84%). Conclusions: In EoE patients, markers of mucosal integrity correlate with esophageal eosinophilia. Additionally, endoscopic mucosal impedance measurements can predict disease activity.
The purpose of this study was to determine effects and mechanisms of vagal nerve stimulation (VNS) and additive effects of electroacupuncture (EA) on colonic inflammation in a rodent model of IBD. Chronic inflammation in rats was induced by intrarectal TNBS (2,4,6-Trinitrobenzenesulfonic acid). The rats were then treated with sham-ES (electrical stimulation), VNS or VNS+EA for 3 weeks. Inflammatory responses were assessed by disease activity index (DAI), macroscopic scores and histological scores of colonic tissues, and plasma levels of TNF-α, IL-1β and IL-6, myeloperoxidase (MPO) activity of colonic tissues. The autonomic function was assessed by the spectral analysis of the heart rate variability (HRV) derived from the electrocardiogram. It was found: 1) The area under curve (AUC) of DAI was substantially decreased with VNS+EA and VNS, and VNS+EA was more effective than VNS (P<0.001); 2) The macroscopic score was 6.43±0.61 in the sham-ES group and reduced to 1.86±0.26 with VNS (P<0.001) and 1.29±0.18 with VNS+EA (P<0.001). The histological score was 4.05±0.58 in the sham-ES group and reduced to 1.93±0.37 with VNS (P<0.001) and 1.36±0.20 with VNS+EA (P<0.001); 4) the plasma levels of TNF-α, IL-1β, IL-6 and MPO were all significantly decreased with VNS and VNS+EA, compared to the sham-ES group; 5) Autonomically, both VNS+EA and VNS substantially increased vagal activity and decreased sympathetic activity compared to sham-EA group (P<0.001, P<0.001, respectively). In conclusions, chronic VNS improves inflammation in TNBS-treated rats by inhibiting pro-inflammatory cytokines via the autonomic mechanism. Addition of noninvasive EA to VNS may enhance the anti-inflammatory effect of VNS.
Serotonin (5-HT), predominantly synthesized and released by enterochromaffin cells, is implicated in gastrointestinal symptoms such as emesis, abdominal pain and diarrhea. Since luminal short-chain fatty acids (SCFAs) release 5-HT from enterochromaffin cells, which express the SCFA receptor FFA2 in rat duodenum, we examined the effects of the selective FFA2 agonist phenylacetamide-1 (PA1) on duodenal 5-HT release with consequent bicarbonate secretion (DBS) and on indomethacin (IND)-induced enteropathy. Intestinal injury was induced by IND (10 mg/kg, sc) with or without PA1. We measured DBS in vivo in a duodenal loop perfused with PA1 while measuring 5-HT released into the portal vein. Duodenal blood flow was measured by laser-Doppler flowmetry. IND induced small intestinal ulcers with duodenal sparing. PA1 given with IND (IND+PA1) dose-dependently induced duodenal erosions. IND+PA1-induced duodenal lesions were inhibited by the FFA2 antagonist GLPG0974, ondansetron or omeprazole, but not by RS23597 or atropine. Luminal perfusion of PA1 augmented DBS accompanied by increased portal blood 5-HT concentrations with ~8X more release at 0.1 mM than at 1 µM, the effects inhibited by co-perfusion of GLPG0974. Luminal PA1 at 1 µM increased, but at 0.1 mM diminished duodenal blood flow. Co-superfusion of PA1 (0.1 mM) decreased acid-induced hyperemia, further reduced by IND pretreatment, but restored by ondansetron. These results suggest that although FFA2 activation enhances duodenal mucosal defenses, FFA2 overactivation during ulcerogenic COX inhibition may increase the vulnerability of the duodenal mucosa to gastric acid via excessive 5-HT release and 5-HT3 receptor activation, implicated in foregut-related symptoms such as emesis and epigastralgia.
The ileum is considered the primary site of inorganic sulfate (SO42-) absorption. In the present study, we explored the contributions of the apical chloride/bicarbonate (Cl-/HCO3-) exchangers DRA (Slc26a3), and PAT1 (Slc26a6), to the underlying transport mechanism. Transepithelial 35SO42- and 36Cl- fluxes were determined across isolated, short-circuited segments of the distal ileum from wild-type (WT), DRA-knockout (KO) and PAT1-KO mice, together with measurements of urine and plasma sulfate. The WT distal ileum supported net sulfate absorption (197.37 ± 13.61 nmol/cm2/h), but neither DRA nor PAT1 directly contributed to the unidirectional mucosal-to-serosal flux (JmsSO4), which was sensitive to serosal (but not mucosal) DIDS, dependent on Cl-, and regulated by cAMP. However, the absence of DRA significantly enhanced net sulfate absorption by one-third via a simultaneous rise in JmsSO4 and a 30 % reduction to the secretory s-to-m flux (JsmSO4). We propose that DRA, together with PAT1, contribute to JsmSO4 by mediating sulfate efflux across the apical membrane. Associated with increased ileal sulfate absorption in vitro, plasma sulfate was 61 % greater, and urinary sulfate excretion (USO4) 2.2-fold higher, in DRA-KO mice compared to WT controls, while USO4 was increased 1.8-fold in PAT1-KO mice. These alterations to sulfate homeostasis could not be accounted for by any changes to renal sulfate handling suggesting the source of this additional sulfate was intestinal. In summary, we characterized transepithelial sulfate fluxes across the mouse distal ileum demonstrating that DRA (and to a lesser extent, PAT1), secrete sulfate with significant implications for intestinal sulfate absorption and overall homeostasis.
Background: Long non-coding RNA (lncRNA) nuclear-enriched abundant transcript 1 (NEAT1) plays an important role in the pathogenesis and development of several types of cancer. However, the functional mechanism of NEAT1 in hepatocellular carcinoma (HCC) remains unclear. Methods: The NEAT1 and miR-129-5p expression in HCC tissues and cell lines were quantified by means of qPCR. The effects of NEAT1 expression inhibition or up-regulation in HCC cell lines were analyzed in terms of cell viability and apoptosis. Biological software was used to predict the binding sites of NEAT1 and miR-129-5p. The expression of the miR-129-5p target molecules VCP and IB was detected by using Western blotting. The effect of NEAT1 on tumor growth was observed in mouse models of transplanted hepatoma. Results: In the present study, it was concluded that the expression of NEAT1 was significantly increased in the HCC tissues and cell lines. Meanwhile, after down-regulating NEAT1 expression in HepG2/Huh7, the cell viability was significantly lowered, while the corresponding rate of apoptosis was significantly increased. Additionally, it was found that the NEAT1 and miR-129-5p expression showed a negative correlation in HCC tissues. It was further proved that there was a certain negative regulatory mechanism between NEAT1 and miR-129-5p, which was related to the expression of VCP and IB. The mouse model experiments confirmed that the interference with NEAT1 expression inhibited tumor growth. Conclusions: The study concluded that the overexpression of NEAT1 inhibited the expression of miR-129-5p by regulating VCP/IB, thereby promoting the proliferation of HCC cells.
Chronic diseases of the biliary tree (cholangiopathies) represent one of the major unmet needs in clinical hepatology and a significant knowledge gap in liver pathophysiology. The common theme in cholangiopathies is that the target of the disease is the biliary tree. After damage to the biliary epithelium, inflammatory changes stimulate a reparative response with proliferation of cholangiocytes and restoration of the biliary architecture, owing to the re-activation of a variety of morphogenetic signals. Chronic damage and inflammation, will ultimately result in pathologic repair, with generation of biliary fibrosis and clinical progression of the disease. The hallmark of pathologic biliary repair is the appearance of reactive ductular cells, a population of cholangiocyte-like epithelial cells of unclear and likely mixed origin, able to orchestrate a complex process that involves a number of different cell types, along with inflammatory and morphogenetic signals. Several questions remain open concerning the histogenesis of reactive ductular cells, their role in liver repair, their mechanism of activation, and the signals exchanged with the other cellular elements cooperating in the reparative process. This review, contributes to the ongoing debate, highlighting a number of new concepts emerging from the study of the pathophysiology of chronic progressive cholangiopathies, such as Congenital Hepatic Fibrosis, Biliary Atresia, and Alagille Syndrome.
The depth of our knowledge regarding mast cells has widened exponentially in the last 20 years. Once thought to be only important for allergy-mediated events, mast cells are now recognized to be important regulators of a number of pathological diseases. The revelation that mast cells can influence organs, tissues, and cells has increased interest in mast cell research during liver disease. The purpose of this review is to refresh the reader on the development, type, and location of mast cells and to review recent work that demonstrates the role of hepatic mast cells during diseased states. This review will focus primarily on liver diseases and mast cells during autoimmune disease, hepatitis, fatty liver disease, liver cancer and aging in the liver. Overall, these studies demonstrate the potential role that mast cells have in disease progression.
CD151, a member of the tetraspanin family of receptors, is a lateral organizer and modulator of activity of several families of transmembrane proteins. It has been implicated in the development and progression of several cancers but its role in chronic inflammatory disease is less well understood. Here we show that CD151 is up-regulated by distinct microenvironmental signals in a range of chronic inflammatory liver diseases and in primary liver cancer where it supports lymphocyte recruitment. CD151 was highly expressed in endothelial cells of the hepatic sinusoids and neovessels developing in fibrotic septa and tumour margins. Primary cultures of human hepatic sinusoidal endothelial cells (HSEC) expressed CD151 at the cell membrane and in intracellular vesicles. CD151 was up-regulated by vascular endothelial growth factor and HepG2 conditioned media but not by proinflammatory cytokines. Confocal microscopy confirmed that CD151 co-localised with the endothelial adhesion molecule/ immunoglobulin superfamily member, vascular cell adhesion molecule-1 (VCAM-1). Functional flow-based adhesion assays with primary human lymphocytes and HSEC demonstrated a 40% reduction of lymphocyte adhesion with CD151 blockade. Inhibition of lymphocyte adhesion was similar between VCAM-1 blockade and a combination of CD151/VCAM-1 blockade suggesting a collaborative role between the two receptors. These studies demonstrate that CD151 is up-regulated within the liver during chronic inflammation where it supports lymphocyte recruitment via liver endothelium. We propose that CD151 regulates the activity of VCAM-1 during lymphocyte recruitment to the human liver and could be a novel anti-inflammatory target in chronic liver disease and hepatocellular cancer prevention.
Irregular mitochondria structure and reduced ATP in some patients with IBD suggests that metabolic stress contributes to disease. Loss-of-function mutations in the NOD2 gene is a major susceptibility trait for IBD. Hence, we assessed if loss of NOD2 further impairs the epithelial barrier function instigated by disruption of mitochondrial ATP synthesis via the hydrogen ionophore, dinitrophenol (DNP). NOD2 protein (virtually undetectable in epithelia under basal conditions) was increased in T84 (human colon cell line) cells treated with non-invasive E. coli + DNP (16h). Increased intracellular bacteria in wild-type (WT) and NOD2 knock-down (KD) cells and colonoids from NOD2-/- mice was mediated by reactive oxygen species (ROS) and the MAPK ERK1/2 pathways as determined by co-treatment with the anti-oxidant, mitoTEMPO, and the ERK inhibitor, U0126: ROS was upstream of ERK1/2 activation. Despite increased E. coli in DNP-treated NOD2 KD compared to WT cells there were no differences in the internalization of fluorescent inert beads or dead E. coli particles. This suggests that lack of killing in the NOD2 KD cells was responsible for the increased numbers of viable intracellular bacteria; a conclusion supported by evidence of reduced autophagy in NOD2 KD T84 epithelia. Thus, in a two-hit hypothesis, decreased barrier function due to dysfunctional mitochondrial is amplified by lack of NOD2 in transporting enterocytes: subsequently, greater numbers of bacteria entering the mucosa would be a significant inflammatory threat especially since individuals with NOD2 mutations and have compromised macrophage and Paneth cell responses to bacteria.
The copper transporter ATP7B is essential for mammalian copper homeostasis. Mutations in ATP7B result in copper accumulation, especially in the liver, and cause Wilson disease (WD). The major role of hepatocytes in WD pathology is firmly established. It is less certain whether the excess Cu in hepatocytes is solely responsible for development of WD. To address this issue, we generated a mouse strain for Cre-mediated deletion of Atp7b and inactivated Atp7b selectively in hepatocytes. Atp7bHep mice accumulate copper in the liver, have elevated urinary copper, lack holo-ceruloplasmin, but show no liver disease for up to 30 weeks. Liver inflammation is muted and markedly delayed compared to the age-matched Atp7b-/- null mice, which show a strong type1 inflammatory response. Expression of metallothioneins is higher in Atp7bHep livers than in Atp7b-/- mice, suggesting better sequestration of excess copper. Characterization of purified cell populations also revealed that non-parenchymal cells in Atp7bHep liver maintain Atp7b expression, have normal copper balance, and remain largely quiescent. The lack of inflammation unmasked metabolic consequences of copper misbalance in hepatocytes. Atp7bHep animals weigh more than controls and have higher levels of liver triglycerides and HMG-CoA reductase. By 45 weeks, all animals develop liver steatosis on a regular diet. Thus, copper misbalance in hepatocytes dysregulates lipid metabolism, whereas development of inflammatory response in WD may depend on copper status of non-parenchymal cells. The implications of these findings for the cell-targeting WD therapies are discussed.
Altered gastric accommodation and intestinal morphology suggests impaired gastrointestinal (GI) transit may occur in the Wistar Kyoto (WKY) rat strain, as common in stress-associated functional GI disorders. Because changes in GI transit can alter microbiota composition, we investigated whether these are altered in WKY rats compared with the resilient Sprague Dawley (SD) rats under basal conditions, and characterized plasma lipid and metabolite differences. Bead transit was tracked by X-ray imaging to monitor: gastric emptying (GE; 4 h), small intestine (SI) transit (9 h) and large intestine transit (12 h). Plasma extracts were analysed by lipid and HILIC LC-MS. Cecal microbial composition was determined by Illumina MiSeq 16S rRNA amplicon sequencing and analysis using the QIIME pipeline. Stomach retention of beads was 77% for WKY compared with 35% for SD rats. GI transit was decreased by 34% (9 h), and 21% (12 h) in WKY compared with SD rats. Excluding stomach retention, transiting beads moved 29% further along the SI over 4-9 h for WKY compared with SD rats. Cecal Ruminococcus, Roseburia, and unclassified Lachnospiraceae genera were less abundant in WKY rats, whereas the minor taxa Dorea, Turicibacter, and Lactobacillus were higher. Diglycerides, triglycerides, phosphatidyl-ethanolamines and phosphatidylserine were lower in WKY rats, whereas cholesterol esters and taurocholic acids were higher. The unexpected WKY rat phenotype of delayed gastric emptying, yet rapid SI transit, was associated with altered lipid and metabolite profiles. The delayed gastric emptying of the WKY phenotype suggests this rat strain may be useful as a model for gastroparesis.
Background: Elevated integrated relaxation pressure (IRP) on esophageal high resolution manometry (HRM) identifies obstructive processes at the esophagogastric junction (EGJ). Aim: To determine if intrabolus pressure (IBP) can identify structural EGJ processes when IRP is normal. Methods: In this observational cohort study, adult patients with dysphagia and undergoing HRM were evaluated for endoscopic evidence of structural EGJ processes (strictures, rings, hiatus hernia) in the setting of normal IRP. HRM metrics (IRP; distal contractile integral, DCI; distal latency, DL; IBP; and EGJ contractile integral, EGJ-CI) were compared between 74 patients with structural EGJ findings (62.8 ± 1.6 yr, 67.6% F), 27 patients with normal EGD (52.9 ± 3.2 yr, 70.3% F), and 21 healthy controls (27.6 ± 0.6 yr, 52.4% F). Findings were validated in 85 consecutive symptomatic patients to address clinical utility. Results: In the primary cohort, mean IBP (18.4 ± 0.9 mmHg) was higher with structural EGJ findings compared to dysphagia with normal EGD (13.5 ± 1.1 mmHg, p=0.002) and healthy controls (10.9 ± 0.9 mmHg, p<0.001). However, mean IRP, DCI, DL, and EGJ-CI were similar across groups (p>0.05 for each comparison). During multiple rapid swallows, IBP remained higher in the structural findings group compared to controls (p=0.02). Similar analysis of the prospective validation cohort confirmed IBP elevation in structural EGJ processes but correlation with dysphagia could not be demonstrated. Conclusions: Elevated IBP predicts the presence of structural EGJ processes even when IRP is normal, but correlation with dysphagia is suboptimal.
Parietal cells play a fundamental role in stomach maintenance, not only by creating a pathogen-free environment through the production of gastric acid, but also by secreting growth factors important for homeostasis of the gastric epithelium. The gastrointestinal hormone gastrin is known to be a central regulator of both parietal cell function and gastric epithelial cell proliferation and differentiation. Our previous gene expression profiling studies of mouse stomach identified parathyroid hormone-like hormone (PTHLH) as a potential gastrin-regulated gastric growth factor. Although PTHLH is commonly overexpressed in gastric tumors, its normal expression, function and regulation in the stomach are poorly understood. In this study we used pharmacologic and genetic mouse models as well as human gastric cancer cell lines to determine the cellular localization and regulation of this growth factor by the hormone gastrin. Analysis of PthlhLacZ/+ knock-in reporter mice localized Pthlh expression to parietal cells in the gastric corpus. Regulation by gastrin was demonstrated by increased Pthlh mRNA abundance after acute gastrin treatment in wild type mice and reduced expression in gastrin-deficient mice. PTHLH transcripts were also observed in normal human stomach as well as in human gastric cancer cell lines. Gastrin treatment of AGS-E gastric cancer cells induced a rapid and robust increase in numerous PTHLH mRNA isoforms. This induction was largely due to increased transcriptional initiation, although analysis of mRNA half-life showed that gastrin treatment also extended the half-life of PTHLH mRNA, suggesting that gastrin regulates expression by both transcriptional and post-transcriptional mechanisms.
Serotonin (5-hydroxytryptamine, 5-HT), an important neurotransmitter and a paracrine messenger in the gastrointestinal (GI) tract, regulates intestinal secretion by its action primarily on 5-HT3 and 5-HT4 receptors. Recent studies highlight the role of gut microbiota in 5-HT biosynthesis. In this study we determine if human-derived gut microbiota affect host secretory response to 5-HT and 5-HT receptor expression. We used proximal colonic mucosa-submucosa preparation from age matched Swiss Webster germ free (GF) and humanized (HM; ex-GF colonized with human gut microbiota) mice. 5-HT evoked a significantly greater increase in short circuit current (Isc) in GF compared to HM mice. Additionally, 5-HT3 receptor mRNA and protein expression was significantly higher in GF compared to HM mice. Ondansetron, a 5-HT3 receptor antagonist, inhibited 5-HT evoked Isc in GF mice but not in HM mice. Furthermore, a 5-HT3 receptor selective agonist, 2-methyl 5-hydroxytryptamine hydrochloride evoked a significantly higher Isc in GF compared to HM mice. Immunohistochemistry in 5-HT3A-GFP mice localized 5-HT3 receptor expression to enterochromaffin cells in addition to nerve fibers. The significant difference in 5-HT evoked Isc between GF and HM mice persisted in the presence of tetrodotoxin (TTX), but was lost after ondansetron application in the presence of TTX. Application of acetate (10 mM) significantly lowered 5-HT3 receptor mRNA in GF mice colonoids. We conclude that host secretory response to 5-HT may be modulated by gut microbiota regulation of 5-HT3 receptor expression via acetate production. Epithelial 5-HT3 receptor may function as a mediator of gut microbiota driven change in intestinal secretion.
Vasoactive intestinal peptide (VIP) is an endogenous neuropeptide with a broad array of physiological functions in many organs including the intestine. Its actions are mediated via G- protein coupled receptors and VPAC1 is the key receptor responsible for majority of VIP's biological activity. The distribution of VPAC1 along the length of the gastrointestinal tract and its sub cellular localization in intestinal epithelial cells has not been fully characterized. The current studies were undertaken to determine VPAC1 distribution and localization so that VIP based therapies can be targeted to specific regions of the intestine. The results indicated that the mRNA levels of VPAC1 showed an abundance pattern of colon> ileum> jejunum in the mouse intestine. In parallel, the VPAC1 protein levels were higher in the mouse colon, followed by the ileum and jejunum. Immuno-fluorescence studies in mouse colon demonstrated that the receptor was specifically localized to the luminal surface as evident by co-localization with the apical marker villin but not with the basolateral marker Na+/K+ ATPase. In the human intestine, VPAC1 mRNA expression exhibited a distribution similar to mouse intestine and was highest in the sigmoid colon. Furthermore, in the human colon, VPAC1 also showed predominantly apical localization. The physiological relevance of the expression and apical localization of VPAC1 remains elusive. We speculate that apical VPAC1 in intestinal epithelial cells may have relevance in recognizing secreted peptides in the intestinal lumen and therefore, supports the feasibility of potential therapeutic and targeting use of VIP formulations via oral route to treat GI diseases.
Mice fed a methionine and choline deficient (MCD) diet develop steatohepatitis that recapitulates key features of nonalcoholic steatohepatitis (NASH) in humans. Phosphatidylcholine is the most abundant phospholipid in the surfactant monolayer that coats and stabilizes lipid droplets within cells, and choline is required for its major biosynthetic pathway. Phosphatidylcholine-transfer protein (PC-TP), which exchanges phosphatidylcholine among membranes, is enriched in hepatocytes. PC-TP also regulates fatty acid metabolism through interactions with thioesterase superfamily member 2. We investigated the contribution of PC-TP to steatohepatitis induced by the MCD diet. Pctp-/- and WT control mice were fed the MCD diet for 5 w and were then sacrificed for histopathologic and biochemical analyses, as well determinations of mRNA and protein expression. Whereas all mice developed steatohepatitis, plasma alanine aminotransferase and aspartate aminotransferase activities were only elevated in WT mice, indicating that Pctp-/- mice were protected from MCD diet-induced hepatocellular injury. Reduced hepatotoxicity due to the MCD diet in the absence of PC-TP expression was further evidenced by decreased activation of c-jun and reduced plasma concentrations of fibroblast growth factor 21. Despite similar total hepatic concentrations of phosphatidylcholines and other lipids, the relative abundance of microvesicular lipid droplets within hepatocytes was reduced in Pctp-/- mice. Considering that the formation of larger lipid droplets may serve to protect against lipotoxicity in NASH, our findings suggest a pathogenic role for PC-TP that could be targeted in the management of this condition.
Inflammatory bowel diseases (IBD) are a group of common and debilitating chronic intestinal disorders for which currently-available therapies are often unsatisfactory. The naturally-occurring secondary bile acid, ursodeoxycholic acid (UDCA), has well-established anti-inflammatory and cytoprotective actions and may therefore be effective in treating IBD. Here, we aimed to investigate regulation of colonic inflammatory responses by UDCA and to determine the potential impact of bacterial metabolism on its therapeutic actions. The anti-inflammatory efficacy of UDCA, a non-metabolisable analogue, 6-methyl-UDCA (6-MUDCA), and its primary colonic metabolite, lithocholic acid (LCA), were assessed in the murine DSS model of mucosal injury. The effects of bile acids on cytokine release (TNF-α, IL-6, Il-1β, IFN-) from cultured colonic epithelial cells and mouse colonic tissue in vivo were investigated. Luminal bile acids were measured by GC-MS. UDCA attenuated release of proinflammatory cytokines from colonic epithelial cells in vitro and was protective against the development of colonic inflammation in vivo. In contrast, although 6-MUDCA mimicked the effects of UDCA on epithelial cytokine release in vitro, it was ineffective in preventing inflammation in the DSS model. In UDCA-treated mice, LCA became the most common colonic bile acid. Finally, LCA treatment more potently inhibited epithelial cytokine release and protected against DSS-induced mucosal inflammation than did UDCA. These studies identify a new role for the primary metabolite of UDCA, LCA, in preventing colonic inflammation and suggest that microbial metabolism of UDCA is necessary for the full expression of its protective actions.
Determination of fecal pancreatic elastase content by ELISA is a reliable, non-invasive clinical test for assessing exocrine pancreatic function. Despite the widespread use of commercial tests, their exact molecular targets remain poorly characterized. This study was undertaken to clarify which human pancreatic elastase isoforms are detected by the ScheBo Pancreatic Elastase 1 Stool Test and whether naturally-occurring genetic variants influence the performance of this test. Using recombinantly expressed and purified human pancreatic proteinases we found that the test specifically measured chymotrypsin-like elastases 3A and 3B (CELA3A and CELA3B) while CELA2A was not detected. Inactive proelastases, active elastases and autolyzed forms were detected with identical efficiency. CELA3B gave approximately four times higher signal than CELA3A and we identified Glu154 in CELA3B as the critical determinant of detection. Common genetic variants of CELA3A and CELA3B had no effect on ELISA signal strength with the exception of the CELA3B variant W79R which increased detection by 1.4-fold. Finally, none of the human trypsin and chymotrypsin isoforms were detected. We conclude that the ScheBo Pancreatic Elastase 1 Stool Test is specific for human CELA3A and CELA3B, with most of the ELISA signal attributable to CELA3B.
Colonic cyclic motor patterns (CMPs) have been hypothesized to act as a brake to limit rectal filling. However, the spatiotemporal profile of CMPs, including anatomical origins and distributions, remains unclear. This study characterized colonic CMPs using high-resolution (HR) manometry (72 sensors, 1 cm resolution) and their relationship with proximal antegrade propagating events. Nine healthy volunteers were recruited. Recordings were performed over 4 h, with a 700 kcal meal given after 2 h. Propagating events were visually identified and analyzed by pattern, origin, amplitude, extent of propagation, velocity, and duration. Manometric data were normalized using anatomical landmarks identified on abdominal radiographs. These were mapped over a three-dimensional anatomical model. CMPs comprised a majority of detected propagating events. Most occurred postprandially and were retrograde propagating events (84.9 ± 26.0 retrograde vs. 14.3 ± 11.8 antegrade events per 2 h, p = 0.004). The dominant sites of initiation for retrograde CMPs were in the rectosigmoid region, with patterns proximally propagating by a mean distance of 12.4 ± 0.3 cm. There were significant differences in the characteristics of CMPs depending on the direction of travel and site of initiation. Association analysis showed that proximal antegrade propagating events occurred independently of CMPs. This study accurately characterized CMPs with anatomical correlation. CMPs were unlikely to be triggered by proximal antegrade propagating events in our study context. However, the distal origin and prominence of retrograde CMPs could still act as a mechanism to limit rectal filling and support the theory of a "rectosigmoid brake."
The magnitude, temporal dynamics, and physiologic effects of intestinal microbiome responses to physiologic stress are poorly characterized. This study used a systems biology approach and multiple-stressor military training environment to determine the effects of physiologic stress on intestinal microbiota composition and metabolic activity, and intestinal permeability (IP). 73 Soldiers were provided three rations/d with or without protein- or carbohydrate-based supplements during a four day cross-country ski march (STRESS). IP was measured before and during STRESS. Blood and stool samples were collected before and after STRESS to measure inflammation, stool microbiota, and stool and plasma global metabolite profiles. IP increased 62%±57% (mean±SD, P<0.001) during STRESS independent of diet group, and was associated with increased inflammation. Intestinal microbiota responses were characterized by increased α-diversity, and changes in the relative abundance of >50% of identified genera, including increased abundances of less dominant taxa at the expense of more dominant taxa such as Bacteroides. Changes in intestinal microbiota composition were linked to 23% of metabolites that were significantly altered in stool after STRESS. Pre-STRESS Actinobacteria relative abundance, and changes in serum IL-6 and stool cysteine concentrations, collectively, accounted for 84% of the variability in the change in IP. Findings demonstrate that a multiple-stressor military training environment induced increases in IP that were associated with alterations in markers of inflammation, and with intestinal microbiota composition and metabolism. Observed associations between IP, the pre-stress microbiota, and microbiota metabolites suggest targeting the intestinal microbiota could provide novel strategies for preserving IP during physiologic stress.
Objective: Gastroesophageal reflux disease (GERD) clinically predisposes to columnar Barrett's metaplasia (BM) in the distal esophagus. We demonstrate evidence supporting cellular origin of BM from reprograming or trans-commitment of resident normal esophageal squamous (NES) epithelial cells in response to acid and bile exposure using an in vitro cell culture model. Design: The hTERT-immortalized NES cell line, NES-B10T, was exposed 5 min/day to Acid and Bile (A+B) mixture for 30 weeks. Morphological changes, mRNA and protein expression levels for inflammatory marker, COX-2; lineage determining transcription factors, TAp63 (Squamous), CDX2 and SOX9 (both columnar); and markers of columnar lineage, Villin, Muc-2, CK8, and mAb Das-1 (incomplete phenotype of intestinal metaplasia) were assessed every 10 weeks. Results: Markers of columnar lineage, and inflammation increased progressively, while squamous lineage determining transcriptional factors were significantly decreased both at the mRNA and/or protein level in the NES-B10T cells at/after acid and bile treatment for 30 weeks. Distinct modifications in morphological features were only observed at/after 30 weeks of A+B exposure. These changes acquired by the NES-B10T 30 week cells were retained even after cessation of A+B exposure for at least 3 weeks. Conclusions: This study provides evidence that chronic exposure to physiological components of gastric refluxate led to repression of the discernable squamous transcriptional factors and activation of latent columnar transcriptional factors. This reflects the alteration in lineage commitment of the precursor-like bi-phenotypic, NES-B10T cells in response to A+B exposure as the possible origin of BM from the resident NES cells.
Despite the fact that many membrane proteins carry extracellular glycans, little is known about whether the glycan chains also affect protein function. We recently demonstrated that the proton-coupled oligopeptide transporter 1 (PEPT1) in the intestine is glycosylated at six asparagine residues (N50, N406, N439, N510, N515, N532). Mutagenesis-induced disruption of the individual N-glycosylation site N50, which is highly conserved among mammals, was detected to significantly enhance the PEPT1 mediated inward transport of peptides. Here, we show for the murine protein, that the inhibition of glycosylation at sequon N50 by substituting N50 with glutamine, lysine or cysteine, or by replacing S52 with alanine, equally altered PEPT1 transport kinetics in oocytes. Further, we provide evidence that the uptake of [14C]-glycyl-sarcosine in immortalized murine small intestinal (Mode-K) or colonic epithelial (PTK-6) cells stably expressing the PEPT1 transporter N50Q is also significantly increased relative to the wild type protein. By using electrophysiological recordings and tracer flux studies, we further demonstrate that the rise in transport velocity observed for PEPT1 N50Q is bidirectional. In line with these findings, we show that attachment of biotin derivatives, comparable in weight to 2-4 monosaccharides, to the PEPT1 N50C transporter slows down the transport velocity. In addition, our experiments provide strong evidence that glycosylation of PEPT1 confers resistance against proteolytic cleavage by proteinase K, while a remarkable intrinsic stability against trypsin, even in absence of N-linked glycans, was detected.
Nutrient sensing triggers responses by the gut-brain axis modulating hormone release, feeding behavior and metabolism that become dysregulated in metabolic syndrome and some cancers. Except for absorptive enterocytes and secretory enteroendocrine cells, the ability of many intestinal cell types to sense nutrients is still unknown, hence we hypothesized that progenitor stem cells (ISC) possess nutrient sensing ability inherited by progenies during differentiation. We directed via modulators of Wnt and Notch signaling, differentiation of precursor mouse intestinal crypts into specialized organoids each containing ISC, enterocyte, goblet or Paneth cells at relative proportions much higher than in situ as determined by mRNA expression and immunocytochemistry of cell type biomarkers. We identified nutrient sensing cell type(s) by increased expression of fructolytic genes in response to a fructose challenge. Organoids comprised primarily of enterocytes, Paneth or goblet but not ISC, cells responded specifically to fructose without affecting nonfructolytic genes. Sensing was independent of Wnt and Notch modulators, and of glucose concentrations in the medium, but required fructose absorption and metabolism. More mature enterocyte- and goblet-enriched organoids exhibited stronger fructose responses. Remarkably, enterocyte organoids, upon forced dedifferentiation to reacquire ISC characteristics, exhibited a markedly extended lifespan and retained fructose sensing ability, mimicking responses of some dedifferentiated cancer cells. Using an innovative approach, we discovered that nutrient sensing is likely repressed in progenitor ISCs then irreversibly derepressed during specification into sensing-competent absorptive or secretory lineages, the surprising capacity of Paneth and goblet cells to detect fructose, and the important role of differentiation in modulating nutrient sensing.
Anoctamin1 (Ano1, TMEM16A) is a calcium-activated chloride channel specifically expressed in interstitial cells of Cajal (ICC) of the gastrointestinal (GI) tract muscularis propria. Ano1 is necessary for normal electrical slow waves and ICC proliferation. The full length human Ano1 sequence includes an additional exon, exon "0," at the N-terminus. Ano1 with exon "0" (Ano1(0)) had a lower EC50 for intracellular calcium ([Ca2+]i) and faster chloride current (ICl) kinetics. The Ano1 alternative splice variant with segment "c" encoding exon 13 expresses on the first intracellular loop four additional amino acid residues, EAVK, which alter ICl at low [Ca2+]i. Exon 13 is expressed in 75-100% of Ano1 transcripts in most human tissues but only 25% in human stomach. Our aim was to determine the effect of EAVK deletion on Ano1(0) ICl parameters. By voltage-clamp electrophysiology, we examined ICl in HEK293 cells transiently expressing Ano1(0) with or without the EAVK sequence (Ano1(0)EAVK). The EC50 values of activating and deactivating ICl for [Ca2+]i was 438±7 and 493±9 nM for Ano1(0) but higher for Ano1(0)EAVK at 746±47 and 761±26 nM, respectively. Meanwhile, the EC50 values for the ratio of instantaneous to steady-state ICl were not different between variants. Congruently, the time constant of activation was slower for Ano1(0)EAVK than Ano1(0) currents at intermediate [Ca2+]i. These results suggest that EAVK decreases the calcium sensitivity of Ano1(0) current activation and deactivation by slowing activation kinetics. Differential expression of EAVK in human stomach may function as a switch to increase sensitivity to [Ca2+]i via faster gating of Ano1.
Background: Esophageal hypersensitivity is important in gastro-esophageal reflux disease (GERD) patients refractory to acid-suppressive therapy. Stress affects visceral sensitivity and exacerbates heartburn in GERD. Peripheral CRH is a key mediator of the gut stress response. We hypothesize that CRH increases esophageal sensitivity and alters esophageal motility in health. Methods: Esophageal sensitivity to thermal, mechanical, electrical and chemical stimuli was assessed in 14 healthy subjects after placebo or CRH (100μg IV). Perception scores were assessed for first perception, pain perception threshold (PPT) and pain tolerance threshold (PTT). Esophageal motility was investigated by high resolution impedance manometry, before and after CRH and evaluated by distal contractile integral (DCI) and intrabolus pressure (IBP). Pressure flow analysis assessed bolus clearance (impedance ratio), degree of pressurization needed to propel bolus onward (IBP slope) and pressure flow (pressure flow index, PFI). Stress and mood were assessed during the study. Results: Sensitivity to mechanical distention was increased after CRH compared to placebo (PPT p=0.0023; PTT p=0.0253). CRH had no influence on the other stimulations. DCI was increased for all boluses (liquid, p=0.0012; semi-solid, p=0.0017; solid, p=0.0107). Impedance ratio for liquid (p<0.0001) and semi-solid swallows (p=0.0327) decreased after CRH. IBP slope increased after CRH for semi-solid (p=0.0041) and solid (p=0.0003) swallows. PFI increased for semi-solid (p=0.0017) and solid swallows (p=0.0031). Conclusion: CRH increased esophageal sensitivity to mechanical distention, not to the other stimulation modalities. CRH increased esophageal contractility and tone, decreased LES relaxation, increased esophageal bolus pressurization, improved esophageal bolus clearance and increased pressure flow.
An important characteristic of intrauterine growth restricted (IUGR) neonate is the impaired intestinal barrier function. Using a pig model, this study was conducted to identify the responsible miRNA for the intestinal damage in IUGR neonates through comparing the miRNA profile of IUGR and normal porcine neonates, and to investigate the regulation mechanism. Compared to the normal ones, we identified 83 upregulated and 76 downregulated miRNAs in the jejunum of IUGR pigs. Notably, IUGR is associated with profoundly increasesd miR-29 family and decreased expression of extracellular matrix (ECM) and tight junction (TJ) proteins in the jejunum. Furthermore, in vitro study using porcine intestinal epithelial cell line (IPEC-1) showed that inhibition of miR-29a expression could improve the monolayer integrity by increasing cell proliferation and transepithelial resistance. Also, overexpression/inhibition of miR-29a in IPEC-1 cells can suppress/increase the expression of integrin β1, collagen I, collagen IV, fibronectin and claudin 1, both at transcriptional and translational levels. Subsequent luciferase reporter assay confirmed a direct interaction between miR-29a and the 3'-untranslated regions of these genes. In conclusion, this study reveals that IUGR impaired intestinal barrier function is associated with downregulated ECM and TJ proteins expression mediated by the upregulation of miR-29a. Keywords: miRNA microarray; extracellular matrix; tight junction; intestinal permeability; IUGR
Microbial dysbiosis and increased intestinal permeability is a target for prevention or reversal of weight gain in high-fat (HF) diet-induced obesity (DIO). Prebiotic milk oligosaccharides (MO) have been shown to benefit the host intestine, but have not been used in DIO. We hypothesized that supplementation with bovine MO would prevent the deleterious effect of HF diet on the gut microbiota and intestinal permeability, and attenuate development of the obese phenotype. C57BL/6 mice were fed a control diet (LF), HF (40% fat/kcal), or HF + prebiotic (6%/Kg BMO or inulin) for 1, 3 or 6 weeks. Gut microbiota and intestinal permeability were assessed in the ileum, cecum and colon. Addition of BMO to the HF diet significantly attenuated weight gain, decreased adiposity and decreased caloric intake; inulin supplementation also lowered weight gain and adiposity, but this did not reach significance. BMO and inulin completely abolished the HF diet-induced increase in paracellular and transcellular permeability in the small and large intestine. Both BMO and inulin increased abundance of beneficial microbes Bifidobacterium and Lactobacillus in the ileum. However, inulin supplementation altered phylogenetic diversity and decreased species richness. We conclude that addition of BMO to the HF diet completely prevented increases in intestinal permeability and microbial dysbiosis and was partially effective to prevent weight gain in DIO.
Purpose: Hydrogen Sulfide (H2S) is an endogenous gasotransmitter that has vasodilatory properties. It may be a novel therapy for intestinal I/R. We hypothesized that: 1) H2S would improve post-ischemic survival, mesenteric perfusion, mucosal injury, and inflammation compared to vehicle, and 2) the benefits of H2S would be mediated through endothelial nitric oxide. Methods: C57Bl6J wild type (WT) and endothelial nitric oxide synthase knock out (eNOS KO) mice were anesthetized and a midline laparotomy performed. Intestines were eviscerated, the small bowel mesenteric root identified, and baseline intestinal perfusion determined using Laser Doppler. Intestinal ischemia was established by temporarily occluding the superior mesenteric artery. Following ischemia, the clamp was removed and the intestines were allowed to recover. Sodium hydrosulfide (NaHS) in 250µl of PBS or the vehicle was injected into the peritoneum. Animals were allowed to recover and were assessed for survival, mesenteric perfusion, mucosal injury, and intestinal cytokines. P-values less than 0.05 were significant. Results: H2S improved survival at lower doses, while the high dose resulted in immediate demise. Mesenteric perfusion and mucosal injury scores were improved following I/R with low and mid-range H2S therapy. In the setting of eNOS ablation, there was no improvement in mesenteric perfusion or mucosal injury. H2S also resulted in lower levels of intestinal cytokines. Conclusion: Although high levels of hydrogen sulfide can result in mortality, appropriate doses can improve mesenteric perfusion and intestinal mucosal injury following I/R. The benefits of H2S appear to be mediated through endothelial nitric oxide dependent pathways.
Dysphagia is caused not only by neurological and/or structural damage but also by medication. We hypothesized memantine, dextromethorphan, diazepam and baclofen, all commonly used drugs with central sites of action, may regulate swallowing function. Swallows were evoked by upper airway (UA)/pharyngeal distension, punctate mechanical stimulation using a von Frey filament, capsaicin or distilled water (DW) applied topically to the vocal folds, and electrical stimulation of a superior laryngeal nerve (SLN) in anesthetized rats and were documented by recording electromyographic activation of the suprahyoid and thyrohyoid muscles and by visualizing laryngeal elevation. The effects of intraperitoneal or topical administration of each drug on swallowing function was studied. Systemic administration of diazepam and baclofen, but not memantine or dextromethorphan, inhibited swallowing evoked by mechanical, chemical and electrical stimulation. Both benzodiazepines and GABAA receptor antagonists diminished the inhibitory effects of diazepam while a GABAB receptor antagonist diminished the effects of baclofen. Topical applied diazepam or baclofen was without effect on swallowing. These data indicate that diazepam and baclofen act centrally to inhibit swallowing in anesthetized rats.
Background: Current understanding of pharyngeal motor function remains incomplete. Among the remaining gaps of knowledge in this regard is the magnitude of variability of pharyngeal peristaltic pressure amplitude. While variability can pose difficulty in interpretation of manometric findings its magnitude can inform the operational range and reserve of the pharyngeal contractile function, Aims: To define the intra-, inter-subject and inter-session variability of select pharyngeal manometric parameters and, using this information, determine the number of swallow repetitions for acquiring reliable pharyngeal manometric data. Methods: We recorded pharyngeal peristalsis in 10 healthy subjects (age: 50 ± 25 yrs, 5 female) by high-resolution manometry during two separate sessions of 20 sequences of 0.5 ml water swallows. Results: Two-way ANOVA showed significant variation in the mean peak peristaltic pressure value across sites (p<0.0001) as well as within the data at each site (p<0.0001). Similarly, the pharyngeal contractile integral (PhCI) exhibited significant inter- (p=0.003) and intra-subject (p<0.001) variability. The Shapiro-Wilk normality test showed mixed results, in that some sites showed normally distributed data while others did not. A robust Monte Carlo simulation showed that the nominal sample size was different for various tested metrics. For a power of 0.8, commonly accepted as an adequate threshold for acceptable statistical power, the optimal sample size for various peristaltic parameters ranged between3-15. Conclusions: There is significant intra- and inter- subject variability in site-specific and integrated parameters of pharyngeal peristalsis. The observed variance indicates a significant operational range and reserve in pharyngeal contractile function while necessitating parameter-specific sample size for reliable results.
Although melatonin attenuates the increases in inflammatory mediators and reduces organ injury during trauma-hemorrhage, the mechanisms remain unclear. This study explored whether melatonin prevents liver injury after trauma-hemorrhage through the p38 mitogen-activated protein kinase (MAPK)-dependent, inducible nitrite oxide (iNOS)/hypoxia-inducible factor (HIF)-1α pathway. After a 5-cm midline laparotomy, male rats underwent hemorrhagic shock (mean blood pressure approximately 40 mmHg for 90 min) followed by fluid resuscitation. At the onset of resuscitation, rats were treated with vehicle, melatonin (2 mg/kg), melatonin plus p38 MAPK inhibitor SB203580 (2 mg/kg), or melatonin plus melatonin receptor antagonist luzindole (2.5 mg/kg). At 2 hours after trauma-hemorrhage, the liver tissue myeloperoxidase activity, malondialdehyde, adenosine triphosphate, serum alanine aminotransferase and asparate aminotransferase levels were significantly increased compared with sham-operated control. Trauma-hemorrhage resulted in a significant decrease in the p38 MAPK activation compared with that in the sham-treated animals. Administration of melatonin after trauma-hemorrhage normalized liver p38 MAPK phosphorylation, iNOS and HIF-1α expression, and attenuated cleaved caspase 3 and receptor interacting protein kinase-1 levels. Co-administration of SB203580 or luzindole abolished the melatonin-mediated attenuation of the trauma-hemorrhage-induced increase of iNOS/HIF-1α protein expression and liver injury markers. Taken together, our results suggest that melatonin prevents trauma-hemorrhage-induced liver injury in rats, at least in part, through melatonin receptor-related, p38 MAPK-dependent iNOS/HIF-1α pathway.
The sequence of events that lead to inflammation and fibrosing nonalcoholic steatohepatitis (NASH) are incompletely understood. We investigated the chronology of whole body, tissue and cellular events that occur during the evolution of diet-induced NASH. Methods: Male C57Bl6 mice were assigned to a fast food (FF, high calorie, high cholesterol, high fructose) or standard chow (SC) diet for 36 weeks (W). Liver histology, body composition, mitochondrial respiration, metabolic rate, gene expression and hepatic lipid content were analyzed. Results: Insulin resistance (HOMA IR) increased ten-fold after 4W. Fibrosing NASH was fully established by 16W. Total hepatic lipids increased by 4W and remained 2-3 fold increased throughout. Hepatic triglycerides declined from six-fold increase at 8W to three-fold increase by 36W. In contrast, hepatic cholesterol levels steadily increased from baseline at 8W to two-fold by 36W. Mitochondrial oxygen flux rates of FF mice diet were persistently lower with all the tested substrates (13-276pmoles/CS µmol/min) than SC mice (17-394pmoles/ml/CS µmol/min) and was accompanied by decreased mitochondrial nuclear gene copy number ratios after 4W. Metabolic rate was lower in FF mice. Mitochondrial glutathione was significantly decreased only at 24W in FF mice. Conclusion: The evolution of NASH is multiphasic, particularly in terms of hepatic lipid composition. Mitochondrial dysfunction and depletion occur after the histological features of NASH are apparent. Collectively these observations provide a unique overview of the sequence of changes that co-evolve with the histological evolution of NASH.
Intestinal inorganic phosphate (Pi) transport seems to be mainly mediated by the sodium/Pi-cotransporter NaPi2b. To verify this role, we have studied the combined effects of pH, phosphonoformate, and Pi deprivation on intestinal Pi transport. Rats were fed, ad libitum, three fodders containing 1.2, 0.6, or 0.1% Pi for 1, 5, or 10 days. Pi deprivation (0.1%) increased both sodium-activated and sodium-independent Pi transport in brush-border membrane vesicles from the duodenum and jejunum, for all three times. Alkaline pH (range 6.0-8.5) inhibited Pi transport, despite the increasing concentration of HPO4= (NaPi2b substrate), while acidity increased transport when the concentration of PiT1/PiT2 substrate, H2PO4-, was at its highest. The effect of Pi deprivation was also maximal at acid pH, but both basal and upregulated transport were inhibited (70%) with phosphonoformate, an inhibitor of NaPi2b. PiT2 and NaPi2b protein abundance increased after 24 hours of Pi deprivation in the duodenum, jejunum, and ileum, while PiT1 required 5-10 days in the duodenum and jejunum. Therefore, while transporter expressions are partially correlated with Pi transport adaptation, the pH effect precludes NaPi2b, and PFA precludes PiT1 and PiT2 as the main transporters. Transport and transporter expression was also inconsistent when feeding was limited to 4 hours daily, because the 1.2% Pi diet paradoxically increased Pi transport in the duodenum and jejunum, but NaPi2b and PiT1 expressions only increased with the 0.1% diet. These findings suggest the presence of a major transporter that carries H2PO4- and is inhibited by phosphonoformate, and its expression correlates with Pi transport
Dicer processes microRNAs (miRs) into active forms in a wide variety of tissues including the liver. To determine the role of Dicer in liver regeneration, we performed a series of in vivo and in vitro studies in a murine 2/3 hepatectomy model. Dicer was downregulated after 2/3 hepatectomy, and loss of Dicer inhibited liver regeneration associated with decreased cyclin A2 and miR-221, and increased levels of the cell cycle inhibitor p27. In vitro, miR-221 inhibited p27 production in primary hepatocytes and increased hepatocyte proliferation. Specific reconstitution of miR-221 in hepatocyte-specific Dicer null mice inhibited p27 and restored liver regeneration. In wild type mice, targeted inhibition of miR-221 using a cholesterol-conjugated anti-miR-221 inhibited hepatocyte proliferation after 2/3 hepatectomy. These results identify Dicer production of miR-221 as an essential component of a miRNA-dependent mechanism for suppression of p27 that controls the rate of hepatocyte proliferation after partial hepatectomy.
Enteropathogenic E. coli (EPEC), one of the diarrheagenic E. coli pathotypes, is among the most important food-borne pathogens infecting children worldwide. Inhibition of serotonin transporter (SERT), that regulates extracellular availability of serotonin (5-HT), has been previously implicated in EPEC-associated diarrhea. EPEC was shown to inhibit SERT via activation of protein tyrosine phosphatases (PTPase), albeit the specific PTPase involved is not known. Current studies aimed to identify EPEC activated PTPase and its role in SERT inhibition. Infection of Caco-2 monolayers with EPEC strain E2348/69 for 30 min increased the activity of SHP2 (Src-Homology-2 Domain containing PTPase) but not SHP1 or PTP1B. Similarly, western blot studies showed increased tyrosine phosphorylation of SHP2 indicative of its activation. Concomitantly, EPEC infection decreased SERT tyrosine phosphorylation levels. This was associated with increased interaction of SHP2 with SERT as evidenced by co-immunoprecipitation studies. To examine whether SHP2 directly influences SERT phosphorylation status or function, SHP2 cDNA plasmid constructs (wild type, constitutively active or dominant negative) were overexpressed in Caco-2 cells by Amaxa electroporation. In the cells overexpressing constitutively active SHP2, SERT polypeptide showed complete loss of tyrosine phosphorylation. In addition, there was a decrease in SERT function as measured by Na+Cl--sensitive 3[H] 5-HT uptake and an increase in association of SERT with SHP2 in Caco-2 cells expressing constitutively active SHP2 compared to dominant negative SHP2. Our data demonstrate that intestinal SERT is a target of SHP2 and reveal a novel mechanism by which a common food-borne pathogen utilizes cellular SHP2 to inhibit SERT.
Statins are the most widely prescribed medications worldwide for the treatment of hypercholesterolemia. They inhibit the activity of 3-hydroxy-3-methylglutaryl co enzyme A reductase (HMG-R) an enzyme involved in cholesterol synthesis in higher organisms and in isoprenoid biosynthesis in some bacteria. We hypothesized that statins may influence the microbial community in the gut through either direct inhibition or indirect mechanisms involving alterations to host responses. We therefore examined the impact of rosuvastatin (RSV) on the community structure of the murine gastrointestinal microbiota. RSV was orally administered to mice and the effects upon the gut microbiota, host bile acid profiles and markers of inflammation were analysed. RSV significantly influenced the microbial community in both the caecum and faeces, causing a significant decrease in alpha diversity in the caecum and resulting in a reduction of several physiologically relevant bacterial groups. RSV treatment of mice significantly affected bile acid metabolism and impacted upon expression of inflammatory markers known to influence microbial community structure (including RegIII and Camp) in the gut. This study suggests that a commonly used statin (RSV) leads to an altered gut microbial composition in normal mice with attendant impacts upon local gene expression profiles, a finding which should prompt further studies to investigate the implications of statins for gut microbiota stability and health in humans.
Non-erosive reflux disease (NERD) is a highly prevalent phenotype of the gastroesophageal reflux disease (GERD). In this study, we developed a novel murine model of NERD in mice with microscopic inflammation and impairment in the epithelial esophageal barrier. Female Swiss mice were subjected to the following surgical procedure: the transitional region between the forestomach and the glandular portion of the stomach was ligated, and a nontoxic ring was placed around the duodenum near the pylorus. The control group underwent sham surgery. The animals were sacrificed at 1, 3, 7, and 14 days post-surgery. Survival and body weight were monitored daily. Esophageal wet weight, macroscopic lesion, histopathological alterations, myeloperoxidase (MPO) activity, cytokines levels, transepithelial electrical resistance (TEER), and mucosal permeability were evaluated. The survival rate was 78% at 14 days, with mild loss in body weight. Surgery did not induce erosive esophagitis, but instead induced microscopic inflammation, increased esophageal wet weight, IL-6 and keratinocyte-derived cytokine (KC) levels, and MPO activity with maximal peak between 3 and 7 days, and resolution at 14 days post-surgery. Epithelial esophageal barrier was evaluated in operated mice at 7 and 14 days post-surgery; decrease in TEER and increase in the esophageal epithelial permeability were observed, compared to sham group. In addition, the inhibition of acid secretion with omeprazole significantly prevented the esophageal inflammation and impairment of barrier function at 7 days post surgery. Thus, we established a novel experimental model of NERD in mice, which can contribute to understanding the pathophysiological events associated with NERD.
Several recent studies have shown that liver injury is associated with the release of DNA from hepatocytes. This DNA stimulates innate immunity and induces sterile inflammation exacerbating the liver damage. Similar mechanisms have been described for acute renal injury. Deoxyribonuclease degrades cell-free DNA and can potentially prevent some of the induced tissue damage. In this study the effects of thioacetamide-induced hepatorenal injury on plasma DNA in rats were analysed. Plasma DNA of both nuclear and mitochondrial origin was higher in thioacetamide-treated animals. Administration of deoxyribonuclease resulted in a mild non-significant decrease of total plasma DNA and plasma DNA of mitochondrial but not of nuclear origin. This was accompanied by a decrease in bilirubin as well as in creatinine and blood urea nitrogen as markers of renal function. In conclusion, our study confirmed the hepatotoxic and nephrotoxic effect of thioacetamide. The associated increase in cell-free DNA seems to be involved in the pathogenesis as treatment with deoxyribonuclease resulted in a partial prevention of the hepatorenal injury. Further experiments will focus on the effects of long-term treatment with deoxyribonuclease in other clinically more relevant models. Clinical studies should test endogenous deoxyribonuclease activity as a potential risk determinant for kidney or liver failure.
Microbiota in the gut is known to play a pivotal role in host physiology by interacting with the immune and neuroendocrine systems in gastrointestinal (GI) tissues. Glucagon-like peptide 1 (GLP-1), a gut hormone, is involved in metabolism as well as GI motility. We examined how gut microbiota affects the link between GLP-1/GLP-1 receptor (GLP-1R) expression and motility of the GI tract. Germ-free (GF) mice (6 weeks old) were orally administered a fecal bacterial suspension prepared from specific pathogen-free (SPF) mice, and then after fecal transplantation (FT) GI tissues were obtained from the GF mice at various time points. The expression of GLP-1 and its receptor was examined by immunohistochemistry, and gastrointestinal transit time (GITT) was measured by administration of carmine red solution. GLP-1 was expressed in endocrine cells in the colonic mucosa, and GLP-1R was expressed in myenteric neural cells throughout the GI wall. GLP-1R-positive cells throughout the GI wall were significantly fewer in GF mice with FT than in GF mice without gut microbiota reconstitution. The GITT was significantly shorter in GF mice with FT than in control GF mice without FT, and correlated with the number of GLP-1R-positive cells throughout the GI wall. The GITT was significantly longer in GF controls than in SPF mice. When those mice were treated with GLP-1 agonist extendin4, GITT was significantly longer in GF mice group. Gut microbiota may accelerate or at least modify GI motility while suppressing GLP-1R expression in myenteric neural cells throughout the GI tract.
Parenteral Nutrition (PN) is a lifesaving therapy that provides intravenous nutrition support to patients who cannot, or should not, feed via the gastrointestinal tract. Unfortunately, PN also carries certain risks related to infection and metabolic complications compared with enteral nutrition. In this review, an overview of PN and gastrointestinal (GI) immune and microbiome changes is provided. PN impacts the gut-associated lymphoid tissue (GALT) functions - especially adaptive immune cells - changes to intestinal epithelium and chemical secretions, and significantly alters the intestinal microbiome. Collectively, these changes functionally result in increased susceptibility to infectious and injurious challenge. Since PN remains necessary in large numbers of patients, the search to improve outcomes by stimulating GI immune function during PN remains of interest. This review closes by describing recent advances in using enteric nervous system (ENS) neuropeptides or microbially derived products during PN, which may improve GI parameters by maintaining immunity and physiology.
Neuronal and inducible NO synthase (nNOS and iNOS) play a protective and damaging role, respectively, on the intestinal neuromuscular function after ischemia and reperfusion (I/R) injury. To uncover the molecular pathways underlying this dichotomy we investigated their possible correlation with orthodenticle homeobox proteins OTX1 and OTX2 in the rat small intestine myenteric plexus after in vivo I/R. Homeobox genes are fundamental for the regulation of the gut wall homeostasis both during development and in pathological conditions (inflammation, cancer). I/R injury was induced by temporary clamping the superior mesenteric artery under anaesthesia, followed by 24 and 48 hours of reperfusion. At 48hr I/R intestinal transit decreased and was further reduced by NPLA, nNOS selective inhibitor. By contrast this parameter was restored to control values by 1400W, iNOS selective inhibitor. In longitudinal muscle myenteric plexus (LMMP) preparations, iNOS, OTX1 and OTX2 mRNA and protein levels increased at 24hr and 48hr I/R. At both time periods, the number of iNOS and OTX immunopositive myenteric neurons increased. nNOS mRNA, protein levels and neurons were unchanged. In LMMPs, OTX1 and OTX2 mRNA and protein up-regulation was reduced by 1400W and NPLA, respectively. In myenteric ganglia OTX1 and OTX2 staining was superimposed with that of iNOS and nNOS, respectively. Thus in myenteric ganglia iNOS and nNOS-derived NO may promote OTX1 and OTX2 up-regulation, respectively. We hypothesize that the neurodamaging and neuroprotective roles of iNOS and nNOS during I/R injury in the gut may involve corresponding activation of molecular pathways downstream of OTX1 and OTX2.
Organoids are 3D structures derived from adult or embryonic stem cells that maintain many structural and functional features of their respective organ. Recently, genome editing based on the bacterial defense mechanism CRISPR/Cas9 has emerged as an easily applicable and reliable lab tool. Combining organoids and CRISPR/Cas9 creates exciting new opportunities to study organ development and human disease in vitro. The potential applications of CRISPR in organoids are only beginning to be explored.
Alcoholism causes an imbalance of endoplasmic reticulum (ER) homeostasis in pancreatic acini. In those cells, the ER is involved in the synthesis and folding of pancreatic enzymes. Ufm1 (Ubiquitin-fold modifier 1) is part of a novel ubiquitin-like modification system involved in maintaining ER homeostasis. Among the components of the Ufm1 system, Regulator of C53 and DDRGK1 (RCAD) has recently been identified as an Ufm1-specific E3 ligase that promotes ufmylation of DDRGK1, a RCAD-interacting protein. We determined the importance of RCAD in the proper synthesis and secretion of pancreatic enzymes using RCAD-deficient mice. The pancreas of RCAD-deficient mice was of normal size and histology. Using quantitative PCR and Western-blotting, we found that amylase was up-regulated in pancreas from RCAD KO mice. The constitutive amylase secretion was much higher in isolated pancreatic acini from RCAD KO mice, while CCK-stimulated amylase secretion was disturbed. RCAD deficiency caused a down-regulation in the expression of ER chaperone BiP, which affected ER homeostasis and activated both apoptosis and trypsin. Both RCAD and DDRGK1 transcript levels were up-regulated in pancreatic acini from alcohol-preferring rats. The elevated expression of RCAD and DDRGK1 was associated with increased ER stress and UPR activation. Caspase 3 and trypsin activation was enhanced in RCAD-deficient pancreatic acini upon treatment with ethanol and CCK. In conclusion, RCAD/BiP pathway is required for the proper synthesis and secretion of pancreatic enzymes. In alcoholism, the increased levels of components of the Ufm1 system could prevent alcohol's deleterious effects in the pancreas by regulating BiP levels.
Background: Glucagon-like peptide-2 (GLP-2) and epidermal growth factor (EGF) treatment enhance intestinal adaptation. To determine whether these growth factors exert synergistic effects on intestinal growth and function, GLP-2 and EGF-containing media (EGF-cm) were administered, alone and in combination, in neonatal piglet with short bowel syndrome (SBS). Methods: Neonatal Landrace/Large White piglets were block-randomized to 75% mid-intestinal (JI group) or distal-intestinal (JC group) resection or sham control, with 7-d infusion of saline (control), intravenous human GLP-2 (11 nmol/kg/day) alone, enteral EGF-cm (80 μg/kg/day) alone, or GLP-2 and EGF-cm in combination. Adaptation was assessed by intestinal length, histopathology, Üssing chamber analysis and RT-qPCR of intestinal growth factors. Results: Combined EGF-cm and GLP-2 treatment increased intestinal length in all three surgical models (p<0.01). EGF-cm alone selectively increased bowel weight per length and jejunal villus height in the JI group. The JC group demonstrated increased intestinal weight and villus height (p<0.01) when given either GLP-2 alone or in combination with EGF-cm, with no effect of EGF-cm alone. Jejunal permeability of mannitol and polyethylene glycol decreased with combination therapy in both SBS groups (p<0.05). No difference was observed in fat absorption or body weight gain. IGF-1 mRNA was differentially expressed in JI versus JC piglets with treatment. Conclusions: Combined treatment with GLP-2 and EGF-cm induced intestinal lengthening and decreased permeability, in addition to the trophic effects of GLP-2 alone. Our findings demonstrate the benefits of novel combination GLP-2 and EGF treatment for neonatal SBS, especially in the JC model representing most human infants with SBS.
Peroxisome proliferator-activated receptor alpha (PPARA) is a nuclear transcription factor and key mediator of systemic lipid metabolism. Prolonged activation in rodents causes hepatocyte proliferation and hepatocellular carcinoma. Little is known about the contribution of non-parenchymal cells (NPCs) to PPARA-mediated cell proliferation. NPC contribution to PPARA agonist-induced hepatomegaly was assessed in hepatocyte (PparaHep)- and macrophage (PparaMac)-specific Ppara null mice. Mice were treated with the agonist Wy-14643 for 14 days and response of conditional null mice was compared to conventional knockout mice (Ppara-/-). Wy-14643 treatment caused weight loss and severe hepatomegaly in wild-type and PparaMac mice and histological analysis revealed characteristic hepatocyte swelling; PparaHep and Ppara-/- mice were protected from these effects. PparaMac serum chemistries, as well as AST and ALT levels, matched wild-type mice. Agonist treated PparaHep mice had elevated serum cholesterol, phospholipids, and triglycerides when compared to Ppara-/- mice indicating a possible role for extrahepatic PPARA in regulating circulating lipid levels. BrdU labeling confirmed increased cell proliferation only in wild-type and PparaMac mice. Macrophage PPARA disruption did not impact agonist-induced upregulation of lipid metabolism, cell proliferation, or DNA damage and repair-related gene expression, while gene expression was repressed in PparaHep mice. Interestingly, downregulation of inflammatory cytokines IL15 and IL18 were dependent on macrophage PPARA. Cell type-specific regulation of target genes was confirmed in primary hepatocytes and Kupffer cells. These studies conclusively show that cell proliferation is mediated exclusively by PPARA activation in hepatocytes and that Kupffer cell PPARA has an important role in mediating the anti-inflammatory effects of PPARA agonists.
Vagal nerve stimulation (VNS) has been shown to limit intestinal inflammation following injury, however, direct connection between vagal terminals and resident intestinal immune cells have yet to be identified. We have previously shown that enteric glia cell (EGC) expression is increased after injury through a vagal-mediated pathway to help restore gut barrier function. We hypothesize that EGCs modulate immune cell recruitment following injury and relay vagal anti-inflammatory signals to resident immune cells in the gut. EGCs were selectively ablated from an isolated segment of distal bowel with topical application of benzalkonium chloride (BAC) in male mice. Three days following BAC application, mice were subjected to an ischemia-reperfusion injury (I/R) by superior mesenteric artery occlusion for 30 minutes. VNS was performed in a separate cohort of animals. EGC+ and EGC- segments were compared utilizing histology, flow cytometry, immunohistochemistry, and intestinal permeability. VNS significantly reduced immune cell recruitment after I/R injury in EGC+ segments with cell percentages similar to sham. VNS failed to limit immune cell recruitment in EGC- segments. Histologic evidence of gut injury was diminished with VNS application in EGC+ segments, whereas EGC- segments showed features of more severe injury. Intestinal permeability increased following I/R injury in both EGC+ and EGC- segments. Permeability was significantly lower after VNS application compared to injury alone in EGC+ segments only (95.1 ± 30.0 vs. 217.6 ± 21.7 μg/mL, p<0.05). Therefore, EGC ablation uncouples the protective effects of VNS, suggesting that vagal-mediated signals are translated to effector cells through EGCs.
WNT-5A is a secreted growth factor that belongs to the non-canonical members of the Wingless-related MMTV-integration family. Previous studies pointed to a connection between WNT-5A and the fibrogenic factor TGF-β warranting further studies into the functional role of WNT-5A in liver fibrosis. Therefore, we studied WNT-5A expressions in mouse and human fibrotic livers and examined the relation between WNT-5A and various fibrosis-associated growth factors, cytokines and extracellular matrix proteins. WNT-5A gene and protein expressions were significantly increased in fibrotic mouse and human livers compared to healthy. Regression or therapeutic intervention in mice resulted in decreased hepatic WNT-5A levels paralleled by lower collagen levels. Immunohistochemical analysis showed WNT-5A staining in fibrotic septa co-localizing with desmin staining indicating WNT-5A expression in myofibroblasts. In vitro studies confirmed WNT-5A expression in this cell-type and showed that TGF-β significantly enhanced WNT-5A expression in contrast to PDGF-BB and pro-inflammatory cytokines IL1β and TNFα. After silencing of WNT-5A, reduced levels of collagen type I, vimentin, and fibronectin in TGF-β-stimulated myofibroblasts were measured as compared to non-silencing siRNA-treated controls. Interestingly, the antifibrotic cytokine IFN suppressed WNT-5A in vitro and in vivo. IFN-treated fibrotic mice showed significantly less WNT-5A expression as compared to untreated fibrotic mice. In conclusion, WNT-5A paralleled collagen I levels in fibrotic mouse and human livers. WNT-5A expression in myofibroblasts is induced by the profibrotic factor TGF-β and plays an important role TGF-β induced regulation of fibrotic matrix proteins, whereas its expression can be reversed upon treatment, both in vitro and in vivo.
BACKGROUND: Nonalcoholic steatohepatitis (NASH) is a lipotoxic disorder, wherein proinflammatory lipids, such as ceramide and its derivative sphingosine 1-phosphate (S1P), contribute to macrophage-associated liver inflammation. For example, we have previously demonstrated a role for S1P in steatotic hepatocyte-derived S1P enriched extracellular vesicles in macrophage chemotaxis in vitro. Therefore, we hypothesized that FTY720, an S1P antagonist, would ameliorate NASH by inhibiting proinflammatory monocyte chemotaxis. METHODS: To test our hypothesis NASH was established in C57BL/6 male mice by feeding a diet high in fructose, saturated fat and cholesterol for 22 weeks. Then mice received daily intraperitoneal injections of FTY720 for 2 weeks prior to analysis of liver injury, inflammation, and fibrosis. RESULTS: FTY720-treated mice with NASH demonstrated improved liver histology with a significant reduction in hepatocyte ballooning and inflammatory foci. Hepatomegaly was reversed and liver triglycerides reduced following FTY720 administration to mice with NASH. Correspondingly, serum ALT levels, hepatic inflammatory macrophage accumulation, and expression of Ly6C expressing recruited myeloid cells was reduced in FTY720 treated mice. Hepatic collagen accumulation and expression of alpha-smooth muscle actin were significantly lowered as well. Body composition, energy consumption and utilization, and hepatic sphingolipid composition remained unchanged following FTY720 administration. CONCLUSIONS: FTY720 ameliorates murine nonalcoholic steatohepatitis. Reduction in liver injury and inflammation is associated with a reduction in hepatic macrophage accumulation, likely due to dampened recruitment of circulating myeloid cells into the liver. Nonalcoholic steatohepatitis may be a novel indication for the therapeutic use of FTY720.
Current treatment for pediatric IBD patients is often ineffective, with serious side effects. Manipulating the gut microbiota via fecal microbiota transplantation (FMT) is an emerging treatment approach but remains controversial. We aimed to assess the composition of the fecal microbiome through a comparison of pediatric IBD patients to their healthy siblings, evaluating risks and prospects for FMT in this setting. A Case-Control (Sibling) Study was conducted analyzing fecal samples of six children with Crohn's Disease (CD), six children with Ulcerative Colitis (UC) and 12 healthy siblings by metagenomic sequencing. In addition, lifetime antibiotic intake was retrospectively determined. Species richness and diversity were significantly reduced in UC patients compared to control (MWU FDR = 0.011). In UC, bacteria positively influencing gut homeostasis e.g. Eubacterium rectale and Faecalibacterium prausnitzii were significantly reduced in abundance (MWU FDR = 0.05). Known pathobionts like Escherichia coli were enriched in UC patients (MWU FDR = 0.084). Moreover, E. coli abundance correlated positively with that of several virulence genes (SCC > 0.65, FDR < 0.1). A shift towards antibiotic resistant taxa in both IBD groups distinguished them from controls (MWU BY FDR = 0.062 in UC, MWU BY FDR = 0.019 in CD). The collected results confirm a microbial dysbiosis in pediatric UC, and to a lesser extent in CD patients, replicating associations found previously using different methods. Taken together, these observations suggest microbiotal remodeling therapy from family donors, at least for children with UC, as a viable option.
It has been hypothesized that apically expressed L-type Ca2+ channel Cav1.3 (encoded by CACNA1D gene) contributes towards an alternative TRPV6-independent route of intestinal epithelial Ca2+ absorption, especially during digestion when high luminal concentration of Ca2+ and other nutrients limit TRPV6 contribution. We and others have implicated altered expression and activity of key mediators of intestinal and renal Ca2+ (re)absorption as contributors to negative systemic Ca2+ balance and bone loss in intestinal inflammation. Here, we investigated the effects of experimental colitis and related inflammatory mediators on colonic Cav1.3 expression. We confirmed Cav1.3 expression within the segments of the mouse and human gastrointestinal tract. Consistent with available microarray data (GEO database) from IBD patients, mouse colonic expression of Cav1.3 was significantly reduced in TNBS colitis. In vitro, IFN most potently reduced Cav1.3 expression. We reproduced these findings in vivo with wild-type and Stat1-/- mice injected with IFN. The observed effect in Stat1-/- suggested a non-canonical transcriptional repression or a post-transcriptional mechanism. In support of the latter, we observed no effect on the cloned Cav1.3 gene promoter activity, and accelerated Cav1.3 mRNA decay rate in IFN-treated HCT116 cells. While the relative contribution of Cav1.3 to intestinal Ca2+ absorption and its value as a therapeutic target remain to be established, we postulate that Cav1.3 downregulation in IBD may contribute to the negative systemic Ca2+ balance, increased bone resorption, and to reduced bone mineral density in IBD patients.
Cecal crypts represent a unique niche that are normally occupied by the commensal microbiota. Due to their density and close proximity to stem cells, microbiota within cecal crypts may modulate epithelial regeneration. Here it is demonstrated that surgical stress, a process that invariably involves a short period of starvation, antibiotic exposure and tissue injury, results in cecal crypt evacuation of their microbiota. Crypts devoid of their microbiota display pathophysiological features characterized by abnormal stem cell activation as judged by Lgr5 staining, abnormal stem cell distribution with cells migrating toward the tips of the crypts, and an increase in TUNEL positive cells. In addition, crypts are devoid of their microbiota also display loss of their regenerative capacity as assessed by their ability to form organoids ex vivo. When a four (4) member human pathogen community isolated from the stool of a critically ill patient is introduced into the cecum of mice with empty crypts, crypts become occupied by the introduced pathogens and develop persistent and abnormal Lgr5 expression and severe crypt cell disruption. Fecal microbiota transplantation restores the cecal crypts' microbiota, normalizes the Lgr5 pattern, and reestablishes its regenerative capacity. Taken together, these findings define an emerging role for the microbiota within cecal crypts to maintain epithelial cell homeostasis in a manner that may enhance recovery in response to the physiological stress imposed by the process of surgery.
Inflammatory bowel diseases (IBDs) are chronic, inflammatory disorders of the gastrointestinal tract with unclear aetiologies. Intestinal epithelial cells (IECs), containing crypt and villus enterocytes, occupy a critical position in the pathogenesis of IBDs and are a major producer of immunoregulatory cytokines and a key component of the intact epithelial barrier. Previously, we have reported that miR-200b is involved in the progression of IBDs and might maintain the integrity of the intestinal epithelial barrier via reducing the loss of enterocytes. In this study, we further investigated the impact of miR-200b on intestinal epithelial inflammation and tight junctions in two distinct differentiated states of Caco-2 cells after TNFα treatment. We demonstrated that TNFα-enhanced IL-8 expression was decreased by miR-200b in undifferentiated IECs. Simultaneously, miR-200b could alleviated TNFα-induced tight junction (TJ) disruption in well-differentiated IECs by reducing the reduction in the TEER, inhibiting the increase in paracellular permeability and preventing the morphological redistribution of TJ proteins Claudin1 and ZO-1. The expression levels of the JNK/C-JUN/AP-1 and MLCK/P-MLC pathways were attenuated in undifferentiated and differentiated enterocytes, respectively. Furthermore, a dual-luciferase reporter gene detection system provided direct evidence that C-JUN and MLCK were the specific targets of miR-200b. Collectively, our results highlighted that miR-200b played a positive role on IECs via suppressing intestinal epithelial IL-8 secretion and attenuating TJ damage in vitro, which suggested that miR-200b might be a promising strategy for IBD therapy.
Gamma-aminobutyric acid (GABA) is produced by various cells through the catalytic activity of glutamic acid decarboxylase (GAD). Activation of type-A GABA receptor (GABAAR) inhibits stem cell proliferation but protects differentiated cells from injures. The present study investigated hepatic GABA signaling system and the role of this system in liver physiology and pathophysiology. RT-PCR and immunoblot assays identified GAD and GABAAR subunits in rat livers and in HepG2 and Clone 9 hepatocytes. Patch-clamp recording detected GABA-induced currents in Clone 9 hepatocytes and depolarization in WITT cholangiocytes. The function of hepatic GABA signaling system in rats was examined using models of D-galactosamine (GalN)-induced acute hepatocytic injury in vivo and in vitro. The expression of GAD increased whereas GABAAR subunits decreased in the liver of GalN-treated rats. Remarkably, treating rats with GABA or the GABAAR agonist muscimol, but not the GABABR agonist baclofen, protected hepatocytes against GalN toxicity and improved liver function. In addition, muscimol treatment decreased the formation of pseudo bile ductules and the enlargement of hepatocytic canaliculi in GalN-treated rats. Our results revealed that a complex GABA signaling system exists in the rat liver. Activation of this intrahepatic GABAergic system protected the liver against toxic injury.
Myenteric plexus interstitial cells of Cajal (ICC-MY) in the small intestine are Kit+ electrical pacemakers that express the Ano1/TMEM16A Ca2+-activated Cl- channel, whose functions in the GI tract remain incompletely understood. In this study, an inducible Cre-LoxP-based approach was used to advance the understanding of Ano1 in ICC-MY of adult mouse small intestine. KitCreERT2/+;Ano1Fl/Fl mice were treated with tamoxifen or vehicle, and small intestines (mucosa-free) examined. Quantitative RT-PCR demonstrated ~50% reduction in Ano1 mRNA in intestines of conditional knock-outs (cKOs) compared to vehicle-treated controls. Whole mount immunohistochemistry showed a mosaic/patchy pattern loss of Ano1 protein in ICC networks. Ca2+ transients in ICC-MY network of cKOs displayed reduced duration compared to highly synchronized controls, and showed synchronized and desynchronized profiles. When matched, the rank order for Ano1 expression in Ca2+ signal imaged field-of-views was: vehicle-controls>>>cKO(synchronized)>cKO(desynchronized). Maintenance of Ca2+ transients' synchronicity despite high loss of Ano1 indicates a large functional reserve of Ano1 in the ICC-MY network. Slow-waves in cKOs displayed reduced duration and increased inter slow-wave interval, and occurred in regular- and irregular-amplitude oscillating patterns. The latter activity suggested ongoing interaction by independent interacting oscillators. Lack of slow-wave and depolarization, previously reported for neonatal constitutive KOs, were also seen. In summary, Ano1 in adults regulates gastrointestinal function by determining Ca2+ transients and electrical activity depending on the level of Ano1 expression. Partial Ano1 loss results in Ca2+ transients and slow-wave displaying reduced duration, while complete and widespread absence of Ano1 in ICC-MY causeslack of slow-wave and desynchronized Ca2+ transients.
The Notch signaling pathway is known to regulate stem cells and epithelial cell homeostasis in gastrointestinal tissues; however, Notch function in the corpus region of the stomach is poorly understood. In this study we examined the consequences of Notch inhibition and activation on cellular proliferation and differentiation and defined the specific Notch receptors functioning in the mouse and human corpus. Notch pathway activity was observed in the mouse corpus epithelium, and gene expression analysis revealed NOTCH1 and NOTCH2 to be the predominant Notch receptors in both mouse and human. Global Notch inhibition for 5 days reduced progenitor cell proliferation in the mouse corpus, as well as in organoids derived from mouse and human corpus tissue. Proliferation effects were mediated through both NOTCH1 and NOTCH2 receptors, as demonstrated by targeting each receptor alone or in combination with Notch receptor inhibitory antibodies. Analysis of differentiation by marker expression showed no change to the major cell lineages; however there was a modest increase in the number of transitional cells co-expressing markers of mucous neck and chief cells. In contrast to reduced proliferation after pathway inhibition, Notch activation in the adult stomach resulted in increased proliferation coupled with reduced differentiation. These findings suggest that NOTCH1 and NOTCH2 signaling promotes progenitor cell proliferation in the mouse and human gastric corpus, which is consistent with previously defined roles for Notch in promoting stem and progenitor cell proliferation in the intestine and antral stomach.
Mammalian cells utilize two transporters for the uptake of ascorbic acid (AA), the sodium-dependent vitamin C transporter-1 & -2 (SVCT-1 and SVCT-2). In the intestine, these transporters are involved in the absorption of AA and are expressed at the apical and basolateral membrane domains of the polarized epithelia, respectively. Little is known about the differential expression of these two transporters along the anterior-posterior axis of the intestinal tract and the molecular mechanism(s) that dictate this pattern of expression. We addressed these issues using mouse and human intestinal cDNAs. The results showed a significantly lower rate of carrier-mediated AA uptake by mouse colon compared to jejunum. This was associated with a significantly lower level of expression of both the SVCT-1 and -2 at the protein, mRNA, and hnRNA levels in colon compared to jejunum, implying the involvement of transcriptional mechanism(s). Similarly, expression of SVCT-1 and -2 mRNA and hnRNA were found to be significantly lower in human colon. We also examined the levels of expression of HNF1α and Sp1 that drive the transcription of the Slc23a1 and Slc23a2 promoters, respectively, and found them to be markedly lower in the colon. Furthermore, significantly lower levels of the activating markers for histone modifications (H3K4me3 and H3K9ac) were observed in the Slc23a1 and Slc23a2 promoters in the colon. These findings show, for the first time, that the SVCT-1 and -2 are differentially expressed along the intestinal tract, and this pattern of expression is, at least in part mediated, via transcriptional/epigenetic mechanisms.
Previous studies have shown that localized delivery of the aquaporin-1 (AQP1) gene to the parotid duct can restore saliva flow in minipigs following irradiation-induced salivary hypofunction. The resulting flow rate and electrochemisty of secreted saliva contradicts current understanding of ductal fluid transport. We hypothesized that changes in expression of ion transport proteins have occurred following AQP1 transfection. We use a mathematical model of ion and fluid transport across the parotid duct epithelial cells to predict the expression profile of ion transporters that are consistent with the experimental measurements of saliva composition and secretion rates. Using a baseline set of parameters, the model reproduces the data for the irradiated, non-AQP1 transfected case. We propose three scenarios which may have occurred after transfection, which differ in the location of the AQP1 gene. The first scenario places AQP1 within non-secretory cells, and requires that epithelial sodium channel (ENaC) expression is greatly reduced (1.3 % of baseline), and ductal bicarbonate concentration is increased from 40.6 mM to 137.0 mM, to drive water secretion into the duct. The second scenario introduces the AQP1 gene into all ductal cells. The final scenario has AQP1 primarily in the proximal duct cells which secrete water under baseline conditions. We find the change in the remaining cells includes a 95.8 % reduction in ENaC expression, enabling us to reproduce all experimental ionic concentrations within 9 mM. These findings provide a mechanistic basis for the observations and will guide the further development of gene transfer therapy for salivary hypofunction.
The gut barrier plays a crucial role by spatially compartmentalizing bacteria to the lumen through the production of secreted mucus and is fortified by the production of sIgA and antimicrobial peptides and proteins. With exception of sIgA the expression of these protective barrier factors is largely controlled by innate immune recognition of microbial molecular ligands. Several specialized adaptations and checkpoints are operating in the mucosa to scale the immune response according to the threat and prevent overreaction to the trillions of symbionts inhabiting the human intestine. A healthy microbiota plays a key role influencing epithelial barrier functions. However, perturbation of gut barrier homeostasis can lead to increased inflammatory signaling, increased epithelial permeability and dysbiosis of the microbiota, which are recognized to play a role in the pathophysiology of gastrointestinal disorders. Additionally, the gut-brain signaling may be affected by prolonged mucosal immune activation, leading to increased afferent sensory signaling and abdominal symptoms. In turn, neuronal mechanisms can affect the intestinal barrier partly by activation of the HPA-axis and both mast cell-dependent as well as mast cell- independent mechanisms. Several biomarkers have been used to measure gut permeability and loss of barrier integrity in patients but there remains a need to explore their use in assessing impact of nutritional factors on gut barrier function. Future studies should aim to establish normal ranges of the available biomarkers and their predictive value for gut health in human cohorts.
The Winnie mouse, carrying a missense mutation in Muc2, is a model for chronic intestinal inflammation demonstrating symptoms closely resembling inflammatory bowel disease (IBD). Alterations to the immune environment, morphological structure and innervation of Winnie mouse colon have been identified; however analyses of intestinal transit and colonic functions have not been conducted. In this study, we investigated in vivo intestinal transit in radiographic studies and in vitro motility of the isolated colon in organ bath experiments. We compared neuromuscular transmission using conventional intracellular recording between distal colon of Winnie and C57BL/6 mice and smooth muscle contractions using force displacement transducers. Chronic inflammation in Winnie mice was confirmed by detection of lipocalin-2 in fecal samples over 4 weeks and gross morphological damage to the colon. Colonic transit was faster in Winnie mice. Motility was altered including decreased frequency and increased speed of colonic migrating motor complexes, increased occurrence of short and fragmented contractions. The mechanisms underlying colon dysfunctions in Winnie mice included inhibition of excitatory and fast inhibitory junction potentials, diminished smooth muscle responses to cholinergic and nitrergic stimulation and increased number of α-smooth muscle actin-immunoreactive cells. We conclude that diminished excitatory responses occur both pre- and post-junctionally, reduced inhibitory purinergic responses are potentially a pre-junctional event, while diminished nitrergic inhibitory responses are probably due to a post-junction mechanism in the Winnie mouse colon. Many of these changes are similar to disturbed motor functions in IBD patients indicating that the Winnie mouse is a model highly representative of human IBD.
The plasticity of gastric chief cells is exemplified by their ability to transdifferentiate into Spasmolytic Polypeptide-expressing Metaplasia (SPEM) after parietal cell loss. We sought to determine if chief cell maturity is a limiting factor in the capacity to transdifferentiate. Mist1-/- mice, previously shown to form only immature chief cells, were treated with DMP-777 or L635 to study the capability of these immature chief cells to transdifferentiate into a proliferative metaplastic lineage after acute parietal cell loss. Mist1-/- mice treated with DMP-777 showed fewer chief cell to SPEM transitions. Mist1-/- mice treated with L635 demonstrated significantly fewer proliferative SPEM cells compared to control mice. Thus, immature chief cells were unable to transdifferentiate efficiently into SPEM after acute parietal cell loss. To determine whether chief cell age affects transdifferentiation into SPEM, we used tamoxifen to induce YFP expression in chief cells of Mist1CreER/+;RosaYFP mice and subsequently treated with L635 to induce SPEM at 1 to 3.5 months after tamoxifen treatment. After L635 treatment to induce acute parietal cell loss, 43% of all YFP-positive cells at 1 month post-tamoxifen were SPEM cells, of which 44% of these YFP-positive SPEM cells were proliferative. By 2 months after tamoxifen induction, only 24% of marked SPEM cells were proliferating. However, by 3.5 months after tamoxifen induction, only 12% of marked chief cells transdifferentiated into SPEM and none were proliferative. Thus, as chief cells age, they lose their ability to transdifferentiate into SPEM and proliferate. Therefore, both functional maturation and age limit chief cell plasticity.
Fecal incontinence (FI) in men is common yet data on gender differences in clinical features, physiology and treatment is scarce. Our aim was to provide insights into FI in males compared to females. Prospectively collected data from 73 men and 596 women with FI in a tertiary referral centre was analysed. Anorectal physiology, clinical characteristics and outcome of instrumented biofeedback (BF) was recorded. 31 men with FI proceeded to BF and were matched with 62 age-matched women with FI who underwent BF. Men with FI had higher resting, squeeze and cough anal sphincter pressures (P<0.001) and were more able to hold a sustained squeeze compared to women (p=0.04). Men with FI had higher rectal pressure and less inadequate rectal pressure on strain, and higher sensory thresholds (p<0.05). Men, but not women, with isolated soiling had higher anal resting and squeeze pressures compared to those with overt FI (p<0.05). Men were less likely to undergo BF when offered compared to women. Baseline symptom severity did not differ between the groups. In men, the absence of an organic cause for the FI and the presence of overt FI, but not isolated soiling, were correlated with improvement in patient satisfaction. The outcomes of 50% reduction in FI episodes, symptom and quality of life scores after BF all significantly improved in men similarly to women. We conclude that men, compared to women, with FI have unique clinical features and physiology and are less likely to have investigations and treatment despite successful outcome with BF.
The bone morphogenetic proteins (BMPs) regulate gastrointestinal homeostasis. We investigated the expression of BMP-4 and the localization and function of BMP signaling during colonic injury and inflammation. Wild type mice and mice expressing the β-galactosidase (β-gal) gene under the control of a BMP responsive element (BRE), BMP-4-β-gal/+ mice, and animals generated by crossing villin-Cre mice to mice with floxed alleles of BMP receptor 1A (villin-Cre;Bmpr1aflox/flox), were treated with DSS to induce colonic injury and inflammation. Expression of BMP-4, β-gal, BMPR1A, IL-8, α-SMA, and phosphorylated Smad1-5-8, were assessed by X-Gal staining, QRT-PCR and immunohistochemistry. Morphology of the colonic mucosa was examined by staining with H&E. The effect of IFN-, TNF-α, IL-1β, and IL-6 on BMP-4 mRNA expression was investigated in human intestinal fibroblasts, while that of BMP-4 on IL-8 was assessed in human colonic organoids. BMP-4 was localized in α-SMA-positive mesenchymal cells while the majority of BMP-generated signals targeted the epithelium. DSS caused injury and inflammation, leading to reduced expression of BMP-4 and BMPR1A mRNAs, and to decreased BMP signaling. Deletion of Bmpr1a enhanced colonic inflammation and damage. Administration of anti-TNF-α antibodies to DSS-treated mice ameliorated colonic inflammation and increased the expression of BMP-4 and BMPR1A mRNAs. TNF-α and IL-1β inhibited both basal and IFN- stimulated BMP-4 expression, while IL-6 had no effect. BMP-4 reduced IL-8 mRNA expression in the organoids. Inflammation and injury inhibit BMP-4 expression and signaling, leading to enhanced colonic damage. These observations underscore the importance of BMP signaling in the regulation of intestinal inflammation and homeostasis.
MUC2 mucin is the major glycoprotein in colonic mucus that separates intestinal microbiota from underlying host cells and serves as a food source for some eubacteria. MUC2 deficiency results in impaired epithelial barrier function, imbalance in gut microbiota and spontaneous colitis. Use of probiotics has been shown to have protective effect against colitis. In this study we tested whether the probiotic mixture VSL#3 required an intact mucin barrier to exert its beneficial effect using Muc2 mucin deficient (Muc2-/-) and Muc2+/+ littermates. VSL#3 treatment alone reduced basal colonic pro-inflammatory cytokine levels and improved epithelial barrier functions in Muc2-/- animals. Likewise, in DSS-induced colitis, VSL#3 administration dampened the pro-inflammatory chemokines KC, MCP-1 and MIP-2 and up regulated tissue regeneration growth factors TGF-β, FGF-1 and VEGF-A that accelerated resolution of colitis symptoms in Muc2-/- animals. Importantly, improved colonic health in VSL#3 treated animals was associated with attenuated reactive oxygen species (ROS) production by peritoneal macrophages, restoration of antimicrobial peptide gene expression in the small intestine and increased abundance of bacterial commensals in the gut. The beneficial effects of VSL#3 in Muc2-/- animals were mediated by acetate, an important short chain fatty acid produced by gut bacteria. These studies provide evidence for the first time that VSL#3 can enhance epithelial barrier function by dampening pro-inflammatory cytokines and chemokine response, accelerating restitution and altering commensal microbiota in the absence of a functional mucus barrier.
We have demonstrated that neuropeptide Y (NPY), abundantly produced by enteric neurons, is an important regulator of intestinal inflammation. However the role of NPY in the progression of chronic inflammation to tumorigenesis is unknown. We investigated whether NPY could modulate epithelial cell proliferation and apoptosis, and thus regulate tumorigenesis. Repeated cycles of dextran sodium sulfate (DSSn) was used to model inflammation-induced tumorigenesis in wild-type (WT) and NPY knockout (NPY-/-) mice. Intestinal epithelial cell lines (T84) were used to assess the effects of NPY (0.1µM) on epithelial proliferation and apoptosis in vitro. DSSn-WT mice exhibited enhanced intestinal inflammation, polyp size and polyp number (7.5 ± 0.8) compared to DSSn-NPY-/- mice (4 ± 0.5, P < 0.01). Accordingly, DSSn-WT mice also showed increased colonic epithelial proliferation (PCNA, Ki67) and reduced apoptosis (TUNEL) compared to DSSn- NPY-/- mice. The apoptosis regulating microRNA, miR-375, was significantly down regulated in the colon of DSSn-WT (2 fold, P < 0.01) compared to DSSn-NPY-/--mice. In vitro studies indicated that NPY promotes cell proliferation (increase in PCNA and β-catenin, P < 0.05) via phosphatidyl-inositol-3-kinase (PI3-K)- β-catenin signaling, suppressed miR-375 expression and reduced apoptosis (increase in phospho-Bad). NPY-treated cells also displayed increased c-myc and cyclin D1 and reduction in p21 (P < 0.05). Addition of miR-375 inhibitor to cells already treated with NPY did not further enhance the effects induced by NPY alone. Our findings demonstrate a novel regulation of inflammation-induced tumorigenesis by NPY-epithelial cross talk as mediated by activation of PI3-K-β-catenin signaling and down regulation of miR-375.
Fibrosis is a complication of chronic inflammatory disorders such as inflammatory bowel disease (IBD), a condition which has limited therapeutic options and often requires surgical intervention. Pharmacologic inhibition of oxygen-sensing prolyl hydroxylases (PHD), which confer oxygen-sensitivity upon the hypoxia inducible factor (HIF) pathway, has recently been shown to have therapeutic potential in colitis, although the mechanisms involved remain unclear. Here, we investigated the impact of hydroxylase inhibition on inflammation-driven fibrosis in a murine colitis model. Mice exposed to dextran sodium sulfate followed by period of recovery developed intestinal fibrosis characterized by alterations in the pattern of collagen deposition and infiltration of activated fibroblasts. Treatment with the hydroxylase inhibitor dimethyloxalylglycine (DMOG) ameliorated fibrosis. TGF-β1 is a key regulator of fibrosis which acts through the activation of fibroblasts. Hydroxylase inhibition reduced TGF-β1-induced expression of fibrotic markers in cultured fibroblasts suggesting a direct role for hydroxylases in TGF-β1 signalling. This was at least in part due to inhibition of non-canonical activation of extracellular signal-regulated kinase (ERK) signalling. In summary, pharmacologic hydroxylase inhibition ameliorates intestinal fibrosis, through suppression of TGF-β1-dependent ERK activation in fibroblasts. We hypothesize that in addition to previously reported immunosupressive effects, hydroxylase inhibitors independently suppress pro-fibrotic pathways.
The impact of omeprazole (OM), a widely used over-the-counter proton pump inhibitor, on weight gain has not been extensively explored. We examined what factors, e.g., diet composition, microbiota, genetic strain and sex, might affect weight gain in mice fed a high caloric diet while on OM. Methods: Inbred C57BL/6J strain, a 50:50 hybrid (B6SJLF1/J) strain and mice on a highly mixed genetic background were fed four diets: standard chow (STD, 6% fat), STD with 200 ppm OM (STD+O), a high-energy chow (HiE, 11% fat) and HiE chow with OM (HiE+O) for 17 weeks. Metabolic analysis, body composition and fecal microbiota composition were analyzed in C57BL/6J mice. Oral glucose tolerance tests were performed using mice on the mixed background. Results: After 8 weeks, female and male C57BL/6J mice on the HiE diets ate less; whereas, males on the HiE diets compared to the STD diets gained weight. All diet treatments reduced energy expenditure in females but in males only those on the HiE+O diet. Gut microbiota composition differed in the C57BL/6J females but not the males. Hybrid B6SJLF1/J mice showed similar weight gain on all test diets. In contrast, mixed strain male mice fed a HiE+O diet gained ~40% more weight than females on the same diet. In addition to increased weight gain, mixed genetic mice on the HiE+O diet cleared glucose normally but secreted more insulin. Conclusion: Sex and genetic background define weight gain and metabolic responses of mice on high caloric diets and OM.
Introduction: It is presumed that extrinsic afferent nerves link the rectum to the central nervous system. However, the anatomical / functional existence of such nerves has never previously been demonstrated in humans. Therefore, we aimed to identify and make electrophysiological recordings in vitro from extrinsic afferents, comparing human rectum to colon. Methods: Sections of normal rectum and colon were procured from anterior resection and right hemicolectomy specimens, respectively. Sections were pinned and extrinsic nerves dissected. Extracellular visceral afferent nerve activity was recorded. Neuronal responses to chemical (capsaicin and 'inflammatory soup' [IS]) and mechanical (Von Frey probing) stimuli were recorded and quantified as peak firing rate [range] in one-second intervals. Results: 28 separate nerve trunks from 8 rectums were studied. Of these, spontaneous multi-unit afferent activity was recorded in 24 nerves. Peak firing rates increased significantly following capsaicin (median 6 [range 3-25] spikes/sec vs. 2 [1-4], P<0.001) and IS (median 5 [range 2-18] spikes/sec vs. 2 [1-4], P<0.001). Mechanosensitive 'hot-spots' were identified in 16 nerves (median threshold 2.0g [range 1.4-6.0g]). In 8 of these, the threshold decreased after IS (1.0g [0.4-1.4g]). By comparison, spontaneous activity was recorded in only 3/30 nerves studied from 10 colons and only one 'hot-spot' (threshold 60g) was identified. Conclusions: This study confirms the anatomical / functional existence of extrinsic rectal afferent nerves and characterizes their chemo- and mechano-sensitivity for the first time in Man. They have different electrophysiological properties to colonic afferents and warrant further investigation in disease states.
Lipopolysaccharide (LPS)-induced microvascular hyperpermeability and hemorrhage play a key role in the development of sepsis, attenuation of which might be an important strategy to prevent sepsis. However, present clinical therapies are proved to be inefficiency in improving the final survival rate for the patients with sepsis. Catalpol, an iridoid glycoside extracted from the roots of Rehmannia, has been reported able to protect against LPS-induced acute lung injury through Tool-like Receptor-4 (TLR-4) mediated NF-B signaling pathway. However, it is still unknown whether Catalpol is effective to ameliorate the LPS-induced microvascular disorder. The present study was aimed to investigate the impact of Catalpol on LPS-induced mesenteric microvascular disorder and its underlying mechanism. Male Wistar rats were challenged by infusion of LPS (10 mg/kg/h) through left femoral vein for 120 min. Post-treatment with Catalpol (10 mg/kg) alleviated the LPS-induced microvascular hyperpermeability and hemorrhage, reduced mortality, ameliorated the alteration in distribution of claudin-5 and JAM-1, and the degradation of collagen IV and laminin, attenuated the increase of TLR-4 level, phosphorylations of Src tyrosine kinase, phosphatidyl inositol 3-kinase, focal adhesion kinase, and Cathepsin B activation. Inhibition of TLR-4 and Src each simulated some but not all effects Catalpol exerted. Besides, surface plasmon resonance showed that Catalpol could directly bind to TLR-4 and Src. These results demonstrated that Catalpol was able to ameliorate the LPS-induced microvascular barrier damage and hemorrhage by targeting both TLR-4 and Src thus attenuating the phosphorylation of Src kinase, phosphatidyl inositol 3-kinase and focal adhesion kinase, as well as Cathepsin B activation.
The relationship between gastric motility and the mixing of liquid food in the stomach is investigated using a numerical analysis. Three parameters of gastric motility are considered: the propagation velocity, frequency, and terminal acceleration of peristaltic contractions. We simulate gastric flow using an anatomically-realistic geometric model of the stomach, considering free-surface flow and moving boundaries. When a peristaltic contraction approaches the pylorus, retropulsive flow is generated in the antrum. Flow separation then occurs behind the contraction. The extent of flow separation depends on the Reynolds number (Re) which quantifies the inertial forces due to the peristaltic contractions relative to the viscous forces of the gastric contents; no separation is observed at low Re, while an increase in reattachment length is observed at high Re. While mixing efficiency is nearly constant for low Re, it increases with Re for high Re because of flow separation. Hence, the effect of the propagation velocity, frequency, or terminal acceleration of peristaltic contractions on mixing efficiency increases with the Reynolds number.
Background: There are no approved treatments for liver fibrosis. To aid development of anti-fibrotic therapies, non-invasive biomarkers that can identify patients with progressive fibrosis and that permit monitoring of the response to anti-fibrotic therapy are much needed. Methods: Samples from a phase II anti-fibrotic trial of the glitazone farglitazar in patients with advanced hepatitis C, with matched follow-up liver biopsies, and from a phase III study of balaglitazone in late stage type 2 diabetics (BALLET study), were analysed for serological Pro-C3 levels in conjunction with other disease parameters. Results: In the farglitazar study, a pre-defined cut-off value for Pro-C3 as selection criterion led to identification of those subjects who a) progressed by histological scores and b) responded to therapy as documented by attenuated fibrosis in liver biopsies. In the BALLET trial, subjects with the highest tertile of Pro-C3 levels responded to balaglitazone with reductions in levels of ALT and Pro-C3, as well as improved insulin sensitivity and lipid profile. Conclusions: Elevated Pro-C3 levels are indicative of active fibrogenesis and structural progression of fibrosis and can potentially identify patients most likely to benefit from anti-metabolic and potential anti-fibrotic treatments. Serum Pro-C3 may facilitate patient selection and could help to speed up anti-fibrotic drug development and validation.
We used spatiotemporal mapping of strain rate to determine the direction of propagation and amplitudes of the longitudinal and circumferential components of antro-corporal contractions and fundal contractions in the rat stomach maintained ex vivo and containing a volume of fluid that was within its normal functional capacity. In the region of the greater curvature the longitudinal and circular components of anterocorporal (AC) contractions propagated synchronously at right angles to the arciform geometric axis of the stomach. However the configuration of AC contractions was u shaped, neither the circular nor the longitudinal component of contractions being evident in the upper proximal corpus. Similarly, in the distal upper antrum of some preparations circumferential components propagated more rapidly than longitudinal components. Ongoing 'high frequency, low amplitude myogenic contractions' i.e. HFLAC were identified in the upper proximal gastric corpus and on the anterior and posterior wall of the fundus. The amplitudes of these contractions were modulated in the occluded stomach by low frequency pressure waves which occurred spontaneously. Hence the characteristics of phasic contractions vary regionally in the antrum and corpus and a previously undescribed high frequency contractile component was identified in the proximal corpus and fundus, the latter being modulated in synchrony with cyclic variation in intrafundal pressure in the occluded fundus.
Introduction: Food antigens are common inflammatory triggers in pediatric eosinophilic esophagitis (EoE). TNF-related apoptosis-inducing ligand (TRAIL) promotes eosinophilic inflammation through the upregulation of Midline (MID)-1 and subsequent downregulation of Protein Phosphatase 2A (PP2A) but the role of this pathway in EoE that is experimentally induced by repeated food antigen challenges has not been investigated. Methods: Esophageal mucosal biopsies were collected from children with EoE and controls and assessed for TRAIL and MID-1 protein and mRNA transcript levels. Wild type and TRAIL deficient (Tnfsf10 -/-) mice were administered subcutaneous ovalbumin (OVA) followed by oral OVA challenges. In separate experiments OVA challenged mice were intraperitoneally administered salmeterol or dexamethasone. Results: Esophageal biopsies from children with EoE revealed increased levels of TRAIL and MID-1 and reduced PP2A activation as compared to controls. Tnfsf10-/- mice with experimental EoE, were largely protected from esophageal fibrosis, eosinophilic inflammation, and the upregulation of TSLP, IL-5, IL-13 and CCL11 when compared with wild type mice. Salmeterol administration to wild type mice with experimental EoE restored PP2A activity and also prevented esophageal eosinophilia, inflammatory cytokine expression and remodeling which was comparable to the treatment effect of dexamethasone. Conclusion: TRAIL and PP2A regulate inflammation and fibrosis in experimental EoE, which can be therapeutically modulated by Salmeterol.
ICC generate electrical slow waves by coordinated openings of ANO1 channels, a Ca2+-activated Cl- (CaCC) conductance. Efflux of Cl- during slow waves must be significant as there is high current density during slow wave currents and slow waves are of sufficient magnitude to depolarize the syncytium of smooth muscle cells and PDGFRα+ cells to which they are electrically coupled. We investigated how the driving force for Cl- current is maintained in ICC. We found robust expression of Slc12a2 (which encodes a Na+K+Cl- cotransporter, NKCC1) and immunohistochemical confirmation that NKCC1 is expressed in ICC. Using the gramicidin permeabilized-patch technique, which is reported to not disturb [Cl-]i, the reversal potential for spontaneous transient inward currents (ESTICs) was -10.5 mV. This value corresponds to the peak of slow waves when they are recorded directly from ICC in situ. Inhibition of NKCC1 with bumetanide shifted ESTICs to more negative potentials within a few minutes and reduced pacemaker activity. Bumetanide had no direct effects on ANO1 or CaV3.2 channels expressed in HEK 293 cells, or L-type Ca2+ currents. Reducing extracellular Cl- to 10 mM shifted ESTICs to positive potentials as predicted by the Nernst equation. The relatively rapid shift in ESTICs when NKCC1 was blocked suggests that significant changes in the transmembrane Cl- gradient occur during the slow wave cycle, possibly within microdomains formed between ER and the plasma membrane in ICC. Recovery of Cl- via NKCC1 might have additional consequences on shaping the waveforms of slow waves via Na+ entry into microdomains.
Angiogenesis is critical for the healing of gastric mucosal injury and is considered to be primarily regulated by vascular endothelial growth factor (VEGF), the fundamental pro-angiogenic factor. The role of nerve growth factor (NGF) in gastric angiogenesis is unknown. We examined: the expression of NGF and its TrkA receptor in endothelial cells (ECs) isolated from gastric mucosa of rats (GMECs); the effect of NGF treatment on in vitro angiogenesis in GMECs; and, the mechanisms underlying NGF's pro-angiogenic actions. Isolated GMECs from Fisher rats were treated with: vehicle, NGF (10-1,000 ng/ml), VEGF (20 ng/ml) or NGF+VEGF. To determine whether and to what extent NGF is critical for angiogenesis in GMECs, we silenced NGF expression using specific siRNA and examined in vitro angiogenesis with and without treatment with exogenous NGF and/or VEGF. Treatment of GMECs with NGF significantly increased in vitro angiogenesis similar to that seen in GMECs treated with VEGF. Silencing of NGF in GMECs abolished angiogenesis and this effect was reversed only by exogenous NGF but not VEGF, which indicates a direct pro-angiogenic action of NGF on GMECs that is at least in part distinct and independent of VEGF. NGF's pro-angiogenic action on GMECs was mediated via PI3 K/Akt signaling. This study showed for the first time that gastric mucosal ECs express NGF and its receptor TrkA, and that NGF is critical for angiogenesis in these cells.
In the current study, we have characterized the global miRNA expression profile in mouse pancreatic acinar cells and during acute pancreatitis using next-generation RNA-Sequencing. We identified 324 known and 6 novel miRNAs being expressed in mouse pancreatic acinar cells. At basal state, miR-148a-3p, miR-375-3p, miR-217-5p, miR-200a-3p were among the most abundantly expressed whereas, miR-24-5p and miR-421-3p were least abundant. Treatment of acinar cells with caerulein (100 nM) and taurolithocholic acid 3-sulfate [TLC-S (250 μM)] induced numerous changes in miRNA expression profile. In particular, we found significant overexpression of miR-21-3p in acini treated with caerulein and TLC-S. We further looked at the expression of miR-21-3p in caerulein, L-arginine and caerulein + LPS induced acute pancreatitis mouse models and found 12, 21 and 50 fold increased expression in pancreas, respectively. In summary, this is the first comprehensive analysis of global miRNA expression profile of mouse pancreatic acinar cells in normal and disease conditions. Our analysis shows that miR-21-3p expression level correlates with severity of the disease.
Previous studies reported that administration of somatostatin (SST) to human patients mitigated their diarrheal symptoms. Octreotide (an analogue of SST) treatment in animals resulted in upregulation of sodium/hydrogen exchanger 8 (NHE8). NHE8 is important for water/sodium absorption in the intestine, and loss of NHE8 function results in mucosal injury. Thus, we hypothesized that NHE8 expression is inhibited during colitis and that SST treatment during pathological conditions can restore NHE8 expression. Our data showed for the first time that NHE8 is expressed in the human colonic tissue and that NHE8 expression is decreased in ulcerative colitis (UC) patients. We also found that octreotide could stimulate colonic NHE8 expression in colitic mice. Furthermore, somatostatin receptor 2 (SSTR2) agonist seglitide and somatostatin receptor 5 (SSTR5) agonist L-817,818 could restore NHE8 expression via its role in suppressing ERK1/2 phosphorylation. Our study uncovered a novel mechanism of SST stimulation of NHE8 expression in colitis.
Biliary atresia (BA) is a pediatric inflammatory disease of the biliary system which leads to cirrhosis and the need for liver transplantation. Various cells types have been reported to participate in the pro-inflammatory response, including Th1, Th2, Th17, CD8+ T cells, or NK cells. The suppressive Treg cells, on the contrary, were not functioning properly. The underlying mechanism is largely unknown. Focusing on why the suppressive function of Treg was impaired, we found out methylation status of CpG islands within the Foxp3 promotor of Tregs in BA patients and murine models were both increased. Moreover, when injecting Aza as DNA-methylation inhibitor to RRV infected mice, jaundice rate were decreased and survival of the mice were prolong. Strikingly, Tregs isolated from Aza treated infected mice had better suppressive function than Tregs from the infected mice without Aza treatment. Besides, IL-17+Treg cells and Th17 cells were both elevated in mice when Foxp3 expression was epigenetically down regulated. Thus we concluded that aberrant increased demethylation status of "Foxp3 promoter" in Treg cells leads to impaired Treg suppressive function and participated in inflammatory injury to extrahepatic and intrathepatic bile duct in BA.
Glycosylation is involved in various pathophysiological conditions. N-Acetylglucosaminyltransferase V (GnT-V), catalyzing β1-6 branching in asparagines-linked oligosaccharides, is one of the most important glycosyltransferases involved in cancer and the immune system. Recent findings indicate that aberrant N-glycan structure can modify lipid metabolism. In this study, we investigated the effects of aberrant glycosylation by GnT-V on high-density lipoprotein cholesterol (HDL) assembly. We used GnT-V transgenic (Tg) mice and GnT-V Hep3B cell (human hepatoma cell line) transfectants. The study also included 96 patients who underwent medical health check-ups. Total serum cholesterol levels, particularly HDL-cholesterol (HDL-C) levels, were significantly increased in Tg versus wild type (WT) mice. Hepatic expression of apolipoprotein AI (ApoAI) and ATP-binding cassette sub-family A member 1 (ABCA1), two important factors in HDL assembly, were higher in Tg mice compared to WT mice. ApoAI and ABCA1 were also significantly elevated in GnT-V transfectants compared with mock-transfected cells. Moreover, ApoAI protein in the cultured media of GnT-V transfectants was significantly increased. Finally, we found a strong correlation between serum GnT-V activity and HDL-C concentration in human subjects. Multivariate logistic analyses demonstrated that GnT-V activity was an independent and significant determinant for serum HDL-C levels even adjusted with age and gender differences. Further analyses represented that serum GnT-V activity had strong correlation especially with the large size HDL particle concentration. These findings indicate that enhanced hepatic GnT-V activity accelerated HDL assembly and could be a novel mechanism for HDL synthesis.
In vertebrates, visceral pain from internal organs is detected by spinal afferents, whose cell bodies lie in dorsal root ganglia (DRG). Until now, all recordings from spinal afferents have been restricted to recording transmission of action potentials along axons, or from cell bodies lying outside their target organ, which is not where sensory transduction occurs. Our aim was to record directly from a major class of spinal afferent within visceral organs, where transduction of sensory stimuli into action potentials takes place. Using novel Calcitonin-Gene-Related-Peptide (CGRP)α-reporter mice, DRG neurons expressed mCherry, including nerve axons within viscera. In colon, a minority of total CGRP immunoreactivity was attributed CGRPα. In isolated un-stretched colon, calcium imaging from CGRPα expressing varicose axons did not detect resolvable calcium transients. However, noxious levels of maintained circumferential stretch to the colon induced repetitive calcium transients simultaneously in multiple neighboring varicosities along single mCherry-expressing axons. Discrete varicosities could generate unitary calcium transients independently of neighboring varicosities. However, axons expressing mCherry only generated coordinated calcium transients when accompanied by simultaneous activation of multiple varicosities along that axon. Simultaneous imaging from different classes of myenteric neurons at the same time as mCherry-expressing axons revealed coordinated calcium transients in multiple myenteric neurons, independent of activity in mCherry-expressing axons. The mCherry-expressing axons preferentially responded to heat, capsaicin and low pH. We show that direct recordings can be made from the major class of peptidergic spinal afferent that contributes to visceral nociception. This approach can provide powerful insights into transduction of stimuli in viscera.
High-resolution (HR) mapping has been used to study gastric slow-wave activation, however the specific characteristics of antral electrophysiology remain poorly defined. This study applied HR mapping and computational modeling to define functional human antral physiology. HR mapping was performed in ten subjects using flexible electrode arrays (128-192 electrodes; 16-24 cm2) arranged from the pylorus to mid corpus. Anatomical registration was by photographs and anatomical landmarks. Slow wave parameters were computed and resultant data incorporated into a computational fluid dynamics (CFD) model of gastric flow to calculate impact on gastric mixing. In all subjects, extracellular mapping demonstrated normal aboral slow-wave propagation and a region of increased amplitude and velocity in the pre-pyloric antrum. On average, the high-velocity region commenced 28 mm proximal to the pylorus and activation ceased 6 mm from the pylorus. Within this region, velocity increased 0.2 mm s-1 per mm of tissue, from mean 3.3 ± 0.1 mm s-1 to 7.5 ± 0.6 mm s-1 (p<0.001), and extracellular amplitude increased from 1.5 ± 0.1 mV to 2.5 ± 0.1 mV (p<0.001). CFD modeling using representative parameters quantified a marked increase in antral recirculation, resulting in an enhanced gastric mixing, due to the accelerating terminal antral contraction. The extent of gastric mixing increased almost linearly with the maximal velocity of the contraction. In conclusion, the human terminal antral contraction is controlled by a short region of rapid high-amplitude slow wave activity. Distal antral wave acceleration plays a major role in antral flow and mixing, increasing particle strain and trituration.
Knockout technology has proven useful for delineating functional roles of specific genes. Here we describe and provide an explanation for striking pathology that occurs in a subset of genetically engineered mice expressing a rat CaVβ2a transgene under control of the cardiac α-myosin heavy chain promoter. Lesions were limited to mice homozygous for transgene and independent of native Cacnb2 genomic copy number. Gross findings included an atrophied pancreas, decreased adipose tissue, thickened, orange intestines, and enlarged liver, spleen and abdominal lymph nodes. Immune cell infiltration and cell engulfment by macrophages were associated with loss of pancreatic acinar cells. Foamy macrophages diffusely infiltrated the small intestine's lamina propria; while similar macrophage aggregates packed liver and splenic red pulp sinusoids. Periodic-acid-Schiff positive, diastase- resistant, iron-negative, Oil Red O positive, and autofluorescent cytoplasm was indicative of a lipid storage disorder. EM analysis revealed liver sinusoids distended by clusters of macrophages containing intracellular myelin "swirls" and hepatocytes with enlarged lysosomes. Additionally, build-up of cholesterol, cholesterol esters and triglycerides along with changes in liver metabolic enzyme levels were consistent with a lipid processing defect. Because of this complex pathology, we examined the transgene insertion site. Multiple transgene copies inserted into chromosome 19; at this same site an approximate 180,000 base pair deletion occurred, ablating cholesterol 25-hydroxylase and partially deleting lysosomal acid lipase and CD95. Loss of gene function can account for the altered lipid processing along with hypertrophy of the immune system, which define this phenotype and serendipitously provides a novel mouse model of lysosomal storage disorder.
Strategies for prevention and treatment of nonalcoholic steatohepatitis (NASH) remain to be established. Here, we evaluated the effect of glycine on metabolic steatohepatitis in genetically obese, diabetic KK-Ay mice. Male KK-Ay mice were fed a diet containing 5% glycine for 4 weeks, and liver pathology was evaluated. Hepatic mRNA levels for lipid-regulating molecules, cytokines/chemokines, and macrophage M1/M2 markers were determined by real time RT-PCR. Hepatic expression of natural killer (NK) T cells was analyzed by flow cytometry. Dietary glycine blunted body weight gain significantly, and prevented the development of hepatic steatosis and inflammatory infiltration remarkably as compared to controls. Indeed, hepatic induction levels of molecules related to lipogenesis were largely blunted in glycine-fed mice. Dietary glycine also blunted elevations in hepatic mRNA levels for TNFα and chemokine (C-C motif) ligand (CCL)2 remarkably in these mice. Further, dietary glycine reversed suppression of hepatic NKT cells in KK-Ay mice, which was accompanied by increases in basal hepatic expression levels of NKT cell-derived cytokines such as IL-4 and IL-13. Moreover, hepatic mRNA levels of arginase-1, a marker of macrophage M2 transformation, were significantly increased in glycine-fed mice. Furthermore, dietary glycine improved glucose tolerance and hyperinsulinemia in KK-Ay mice. These observations clearly indicate that glycine prevents maturity-onset obesity and metabolic steatohepatitis in genetically diabetic KK-Ay mice. The underlying mechanisms most likely include normalization in hepatic innate immune responses involving NKT cells and M2 transformation of Kupffer cells. It is proposed that glycine is a promising immuno-nutrient for prevention and treatment of metabolic syndrome-related NASH.
Inhibitory motor neurons regulate several gastric motility patterns including receptive relaxation, gastric peristaltic motor patterns and pyloric sphincter opening. Nitric oxide (NO) and purines have been identified as likely candidates that mediate inhibitory neural responses. However, the contribution from each neurotransmitter has received little attention in the distal stomach. The aims of this study were to identify the roles played by NO and purines in inhibitory motor responses in the antrums of mice and monkeys. By using wildtype mice and mutants with genetically deleted neural nitric oxide synthase (Nos1-/-) and P2Y1 receptors (P2ry1-/-) we examined the roles of NO and purines in post-junctional inhibitory responses in the distal stomach and compared these responses to those in primate stomach. Activation of inhibitory motor nerves using electrical field stimulation (EFS) produced frequency-dependent inhibitory junction potentials (IJPs) that produced muscle relaxations in both species. Stimulation of inhibitory nerves during slow waves terminated pacemaker events and associated contractions. In Nos1-/- mice IJPs and relaxations persisted whereas in P2ry1-/- mice IJPs were absent but relaxations persisted. In the gastric antrum of the non-human primate model Macaca fascicularis, similar NO and purine neural components contributed to inhibition of gastric motor activity. These data support a role of convergent inhibitory neural responses in the regulation of gastric motor activity across diverse species.
Non-alcoholic steatohepatitis (NASH), especially as part of the metabolic syndrome (MS), is an increasing burden in western countries. Statins are already used in metabolic syndrome and seem to be beneficial in liver diseases. The aim of this study was to investigate the molecular mechanisms underlying pleiotropic effects on small GTPases of statins in NASH. NASH within MS was induced in 12-week-old ApoE-/- mice after 7 weeks of western diet (NASH mice). Small GTPases were inhibited by activated simvastatin (SMV), NSC23766 (NSC) or Clostridium sordellii lethal toxin (LT) using subcutaneous osmotic mini-pumps. Hepatic steatosis, inflammation and fibrosis were assessed by histology, Western blot and RT-PCR, measurements of cholesterol and hydroxyproline content. SMV treatment significantly decreased hepatic inflammation and fibrosis, but had no significant effect on steatosis and hepatic cholesterol content in NASH. SMV blunted fibrosis due to inhibition of both RhoA/Rho-kinase and Ras/ERK pathways. Interestingly, inhibition of RAC1 and Ras (by LT) failed to decrease fibrosis to the same extent. Inhibition of RAC1 (by NSC) showed no significant effect at all. Inhibition of RhoA and Ras downstream signaling by statins is responsible for the beneficial hepatic effects in NASH.
Impaired absorption of electrolytes is a hallmark of diarrhea associated with inflammation or enteric infections. Intestinal epithelial luminal membrane NHE3 (Na+/H+ exchanger 3) and DRA (Cl-/HCO3- exchanger) play key roles in mediating electroneutral NaCl absorption. We have previously shown decreased NHE3 and DRA function in response to short-term infection with enteropathogenic E. coli (EPEC), a diarrheal pathogen. Recent studies have also shown substantial downregulation of DRA expression in diarrheal model of infection with Citrobacter rodentium, the mouse counterpart of EPEC. Since our previous studies showed that the probiotic Lactobacillus acidophilus (LA) increased DRA and NHE3 function and expression and conferred protective effects in experimental colitis, we sought to evaluate the efficacy of LA in counteracting NHE3 and DRA inhibition and ameliorating diarrhea in a model of C rodentium infection. FVBN mice challenged with C. rodentium (1x109 CFU) with or without administration of live LA (3x109 CFU) were assessed for NHE3 and DRA mRNA and protein expression, mRNA levels of carbonic anhydrase, diarrheal phenotype (assessed by colonic weight/length ratio), myeloperoxidase (MPO) activity and proinflammatory cytokines. LA counteracted C. rodentium-induced inhibition of colonic DRA, NHE3 and carbonic anhydrase I and IV expression, attenuated diarrheal phenotype and MPO activity. Further, LA completely blocked C. rodentium induction of IL-1β, IFN- and CXCL1 mRNA and C. rodentium-induced STAT3 phosphorylation. In conclusion, our data provide mechanistic insights into anti-diarrheal effects of LA in a model of infectious diarrhea and colitis.
A comprehensive -omic, computational, and physiological approach was employed to examine the (previously unexplored) role of microRNAs (miRNAs) as regulators of IAS smooth muscle contractile phenotype and basal tone. MicroRNA profiling, genome wide expression, validation and network analyses were employed to assess changes in mRNA and miRNA expression in IAS smooth muscles from young vs. aging rats. Multiple miRNAs, including rno-miR-1, rno-miR-340-5p, rno-miR-185, rno-miR-199a-3p, rno-miR-200c, rno-miR-200b, rno-miR-31, rno-miR-133a and rno-miR-206 were found to be up-regulated in aging IAS. qRT-PCR confirmed the up-regulated expression of these miRNAs and down regulation of multiple, predicted targets (Eln, Col3a1, Col1a1, Zeb2, Myocd, SRF, Smad1, Smad2, RhoA/ROCK2, Fn1, Sm22-v2, Klf4, and Acta2) involved in regulation of SM contractility. Subsequent studies demonstrated an aging-associated increase in the expression of miR-133a, corresponding decreases in RhoA, ROCK2, MYOCD, SRF and SM22α protein expression, RhoA-signaling, and a decrease in basal and agonist (U-46619 (thromboxane A2 analog))-induced increase in the IAS tone. Moreover, in vitro transfection of miR-133a caused a dose-dependent increase of IAS tone in strips, which was reversed by anti-miR-133a. Lastly, in vivo perianal injection of anti-miR-133a reversed the loss of IAS tone associated with age. This work establishes the important regulatory effect of miRNA-133a on basal and agonist-stimulated IAS tone. Moreover, reversal of age-associated loss of tone via anti-miR delivery strongly implicates miR dysregulation as a causal factor in the aging-associated decrease in IAS tone, and suggests miR-133a is feasible therapeutic target in aging-associated rectoanal incontinence.
Stimulation of the esophagus activates the pharyngeal swallow response (EPSR) in human infants and animals. The aims of this study were to characterize the stimulus and response of the EPSR, and to determine the function and mechanisms generating the EPSR. Studies were conducted in 46 decerebrate cats in which pharyngeal, laryngeal and esophageal motility was monitored using EMG, strain gauges, or manometry. The esophagus was stimulated by balloon distension or luminal fluid infusion. We found that esophageal distension increased the chance of occurrence of the EPSR, but the delay was variable. The chance of occurrence of the EPSR was related to the position, magnitude, and length of the stimulus in the esophagus. The most effective stimulus was long, strong and situated in the cervical esophagus. Acidification of the esophagus activated pharyngeal swallows and sensitized the receptors which activate the EPSR. The EPSR was blocked by local anesthesia applied to the esophageal lumen and electrical stimulation of the RLNc activated the pharyngeal swallow response. We conclude that the EPSR is activated in a probabilistic manner. The receptors mediating the EPSR are probably mucosal slowly adapting tension receptors. The sensory neural pathway includes the RLNc and SLN. We hypothesize that since the EPSR is observed in human infants and animals, but not human adults, activation of EPSR is related to the elevated position of the larynx. In this situation, the EPSR occurs rather than secondary peristalsis, to prevent supra-esophago reflux when the esophageal bolus is in the proximal esophagus.
Liver regeneration is a clinically significant tissue repair process, which is suppressed by chronic alcohol intake through poorly understood mechanisms. Recently, miR-21 has been suggested to serve as a crucial microRNA regulator driving hepatocyte proliferation after partial hepatectomy (PHx) in mice. However, we reported recently that miR-21 is significantly upregulated in ethanol-fed rats at 24h after PHx despite inhibition of cell proliferation, suggesting a more complex role for this microRNA. Here, we investigate how inhibition of miR-21 in vivo affects the early phase of liver regeneration in ethanol-fed rats. Chronically ethanol-fed rats and pair-fed control animals were treated with AM21, a mixed locked-nucleic acid/DNA analogue antisense to miR-21 that inhibited miR-21 in vivo to undetectable levels. Liver regeneration after PHx was followed by cell proliferation markers, gene expression and microRNA profiling, and cell signaling pathway analysis. Although liver regeneration was not significantly impaired by AM21 in chow-fed rats, AM21 treatment in ethanol-fed animals completely restored regeneration and enhanced PHx-induced hepatocyte proliferation to levels comparable to untreated or chow-fed animals. In addition, a marked deposition of α-smooth muscle actin, a marker of stellate cell activation, which was evident in ethanol-treated animals after PHx, was effectively suppressed by AM21 treatment. Gene expression analysis further indicated that suppression of stellate cell-specific pro-fibrogenic profiles and NOTCH signaling contributed to AM21-mediated rescue from deficient hepatocyte proliferation in ethanol-fed animals. Our results indicate that the impact of miR-21 balances pro-proliferative effects with anti-proliferative pro-fibrogenic actions in regulating distinctive regenerative responses in normal versus disease conditions.
Stearoyl CoA Desaturase (SCD-1) is a lipogenic enzyme involved in the de novo biosynthesis of oleate (C18:1, n9), a major fatty acid in the phospholipids of lipid bilayers of cell membranes. Accordingly, Scd1KO mice display substantially reduced oleate in cell membranes. Altered SCD-1 level was observed during intestinal inflammation; however, its role in modulating inflammatory bowel disease remains elusive. Herein, we investigated the colitogenic capacity of Scd1KO effector T cells by employing the adoptive T-cell transfer colitis model. Splenic effector T-cells (CD4+CD25-) from age- and sex-matched WT and Scd1KO mice were isolated by FACS and intraperitoneally administered to Rag1KO mice, which were monitored for the development of colitis. At day 60 post-cell transfer, Rag1KO mice which received Scd1KO CD4+CD25- T cells displayed accelerated and exacerbated colitis than mice receiving WT cells. Intriguingly, Scd1KO CD4+CD25- T cells display augmented inflammatory cytokine profile and cellular membrane fluidity with concomitant increase in pro-inflammatory saturated fatty acids, which we postulate to potentially underlie their augmented colitogenic potential.
Rat proximal and distal colon are net K+ secretory and net K+ absorptive epithelia, respectively. Chronic dietary K+ loading increases net K+ secretion in the proximal colon, and transforms net K+ absorption to net K+ secretion in the distal colon, but changes in apical K+ channel expression are unclear. We evaluated expression/activity of apical K+ (BK) channels in surface colonocytes in proximal and distal colon of control and K+ loaded animals using patch-clamp recording, immunohistochemistry and western blot analyses. In controls, BK channels were more prevalent in surface colonocytes from K+ secretory proximal colon (39% of patches) than in those from K+ absorptive distal colon (12% of patches). Immunostaining demonstrated more pronounced BK channel α-subunit protein expression in surface cells and cells in the upper 25% of crypts in proximal colon, compared with distal colon. Dietary K+ loading had no clear-cut effects on the prevalence, immunolocalization or expression of BK channels in proximal colon. By contrast, in distal colon, K+ loading (i) increased BK channel prevalence in patches from 12% to 41%; (ii) increased density of immunostaining in surface cells, which extended along the upper 50% of crypts; and (iii) increased expression of BK channel α-subunit protein when assessed by western blotting (P<0.001). Thus, apical BK channels are normally more abundant in K+ secretory proximal colon than in K+ absorptive distal colon, and apical BK channel expression in distal (but not proximal) colon is greatly stimulated as part of the enhanced K+ secretory response to dietary K+ loading.
Obesity presents a significant public health concern given its association with increased cancer incidence, unfavorable prognosis, and metastasis. However, there is very little literature on the effects of weight-loss, following obesity, on risk for colon cancer or liver cancer. Therefore, we sought to study whether intentional weight loss through diet manipulation was capable of mitigating colon and liver cancer in mice. We fed mice with a high-fat diet (HFD) comprised of 47% carbohydrates, 40% fat, and 13% protein for 20 weeks to mimic human obesity. Subsequently, azoxymethane (AOM) was used to promote colon and liver carcinogenesis. A subset of obese mice was then switched to a low-fat diet (LFD) containing 67.5% carbohydrate, 12.2% fat, and 20% protein in order to promote intentional weight loss. Body weight loss and excess fat reduction did not protect mice from colon cancer progression and liver dysplastic lesion in the AOM-chemical-cancer model even though these mice had improved blood glucose and leptin levels. Intentional weight loss in AOM-treated mice actually produced histological changes that resemble dysplastic alterations in the liver and presented a higher percentage of F4/80+CD206+ macrophages and activated T cells (CD4+CD69+) in the spleen and lymph nodes, respectively. In addition, the liver of AOM-treated mice exposed to a HFD during the entire period of the experiment exhibited a marked increase in proliferation and pNFkB activation. Altogether, these data suggest that intentional weight loss following chemical-induced carcinogenesis does not affect colon tumorigenesis but may in fact negatively impact liver repair mechanisms.
Epidemiological studies support strong links between obesity, diabetes and pancreatic disorders including pancreatitis and pancreatic adenocarcinoma (PDAC). Type 2 diabetes (T2DM) is associated with insulin resistance, hyperglycemia and hyperinsulinemia, the latter due to increased insulin secretion by pancreatic beta-cells. We reported that high fat diet-induced PDAC progression in mice is associated with hyperglycemia, hyperinsulinemia and activation of pancreatic stellate cells (PaSC). We investigated here the effects of high concentrations of insulin and glucose on mouse and human PaSC growth and fibrosing responses. We found that compared to normal, pancreata from T2DM patients displayed extensive collagen deposition and activated PaSC in islet and peri-islet exocrine pancreas. Mice fed a high-fat diet for up to 12 months similarly displayed increasing peri-islet fibrosis compared to mice fed control diet. Both quiescent and activated PaSC co-express insulin (IR; mainly A type) and IGF (IGF-1R) receptors, and both insulin and glucose modulate receptor expression. In cultured PaSC, insulin induced rapid tyrosine autophosphorylation of IR/IGF-1R at specific kinase domain activation loop sites, activated Akt/mTOR/p70S6K signaling and inactivated FoxO1, a transcription factor that restrains cell growth. Insulin did not promote activation of quiescent PaSC in either 5 mM or 25 mM glucose containing media. However, in activated PaSC, insulin enhanced cell proliferation and augmented production of extracellular matrix proteins, and these effects were abolished by specific inhibition of mTORC1 and mTORC2. In conclusion, our data supports the concept that increased local glucose and insulin concentrations associated with obesity and T2DM promote PaSC growth and fibrosing responses.
Background: Total parenteral nutrition (TPN) leads to a shift in small intestinal microbiota with a characteristic dominance of Proteobacteria. This study examined how metabolomic changes within the small bowel support an altered microbial community in enterally deprived mice. Methods: C57BL/6 mice were given TPN or enteral chow. Metabolomic analysis of jejunal contents was performed by liquid chromatography/mass spectrometry (LC/MS). In some experiments, leucine in TPN was partly substituted with 13C-leucine. Additionally, jejunal contents from TPN dependent and enterally fed mice were gavaged into germ-free mice to reveal if the TPN phenotype was transferrable. Results: Small bowel contents of TPN mice maintained an amino acid composition similar to that of the TPN solution. Mass spectrometry analysis of small bowel contents of TPN dependent mice showed increased concentration of 13C compared to fed mice receiving saline enriched with 13C-leucine. 13C-leucine added to the serosal side of Ussing chambers showed rapid permeation across TPN-dependent jejunum, suggesting increased transmucosal passage. Single-cell analysis by fluorescence in situ hybridization (FISH) - NanoSIMS demonstrated uptake of 13C-leucine by TPN-associated bacteria, with preferential uptake by Enterobacteriaceae. Gavage of small bowel effluent from TPN mice into germ-free, fed mice resulted in a trend toward the pro-inflammatory TPN-phenotype with loss of epithelial barrier function. Conclusions: TPN-dependence leads to increased permeation of TPN-derived nutrients into the small intestinal lumen, where they are predominately utilized by Enterobacteriaceae. The altered metabolomic composition of the intestinal lumen during TPN promotes dysbiosis.
Enteric inhibitory motorneurons use nitric oxide (NO) and a purine neurotransmitter to relax gastrointestinal smooth muscle. Enteric P/Q type Ca2+ channels contribute to excitatory neuromuscular transmission; their contribution to inhibitory transmission is less clear. We used the colon from tottering mice (tg/tg, loss of function mutation in the α1A pore-forming subunit of P/Q-type Ca2+ channels) to test the hypothesis that P/Q-type Ca2+ channels contribute to inhibitory neuromuscular transmission and colonic propulsive motility. Fecal pellet output in vivo and the colonic migrating motor complex (CMMC, ex vivo) were measured. Neurogenic circular muscle relaxations and inhibitory junction potentials (IJPs) were also measured ex vivo. Colonic propulsive motility in vivo and ex vivo was slightly impaired in tg/tg mice. IJPs were either unchanged or somewhat larger in tissues from tg/tg compared to WT mice. Nifedipine (L-type Ca2+ channel antagonist) inhibited IJPs by 35% and 14% in tissues from tg/tg and WT mice, respectively. The contribution of N- and R-type channels to neuromuscular transmission was larger in tissues from tg/tg compared to WT mice. The resting membrane potential of circular muscle cells was similar in tissues from tg/tg and WT mice. Neurogenic relaxations of circular muscle from tg/tg and WT mice were similar. These results demonstrate that a functional deficit in P/Q-type channels does not alter propulsive colonic motility. Myenteric neuron L-type Ca2+ channel function increases to compensate for loss of functional P/Q type Ca2+ channels. This compensation maintains inhibitory neuromuscular transmission and normal colonic motility.
Epidermal growth factor receptor (EGFR) and its ligands have been implicated in liver fibrosis. However, it has not been directly shown that hepatocellular genetic ablation of either this receptor tyrosine kinase or ERBB3, its interactive signaling partner, affects hepatic fibrosis. Carbon tetrachloride (CCl4)-induced liver fibrosis in hepatocyte-specific (HS) mouse models of EGFR and ERBB3 ablation was evaluated in both single gene knockouts and a HS-EGFR-ERBB3 double knockout. Loss of hepatocellular EGFR or ERBB3 did not impact cytochrome P450-2E1 expression, the extent of centrilobular injury, or the initial regenerative response, but it did diminish liver fibrosis induced by chronic intraperitoneal administration of CCl4. The reduction of liver fibrosis correlated with reduced α-smooth muscle actin expression. Maximal impact in fibrogenesis occurred in the ERBB3 and EGFR-ERBB3 DKO models, suggesting that EGFR-ERBB3 heterodimeric signaling in damaged hepatocytes may play a more important role in liver fibrosis than EGFR-EGFR homodimeric signaling. Immunohistochemical analyses of phospho-EGFR and phospho-ERBB3 isoforms revealed clear staining in hepatocytes, activated stellate cells and macrophages. Our results support a role for the hepatocellular ERBB tyrosine kinases in fibrogenesis and suggest that pharmacologic inhibition of EGFR-ERBB3 signaling may reverse or retard hepatic fibrosis.
The therapeutic and preventive application of probiotics for necrotizing enterocolitis (NEC) has been supported by more and more experimental and clinical evidence in which Toll-like receptor 4 (TLR4) exerts a significant role. In immune cells, probiotics not only regulate the expression of TLR4 but also use the TLR4 to modulate the immune response. Do probiotics also use the TLR4 in immature enterocytes for anti-inflammation? Here we demonstrate that probiotic conditioned media (PCM) from Bifidobacterium longum supp infantis but not isolated organisms attenuates interleukin-6 (IL-6) induction in response to IL-1β by using TLR4 in a human fetal small intestinal epithelial cell line (H4 cells), human fetal small intestinal xenografts, mouse fetal small intestinal organ culture tissues and primary NEC enterocytes. Furthermore, we show that PCM, using TLR4, down regulates the mRNA expression of interleukin -1 receptor - associated kinase 2 (IRAK2), a common adapter protein shared by IL-1β and TLR4 signaling. PCM also reduces the phosphorylation of the activator-protein 1(AP-1) transcription factors c-Jun and c-Fos in response to IL-1β stimulation in a TLR4-dependent manner. This study suggests that PCM may use TLR4 through IRAK2, and via AP-1 to prevent IL-1β-induced IL-6 induction in immature enterocytes. Based on these observations, the combined use of probiotics and anti-TLR4 therapy to prevent NEC may not be a good strategy.
Anti-TNFα therapy decreases inflammation in Crohn's disease (CD). However, its ability to decrease fibrosis and alter the natural history of CD is not established. Anti-TNFα prevents inflammation and fibrosis in the peptidoglycan-polysaccharide (PG-PS) model of CD. Here we studied anti-TNFα in a treatment paradigm. PG-PS or HSA (control) was injected into bowel wall of anesthetized Lewis rats at laparotomy. Mouse anti-mouse TNFα or vehicle treatment was begun d1, d7, or d14 post-laparotomy. Rats were euthanized d21-23. Gross abdominal and histologic findings were scored. Cecal levels of relevant mRNAs were measured by quantitative real-time PCR. There was a stepwise loss of responsiveness when anti-TNFα was begun on d7 and d14 compared with d1 that was seen in the % decrease in the median gross abdominal score and histologic inflammation score in PG-PS-injected rats [as % decrease; gross abdominal score: d1=75% (p=0.003), d7=57% (p=0.18), d14=no change (p=0.99); histologic inflammation: d1=57% (p=0.006), d7=50% (p=0.019), d14=no change (p=0.99)]. This was also reflected in changes in IL-1β, IL-6, TNFα, IGF-I, TGF-β1, procollagen I, and procollagen III mRNAs that were decreased or trended downward in PG-PS-injected animals given anti-TNFα beginning d1 or d7 compared to vehicle-treated rats; there was no effect if anti-TNFα was begun d14. This change in responsiveness to anti-TNFα therapy was coincident with a major shift in the cytokine milieu observed on d14 in the PG-PS injected rats (vehicle-treated). Our data are consistent with the clinical observation that improved outcomes occur when anti-TNFα therapy is initiated early in the course of Crohn's disease.
Altered gut microbial diversity has been associated with several chronic disease states, including heart failure. Stimulation of the vagus nerve, which innervates the heart and abdominal organs, is proving to be an effective therapeutic in heart failure. We hypothesized that cervical vagus nerve stimulation (VNS) could alter fecal flora and prevent aberrations observed in fecal samples from heart failure animals. To determine if microbial abundances were altered by pressure overload (PO) leading to heart failure and VNS therapy, a VNS pulse generator was implanted with a stimulus lead on either the left or right vagus nerve prior to creation of PO by aortic constriction. Animals received intermittent, open-loop stimulation or sham treatment and their heart function was monitored by echocardiography. Left ventricular end systolic and diastolic volumes and cardiac output were impaired in PO animals compared to baseline. VNS mitigated these effects. Metagenetic analysis was then performed using 16S rRNA sequencing to identify bacterial genera present in fecal samples. The abundance of ten genera was significantly altered by PO; eight of which were mitigated in animals receiving either left- or right-sided VNS. Metatranscriptomics analyses indicate that the abundance of genera that express genes associated with ATP-binding cassette (ABC) transport and amino sugar/nitrogen metabolism was significantly changed following PO. These gut flora changes were not observed in PO animals subjected to VNS. These data suggest that VNS prevents aberrant gut flora following PO, which could contribute to its beneficial effects in heart failure patients.
The impact of the dietary protein level on the process of colonic mucosal inflammation and subsequent recovery remains largely unknown. In this study, we fed DSS-treated mice with either a normoproteic (NP) or a high-protein (HP) isocaloric diet from the beginning of the 5-day DSS treatment to 14 days later. Measurements of colitis indicators (colon weight:length ratio, myeloperoxidase activity, cytokine expressions) showed a similar level of colonic inflammation in both DSS groups during the colitis induction phase. However, during the colitis resolution phase, inflammation intensity was higher in the DSS-HP group than in the DSS-NP group as evidenced by higher inflammatory score and body weight loss. This coincided with a higher mortality rate. In surviving animals, an increase in colonic crypt height associated with a higher number of colon epithelial cells per crypt and TGF-β3 content was observed in the DSS-HP vs. DSS-NP group. Moreover, colonic expression patterns of tight junction proteins and E-cadherin were also different according to the diet. Altogether, our results indicate that the HP diet, when given during both the induction and resolution periods of DSS-induced colitis, showed deleterious effects during the post-induction phase. However, HP diet ingestion was also associated with morphological and biochemical differences compatible with higher colonic epithelium restoration in surviving animals, indicating an effect of the dietary protein level on colonic crypt repair after acute inflammation. These data highlight the potential impact of the dietary protein amount during the colitis course.
The endocannabinoid system mainly consists of endogenously produced cannabinoids (endocannabinoids) and two G protein-coupled receptors (GPCRs), cannabinoid receptors 1 and 2 (CB1 and CB2). This system also includes enzymes responsible for the synthesis and degradation of endocannabinoids and molecules required for the uptake and transport of endocannabinoids. In addition, endocannabinoid-related lipid mediators and other putative endocannabinoid receptors, such as transient receptor potential channels and other GPCRs have been identified. Accumulating evidence indicates that the endocannabinoid system is a key modulator of gastrointestinal physiology, influencing satiety, emesis, immune function, mucosal integrity, motility, secretion, and visceral sensation. In light of therapeutic benefits of herbal and synthetic cannabinoids, the vast potential of the endocannabinoid system for the treatment of gastrointestinal diseases has been demonstrated. This review focuses on the role of the endocannabinoid system in gut homeostasis and in the pathogenesis of intestinal disorders associated with intestinal motility, inflammation and cancer. Finally, links between gut microorganisms and the endocannabinoid system are briefly discussed.
Pancreatitis is an inflammatory disease of the pancreas characterized by dysregulated activity of digestive enzymes, necrosis, immune infiltration, and pain. Repeated incidence of pancreatitis is an important risk factor for pancreatic cancer. Legumain, a lysosomal cysteine protease, has been linked to inflammatory diseases such as atherosclerosis, stroke, and cancer. Until now, legumain activation has not been studied during pancreatitis. We used a fluorescently quenched activity-based probe to assess legumain activation during caerulein-induced pancreatitis in mice. We detected activated legumain by ex vivo imaging, confocal microscopy and gel electrophoresis. Compared to healthy controls, legumain activity in the pancreas of caerulein-treated mice was increased in a time-dependent manner. Legumain was localized to CD68+ macrophages and was not active in pancreatic acinar cells. Using a small-molecule inhibitor of legumain, we found that this protease is not essential for the initiation of pancreatitis. However, it may serve as a biomarker of disease, since patients with chronic pancreatitis show strongly increased legumain expression in macrophages. Moreover, the occurrence of legumain-expressing macrophages in regions of acinar-to-ductal metaplasia suggests that this protease may influence reprogramming events that lead to inflammation-induced pancreatic cancer.
Bifidobacterium breve and other Gram-positive gut commensal microbes protect the gastrointestinal epithelium against inflammation-induced stress. However, the mechanisms whereby these bacteria accomplish this protection are poorly understood. In this study, we examined soluble factors derived from Bifidobacterium breve and their impact on the two major protein degradation systems within intestinal epithelial cells, proteasomes and autophagy. Conditioned media from gastrointestinal Gram-positive, but not Gram-negative, bacteria activated autophagy and increased expression of the autophagy proteins Atg5 and Atg7 along with the stress response protein heat shock protein 27 (Hsp27). Specific examination of media conditioned by the Gram-positive bacterium Bifidobacterium breve (Bb-CM) showed that this microbe produces small molecules (<3kD) that increase expression of the autophagy proteins Atg5 and Atg7, activate autophagy, and inhibit proteasomal enzyme activity. Upregulation of autophagy by Bb-CM was mediated through MAP kinase signaling. In vitro studies using C2BBe1 cells silenced for Atg7 and in vivo studies using mice conditionally deficient in intestinal epithelial cell Atg7 showed that Bb-CM induced cytoprotection is dependent upon autophagy. Therefore, this work demonstrates that Gram-positive bacteria modify protein degradation programs within intestinal epithelial cells to promote their survival during stress. It also reveals the therapeutic potential of soluble molecules produced by these microbes for prevention and treatment of gastrointestinal disease.
The cholinergic anti-inflammatory pathway reduces systemic TNF via acetylcholine producing memory T-cells in the spleen. These choline acetyltransferase (ChAT) expressing T-cells are also found in the intestine, where their function is unclear. We aimed to characterize these cells in mouse and human intestine and delineate their function. We made use of the ChAT-eGFP reporter mice. CD4Cre mice were crossed to ChATfl/fl mice to achieve specific deletion of ChAT in CD4+ T-cells. We observed that the majority of ChAT expressing T-cells in the human and mouse intestine have characteristics of Th17 cells, and co-express IL17A, IL22 and RORC. The generation of ChAT expressing T-cells was skewed by dendritic cells after activation of their adrenergic receptor β2. To evaluate ChAT T-cell function, we generated CD4 specific ChAT deficient mice. CD4ChAT-/- mice showed a reduced level of epithelial antimicrobial peptides (AMP) lysozyme, defA, and ang4, which was associated with an enhanced bacterial diversity and richness in the small intestinal lumen in CD4ChAT-/- mice. We conclude that ChAT expressing T-cells in the gut are stimulated by adrenergic receptor activation on dendritic cells. ChAT expressing T-cells may function to mediate the host AMP secretion, microbial growth and expansion.
Background: C. difficile infection (CDI) is a common debilitating nosocomial infection associated with high mortality. Several CDI outbreaks have been attributed to ribotypes 027, 017, and 078. Clinical and experimental evidence indicates that the non-pathogenic yeast Saccharomyces boulardii CNCM I-745 (S.b) is effective for the prevention of CDI. However, there is no current evidence suggesting this probiotic can protect from CDI caused by outbreak-associated strains. Methods: We used established hamster models infected with outbreak-associated C. difficile strains to determine whether oral administration of live or heat-inactivated S.b can prevent cecal tissue damage and inflammation. Results: Hamsters infected with C. difficile strain VPI10463 (ribotype 087) and outbreak-associated strains ribotype 017, 027 and 078 developed severe cecal inflammation with mucosal damage, neutrophil infiltration, edema, increased NF-kappaB phosphorylation, and increased pro-inflammatory cytokine tumor necrosis factor alpha (TNFalpha) protein expression. Oral gavage of live, but not heated, S.b starting 5 days before C. difficile infection significantly reduced cecal tissue damage, NF-kappaB phosphorylation, and TNFalpha protein expression caused by infection with all strains. Moreover, S.b conditioned medium reduced cell rounding caused by filtered supernatants from all C. difficile strains. S.b conditioned medium also inhibited toxin A- and B-mediated actin cytoskeleton disruption. Conclusions: S.b is effective in preventing C. difficile infection by outbreak-associated via inhibition of the cytotoxic effects of C. difficile toxins.
Non-Alcoholic Fatty Liver Disease (NAFLD) is a common causes of chronic liver disease and is increasing with the rising rate of obesity in the developed world. Signaling pathways known to influence the rate of lipid deposition in liver, known as hepatic steatosis, include the Transforming Growth Factor (TGF) superfamily, which function through the SMAD second messengers. The Kielin/Chordin-like Protein (KCP) is a large secreted protein that can enhance Bone Morphogenetic Protein (BMP) signaling while suppressing TGF-β signaling in cells and in genetically modified mice. In this report, we show that aging KCP mutant (Kcp-/-) mice are increasingly susceptible to developing hepatic steatosis and liver fibrosis. When young mice are put on a high fat diet, Kcp-/- mice are also more susceptible to developing liver pathology, compared to their wild-type littermates. Furthermore, mice that express a Pepck-KCP transgene (KcpTg) in the liver are resistant to developing liver pathology even when fed a high fat diet. Analyses of liver tissues reveal a significant reduction of P-Smad3, consistent with a role for KCP in suppressing TGF-β signaling. Transcriptome analyses show that livers from Kcp-/- mice fed a normal diet are more like wild-type livers from mice fed a high fat diet. However, the KCP transgene can suppress many of the changes in liver gene expression that are due to a high fat diet. These data demonstrate that shifting the TGF-β signaling paradigm with the secreted regulatory protein KCP can significantly alter the liver pathology in aging mice and in diet induced NAFLD.
It is not well understood of how monosodium glutamate affects gastrointestinal physiology, especially regarding the absorption and the subsequent transport of dietary lipids into lymph. Thus far, there is little information about how the ingestion of MSG affects the lipid lipolysis, uptake, intracellular esterification, and formation and secretion of chylomicrons. Using lymph fistula rats treated with the infusion of a 2% MSG prior solution prior to a continuous infusion of triglyceride, we show that MSG causes a significant decrease in both triglyceride and cholesterol secretion into lymph. Intriguingly, the diminished lymphatic transport of triglyceride and cholesterol was not caused by an accumulation of these labeled lipids in the intestinal lumen nor in the intestinal mucosa. Rather, it is a result of increased portal transport in the animals fed acutely the lipid plus 2% MSG in the lipid emulsion. This is a first demonstration of MSG on intestinal lymphatic transport of lipids.
Mechanosensory neurons detect physical events in the local environments of the tissues that they innervate. Studies of mechanosensitivity of neurons or nerve endings in the gut have related their firing to strain, wall tension or pressure. Digital Image Correlation (DIC) is a technique from materials engineering that can be adapted to measure the local physical environments of afferent neurons at high resolution. Flat sheet preparations of guinea pig distal colon were set up with arrays of tissue markers, in vitro. Firing of single viscerofugal neurons was identified in extracellular colonic nerve recordings. The locations of viscerofugal nerve cell bodies were inferred by mapping firing responses to focal application of the nicotinic receptor agonist, DMPP. Mechanosensory firing was recorded during load-evoked uni-axial or bi-axial distensions. Distension caused movement of surface markers which was captured using video imaging. DIC tracked the markers, interpolating the mechanical state of the gut at the location of the viscerofugal nerve cell body. This technique revealed heterogeneous load-evoked strain within preparations. Local strains at viscerofugal nerve cell bodies were usually smaller than global strain measurements and correlated more closely with mechanosensitive firing. Both circumferential and longitudinal strain activated viscerofugal neurons. Simultaneous loading in circumferential and longitudinal axes, caused the highest levels of viscerofugal neuron firing. Multiaxial strains, reflecting tissue shearing and changing area, linearly correlated with mechanosensory firing of viscerofugal neurons. Viscerofugal neurons were mechanically sensitive to both local circumferential and local longitudinal gut strain, and appear to lack directionality in their stretch sensitivity.
BACKGROUND & AIMS Autophagy and the unfolded protein response (UPR) are key cellular homeostatic mechanisms and are both involved in liver diseases, including non-alcoholic fatty liver disease (NAFLD). Although increasing but conflicting results link these mechanisms to lipid metabolism, their role and potential crosstalk herein has been poorly investigated. Therefore, we assessed the effects of hepatocyte-specific autophagy-deficiency on liver parenchyma, the UPR and lipid metabolism. METHODS Adult hepatocellular-specific autophagy-deficient mice (Atg7F/FAlb-Cre+) were compared with their autophagy-competent littermates (Atg7+/+Alb-Cre+). Livers were analysed by electron microscopy, histology, real-time qPCR and Western blotting. RESULTS Atg7F/FAlb-Cre+ mice developed hepatomegaly with significant parenchymal injury as evidenced by inflammatory infiltrates, hepatocellular apoptosis, pericellular fibrosis and a pronounced ductular reaction. Surprisingly, the UPR exhibited a pathway-selective pattern upon autophagy-deficiency. The activity of the adaptive ATF6 pathway was abolished, whereas the pro-apoptotic PERK pathway was increased compared to Atg7+/+Alb-Cre+ mice. The IRE1α signal was unaltered. Fasting-induced steatosis was absent in Atg7F/FAlb-Cre+ mice. Remarkably, some isolated islands of fat-containing and autophagy-competent cells were observed in these livers. CONCLUSIONS Hepatocellular autophagy is essential for parenchymal integrity in mice. Moreover, in case of autophagy-deficiency, the three different UPR branches are pathway-selectively modulated. Attenuation of the ATF6 pathway might explain the observed impairment of fasting-induced steatosis. Finally, autophagy and lipid droplets are directly linked to each other.
Development of the infant small intestine is influenced by bacterial colonization. To promote establishment of optimal microbial communities in preterm infants, knowledge of the beneficial functions of the early gut microbiota on intestinal development is needed. The purpose of this study was to investigate the impact of early preterm infant microbiota on host gut development using a gnotobiotic mouse model. Histological assessment of intestinal development was performed. The differentiation of four epithelial cell lineages (enterocytes, goblet cells, Paneth cells, enteroendocrine cells) and tight junction (TJ) formation was examined. Using weight gain as a surrogate marker for health, we found that early microbiota from a preterm infant with normal weight gain (MPI-H) induced increased villus height and crypt depth, increased cell proliferation, increased numbers of goblet cells and Paneth cells, and enhanced TJs compared to the changes induced by early microbiota from a poor weight gain preterm infant (MPI-L). Laser capture microdissection (LCM) plus qRT-PCR further revealed, in MPI-H mice, a higher expression of stem cell marker Lgr5 and Paneth cell markers Lyz1 and Cryptdin5 in crypt populations; along with higher expression of the goblet cell and mature enterocyte marker Muc3 in villus populations. In contrast, MPI-L microbiota failed to induce the aforementioned changes and presented intestinal characteristics comparable to a germ free host. Our data demonstrate that microbial communities have differential effects on intestinal development. Future studies to identify pioneer settlers in neonatal microbial communities necessary to induce maturation may provide new insights for preterm infant microbial ecosystem therapeutics.
High throughput technologies revealed new categories of genes, including the long non-coding RNAs (lncRNAs), involved in the pathogenesis of human disease; however the role of lncRNAs in the ulcerative colitis (UC) has not been evaluated. Gene expression profiling was used to develop lncRNA signatures in UC samples. Jurkat T cells were activated by PMA/ionomycin subsequently interferon-gamma (IFNG) and tumor necrosis factor (TNF)-alpha protein levels were assessed by ELISA. Anti-sense molecules were designed to block IFNG-AS1 expression. A unique set of lncRNAs was differentially expressed between UC and control samples. Of these, IFNG-AS1 was among the highest statistically significant lncRNAs (fold change: 5.27, p-value: 7.07E-06). Bioinformatic analysis showed that IFNG-AS1 was associated with the IBD susceptibility loci SNP rs7134599 and its genomic location is adjacent to the inflammatory cytokine, IFNG. Using mouse models of colitis, active colitis samples had increased colonic expression of this lncRNA. Utilizing the Jurkat T cell model, IFNG-AS1 was found to positively regulate IFNG expression. Novel lncRNA signatures differentiate UC patients with active disease, in remission and control subjects. A subset of these lncRNAs was found to be associated with the clinically validated IBD susceptibility loci. IFNG-AS1 was one of these differentially expressed lncRNAs in UC patients and found to regulate the key inflammatory cytokine, IFNG, in CD4 T cells. Taken together, our study revealed novel lncRNA signatures deregulated in ulcerative colitis and identified IFNG-AS1 as a novel regulator of IFNG inflammatory responses, suggesting the potential importance of non-coding RNA mechanisms on regulation of IBD-related inflammatory responses.
Utilizing a conditional (intestinal-specific) knockout (cKO) mouse model, we have recently shown that the SMVT (SLC5A6) is the only biotin uptake system that operates in the gut and that its deletion leads to biotin deficiency. Unexpectedly, we also observed that all SMVT-cKO mice develop chronic active inflammation, especially in the cecum. Our aim here was to examine the role of SMVT in the maintenance of intestinal mucosal integrity [permeability and expression of tight junction (TJ) proteins]. Our results showed that knocking out the mouse intestinal SMVT is associated with a significant increase in gut permeability and with changes in level of expression of TJ proteins. To determine whether these changes are related to the state of biotin deficiency that develops in SMVT-cKO mice, we induced (by dietary means) biotin deficiency in wild-type (WT) mice and examined its effect on the above-mentioned parameters. The results showed that dietary-induced biotin deficiency leads to a similar development of chronic active inflammation in the cecum with an increase in level of expression of pro-inflammatory cytokines, as well as an increase in intestinal permeability and changes in level of expression of TJ proteins. We also examined the effect of chronic biotin deficiency on permeability and expression of TJ proteins in confluent intestinal epithelial Caco-2 monolayers but observed no changes in these parameters. These results show that the intestinal SMVT plays an important role in the maintenance of normal mucosal integrity, most likely via its role in providing biotin to different cells of the gut mucosa.
Barrier dysfunction is a characteristic of the inflammatory bowel diseases (IBD), Crohn's disease and ulcerative colitis. Understanding how the tight junction is modified to maintain barrier function may provide avenues for treatment of IBD. We have previously shown that the apical addition of serine proteases to intestinal epithelial cell lines causes a rapid and sustained increase in transepithelial electrical resistance (TER), but the mechanisms are unknown. We hypothesized that serine proteases increase barrier function through trafficking and insertion of tight junction proteins into the membrane, and this could enhance recovery of a disrupted monolayer after calcium switch or cytokine treatment. In the canine epithelial cell line, SCBN, we showed that matriptase, an endogenous serine protease, could potently increase TER. Using detergent solubility-based cell fractionation, we found that neither trypsin nor matriptase treatment changed levels of tight junction proteins at the membrane. In a fast calcium switch assay, serine proteases did not enhance the rate of recovery of the junction. In addition, serine proteases could not reverse barrier disruption induced by IFN and TNFα. We knocked down occludin in our cells using siRNA and found this prevented the serine protease-induced increase in TER. Using fluorescence recovery after photobleaching (FRAP), we found serine proteases induce a greater mobile fraction of occludin in the membrane. These data suggest that a functional tight junction is needed for serine proteases to have an effect on TER, and that occludin is a crucial tight junction protein in this mechanism.
During sepsis bacterial products particularly LPS trigger injury in organs such as the liver. This common condition remains largely untreatable in part due to a lack of understanding of how high concentrations of LPS cause cellular injury. In the liver, the lysosomal degradative pathway of autophagy performs essential hepatoprotective functions and is induced by LPS. We therefore examined whether hepatocyte autophagy protects against liver injury from septic levels of LPS. Mice with an inducible hepatocyte-specific knockout of the critical autophagy gene Atg7 were examined for their sensitivity to high-dose LPS. Increased liver injury occurred in knockout mice as determined by significantly increased serum alanine aminotransferase levels, histological evidence of liver injury, terminal deoxynucleotide transferase-mediated deoxyuridine triphosphate nick end-labeling and effector caspase 3 and 7 activation. Hepatic inflammation and proinflammatory cytokine induction were unaffected by the decrease in hepatocyte autophagy. Although knockout mice had normal NF-B signaling, hepatic levels of Akt1 and Akt2 phosphorylation in response to LPS were decreased. Cultured hepatocytes from knockout mice displayed a generalized defect in Akt signaling in response to multiple stimuli including LPS, TNF and IL-1β. Akt activation mediates hepatocyte resistance to TNF cytotoxicity, and anti-TNF antibodies significantly decreased LPS-induced liver injury in knockout mice, indicating that the loss of autophagy sensitized to TNF-dependent liver damage. Hepatocyte autophagy therefore mediates hepatic resistance to LPS-induced liver injury. Conditions such as aging and steatosis that impair hepatic autophagy may predispose to poor outcomes from sepsis through this mechanism.
Gluten are proline- and glutamine-rich proteins present in wheat, barley and rye, and contain the immunogenic sequences that drive celiac disease (CD). Rothia mucilaginosa, an oral microbial colonizer, can cleave these gluten epitopes. The aim was to isolate and identify the enzymes and evaluate their potential as novel enzyme therapeutics for CD. The membrane-associated R. mucilaginosa proteins were extracted and separated by DEAE chromatography. Enzyme activities were monitored with paranitroanilide derivatized- and FRET peptide substrates, and by gliadin zymography. Epitope elimination was determined in R5 and G12 ELISA assays. The gliadin-degrading Rothia enzymes were identified by LC-ESI-MS/MS as hypothetical proteins ROTMU0001_0241 (C6R5V9_9MICC), ROTMU0001_0243 (C6R5W1_9MICC) and ROTMU0001_240 (C6R5V8_9MICC). A blast search revealed that these are subtilisin-like serine proteases belonging to the peptidase S8 family. Alignment of the major Rothia subtilisins indicated that all contain the catalytic triad with Asp (D), His (H) and Ser (S) in the D-H-S order. They cleaved succinyl-Ala-Ala-Pro-Phe-paranitroanilide, a substrate for subtilisin with Pro in the P2 position, as in Tyr-Pro-Gln and Leu-Pro-Tyr in gluten, which are also cleaved. Consistently, FRET substrates of gliadin immunogenic epitopes comprising Xaa-Pro-Xaa motives were rapidly hydrolyzed. The Rothia subtilisins as well as two subtilisins from Bacillus licheniformis, subtilisin A and the food-grade Nattokinase efficiently degraded the immunogenic gliadin-derived 33-mer peptide as well as the immunodominant epitopes recognized by the R5 and G12 antibodies. This study identified Rothia and food-grade Bacillus subtilisins as promising new candidates for enzyme therapeutics in CD.
Opioids are one of the most prescribed drug classes for treating acute pain. However chronic use is often associated with tolerance as well as debilitating side effects including nausea and dependence, which are mediated by the central nervous system, as well as constipation emerging from effects on the enteric nervous system. These gastrointestinal side effects limit the usefulness of opioids in treating pain in many patients. Understanding the mechanism(s) of action of opioids on the nervous system that show clinical benefit as well as those that have unwanted effects is critical for the improvement of opioid drugs. The opioidergic system comprises 3 classical receptors (µ,, ) and a non-classical receptor (Nociceptin) and each of these receptors are expressed to varying extents by the enteric and intestinal extrinsic sensory afferent nerves. The purpose of this review is to discuss the role that the opioidergic system has on enteric and extrinsic nerves in the lower GI tract in health and diseases of the lower GI tract, particularly Inflammatory Bowel Disease and Irritable Bowel Syndrome, and the implications opioid treatment has on clinical outcomes. Consideration is also given to emerging developments in our understanding of the immune system as a novel source of endogenous opioids and the mechanisms underlying opioid tolerance including the potential influence of opioid receptor splice variants and heteromeric complexes.
Prior studies in IBS-D patients showed immune activation, secretion and barrier dysfunction in jejunal or colorectal mucosa. We measured mRNA expression by RT-PCR of 91 genes reflecting tight junction proteins, chemokines, innate immunity, ion channels, transmitters, housekeeping genes, and controls for DNA contamination and PCR efficiency in small intestinal mucosa from 15 IBS-D and 7 controls (biopsies negative for celiac disease). Fold change was calculated using 2(-, CT) formula. Nominal p values (p<0.05) were interpreted with FDR correction (q value). Cluster analysis with LENS explored connectivity of mechanisms. Upregulated genes (uncorrected p<0.05) were related to ion transport (INADL, MAGI1 and SONS1), barrier (TJP1, 2, 3 and CLDN) or immune functions (TLR3, IL15, MAPKAPK5), or histamine metabolism (HNMT); downregulated genes were related to immune function (IL-1β, TGF β 1 and CCL20) or antigen detection (TLR1 and 8). The following genes were significantly upregulated (q<0.05) in IBS-D: INADL, MAGI1, PPP2R5C, MAPKAPK5, TLR3, IL-15. Among the 14 nominally upregulated genes, there was clustering of barrier and PDZ domains (TJP1, TJP2, TJP3, CLDN4, INADL, MAGI1) and clustering of downregulated genes (CCL20, TLR1, IL1B, and TLR8). Protein expression of PPP2R5C in nuclear lysates was greater in patients with IBS-D and controls. There was increase in INADL protein (median 9.4ng/mL) in patients with IBS-D relative to controls (median 5.8ng/mL, p>0.05). In conclusion, altered transcriptome (and to lesser extent protein) expression of ion transport, barrier, immune and mast cell mechanisms in small bowel may reflect different alterations in function and deserves further study in IBS-D.
Hyperphagic Otsuka Long-Evans Tokushima Fatty (OLETF) rats develop obesity, insulin resistance, and nonalcoholic fatty liver disease (NAFLD) but lifestyle modifications, such as caloric restriction (CR), can prevent these conditions. PURPOSE: We sought to determine if prior CR had protective effects on metabolic health and NAFLD development following a 4 week return to ad libitum (AL) feeding. METHODS: Four-week old male OLETF rats (n=8-10/group) were fed AL for 16 weeks (O-AL), CR for 16 weeks (O-CR; ~70% kcal of O-AL), or CR for 12 weeks followed by 4 weeks of AL feeding (O-AL4wk). RESULTS: CR-induced benefits in the prevention of NAFLD, including reduced hepatic steatosis, inflammation, and markers of Kupffer cell activation/number, were largely lost in AL4wk rats. These findings occurred in conjunction with a partial loss in CR-induced benefits on obesity and serum triglycerides in O-AL4wk but in the absence of changes in serum glucose or insulin. CR-induced increases in hepatic mitochondrial respiration remained significantly elevated (p<0.01) in O-AL4wk rats compared with O-AL rats, while mitochondrial [1-14C] palmitate oxidation, citrate synthase activity, and β-HAD activity did not differ among OLETF groups. NAFLD development in O-AL4wk rats was accompanied by increases in the protein content of the de novo lipogenesis markers FAS and SCD-1, and decreases in pACC/ACC vs O-CR (p<0.05 for each). CONCLUSION: The beneficial effects of chronic CR on NAFLD development were largely lost with 4 weeks of ad libitum feeding in the hyperphagic OLETF rat, highlighting the importance of maintaining energy balance in the prevention of NAFLD.
Macrophage colony-stimulating factor (CSF1) is an essential growth and differentiation factor for cells of the macrophage lineage. To explore the practical applications of CSF1 therapy, we developed a bioactive protein with a longer half-life in circulation by fusing pig CSF1 with Fc region of pig IgG1a. Three consecutive daily doses of CSF1-Fc in 8 week old pigs expanded macrophage populations in blood and organs, accelerated maturation of macrophage populations in peripheral blood monocytes, and expanded progenitor pools in bone marrow. The hepatosplenomegaly present after CSF1-Fc administration was partially due to a substantial increase in macrophage numbers in both organs as well as an increase in proliferating cells in the liver. Despite the large influx of macrophages a panel of biochemical tests to measure liver damage demonstrated normal function in CSF1-Fc treated pigs. Additionally microarray data confirmed that the increase in total liver weight must be primarily due to the extensive proliferation and subsequent increase in hepatocyte numbers. In the current study we have extended our previous finding that CSF1-Fc increased the size of the liver in mice to the domestic pig, an animal that is considerably more human-like in size and vascular biology. CSF1-dependent monocyte recruitment is both necessary and sufficient to drive hepatic proliferation and CSF1-Fc treatment can push it beyond the homeostatic limits even in a large animal. Treatment confirms the existence of a homeostatic feedback loop linking macrophages of the liver with bone marrow and blood monocytes which together regulate the physiological hepatostat as a "macrostat".
Mother's own milk is the optimal first diet for preterm infants, but donor human milk (DM) or infant formula (IF) are used when supply is limited. We hypothesized that a gradual introduction of bovine colostrum (BC) or DM improves gut maturation, relative to IF during the first 11 days after preterm birth. Preterm pigs were fed gradually advancing doses of BC, DM or IF (3-15 mL/kg/3 h, n=14-18) before measurements of gut structure, function, microbiology and immunology. The BC pigs showed higher body growth, intestinal hexose uptake and transit time, and reduced diarrhea and gut permeability, relative to DM and IF pigs (P<0.05). Relative to IF pigs, BC pigs also had lower density of mucosa-associated bacteria and of some putative pathogens in colon, together with higher intestinal villi, mucosal mass, brush-border enzyme activities, colonic short chain fatty acid levels and bacterial diversity, and an altered expression of immune-related genes (higher TNFα, IL17; lower IL8, TLR2, TFF, MUC1, MUC2) (all P<0.05). Values in DM pigs were intermediate. Severe necrotizing enterocolitis (NEC) was observed in >50% of IF pigs, while only sub-clinical intestinal lesions were evident from DM and BC pigs. Bovine colostrum, and to some degree donor human milk, are superior to preterm infant formula in stimulating gut maturation and body growth, using a gradual advancement of enteral feeding volume over the first 11 days after preterm birth in piglets. Whether the same is true in preterm infants remains to be tested.
Muscularis propria throughout the entire gastrointestinal tract including the esophagus is comprised of circular and longitudinal muscle layers. Based on the studies conducted in the colon and the small intestine, for more than a century, it has been debated whether the two muscle layers contract synchronously or reciprocally during the ascending contraction, and descending relaxation of the peristaltic reflex. Recent studies in the esophagus and colon prove that the two muscle layers indeed contract and relax together in almost perfect synchrony during ascending contraction, and descending relaxation of the peristaltic reflex, respectively. Studies in patients with various types of esophageal motor disorders reveal temporal disassociation between the circular and longitudinal muscle layers. We suggest that the discoordination between the two muscle layers plays a role in the genesis of esophageal symptoms, i.e., dysphagia and esophageal pain. Certain pathologies may selectively target one and not the other muscle layer, e.g. in eosinophilic esophagitis there is a selective dysfunction of the longitudinal muscle layer. In achalasia esophagus, swallows are accompanied by the strong contraction of the longitudinal muscle without circular muscle contraction. The possibility that the discoordination between two muscle layers plays a role in the genesis of esophageal symptoms, i.e., dysphagia and esophageal pain is discussed. The purpose of this review is to summarize the regulation and dysregulation of peristalsis by the coordinated and discoordinated function of circular and longitudinal muscle layers in health and diseased states.
Chemotherapy-induced gastrointestinal (GI) toxicity is a common adverse effect of cancer treatment. We used preweaned piglets as models to test our hypothesis that the immunomodulatory and gastrointestinal trophic effects of bovine colostrum would reduce the severity of GI complications associated with doxorubicin treatment. Five-day-old pigs were administered doxorubicin (DOX, 1 x 100 mg/m2) or an equivalent volume of saline (SAL) and either fed formula (DOX-Form, n=9 or SAL-Form, n=7) or bovine colostrum (DOX-Colos, n=9 or SAL-Colos, n=7). Pigs were euthanized five days after initiation of chemotherapy to assess markers of small intestinal function and inflammation. All DOX-treated animals developed diarrhea, growth deficits and leukopenia. However, the intestines of DOX-Colos pigs had lower intestinal permeability, longer intestinal villi with higher activities of brush border enzymes, and lower tissue IL-8 levels compared with DOX-Form (all P<0.05). DOX-Form pigs, but not DOX-Colos pigs, had significantly higher plasma C-reactive protein (CRP), compared with SAL-Form. Plasma citrulline was not affected by DOX treatment or diet. Thus, a single dose of doxorubicin induces intestinal toxicity in preweaned pigs and may lead to a systemic inflammatory response. The toxicity is affected by type of enteral nutrition with more pronounced GI toxicity when formula is fed compared with bovine colostrum. The results indicate that bovine colostrum may be a beneficial supplementary diet for children subjected to chemotherapy and subsequent intestinal toxicity.
Intestinal wounds often occur during inflammatory and ischemic disorders of the gut. To repair damage, intestinal epithelial cells must rapidly spread and migrate to cover exposed lamina propria, events that involve redox signaling. Wounds are subject to extensive redox alterations, particularly resulting from H2O2 produced in the adjacent tissue by both the epithelium and emigrating leukocytes. The mechanisms governing these processes are not fully understood, particularly at the level of protein signaling. Crk-associated substrate, or Cas, is an important signaling protein known to modulate focal adhesion and actin cytoskeletal dynamics, and whose association with Crk is regulated by Abl kinase, a ubiquitously expressed tyrosine kinase. We sought to evaluate the role of Abl regulation of Cas at the level of cell spreading and migration during wound closure. As a model, we used intestinal epithelial cells exposed to H2O2 or scratch-wounded to assess the Abl-Cas signaling pathway. We characterized the localization of phosphorylated Cas in mouse colonic epithelium under baseline conditions and after biopsy wounding the mucosa. Analysis of actin and focal adhesion dynamics by microscopy or biochemical analysis after manipulating Abl kinase revealed that Abl controls redox dependent Cas phosphorylation and localization to influence cell spreading and migration. Collectively, our data sheds new light on redox sensitive protein signaling modules controlling intestinal wound healing.
The technically easier one-anastomosis (mini) gastric bypass (MGB) is associated with similar metabolic improvements and weight loss as the Roux-en-Y gastric bypass (RYGB). However, MGB is controversial and suspected to result in greater malabsorption than RYGB. In this study, we compared macronutrient absorption and intestinal adaptation after MGB or RYGB in rats. Body weight and food intake were monitored and glucose tolerance tests were performed in rats subjected to MGB, RYGB, or sham surgery. Carbohydrate, protein, and lipid absorption was determined by fecal analyses. Intestinal remodeling was evaluated by histology and immunohistochemistry. Peptide and amino acid transporter mRNA levels were measured in the remodeled intestinal mucosa and those of anorexigenic and orexigenic peptides in the hypothalamus. The MGB and RYGB surgeries both resulted in a reduction of body weight and an improvement of glucose tolerance relative to sham rats. Hypothalamic orexigenic neuropeptide gene expression was higher in MGB rats than in RYGB or sham rats. Fecal losses of calories and proteins were greater after MGB than RYGB or sham surgery. Intestinal hyperplasia occurred after MGB and RYGB with increased jejunum diameter, higher villi, and deeper crypts than in sham rats. Peptidase and peptide or amino acid transporter genes were overexpressed in jejunal mucosa from MGB rats but not RYGB rats. In rats, MGB led to greater protein malabsorption and energy loss than RYGB. This malabsorption was not compensated by intestinal overgrowth and increased expression of peptide transporters in the jejunum.
Nitric oxide alters gastric blood flow, improves vascular function and mediates glucose uptake within the intestines and skeletal muscle. Dietary nitrate, acting as a source of nitric oxide, appears to be a potential low cost therapy that may help maintain glucose homeostasis. In a randomised, double-blind, placebo-controlled crossover study, 31 young and older adult participants had a standardised breakfast, supplemented with either nitrate rich beetroot juice (11.91 mmol nitrate) or nitrate depleted beetroot juice as placebo (0.01 mmol nitrate). MRI was used to assess apparent diffusion coefficient (ADC), portal vein flux and velocity. Plasma glucose, incretin and C-peptide concentrations and BP were assessed. Outcome variables were measured at baseline and hourly for 3 hours. Compared with a placebo, beetroot juice resulted in a significant elevation in plasma nitrate and plasma nitrite concentration. No differences were seen for the young or older adult cohorts between placebo and beetroot juice for ADC, or portal vein flux. There was an interaction effect in the young adults, which was absent in the older adults between visits for portal vein velocity. Nitrate supplementation did not reduce plasma glucose active GLP-1, total GLP-1 or plasma C-peptide concentrations for the young or older adult cohorts. Despite a significant elevation in plasma nitrite concentration following an acute dose of 11.91 mmol of nitrate, there was no effect on hepatic blood flow, plasma glucose, C-peptide, or incretin concentration in healthy adults.
Divalent metal-ion transporter-1 (DMT1), the principal mechanism by which nonheme iron is taken up at the intestinal brush border, is energized by the H+-electrochemical potential gradient. The provenance of the H+ gradient in vivo is unknown so we have explored a role for brush-border Na+/H+ exchangers by examining iron homeostasis and intestinal iron handling in mice lacking Na+/H+ exchanger-2 (NHE2) or Na+/H+ exchanger-3 (NHE3). We observed modestly depleted liver iron stores in NHE2-null (NHE2-/-) mice stressed on a low-iron diet but no change in hematological or blood-iron variables, or the expression of genes associated with iron metabolism, compared with wildtype mice. Ablation of NHE3 strongly depleted liver iron stores regardless of diet. We observed decreases in blood-iron variables but no overt anemia in NHE3-/- mice on a low-iron diet. Intestinal expression of DMT1, the apical surface ferrireductase Cybrd1, and the basolateral iron exporter ferroportin was upregulated in NHE3-/- mice, and expression of liver Hamp1 (hepcidin) suppressed, compared with wildtype. Absorption of 59Fe from an oral dose was substantially impaired in NHE3-/- mice compared with wildtype. Our data point to an important role for NHE3 in generating the H+ gradient that drives DMT1-mediated iron uptake at the intestinal brush border.
The mu opioid receptor (MOR) is a major regulator of gastrointestinal motility and secretion, and mediates opiate-induced bowel dysfunction. Although MOR is of physiological and therapeutic importance to gut function, the cellular and subcellular distribution and regulation of MOR within the enteric nervous system is largely undefined. Herein, we defined the neurochemical coding of MOR expressing neurons in the guinea pig gut, and examined the effects of opioids on MOR trafficking and regulation. MOR expression was restricted to subsets of enteric neurons. In the stomach MOR was mainly localized to nitrergic neurons (~88%), with some overlap with NPY and no expression by cholinergic neurons. These neurons are likely to have inhibitory motor and secretomotor functions. MOR was restricted to non-cholinergic secretomotor neurons (VIP-positive) of the ileum and distal colon submucosal plexus. MOR was mainly detected in nitrergic neurons of the colon (NOS-positive, 87%), with some overlap with ChAT. No expression of MOR by intrinsic sensory neurons was detected. DAMGO, morphiceptin and loperamide induced MOR endocytosis in myenteric neurons. After stimulation with DAMGO and morphiceptin MOR recycled, whereas MOR was retained within endosomes following loperamide treatment. Herkinorin or the delta opioid receptor agonist DADLE did not evoke MOR endocytosis. In summary, we have identified the neurochemical coding of MOR positive enteric neurons and have demonstrated differential trafficking of MOR in these neurons in response to established and putative MOR agonists.
Ischemia-reperfusion (IR) injury is a major clinical problem and is associated with numerous adverse effects. GGsTop [2-amino-4{[3-(carboxymethyl)phenyl](methyl)phosphono}butanoic acid] is a highly specific and irreversible -glutamyl transpeptidase (-GT) inhibitor. We studied the protective effects of GGsTop on IR induced hepatic injury in rats. Ischemia was induced by clamping the portal vein and hepatic artery of left lateral and median lobes of the liver. Before clamping, saline (IR group) or saline containing 1 mg/kg body weight of GGsTop (IR-GGsTop group) was injected into the liver through inferior vena cava. At 90 min of ischemia, blood flow was restored. Blood was collected before induction of ischemia and prior to restoration of blood flow, and at 12, 24, and 48 h after reperfusion. All the animals were sacrificed at 48 h after reperfusion and the livers were harvested. Serum levels of ALT, AST, and -GT were significantly lower after reperfusion in IR-GGsTop group compared to IR group. Massive hepatic necrosis was present in IR group, while only few necroses were present in IR-GGsTop group. Treatment with GGsTop increased hepatic GSH content which was significantly reduced in IR group. Furthermore, GGsTop prevented increase of hepatic -GT, malondialdehyde, 4-hydroxynonenal, and TNF-α while all these molecules significantly increased in the IR group. In conclusion, treatment with GGsTop increased glutathione levels and prevented formation of free radicals in the hepatic tissue that lead to decreased IR-induced liver injury. GGsTop could be used as a pharmacological agent to prevent IR-induced liver injury and the related adverse events.
Pericytes and glial cells are accessory cells of the neurovascular networks, which have been reported to participate in scar formation after tissue injury. However, it remains unclear whether similar reactive cellular responses occur in pancreatic intraepithelial neoplasia (PanIN). Here, we develop 3-dimensional (3-D) duct lesion histology to investigate PanIN and the associated pericyte, glial, and islet remodeling. Transparent mouse pancreata with KrasG12D mutation were used to develop 3-D duct lesion histology. Deep-tissue, tile-scanning microscopy was performed to generate panoramic views of the diseased pancreas for global examination of the early-stage and advanced duct lesion formation. Fluorescence signals of the ductal and neurovascular networks were simultaneously detected to reveal the associated remodeling. Significantly, in the KrasG12D-mutant mice, when the low-grade PanINs emerge, duct lesions appear as epithelial buds with peri-lesional pericyte and glial activation. When PanINs occur in large-scale (induced by cerulein injections to the mutant mice), the 3-D image data identifies: i) aggregation of PanINs in clusters in space, ii) overexpression of the pericyte marker NG2 in the PanIN microenvironment, and iii) epithelial ingrowth to islets, forming the PanIN-islet complexes. Particularly, the PanIN-islet complexes associate with the proliferating epithelial and stromal cells and receive substantial neurovascular supplies, making them landmarks in the atrophic lobe. Overall, the peri-lesional pericyte and glial activation and formation of PanIN-islet complex underline the cellular heterogeneity in the duct lesion microenvironment. The results also illustrate the advantage of using 3-D histology to reveal the previously unknown details of the neurovascular and endocrine links to the disease.
The IL-33/ST2 axis plays a protective role in T cell-mediated hepatitis, but little is known about the functional impact of endogenous IL-33 on liver immunopathology. We used IL33-deficient mice to investigate the functional effect of endogenous IL-33 in concanavalin A (Con A)-hepatitis. IL-33-/- mice displayed more severe Con A liver injury than WT mice, consistent with a hepatoprotective effect of IL-33. The more severe hepatic injury in IL-33-/- mice was associated with significantly higher levels of TNF-α and IL-1-β and a larger number of NK cells infiltrating the liver. The expression of Th2 cytokines (IL-4, IL10) and IL-17 was not significantly varied between WT and IL-33-/- mice following Con A-hepatitis. The percentage of CD25+ NK cells was significantly higher in the livers of IL-33-/- mice than in WT mice in association with up-regulated expression of CXCR3 in liver. Regulatory T cells (Treg cells) strongly infiltrated the liver in both WT and IL-33-/- mice, but Con A treatment increased their membrane expression of ST2 and CD25 only in WT mice. In vitro, IL-33 had a significant survival effect, increasing the total number of splenocytes, including B cells, CD4+ and CD8+ T cells, and the frequency of ST2+ Treg cells. In conclusion, IL-33 acts as a potent immune modulator protecting the liver through activation of ST2+Treg cells and control of NK cells.
We have previously shown that the sequential transcription factors Xbp1->Mist1 (Bhlha15) govern the ultrastructural maturation of the secretory apparatus in enzyme-secreting zymogenic chief cells (ZCs) in the gastric unit. Here we sought to identify transcriptional regulators upstream of XBP1 and MIST1. We used immunohistochemistry to characterize Hnf4αflox/flox in adult mouse stomach after tamoxifen-induced deletion of Hnf4α. We used qRT-PCR, western blotting, and chromatin immunoprecipitation to define the molecular interaction between HNF4α and Xbp1 in mouse stomach and human gastric cells. We show that HNF4α protein is expressed in pit (foveolar) cells, mucous neck cells, and zymogenic chief cells (ZCs) of the corpus gastric unit. Loss of HNF4α in adult mouse stomach led to reduced ZC size and ER content, phenocopying previously characterized effects of Xbp1 deletion. However, HNF4α/ stomachs also exhibited additional phenotypes including increased proliferation in the isthmal stem cell zone and altered mucous neck cell migration, indicating a role of HNF4α in progenitor cells as well as in ZCs. HNF4α directly occupies the Xbp1 promoter locus in mouse stomach, and forced HNF4α expression increased abundance of XBP1 mRNA in human gastric cancer cells. Finally, as expected, loss of HNF4α caused decreased Xbp1 and Mist1 expression in mouse stomachs. We show that HNF4α regulates homeostatic proliferation in the gastric epithelium and is both necessary and sufficient for the upstream regulation of the Xbp1->Mist1 axis in maintenance of ZC secretory architecture.
Background: The metabolic benefits induced by gastric bypass - currently the most effective treatment for morbid obesity - are associated with BA delivery to the distal intestine. However, mechanistic insights into BA signaling in the mediation of metabolic benefits remain an area of focus. Methods: The BD mouse model, in which the gallbladder is anastomosed to the distal jejunum, was utilized to test BA specific role in the regulation of glucose and lipid homeostasis. Metabolic phenotype, including body weight and composition, glucose tolerance, energy expenditure, thermogenesis genes, total BA and BA composition in the circulation and portal vein, and gut microbiota were examined. Results: BD improves metabolic phenotype in line with increased circulating primary BAs and regulation of enterohormones. BD-induced hypertrophy of the proximal intestine in the absence of BAs was reversed by BA oral gavage, but without influencing BD metabolic benefits. BD enhanced energy expenditure was associated with elevated TGR5, D2 and thermogenic genes, including UCP1, PRDM16, PGC1α/β and PDGFRα, in epididymal white adipose tissue (eWAT) and inguinal WAT (iWAT), but not in brown adipose tissue (BAT). BD altered the gut microbiota profile, i.e. decreasing Firmicutes and increasing Bacteriodetes and Akkermansia being positively correlated with increased circulating primary BAs. Conclusions: Our study demonstrates that enhancement of BA signaling regulates glucose and lipid homeostasis, promotes thermogenesis, and modulates the gut microbiota, which collectively resulted in an improved metabolic phenotype.
Monocytes are critical to the pathogenesis of inflammatory bowel disease (IBD) as they infiltrate the mucosa and release cytokines that drive the inflammatory response. Ursodeoxycholic acid (UDCA), a naturally-occurring bile acid with anti-inflammatory actions, has been proposed as a potential new therapy for IBD. However, its effects on monocyte function are not yet known. Primary monocytes from healthy volunteers or cultured U937 monocytes were treated with either the pro-inflammatory cytokine, TNFα (5 ng/mL) or the bacterial endotoxin, lipopolysaccharide (LPS; 1 µg/ml) for 24 hrs, in the absence or presence of UDCA (25 - 100 μM). IL-8 release into the supernatant was measured by ELISA. mRNA levels were quantified by qPCR and changes in cell signalling proteins were determined by western blotting. Toxicity was assessed by measuring lactate dehydrogenase (LDH) release. UDCA treatment significantly attenuated TNFα-, but not LPS-driven, release of IL-8 from both primary and cultured monocytes. UDCA inhibition of TNFα-driven responses was associated with reduced IL-8 mRNA expression. Both TNFα and LPS stimulated NFB activation in monocytes, while IL-8 release in response to both cytokines was attenuated by an NFB inhibitor, BMS-345541. Interestingly, UDCA inhibited TNFα-, but not LPS-stimulated, NFB activation. Finally, TNFα, but not LPS, induced phosphorylation of TNF receptor associated factor (TRAF2), while UDCA co-treatment attenuated this response. We conclude that UDCA specifically inhibits TNFα-induced IL-8 release from monocytes by inhibiting TRAF2 activation. Since such actions would serve to dampen mucosal immune responses in vivo, our data and support the therapeutic potential of UDCA for IBD.
Protease-activated receptors PAR1 and PAR2 play an important role in the control of epithelial cell proliferation and migration. However, the survival of normal and tumor intestinal stem/progenitor cells promoted by proinflammatory mediators may be critical in oncogenesis. Glycogen synthase kinase 3β (GSK3β) pathway is over-activated in colon cancer cells and promotes their survival and drug resistance. We thus aimed to determine PAR1 and PAR2 effects on normal and tumor intestinal stem/progenitor cells and whether they involved GSK3β. First, PAR1 and PAR2 were identified in colon stem/progenitor cells by immunofluorescence. In 3D cultures of murine crypt units or single tumor Caco-2 cells, PAR2 activation decreased numbers and size of normal or cancerous spheroids, and PAR2-deficient spheroids showed increased proliferation, indicating that PAR2 represses proliferation. PAR2-stimulated normal cells were more resistant to stress (serum starvation or spheroid passaging), suggesting pro-survival effects of PAR2. Accordingly, active Caspase 3 was strongly increased in PAR2-deficient normal spheroids. PAR2 but not PAR1 triggered GSK3β activation through serine 9-dephosphorylation in normal and tumor cells. The PAR2-triggered GSK3β activation implicates an arrestin/PP2A/GSK3β complex which is dependent on the Rho kinase activity. Loss of PAR2 was associated with high levels of GSK3β non-active form, strengthening the role of PAR2 in GSK3β activation. GSK3 pharmacological inhibition impaired the survival of PAR2-stimulated spheroids and serum-starved cells. Altogether our data identify PAR2/GSK3β as a novel pathway that plays a critical role in the regulation of stem/progenitor cell survival and proliferation in normal colon crypts as well as in colon cancer.
Lymphatic failure is a histopathological feature of IBD. Recent studies show that the interaction between platelets and podoplanin on the lymphatic endothelial cells (LECs) suppresses lymphangiogenesis. We aimed to investigate the role of platelets in the inflammatory process of colitis, which is likely to be through modulating lymphangiogenesis. Lymphangiogenesis in the colonic mucosal specimens from IBD patients was investigated by studying the mRNA expression of lymphangiogenic factors and lymphatic vessel (LV) densities histologically. The involvement of lymphangiogenesis in intestinal inflammation was studied by administering VEGFR-3 inhibitors to the mouse model of DSS colitis and evaluating platelet migration to LV. Inhibitory effect of platelets on lymphangiogenesis was investigated in vivo by administering anti-platelet antibody to the colitis mouse model and in vitro by co-culturing platelets with LECs. Although mRNA expressions of lymphangiogenic factors such as VEGFR-3 and podoplanin increased significantly in the inflamed mucosa of IBD patients compared to those in the quiescent mucosa, there was no difference in LV densities between them. In the colitis model, VEGFR-3 inhibition aggravated colitis, decreased the lymphatic density, and increased platelet migration to LV. Administration of anti-platelet antibody increased the LV densities and significantly ameliorated colitis. Co-culture with platelets inhibited proliferation of LECs in vitro. Our data suggest that, in spite of elevated lymphoangiogenic factors during colonic inflammation, platelet migration to LV suppressed lymphangiogenesis, leading to the aggravation of colitis by blocking the clearance of inflammatory cells. Modulating the interaction between platelets and LV could be a new therapeutic means for treating IBD.
We tested the hypothesis that colonic enteric neurotransmission and smooth muscle cell (SMC) function are altered in mice fed a high fat diet (HFD). We used wild type (WT) mice and mice lacking the β1-subunit of the BK channel (BKβ1-/-). WT mice fed a HFD had increased myenteric plexus oxidative stress, a 28% decrease in nitrergic neurons and a 20% decrease in basal nitric oxide (NO) levels. Circular muscle inhibitory junction potentials (IJPs) were reduced in HFD WT mice. The NO synthase inhibitor nitro-L-arginine (NLA) was less effective at inhibiting relaxations in HFD compared to control diet (CD) WT mice (11% vs 37%, P<0.05). SMCs from HFD WT mice had depolarized membrane potentials (-47±2 mV) and continuous action potential firing compared to CD WT mice (-53±2 mV, P<0.05), which showed rhythmic firing. SMCs from HFD or CD fed BK β1-/- mice fired action potentials continuously. NLA depolarized membrane potential and caused continuous firing only in SMCs from CD WT mice. Sodium nitroprusside (NO donor) hyperpolarized membrane potential and changed continuous to rhythmic action potential firing in SMCs from HFD WT and BK β1-/- mice. Migrating motor complexes were disrupted in colons from BK β1-/- mice and HFD WT mice. BK channel α-subunit protein and β1-subunit mRNA expression were similar in CD and HFD WT mice. We conclude that HFD induced obesity disrupts inhibitory neuromuscular transmission, SMC excitability and colonic motility by promoting oxidative stress, loss of nitrergic neurons and SMC BK channel dysfunction.
Gut barrier dysfunction is the major trigger for multi-organ failure associated with hemorrhagic shock (HS). Although the molecular mediators responsible for this dysfunction are unclear, oxidative stress-induced disruption of proteostasis contributes to the gut pathology in HS. The objective of this study was to investigate whether resuscitation with nanoparticulate liposome-encapsulated hemoglobin (LEH) is able to restore the gut proteostatic mechanisms. Sprague Dawley rats were recruited in four groups: control, HS, HS+LEH, and HS+saline. HS was induced by withdrawing 45% blood, and isovolemic LEH or saline was administered after 15 min of shock. The rats were euthanized at 6 h to collect plasma and ileum for measurement of the markers of oxidative stress, unfolded protein response (UPR), proteasome function, and autophagy. HS significantly increased the protein and lipid oxidation, trypsin-like proteasome activity and plasma levels of IFN. These effects were prevented by LEH resuscitation. However, saline was not able to reduce protein oxidation and plasma IFN in hemorrhaged rats. Saline resuscitation also suppressed the markers of UPR and autophagy below the basal levels; the HS or LEH groups showed no effect on the UPR and autophagy. Histological analysis showed that LEH resuscitation significantly increased the villi height and thickness of the submucosal and muscularis layers as compared to the HS and saline groups. Overall, the results showed that LEH resuscitation was effective in normalizing the indicators of proteostasis stress in ileal tissue. On the other hand, saline-resuscitated animals showed a decoupling of oxidative stress and cellular protective mechanisms.
Our laboratory has reported that mice deficient in Programmed Cell Death Receptor (PD-1) have increased bacterial clearance and improved survival in experimental sepsis induced by cecal ligation and puncture (CLP). In response to infection, the liver clears the blood of bacteria and produces cytokines. Kupffer cells (KC), the resident macrophages in the liver, are strategically situated to perform the above functions. However, it is not known if PD-1 expression on KCs is altered by septic stimuli, let alone if PD-1 ligation contributes to the altered microbial handling seen. Here we report that PD-1 is significantly up-regulated on KCs during sepsis. PD-1 deficient septic KCs displayed markedly enhanced phagocytosis and restoration of the expression of MHCII and CD86, but reduced CD80 expression when compared with septic wild type (WT) mouse KCs. In response to ex vivo LPS stimulation, the cytokine productive capacity of KCs derived from PD-1-/- CLP mice exhibited a marked, albeit partial, restoration of the release of IL-6, IL-12, IL-1β, MCP-1 and IL-10 when compared with septic WT mouse KCs. In addition, PD-1 gene deficiency decreased LPS-induced apoptosis of septic KCs, as indicated by decreased levels of cleaved caspase-3 and reduced TUNEL-positive cells. Exploring the signal pathways involved, we found that after ex vivo LPS stimulation, septic PD-1-/- mouse KCs exhibited an increased Akt phosphorylation and a reduced p38 phosphorylation in comparison to septic WT mouse KCs. Together, these results indicate that PD-1 appears to play an important role in regulating the development of KC dysfunction seen in sepsis.
Non-invasive measurement of liver stiffness (LS) has been established to screen for liver fibrosis. Since LS is also elevated in response to pressure-related conditions such as liver congestion, this study was undertaken to learn more about the role of arterial pressure on LS. LS was measured using transient elastography (µFibroscan platform, Echosens, Paris) during single i.v. injections of catecholamines in anaesthetized rats with and without TAA-induced fibrosis. The effect of vasodilating glycerol trinitrate (GTN) on LS was also studied. Pressures in the abdominal aorta, caval and portal vein were measured in real-time using the Powerlab device (AD Instruments, New Zealand). Baseline LS values in all rats (3.8±0.5 kPa, n=25) did not significantly differ from those in humans. Epinephrine and norepinephrine drastically increased mean arterial pressure (MAP) from 82 to 173 and 156 mmHg. Concomitantly, LS almost doubled from 4 to 8 kPa, while central venous pressure (CVP) remained unchanged. Likewise, portal pressure only showed a slight and delayed increase. In the TAA-induced fibrosis model, LS increased from 9.5±1.0 to 25.6±14.7 kPa upon epinephrine injection and could efficiently be decreased by GTN. We finally show a direct association in humans in a physiological setting of elevated cardiac output and MAP. During continuous spinning at 200 W, MAP increased from 84±8 to 99±11 mmHg while LS significantly increased from 4.4±1.8 to 6.7±2.1 kPa. In conclusion, our data show that arterial pressure suffices to increase LS. Moreover, lowering MAP efficiently decreases LS in fibrotic livers that are predominantly supplied by arterial blood.
Poor aerobic fitness is linked to nonalcoholic fatty liver disease and increased all-cause mortality. We previously found that low capacity running (LCR) rats fed acute high fat diet (HFD; 45% kcal from fat) for 3 days resulted in positive energy balance and increased hepatic steatosis compared with high aerobically fit, high capacity running (HCR) rats. Here, we tested the hypothesis that poor physiological outcomes in LCR rats following acute HFD feeding are associated with alterations in cecal microbiota. LCR rats exhibited greater body weight, feeding efficiency, 3 d body weight change, and liver triglycerides after acute HFD feeding compared with HCR rats. Furthermore, LCR rats exhibited reduced expression of intestinal tight junction proteins compared with HCRs. Cecal bacterial 16S rDNA revealed that LCR rats had reduced cecal Proteobacteria compared with HCR rats. Microbiota of HCR rats had greater, Desulfovibrionaceae, unassigned genera within Desulfovibrionaceae, suggesting increased reduction of endogenous mucins and proteins. While acute HFD feeding reduced Firmicutes in both strains, short-chain fatty acid-producing Phascolarctobacterium was reduced in LCR rats. In addition, Ruminococcae and Ruminococcus were negatively correlated with energy intake in the LCR/HFD rats. Predicted metagenomic function suggests that LCR rats have a greater metabolic capacity to metabolize carbohydrate and energy compared with HCR rats. Overall, these data suggest that the populations and metabolic capacity of the microbiota in low aerobically fit LCR rats may contribute to their susceptibility to acute HFD-induced hepatic steatosis and poor physiologic outcomes.
The Hippo pathway effector, Yes-Associated Protein (YAP), regulates liver size by promoting cell proliferation and inhibiting apoptosis. However, recent in vivo studies suggest that YAP has important cellular functions other than controlling proliferation and apoptosis. Transgenic YAP expression in mouse hepatocytes results in severe jaundice. A possible explanation for the jaundice could be defects in adherens junctions that prevent bile from leaking into the blood stream. Indeed, immunostaining of E-cadherin and electron microscopic examination of bile canaliculi of Yap transgenic livers revealed abnormal adherens junction structures. Using primary hepatocytes from Yap transgenic livers and Yap knockout livers, we found that YAP antagonizes E-cadherin-mediated cell-cell junction assembly by regulating the cellular actin architecture, including its mechanical properties (elasticity and cortical tension). Mechanistically, we found that YAP promoted contractile actin structure formation by upregulating non-muscle myosin light chain expression and cellular ATP generation. Thus, by modulating actomyosin organization, YAP may influence many actomyosin-dependent cellular characteristics, including adhesion, membrane protrusion, spreading, morphology, and cortical tension and elasticity, which in turn determine cell differentiation and tissue morphogenesis.
Loss of function mutations in the actin motor Myosin Vb (Myo5b) lead to Microvillus Inclusion Disease (MVID) and death in newborns and children. MVID results in secretory diarrhea (SD), brush border (BB) defects, villus atrophy and microvillus inclusions (MVIs) in enterocytes. How loss of Myo5b results in increased stool loss of chloride (Cl-) and sodium (Na+) is unknown. The current study used Myo5b loss of function human MVID intestine, polarized intestinal cell models of secretory crypt (T84) and villus resembling (CaCo2BBe, C2BBe) enterocytes lacking Myo5b in conjunction with immunofluorescence confocal gSTED imaging, immunohistochemical staining, TEM, shRNA silencing, immunoblots, and electrophysiological approaches to examine the distribution, expression and function of the major BB ion transporters NHE3 (Na+), CFTR (Cl-) and SLC26A3 (DRA) (Cl- /HCO3-) that control intestinal fluid transport. We hypothesized that enterocyte maturation defects lead villus atrophy with immature secretory crypt like enterocytes in the MVID epithelium. We investigated the role of Myo5b in enterocyte maturation. NHE3 and DRA localization and function were markedly reduced on the BBM of human MVID enterocytes and Myo5bKD C2BBe cells, while CFTR localization was preserved. Forskolin-stimulated CFTR ion transport in Myo5bKD T84 cells resembled that of control. Loss of Myo5b led to YAP1 nuclear retention, retarded enterocyte maturation and a crypt-like phenotype. We conclude that preservation of functional CFTR in immature enterocytes, reduced functional expression of NHE3 and DRA contribute to Cl- and Na+ stool loss in MVID diarrhea.
Mucin-type O-glycans, primarily core 1- and core 3-derived O-glycans (O-glycans), are the major mucus barrier components throughout the gastrointestinal tract. Previous reports identified the biological role of O-glycans in the stomach and colon. However, the biological function of O-glycans in the small intestine remains unknown. Using mice lacking intestinal core 1- and core 3-derived O-glycans (IEC C1galt1-/-;C3GnT-/- or DKO), we found that DKO mice developed spontaneous duodenal tumors around 1 year of age. Tumor incidence did not increase with age; however, tumors advanced in aggressiveness by 20 months. Duodenal tumor development and O-glycan deficiency was associated with reduced luminal mucus in DKO mice prior to and within tumors. Altered intestinal epithelial homeostasis with enhanced baseline crypt proliferation characterizes these phenotypes as assayed by Ki67 staining. In addition, FISH analysis reveals a significantly lower bacterial burden in the duodenum compared to the large intestine. This phenotype is not reduced with antibiotic treatment, implying O-glycosylation defects, rather than bacterial-induced inflammation, causes spontaneous duodenal tumorigenesis. Moreover, inflammatory responses in DKO duodenal mucosa are mild as assayed with histology, qPCR for inflammation-associated cytokines, and immunostaining for immune cells. Importantly, inducible deletion of intestinal O-glycans in adult mice leads to analogous spontaneous duodenal tumors, though with higher incidence and heightened severity compared to mice with O-glycans constitutive deletion. In conclusion, these studies reveal O-glycans within the small intestine are critical determinants of duodenal cancer risk.
Our review focuses on the colonic macrophage, a monocyte-derived tissue-resident macrophage, and the role it plays in health and disease, specifically in inflammatory conditions such as Inflammatory Bowel Disease and cancer of the colon and rectum. We give special emphasis to macrophage polarization, or phenotype, in these different states. We focus on macrophages because they are one of the most numerous leukocytes in the colon and because they normally contribute to homeostasis through an anti-inflammatory phenotype. However, in conditions such as Inflammatory Bowel Disease, proinflammatory macrophages are increased in the colon and have been linked to disease severity and progression. In colorectal cancer, tumor cells may employ anti-inflammatory macrophages to promote tumor growth and dissemination, whereas proinflammatory macrophages may antagonize tumor growth. Given the key roles that this cell type plays in homeostasis, inflammation, and cancer, the colonic macrophage is an intriguing therapeutic target. As such, potential macrophage-targeting strategies are discussed.
Alcohol-induced intestinal hyperpermeability (AIHP) is a known risk factor for alcoholic liver disease (ALD), but only 20-30% of heavy alcoholics develop AIHP and ALD. The hypothesis of this study is that circadian misalignment would promote AIHP. We studied two groups of healthy subjects on a stable work schedule for 3 months - day workers (DW) and night workers (NW). Subjects underwent two circadian phase assessments with sugar challenge to access intestinal permeability between which they drank 0.5 g/kg alcohol daily for 7 days. Sleep architecture by actigraphy did not differ at baseline or after alcohol between either group. After alcohol, the dim light melatonin onset (DLMO) in DW group did not significantly change, but in the NW group there was a significant 2 hour phase delay. Both the NW and DW group had no change in small bowel permeability with alcohol, but only in the NW group was there an increase in colonic and whole gut permeability. A lower area under the curve of melatonin inversely correlated with increased colonic permeability. Alcohol also altered peripheral clock gene amplitude of in peripheral blood mononuclear cells in CLOCK, BMAL, PER1, CRY1, and CRY2 in both groups, and inflammatory markers LBP, LPS, and IL-6, had an elevated mesor at baseline in NW vs DW and became arrhythmic with alcohol consumption. Together, our data suggests that central circadian misalignment is a previously unappreciated risk factor for AIHP, and that night workers may be at increased risk for developing liver injury with alcohol consumption.
Acid reflux episodes that extend to the proximal esophagus are more likely to be perceived. This suggests that the proximal esophagus is more sensitive to acid than the distal esophagus, which could be caused by impaired mucosal integrity. Our aim was to explore sensitivity to acid and mucosal integrity in different segments of the esophagus. A prospective study including 12 patients with gastroesophageal reflux disease. Two procedures were performed: an acid perfusion test and an upper endoscopy with electrical tissue impedance spectroscopy and esophageal biopsies. Proximal and distal acid sensitivity and tissue impedance were measured in vivo, and mucosal integrity at different esophageal levels was measured in vitro. Mean lag time to heartburn perception was shorter after proximal acid perfusion (0.8 minutes) than after distal acid perfusion (3.9 minutes); p = 0.02. Median in vivo tissue impedance was significantly lower in the distal esophagus (4563 •m) compared to the proximal esophagus (8170 •m); p = 0.002. Transepithelial permeability, measured by the median fluorescein flux was significantly higher in the distal (2051 nmol/cm2/h) than the proximal segment (368 nmol/cm2/h); p = 0.033. Intercellular space ratio was not significantly different between the proximal and distal esophagus. Conclusion In GERD patients off acid secretion-inhibiting medication, acid exposure in the proximal segment of the esophagus provokes symptoms earlier than acid exposure in the distal esophagus, whereas mucosal integrity is impaired more in the distal esophagus. These findings indicate that the enhanced sensitivity to proximal reflux episodes is not explained by increased mucosal permeability.
Background: Oropharyngeal dysphagia due to UES dysfunction is commonly encountered in the clinical setting. Selective experimental perturbation of various components of deglutitive apparatus can provide an opportunity to improve our understanding of the swallowing physiology and pathophysiology. The aim is to characterize the pharyngeal and UES deglutitive pressure phenomena in an experimentally induced restriction of UES opening in humans. Methods: We studied 14 volunteers without any dysphagic symptoms (7 male, 66±11 years) but with various supraesophageal reflux symptoms. To induce UES restriction, we used a handmade device that with adjustment could selectively apply 0, 20, 30, 40 mmHg pressure perpendicularly to the cricoid cartilage. Deglutitive pharyngeal and UES pressure phenomena were determined during dry, 5 and 10ml water swallows x 3 for each of the UES perturbations. Results: Increased restrictive pressure against UES resulted in a significant increase in hypopharyngeal intra-bolus pressure and UES nadir deglutitive relaxation pressure for all tested swallowed volumes (p<0.05). Application of external cricoid pressure increased the length of the UES high pressure zone from 2.5± 0.2 cm to 3.1± 0.2, 3.5± 0.1 and 3.7± 0.1 cm for 20, 30, 40mmHg cricoid pressure, respectively (p<0.05). External cricoid pressure had no significant effect on pharyngeal peristalsis. On the other hand, irrespective to external cricoid pressure deglutitive velopharyngeal contractile integral progressively increased with increased swallowed volumes (p<0.05). Conclusion: Acute experimental restriction of UES opening by external cricoid pressure manifests the pressure characteristics of increased resistance to UES trans-sphincteric flow observed clinically without affecting the pharyngeal peristaltic contractile function.
Barrett's esophagus (BE) is considered to be the most severe complication of gastro-esophageal reflux disease (GERD), in which the prolonged, repetitive episodes of combined acidic and biliary reflux result in the replacement of the squamous esophageal lining by columnar epithelium. Therefore, acid extruding mechanisms of esophageal epithelial cells (EECs) may play an important role in the defence. Our aim was to identify the presence of acid/base transporters on EECs and to investigate the effect of bile acids on their expressions and functions. Human EEC lines (CP-A and CP-D) was acutely exposed to bile acid cocktail (BAC) and the changes in intracellular pH (pHi) and Ca2+ concentration ([Ca2+]i) were measured by microfluorometry. mRNA and protein expression of ion transporters were investigated by RT-PCR, Western Blot and immunohistochemistry. We have identified the presence of Na+/H+ exchanger (NHE), Na+/HCO3- cotransporter (NBC) and a Cl- dependent HCO3- secretory mechanism in CP-A and CP-D cells. Acute administration of BAC stimulated HCO3- secretion in both cell lines and the NHE activity in CP-D cells by an IP3-dependent calcium release. Chronic administration of BAC to EECs increased the expression of ion transporters compared to non-treated cells. Similar expression pattern was observed in biopsy samples from BE compared to normal epithelium. We have shown that acute administration of bile acids differently alters ion transport mechanisms of EECs, whereas chronic exposure to bile acids increases the expression of acid/base transporters. We speculate that these adaptive processes of EECs, represent an important mucosal defence against the bile acid-induced epithelial injury.
Abnormal glucose metabolism is present in almost 40% of patients after acute pancreatitis but its pathophysiology has been poorly investigated. Pancreatic hormone derangements have been sparingly studied to date and their relationship with abnormal glucose metabolism is largely unknown. The aim was to investigate the associations between pancreatic hormones and glucose metabolism after acute pancreatitis, including the effect of potential confounders. This was a cross-sectional study of 83 adult acute pancreatitis patients. Fasting venous blood was collected from all patients and used for analysis of insulin, glucagon, pancreatic polypeptide, amylin, somatostatin, C-peptide, glucose and haemoglobin A1c. Statistical analyses were conducted using the modified Poisson regression, multivariable linear regression, and Spearman's correlation. Age, sex, BMI, recurrence of acute pancreatitis, duration from first attack, severity, aetiology, etc. were adjusted for. Increased insulin was significantly associated with abnormal glucose metabolism after acute pancreatitis, in both unadjusted (p = 0.038) and adjusted (p = 0.001) analyses. Patients with abnormal glucose metabolism also had significantly decreased pancreatic polypeptide (p = 0.001), and increased amylin (p = 0.047), in adjusted analyses. Somatostatin, C-peptide, and glucagon were not changed significantly in both unadjusted and adjusted analyses. Increased insulin resistance and reduced insulin clearance may be important components of hyperinsulinaemic compensation in acute pancreatitis patients, and increased amylin and reduced pancreatic polypeptide fasting levels characterize impaired glucose homeostasis. Clinical studies investigating islet-cell hormonal responses to mixed-nutrient meal testing, euglycaemic-hyperinsulinaemic clamps are warranted for further insights into role of pancreatic hormones in glucose metabolism derangements secondary to pancreatic diseases.
The Compounds of sphingomyelin-ceramide-glycosphingolipid pathways have been studied as a potential secondary messenger molecules in various systems, along with liver function and insulin resistance. Secondary messenger molecules act directly or indirectly to affect cell organelles and intercellular interactions. Their potential role in the pathogenesis of steatohepatitis and diabetes has been suggested. Data samples collected from patients with Gaucher's disease (GD), having a high level of glucocerebroside, support a role for compounds from these pathways as a messenger molecules in the pathogenesis of fatty liver disease and diabetes. The present review summarizes some of the recent data on the role of glycosphingolipid molecules as messenger molecules in various physiological and pathological conditions, more specifically including insulin resistance and fatty liver disease.
Despite increased appreciation for the role of nicotinic receptors in the modulation of and response to inflammation, the contribution of muscarinic receptors to mucosal homeostasis, clearance of enteric pathogens, and modulation of immune cell function remains relatively undefined. Uninfected and Nippostrongylus brasiliensis-infected WT and type 3 muscarinic receptor (M3R)-deficient (Chrm3-/-) mice were studied to determine the contribution of M3R to mucosal homeostasis as well as host defense against the TH2-eliciting enteric nematode N. brasiliensis. Intestinal permeability and expression of TH1/TH17 cytokines were increased in uninfected Chrm3-/- small intestine. Notably, in Chrm3-/- mice infected with N. brasiliensis, small intestinal up-regulation of TH2 cytokines was attenuated and nematode clearance was delayed. In Chrm3-/- mice, TH2-dependent changes in small intestinal function including smooth muscle hypercontractility, increased epithelial permeability, decreased epithelial secretion and absorption, and goblet cell expansion were absent despite N. brasiliensis infection. These findings identify an important role for M3R in host defense and clearance of N. brasiliensis, and support the expanding role of cholinergic muscarinic receptors in maintaining mucosal homeostasis.
Hepatocellular carcinoma (HCC) is the most common hepatic malignancy and the third leading cause of cancer related deaths. Previous studies have implicated bile acids in pathogenesis of HCC but the mechanisms are not known. We investigated the mechanisms of HCC tumor promotion by bile acids the diethylnitrosamine (DEN)- initiation-cholic acid (CA)-induced tumor promotion protocol in mice. The data show that 0.2% CA treatment resulted in 3-fold increase in number and size of DEN-induced liver tumors. All tumors observed in DEN-treated mice were well-differentiated HCCs. The HCCs observed in DEN-treated CA-fed mice exhibited extensive CD3, 20 and 45 positive inflammatory cell aggregates. Microarray based global gene expression studies combined with Ingenuity Pathway Analysis revealed significant activation of NF-B and Nanog in the DEN-treated 0.2% CA-fed livers. Further studies showed significantly higher TNFα and IL-1β mRNA, a marked increase in total and phosphorylated-p65 and phosphorylated IBα (degradation form) in livers of DEN-treated 0.2% CA-fed mice. Treatment of primary mouse hepatocytes with various bile acids showed significant induction of stemness genes including Nanog, KLF4, Sox2 and Oct4. Quantification of total and twenty specific bile acids in liver and serum revealed a tumor associated bile acid signature. Finally, quantification of total serum bile acids in normal, cirrhotic and HCC human samples revealed increased bile acids in serum of cirrhotic and HCC patients. Taken together, these data indicate that bile acids are mechanistically involved pathogenesis of HCC and may promote HCC formation via activation of inflammatory signaling.
Pancreatic ductal adenocarcinoma (PDAC) is a very aggressive cancer, with low survival rates and limited therapeutic options. Thus, the elucidation of signaling pathways involved in PDAC pathogenesis is essential to identify novel potential therapeutic gene targets. Here, we used a systems approach by integrating gene and microRNA profiling analyses together with CRISPR/Cas9 technology, to identify novel transcription factors involved in PDAC pathogenesis. FOXA2 transcription factor was found to be significantly down-regulated in PDAC relative to control pancreatic tissues. Functional experiments revealed that FOXA2 has a tumor suppressor function through inhibition of pancreatic cancer cell growth, migration, invasion and colony formation. In situ hybridization analysis revealed miR-199a significantly upregulated in pancreatic cancer. Bioinformatics and luciferase analyses showed that miR-199a negatively regulates directly FOXA2 expression, through binding in its 3' untranslated region (UTR). Evaluation of the functional importance of miR-199 on pancreatic cancer revealed that miR-199 acts as an inhibitor of FOXA2 expression, inducing an increase in pancreatic cancer cell proliferation, migration and invasion. Additionally, gene ontology and network analyses in PANC-1 cells treated with an siRNA against FOXA2 revealed an enrichment for cell invasion mechanisms through PLAUR and ERK activation. FOXA2 deletion (FOXA2) by using two CRISPR/Cas9 vectors in PANC-1 cells, induced tumor growth in vivo, resulting in up-regulation of PLAUR and ERK pathways in FOXA2 xenograft tumors. Taken together, we have identified FOXA2 as a novel tumor suppressor in pancreatic cancer, regulated directly by miR-199a, enhancing our understanding on how microRNAs interplay with the transcription factors to affect pancreatic oncogenesis.
Gastric hypersensitivity (GHS) and anxiety are prevalent in Functional Dyspepsia patients; their underlying mechanisms remain unknown largely because of lack of availability of live visceral tissues from human subjects. Recently, we demonstrated in a preclinical model that rats subjected to neonatal colon inflammation show increased basal plasma norepinephrine, which contributes to GHS through the up-regulation of nerve growth factor (NGF) expression in the gastric fundus. We tested the hypothesis that neonatal colon inflammation increases anxiety-like behavior and sympathetic nervous system (SNS) activity, which upregulates the expression of NGF to induce GHS in adult-life (GHS-rats). Chemical sympathectomy, but not adrenalectomy, suppressed the elevated NGF expression in the fundus muscularis externa and gastric hypersensitivity. The measurement of heart rate variability showed a significant increase in the LF/HF ratio in GHS vs. the control rats. Stimulus-evoked release of NE from the fundus muscularis externa strips was significantly greater in GHS vs. the control rats. Tyrosine hydroxylase expression was increased in the celiac ganglia of the GHS vs. the control rats. We found an increase in trait but not stress-induced anxiety-like behavior in GHS-rats in elevated plus maze. We concluded that neonatal programming triggered by colon inflammation upregulates tyrosine hydroxylase (TH) in the celiac ganglia, which upregulates the release of NE in the gastric fundus muscularis externa. The increase of NE release from the sympathetic nerve terminals concentration-dependently upregulates NGF, which proportionately increases the visceromotor response to gastric distension. Neonatal programming concurrently increases anxiety-like behavior in GHS-rats.
The human microbiota consists of 100 trillion microorganisms that provide important metabolic and biological functions benefiting the host. However, the presence in host plasma of a gut derived-bacteria component, the lipopolysaccharide (LPS), has been identified as a causal or complicating factor in multiple serious diseases like sepsis and septic shock and more recently obesity-associated metabolic disorders. Understanding the precise mechanisms by which gut-derived LPS is transported from the gut lumen to the systemic circulation is crucial to advance our knowledge in LPS-associated diseases and elaborate targeted strategies for their prevention. The aim of this review is to synthetize the current knowledge on the host mechanisms limiting the entry and dissemination of LPS into the systemic circulation. To prevent bacterial colonization and penetration, the intestinal epithelium harbors multiple defense mechanisms including the secretion of anti-microbial peptides and mucins as well as detoxification enzymes. Despite this first line of defense, LPS can reach the apical site of intestinal epithelial cells (IEC) and, due to its large size, likely crosses IEC via a transcellular transport, either lipid raft or clathrin-mediated endocytosis or goblet-cell associated passage. However, the precise pathway remains poorly described. Finally, if LPS crosses the gut mucosa, it is directed to the liver via the portal vein where major detoxification processes occur by deacetylation and excretion through the bile. If this disposal process is not sufficient, LPS enters the systemic circulation where it is handled by numerous transport proteins that clear it back to the liver for further excretion.
Exposure to remifentanil contributes to an increased risk of pulmonary aspiration, likely through reduced pharyngeal contractile vigour and diminished bolus propulsion during swallowing. Here, we employed a novel high resolution pressure-flow analysis to quantify the biomechanical changes across the upper esophageal sphincter (UES). Eleven healthy young participants (mean age 23.3±3.1 years, 7 male) received remifentanil via intravenous target controlled infusion with an effect-site concentration of 3 ng/ml. Before and 30 min following commencement of remifentanil administration, participants performed ten 10 ml saline swallows while pharyngo-esophageal manometry and electrical impedance data were recorded using a 4.2 mm diameter catheter housing 36 circumferential pressure sensors. Remifentanil significantly shortened the time period of UES opening (p<0.001) and increased residual UES pressure (p=0.003). At the level of the hypopharynx, remifentanil significantly shortened the time latency from maximum bolus distension to peak contraction (p=0.004) and significantly increased intrabolus distension pressure (p=0.024). Novel mechanical states analysis revealed that the latencies between the different phases of the stereotypical UES relaxation sequence were shortened by remifentanil. Reduced duration of bolus flow during shortened UES opening in concert with increased hypopharyngeal distension pressures are mechanically consistent with increased flow resistance due to a more rapid bolus flow rate. These biomechanical changes are congruent with modification of the physiologic neuro-regulatory mechanism governing accommodation to bolus volume.
Intracellular zinc is required for a variety of cell functions, but its precise roles in the maintenance of the intestinal tight junction (TJ) barrier remain unclear. The present study investigated the essential roles of intracellular zinc in the preservation of intestinal TJ integrity and the underlying molecular mechanisms. Depletion of intracellular zinc in both intestinal Caco-2 cells and mouse colons through the application of a cell-permeable zinc chelator, N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), induced a disruption of the TJ barrier, as indicated by increased FITC-labeled dextran flux and decreased transepithelial electrical resistance. The TPEN-induced TJ disruption is associated with downregulation of 2 TJ proteins, occludin and claudin-3. Biotinylation of cell surface proteins revealed that the zinc depletion induced the proteolysis of occludin, but not claudin-3. Occludin proteolysis was sensitive to the inhibition of calpain activity, and increased calpain activity was observed in the zinc-depleted cells. Although qPCR analysis and promoter reporter assay have demonstrated that the zinc depletion-induced claudin-3 downregulation occurred at transcriptional levels, a site-directed mutation in the egr1 binding site in the claudin-3 promoter sequence induced loss of both the basal promoter activity and the TPEN-induced decreases. Reduced egr1 expression by a specific siRNA also inhibited claudin-3 expression and TER maintenance in cells. This study shows that intracellular zinc has an essential role in the maintenance of the intestinal epithelial TJ barrier through regulation of occludin proteolysis and claudin-3 transcription.
Celiac disease (CD) is an immune-mediated enteropathy triggered by gluten in genetically susceptible individuals. Innate immunity contributes to the pathogenesis of CD, but the mechanisms remain poorly understood. Although previous in vitro work suggests that gliadin peptide p31-43 acts as an innate immune trigger, the underlying pathways are unclear and have not been explored in vivo. Here we show that intraluminal delivery of p31-43 induces morphological changes in the small intestinal mucosa of normal mice consistent with those seen in CD, including increased cell death and expression of inflammatory mediators. The effects of p31-43 were dependent on MyD88 and Type I IFNs, but not TLR4, and were enhanced by co-administration of the TLR3 agonist poly I:C. Together these results indicate that gliadin peptide p31-43 activates the innate immune pathways in vivo, such as IFN-dependent inflammation, relevant to CD. Our findings also suggest a common mechanism for the potential interaction between dietary gluten and viral infections in the pathogenesis of CD.
Background: Feeding intolerance is a common issue in the care of preterm neonates. The condition manifests as delayed gastric content emptying and represents a therapeutic challenge, since the factors accounting for its manifestations are unknown. The main goal of this study was to comparatively investigate the age-related rat gastric and pyloric smooth muscle function and their putative regulators. We hypothesized that a reduced gastric muscle contraction potential early in life, contributes to the delayed gastric emptying of the newborn. Methodology: Newborn and adult rat gastric (fundal) and pyloric sphincter tissues were comparatively studied in vitro. The tissue-specific dissociated smooth muscle cells (SMC) shortening properties were evaluated and the key regulatory proteins Rho-associated kinase 2 (ROCK-2) and myosin light chain kinase (MLCK) expression determined. Results: Adult gastric and pyloric SMC shortening was significantly greater, when compared with the respective newborn counterpart. MLCK and ROCK-2 expression were developmentally regulated and increased with age. The newborn pyloric sphincter muscle expresses a higher neuronal nitric oxide synthase and phosphorylated vasodilator-stimulated phosphoprotein content, when compared with adult tissue. Conclusions: As compared with later in life, the newborn rat gastropyloric muscle has a Ca2+-related reduced potential for contraction and the pyloric sphincter relaxation-dependent modulators are overexpressed. To the extent that these rodent data can be extrapolated to humans, the neonatal delayed gastric emptying reflects the reduced stomach muscles contraction potential, as opposed to increased pyloric sphincter tone.
Liver fibrosis can progress to cirrhosis and result in serious complications of liver disease. The pathogenesis of liver fibrosis involves the activation of hepatic stellate cells (HSCs), for which the underlying mechanisms are not fully known. Emerging evidence suggests that the classic histone deacetylases play a role in liver fibrosis, but the role of another subfamily of histone deacetylases, the Sirtuins, in the development of hepatic fibrosis remains unknown. In this study, we found that blocking the activity of Sirtuin 2 (SIRT2) using inhibitors or shRNAs significantly suppressed the fibrogenic gene expression in HSCs. We further demonstrated that inhibition of SIRT2 results in the degradation of c-MYC, which is important for HSC activation. In addition, we discovered that inhibition of SIRT2 suppresses the phosphorylation of ERK, which is critical for stabilization of c-MYC. Moreover, we found that Sirt2 deficiency attenuates the hepatic fibrosis induced by carbon tetrachloride (CCl4) and thioacetamide (TAA). Furthermore, we showed that SIRT2, p-ERK and c-MYC proteins are overexpressed in human hepatic fibrotic tissues. These data suggest a critical role for the SIRT2/ERK/c-MYC axis in promoting hepatic fibrogenesis. Inhibition of the SIRT2/ERK/c-MYC axis represents a novel strategy to prevent and to potentially treat liver fibrosis and cirrhosis.
Keratins (K) are intermediate filament proteins important in protection from stress. The roles of keratins in the intestine are not clear, but K8 knockout (K8-/-) mice develop a Th2-type colonic inflammation, epithelial hyperproliferation, and mild diarrhea caused by a keratin level-dependent decrease in short circuit current and net sodium and chloride absorption in the distal colon. The lack of K8 leads to mistargeting or altered levels of membrane proteins in colonocytes, however, the main transporter responsible for the keratin-related ion transport defect is unknown. We here analyzed protein and mRNA levels of candidate ion transporters CFTR, PAT-1, NHE-3 and DRA in ileum, caecum, proximal and distal colon. While no differences were observed for CFTR, PAT-1 or NHE-3, DRA mRNA levels were decreased by 3-4-fold and DRA protein was almost entirely lost in K8-/- caecum, proximal and distal colon compared to K8+/+, while the levels in ileum were normal. In K8+/- mice, DRA mRNA-levels were unaltered while decreased DRA protein was detected in the proximal colon. Immunofluorescence staining confirmed the loss of DRA in K8-/- distal colon while K8+/- displayed a similar but more patchy apical DRA distribution compared to K8+/+. DRA was similarly decreased when K8 was knocked-down in Caco-2 cells, confirming that K8 levels modulate DRA levels in an inflammation-independent manner. Taken together, the loss of DRA in the K8-/- mouse colon and caecum explains the dramatic chloride transport defect and diarrheal phenotype after K8 inactivation and identifies K8 as a novel regulator for DRA.
The bile acid, nor-ursodeoxycholic acid (norUDCA), has many biological actions, including anti-apoptotic effects. Homozygous PIZZ alpha-1-antitrypsin (A1AT) deficient humans are known to be at risk for liver disease, cirrhosis, and liver cancer as a result of the accumulation of the toxic, A1AT mutant Z protein within hepatocytes. This accumulation triggers cell death in the hepatocytes with the largest mutant Z protein burdens, followed by compensatory proliferation. Proteolysis pathways within the hepatocyte, including autophagy, act to reduce the intracellular burden of A1AT Z protein. We hypothesized that norUDCA would reduce liver cell death and injury in A1AT deficiency. We treated groups of PiZ transgenic mice and WT mice with norUDCA or vehicle, orally, and examined the effects on the liver. The PiZ mouse is the best model of A1AT liver injury and recapitulates many features of the human liver disease. Results: Mice treated with norUDCA demonstrated reduced hepatocellular death by compensatory hepatocellular proliferation as determined by BrdU incorporation (3.8% control, 0.88% treated, p<0.04). Ki-67 staining as a marker for hepatocellular senescence and death was also reduced (p<0.02). Reduced apoptotic signaling was associated with norUDCA, including reduced cleavage of caspases 3, 7, and 8 (all p<0.05). We determined that norUDCA was associated with a >70% reduction in intrahepatic mutant Z protein (p<0.01). A 32% increase in hepatic autophagy associated with norUDCA was the likely mechanism. Conclusions: norUDCA administration is associated with increased autophagy, reduced A1AT protein accumulation and reduced liver injury in a model of A1AT deficiency.
TMEM16A is a newly identified Ca2+-activated Cl- channel in biliary epithelial cells (BECs) that is important in biliary secretion. While extracellular ATP stimulates TMEM16A via binding P2 receptors and increasing [Ca2+]i, the regulatory pathways have not been elucidated. Protein Kinase C (PKC) contributes to ATP-mediated secretion in BECs, though its potential role in TMEM16A regulation is unknown. To determine whether PKCα regulates the TMEM16A-dependent membrane Cl- transport in BECs, studies were performed in human biliary Mz-cha-1 cells. Addition of extracellular ATP induced a rapid translocation of PKCα from the cytosol to the plasma membrane and activation of whole cell Ca2+-activated Cl- currents. Currents demonstrated outward rectification, reversal at 0 mV (properties consistent with TMEM16A) and were inhibited by either molecular (siRNA) or pharmacologic (PMA or Gö6976) inhibition of PKCα. Intracellular dialysis with recombinant PKCα activated Cl- currents with biophysical properties identical to TMEM16A in control cells, but not in cells after transfection with TMEM16A siRNA. In conclusion, our studies demonstrate that PKCα is coupled to ATP-stimulated TMEM16A activation in BECs. Targeting this ATP-Ca2+-PKCα signaling pathway may represent a therapeutic strategy to increase biliary secretion and promote bile formation.
Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR), an anion channel providing a major pathway for Cl- and HCO3- efflux across the apical membrane of epithelium. In intestine, CF manifests as obstructive syndromes, dysbiosis, inflammation and an increased risk for gastrointestinal cancer. Cftr knockout (Cftr KO) mice recapitulate CF intestinal disease including intestinal hyperproliferation. Previous studies using Cftr KO intestinal organoids (enteroids) indicated that the crypt epithelium maintains an alkaline intracellular pH (pHi). We hypothesized that Cftr has a cell-autonomous role in down-regulating pHi that is incompletely compensated by acid-base regulation in its absence. Here, BCECF microfluorimetry of enteroids showed that Cftr KO crypt epithelium sustains an alkaline pHi and resistance to cell acidification relative to wild-type (WT). Quantitative real-time PCR revealed that Cftr KO enteroids exhibit down-regulated transcription of base (HCO3-)-loading proteins and up-regulation of the basolateral membrane HCO3--unloader anion exchanger 2 (Ae2). Although Cftr KO crypt epithelium had increased Ae2 expression and Ae2-mediated Cl-/HCO3- exchange with maximized gradients, it also had increased intracellular Cl- concentration ([Cl-]i) relative to WT. Pharmacological reduction of [Cl-]i in Cftr KO crypt epithelium normalized pHi which was largely Ae2 dependent. We conclude that Cftr KO crypt epithelium maintains an alkaline pHi as a consequence of losing both Cl- and HCO3- efflux which impairs pHi regulation by Ae2. Retention of Cl- and an alkaline pHi in crypt epithelium may alter several cellular processes in the proliferative compartment of the Cftr KO intestine.
Glucagon like peptide-1 is a glucoincretin hormone that can act through its receptor (GLP-1R) on pancreatic β cells and increase insulin secretion and production. GLP-1R agonists are being used clinically to treat type 2 diabetes. GLP-1 may also regulate the exocrine pancreas at multiple levels including inhibition through the CNS, stimulation indirectly through insulin and stimulation directly on the acinar cells. However, it has been unclear whether GLP-1R is present in pancreatic acini and what physiological functions these receptors regulate. In the current study, we utilized the GLP-1R knockout mice to study the role of GLP-1R in acinar cells. RNA expression of GLP-1R was detected in acutely isolated pancreatic acini. Acinar cell morphology and expression of digestive enzymes were not affected by loss of GLP-1R. GLP-1 induced amylase secretion in WT acini. In GLP-1R knockout (KO) mice, this effect was abolished, whereas VIP induced amylase release in KO acini showed a similar pattern to that in WT acini. GLP-1 stimulated cyclic AMP production and increased PKA-mediated protein phosphorylation in WT acini, and these effects were absent in KO acini. These data show that GLP-1R is present in pancreatic acinar cells and that GLP-1 can regulate secretion through its receptor and cAMP signaling pathway.
Recent studies have implicated a pathogenic role for Matrix Metalloproteinases 9 (MMP-9) in inflammatory bowel disease. Though loss of epithelial barrier function has been shown to be a key pathogenic factor for the development of intestinal inflammation, the role of MMP-9 in intestinal barrier function remains unclear. The aim of this study was to investigate the role of MMP-9 in intestinal barrier function and intestinal inflammation. Wild type (WT) and MMP-9-/- mice were subjected to experimental dextran sodium sulfate (DSS) colitis by administration of 3% DSS in drinking water for 7 days. The mouse colonic permeability was measured in vivo by recycling perfusion of the entire colon using fluorescent labeled dextran. The DSS-induced increase in the colonic permeability was accompanied by an increase in intestinal epithelial cell MMP-9 expression in WT mice. The DSS-induced increase in intestinal permeability and the severity of DSS colitis was found to be attenuated in MMP-9-/- mice. The colonic protein expression of myosin light chain kinase (MLCK), and phospho-MLC was found to be significantly increased after DSS administration in WT mice but not in MMP-9-/- mice. The DSS-induced increase in colonic permeability and colonic inflammation was attenuated in MLCK-/- mice and MLCK inhibitor ML-7 treated WT mice. DSS-induced increase in colonic surface epithelial cell MLCK mRNA was abolished in MMP-9-/- mice. Lastly, increased MMP-9 protein expression was detected within the colonic surface epithelial cells in ulcerative colitis cases. This data suggest role of MMP-9 in modulation of colonic epithelial permeability and inflammation via MLCK.
Injury and inflammation in the gastric epithelium can cause disruption of the pathways that guide the differentiation of cell lineages, which in turn can cause persistent alterations in differentiation patterns, known as metaplasia. Metaplasias that occur in the stomach are associated with increased risk for cancer. Methods for isolating distinct gastric epithelial cell populations would facilitate dissection of the molecular and cellular pathways that guide normal and metaplastic differentiation. Here, we identify Alanyl Aminopeptidase (ANPEP; aka CD13) as a specific, surface marker of zymogenic chief cells (ZCs) in the gastric epithelium. We show that: 1) among gastric epithelial cells, ANPEP expression is confined to mature ZCs, and 2) its expression is lost en route to metaplasia in both mouse and human stomachs. With this new marker coupled with new techniques we introduce for dissociating gastric epithelial cells and overcoming their constitutive autofluorescence, we are able to reliably isolate pure populations of ZCs, and observe changes in ZC differentiation ex vivo in response to epithelial damage.
We tested whether the Th2 agonist and allergenic ligand IL-33, was associated with eosinophilic esophagitis (EoE) development in a paediatric cohort, and whether IL-33 protein could induce disease symptoms in mice. Biopsies from eosinophilic esophagitis patients or controls were used to measure IL-33 mRNA and protein expression. Increased expression of IL-33 mRNA was found in the esophageal mucosa in EoE. IL-33 protein was detected in cells negative for CD45, mast and epithelial cell markers, near blood vessels. Circulating levels of IL-33 were not increased. The time-course for IL-33 gene expression was quantified in an established Aspergillus fumigatus allergen mouse model of EoE. Because IL-33 induction was transient in this model, and chronicity of IL-33 expression was demonstrated in humans, naive mice were treated with recombinant IL-33 for 1 week, and esophageal pathology evaluated. IL-33 application produced changes consistent with phenotypically early EoE, including transmural eosinophilia, mucosal hyperproliferation, and up-regulation of eosinophilic genes and chemokines. Th2 cytokines, including IL-13, were increased after IL-33 application, along with ILC2, Th1/17 and M2 macrophage marker genes. IL-33-induced eosinophilia was ablated in IL-13 null mice. In addition, IL-33 induced a profound inhibition of the regulatory T cell gene signature. We conclude that IL-33 gene expression is associated with paediatric EoE development, and that application of recombinant protein in mice phenocopies the early clinical phase of the human disease in an IL-13-dependent manner. IL-33 inhibition of esophageal regulatory T cell function may induce loss of antigenic tolerance, thereby providing a mechanistic rationale for EoE development.
Epidermal growth factor receptor (EGFR) and ERBB3 have been implicated in hepatocellular carcinogenesis (HCC). However, it is not known whether altering the activity of either EGFR or ERBB3 affects HCC development. We now show that EgfrDsk5mutant mice, which have a gain-of-function allele that increases basal EGFR kinase activity, develop spontaneous HCC by 10 months of age. Their tumors show increased activation of EGFR, ERBB2, and ERBB3 as well as Akt and ERK 1,2. Hepatocyte-specific (HS) models of EGFR and ERBB3 gene ablation were generated to evaluate how the loss of these genes affected tumor progression. Loss of either receptor tyrosine kinase did not alter liver development or regenerative liver growth following carbon tetrachloride injection. However, using a well-characterized model of HCC in which N-nitrosodiethylamine (DEN) is injected into 14 day old mice, we discovered that loss of hepatocellular ERBB3 but not EGFR, which occurred after tumor initiation, retarded liver tumor formation and cell proliferation. We found no evidence that this was due to increased apoptosis or diminished phosphatidylinositol-3-kinase (PI3K) activity in the ERBB3 null cells. However, the relative amount of phospho-STAT3 was diminished in tumors derived from these mice, suggesting that ERBB3 may promote HCC through STAT3 activation.
Entero-pancreatic hormone secretion is thought to include a cephalic phase but the evidence in humans is ambiguous. We studied vagally-induced gut hormone responses with and without muscarinic blockade in ten glucose-clamped healthy males (age: 24.5±0.6 years(mean±standard error of the mean (SEM); body mass index: 24.0±0.5kg/m2; HbA1c: 5.1±0.1% /31.4±0.5mmol/mol). Cephalic activation was elicited by modified sham feeding (MSF, aka "chew and spit") with or without atropine (1mg bolus 45min before MSF + 80ng/kg/min for 2h). To mimick incipient prandial glucose excursions, glucose levels were clamped at 6mmol/L on all days. The meal stimulus for the MSF consisted of an appetizing breakfast. Participants (9/10) also had a 6mmol/L-glucose clamp without MSF. PP levels rose from 6.3±1.1 to 19.9±6.8pmol/L(means±SEM) in response to MSF and atropine lowered basal PP levels and abolished the MSF response. Neither insulin, C-peptide, glucose-dependent insulinotropic polypeptide(GIP) nor glucagon-like peptide-1(GLP-1) levels changed in response to MSF or atropine. Glucagon and ghrelin levels were markedly attenuated by atropine prior to and during the clamp: at t=105min on the ATR+CLA+MSF compared to the SAL+CLA and SAL+CLA+MSF days; baseline-subtracted glucagon levels were -10.7±1.1 vs. -4.0±1.1 and -4.7±1.9pmol/L(mean±SEM),P<0.0001), respectively; corresponding baseline-subtracted ghrelin levels were 303±36 vs. 39±38 and 3.7±21pg/mL(mean±SEM), P<0.0001. Glucagon and ghrelin levels were unaffected by MSF. In spite of adequate PP responses, a cephalic phase response was absent for insulin, glucagon, GLP-1, GIP and ghrelin.
There are two major stem cell populations in the intestinal crypt region that express either Bmi1 or Lgr5; however, it has been shown that other populations in the crypt can regain stemness. In this study, we demonstrate that the transcription factor Nkx2.2 is expressed in enteroendocrine cells located in the villus and crypt of the intestinal epithelium and is co-expressed with the stem cell markers Bmi1 and Lgr5 in a subset of crypt cells. To determine whether Nkx2.2-expressing enteroendocrine cells display cellular plasticity and stem cell potential, we performed genetic lineage-tracing of the Nkx2.2-expressing population using Nkx2.2Cre/+;R26RTomato mice. These studies demonstrated that Nkx2.2+ cells are able to give rise to all intestinal epithelial cell types in basal conditions. The proliferative capacity of Nkx2.2-expressing cells was also demonstrated in vitro using crypt organoid cultures. Injuring the intestine with irradiation, systemic inflammation and colitis did not enhance the lineage potential of Nkx2.2-expressing cells. These findings demonstrate that a rare mature enteroendocrine cell subpopulation that is demarcated by Nkx2.2 expression, display stem cell properties during normal intestinal epithelial homeostasis, but is not easily activated upon injury.
Fatty liver is associated with endoplasmic reticulum stress and activation of the hepatic Unfolded Protein Response (UPR). Reduced hepatic expression of the UPR regulator X-box binding protein 1 spliced (XBP1s) is associated with human NASH, and feeding mice a high fat diet with fructose/sucrose causes progressive, fibrosing steatohepatitis. This study examines the role of XBP1 in non-alcoholic fatty liver injury and fatty acid-induced cell injury. Hepatocyte-specific Xbp1-deficient (Xbp1-/-) mice were fed a high fat/sugar (HFS) diet for up to 16 weeks. HFS-fed Xbp1-/- mice exhibited higher serum alanine aminotransferase levels compared to Xbp1fl/fl controls. RNA-Sequencing and Gene Ontogeny pathway analysis of hepatic mRNA revealed that apoptotic process, inflammatory response and extracellular matrix structural constituent pathways had enhanced activation in HFS-fed Xbp1-/- mice. Liver histology demonstrated enhanced injury and fibrosis but less steatosis in the HFS-fed Xbp1-/- mice. Hepatic Col1a1 and Tgfβ1 gene expression, as well as Chop and phosphorylated JNK (p-JNK), were increased in Xbp1-/- compared to Xbp1fl/fl mice after HFS feeding. In vitro, stable XBP1-knockdown Huh7 cells (Huh7-KD) and scramble control cells (Huh7-SCR) were generated and treated with palmitic acid (PA) for 24 hrs. PA-treated Huh7-KD cells had increased cytotoxicity measured by LDH release, apoptotic nuclei and caspase3/7 activity assays compared to Huh7-SCR cells. CHOP and p-JNK expression was also increased in Huh7-KD cells following PA treatment. In conclusion, loss of XBP1 enhances injury in both in vivo and in vitro models of fatty liver injury. We speculate that hepatic XBP1 plays an important protective role in pathogenesis of NASH.
Krüppel-like factor (KLF)-10 is an important transcriptional regulator of TGF-β1 signaling in both CD8+ and CD4+ T lymphocytes. In the current study, we demonstrate a novel role for KLF10 in the regulation of TGFβRII expression with functional relevance in macrophage differentiation and activation. We first show that transfer of KLF10-/- BMDM into wt mice leads to exacerbation of experimental colitis. At the cell biological level, using two phenotypic strategies, we show that KLF10-deficient mice have an altered colonic macrophage phenotype with higher frequency of pro-inflammatory LyC6+MHCII+ cells and a reciprocal decrease of the anti-inflammatory LyC6-MCHII+ subset. Additionally, the anti-inflammatory CD11b+CX3CR1hi subset of colonic macrophages is significantly decreased in KLF10-/- compared to wt mice under inflammatory conditions. Molecularly, CD11b+ colonic macrophages from KLF10-/- mice exhibit a pro-inflammatory cytokine profile with increased production of TNF-α and lower production of IL-10 in response to LPS stimulation. Because KLF10 is a transcription factor, we explored how this protein may regulate macrophage function. Consequently, we analyzed the expression of TGFβRII expression in colonic macrophages and found that in the absence of KLF10, macrophages express lower levels of TGFβRII and display an attenuated Smad-2 phosphorylation following TGF-β1 stimulation. We further show that KLF10 directly binds to the TGFβRII promoter in macrophages leading to enhanced gene expression through histone H3 acetylation. Collectively, our data reveal a critical role for KLF10 in the epigenetic regulation of TGFβRII expression in macrophages and the acquisition of a "regulatory" phenotype that contributes to intestinal mucosal homeostasis.
Background: Liver transplantation and cholangiocarcinoma induce biliary dysfunction following ischemia reperfusion (IR). The function of the intrahepatic biliary tree is regulated by both autocrine and paracrine factors. The aim of the study was to demonstrate that IR-induced damage of cholangiocytes is associated with altered expression of biliary angiogenic factors. Methods: Normal and BDL rats underwent 24 hr sham or hepatic reperfusion after 30 minutes of transient occlusion of the hepatic artery (HAIR) or portal vein (PVIR) before collecting liver blocks and cholangiocyte RNA or protein. We evaluated liver histology, biliary apoptosis, proliferation and expression of VEGF-A/C, VEGFR-2/3, Ang-1/2 and Tie-1/2 in liver sections and isolated small and large cholangiocytes. Normal rat intrahepatic cholangiocyte cultures (NRICC) were maintained under standard conditions in normoxic or under a hypoxic atmosphere for 4 hr and then transferred to normal conditions for selected times. Subsequently, we measured changes in biliary proliferation and apoptosis and the expression of VEGF-A/C and VEGFR-2/3. Results: In vivo, HAIR (but not PVIR) induced damage of large bile ducts and decreased proliferation and secretin-stimulated cAMP levels. HAIR-induced damage of large bile ducts was associated with increased expression of VEGF-A/C, VEGFR-2/3, Ang-1/2 and Tie-1/2. In vitro, under hypoxic conditions, there was increased apoptosis and reduced proliferation of NRICC concomitant with enhanced expression of VEGF-A/C and VEGFR-2/3. Conclusion: The functional damage of large bile ducts by HAIR and hypoxia is associated with increased expression of angiogenic factors in small cholangiocytes, presumably due to a compensatory mechanism in response to biliary damage.
Early life stress and adversity are major risk factors in the later life onset and severity of gastrointestinal (GI) disease in humans. The mechanisms by which early life stress leads to increased GI disease susceptibility in adult life remain poorly understood. Animal models of early life stress have provided a foundation from which to gain a more fundamental understanding of this important GI disease paradigm. This review will focus on animal models of early life stress-induced GI disease, with a specific emphasis on translational aspects of each model to specific human GI disease states. Early postnatal development of major GI systems and the consequences of stress on their development will be discussed in detail. Relevant translational differences between species and models will be highlighted.
Hyaluronic acid, a glycosaminoglycan in the extracellular matrix, binds to CD44 and TLR4. We previously addressed the role of hyaluronic acid in small intestinal and colonic growth in mice. We addressed the role of exogenous hyaluronic acid by giving hyaluronic acid IP and the role of endogenous hyaluronic acid by giving PEP-1, a peptide that blocks hyaluronic acid binding to its receptors. Exogenous hyaluronic acid increased epithelial proliferation but had no effect on intestinal length. PEP-1 resulted in a shortened small intestine and colon and diminished epithelial proliferation. In the current study we sought to determine if the effects of hyaluronic acid on growth were mediated by signaling through CD44 or TLR4 by giving exogenous hyaluronic acid or PEP-1 twice a week from 3-8 weeks of age to wild type, CD44-/- and TLR4-/- mice. These studies demonstrated that signaling through both CD44 and TLR4 were important in mediating the effects of hyaluronic acid on growth in the small intestine and colon. Extending our studies to early postnatal life we assessed the effects of exogenous hyaluronic acid and PEP-1 on Lgr5+ stem cell proliferation and crypt fission. Administration of PEP-1 to Lgr5+ reporter mice from postnatal day 7 to day 14 decreased Lgr5+ cell proliferation and decreased crypt fission. These studies indicate that endogenous hyaluronic acid increases Lgr5+ stem cell proliferation, crypt fission and intestinal lengthening and that these effects are dependent on signaling through CD44 and TLR4.
Background: Celiac disease (CD) is an inflammatory disorder triggered by ingested gluten, causing immune-mediated damage to the small-intestinal mucosa. Gluten proteins are strikingly similar in amino acid composition and sequence to proline-rich proteins (PRPs) in human saliva. Based on this feature and their shared destination in the gastro-intestinal tract, we hypothesized that salivary PRPs may modulate gluten-mediated immune responses in CD. Methods: Parotid salivary secretions were collected from CD patients, refractory CD patients, non-CD patients with functional gastrointestinal complaints (GI), and healthy controls (HC). Structural similarities of PRPs with gluten were probed with anti-gliadin antibodies. Immune responses to PRPs were investigated towards CD patient-derived peripheral blood mononuclear cells and in a humanized transgenic HLA-DQ2/DQ8 mouse model for CD. Results: Anti-gliadin antibodies weakly cross-reacted with salivary amylase but not with PRPs. Likewise, the R5 antibody, recognizing potential antigenic gluten epitopes, showed negligible reactivity to salivary proteins from all groups. Inflammatory responses in PBMCs were provoked by gliadins while responses to PRPs were similar to control levels, and PRPs did not compete with gliadins in immune stimulation. In vivo, PRP peptides were well-tolerated and non-immunogenic in the transgenic HLA-DQ2/DQ8 mouse model. Conclusions: Although structurally similar to dietary gluten, salivary RPRs were non-immunogenic in CD patients and in a transgenic HLA-DQ2/DQ8 mouse model for CD. It is possible that salivary PRPs play a role in tolerance induction to gluten early in life. Deciphering the structural basis for the lack of immunogenicity of salivary PRPs may further our understanding of the toxicity of gluten.
Apolipoprotein (apo) A-V is a protein synthesized only in the liver that dramatically modulates plasma triglyceride levels. Recent studies suggest a novel role for hepatic apoA-V in regulating the absorption of dietary triglycerides, but its mode of action on the gut remains unknown. The aim of this study was to test for apoA-V in bile and to determine if its secretion is regulated by dietary lipids. After an overnight recovery, adult male Sprague-Dawley bile fistula rats indeed secreted apoA-V into bile at a constant rate under fasting conditions. An intraduodenal bolus of intralipid (n=12) increased the biliary secretion of apoA-V but not of other apolipoproteins, such as A-I, A-IV, B and E. The lipid-induced increase of biliary apoA-V was abolished under conditions of poor lymphatic lipid transport, suggesting that the stimulation is regulated by the magnitude of lipids associated with chylomicrons transported into lymph. We also studied the secretion of apoA-V into bile immediately following bile duct cannulation. Biliary apoA-V increased over time (~6-fold increase at hour 16, n=8) but the secretions of other apolipoproteins remained constant. Replenishing luminal phosphatidylcholine and taurocholate (n=9) only enhanced apoA-V secretion in bile, suggesting that the increase was not due to depletion of phospholipids or bile salts. This is the first study to demonstrate that apoA-V is secreted into bile, introducing a potential route of delivery of hepatic apoA-V to the gut lumen. Our study also reveals the uniqueness of apoA-V secretion into bile that is regulated by mechanisms different from other apolipoproteins.
The newest member of the Na+/H+ exchanger (NHE) family, NHE8 is abundantly expressed at the apical membrane of the intestinal epithelia. We previously reported that Muc2 expression was significantly decreased in the colon in NHE8-/- mice, suggesting NHE8 is involved in intestinal mucosal protection. In this study, we further evaluated the role of NHE8 in intestinal epithelial protection after DSS challenge. Compared with wild-type mice, NHE8-/- mice have increased bacterial adhesion and inflammation, especially in the distal colon. NHE8-/- mice are also susceptible to DSS treatment. Real-time PCR detected a remarkable increase in the expression of IL-1β, IL-6, TNFα and IL-4 in DSS-treated NHE8-/- mice compared with DSS-treated wild-type littermates. Immunohistochemistry showed a disorganized epithelial layer in the colon of NHE8-/- mice. Periodic Acid-Schiff staining showed a reduction in the number of mature goblet cells and the area of the goblet cell theca in NHE8-/- mice. Phyloxine/tartrazine staining revealed a decrease in functional Paneth cell population in the NHE8-/- small intestinal crypt. The expression of enteric defensins was also decreased in NHE8-/- mice. The reduced mucin production in goblet cells and antimicrobial peptides production in Paneth cells lead to disruption of the intestinal mucosa protection. Therefore, NHE8 may be involved in the establishment of intestinal mucosal integrity by regulating the functions of goblet and Paneth cells.
The igf1 gene is alternatively spliced as IGF-IEa and IGF-IEc variants in humans. In fibrostenotic Crohn's disease, the fibrogenic cytokine TGF-β1 induces IGF-IEa expression, IGF-I production in intestinal smooth muscle and results in muscle hyperplasia and collagen I production that contribute to stricture formation. MGF derived from IGF-IEc induces skeletal and cardiac muscle hypertrophy following stress. We hypothesized that increased IGF-IEc expression and MGF production mediated smooth muscle hypertrophy also characteristic of fibrostenotic Crohn's disease. IGF-IEc transcripts and MGF protein were increased in muscle cells isolated from fibrostenotic intestine under regulation by endogenous TGF-β1. Erk5 and MEF-2C were phosphorylated in vivo in fibrostenotic muscle; both were phosphorylated and co-localized to nucleus in response to synthetic MGF in vitro. Smooth muscle-specific protein expression: α-smooth muscle actin, -smooth muscle actin, desmin, and smoothelin was increased in affected intestine. Erk5 inhibition or MEF2C siRNA blocked smooth muscle-specific gene expression and hypertrophy induced by synthetic MGF. Conditioned media of cultured fibrostenotic muscle induced muscle hypertrophy that was inhibited by immunoneutralization of endogenous MGF or pro-IGF-IEc. The results indicate that TGF-β1-dependent IGF-IEc expression and MGF production in patients with fibrostenotic Crohn's disease regulates smooth muscle cell hypertrophy a critical factor that contributes to intestinal stricture formation.
Background: Stabilization of mast cell (MC) degranulation has been proposed to prevent postoperative ileus (POI). Nerve growth factor (NGF) mediates MC degranulation. The aim of the study was to evaluate whether NGF receptor antagonist K252a acts as a MC stabilizer in vitro and in vivo model of POI. Methods: Peritoneal mast cells (PMCs) were obtained from Sprague-Dawley rats and were incubated with K252a and exposed to NGF or Compound 48/80 (C48/80). MC degranulation was assessed by β-hexosaminidase assay. POI was induced in rats by intestinal manipulation (IM). Rats were pre-treated with K252a (100µg/kg, sc) 20 min prior POI induction. 20 min after IM, release of rat mast cell protease 6 (RMCP-6) was evaluated in peritoneal lavage. At 24h, intestinal transit (IT) and gastric emptying (GE) were evaluated. Ileal inflammation was assessed by myeloperoxidase (MPO) activity, expression of IL-6, NGF, TrkA, RMCP-2 and 6 and MC density within the full thickness ileum. Results: C48/80 and NGF evoked degranulation of PMCs in a dose dependent manner. K252a prevented NGF-evoked, but not C48/80-evoked MC degranulation. IM evoked the release of peritoneal RMCP-6 and subsequently delayed IT and GE. IM increased MPO activity and expression of IL-6. In IM rats, K252a prevented up-regulation of IL-6 expression and reduced TrkA. IT, GE and inflammation were not affected by K252a. Conclusions: K252a inhibited NGF-evoked degranulation of PMCs in vitro. In vivo, K252a decreased IL-6 and PMC degranulation. This may be of relevance for the development of new therapeutic targets for POI.
The incretin hormones, GIP and GLP-1, enhance postprandial insulin secretion, promote adipogenesis, and regulate gastrointestinal motility and food intake. To date, a consensus on how the incretin response is altered in obesity is lacking. We investigated the effects of chronic high-fat-feeding on incretin secretion in the lymph fistula rat model. Male Sprague-Dawley rats (8 wk) were provided a semi-purified AIN93M high-fat (HF) or low-fat (LF) diet ad libitum for 3 or 13 wk; a high-fat pair-fed (HF-PF) group was included as a control during the 3 wk feeding trial. Energy intake, body weight, and body composition were regularly monitored. At the culmination of the feeding period, an intestinal lymphatic duct cannula and duodenal infusion tube were installed. All animals were challenged with a 3 ml Ensure bolus (3.125 kcal/animal) to measure lymphatic incretin secretion. Despite a significantly higher energy intake, both the 3 wk and 13 wk HF-fed animals did not have an increase in body weight and only a slight increase in body fat compared to LF-fed rats. Following the duodenal Ensure challenge, the 3 wk and 13 wk HF-fed rats had significantly greater lymphatic GIP and GLP-1 secretion than the LF-fed animals. Additionally, the HF-PF group displayed a secretion profile similar to the HF-fed animals for GIP but a similar pattern to the LF-fed animals for GLP-1. The HF-PF data suggest that the increased GIP secretion is driven by the greater percentage of fat intake, whereas the increased GLP-1 secretion is driven by the excess caloric intake.
Bilirubin is thought to exert anti-inflammatory effects by inhibiting vascular cell adhesion molecule-1 (VCAM-1)-dependent leukocyte migration and by suppressing the expression of inducible nitric oxide synthase (iNOS). As VCAM-1 and iNOS are important mediators of tissue injury in the dextran sodium sulfate (DSS) murine model of inflammatory colitis, we examined whether bilirubin prevents colonic injury in DSS-treated mice. Methods: Male C57BL/6 mice were administered 2.5% DSS in the drinking water for 7 days, while simultaneously receiving i.p. injections of bilirubin (30 mg/kg) or potassium phosphate vehicle. Disease activity was monitored, peripheral blood counts and serum nitrate levels determined, and intestinal specimens analyzed for histological injury, leukocyte infiltration, and iNOS expression. The effect of bilirubin on IL-5 production by HSB-2 cells and on Jurkat cell transendothelial migration also was determined. Results: DSS-treated mice that simultaneously received bilirubin lost less body weight, had lower serum nitrate levels, and exhibited reduced disease severity than vehicle-treated animals. Concordantly, histopathological analyses revealed that bilirubin-treated mice manifested significantly less colonic injury, including reduced infiltration of eosinophils, lymphocytes, and monocytes, and diminished iNOS expression. Bilirubin administration also was associated with decreased eosinophil and monocyte infiltration into the small intestine, with a corresponding increase in peripheral blood eosinophilia. Bilirubin prevented Jurkat migration, but did not alter IL-5 production. Conclusion: Bilirubin prevents DSS-induced colitis by inhibiting the migration of leukocytes across the vascular endothelium and by suppressing iNOS expression.
The p66Shc protein mediates oxidative stress-related injury in multiple tissues. Steatohepatitis is characterized by enhanced oxidative stress-mediated cell damage. The role of p66Shc in redox signaling was investigated in human liver cells and alcoholic steatohepatitis. HepG2 cells with overexpression of wild-type or mutant p66Shc, with Ser36 replacement by Ala, were obtained through infection with recombinant adenoviruses. Reactive oxygen species and oxidation-dependent DNA damage were assessed by measuring dihydroethidium oxidation and 8-hydroxy-2'-deoxyguanosine accumulation into DNA, respectively. mRNA and protein levels of signaling intermediates were evaluated in HepG2 cells and liver biopsies from control and alcoholic steatohepatitis subjects. Exposure to H2O2 increased reactive oxygen species and phosphorylation of p66Shc on Ser36 in HepG2 cells. Overexpression of p66Shc promoted reactive oxygen species synthesis and oxidation-dependent DNA damage, which were further enhanced by H2O2. p66Shc activation also resulted in increased Erk-1/2, Akt and FoxO3a phosphorylation. Blocking of Erk-1/2 activation inhibited p66Shc phosphorylation on Ser36. Increased p66Shc expression was associated with reduced mRNA levels of anti-oxidant molecules, such as NF-E2-related factor 2 and its target genes. In contrast, overexpression of the phosphorylation defective p66Shc Ala36 mutant inhibited p66Shc signaling, enhanced anti-oxidant genes, and suppressed reactive oxygen species and oxidation-dependent DNA damage. Increased p66Shc protein levels and Akt phosphorylation were observed in liver biopsies from alcoholic steatohepatitis compared to control subjects. Conclusions: in human alcoholic steatohepatitis, increased hepatocyte p66Shc protein levels may enhance susceptibility to DNA damage by oxidative stress by promoting reactive oxygen species synthesis and repressing anti-oxidant pathways.
Pituitary adenylate cyclase-activating peptide (PACAP) is expressed within the gastroenteric system where it has profound physiological effects. PACAP was shown to regulate food intake and thermogenesis centrally; however, PACAP peripheral regulation of appetite and feeding behavior is unknown. Therefore, we studied PACAP's effect on appetite and food intake control by analyzing feeding behavior and metabolic hormones in PAC1 deficient (PAC1-/-) and age-matched WT mice, intraperitoneally injected with PACAP1-38 or PACAP1-27 prior to the dark phase of feeding. Food intake and feeding behavior were analyzed using the BioDAQ system. Active-ghrelin, glucagon-like peptide-1 (GLP-1), leptin, peptide YY (PYY), pancreatic polypeptide (PP) and insulin were measured following PACAP1-38 administration in fasted WT mice. PACAP1-38/PACAP1-27 injected into WT significantly decreased in a dose-dependent manner cumulative food intake and reduced bout and meal feeding parameters. Conversely, PACAP1-38 injected into PAC1-/-, failed to significantly change food intake. Importantly, PACAP1-38 reduced plasma levels of active-ghrelin compared to vehicle in WT mice. In PAC1-/- mice, fasting levels of active-ghrelin, GLP-1, insulin and leptin and postprandial levels of active-ghrelin and insulin were significantly altered compared to WT. Therefore, PAC1 is a novel regulator of appetite/satiety. PACAP1-38/PACAP1-27 significantly reduced appetite and food intake through PAC1. In PAC1-/- mice, the regulation of anorexigenic/orexigenic hormones was abolished, while active-ghrelin remained elevated even postprandially. PACAP significantly reduced active-ghrelin in fasting conditions. These results establish a role for PACAP via PAC1 in the peripheral regulation of appetite/satiety and suggest future studies to explore a therapeutic use of PACAP or PAC1 agonists for obesity treatment.
End stage liver disease is responsible for 30,000 deaths per year in the US alone, and is continuing to increase every year. With liver transplantation the only curative treatment currently available, new therapies are in great demand. Mesenchymal Stem Cells (MSC) offer an opportunity to both treat liver inflammatory damage as well as reverse some of the changes that occur following chronic liver injury. With the ability to regulate both the innate and adaptive immune system, as well as both inhibit and promote apoptosis of effector inflammatory cells, there are numerous therapeutic opportunities for MSC in acute and chronic liver disease. This article critically appraises the potential roles of MSC in liver disease, as well as the barriers to their adoption into clinical practice.
The Hippo signaling pathway has been implicated in mammalian organ size regulation and tumor suppression. Specifically, the Hippo pathway plays a critical role regulating the activity of transcriptional co-activator Yes-associated protein (YAP) which modulates a proliferative transcriptional program. Recent investigations have demonstrated that while this pathway is activated in quiescent livers, its inhibition leads to liver overgrowth and tumorigenesis. However, the role of the Hippo pathway during the natural process of liver regeneration remains unknown. Here we investigated alterations in the Hippo signaling pathway and YAP activation during liver regeneration using a 70% partial hepatectomy (PH) rat model. Our results indicate an increase in YAP activation by 1 day following PH as demonstrated by increased YAP nuclear localization and increased YAP target gene expression. Investigation of the Hippo pathway revealed a decrease in the activation of core kinases Mst1/2 by 1 day as well as Lats1/2 and its adapter protein Mob1 by 3 days following PH. Evaluation of liver-to-body weight ratios indicated that the liver reaches its near normal size by 7 days following PH that correlated with a return to baseline YAP nuclear levels and target gene expression. Additionally, when liver size was restored, Mst1/2 kinase activation returned to levels observed in quiescent livers indicating reactivation of the Hippo signaling pathway. These findings illustrate the dynamic changes in the Hippo signaling pathway and YAP activation during liver regeneration that stabilizes when the liver-to-body weight ratio reaches homeostatic levels.
Eosinophilic esophagitis (EoE) is characterized with eosinophils and mast cells predominated allergic inflammation in the esophagus and present with esophageal dysfunctions such as dysphagia, food impaction, and heartburn. But the underlying mechanism of esophageal dysfunctions is unclear. This study aims to determine whether neurons in the vagal sensory ganglia are modulated in a guinea pig model of EoE. Animals were actively sensitized by ovalbumin (OVA) then challenged with aerosol OVA inhalation for two weeks. This results in a mild esophagitis with increases in mast cells and eosinophils in the esophageal wall. Vagal nodose and jugular neurons were disassociated and their responses to acid, capsaicin, and TRPV1 antagonist AMG9810 were studied by calcium imaging and whole-cell patch clamp recording. Compared to naïve animals, antigen challenge significantly increased acid responsiveness in both nodose and jugular neurons. Their responses to capsaicin were also increased after antigen challenge. AMG9810, at a concentration that blocked capsaicin-evoked calcium influx, abolished the increase in acid-induced activation in both nodose and jugular neurons. Vagotomy strongly attenuated those increased responses of nodose and jugular neurons to both acid and capsaicin induced by antigen challenge. This data for the first time demonstrated that prolonged antigen challenge significantly increases acid responsiveness in vagal nodose and jugular ganglia neurons. This sensitization effect is mediated largely through TRPV1 and initiated at sensory nerve endings in the peripheral tissues. Allergen-induced enhancement of responsiveness to noxious stimulation by acid in sensory nerve may contribute to the development of esophageal dysfunctions such as heartburn in EoE.
IFN--driven and CD8+ T cells-dependent inflammatory injury to extrahepatic biliary epithelium (EHBE) is likely to be involved in the development of biliary atresia (BA). We previously showed that viral protein NSP4 is the pathogenic immunogen which causes biliary injury in BA. In this study, NSP4 or four synthetic NSP4 (NSP4157-170, NSP4144-152, NSP493-110, NSP424-32) identified by computer analysis as candidate CD8+ T cells epitopes were injected into neonatal mice. The pathogenic NSP4 epitopes were confirmed by studying extrahepatic bile duct injury, IFN- release and CD8+ T cell response against EHBE. The results revealed, at 7 days post-injection (dpi), inoculation of GST-NSP4 caused EHBE injury and BA in neonatal mice. At 7 or 14 dpi, inoculation of GST-NSP4, NSP4144-152 or NSP4157-170 increased IFN- release by CD8+ T cells, elevated the population of hepatic memory CD8+ T cells, and augmented cytotoxicity of CD8+ T cells to rhesus rotavirus (RRV)-infected or naïve EHBE cells. Furthermore, depletion of CD8+ T cells in mice abrogated the elevation of GST-NSP4-induced serum IFN-. Lastly, parenteral immunization of mouse dams with GST-NSP4, NSP4144-152 or NSP4157-170 decreased the incidence of RRV-induced BA in their offspring. Overall, this study reports the CD8+ T cell response against EHBE is activated by epitopes within rotavirus NSP4 in experimental BA. Neonatal passive immunization by maternal vaccination against NSP4144-152 or NSP4157-170 is effective in protecting neonates from developing RRV- related BA.
The hypoxic response is mediated by two transcription factors, HIF-1α and HIF-2α. These highly homologous transcription factors are induced in hypoxic foci and regulate cell metabolism, angiogenesis, cell proliferation and cell survival. HIF-1α and HIF-2α are activated early in cancer progression and are important in several aspects of tumor biology. HIF-1α and HIF-2α have overlapping and distinct functions. In the intestine, activation of HIF-2α increases inflammation and colon carcinogenesis in mouse models. Interestingly, in ischemic and inflammatory diseases of the intestine, activation of HIF-1α is beneficial, and can reduce intestinal inflammation. HIF-1α is a critical transcription factor regulating epithelial barrier function following inflammation. The beneficial value of pharmacological agents that chronically activate HIF-1α are decreased due to the tumorigenic potential of HIFs. The present study tested the hypothesis that chronic activation of HIF-1α may enhance colon tumorigenesis. Two models of colon cancer were assessed, a sporadic and a colitis-associated colon cancer model. Activation of HIF-1α in intestinal epithelial cells does not increase carcinogenesis or progression of colon cancer. Together, the data provides proof of principle that pharmacological activation of HIF-1α could be a safe therapeutic strategy for inflammatory bowel disease.
Type 2 diabetes is associated with elevated circulating levels of the chemokine RANTES and with decreased plasma levels of the incretin hormone glucagon-like peptide 1 (GLP-1). GLP-1 is a peptide secreted from intestinal L-cells upon nutrient ingestion. It enhances insulin secretion from pancreatic ß-cells and protects from ß-cell loss but also promotes satiety and weight loss. In search of chemokines that may reduce GLP-1 secretion we identified RANTES and show that it reduces glucose-stimulated GLP-1 secretion in the human enteroendocrine cell line NCI-H716 - blocked by the antagonist Met-RANTES - and in vivo in mice. RANTES exposure to mouse intestinal tissues lowers transport function of the intestinal glucose transporter SGLT1 and administration in mice reduces plasma GLP-1 and GLP-2 levels after an oral glucose load and thereby impairs insulin secretion. These data show that RANTES is involved in altered secretion of glucagon-like peptide hormones most probably acting through SGLT1 and our study identifies the RANTES-receptor CCR1 as a potential target in diabetes therapy.
Although human liver fatty acid binding protein (FABP1) T94A variant has been associated with non-alcoholic fatty liver disease (NAFLD) and reduced ability of fenofibrate to lower serum triglycerides (TG) to target levels, molecular events leading to this phenotype are poorly understood. Cultured primary hepatocytes from female human subjects expressing the FABP1 T94A variant exhibited increased neutral lipid (TG, cholesteryl ester) accumulation associated with: 1) upregulation of total FABP1, a key protein stimulating GPAM the rate limiting enzyme in lipogenesis; 2) increased mRNA expression of key enzymes in lipogenesis (GPAM, LPIN2) in heterozygotes; 3) decreased mRNA expression of microsomal triglyceride transfer protein (MTTP); 4) increased secretion of ApoB100 but not TG; 5) decreased LCFA β-oxidation. TG accumulation was not due to any increase in long chain fatty acid (LCFA) uptake, de novo lipogenesis, or the alternate MOGAT pathway in lipogenesis. Despite increased expression of total FABP1 mRNA and protein, fenofibrate mediated FABP1 redistribution to nuclei and ligand-induced PPARα transcription of LCFA β-oxidative enzymes (CPT1A, CPT2, and ACOX1) were attenuated in FABP1 T94A hepatocytes. While the phenotype of FABP1 T94A variant human hepatocytes exhibits some similarities to that of FABP1 null or PPARα null hepatocytes and mice, expression of FABP1 T94A variant did not abolish or reduce ligand binding. Thus, the FABP1 T94A variant represents an altered/reduced function mutation resulting in TG accumulation.
The glucagon like peptide 2 (GLP-2) is an intestinotrophic hormone with growth promoting and anti-inflammatory actions. However, the full biological functions of GLP-2 and the localization of its receptor (GLP-2R) remain controversial. Among cell lines tested, the expression of GLP-2R transcript was detected in human colonic myofibroblasts (CCD-18Co) and in primary culture of rat enteric nervous system but not in intestinal epithelial cell lines, lymphocytes, monocytes, and endothelial cells. Surprisingly, GLP-2R was expressed in murine (GLUTag), but not human (NCI-H716) enteroendocrine cells. The screening of GLP-2R mRNA in mice organs revealed an increasing gradient of GLP-2R toward the distal gut. An unexpected expression was detected in the mesenteric fat, mesenteric lymph nodes, bladder, spleen, and in the liver, particularly in hepatocytes. In two mice models of TNBS- and DSS-induced colitis, the colonic expression of GLP-2R mRNA was decreased by 60% compared to control mice. Also, GLP-2R mRNA was significantly downregulated in intestinal tissues of IBD patients. Therapeutically, GLP-2 showed a weak restorative effect on intestinal inflammation during TNBS-induced colitis as assessed by macroscopic score and inflammatory markers. Finally, GLP-2 treatment accelerated mice liver regeneration following partial hepatectomy as assessed by histological and molecular analyses. In conclusion, the limited therapeutic effect of GLP-2 on colonic inflammation suspects its utility in the management of severe inflammatory intestinal disorders. However, the role of GLP-2 in liver regeneration is a novelty that might introduce GLP-2 into the management of liver diseases and emphasizes on the importance of elucidating other extra-intestinal functions of GLP-2.
Adenosine is a purine metabolite that can mediate anti-inflammatory responses in the digestive tract through the A2A adenosine Receptor (A2AAR). Here we examined the role of this receptor in the control of inflammation in the adoptive transfer model of colitis. Infection of A2AAR-/- mice with Helicobacter hepaticus increased colonic inflammation scores compared to uninfected A2AAR controls. Comparison of T cell subsets in wildtype and A2AAR-/- mice revealed differences in markers associated with activated helper T (Th) cells and Treg. Past studies have shown that expression of A2AAR on CD45RBHI and CD45RBLO Th cells is essential for the proper regulation of colonic inflammation. Adoptive transfer of CD45RBHI with CD45RBLO from wildtype mice into RAG1-/-/A2AAR-/- mice induced severe disease within 3 weeks, although transfer of the same subsets into RAG1-/- mice does not induce colitis. This suggests that the presence of A2AAR on recipient cells is also important for controlling colitis. To investigate the role of A2AAR on myeloid cells, chimeric recipients were generated by giving RAG1-/- or RAG1-/-/A2AAR-/- bone marrow to irradiated RAG1-/- mice. After adoptive transfer, these recipients did not develop colitis regardless of the A2AAR expression by the donor. Together, our results suggest that the control of inflammation in vivo is dependent on A2AAR signaling through multiple cell types that collaborate in the regulation of colitis by responding to extracellular adenosine.
We examined the effects of two over-the-counter H1-antihistamines on the progression of fatty liver disease in male C57Bl/6 wild-type and ApoE-/- mice. Mice were fed a high-fat diet (HFD) for 3 months, together with administration of either cetirizine (4 mg/kg b.w.) or fexofenadine (40 mg/kg b.w.) in drinking water. Antihistamine treatments increased body weight gain, gonadal fat deposition, liver weight and hepatic steatosis in wild-type mice, but not in ApoE-/- mice. Lobular inflammation, acute inflammation and necrosis were not affected by H1-antihistamines in either genotype. Serum biomarkers of liver injury tended to increase in antihistamine-treated wild-type mice. Serum level of glucose was increased by fexofenadine, while lipase was increased by cetirizine. H1-antihistamines reduced the mRNA expression of ApoE and carbohydrate response element binding protein (ChREBP) in wild-type mice, without altering the mRNA expression of sterol regulatory element binding protein 1c (SREBP1c), fatty acid synthase (FAS), or ApoB100, in either genotype. Fexofenadine increased triglycerides, while cetirizine increased cholesterol ester in liver, with a concomitant decrease in serum triglycerides by both antihistamines in wild-type mice. Antihistamines increased hepatic levels of conjugated bile acids in wild type mice, with the effect being significant in fexofenadine-treated animals. The increase was associated with changes in the expression of organic anion transport polypeptide 1b2 (Oatp1b2) and bile salt export pump (Bsep). These results suggest that H1-antihistamines increase the progression of fatty liver disease on wild-type mice, and there seems to be an association between the severity of disease, presence of ApoE and increase in hepatic bile acid levels.
Necrotizing enterocolitis (NEC) is an inflammatory disease with evidence of increased production of proinflammatory cytokines in the intestinal mucosa. Lactobacillus reuteri DSM17938 (LR17938) has been shown to have anti-inflammatory activities in an experimental model of NEC. Activated effector lymphocyte recruitment to sites of inflammation requires the sequential engagement of adhesion molecules such as CD44. The phenotype of CD44+CD45RBlo separates T effector/memory (Tem) cells from naïve cells (CD44-CD45RBhi). It is unknown whether these Tem cells participate in the inflammation associated with NEC and can be altered by LR17938. NEC was induced in 8-10-day old C57BL/6J mice by gavage feeding with formula and exposure to hypoxia and cold stress for 4 days. Survival curves and histological scores were analyzed. Lymphocytes isolated from mesenteric lymph nodes (MLNs) and ileum were labeled for CD4, CD44, CD45RB, intracellular Foxp3, and Helios and were subsequently analyzed by flow cytometry. LR17938 decreased the mortality, and incidence and severity of NEC. The percentage of Tem cells in the ileum and MLNs was increased in NEC but was decreased by LR17938. Conversely, the CD4+Foxp3+ regulatory T cells (Tregs) in the intestine decreased during NEC and were restored to normal by LR17938. The majority of the Tregs preserved by LR17938 were Helios+ subsets, possibly of thymic origin. In conclusion, LR17938 may represent a useful treatment to prevent NEC. The mechanism of protection by LR17938 involves modulation of the balance between Tem and Treg cells. These T cell subsets might be potential biomarkers and therapeutic targets during intestinal inflammation.
Hydrophobic bile acids like deoxycholic acid (DCA), which cause oxidative DNA damage and activate NF-B in Barrett's metaplasia, might contribute to carcinogenesis in Barrett's esophagus. We have explored mechanisms whereby ursodeoxycholic acid (UDCA, a hydrophilic bile acid) protects against DCA-induced injury in vivo in patients and in vitro using non-neoplastic, telomerase-immortalized Barrett's cell lines. We took biopsies of Barrett's esophagus from 21 patients before and after esophageal perfusion with DCA (250 µM) at baseline, and after 8 weeks of oral UDCA treatment. DNA damage was assessed by phospho-H2AX expression, neutral comet assay and phospho-H2AX nuclear foci formation. Quantitative PCR was performed for antioxidants including catalase and GPX1. Nrf2, catalase, and GPX1 were knocked down with siRNAs. Reporter assays were performed using a plasmid construct containing antioxidant responsive element. In patients, baseline esophageal perfusion with DCA significantly increased phospho-H2AX and phospho-p65 in Barrett's metaplasia. Oral UDCA increased GPX1 and catalase levels in Barrett's metaplasia, and prevented DCA perfusion from inducing DNA damage and NF-B activation. In cells, DCA-induced DNA damage and NF-B activation was prevented by 24-hour pretreatment with UDCA, but not by mixing UDCA with DCA. UDCA activated Nrf2 signaling to increase GPX1 and catalase expression, and protective effects of UDCA pretreatment were blocked by siRNA knockdown of these antioxidants. UDCA increases expression of antioxidants that prevent toxic bile acids from causing DNA damage and NF-B activation in Barrett's metaplasia. Elucidation of this molecular pathway for UDCA protection provides rationale for clinical trials on UDCA for chemoprevention in Barrett's esophagus.
This study aimed to develop and validate a method to measure bolus flow time (BFT) through the EGJ using a HRIM sleeve. Methods: 10 healthy subjects were studied with concurrent HRIM and videofluoroscopy while another 15 controls underwent HRIM alone. HRIM studies were performed using a 4.2 mm outer diameter assembly with 36 pressure sensors (1-cm intervals) and 18 impedance segments (2-cm intervals) (Given Imaging, Los Angeles, CA). HRIM and fluoroscopic data from 4 barium swallows (2 supine; 2 upright) were analyzed to create a customized MATLAB program to calculate the BFT using a HRIM sleeve comprised of 3 sensors positioned at the crural diaphragm (CD). Results: Bolus transit through the EGJ measured during blinded review of fluoroscopy was almost identical to the BFT calculated with the HRIM sleeve using the nadir impedance deflection point as the signature of bolus presence. Good correlation existed between videofluoroscopy for measurement of upper sphincter relaxation to beginning flow (supine, R=0.97, P<0.001; upright, R=0.77, P<0.01) and time to end of flow (supine. R=0.95, P<0.001; upright, R=0.82, P<0.01). The median (IQR) of Flow Time though the EGJ in 15 healthy subjects calculated using the virtual sleeve was 3.5 s (2.3-3.9 s) in supine position and 3.2 s (2.3-3.6 s). Conclusions: BFT is a new metric that provides important information on bolus transit through the EGJ. An assessment of BFT will determine the time period where the EGJ is open and also provide a useful method to accurately assess trans-EGJ pressure gradients during flow.
Evidence has grown to support the efficacy of probiotics in the management of gastrointestinal disorders, many of which are associated with dysregulated fluid and electrolyte transport. A growing body of evidence now suggests that the host microbiota and probiotics can influence intestinal ion transport, and that these effects often occur in a strain-dependent manner. In this study, we sought to investigate the effects of two therapeutically relevant organisms, Bifidobacterium infantis 35624 and Lactobacillus salivarius UCC118, on small intestinal transit, fecal output and water content, transepithelial resistance (TER) and colonic secretomotor function. Mice fed either strain displayed significantly reduced small intestinal transit in vivo, though neither strain influenced fecal pellet output or water content. Colon from mice fed both organisms displayed increased colonic TER, without a concomitant change in the gene expression of the tight junction proteins, claudin 1 and occludin. However, L. salivarius UCC118 selectively inhibited neurally-evoked ion secretion in tissues from animals fed this particular probiotic. Consistent with this finding, the neurotoxin, tetrodotoxin (TTx), significantly inhibited the short-circuit current response induced by L. salivarius UCC118 following addition to colonic preparations in Ussing chambers. Responses to B. infantis 35624 also displayed sensitivity to TTx, though to a significantly lesser degree than L. salivarius UCC118. Both strains similarly inhibited cholinergic-induced ion transport after addition to Ussing chambers. Taken together, these data suggest that B. infantis 35624 and L. salivarius UCC118 may be indicated in disorders associated with increased small intestinal transit, and, in particular for L. salivarius UCC118, neurally-mediated diarrhea.
Nitric oxide (NO) is a major inhibitory neurotransmitter in the gastrointestinal (GI) tract. Its main effector, NO-sensitive guanylyl cyclase (NO-GC), is expressed in several GI cell types including smooth muscle cells (SMC), interstitial cells of Cajal (ICC) and fibroblast-like cells. Up to date the interplay between neurons and these cells to initiate a nitrergic inhibitory junction potential (IJP) is unclear. Here, we investigate the origin of the nitrergic IJP in murine fundus and colon. IJPs were determined in fundus and colon SMC of mice lacking NO-GC globally (GCKO) and specifically in SMC (SM-GCKO), ICC (ICC-GCKO) and both SMC/ICC (SM/ICC-GCKO). Nitrergic IJP was abolished in ICC-GCKO fundus and reduced in SM-GCKO fundus. In colon, the amplitude of nitrergic IJP was reduced in ICC-GCKO whereas nitrergic IJP in SM-GCKO was reduced in duration. These results were corroborated by loss of the nitrergic IJP in global GCKO. In conclusion, our results prove the obligatory role of NO-GC in ICC for the initiation of an IJP. NO-GC in SMC appears to enhance the nitrergic IJP resulting in a stronger and a prolonged hyperpolarization in fundus and colon SMC, respectively. Thus, NO-GC in both cell types is mandatory to induce a full nitrergic IJP. Our data from colon clearly reveal the nitrergic IJP to be biphasic resulting from individual inputs of ICC and SMC.
Retinoic acid (RA) has diverse biological effects. The liver stores vitamin A, generates RA, and expresses receptors for RA. The current study examines the hepatic binding profile of two RA receptor isoforms, RARA (RARα) and RARB (RARβ), in response to RA treatment in mouse livers. Our data uncovered 35,521, and 14,968 genomic bindings for RARA and RARB, respectively. Each expressed unique and common bindings, implying their redundant and specific roles. RARB has higher RA responsiveness than RARB. RA treatment generated 18,821 novel RARB bindings, but only 14,798 of RARA bindings, compared with the control group. RAR frequently bound the consensus hormone response element (HRE, (A/G)G(G/T)TCA), which often contained the motifs assigned to SP1, GABPA, and FOXA2, suggesting potential interactions between those transcriptional factors. Functional annotation coupled with principle component analysis (PCA) revealed that the function of RAR target genes were motif-dependent. Taken together, the cistrome of RARA and RARB revealed their extensive biological roles in the mouse liver. RAR target genes are enriched in various biological processes. The hepatic RAR genome-wide binding data can help us understand the global molecular mechanisms underlying RAR and RA-mediated gene and pathway regulation.
Regular "mucosal block" is characterized by decreased uptake of a normal iron load 3-72 h after the administration of excess iron (generally 10 mg) to iron deficient animals. We found that short-acting mucosal block could be induced by much lower iron concentration and much shorter induction time than previously reported, without affecting levels of gene expression. A rapid endocytic mechanism was reported to decrease intestinal iron absorption after a high iron load, but the activating iron load and the time to decreased absorption were undetermined. We assessed the effects of 30-2,000 μg iron load on iron uptake in the duodenal loop of iron-deficient and iron-sufficient rats under anesthesia. One hour later, mucosal cellular iron uptake in iron-deficient rats administered 30 μg iron was 76.1%, decreasing to 25-50.7% in rats administered 2,000 μg iron. In contrast, iron uptake by iron-sufficient rats was 63% (range, 60.3-65.5%) regardless of iron load. Duodenal mucosal iron concentration was significantly lower in iron-deficient than in iron-sufficient rats. Iron levels in portal blood were consistently higher in iron-deficient rats regardless of iron load, in contrast to the decreased iron uptake on the luminal side. Iron loading blocked mucosal uptake of marginally excess iron (1,000 μg), with a greater effect at 15 min than at 30 min. The rapid induction of short-acting mucosal block only in iron-deficient rats suggests DMT1 internalization.
Autophagy is a catabolic process involved in homeostatic and regulated cellular protein recycling and degradation via the lysosomal degradation pathway. Emerging data associates impaired autophagy, increased activity in the endocannabinoid system and upregulation of suppressor of cytokine signaling (SOCS)-3 protein expression during intestinal inflammatory states. We have investigated whether these three processes are linked. By assessing the impact of phyto-cannabinoid cannabidiol (CBD), synthetic cannabinoid (ACEA) and endocannabinoid (AEA) on autophagosome formation, we explored whether these actions were responsible for cyclic SOCS3 protein levels. Our findings show that all three cannabinoids induce autophagy in a dose-dependent manner in fully differentiated CaCo2 cells, a model of mature intestinal epithelium. ACEA and AEA induced canonical autophagy, which was cannabinoid receptor (CB)-1 mediated. In contrast, CBD was able to bypass both the CB1 receptor and the canonical pathway to induce autophagy, albeit to a lesser extent. Functionally, all three cannabinoids reduced SOCS3 protein expression, which was reversed by blocking both early and late autophagy. In conclusion, the regulatory protein, SOCS3, is itself regulated by autophagy and cannabinoids play a role in this process, which could be important when considering therapeutic applications for the cannabinoids in inflammatory conditions.
Individuals with Familial Adenomatous Polyposis (FAP) harbor a germline mutation in APC. The major clinical manifestation is development of multiple colonic tumors at a young age due to stochastic loss of the remaining APC allele. Extra-colonic features may occur, including periampullary tumors, gastric abnormalities, and congenital hypertrophy of the retinal pigment epithelium (CHRPE). The objective of this study was to develop a mouse model that simulates these features of FAP. To accomplish this, we combined our Lrig1-CreERT2/+ mice with Apcfl/+ mice, eliminated one copy of Apc in Lrig1+ progenitor cells with tamoxifen injection and monitored tumor formation in the colon by colonoscopy and positron emission tomography (PET). Initial loss of one Apc allele in Lrig1+ cells results in a predictable pattern of preneoplastic changes, culminating in multiple distal colonic tumors within 50 days of induction, as well as the extra-colonic manifestations of FAP mentioned above. We show that tumor formation can be monitored by noninvasive PET imaging. This inducible stem cell-driven model recapitulates features of FAP and offers a tractable platform to monitor therapeutic interventions over time by colonoscopy and non-invasive imaging.
Sestrins (Sesns) are a family of stress-sensitive genes that have been suggested to regulate lipid metabolism. Chronic ethanol feeding is known to cause lipid accumulation in hepatocytes. This study was designed to investigate the role of Sesn3 in the pathogenesis of alcohol-induced hepatic steatosis. We demonstrated that ethanol inhibited the expression of Sesn3 in VL-17A cells. Over-expression of Sesn3 ameliorated TG accumulation; down-regulation using shRNA significantly deteriorated TG accumulation in these cells. The expression of Sesn3 was also reduced in mice fed with ethanol for 4 weeks. Overexpression of Sesn3 prevented hepatic steatosis whereas knockdown of Sesn3 worsened hepatic steatosis in ethanol-fed mice. Overexpression of Sesn3 significantly reduced the expression of genes encoding for lipid synthesis through AMPK pathway. Overexpression of Sesn3 augmented the effect of ethanol on phopspho-p70 S6 kinase. The levels of hepatic LC3-a marker for autophagy-- expression were significantly decreased in ethanol-fed mice after Sesn3 gene was knocked down. Our findings suggest that inhibitory effect of ethanol on Sesn3 may play an important role in the development of ethanol-induced fatty liver.
Background: It has been shown, in animal models, that the gastrointestinal tract (GIT) motility is influenced by temperature; nevertheless the basic mechanism governing thermal GIT smooth muscle responses has not been fully investigated. Studies based on physiologically tuned mathematical models predict that thermal inhomogeneity may induce an electrochemical destabilization of the peristaltic activity. Herewith, the effect of thermal cooling on human colonic muscle strip (HCMS) contractility was studied. Methods: HCMSs were obtained from disease-free margins of resected segments for cancer. After removing the mucosa and serosa layers, strips were mounted in separate chambers. After 30 minutes, spontaneous contractions developed, which were measured using force displacement transducers. Temperature was changed every hour (37, 34, 31°C). The effect of cooling was analyzed on mean contractile activity, oscillations amplitude, frequency and on contraction to Acetylcholine (Ach, 10-5M). Key Results: At 37°C HCMSs developed a stable phasic contraction (~ 0.02 Hz) with a significant Ach-elicited mean contractile response (31%, 22% compared to baseline in circular and longitudinal axis, respectively). Lowering bath temperature, higher mean contractile amplitude was observed and it increased in presence of Ach (78% and 43% higher than the basal tone in the circular and longitudinal axis, respectively, at 31 °C). A simplified thermo-chemo-mechanical model is tuned on experimental data characterizing the stress state coupling the intracellular calcium concentration to tissue temperature. Conclusions: Acute thermal cooling affects colonic muscular function. Further studies are needed to establish the mechanisms involved in order to better understand clinical consequences of hypothermia on intestinal contractile activity.
The presence of negatively charged, impermeant proteins in the plasma space alters the distribution of diffusible ions in the plasma and interstitial fluid compartments in order to preserve electroneutrality and is known as Gibbs-Donnan equilibrium. In patients with hypoalbuminemia due to underlying cirrhosis, the decrease in the plasma water albumin concentration ([Alb–]pw) would be expected to result in a decrease in the plasma water sodium concentration ([Na+]pw) due to an alteration in the distribution of Na+ ions between the plasma and ISF. In addition, cirrhosis-associated hyponatremia may be due to the renal diluting defect resulting from the intravascular volume depletion due to gastrointestinal losses and overdiuresis and/or decreased effective circulatory volume secondary to splanchnic vasodilatation. Therefore, albumin infusion may result in correction of the hyponatremia in cirrhotic patients either by modulating the Gibbs-Donnan effect or by restoring intravascular volume in patients with intravascular volume depletion. However, the differential role of albumin infusion in modulating the [Na+]pw in these patients has not previously been analyzed quantitatively. In the present study, we developed an in vitro assay system to examine the quantitative effect of changes in [Alb–] on the distribution of Na+ ions between two compartments separated by a semipermeable membrane. Our findings demonstrated that changes in [Alb–]pw are linearly related to changes in Na+]pw as predicted by Gibbs-Donnan equilibrium. However, based on our findings, we predict that the improvement in cirrhosis-associated hyponatremia due to intravascular volume depletion results predominantly from the restoration of intravascular volume rather than alterations in Gibbs-Donnan equilibrium.
Decreased extracellular pH is observed in a number of clinical conditions and can sensitize to the development and worsen the severity of acute pancreatitis. Because intercellular communication through gap junctions is pH sensitive and modulates pancreatitis responses, we evaluated the effects of low pH on gap junctions in the rat pancreatic acinar cell. Decreasing extracellular pH from 7.4 to 7.0 significantly inhibited gap junctional intracellular communication. Acidic pH also significantly reduced levels of connexin32 (Cx32), the predominant gap junction protein in acinar cells, and altered its localization. Increased degradation through the proteasomal, lysosomal, and autophagic pathways mediated the decrease in Cx32 under low pH conditions. These findings provide the first evidence that low extracellular pH can regulate gap junctional intercellular communication by enhancing connexin degradation.
The calcium-sensing receptor (CaSR) is expressed in various tissues, including the gastrointestinal tract. To investigate the role of gut CaSR on glycemic control, we examined whether single oral administration of CaSR agonist peptides affected the glycemic response in rats. Glucose tolerance tests were performed under oral or duodenal administration of various CaSR agonist peptides (Glu-Cys [EC], protamine, and poly-lysine) in conscious rats. Involvement of CaSR was determined by using a CaSR antagonist. Signaling pathways underlying CaSR agonist-modified glycemia were investigated using gut hormone receptor antagonists. The gastric emptying rate after the administration of CaSR agonist peptides was measured by phenol red recovery method. Oral and duodenal administration of CaSR agonist peptides respectively attenuated glycemic responses under the oral glucose tolerance test, but the administration of casein did not. The promotive effect on glucose tolerance was weakened by luminal pretreatment with a CaSR antagonist. Treatment with a 5-HT3 receptor antagonist partially diminished the glucose-lowering effect of peptides. Furthermore, the gastric emptying rate was decreased by duodenal administration of CaSR agonist peptides. These results demonstrate that activation of the gut CaSR by peptide agonists promotes glucose tolerance in conscious rats. 5-HT3 receptor and the delayed gastric emptying rate appear to be involved in the glucose-lowering effect of CaSR agonist peptides. Thus, activation of gut CaSR by dietary peptides reduces glycemic responses, so that gut CaSR may be a potential target for the improvement of postprandial glycemia.
We have previously reported that epiregulin is a growth factor that seems to act on liver progenitor cells (LPCs) during liver regeneration. However, the relationship between epiregulin and LPCs has remained unclear. The aim of the present study was to clarify the role of epiregulin during liver regeneration. [Methods] The serum levels of epiregulin in patients with acute liver failure were examined. A liver injury model was developed using mice fed a diet containing 0.1% 3.5-diethoxycarbonyl-1.4-dihydrocollidine (DDC) to induce LPCs. We then evaluated the expression of epiregulin and LPCs in these mice. The proliferation of EpCAM+ LPCs cultured with epiregulin was examined in vitro, and finally epiregulin was overexpressed in mouse liver. [Results] In patients with acute liver failure, serum epiregulin levels were elevated significantly. In DDC mice, LPCs emerged around the portal area. Epiregulin was also detected around the portal area during the course of DDC-induced liver injury, and was partially co-expressed with Thy1. Serum epiregulin levels in DDC mice were also significantly elevated. Recombinant epiregulin augmented the proliferative capacity of the LPCs in a dose-dependent manner. In mice showing overexpression of epiregulin, the expression of PCNA on hepatocytes was increased significantly. Finally, LPCs emerged around the portal area after epiregulin gene delivery. [Summary] Epiregulin promotes the proliferation of LPCs and DNA synthesis by hepatocytes, and is upregulated in the serum of patients with liver injury. Furthermore, induction of epiregulin leads to the appearance of LPCs. Epiregulin would be a useful biomarker of liver regeneration.
Plasma levels of the orexigenic hormone ghrelin are suppressed by meals with an efficacy dependent on their macronutrient composition. We hypothesized that heterogeneity in osmolarity among macronutrient classes contributes to these differences. In three studies, the impact of small-intestinal hyperosmolarity was examined in Sprague-Dawley rats. In Study 1, isotonic, 2.5x and 5x hypertonic solutions of several agents with diverse absorption and metabolism properties were infused duodenally at physiologic rates (3 ml/10 min). Jugular-vein blood was sampled before and at 30, 60, 90, 120, 180, 240 and 300 minutes after infusion. Plasma ghrelin was suppressed dose-dependently and most strongly by glucose. Hyperosmolar infusions of lactulose, which transits the small intestine unabsorbed, and 3-O-methylglucose (3-O-MG), which is absorbed like glucose but remains unmetabolized, also suppressed ghrelin. Glucose, but not lactulose or 3-O-MG infusions increased plasma insulin. In Study 2, intestinal infusions of hyperosmolar NaCl suppressed ghrelin, a response that was not attenuated by co-infusion with the neural blocker lidocaine. In Study 3, we re-confirmed that the low-osmolar lipid emulsion Intralipid suppresses ghrelin more weakly than isocaloric (but hypertonic) glucose. Importantly, raising Intralipid's osmolarity to that of the glucose solution by non-absorbable lactulose supplementation enhanced ghrelin suppression to that seen after glucose. Hyperosmolar ghrelin occurred particularly during the initial 3 post-infusion hours. We conclude that small-intestinal hyperosmolarity: (a) is sufficient to suppress ghrelin; (b) may combine with other post-prandial mechanisms to suppress ghrelin; (c) might contribute to altered ghrelin regulation after gastric bypass surgery; and (d) may inform dietary modifications for metabolic health.
Gastrointestinal ulcers and bleeding are serious complications of non-steroidal anti-inflammatory drug (NSAID) use. While administration of antibiotics and Toll-like receptor 4 knockdown mitigate NSAID-induced enteropathy, the molecular mechanism of these effects is poorly understood. Intestinal hyperpermeability is speculated to trigger the initial damage due to NSAID use. Transient receptor potential vanilloid 4 (TRPV4) is a non-selective cation channel expressed throughout the gastrointestinal (GI) tract epithelium, which is activated by temperature, extension, and chemicals such as 5,6-epoxyeicosatrienoic acid (5,6-EET). The aim of this study was to investigate the possible role of TRPV4 in NSAID-induced intestinal damage. TRPV4 mRNA and protein expression were confirmed by RT-PCR and immunochemistry, respectively, in mouse and human tissues while TRPV4 channel activity of the intestinal cell line IEC-6 was assessed by Ca2+-imaging analysis. TRPV4 activators or the NSAID indomethacin significantly decreased transepithelial resistance (TER) in IEC-6 cells, and indomethacin-induced TER decreases were inhibited by specific TRPV4 inhibitors or siRNA TRPV4 knockdown, as well as by the epoxygenase inhibitor N-(methylsulfonyl)-2-(2-propynyloxy)-benzenehexanamide (MS-PPOH), which decreased 5,6-EET levels. In TRPV4 knockout mice (TRPV4KO), indomethacin-induced intestinal damage was significantly reduced compared to WT mice. Taken together, these results show that TRPV4 activation in the intestinal epithelium caused epithelial hyperpermeability in response to NSAID-induced arachidonic acid metabolites and contributed to NSAID-induced intestinal damage. Thus, TRPV4 could be a promising new therapeutic target for the prevention of NSAID-induced intestinal damage.
Background and Objectives: Central and peripheral neural regulation of swallowing and aero-digestive reflexes is unclear in human neonates. Functional near infrared spectroscopy (NIRS) is a noninvasive method to measure changes in oxy-hemoglobin (HbO) and deoxy-hemoglobin (HbD). Pharyngo-esophageal manometry permits evaluation of aero-digestive reflexes. Modalities were combined to investigate feasibility and to test neonatal fronto-parietal cortical changes during pharyngo-esophageal (visceral) stimulation and/or swallowing. Methods: Ten neonates (45.6±3.0wks PMA, 4.1±0.5kg) underwent novel pharyngo-esophageal manometry concurrent with NIRS. To examine esophagus-brain interactions, we analyzed cortical hemodynamic response (HDR) latency and durations during aerodigestive provocation and esophageal reflexes. Data are presented as mean ± SE or %. Results: HDR rates were 8.84 times more likely with basal spontaneous deglutition compared to sham stimuli (P=0.004). Of 182 visceral stimuli, 95% were analyzable for esophageal responses, 38% for HDR, and 36% for both. Of analyzable HDR (N=70): a) HbO concentration (µmol/L) baseline 1.5±0.7 vs. 3.7±0.7 post- stimulus was significant (P=0.02) b) HbD concentration (µmol/L) between baseline 0.1±0.4 vs. post-stimulus -0.5±0.4 was not significant (P=0.73). c) Hemispheric lateralization was 21% left only, 29% right only, and 50% bilateral. During concurrent esophageal and NIRS responses (N=66): a) Peristaltic reflexes were present in 74% and HDR in 61%. b) HDR was 4.75 times more likely with deglutition reflex vs. secondary peristaltic reflex (P=0.016). Conclusions: Concurrent NIRS with visceral stimulation is feasible in neonates, and fronto-parietal cortical activation is recognized. Deglutition contrasting with secondary peristalsis is related to cortical activation; thus, implicating higher hierarchical aerodigestive protective functional neural networks.
The interstitial cells of Cajal (ICC) drive rhythmic pacemaking contractions in the gastro-intestinal system. The ICC generate pacemaking signals by membrane depolarizations associated with the release of intracellular calcium (Ca2+) in the endoplasmic reticulum (ER) through inositol-trisphosphate (IP3) receptors (IP3R) and uptake by mitochondria (MT). This Ca2+ dynamic is hypothesized to generate pacemaking signals by calibrating ER Ca2+ store depletions and membrane depolarization with ER store operated Ca2+ entry (SOCE) mechanisms. Using a biophysically based spatio-temporal model of integrated Ca2+ transport in the ICC, we determine the feasibility of ER depletion timescale correspondence with experimentally observed pacemaking frequencies while considering the impact of IP3R Ca2+ release and MT uptake on bulk cytosolic Ca2+ levels, since persistent elevations of free intracellular Ca2+ are toxic to the cell. MT densities and distributions are varied in the model geometry to observe MT influence on free cytosolic Ca2+ and the resulting frequencies of ER Ca2+ store depletions, and the sarco-endoplasmic reticulum Ca2+ ATP-ase (SERCA) and IP3 agonist concentrations are also varied. Our simulations show high MT densities observed in the ICC are more relevant to ER establishing Ca2+ depletion frequencies than protection of the cytosol from elevated free Ca2+, while the SERCA pump is more relevant to containing cytosolic Ca2+ elevations. Our results further suggest that the level of IP3 agonist stimulating ER Ca2+ release, subsequent MT uptake and eventual activation of ER store-operated Ca2+ entry may determine frequencies of rhythmic pacemaking exhibited by the ICC across species and tissue types.
Bone marrow-derived mesenchymal stem cells (MSCs) sustain cancer cells by creating a microenvironment favorable for tumor growth. In particular, MSCs have been implicated in gastric cancer development. There is extensive evidence suggesting that Hedgehog signaling regulates tumor growth. However, very little is known regarding the precise roles of Hedgehog signaling and MSCs in tumor development within the stomach. The current study tests that hypothesis that Shh, secreted from MSCs, provides a proliferative stimulus for the gastric epithelium in the presence of inflammation. RFP-expressing MSCs transformed in vitro (stMSCs) were transduced with lentiviral constructs containing a vector control (stMSCvect) or shRNA targeting the Shh gene (stMSCShhKO). Gastric submucosal transplantation of wild type MSCs (wtMSCs), wild type MSCs over-expressing Shh (wtMSCShh), stMSCvect or stMSCShhKO cells in C57BL/6 control (BL/6) or gastrin-deficient (GKO) mice was performed and mice analyzed 30 and 60 days post-transplantation. Compared to BL/6 mice transplanted with wtMSCShh and stMSCvect cells, inflamed GKO mice developed aggressive gastric tumors. Tumor development was not observed in mouse stomachs transplanted with wtMSC or stMSCShhKO cells. Compared to stMSCShhKO transplanted mice, within the inflamed GKO mouse stomach Shh-expressing stMSCsvect and wtMSCsShh induced proliferation of CD44-positive cells. CD44-positive cells clustered in gland-like structures within the tumor stroma and were positive for Ptch expression. We conclude that Shh, secreted from MSCs, provides a proliferative stimulus for the gastric epithelium that is associated with tumor development, a response that is sustained by chronic inflammation.
Our aim was to conduct a pilot case-control study of RNA expression profile using RNA sequencing of rectosigmoid mucosa of 9 females with irritable bowel syndrome-diarrhea [IBS-D] with accelerated colonic transit and 9 female healthy controls. Mucosal total RNA was isolated, purified, and next generation pair-end sequencing was performed using Illumina TruSeq. Analysis was carried out using: a targeted approach towards 12 genes previously associated with IBS, and a hypothesis-generating approach. Of the 12 targeted genes tested, patients with IBS-D had decreased mRNA expression of TNFSF15 (fold change controls to IBS-D: 1.53, p=0.01). Overall, up- and downregulated mRNA expressions of 21 genes (p values 10-5 to 10-8; p values with FDR shown) were potentially relevant to IBS-D: neurotransmitters [P2RY4 (p=0.001), VIP (p=0.02)]; cytokines [CCL20 (p=0.019)]; immune function [C4BPA complement cascade (p=0.0187)], and IFIT3 [interferon-related, p=0.016]; mucosal repair and cell adhesion [TFF1 (trefoil protein, p= 0.012)], RBP2 (retinol binding protein, p=0.017), FN1 (fibronectin, p=0.009)]; and ion channel functions [GUCA2B (guanylate cyclase, p= 0.017), PDZD3 (PDZ domain-containing protein 3, p=0.029)]. Ten genes associated with functions related to pathobiology of IBS-D were validated by RT-PCR: There was significant correlation in fold changes of the selected genes (Rs=0.73, p=0.013). Up- or downregulation of P2RY4, GUC2AB, RBP2, FNI and C4BPA genes were confirmed on RT-PCR, which also revealed upregulation of FXR and ASBT. RNA seq and RT-PCR analysis of rectosigmoid mucosa in IBS-D show transcriptome changes that provide the rationale for validation studies to explore the role of mucosal factors in the pathobiology of IBS-D.
Flaxseed (FS) is a dietary oilseed containing a variety of anti-inflammatory bioactives including fermentable fiber, phenolic compounds (lignans), and the n3-polyunsaturated fatty acid (n3-PUFA) α-linolenic acid. The objective of this study was to determine the effects of FS, and its n3-PUFA-rich kernel or lignan- and soluble fibre-rich hull, on colitis severity in a mouse model of acute colonic inflammation. C57Bl/6 male mice were fed basal diet (NEG), or basal diet supplemented with 10% FS, 6% kernel, or 4% hull for 3 weeks prior to and during colitis induction via 5 day dextran sodium sulfate (DSS; 2% w/v) in their drinking water (n=12/group). Although consumption of all FS-based diets increased anti-inflammatory metabolites (hepatic n3-PUFAs, serum mammalian lignans, and cecal short chain fatty acids (SCFAs)), this was not associated with anti-inflammatory effects in DSS-exposed mice. In fact, dietary FS exacerbated DSS-induced acute colitis as indicated by a heightened disease activity index and increased colonic injury and inflammatory biomarkers (histological damage, apoptosis, myeloperoxidase, inflammatory cytokines (IL-6 and IL-1β), and NF-B signalling-related genes (Nfkb1, Ccl5, Bcl2a1a, Egfr, Relb, Birc3 and Atf1)). Additionally, the adverse effect of FS diet was extended systemically, as serum cytokines (IL-6, IFN-, and IL-1β) and hepatic cholesterol levels were increased. The adverse effects of FS were not associated with alterations in fecal microbial load or systemic bacterial translocation (endotoxemia). Collectively, this study demonstrates that although consumption of a 10% FS diet enhanced the levels of n3-PUFAs, SCFAs, and lignans in mice, it exacerbated DSS-induced colonic injury and inflammation.
The gastrointestinal (GI) tract of mammals is inhabited by trillions of microorganisms, resulting in exceedingly complex networking. The interaction between distinct bacterial species and the host immune system is essential in maintaining homeostasis in the gut ecosystem. For instance, the gut commensal microbiota dictates intestinal mucosa maturation and its abundant immune components, such as cytoprotective heat shock proteins (HSP). Here we examined physiological expression of HSP in the normal porcine GI tract and found it to be gut region- and cell type-specific in response to dietary components, microbes and microbial metabolites to which the mucosa surface is exposed. Correlations between HSP72 expression and ileal Lactobacillus spp. and colonic clostridia species, and between HSP27 expression and uronic acid ingestion, were important interplays identified here. Thus, this study provides novel insights into host-microbe interactions shaping the immune system that are modifiable by dietary regime.
PNPLA3 was recently associated with the susceptibility to nonalcoholic fatty liver disease, a common cause of chronic liver disease characterized by abnormal triglyceride accumulation. While it is established that PNPLA3 has both triacylglycerol lipase and acylglycerol O-acyltransferase activities, is still unknown whether the gene has any additional role in the modulation of the human liver metabolome. To uncover the functional role of PNPLA3 on liver metabolism, we performed high-throughput metabolic profiling of PNPLA3 siRNA-silencing and overexpression of wild type and mutant Ile148Met variants (isoleucine/methionine substitution at codon 148) in Huh-7 cells. Metabolomic analysis was performed using GC/MS and LC/MS platforms. Silencing of PNPLA3 was associated with a global perturbation of Huh-7 hepatoma cells that resembled a catabolic response associated with protein breakdown. A significant decrease in amino- and gamma-glutamyl-amino acids and dipetides, and a significant increase in cysteine sulfinic acid, myo-inositol, along with lysolipids, sphingolipids and poly-unsaturated fatty acids were observed. Overexpression of the PNPLA3 Met148 variant mirrored many of the metabolic changes observed during gene silencing, but in opposing direction. These findings were replicated by the exploration of canonical pathways associated with PNPLA3 silencing and Met148 overexpression. Overexpression of the PNPLA3 Met148 variant was associated with a 1.75-fold increase in lactic acid suggesting a shift to anaerobic metabolism and mitochondrial dysfunction. Together, these results suggest a critical role of PNPLA3 in the modulation of liver metabolism beyond its classical participation in triacylglycerol remodeling.
Engineering cancer cells to express heterologous antigen α-gal and induce the destruction of tumor cells depending on the complement cascade may be a promising strategy of tumor therapy. However, the feasibility and effect of using α-gal to induce colorectal adenocarcinoma cell lines cytolysis is not yet known. In this study, we evaluated α-gal expression's ability to sensitize human colorectal adenocarcinoma cell lines to complement attack in cell lines LoVo, SW620 and Ls-174T. Nearly all α-gal -expressing LoVo and SW620 cells were killed by human normal serum (NHS), but α-gal expression Ls-174T cells showed no significant lysis. We analyzed the expression levels of membrane-bound complement regulatory proteins (mCRPs) on the three cell lines, and their protective role in α-gal-mediated activation of the complement. LoVo show no expression of any of the three proteins. CD59 was strongly expressed by SW620 and Ls-174T. CD46 and CD55 varied between the two cell lines, CD46 on SW620 was only half the intensity of CD46 on Ls-174T. Ls-174T showed a notable expression of CD55, while expression of CD55 on SW620 was not detected. The sensitivity of Ls-174T expressing α-gal to NHS greatly increased following the down-regulation of CD46 and CD55 with shRNA. However, there is no increase in cell killing when CD59 expression was diminished. Our findings suggest that the use of α-gal as antigen to induce tumor cell killing may be a potential therapeutic strategy in colon cancer, and that CD55 plays a primary role in conferring resistance to lysis.
The intestinal epithelium forms a selective barrier maintained by tight junctions (TJs) and separating the luminal environment from the submucosal tissues. N-acylhomoserine lactone (AHL) quorum sensing molecules produced by Gram-negative bacteria in the gut can influence homeostasis of the host intestinal epithelium. In the present study, we evaluated the regulatory mechanisms affecting the impact of two representative long- and short-chain AHLs, N-3-(oxododecanoyl)-homoserine lactone (C12-HSL) and N-butylryl homoserine lactone (C4-HSL), on barrier function of human intestinal epithelial Caco-2 cells. Treatment with C12-HSL, but not with C4-HSL, perturbed Caco-2 barrier function; the effect that was associated with decreased levels of TJ proteins, occludin and tricellulin, and their delocalization from the TJs. C12-HSL induced also matrix metalloprotease (MMP)-2 and MMP-3 activation via lipid raft- and protease-activated receptor (PAR)-dependent signaling. Pretreatment with lipid raft disruptors, PAR antagonists, or MMP inhibitors restored the C12-HSL-induced loss of the TJ proteins and increased permeability of Caco-2 cell monolayers. These results indicate that PAR/lipid raft-dependent MMP-2 and -3 activation, followed by degradation of occludin and tricellulin are involved in C12-HSL-induced alterations of epithelial paracellular barrier functions.
Introduction. Pancreatic polypeptide (PP) is an anorexigenic hormone released from pancreatic F-cells upon food intake. We aimed to determine the effect of PP on gastric accommodation and gastric emptying in conscious Wistar HAN rats to investigate whether effects on motor function could contribute to its anorexigenic effects. Methods. Intragastric pressure (IGP) was measured through a chronically implanted gastric fistula during the infusion of a nutrient meal (Nutridrink; 0.5 ml min-1). Rats were treated with PP (0, 33 and 100 pmol kg-1 min-1) in combination with L-NG-arginine methyl ester (L-NAME; 180 mg kg-1 h-1), atropine (3 mg kg-1 h-1) or vehicle. Furthermore, the effect of PP was tested after subdiaphragmal vagotomy of the stomach. Gastric emptying of a non-caloric and a caloric meal after treatment with 100 pmol kg-1 min-1 PP or vehicle was compared using X-rays. Results. PP significantly increased IGP during nutrient infusion compared to vehicle (p<0.01). L-NAME and atropine significantly increased IGP during nutrient infusion as compared to vehicle treatment (p<0.005 and <0.01 respectively). The effect of PP on IGP during nutrient infusion was abolished in the presence of L-NAME and in the presence of atropine. In vagotomized rats, PP increased IGP compared to intact controls (p<0.05). PP significantly delayed gastric emptying of both a non-caloric (p<0.05) and a caloric meal (p<0.005). Conclusion. PP inhibits gastric accommodation and delays gastric emptying, probably through inhibition of nitric oxide release. These results indicate that, besides the well-known centrally-mediated effects, PP might decrease food intake through peripheral mechanisms.
Purpose: We previously demonstrated increased villus height following Retinoblastoma protein (Rb) deletion in the intestinal epithelium (Rb-IKO). Here we determined the functional consequences of augmented mucosal growth on intestinal fat absorption. Methods: Mice with constitutively disrupted Rb expression in the intestinal epithelium (Rb-IKO) along with their floxed (WT) littermates were placed on a high fat diet (HFD, 42% kcal fat). Mice were weighed weekly, and fat absorption determined, along with indirect calorimetry and MRI body composition. Rb-IKO mice were also subjected to small bowel resections (SBR), followed by HFD feeding. In separate experiments, we examined intestinal fat absorption in mice with conditional (tamoxifen inducible) intestinal Rb (inducible Rb-IKO) deletion. Results: Constitutive Rb-IKO mice demonstrated greater mucosal surface area, yet manifested paradoxically impaired intestinal long chain triglyceride absorption and decreased cholesterol absorption. Despite attenuated lipid absorption, there were no differences in metabolic rate, body composition, and weight gain in Rb-IKO and WT mice at baseline as well as following SBR. We also confirmed fat malabsorption in inducible Rb-IKO mice. Conclusion: Despite an expanded mucosal surface area, Rb-IKO mice demonstrate impaired lipid absorption without compensatory alterations in energy homeostasis or body composition. These findings underscore the importance of delineating structural/functional relationships in the gut and suggest a previously unknown role for Rb in the regulation of intestinal lipid absorption.
Background & Aims: Internal anal sphincter (IAS) tone plays an important role in rectoanal incontinence (RI). The IAS tone may be compromised during aging leading to RI in certain patients. Herein, we examined the influence of oxidative stress in the aging-associated decrease in the IAS tone (AADI). Methods: Using two different age groups of adult (4- 6 months (Ad)) vs. aging (24 to 30 months (Ag)) rats, we determined the effect of oxidative stress on IAS tone, and the regulatory RhoA/ROCK signal transduction cascade. We determined the effect of an oxidative stress inducer LY83583 (that produces superoxide anions or O2-), on the basal and stimulated IAS tone, before and after O2- scavenger superoxide dismutase (SOD) using intact smooth muscle (SM) strips and SMCs. Results: Data showed that AADI was associated with decrease in RhoA/ROCK expression at the transcriptional and translational levels. Oxidative stress with LY83583-mediated decrease in the IAS tone and relaxation of the IAS SMCs were associated with the decrease in RhoA/ROCK signal transduction, reversible by SOD. In addition, LY83583 caused a significant decrease in the IAS contraction produced by the RhoA activator, and a known RhoA/ROCK agonist U46619, also reversible by SOD. The inhibitory effects of LY83583 and ROCK inhibitor Y27632 on U46619-induced increase in the IAS tone were similar. Conclusion: An increase in oxidative stress plays an important role in the AADI in the elderly, and may be one of the underlying mechanisms for the RI in certain aging patients.
Extracellular acidification has been observed in allergic inflammatory diseases. Recently, we demonstrated that the proton-sensing receptor GPR65 regulates eosinophil survival in an acidic environment in vitro and eosinophil accumulation in an allergic lung inflammation model. For mast cells, another inflammatory cell type critical for allergic responses, it remains unknown whether GPR65 is expressed and/or regulates mast cell viability. Thus, in the present study, we employed in vitro experiments and an intestinal anaphylaxis model in which both mastocytosis and eosinophilia can be observed, particularly in the gastrointestinal tract, to enable us to directly compare the effect of GPR65 expression on these two cell types. We identified GPR65 expression on mast cells; however, unlike eosinophil viability, mast cell viability in vitro is not affected by acidification or GPR65 expression. Mechanistically, we determined that mast cells do not respond to extracellular acidification with increased cAMP levels. Further, in the intestinal anaphylaxis model, we observed a significant reduction of eosinophils (59.1% ± 9.2% decrease) in the jejunum of allergen-challenged GPR65-deficient mice compared with allergen-challenged wild type (WT) mice, despite the degree of antigen sensitization and the expression levels of Th2 cytokines (Il4, Il13) and eosinophil chemokines (Ccl11, Ccl24) in the jejunum being comparable. In contrast, the accumulation of mast cells in allergen-challenged mice was not affected by GPR65 deficiency. In conclusion, our study demonstrates differential regulation of eosinophils and mast cells in inflammatory tissue, with mast cell viability and accumulation being independent of GPR65.
Pharmacotherapy based on 5-aminosalicylic acid (5-ASA) is a preferred treatment for ulcerative colitis, but variable patient response to this therapy is observed. Inflammation can affect therapeutic outcomes by regulating the expression and activity of drug-metabolizing enzymes; its effect on 5-ASA metabolism by the colonic arylamine N-acetyltransferase (NAT) enzyme isoforms is not firmly established. We examined if inflammation affects the capacity for colonic 5-ASA metabolism and NAT enzyme expression. 5-ASA metabolism by colonic mucosal homogenates was directly measured with a novel fluorimetric rate assay. 5-ASA metabolism reported by the assay was dependent on Ac-CoA, inhibited by alternative NAT substrates (isoniazid, p-aminobenzoylglutamate [p-ABG]), and saturable with Km (5-ASA) = 5.8 µM. A mouse model of acute dextran sulfate sodium (DSS) colitis caused pronounced inflammation in central and distal colon, and modest inflammation of proximal colon, defined by myeloperoxidase activity and histology. DSS colitis reduced capacity for 5-ASA metabolism in central and distal colon segments by 52% and 51%, respectively. Use of selective substrates of NAT isoforms to inhibit 5-ASA metabolism suggested that mNAT2 mediated 5-ASA metabolism in normal and colitis conditions. Western blot and real-time RT-PCR identified that proximal and distal mucosa had a decreased mNAT2 protein/mRNA ratio after DSS. In conclusion, an acute colonic inflammation impairs the expression and function of mNAT2 enzyme, thereby diminishing the capacity for 5-ASA metabolism by colonic mucosa.
Hepatosteatosis, the ectopic accumulation of lipid in the liver, is one of the earliest clinical signs of alcoholic liver disease (ALD). Alcohol-dependent deregulation of liver ceramide levels as well as inhibition of AMPK and PPARα activity are thought to contribute to hepatosteatosis development. Adiponectin can regulate lipid handling in the liver, and has been shown to reduce ceramide levels and activate AMPK and PPARα. However, the mechanisms by which adiponectin prevents alcoholic hepatosteatosis remain incompletely characterized. To address this question, we assessed ALD progression in wild-type (WT) and adiponectin knockout (KO) mice fed an ethanol containing liquid diet or isocaloric control diet. Adiponectin KO mice relative to WT had increased alcohol-induced hepatosteatosis and hepatomegaly, similar modest increases in serum ALT and reduced liver TNFα. Restoring circulating adiponectin levels using recombinant adiponectin ameliorated alcohol-induced hepatosteatosis and hepatomegaly in adiponectin KO mice. Alcohol-fed WT and adiponectin KO animals had equivalent reductions in AMPK protein and PPARα DNA binding activity compared to control-fed animals. No difference in P-AMPK/AMPK ratio was detected, suggesting alcohol-dependent deregulation of AMPK and PPARα in the absence of adiponectin are not primary causes of the observed increase in hepatosteatosis in these animals. By contrast, alcohol treatment increased liver ceramide levels in adiponectin KO but not WT mice. Importantly, pharmacological inhibition of de novo ceramide synthesis in adiponectin KO mice abrogated alcohol-mediated increases in liver ceramides, steatosis and hepatomegaly. This data suggests adiponectin reduces alcohol-induced steatosis and hepatomegaly through regulation of liver ceramides but does not prevent alcohol-induced liver damage.
Inflammatory bowel diseases (IBD) are chronic inflammatory disorders of the intestine that result in painful and debilitating complications. Currently no cure exists for IBD and treatments are primarily aimed at reducing inflammation to alleviate symptoms. Genome-wide linkage studies have identified the Ron receptor tyrosine kinase and its ligand, hepatocyte growth factor-like protein (HGFL), as genes highly associated with IBD. However, only scant information exists on the role of Ron or HGFL in IBD. Based on the linkage of Ron to IBD, we directly examined the biological role of Ron in colitis. Wild type mice and mice lacking the tyrosine kinase signaling domain of Ron (TK-/- mice) were utilized in a well-characterized model of chronic colitis induced by cyclic exposure to dextran sulfate sodium. In this model, TK-/- mice were more susceptible to injury as judged by increased mortality compared to control mice and developed more severe colitis. Loss of Ron led to significantly reduced body weights and more aggressive clinical and histopathologies. Ron loss also resulted in a dramatic reduction in colonic epithelial cell proliferation and increased proinflammatory cytokine production, which was associated with alterations in important signaling pathways known to regulate IBD. Examination of human gene expression data further supports the contention that loss of Ron signaling is associated with IBD. In total, our studies point to important functional roles for Ron in IBD by regulating healing of the colonic epithelium and by controlling cytokine secretion.
Necrotizing enterocolitis (NEC) is the leading cause of death from gastrointestinal disease in premature infants, and develops partly from an exaggerated intestinal epithelial immune response to indigenous microbes. There has been interest in administering probiotic bacteria to reduce NEC severity, yet concerns exist regarding infection risk. Mechanisms of probiotic activity in NEC are unknown, although activation of the microbial DNA receptor Toll-like receptor-9 (TLR9) has been postulated. We now hypothesize that the Gram-positive bacterium Lactobacillus rhamnosus HN001 can attenuate NEC in small and large animal models, that its microbial DNA is sufficient for its protective effects, and that protection requires activation of the Toll-like receptor-9 (TLR9). We now show that oral administration of live or UV-inactivated Lactobacillus rhamnosus HN001 attenuates NEC severity in newborn mice and premature piglets, as manifest by reduced histology score, attenuation of mucosal cytokine response, and improved gross morphology. TLR9 was required for Lactobacillus rhamnosus-mediated protection against NEC in mice, as the selective decrease of TLR9 from the intestinal epithelium reversed its protective effects. Strikingly, DNA of Lactobacillus rhamnosus HN001 (Lr-DNA) reduced the extent of pro-inflammatory signaling in cultured enterocytes and in samples of resected human ileum ex vivo, suggesting the therapeutic potential of this probiotic in clinical NEC. Taken together, these findings illustrate that Lactobacillus rhamnosus HN001 is an effective probiotic for NEC via activation of the innate immune receptor TLR9, and that Lactobacillus rhamnosus DNA is sufficient for its protective effects, potentially reducing concerns regarding the infectious risk of this novel therapeutic approach.
The intestinal microbiota plays a pivotal role in maintaining human health and well-being. Previously, we have shown that mice deficient in the brush-border enzyme intestinal alkaline phosphatase (IAP) suffer from dysbiosis and that oral IAP supplementation normalizes the gut flora. Here we aimed to decipher the molecular mechanism by which IAP promotes bacterial growth. We used an isolated mouse intestinal loop model to directly examine the effect of exogenous IAP on the growth of specific intestinal bacterial species. We studied the effects of various IAP targets on the growth of stool aerobic and anaerobic bacteria as well as on a few specific gut organisms. We determined the effects of ATP and other nucleotides on bacterial growth. Further, we examined the effects of IAP on reversing the inhibitory effects of nucleotides on bacterial growth. We have confirmed that local IAP bioactivity creates a luminal environment that promotes the growth of a wide range of commensal organisms. IAP promotes the growth of stool aerobic and anaerobic bacteria and appears to exert its growth promoting effects by inactivating (dephosphorylating) luminal ATP and other luminal nucleotide triphosphates. We observed that compared to wild-type mice, IAP-knockout mice have more ATP in their luminal contents, and exogenous IAP can reverse the ATP-mediated inhibition of bacterial growth in the isolated intestinal loop. In conclusion, IAP appears to promote the growth of intestinal commensal bacteria by inhibiting the concentration of luminal nucleotide triphosphates.
The intestinal epithelium is subjected to various types of mechanical stress. In this study, we investigated the impact of cyclic stretch on tight junction and adherens junction integrity in Caco-2 cell monolayers. Stretch for 2 hours resulted in dramatic modulation of tight junction protein distribution from linear organization into wavy structure. Continuation of cyclic stretch for 6 hours led to redistribution of tight junction proteins from the intercellular junctions into intracellular compartment. Disruption of tight junctions was associated with redistribution of adherens junction proteins, E-cadherin and β-catenin, and dissociation of the actin cytoskeleton at the actomyosin belt. Stretch activates JNK2, c-Src and MLCK. Inhibition of JNK, Src kinase or MLCK activity and knockdown of JNK2 or c-Src attenuated stretch-induced disruption of tight junctions, adherens junctions and actin cytoskeleton. Stretch increased tyrosine phosphorylation of occludin, ZO-1, E-cadherin and β-catenin. Inhibition of JNK or Src kinase attenuated stretch-induced occludin phosphorylation. Immunofluorescence localization indicated that phospho-MLC co-localizes with the vesicle-like actin structure at the actomyosin belt in stretched cells. On the other hand, phospho-c-Src co-localizes with the actin at the apical region of cells. This study demonstrates that cyclic stretch disrupts tight junctions and adherens junctions by a JNK2, c-Src and MLCK-dependent mechanism.
Proton pump inhibitors reduce gastric acid secretion and are commonly utilized in the management of gastroesophageal reflux disease (GERD) across all ages. Yet a decrease in lower esophageal sphincter tone has been reported in vitro in rats through an unknown mechanism, however their effect on the gastroesophageal muscle tone early in life was never studied. Hypothesizing that proton pump inhibitors also reduce gastroesophageal muscle contraction in newborn and juvenile rats, we evaluated the in vitro effect of pantoprazole on gastric and lower esophageal sphincter muscle tissue. Electrical field stimulation (EFS) and carbachol-induced force were significantly (P<0.01) reduced in the presence of pantoprazole, whereas the drug had no effect on the neuromuscular-dependent relaxation. When administered in vivo, pantoprazole (9 mg/kg) significantly (P<0.01) reduced gastric emptying time at both ages. In order to ascertain the signal transduction pathway responsible for the reduction in muscle contraction, we evaluated the tissue rho-associated kinase 2 (ROCK-2) and CPI-17 activity. Pantoprazole reduced myosin light chain phosphatase MYPT-1, but not CPI-17 phosphorylation of gastric and lower esophageal sphincter tissue strongly suggesting that it is a ROCK-2 inhibitor. To the extent that these findings can be extrapolated to human neonates, the use of pantoprazole may impair gastric and lower sphincter muscle tone and thus paradoxically exacerbate esophageal reflux. Further studies addressing the effect of proton pump inhibitors on gastroesophageal muscle contraction are warranted to justify its therapeutic use in GERD.
Impaired insulin receptor (IR) activity has been found in various models of insulin resistance, including models of injury or critical illness, and Type 2 diabetes. However, mechanisms that modulate IR function remain unclear. With an animal model of critical illness diabetes, we found insulin-induced IR tyrosine phosphorylation in the liver was impaired as early as 15 minutes following trauma and hemorrhage. Possible mechanisms for this defect were examined, including IR protein levels and IR post-translational modifications. The total amounts of hepatic IR α and β subunits and the membrane localization of the IR were not altered by trauma and hemorrhage, and, likewise, no change in IR tyrosine nitration was found in the liver. However, there was a decrease in the level of protein O-linked beta-N-acetlyglucosamine (O-GlcNac) modification on Ser/Thr in the liver following trauma and hemorrhage. Inhibition of c-jun N-terminal kinase (JNK) increased IR O-GlcNac modification implicating an involvement of JNK. These findings suggest that a balance between O-GlcNac modification and JNK-induced phosphorylation may exist, with decreased Ser/Thr O-GlcNac modification following trauma and hemorrhage, allowing JNK to phosphorylate the IR on neighboring Ser/Thr residues, which subsequently inhibits IR activity. The present studies suggest potential mechanisms of hemorrhage-induced defects in IR activity, and a potential role for acutely decreased O-GlcNac and increased serine phosphorylation of the IR.
Alcohol abuse with/without cirrhosis is associated with an impaired gut barrier and inflammation. Gut microbiota can transform primary bile acids(BA) to secondary BAs which can adversely impact the gut barrier. Aim: Define the effect of active alcohol intake on fecal BA levels, ileal & colonic inflammation in cirrhosis. Methods: Five age-matched groups; two non-cirrhotic (control and drinkers) & three cirrhotic [(NAlc:non-alcoholic (non-drinkers),AbsAlc: abstinent alcoholic for >3mths & CurrAlc:(currently drinking)] were included. Fecal and serum BA analysis, serum endotoxin and stool microbiota using pyro-sequencing were performed. A subgroup of controls, NAlc and CurrAlc underwent ileal and sigmoid colonic biopsies on which mRNA expression of TNF-α, IL1β,IL6 and Cox-2 were performed. Results: 103 patients (19 healthy, 6 non-cirrhotic drinkers, 10 CurrAlc, 38 AbsAlc and 30 NAlc, age 56 yrs, median MELD:10.5) were included. Five each of healthy, CurrrAlc and NAlc underwent ileal/colonic biopsies. Endotoxin, serum conjugated DCA and stool total BAs and secondary/primary BA ratios were highest in current drinkers. On biopsies, a significantly higher mRNA expression of TNF-α, IL1β,IL6 and Cox-2 in colon but not ileum was seen in CurrAlc compared to NAlc & controls. Conclusion: Active alcohol use in cirrhosis is associated with a significant increase in the secondary BA formation compared to abstinent alcoholic cirrhotics and non-alcoholic cirrhotics. This increase in secondary BAs is associated with a significant increase in expression of inflammatory cytokines in colonic mucosa but not ileal mucosa, which may contribute to alcohol-induced gut barrier injury.
Mechanisms whereby acid reflux may accelerate the progression from Barrett's esophagus (BE) to esophageal adenocarcinoma (EA) are not fully understood. Acid and reactive oxygen species (ROS) have been reported to cause DNA damage in Barrett's cells. We have previously shown that NADPH oxidase NOX5-S is responsible for acid-induced H2O2 production in Barrett's cells and in EA cells. In this study we examined the role of intracellular calcium and NADPH oxidase NOX5-S in acid-induced DNA damage in a Barrett's EA cell line FLO and a Barrett's cell line CP-A. We found that pulsed acid treatment significantly increased tail moment in FLO and CP-A cells and histone H2AX phosphorylation in FLO cells. In addition, acid treatment significantly increased intracellular Ca2+ in FLO cells, an increase which is blocked by Ca2+ free medium with EGTA and thapsigargin. Acid-induced increase in tail moment was significantly decreased by NADPH oxidase inhibitor diphenylene iodonium in FLO cells, and by blockade of intracellular Ca2+ increase or knockdown of NOX5-S with NOX5 siRNA in FLO and CP-A cells. Acid-induced increase in histone H2AX phosphorylation was significantly decreased by NOX5 siRNA in FLO cells. Conversely, overexpression of NOX5-S significantly increased tail moment and histone H2AX phosphorylation in FLO cells. We conclude that pulsed acid treatment causes DNA damage via increase of intracellular calcium and activation of NOX5-S. It is possible that in Barrett's esophagus acid reflux increases intracellular calcium, activates NOX5-S and increases ROS production, which causes DNA damage, thereby contributing to the progression from BE to EA.
Fibroblast growth factor (FGF) 15 (human homologue, FGF19) is an endocrine FGF highly expressed in the small intestine of mice. Emerging evidence suggests that FGF15 is critical for regulating hepatic functions; however, the role of FGF15 in liver regeneration is unclear. This study assessed whether liver regeneration is altered in FGF15 KO mice following 2/3 partial hepatectomy (PHx). The results showed that FGF15 KO mice had marked mortality, with the survival rate influenced by genetic background. Compared to WT mice, the KO mice displayed extensive liver necrosis and marked elevation of serum bile acids and bilirubin. Furthermore, hepatocyte proliferation was reduced in the KO mice due to impaired cell cycle progression. After PHx, the KO mice had weaker activation of signaling pathways that are important for liver regeneration, including STAT3, NF-B and MAPK. Examination of the KO mice at early time points after PHx revealed a reduced and/or delayed induction of immediate-early response genes, including growth-control transcription factors that are critical for liver regeneration. In conclusion, the results suggest that FGF15 deficiency severely impairs liver regeneration in mice after PHx. The underlying mechanism is likely due to disrupted bile acid homeostasis and impaired priming of hepatocyte proliferation.
The leucine-to-glutamate(Leu-Glu)pathway, which metabolizes the carbon atoms of L-leucine to form L-glutamate, was studied by incubating rat tissue segments with L-[U-14C]leucine and estimating the [14C]glutamate formed. Metabolism of the leucine carbon chain occurs in most rat tissues, but maximal activity of the Leu->Glu for glutamate formation is limited to the thoracic aorta and pancreas. In rat aorta the Leu->Glu functions to relax the underlying smooth muscle; its functions in pancreas are unknown. This report characterizes the Leu->Glu of rat pancreas and develops methods to examine its functions. Pancreatic segments effect net formation of glutamate on incubation with L-leucine, L-glutamine or a mix of 18 other plasma amino acids at their concentrations in rat plasma. Glutamate formed from leucine remains mainly in the tissue, whereas that from glutamine enters the medium. The pancreatic Leu->Glu pathway uses the leucine carbons for net glutamate formation, the alpha-amino group is not used; the stoichiometry is 1 mole of leucine yields 2 moles of glutamate (2 leucine carbons per glutamate) plus 2 moles of CO2. Comparison of the Leu->Glu pathway in preparations of whole pancreatic segments, isolated acini, and islets of Langerhans localize it in the acini; relatively high activity is found in cultures of the AR42J cell line and very little in the INS-1 832/13 cell line. Pancreatic tissue glutamate concentration is homeostatically regulated in the range of 1-3 umol/g wet weight. L-valine and leucine ethyl, -benzyl, and -ter-butyl esters inhibit the Leu->Glu without decreasing total tissue glutamate.
Background & Aims: Substance P (SP) is commonly co-expressed with acetylcholine in enteric motor neurons and according to the classical paradigm, both these neurotransmitters excite smooth muscle via parallel pathways. We hypothesized that in addition, SP was responsible for maintaining the muscular responsiveness to acetylcholine (ACh). We tested this hypothesis by using botulinum toxin (BoNT/A), a known blocker of vesicular release of neurotransmitters including acetylcholine and neuropeptides. Methods: BoNT/A was injected into rat pyloric sphincter in different doses; as control we used boiled BoNT/A. At the desired time point, the rats were sacrificed and pyloric contractility was measured ex vivo in an organ bath and by measuring phosphorylation of myosin light chain 20 (MLC20). Results: BoNT/A (10 IU) significantly reduced the response of pyloric muscle to exogenous ACh, an effect that was accompanied by reduced MLC20 phopshorylation in the muscle. Both effects were reversed by exogenous SP. CP-96345, a NK1 receptor antagonist, blocked the ability of exogenous SP to reverse the cholinergic hyporesponsiveness as well as the reduction in MLC20 phosphorylation induced by BoNT/A. Conclusion: We have identified a novel role for SP as a co-neurotransmitter that appears to be important for the maintenance of muscular responsiveness to the principal excitatory neurotransmitter, acetylcholine. These results also provide new insight into the effects of botulinum toxin on the enteric nervous system and gastrointestinal smooth muscle.
Pancreatic cancer is a devastating disease with a survival rate of less than 5%. Moreover, pancreatic cancer aggressiveness is closely related to high levels of pro-survival mediators, which can ultimately lead to rapid disease progression. One of mechanisms enabling tumor cells to evade cellular stress and promote unhindered proliferation is the ER stress response. Disturbances in the normal functions of the ER lead to an evolutionarily conserved cell stress response, the unfolded protein response (UPR). The UPR initially compensates for damage but eventually triggers cell death if ER dysfunction is severe or prolonged. Triptolide, a diterpene triepoxide has been shown to be an effective compound against pancreatic cancer. Our results show that triptolide induces unfolded protein response (UPR) by activating the PERK-eIF2α axis and the Ire1α-Xbp-1axis of unfolded protein response and leads to chronic ER stress in pancreatic cancer. Our results further show that GRP78, one of the major regulators of ER stress is downregulated by triptolide leading to cell death by apoptosis in MIA-PaCa2 cells and autophagy in S2-VP10 cells.
The intermediate-conductance Ca2+-activated K+- channel KCa3.1/KCNN4 plays an important role in the modulation of Ca2+ signaling through the control of the membrane potential in T-lymphocytes. Here, we study the involvement of KCa3.1 in the enlargement of the mesenteric lymph nodes (MLNs) in mouse model of inflammatory bowel disease (IBD). The mouse model of IBD was prepared by exposing male C57BL/6J mice to 5 % dextran sulfate sodium for 7 days. Inflammation-induced changes in KCa3.1 activity and the expressions of KCa3.1 and its regulators in MLN CD4+ T-lymphocytes were monitored by real-time PCR, Western blot, voltage-sensitive dye imaging, patch-clamp and flow cytometric analyses. Concomitant with an up-regulation of KCa3.1a and NDPK-B, a positive KCa3.1 regulator, an increase in KCa3.1 activity was observed in MLN CD4+ T-lymphocytes in the IBD model. Pharmacological blockade of KCa3.1 elicited the following results. 1) A significant decrease in IBD disease severity, as assessed by diarrhea, visible fecal blood, inflammation and crypt damage of the colon and MLN enlargement compared with control mice, and 2) the restoration of the expression levels of KCa3.1a, NDPK-B, and Th1 cytokines in IBD model MLN CD4+ T-lymphocytes. These findings suggest that the increase in KCa3.1 activity induced by the up-regulation of KCa3.1a and NDPK-B may be involved in the pathogenesis of IBD by mediating the enhancement of the proliferative response in MLN CD4+ T-lymphocyte and therefore, that the pharmacological blockade of KCa3.1 may decrease the risk of IBD.
A well-developed myenteric plexus exists in the esophagus composed of striated muscle layers, but its functional role in controlling peristaltic movements remains to be clarified. The purpose of this study was to clarify the role of a local neural reflex consisting of capsaicin-sensitive primary afferent neurons and intrinsic neurons in esophageal peristalsis. We firstly devised a method to measure peristaltic movement of esophagus in vivo in rats. Rats were anesthetized with urethane, and esophageal intraluminal pressure and propelled intraluminal liquid volume were recorded. In the experimental system, an intraluminal pressure stimulus evoked periodic changes in intraluminal pressure of the esophagus, which were consistently accompanied by intraluminal liquid propulsion. Bilateral vagotomy abolished changes in intraluminal pressure as well as liquid propulsion. These results indicate that the novel method is appropriate for inducing peristalsis in the esophagus composed of striated muscles. Then, by using the method, we examined functional roles of the local reflex in esophageal peristalsis. For that purpose, we used rats in which capsaicin-sensitive neurons had been destroyed. The esophagus of capsaicin-treated rats showed a multi-phasic rise in intraluminal pressure, which may due to non-coordinated contractions of esophageal muscles, whereas a mono-phasic response was observed in the intact rat esophagus. In addition, destruction of capsaicin-sensitive neurons increased the propelled liquid volume and lowered the pressure threshold for initiating peristalsis. These results suggest that the local neural reflex consisting of capsaicin-sensitive neurons and intrinsic neurons contributes to coordination of peristalsis and suppresses mechanosensory function of vagal afferents in the esophagus.
We previously showed that dietary sphingomyelin (SM) inhibited cholesterol absorption in animals. The key enzyme hydrolyzing SM in the gut is alkaline sphingomyelinase (alk-SMase, NPP7). Here using fecal dual-isotope ratio method we compared cholesterol absorption in the wild type (WT) and alk-SMase knockout (KO) mice. The animals were fed an emulsion containing 14C-cholesterol and 3H-sitosterol. The radioactivities in the lipids of the fecal samples collected 4, 8 and 24 h thereafter were determined and the ratio of 14C/3H calculated. We found that the fecal 14C-cholesterol recovery in the KO mice was significantly higher than in the WT mice. Maximal 92% increase occurred 8 h after feeding. Recovery of 3H-sitosterol did not differ between the two groups. Accordingly the 14C/3H ratio of fecal lipids was 133% higher at 8 h and 75% higher at 24 h in the KO than in the WT mice. Decreased 14C-cholesterol was also found in the serum of the KO mice 4 h after feeding. Supplement of SM in the emulsion reduced the differences in fecal 14C-cholesterol recovery between the WT and KO mice due to a greater increase of 14C-cholesterol recovery in the WT mice. Without treatment the KO mice had significantly higher SM levels in the intestinal content and feces, but not in the intestinal mucosa or serum. The expression of NPC1L1 in the small intestine was not changed. In conclusion, alk-SMase is a physiological factor promoting cholesterol absorption by reducing SM levels in the intestinal lumen.
Vascular endothelial growth factor (VEGF) is crucial for vascular development in several organs. However, the specific contribution of epithelial-VEGF signaling in the liver has not been tested. We utilized a mouse model to specifically delete Vegf from the liver epithelial lineages during mid-gestational development and assessed the cell identities and architectures of epithelial and endothelial tissues. We find that without epithelial-derived VEGF, the zonal endothelial and hepatocyte cell identities are altered. We also find decreased portal vein and hepatic artery branching coincident with an increase in hepatic hypoxia postnatally. Together, these data indicate that VEGF secreted from the hepatic epithelium is required for normal differentiation of cells, and establishment of 3-dimensional vascular branching and zonal architectures in both epithelial and endothelial hepatic tissues.
Mirtazapine is a noradrenergic and specific serotonergic antidepressant. The aim of this study was to investigate the effects of mirtazapine on gastrointestinal motility in dogs, including solid gastric emptying, antral and small intestinal contractions, small intestinal and colonic transit. Methods: Six dogs were implanted with two cannulas located at the duodenum and the ascending colon; Another 6 dogs were implanted with gastric cannula 6cm proximal to the pylorus. Mirtazapine 45mg was administered orally 90 minutes before the study. Results: 1). Mirtazapine accelerated gastric emptying during the entire 3 hours in normal dogs (P < 0.04) and accelerated delayed gastric emptying induced by rectal distention (P < 0.04). 2). Mirtazapine restored impaired gastric tone and accommodation induced by rectal distention (P < 0.05) 3) No significant changes were noted in small intestinal contractions or transit with mirtazapine (P > 0.1). 4). Mirtazapine accelerated colonic transit at 2 and 4 hours but not 6 hours. The geometric center (GC) was increased from 1.9 ± 0.6 to 3.0 ± 0.5, 3.9 ± 0.5 to 4.7 ± 0.1 at 2 and 4 hours respectively (P = 0.04 vs. corresponding control). Conclusions: Mirtazapine improves gastric emptying in healthy dogs and normalizes rectal distention-induced delay in gastric emptying and accelerates colon but not small intestinal transit in healthy dogs. Clinical studies are warranted to assess the effects of mirtazapine on gastrointestinal motility and sensory functions in patients with functional GI diseases.
Necrotizing enterocolitis (NEC) is associated with a high morbidity and mortality in very low birth weight infants. Several hypotheses regarding the pathogenesis of NEC have been proposed but to date no effective treatment is available. Previous studies suggest that probiotic supplementation is protective. We recently reported that probiotic (Bifidobacterium infantis) conditioned media (PCM) has an anti-inflammatory effect in cultured fetal human intestinal cells (H4) and fetal intestine explants. In this study, we tested in vivo if PCM protects neonatal mice from developing intestinal inflammation induced by exposure to Cronobacter sakazakii (C. sakazakii), an opportunistic pathogen associated with NEC. We found that infected neonatal mice had a significantly lower body weight than control groups. Infection led to ileal tissue damage including villous rupture, disruption of epithelial cell alignment, intestinal inflammation, apoptotic cell loss and decreased mucus production. Pre-treatment with PCM prevented infection decrease in body weight, attenuated enterocyte apoptotic cell death, mitigated reduced mucin production and maintained ileal structural . Infected ileum expressed reduced levels of IBα which could be restored upon pretreatment with PCM. We also observed a nuclear translocation of NF-B p65 in H4 cells exposed to, which was prevented in PCM pretreated cells. Finally, treatment of neonatal mice with PCM prior to infection sustained the capacity of ileal epithelial proliferation. This suggests that an active component(s) released into the culture media by Bifidobacterium infantis may prevent ileal damage by a pathogen linked to NEC.
Communication between neurons and glia in the dorsal root ganglia (DRG) and the central nervous system (CNS) is critical for nociception. Both Glial activation and proinflammatory cytokine induction underlie this communication. We investigated whether satellite glial cell (SGCs) and tumor necrosis factor alpha (TNF-α) activation in DRG participate in a 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced rat model of visceral hyperalgesia. In TNBS-treated rats, TNF-α expression increased in DRG and was colocalized to SGCs enveloping a given neuron. These SGCs were activated as visualized under electron microscopy: they had more elongated processes projecting into the connective tissue space and more gap junctions. When nerves attached to DRG (L6-S1) were stimulated with a series of electrical stimulations, TNF-α were released from DRG in TNBS treated animals compared to controls. Using a current clamp, we noted that exogenous TNF-α (2.5 ng/ml) increased DRG neuron activity and visceral pain behavioral responses were reversed by intrathecal administration of anti-TNF-α (10μg/kg per day). Based on our findings, TNF-α and SGC activation in neuron-glial communication of DRG and are critical in inflammatory visceral hyperalgesia.
Nausea is the subjective unpleasant sensation that immediately precedes vomiting. Studies using barostats suggest that gastric fundus and lower esophageal sphincter (LES) relaxation precede vomiting. Unlike barostat, high-resolution manometry (HRM) allows less invasive, detailed measurements of fundus pressure (FP) and axial movement of the gastro-esophageal junction (GEJ). Nausea was induced in 12 healthy volunteers by a motion video and rated on a visual analogue scale. FP was measured as the mean value of the five pressure channels that were clearly positioned below the LES. After intubation, a baseline (BL) recording of 15 min was obtained. This was followed by presentation of the motion video (at least 10 min, max. 20 min) followed by 30min recovery recording. Throughout the experiment we recorded autonomic nervous system (ANS) parameters (blood pressure, heart rate (HR) and cardiac vagal tone (CVT) which reflects efferent vagal activity). 10/12 subjects showed a drop in FP during peak nausea compared to BL (-4.0±0.8mmHg; p=0.005) and8/10 subjects showed a drop in LES pressure (-8.8±2.5mmHg; p=0.04). Peak nausea preceded peak fundus and LES pressure drop.Nausea was associated with configuration changes at the GEJ such as LES shortening and esophageal lengthening. During nausea we observed a significantly increased HR and decreased CVT. In conclusion, nausea is associated with a drop in fundus and LES pressure, configuration changes at the GEJ as well as changes in the ANS activity such as an increased sympathetic tone (increased HR) and decreased parasympathetic tone (decreased CVT).
BACKGROUND: Substance P (SP) promotes cholangiocyte growth during cholestasis by activating its receptor, NK1R. SP is a proteolytic product of tachykinin (Tac1) and is deactivated by membrane metalloendopeptidase (MME). AIM: To evaluate the functional role of SP in the regulation of cholangiocarcinoma (CCA) growth. METHODS: NK1R, Tac1 and MME expression and SP secretion were assessed in human CCA cells and non-malignant cholangiocytes. The proliferative effects of SP (in the absence/presence of the NK1R inhibitor, L-733,060) and L-733,060 were evaluated. In vivo, the effect of L-733,060 treatment or MME overexpression on tumor growth was evaluated using a xenograft model of CCA in nu/nu nude mice. The expression of Tac1, MME, NK1R, PCNA, CK-19, and VEGF-A was analyzed in the resulting tumors. RESULTS: Human CCA cell lines had increased expression of Tac1 and NK1R, along with reduced levels of MME compared to non-malignant cholangiocytes, resulting in a subsequent increase in SP secretion. SP treatment increased CCA cell proliferation in vitro, which was blocked by L-733,060. Treatment with L-733,060 alone inhibited CCA proliferation in vitro and in vivo. Xenograft tumors derived from MME-overexpressed human Mz-ChA-1 CCA cells had a slower growth rate than those derived from control cells. Expression of PCNA, CK-19 and VEGF-A decreased, whereas MME expression increased in the xenograft tumors treated with L-733,060 or MME-overexpressed xenograft tumors compared to controls. CONCLUSION: The study suggests that SP secreted by CCA promotes CCA growth via autocrine pathway. Blockade of SP secretion and NK1R signaling may be important for the management of CCA.
Background: Toll-like receptors (TLRs) play a central role in the recognition and response to microbial pathogens, and in the maintenance and function of the epithelial barrier integrity in the gut. The protein MyD88 adaptor-like (Mal/TIRAP) serves as a bridge between TLR2/TLR4 and MyD88 mediated signalling to orchestrate downstream inflammatory responses. While MyD88 has an essential function in the maintenance of intestinal homeostasis, a role for Mal in this context is less well described. Methods: Colitis was induced in wild type (WT) and Mal-deficient (Mal-/-) mice by administration of dextran sodium sulfate (DSS). Colitis-associated cancer was induced by DSS and azoxymethane (AOM) treatment. Chimeric mice were generated by total-body gamma irradiation followed by transplantation of bone marrow cells. Results: In the DSS model of colon epithelial injury, Mal-/- mice developed increased inflammation and severity of colitis relative to WT mice. Mal-/- mice demonstrated the presence of inflammatory cell infiltrates, increased crypt proliferation, and presence of neo-formations. Furthermore, in the AOM/DSS model, Mal-/- mice had greater incidence of tumours. Mal-/- and WT bone marrow chimeras demonstrated that non-haematopoietic cell expression of Mal had an important protective role in the control of intestinal inflammation and inflammation-associated cancer. Conclusions: Mal is essential for the maintenance of intestinal homeostasis and expression of Mal in non-haematopoietic cells prevents chronic intestinal inflammation that may predispose to colon neoplasia.
In the gastrointestinal tract, vasoactive intestinal peptide (VIP) is found exclusively within neurons. VIP regulates intestinal motility via neurally-mediated and direct actions on smooth muscle, and secretion by a direct mucosal action and via actions on submucosal neurons. VIP acts via VPAC1 and VPAC2 receptors, however, the subtype involved in its neural actions is unclear. The neural roles of VIP and VPAC1 receptors (VPAC1R) were investigated in intestinal motility and secretion in guinea-pig jejunum. Expression of VIP receptors across the jejunal layers was examined using RT-PCR. Submucosal and myenteric neurons expressing VIP receptor subtype VPAC1 and/or various neurochemical markers were identified immunohistochemically. Isotonic muscle contraction was measured in longitudinal muscle-myenteric plexus preparations. Electrogenic secretion across mucosa-submucosa preparations was measured in Ussing chambers by monitoring short-circuit current. Calretinin+ excitatory longitudinal muscle motor neurons expressed VPAC1R. Most cholinergic submucosal neurons, notably NPY+ secretomotor neurons, expressed VPAC1R. VIP (100 nM) induced longitudinal muscle contraction that was inhibited by TTX (1 µM), PG97-269 (VPAC1 antagonist; 1 µM), and hyoscine (10 µM), but not by hexamethonium (200 µM). VIP (50 nM) evoked secretion was depressed by hyoscine or PG97-269 and involved a small TTX-sensitive component. PG97-269 and TTX combined did not further depress the VIP response observed in the presence of PG97-269 alone. We conclude that VIP stimulates acetylcholine-mediated longitudinal muscle contraction via VPAC1R on cholinergic motor neurons. VIP induces Cl- secretion directly via epithelial VPAC1R, and indirectly via VPAC1R on cholinergic secretomotor neurons. No evidence was obtained for involvement of other neural VIP receptors.
Others and we have characterized several Gβ-dependent effectors in smooth muscle, including GRK2, PLCβ3, and PI 3-kinase , and have identified various signaling targets downstream of PI 3-kinase , including cSrc, integrin-linked kinase, and Rac1-Cdc42/p21-activated kinase/p38 MAP kinase. This study identified a novel mechanism whereby Gβ acting via PI 3-kinase and cSrc exerts an inhibitory influence on Gαi activity. The Gi2-coupled opioid receptor agonist, DPDPE, activated cSrc, stimulated tyrosine phosphorylation of Gαi2, and induced Gαi2:RGS12 association; all three events were blocked by PI 3-kinase (LY294002) and cSrc (PP2) inhibitors and by expression of the carboxyl terminal sequence of GRK2(495-689), a Gβ-scavenging peptide. Inhibition of forskolin-stimulated cAMP and muscle relaxation by DPDPE was augmented by PP2, LY294002 and a selective PI 3 kinase inhibitor, AS605420. Expression of tyrosine-deficient Gαi2 mutant (Y69F/Y231F/Y321F) or knockdown of RGS12 blocked Gαi2 phosphorylation and Gαi2:RGS12 association, and caused greater inhibition of cAMP. Parallel studies using somatostatin, cyclopentyl adenosine, or acetylcholine to activate, respectively, Gi1-coupled sstr3 receptors, and Gi3-coupled adenosine A1 or m2 receptors elicited cSrc activation, Gαi1 or Gαi3 phosphorylation, and Gαi1:RGS12 or Gαi3:RGS12 association, and inhibition of cAMP. Inhibition of cAMP and muscle relaxation was greatly increased by AS-605240 and by PP2. The results demonstrate that Gβ-dependent tyrosine phosphorylation of Gαi1/2/3 by cSrc facilitated recruitment of RGS12, a Gαi-specific RGS protein with a unique phospho-tyrosine-binding (PTB) domain, resulting in rapid deactivation of Gαi and facilitation of smooth muscle relaxation.
The main roles of the colonic mucosa are the absorption of water and electrolytes and the barrier function that preserves the integrity of the colonic wall. The mediators and mechanisms to accomplish these functions are under continuous investigation but little attention has been paid to a possible control of colonic motility by the mucosa that would fine tune the relationship between absorption and motility. The purpose of this study was to establish the role of the mucosa in the control of induced colonic contractility. Young ICR-CD1 mice (3-5 mo-old) were studied. Isometric tension transducers were used to record contractility in full thickness (FT) and mucosa free (MF) strips from proximal colon. Proximal FT strips showed lower KCl- and bethanechol-induced responses than MF strips. The difference was not due to mechanical artefacts since the contractile response of FT strips to electrical field stimulation was around 50% lower than in MF. The inhibitory effects of the mucosa on FT strips was mimicked by immersion of separate strips of mucosa in the organ bath but not by addition of mucosal extract, suggesting gaseous molecules as mediator of this effect. Incubation of MF strips with synthase inhibitors of nitric oxide (NO), carbon monoxide (CO) and hydrogen sulphide (H2S) abolished the inhibition caused by addition of the mucosal strip, indicating that mucosal gasotransmitters are the mediators of these effects. This suggests that the control of colonic motility exerted by the mucosa could fine tune the balance between transit and absorption.
Little is known about the time-course of aging on interstitial cells of Cajal (ICC) of colon. The aim of this study was to investigate the change of morphology, ICC and neuronal nitric oxide synthase (nNOS) immunoreactive cells in the aged rat. The proximal colon of 344 Fischer rats at four different ages (6, 31, 74 weeks, and 2 years) were studied. The immunoreactivity of c-Kit, nNOS, anti-protein gene product 9.5(PGP 9.5) and synaptophysin were counted after immunohistochemistry. The c-kit, SCF (stem cell factor; ligand of Kit) and nNOS mRNA were measured by real-time PCR. c-Kit and nNOS protein were assessed by Western blot. Isovolumetric contractile force measurement and electrical field stimulation (EFS) were conducted. The area of intramuscular fat deposition significantly increased with age after 31 weeks. c-Kit immunoreactive ICC and nNOS immunoreactive neurons and nerve fibers significantly declined with age. mRNA and protein expression of c-kit and nNOS decreased with aging. The functional study showed that the spontaneous contractility was decreased in aged rat, whereas EFS responses in the presence of atropine and L-NG-Nitroarginine methyl ester were increased in aged rat. In conclusion, the decrease of proportion of proper smooth muscle, the density of ICC and nNOS immunoreactive neuronal fibers and the number of nNOS immunoreactive neurons during the aging process may explain the aging-associated colonic dysmotility.
Nutrients often stimulate gut hormone secretion, but the effects of fructose are incompletely understood. We studied the effects of fructose on a number of gut hormones with particular focus on GLP-1 and GIP. In healthy humans, fructose intake caused a rise in blood glucose and plasma insulin and GLP-1, albeit to a lower degree than isocaloric glucose. CCK secretion was stimulated similarly by both carbohydrates, but neither PYY3-36 nor glucagon secretion was affected by either treatment. Remarkably, while glucose potently stimulated GIP release, fructose was without effect. Similar patterns were found in the mouse and rat, with both fructose and glucose stimulating GLP-1 secretion, while only glucose caused GIP secretion. In GLUTag cells, a murine cell line used as model for L-cells, fructose was metabolized and stimulated GLP-1 secretion dose-dependently (EC50 = 0.155 mM) by KATP-channel closure and cell depolarization. Because fructose elicits GLP-1 secretion without simultaneous release of glucagonotropic GIP, the pathways underlying fructose-stimulated GLP-1 release might be useful targets for T2DM and obesity drug development.
Histone deacetylases (Hdac) remove acetyl groups from proteins, influencing global and specific gene expression. Hdacs control inflammation, as shown by Hdac inhibitor-dependent protection from DSS-induced murine colitis. While tissue-specific Hdac knockouts show redundant and specific functions, little is known of their intestinal epithelial cell (IEC) role. We have shown previously that dual Hdac1/Hdac2 IEC-specific loss disrupts cell proliferation and determination, with decreased secretory cell numbers and altered barrier function. We thus investigated how compound Hdac1/Hdac2 or Hdac2 IEC-specific deficiency alters the inflammatory response. Floxed Hdac1 and Hdac2 and villin-Cre mice were interbred. Compound Hdac1/Hdac2 IEC-deficient mice showed chronic basal inflammation, with increased basal Disease Activity Index (DAI) and deregulated Reg gene colonic expression. DSS-treated dual Hdac1/Hdac2 IEC-deficient mice displayed increased DAI, histological score, intestinal permeability and inflammatory gene expression. In contrast to double knockouts, Hdac2 IEC-specific loss did not affect IEC determination and growth, nor result in chronic inflammation. However, Hdac2 disruption protected against DSS colitis, as shown by decreased DAI, intestinal permeability and caspase-3 cleavage. Hdac2 IEC-specific deficient mice displayed increased expression of IEC gene subsets, such as colonic antimicrobial Reg3b and Reg3g mRNAs, and decreased expression of immune cell function-related genes. Our data show that Hdac1 and Hdac2 are essential IEC homeostasis regulators. IEC-specific Hdac1 and Hdac2 may act as epigenetic sensors and transmitters of environmental cues and regulate IEC-mediated mucosal homeostatic and inflammatory responses. Different levels of IEC Hdac activity may lead to positive or negative outcomes on intestinal homeostasis during inflammation.
Pancreatic acinar cells (PAC) obtain thiamin from the circulation via carrier-mediated process that involves thiamin transporters 1 and 2 (THTR-1 and THTR-2; products of SLC19A2 and SLC19A3, respectively). Chronic alcohol exposure of PAC inhibits thiamin uptake, and based on in vitro studies this inhibition appears to be transcriptionally mediated. The aim of this study was to confirm the involvement of a transcriptional mechanism in mediating the chronic alcohol effect in in vivo settings, and to delineate the molecular mechanisms involved. Using transgenic mice carrying full-length SLC19A2 and SLC19A3 promoters, we found that chronic alcohol feeding lead to a significant reduction in the activity of SLC19A2 and SLC19A3 promoters (as well as in thiamin uptake and expression of THTR-1 and -2). Similar findings were seen in 266-6 cells chronically exposed to alcohol in vitro. In latter studies, the alcohol inhibitory effect was found to be mediated via the minimal SLC19A2 and SLC19A3 promoters, and involved the cis-regulatory elements SP1/GKLF and SP1-GG box and SP1/GC respectively. Chronic alcohol exposure of PAC also lead to a significant reduction in the expression of the SP1 transcription factor, which upon correction (via expression) not only led to the prevention of alcohol inhibitory effects on the activity of SLC19A2 and SLC19A3 promoters but also on the expression of THTR-1 and -2 mRNA and thiamin uptake. These results demonstrate that the inhibitory effect of chronic alcohol exposure on physiological/molecular parameters of thiamin uptake by PAC is mediated via specific cis-regulatory elements in SLC19A2 and SLC19A3 minimal promoters.
Intestinal homeostasis is maintained by a hierarchy of immune defences acting in concert to minimize contact between luminal microorganisms and the intestinal epithelial cell surface. The intestinal mucus layer, covering the gastrointestinal (GI) tract epithelial cells, contributes to mucosal homeostasis by limiting bacterial invasion. In this study, we used T-cell-deficient (TCR-/-) mice to examine whether and how T-cells modulate the properties of the intestinal mucus layer. Increased susceptibility of TCR-/- mice to dextran sodium sulphate (DSS)-induced colitis is associated with a reduced number of goblet cells. Alterations in the number of goblet cells and crypt lengths were observed in the small intestine (SI) and colon of TCR-/- mice compared to C57BL/6 wt mice. Addition of keratinocyte growth factor (KGF) to small intestinal organoid cultures from TCR-/- mice showed a marked increase in crypt growth, and both goblet cell number and redistribution along the crypts. There was no apparent difference in the thickness or organisation of the mucus layer between TCR-/- and wt mice, as measured in vivo. However, T-cell deficiency led to reduced sialylated mucins in association with increased gene expression of gel-secreting Muc2 and membrane-bound mucins, including Muc13 and Muc17. Collectively, these data provide evidence that T cells play an important role in maintenance of mucosal homeostasis by regulating mucin expression and promoting goblet cell function in the small intestine.
Background Dendritic cells (DC) transfected with interleukin-10 (IL-10) and transforming growth factor-β1 (TGF-β1) enhance T cell immunity and tolerance. However, no quantitative studies have investigated the suppressive functions of immature dendritic cells (imDC) co-transfected with IL-10 and TGF-β1. Methods: Effects of imDC co-transfected with IL-10 and TGF-β1 (IL-10-TGF-β1-imDC) on immune tolerance induction in a rat transplantation model were investigated. In addition, effects of IL-10-TGF-β1-imDC relative to IL-10 transfected imDC (IL-10-imDC) and TGF-β1-transfected imDC (TGF-β1-imDC) were compared. Results: The infusion of IL-10-TGF-β1-imDC into recipients prolonged liver graft survival, which were sustained for more than 90 days. IL-12 serum levels decreased, whereas alanine transaminase (ALT) and total bilirubin (TBIL) slightly increased in rats infused with IL-10-TGF-β1-imDC when compared to the IL-10-imDC and TGF-β1-imDC groups. Furthermore, a higher percentage of TUNEL positive cells were observed and histological analysis of the allografts indicated a rejection activity index (RAI) of mild acute rejection. Conclusion: Our results suggest infusion of IL-10 and TGF-β1 co-transfected imDC induces alloantigen-specific T cell hypo-responsiveness, inhibits antigen-specific immunological responses to liver allografts, prolongs liver allograft survival, and enhances the immune tolerance. This approach may provide a promising and alternative for enhancing donor-specific tolerance during liver transplantation.
Acute H. pylori infection of gastric epithelial cells induces CagA oncoprotein- and peptidoglycan (SLT)-dependent mobilization of NF-B p50 homodimers that bind to H,K-ATPase α subunit (HKα) promoter and repress HKα gene transcription. This process may facilitate gastric H. pylori colonization by induction of transient hypochlorhydria. We hypothesized that H. pylori also regulates HKα expression post-transcriptionally by miRNA interaction with HKα mRNA. In silico analysis of HKα 3' untranslated region (UTR) identified miR-1289 as a highly-conserved putative HKα-regulatory miRNA. H. pylori infection of AGS cells transfected with HKα 3' UTR-Luc reporter construct repressed luciferase activity by 70%, while cagA or slt H. pylori infections partially abrogated repression. Transfection of AGS cells expressing HKα 3' UTR-Luc construct with an oligoribonucleotide mimetic of miR-1289 induced maximal repression (54%) of UTR activity within 30 min; UTR activity was unchanged by non-targeting siRNA transfection. Gastric biopsies from patients infected with cagA+ H. pylori showed a significant increase in miR-1289 expression compared to uninfected patients or those infected with cagA- H. pylori. Finally, miR-1289 expression was necessary and sufficient to attenuate biopsy HKα protein expression in the absence of infection. Taken together, these data indicate that miR-1289 is upregulated by H. pylori in a CagA and SLT-dependent manner and targets HKα 3' UTR, affecting HKα mRNA translation. The sensitivity of HKα mRNA 3' UTR to binding of miR-1289 identifies a novel regulatory mechanism of gastric acid secretion, and offers new insights into mechanisms underlying transient H. pylori-induced hypochlorhydria.
Current literature consolidates the view of Crohn's disease (CD) as a form of immunodeficiency highlighting dysregulation of intestinal innate immunity in the pathogenesis of CD. Intestinal macrophages derived from blood monocytes play a key role in sustaining the innate immune homeostasis in the intestine suggesting that the monocyte/macrophage compartment might be an attractive therapeutic target for the management of CD. Granulocyte macrophage colony-stimulating factor (GM-CSF) is a hematopoietic growth factor which also promotes myeloid cell activation, proliferation, and differentiation. GM-CSF has a protective effect in human CD and mouse models of colitis. However, the role of GM-CSF in immune and inflammatory reactions in the intestine is not well defined. Beneficial effects exerted by GM-CSF during intestinal inflammation could relate to modulation of the mucosal barrier function in the intestine, including epithelial cell proliferation, survival, restitution and immunomodulatory actions. The aim of this review is to summarize potential mechanistic roles of GM-CSF in intestinal innate immune cell homeostasis and to highlight its central role in maintenance of the intestinal immune barrier in the context of immunodeficiency in CD.
Squamous esophageal epithelium adapts to acid reflux-mediated injury by proliferation and differentiation. Induction of the Wnt-antagonist, Dickkopf-1 (Dkk1) is involved in tissue repair during inflammation and cellular injury. In this study, we aimed to identify the biological role of Dkk1 in human reflux-esophagitis with respect to cell growth and regulation of Wnt-signaling. Esophageal biopsies from reflux-esophagitis patients and healthy individuals were characterized for Dkk1 expression. Role of Dkk1 in response to acid-mediated epithelial injury was analyzed by utilizing squamous esophageal epithelial cell lines (EPC1-, EPC2-hTERT and HEEC). Dkk1 was significantly overexpressed in human reflux-esophagitis tissue compared to healthy esophageal mucosa at transcriptional and translational level. Following acute and chronic acid exposure (pH 4), esophageal squamous epithelial cell lines expressed and secreted high levels of Dkk1 as response to stress-associated DNA injury. Human recombinant Dkk1 inhibited epithelial cell growth and induced cellular senescence as demonstrated by reduced cell proliferation, G0/G1 cell cycle arrest, elevated senescence-associated β-galactosidase activity (SA-β-gal) and up-regulation of p16. Acid pulsing induced Dkk1-mediated senescence, which was directly linked to the ability of Dkk1 to antagonize the canonical Wnt/β-catenin signaling. In healthy esophageal mucosa, Dkk1 expression was associated with low expression of transcriptional active β-catenin, while in reflux-esophagitis tissue, Dkk1 overexpression associated with increased SA-β-gal activity and p16 up-regulation. These data indicate that in human reflux-esophagitis, Dkk1 functions as a secreted growth-inhibitor by suppressing Wnt/β-catenin signaling and promoting cellular senescence. Taken together, our findings suggest a significant role for Dkk1 and cellular senescence in esophageal homeostasis during reflux-esophagitis.
Deficiency of ABCB4 is associated with several forms of cholestasis in humans. Abcb4-/- mice also develop cholestasis, but it remains uncertain what role other canalicular transporters play in the development of this disease. We examined the expression of these transporters in Abcb4-/- mice, compared to their wild-type littermate controls at ages of 10 days, 3, 6 and 12 weeks. Elevated plasma bile acid levels were already detected at 10 days and at all ages thereafter in Abcb4-/- mice. The expression of Bsep, Mrp2, Atp8b1, Abcg5 and Abcg8 liver proteins did not change at 10 days, but Bsep, Mrp2, and Atp8b1 were reduced, while Abcg5 and Abcg8 expression were increased in Abcb4-/- mice at all later ages. Lower bile acid concentrations were also detected in the bile of 6 weeks Abcb4-/- mice. Immunofluorescence labeling revealed distorted canalicular architecture in the liver tissue by 12 weeks in Abcb4-/- mice. While Bsep and Mrp2 remained associated with the apical membrane, Atp8b1 was now localized in discrete punctuate structures adjacent to the canalicular membrane in these mice. Expression of Bsep mRNA was increased in the livers of 10 day old Abcb4-/- mice, whereas Ostα was decreased. By 12 weeks, Bsep, Mrp2, and Abcg5 mRNA were all increased, while Ostα and Ntcp were reduced. These findings indicate that canalicular transporters that determine the formation of bile are altered early in the development of cholestasis in Abcb4-/- mice and may contribute to the pathogenesis of cholestasis in this disorder.
Obesity is a growing epidemic with limited effective treatments. The neurotrophic factor glial cell line derived neurotrophic factor (GDNF) was recently shown to enhance β-cell mass and improve glucose control in rodents. It's role in obesity is, however, not well characterized. In this study, we investigated the ability of GDNF to protect against high fat diet (HFD)-induced obesity. GDNF transgenic (Tg) mice, that over express GDNF under the control of the glial fibrillary acidic protein promoter, and wild-type (WT) littermates were maintained on a HFD or regular rodent diet (RD) for 11 weeks, and weight gain, energy expenditure and insulin sensitivity monitored. Differentiated mouse brown adipocytes and 3T3-L1 white adipocytes were used to study the effects of GDNF in vitro. Tg mice resisted the HFD-induced weight gain, insulin resistance, dyslipidemia, hyperleptinemia and hepatic steatosis seen in WT mice despite similar food intake and activity levels. They exhibited significantly (P<0.001) higher energy expenditure than WT mice and increased expression in skeletal muscle and brown adipose tissue of peroxisome proliferator activated receptor-α and β1- and β3-adrenergic receptor genes which are associated with increased lipolysis and enhanced lipid β-oxidation. In vitro, GDNF enhanced β-adrenergic-mediated cAMP release in brown adipocytes and suppressed lipid accumulation in differentiated 3T3L-1 cells through a p38MAPK signaling pathway. Our studies demonstrate a novel role for GDNF in the regulation of high fat diet-induced obesity through increased energy expenditure. They show that GDNF and its receptor agonists may be potential targets for the treatment or prevention of obesity.
Background: Water-perfused high-resolution manometry (HRM) catheters with 36 unidirectional pressure channels have recently been developed but normal values are not yet available. Furthermore, the technique has not been validated and compared with solid-state HRM. We therefore aimed to develop normal values for water-perfused HRM and to assess the level of agreement between water-perfused HRM and solid-state HRM. Methods: We included 50 healthy volunteers (Mean age: 35 years, range: 21 - 64 years, 15 female). Water-perfused HRM and solid-state HRM were performed in a randomized order. Normal values were calculated as 5th and 95th percentile ranges and agreement between the two systems was assessed using intra-class correlations coefficient (ICC) statistics. Results: The 5-95th percentile range for contractile front velocity (CFV) was 3.0 - 6.6 cm s-1, for distal contractile integral (DCI) 141.6 - 3674 mmHg*s*cm, for distal contraction latency (DL) 6.2 - 8.7 s and for integrated relaxation pressure (IRP4s) 1.0 - 18.8 mmHg. Mean (SD) and ICC for water-perfused HRM and solid-state HRM were for CFV 4.4 (1.1) vs 3.9 (0.9) cm s-1, ICC: 0.49, for DCI 1189 (1023) vs 1092 (1019) mmHg*s*cm, ICC: 0.90, for DL 7.4 (0.8) vs 6.9 (0.9) s, ICC: 0.50 and for IRP4s 8.1 (4.8) vs 7.9 (5.1), ICC:0.39. Conclusions: The normal values for this water-perfused HRM system are only slightly different from previously published values with solid state HRM and moderate to good agreement was observed between the two systems with only small differences in outcome measures.
Interstitial cells of Cajal (ICC) act as pacemaker cells in the gastrointestinal tract by generating electrical slow waves to regulate rhythmic smooth muscle contractions. Intrinsic Ca2+ oscillations in ICC appear to produce the slow waves by activating pacemaker currents, currently thought to be carried by the Ca2+-activated Cl- channel, anoctamin 1 (Ano1). In this paper we present a novel model of small intestinal ICC pacemaker activity that incorporates store-operated Ca2+ entry and a new model of Ano1 current. A series of simulations were carried out with the ICC model to investigate current controversies about the reversal potential of the Ano1 Cl- current in ICC, and to predict the characteristics of the other ion channels that are necessary to generate slow waves. The model results show that Ano1 is a plausible pacemaker channel when coupled to a store-operated Ca2+ channel, but suggest that small cyclical depolarizations may still occur in ICC in Ano1 knockout mice. The results predict that voltage-dependent Ca2+ current is likely to be negligible during the slow wave plateau phase. The model shows that the Cl- equilibrium potential is an important modulator of slow wave morphology, highlighting the need for a better understanding of Cl- dynamics in ICC.
Objectives: The current treatment for celiac disease is strict gluten-free diet. Technical processing may render gluten-containing foods safe for consumption by celiac patients, but so far in vivo safety testing can only be performed on patients. We modified a celiac disease mouse model to test antigenicity and inflammatory effects of germinated rye sourdough, a food product characterized by extensive prolamin hydrolysis. Methods: Lymphopenic Rag1-/- or nude mice were injected with splenic CD4+CD62L-CD44high memory T cells from gliadin- or secalin-immunized wildtype donor mice. Results: 1) Rag1-/- recipients challenged with wheat or rye gluten lost more body weight and developed more severe histological duodenitis than mice on gluten-free diet. This correlated with increased secretion of IFN, IL-2 and IL-17 by secalin-restimulated splenocytes. 2) In vitro gluten testing using competitive R5 ELISA demonstrated extensive degradation of the gluten R5 epitope in germinated rye sourdough. 3) However, in nude recipients challenged with germinated rye sourdough (vs. native rye sourdough), serum anti-secalin IgG/Th1-associated IgG2c titers were only reduced, but not eliminated. In addition, there were no reductions in body weight loss, histological duodenitis or T cell cytokine secretion in Rag1-/- recipients challenged accordingly. Conclusions: 1) Prolamin-primed CD4+CD62L-CD44high memory T cells induce gluten-sensitive enteropathy in Rag1-/- mice. 2) Hydrolysis of secalins in germinated-rye sourdough remains incomplete. Secalin peptides retain B and T cell stimulatory capacity, and remain harmful to the intestinal mucosa in this celiac disease model. 3) Current antibody-based prolamin detection methods may fail to detect antigenic gluten fragments in processed cereal food products.
Intraamniotic exposure to pro-inflammatory agonists results in chorioamnionitis and fetal gut inflammation. Fetal gut inflammation is associated with mucosal injury and impaired gut development. We tested whether this detrimental inflammatory response of the fetal gut results from a direct local (gut derived), or an indirect inflammatory response mediated by the chorioamnion/skin or lung, since these organs are also in direct contact with the amniotic fluid. The gastrointestinal tract was isolated from the respiratory tract and the amnion/skin epithelia by fetal surgery in time-mated ewes. Lipopolysaccharide (LPS) or saline (controls) was selectively infused in the gastrointestinal tract, trachea or amniotic compartment at 2 or 6d prior to preterm delivery at 124d gestation (term 150d). Gastrointestinal and intratracheal LPS exposure caused distinct inflammatory responses in the fetal gut. Inflammatory responses could be distinguished by the influx of leukocytes (MPO+, CD3+ and FoxP3+ cells), TNF-α and IFN- expression, and different mRNA levels of TLR 1,2,4,6. Fetal gut inflammation after direct intestinal LPS exposure resulted in severe loss of the tight junctional protein ZO-1 and increased mitosis of intestinal epithelial cells. Inflammation of the fetal gut after selective LPS instillation in the lungs was accompanied by only mild disruption of ZO-1, loss in epithelial cell integrity and impaired epithelial differentiation. LPS exposure of the amnion/skin epithelia did not result in gut inflammation and subsequent morphological, structural or functional changes. Our results indicate that the detrimental consequences of chorioamnionitis on fetal gut development are the combined result of local gut and lung mediated inflammatory responses.
External anal sphincter may be injured in 25 - 35 % of women during the first and subsequent vaginal child births and is likely the most common cause of anal incontinence. Since its first description, almost 300 years ago, EAS is believed to be a circular or a "donut-shaped" structure. We performed 3D-transperineal ultrasound imaging, magnetic resonance imaging, diffusion tensor imaging and muscle fiber tracking to delineate various components of EAS and their muscle fiber directions. These novel imaging techniques suggest "purse-string" morphology, with "external anal sphincter" muscles crossing contra-laterally in the perineal body to contra-lateral transverse pernei & bulbospongiosus muscle thus attaching "external anal sphincter" to the pubic rami. Spin-tag magnetic resonance imaging demonstrated "purse-string" action of the external anal sphincter muscle. Electromyography of transverse perinei/bulbospongiosus and external anal sphincter muscle revealed their simultaneous contraction and relaxation. Lidocaine injection into transverse perinei/bulbospongiosus muscle caused significant reduction in the anal canal pressure. These studies support "purse-string" morphology of external anal sphincter to constrict/close the anal canal opening. Our findings have implications for the effect of episiotomy on anal closure function and currently used surgical technique (overlapping sphincteroplasty) for the external anal sphincter reconstructive surgery to treat anal incontinence.
Variations in the serine protease 1 (PRSS1) gene encoding human cationic trypsinogen have been conclusively associated with autosomal dominant hereditary pancreatitis and sporadic non-alcoholic chronic pancreatitis. Most high-penetrance PRSS1 variants increase intra-pancreatic trypsin activity by stimulating trypsinogen autoactivation and/or by inhibiting chymotrypsin C dependent trypsinogen degradation. Alternatively, some PRSS1 variants can cause trypsinogen misfolding, which results in intracellular retention and degradation with consequent endoplasmic reticulum stress. However, not all PRSS1 variants are pathogenic and clinical relevance of rare variants is often difficult to ascertain. Here we review the PRSS1 variants published since 1996; discuss their functional properties and role in chronic pancreatitis.
The small intestinal epithelium develops from embryonic endoderm into a highly specialized layer of cells perfectly suited for the digestion and absorption of nutrients. The development, differentiation, and regeneration of the small intestinal epithelium require complex gene regulatory networks involving multiple context-specific transcription factors. The evolutionarily conserved GATA family of transcription factors, well known for its role in hematopoiesis, is essential for the development of endoderm during embryogenesis and the renewal of the differentiated epithelium in the mature gut. Here, we review the role of GATA factors in the evolution and development of endoderm, and summarize our current understanding of the function of GATA factors in the mature small intestine. We offer perspective on the application of epigenetics approaches to define the mechanisms underlying context-specific GATA gene regulation during intestinal development.
Liver steatosis in nonalcoholic fatty liver disease is affected by genetics, diet, and associated with insulin resistance (IR) in hepatic and peripheral tissues. Here, we aimed to characterize the severity of diet-induced steatosis, obesity, and IR in two phylogenetically distant mouse strains, C57BL/6J and DBA/2J. To this end, mice (male, 8 weeks) were fed a high fat and high carbohydrate (HFHC) or control diet for 16 weeks followed by applying a combination of classic physiological, biochemical and pathological studies to determine obesity and hepatic steatosis. Peripheral IR was characterized by measuring blood glucose level, serum insulin level, HOMA-IR, glucose intolerance, insulin intolerance, and AKT phosphorylation in adipose tissues, whereas the level of hepatic IR was determined by measuring insulin-triggered hepatic AKT phosphorylation. We discovered that both C57BL/6J and DBA/2J mice developed obesity to a similar degree without feature of liver inflammation after feeding HFHC-diet for 16 weeks. C57BL/6J mice in the HFHC-diet group exhibited severe pan-lobular steatosis, a marked increase in hepatic triglyceride levels, and profound peripheral IR. In contrast, DBA/2J mice in the HFHC-diet group developed only a mild degree of pericentrilobular hepatic steatosis that was associated with moderate changes in peripheral IR. Interestingly, both C57BL/6J and DBA/2J developed severe hepatic IR after HFHC-diet treatment. Collectively, these data suggest the severity of diet-induced hepatic steatosis is correlated to the level of peripheral IR, not with the severity of obesity and hepatic insulin resistance. Peripheral rather than hepatic IR is a dominant factor of pathophysiology in nonalcoholic fatty liver disease.
Dsk5 mice have a gain-of-function in the epidermal growth factor receptor (EGFR), caused by a point mutation in the kinase domain. We analyzed the effect of this mutation on liver size, histology, and composition. We found that the livers of 12-week old male Dsk5 heterozygotes (+/Dsk5) were 62% heavier compared to those of wild-type controls (+/+). The livers of the +/Dsk5 mice compared to +/+ mice had larger hepatocytes with prominent, polyploid nuclei and showed modestly increased cell proliferation indices in both hepatocytes and non-parenchymal cells. An analysis of total protein, DNA, and RNA (expressed relative to liver weight) revealed no differences between the mutant and wild-type mice. However, the livers of the +/Dsk5 mice had more cholesterol but less phospholipid and fatty acid. Circulating cholesterol levels were twice as high in adult male +/Dsk5 mice, but not in post-weaned young male or female mice. The elevated total plasma cholesterol resulted mainly from an increase in low-density lipoprotein (LDL). The +/Dsk5 adult mouse liver expressed markedly reduced protein levels of low-density lipoprotein receptor (LDLR), no change in proprotein convertase subtilisin/kexin type 9 (PCSK9), and a markedly increased fatty acid synthase (FAS) and 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMG-CoA R. Increased expression of transcription factors associated with enhanced cholesterol synthesis was also observed.Together, these findings suggest that the EGFR may play a regulatory role in hepatocyte proliferation and lipid metabolism in adult male mice, explaining why elevated levels of EGF or EGF-like peptides have been positively correlated to increased cholesterol levels in human studies.
We previously reported that Rab1a is associated with asialoorosomucoid (ASOR) containing early endocytic vesicles where it is required for their microtubule-based motility. In Rab1a knockdown cell lines, ASOR failed to segregate from its receptor and consequently did not reach lysosomes for degradation, indicating a defect in early endosome sorting. Although Rab1 is required for Golgi/ER trafficking, this process was unaffected, likely due to retained expression of Rab1b in these cells. The present study shows that Rab1a has a more general role in endocytic vesicle processing that extends to epidermal growth factor (EGF) and transferrin (Tfn) trafficking. As compared to results in control Huh7 cells, EGF accumulated in aggregates within Rab1a KD cells, failing to reach lysosomal compartments. Tfn, a prototypical example of recycling cargo, accumulated in a Rab11-mediated slow recycling compartment in Rab1a KD cells in contrast to control cells that sort Tfn into a fast recycling Rab4 compartment. These data indicate that Rab1a is an important regulator of early endosome sorting for multiple cargo species. The effectors and accessory proteins recruited by Rab1a to early endocytic vesicles include the minus end directed kinesin motor, KifC1 while others remain to be discovered.
Feeding intolerance resulting from delayed gastric emptying is common in premature neonates. Metoclopramide (MCP), the most frequently used prokinetic drug in neonates, enhances gastric muscle contractility through inhibition of dopamine receptors. Although its therapeutic benefit is established in adults, limited data are available to support its clinical use in infants. Hypothesizing that developmentally-dependent differences are present, we comparatively evaluated the effect of MCP on fundus muscle contractility in newborn, juvenile and adult rats. The muscle strips were either contracted with electrical field stimulation (EFS) to induce cholinergic nerve-mediated acetylcholine release or carbachol, a cholinergic agonist acting directly on the muscarinic receptor. Although in adult rats MCP increased EFS induced contraction by 294±122% of control (P<0.01), no significant effect was observed in newborn fundic muscle. MCP had no effect on the magnitude of the carbachol-induced and/or bethanechol-induced gastric muscle contraction at any age. In response to dopamine, a 80.7± 5.3% relaxation of adult fundic muscle was observed, as compared with only a 8.4 ± 8.7%, response in newborn tissue (P<0.01). Dopamine D2 receptor expression was scant in neonates and significantly increased in adult gastric tissue (P<0.01). In conclusion the lack of MCP effect on the newborn fundic muscle contraction potential relates to developmental differences in dopamine D2 receptor expression. To the extent that this novel data can be extrapolated to neonates, the therapeutic value of MCP as a prokinetic agent early in life requires further evaluation.
Intestinal intussusception (ISS) commonly causes intestinal obstruction in children. One proposed mechanism of ISS is that inflammation-induced alterations of intestinal motility cause ISS. We investigated whether innate inflammatory factors or altered motility are required for induction of ISS by LPS. We compared rates of ISS among BALB/c and C57Bl/6 mice, mice lacking lymphocytes or depleted of phagocytes, or mice with defects in the TLR4 signaling pathway following administration of LPS or the calcium analog MnCl2. Six or two hours after administration of LPS or MnCl2 respectively, mice underwent image analysis to assess intestinal contraction rate or laparotomy to identify ISS. LPS-induced ISS (LPS-ISS) was observed in BALB/c mice but not in C57BL/6 mice or in any BALB/c mice with disruptions in TLR4 signaling. LPS-induced serum TNF-α, IL-6, and NO and intestinal NO levels were similar between BALB/c and C57BL/6 mice. The rate of LPS-ISS was significantly reduced in phagocyte-depleted but not in lymphocyte-deficient mice. Intestinal contraction rates were reduced in LPS-ISS susceptible BALB/c but not in LPS-ISS resistant C57BL/6 or TLR4 mutant mice, suggesting a role for reduced intestinal contraction rate in LPS-ISS susceptibility. This was tested with MnCl2, a calcium antagonist that reduced intestinal contraction rates and induced ISS irrespective of mouse strain. Therefore, LPS-ISS is initiated by innate immune signaling that requires TLR4 and phagocytes but may be independent from TNF-α, IL-6, and NO levels. Further, alteration in intestinal motility, specifically reduced intestinal contraction rate, is a key factor in the development of ISS.
Background: Fetal swallowing of amniotic fluid, which contains several cytokines and growth factors, plays a key role in gut mucosal development. Preterm birth interrupts this exposure to amniotic fluid-borne growth factors, possibly contributing to the increased risk of necrotizing enterocolitis (NEC) in premature infants. We hypothesized that supplementation of formula feeds with amniotic fluid might provide amniotic fluid-borne growth factors and prevent experimental NEC in rat pups. Methods: We compared NEC-like injury in rat pups fed with infant formula vs. formula supplemented either with 30% amniotic fluid or recombinant hepatocyte growth factor (rHGF). Cytokines/growth factors in amniotic fluid were measured by immunoassays. Amniotic fluid and HGF effects on enterocyte migration, proliferation, and survival were measured in cultured IEC-6 cells. Finally, we used an antibody array to investigate receptor tyrosine kinase (RTK) activation and immunoblots to measure phosphoinositide 3-kinase (PI3K) signaling. Results: Amniotic fluid supplementation of oral feeds protected rat pups against NEC-like injury. HGF was the most abundant growth factor in rat amniotic fluid in our panel of analytes. Amniotic fluid increased cell migration, proliferation, and cell survival in vitro. These effects were reproduced by rHGF and blocked by anti-HGF antibody or a PI3K inhibitor. rHGF transactivated several RTKs in IEC-6 cells, indicating that its effects extended to multiple signaling pathways. Finally, similar to amniotic fluid, rHGF also reduced the frequency and severity of NEC-like injury in rat pups. Conclusions: Amniotic fluid supplementation protects rat pups against experimental NEC, which is mediated, at least in part, by HGF.
We examined whether CB1 receptors in smooth muscle conform to the signaling pattern observed with other Gi-coupled receptors that stimulate contraction via two Gβ-dependent pathways (PLC-β3 and PI 3-kinase/integrin-linked kinase). Here we show that the anticipated Gβ-dependent signaling was abrogated. Except for inhibition of adenylyl cyclase via Gαi, signaling resulted from Gβ-independent phosphorylation of CB1 receptors by GRK5, recruitment of β-arrestin1/2, and activation of ERK1/2 and Src kinase. Neither uncoupling of CB1 receptors from Gi by PTx or Gi minigene, nor expression of a Gβ-scavenging peptide had any effect on ERK1/2 activity. The latter was abolished in muscle cells expressing β-arrestin1/2 siRNA. CB1 receptor internalization and both ERK1/2 and Src kinase activities were abolished in cells expressing kinase-deficient GRK5(K215R). Activation of ERK1/2 and Src kinase endowed CB1 receptors with the ability to inhibit concurrent contractile activity. We identified a consensus sequence (102KSPSKLSP109) for phosphorylation of RGS4 by ERK1/2 and showed that expression of a RGS4 mutant lacking Ser103/Ser108 blocked the ability of anandamide to inhibit acetylcholine-mediated PI hydrolysis or enhance Gαq:RGS4 association and inactivation of Gαq. Activation of Src kinase by anandamide enhanced both M-RIP:RhoA and M-RIP:MYPT1 association and inhibited Rho kinase activity leading to increase of MLC phosphatase activity and inhibition of sustained muscle contraction. Thus, unlike other Gi-coupled receptors in smooth muscle, CB1 receptors did not engage Gβ but signaled via GRK5/beta-arrestin activation of ERK1/2 and Src kinase: ERK1/2 accelerated inactivation of Gαq by RGS4, and Src kinase enhanced MLC phosphatase activity leading to inhibition of ACh-stimulated contraction.
Colorectal carcinoma (CRC) is one of the most common cancers in the world, and identification of new CRC biomarkers will be helpful for the diagnosis and treatment of CRC. For isobaric tags for relative and absolute quantitation (iTRAQ) analysis, fresh CRC and adjacent, colonic adenoma, ulcerative colitis, Crohn's disease and noncancerous colonic epithelial tissue were obtained from patients at the 2nd Xiangya Hospital of Central South University, China. The function of heterogeneous nuclear ribonucleoprotein M (HnRNP M) during the proliferation, invasion and metastasis of colorectal carcinoma cells in vitro were evaluated. One hundred and twenty-six differentially expressed proteins were identified by iTRAQ analysis. The expression of HnRNP M exhibited progressive changes during the carcinogenic process, and was validated by western blot. The up-regulation of HnRNP M correlated with cancer recurrence and regional lymph nodes. Furthermore, biological role exploring suggests that HnRNP M positively regulates cell cycle progression, promotes cell growth and invasion in vitro and increases the colony-forming ability of LS174T cells. The present data demonstrate that the up-regulation of HnRNP M is involved in human colorectal epithelial carcinogenesis and may serve as a carcinoma biomarker for CRC.
The organic solute transporter OSTα-OSTβ is a key transporter for the efflux of bile acids across the basolateral mem-brane of ileocytes and the subsequent re-turn of bile acids to the liver. Ostα-/- mice exhibit reduced bile acid pools and im-paired lipid absorption. In this study, wild type and Ostα-/- mice were characterized at 5 and 12 months. Ostα-/- mice were re-sistant to age-related weight gain, body fat accumulation, and liver and muscle lipid accumulation, and the males lived slightly longer than wild type mice. Caloric intake and activity levels were similar for Ostα-/- and wild type males. Fecal lipid excretion was increased in Ostα-/- mice, indicating that a defect in lipid absorption contributes to decreased fat accumulation. Analysis of genes involved in intestinal lipid absorption revealed changes consistent with de-creased dietary lipid absorption in Ostα-/- animals. Hepatic expression of cholesterol synthetic genes was upregulated in the Ostα-/- mice, showing that reduced dietary cholesterol absorption was partially compen¬sated for by increased cholesterol synthesis. Male Ostα-/- mice had improved glucose tolerance and both male and fe-male knockout mice had improved insulin sensitivity. Akt phosphorylation was meas-ured in liver and muscle tissue from mice after acute administration of insulin. Male and female Ostα-/- mice showed signifi-cantly larger insulin responses than wild type mice. These findings indicate that loss of OSTα-OSTβ protects against age-related weight gain and insulin resistance.
We have previously reported that doublecortin-like kinase 1 (Dclk1) is a putative intestinal stem cell (ISC) marker. In this report, we evaluated the use of Dclk1 as a marker of surviving ISCs in response to treatment with high-dose total body irradiation (TBI). Both apoptotic and mitotic Dclk1+ cells were observed 24 h post TBI associated with a corresponding loss of intestinal crypts observed at 84 h post TBI. Although the Notch signaling pathway plays an important role in regulating proliferation and lineage commitment within the intestine, its role in ISC function in response to severe genotoxic injury is not yet fully understood. We employed the microcolony assay to functionally assess the effects of Notch inhibition with DAPT on intestinal crypt stem cell survival following severe (> 8 Gy) radiation injury. We observed a nearly 50% reduction in the number of surviving Dclk1+ crypt epithelial cells at 24 h after TBI and similar reduction in the number of surviving small intestinal crypts at 84 h following pre-treatment with DAPT compared to radiation alone. These data demonstrate that inhibition of Notch signaling decreases ISC survival following radiation injury, suggesting that the Notch signaling pathway plays an important role in ISC mediated crypt regeneration. These results also suggest that crypt epithelial cell Dclk1 expression can be used as a surrogate marker to evaluate the early survival of ISCs following severe radiation injury.
Intestinal ischemia and reperfusion (I/R) is a clinical problem occurred for diverse causes with high mortality. Prophylaxis and treatment of intestinal I/R remains a challenge for clinician. The present study was to explore the role of Notoginsenoside R1 (R1), a major component form Panax. Notoginseng, in management of intestinal I/R injury. Intestinal I/R was induced in male Sprague-Dawley rats by clamping the superior mesenteric artery for 90 min followed by reperfusion for 60 min or 3 days. R1 (10 mg/kg/h) was administered either 20 min before ischemia or 20 min after reperfusion. Intestinal microcirculation was evaluated by intravital microscopy over 60 min reperfusion. Sixty min or 3 days after reperfusion, rats were killed for histological examination of the jejunum tissue and immunohistochemical localization of myeloperoxidase and CD68. ATP, ADP and AMP content in jejunum tissue was assessed by ELISA. Activation of NF-B, expression of ATP5D and tight junction proteins were determined by Western blotting. The results demonstrated that R1 is capable of attenuating intestinal I/R-induced microvascular hyperpermeability, inflammatory cytokine production, NF-B activation and loss of tight junction proteins, as well as improving energy metabolism during I/R. The results of the present study suggest R1 as an option in protecting against intestinal I/R injury.
A defect in the gene for the lysosomal enzyme α-galactosidase A (Gla) results in globotriaosylceramide (Gb3) accumulation in Fabry disease and leads to premature death from cardiac and cerebrovascular events. However, gastrointestinal symptoms are often first observed during childhood in these patients, and are not well understood. In this study, we demonstrate an age-dependent microvasculopathy of the mesenteric artery (MA) in a murine model of Fabry disease (Gla-knockout) resulting from dysregulation of the vascular homeostatic enzyme, endothelial nitric oxide synthase (eNOS). The progressive accumulation of Gb3 in MA was confirmed by thin layer chromatographic analysis. A total absence of endothelium-dependent dilatation was present in MA of 8 month old Fabry mice, while suppression in acetylcholine-mediated vasodilatation was evident from 2 months of age. Endothelium-independent dilatation with sodium nitroprusside was normal compared with age-matched WT mice. The microvascular defect in the Fabry MA was endothelium-dependent and associated with a suppression of the active homodimer of eNOS. Phosphorylation of eNOS at the major activation site (Ser1179) was significantly down-regulated while phosphorylation of the major inhibitory site (Thr495) was remarkably enhanced in aged Fabry mouse MA. These profound alterations in eNOS bioavailability at 8 months were observed in parallel with high levels of 3-nitrotyrosine suggesting increased reactive oxygen species along with eNOS uncoupling in this vascular bed. Overall, the mesenteric microvessels in the setting of Fabry disease were observed to have an early and profound endothelial dysfunction associated with elevated reactive nitrogen species and decreased nitric oxide (NO) bioavailability.
Paeonia lactiflora Pall is one of the most well-known herbs in China, Korea, and Japan for more than 1200 years. Paeoniflorin, the major bioactive component of paeony root, has recently been reported to have anti-colitic activity. However, the underlying molecular mechanism is unclear. The present study was to explore the possible mechanism of Paeoniflorin in attenuating dextran sulfate sodium (DSS)-induced colitis. Pre- and co-administration of Paeoniflorin significantly reduced the severity of colitis and resulted in downregulation of several inflammatory parameters in the colon, including the activity of myeloperoxidase (MPO), the levels of TNF-α and IL-6, and the mRNA expression of pro-inflammatory mediators (MCP-1, Cox2, IFN-, TNF-α, IL-6 and IL-17). The decline in the activation of NF-B p65, ERK, JNK and p38 MAPK correlated with a decrease in mucosal Toll-like receptor 4 (TLR4) but not TLR2 or TLR5 expression. In accordance with the in vivo results, Paeoniflorin downregulated TLR4 expression, blocked nuclear translocation of NF-B p65, and reduced the production of IL-6 in LPS-stimulated mouse macrophage RAW264.7 cells. Transient transfection assay performed in LPS-stimulated human colon cancer HT-29 cells indicated that Paeoniflorin inhibits NF-B transcriptional activity in a dose-dependent manner. TLR4 knockdown and overexpression experiments demonstrated a requirement for TLR4 in Paeoniflorin-mediated downregulation of inflammatory cytokines. Thus, for the first time, the present study indicates that Paeoniflorin abrogates DSS-induced colitis via decreasing the expression of TLR4 and suppressing the activation of NF-B and MAPK pathways.
Pulmonary vascular dilation and angiogenesis underlie experimental hepatopulmonary syndrome (HPS) induced by common bile duct ligation (CBDL) and may respond to receptor tyrosine kinase (RTK) inhibition. Vascular endothelial growth factor-A (VEGF-A) expression occurs in proliferating cholangiocytes and pulmonary intravascular monocytes after CBDL, the later contributing to angiogenesis. CBDL cholangiocytes also produce endothelin-1 (ET-1) which triggers lung vascular endothelin B receptor-mediated endothelial nitric oxide synthase (eNOS) activation and pulmonary intravascular monocyte accumulation. However, whether RTK pathway activation directly regulates cholangiocyte and pulmonary microvascular alterations in experimental HPS is not defined. We assessed RTK pathway activation in cholangiocytes and lung after CBDL and the effects of the type II RTK inhibitor, sorafenib in experimental HPS. Cholangiocyte VEGF-A expression and ERK activation accompanied proliferation and increased hepatic and circulating ET-1 levels after CBDL. Sorafenib decreased each of these events and led to a reduction in lung eNOS activation and intravascular monocyte accumulation. Lung monocyte VEGF-A expression and microvascular Akt and ERK activation were also found in vivo after CBDL and VEGF-A activated Akt and ERK and angiogenesis in rat pulmonary microvascular endothelial cells in vitro. Sorafenib inhibited VEGF-A mediated signaling and angiogenesis in vivo and in vitro and improved arterial gas exchange and intrapulmonary shunting. RTK activation in experimental HPS upregulates cholangiocyte proliferation and ET-1 production leading to pulmonary microvascular eNOS activation, intravascular monocyte accumulation and VEGF-A mediated angiogenic signaling pathways. These findings identify a novel mechanism in cholangiocytes through which RTK inhibition ameliorates experimental HPS.
Study objectives were to: mine the complete exome to identify putative rare single nucleotide variants (SNVs) associated with irritable bowel syndrome-diarrhea (IBS D) phenotype; assess genes that regulate bile acids in IBS-D; explore univariate associations of SNVs with symptom phenotype and quantitative traits in independent IBS cohort. Using principal component analysis, we identified 2 groups of IBS-D (n=16) with increased fecal bile acids: rapid colonic transit or high bile acids synthesis. DNA was sequenced indepth, analyzing SNVs in bile acids genes (ASBT, FXR, OSTα/β, FGF19, FGFR4, KLB, SHP, CYP7A1, LRH-1, and FABP6). Exome findings were compared to 50 similar ethnicity controls. We assessed univariate associations of each SNV with quantitative traits and principal component analysis and associations between SNVs in KLB and FGFR4 and symptom phenotype in 405 IBS, 228 controls and colonic transit in 70 IBS-D, 71 IBS-C. Mining the complete exome did not reveal significant associations with IBS-D over controls. There were 54 SNVs in 10/11 bile acids-regulating genes, with no SNVs in FGF19; 15 non-synonymous SNVs were identified in similar proportions of IBS-D and controls. Variations in KLB (rs1015450, downstream) and FGFR4 (rs434434 [intronic], rs1966265, and rs351855 [non-synonymous]) were associated with colonic transit (rs1966265; P=.043), fecal bile acids (rs1015450; P=.064), and PCA groups (all 3 FGFR4 SNVs; P<.05). In the 633 person cohort, FGFR4 rs434434 was associated with symptom phenotype (P=.027) and rs1966265 with 24h colonic transit (P=.066). Thus, exome sequencing identified additional variants in KLB and FGFR4 associated with bile acids or colonic transit in IBS-D.
It is known that chronic ethanol significantly impairs liver regeneration. However, the effect of acute ethanol exposure on liver regeneration remains largely unknown. To address this question, C57Bl6/J mice were exposed to acute ethanol (6 g/kg i.g.) for 3 days and partial hepatectomy (PHx) was performed 24 h after the last dose. Surprisingly, acute ethanol preexposure promoted liver regeneration. This effect of ethanol did not correlate with changes in expression of cell cycle regulatory genes (e.g., cyclin D1, p21 and p27), but did correlate with protection against the effect of PHx on indices of impaired lipid and carbohydrate metabolism. Ethanol preexposure protected against inhibition of the oxidant-sensitive mitochondrial enzyme, aconitase. The activity of aldehyde dehydrogenase 2 (ALDH2) was significantly increased by ethanol preexposure. The effect of ethanol was blocked by inhibiting (daidzin) and was mimicked by activating (alda-1) ALDH2. Lipid peroxides are also substrates for ALDH2; indeed, alcohol preexposure blunted the increase in lipid peroxidation (4OH-nonenal adducts) caused by PHx. Taken together, these data suggest that acute preoperative ethanol exposure 'preconditions' the liver to respond more rapidly to regenerate after PHx by activating mitochondrial ALDH2, which prevents oxidative stress in this compartment.
SLC26A3 (Down-regulated in adenoma, DRA) is a Cl-/HCO3- exchanger involved in electroneutral NaCl absorption in the mammalian intestine. Altered DRA expression levels are associated with infectious and inflammatory diarrheal diseases. Therefore, it is critical to understand the regulation of DRA expression. MicroRNAs (miRNAs) are endogenous, small RNAs that regulate protein expression via blocking the translation and/or promoting mRNA degradation. In order to investigate potential modulation of DRA expression by microRNA, five different in silico algorithms were used to predict the miRNAs that target DRA. Of these miRNAs, miR-494 was shown to have a highly conserved putative binding site in the DRA 3'untranslated region (3'UTR) compared to other DRA-targeting miRNAs in vertebrates. Transfection with pmirGLO dual luciferase vector containing DRA 3'UTR (pmirGLO-3'UTR DRA) resulted in a significant decrease in relative luciferase activity as compared to empty vector. Co-transfection of the DRA 3'UTR luciferase vector with a miR-494 mimic further decreased luciferase activity as compared to cells transfected with negative control. The transfection of a miR-494 mimic into Caco2 and T-84 cells significantly increased the expression of miR-494 and concomitantly decreased the DRA protein expression. Mutation of the seed sequences for miR-494 in 3'UTR of DRA abrogated the effect of miR-494 on 3'UTR. These data demonstrate a novel regulatory mechanism of DRA expression via miR-494 and indicate that targeting this microRNA may serve to be a potential therapeutic strategy for diarrheal diseases.
Background & Aims: Farnesoid X receptor (FXR) is a bile acid nuclear receptor described through mouse knockout studies as a tumor suppressor for the development of colon adenocarcinomas. This study investigates the regulation of FXR in the development of human colon cancer. Methods: Immunohistochemistry of FXR in normal (n=238), polyps (n=32), and adenocarcinomas, staged I-IV (n= 43, 39, 68, and 9), of the colon and RT-PCR, reverse phase protein array (RPPA) and western blot analysis in 15 colon cancer cell lines, NR1H4 promoter methylation and mRNA expression in colon cancer samples from The Cancer Genome Atlas (TCGA), DNA methyltransferase inhibition, methyl-DNA immunoprecipitation (MeDIP), bisulfite sequencing and KRAS knock-down assessed were used to investigate FXR regulation in colon cancer development. Results: IHC and RT-qPCR revealed the expression and function of FXR was reduced in precancerous lesions and silenced in majority of stage I-IV tumors. FXR expression negatively correlated with PI3-kinase signaling and the epithelial-to-mesenchymal transition (EMT). The NR1H4 promoter is methylated in ~12% colon cancer TCGA samples, and methylation patterns segregate with tumor sub-types. Inhibition of DNA methylation and KRAS silencing both increased FXR expression. Conclusion: FXR expression is decreased early in human colon cancer progression and both DNA methylation and KRAS signaling may be contributing factors to FXR silencing. FXR potentially suppresses EMT and other oncogenic signaling cascades and restoration of FXR activity, by blocking silencing mechanisms or increasing residual FXR activity, represents promising therapeutic options for the treatment of colon cancer.
Fructose consumption by Americans has increased markedly whereas Ca2+ intake has decreased below recommended levels. Since fructose metabolism decreases enterocyte ATP concentrations, we tested the hypothesis that luminal fructose acutely reduces active, diet-inducible Ca2+ transport in the small intestine. We confirmed that the decrease in ATP concentrations was indeed greater in fructose- compared to glucose-incubated mucosal homogenates from wildtype, and was prevented in fructose-incubated homogenates from ketohexokinase (KHK)-/-, mice. We then induced active Ca2+ transport by chronically feeding wildtype, fructose transporter GLUT5-/-, as well as KHK-/- mice, a low Ca2+ diet, and measured transepithelial Ca2+ transport in everted duodenal sacs incubated in solutions containing glucose, fructose or their nonmetabolizable analogs. The diet-induced increase in active Ca2+ transport was proportional to dramatic increases in expression of the Ca2+-selective channel TRPV6 as well as of the Ca2+ binding protein CaBP9k, but not that of the voltage-dependent L-type channel Ca(v)1.3. Crypt-villus distribution of CaBP9k seems heterogeneous, but low Ca2+ diets induce expression in more cells. In contrast, KHK distribution is homogeneous, suggesting that fructose metabolism can occur in all enterocytes. Diet-induced Ca2+ transport was not enhanced by addition of the enterocyte fuel glutamine, and was always greater in sacs of wildtype, GLUT5-/- and KHK-/- mice incubated with fructose or nonmetabolizable sugars than those incubated with glucose. Thus, duodenal Ca2+ transport is not affected by fructose and enterocyte ATP concentrations but instead may decrease with glucose metabolism, as Ca2+ transport remains high with 3-O-methylglucose that is also transported by the glucose transporter SGLT1, but cannot be metabolized.
The gastrointestinal peptide cholecystokinin (CCK) causes the release of pancreatic digestive enzymes and growth of the normal pancreas. Exogenous CCK administration has been used in animal models to study pancreatitis and also as a promoter of carcinogen-induced or Kras-driven pancreatic cancer. Defining CCK receptors in normal human pancreas has been problematic due to its retroperitoneal location, high concentrations of pancreatic proteases, and endogenous RNase. Most studies indicate that the predominant receptor in human pancreas is the CCK-B type, and CCK-A is the predominant form in rodent pancreas. In pancreatic cancer cells and tumors, the role of CCK is better established because receptors are often over-expressed by these cancer cells and stimulation of such receptors promotes growth. Furthermore, in established cancer, endogenous production of CCK and/or gastrin occurs and their actions stimulate the synthesis of more receptors plus growth by an autocrine mechanism. Initially it was thought that the mechanism by which CCK served to potentiate carcinogenesis was by interplay with inflammation in the pancreatic microenvironment. But with the recent findings of CCK receptors on early PanIN lesions and on stellate cells, the question has been raised that perhaps CCK actions are not the result of cancer but an early driving promoter of cancer. This review will summarize what is known regarding CCK, its receptors, and pancreatic cancer, and also what is unknown and requires further investigation to determine which comes first, the chicken or the egg; 'CCK or the cancer."
Caveolae are specialized regions of the plasma membrane that concentrate receptors and associated signaling molecules critical in regulation of cellular response to transmitters and hormones. We have determined the effects of caveolin-1 (Cav-1) deletion, caveolin-1 siRNA, and caveolar disruption in mice on the signaling pathways that mediate contraction and relaxation in colonic smooth muscle, and on the components of the peristaltic reflex in isolated tissue and propulsion in intact colonic segments. In Cav-1-/- mice, both relaxation and contraction were decreased in smooth muscle cells and muscle strips, as well as during both phases of the peristaltic reflex and colonic propulsion. The decrease in relaxation in response to the nitric oxide (NO) donor was accompanied by a decrease in cGMP levels and an increase in phosphodiesterase 5 (PDE5) activity. Relaxation by a PDE5-resisitant cGMP analog was not affected in smooth muscle of Cav-1-/- mice suggesting that inhibition of relaxation was due to augmentation of PDE5 activity. Similar effects on relaxation, PDE5 and cGMP were obtained in muscle cells upon disruption of caveolae by methyl-β-cyclodextrin or suppression of Cav-1. Sustained contraction mediated via inhibition of MLCP activity is regulated by Rho kinase and PKC via phosphorylation of two endogenous inhibitors of MLCP; MYPT1 and CPI-17 respectively. The activity of both enzymes and phosphorylation of MYPT1 and CPI-17 were decreased in smooth muscle from Cav-1-/- mice. We conclude that the integrity of caveolae is essential for contractile and relaxant activity in colonic smooth muscle and the maintenance of neuromuscular function at organ level.
Preterm birth, bacterial colonization and formula feeding predispose to necrotizing enterocolitis (NEC). Antibiotics are commonly administered to prevent sepsis in preterm infants, but it is not known whether this affects intestinal immunity and NEC resistance. We hypothesized that broad-spectrum antibiotics treatment improves NEC resistance and intestinal structure, function and immunity in neonates. Caesarean-delivered preterm pigs were fed 3 d of parenteral nutrition followed by 2 d of enteral formula. Immediately after birth they were assigned to receive either antibiotics (oral and parenteral doses of gentamycin, ampicillin and metronidazole, ANTI, n=11) or saline in the control group (CON, n=13), given twice daily. NEC-lesions and intestinal structure, function, microbiology and immunity markers were recorded. None of the ANTI but 85% of the CON pigs developed NEC lesions by d 5 (0/11 vs. 11/13, P<0.05). ANTI pigs had higher intestinal villi (+60%), digestive enzyme activities (+53-73%), goblet cell densities (+110%), and lower myeloperoxidase (-51%) and colonic microbial density (105 vs. 1010 CFU, all p<0.05). Microarray transcriptomics showed strong down-regulation of genes related to inflammation and innate immune response to microbiota and marked up-regulation of genes related to amino acid metabolism, in particular threonine, glucose transport systems and cell cycle in 5 d-old ANTI pigs. In a follow-up experiment, 5 d of antibiotics prevented NEC at least until day 10. Neonatal prophylactic antibiotics effectively reduced gut bacterial load, prevented NEC, intestinal atrophy, dysfunction and inflammation, and enhanced expression of genes related to gut metabolism and immunity in preterm pigs.
Recent studies indicate the metabolic oxidative stress, autophagy and inflammation are hallmarks of nonalcoholic steatohepatitis (NASH) progression. However the molecular mechanisms that link these important events in NASH remain unclear. In this study we investigated the mechanistic role of purinergic receptor X7 in modulating autophagy and resultant inflammation in NASH in response to metabolic oxidative stress. The study uses two rodent models of NASH. In one of them, a CYP2E1 substrate bromodichloromethane is used to induce metabolic oxidative stress and NASH. Methyl choline deficient diet feeding is used for the other NASH model. CYP2E1 and P2X7 receptor gene deleted mice are used to establish their roles in regulating metabolic oxidative stress and autophagy. Autophagy gene expression, protein levels, confocal microscopy based-immunolocalization of LAMP2A and histopathological analysis were performed. CYP2E1-dependent metabolic oxidative stress induced increases in P2X7 receptor expression, chaperone-mediated autophagy markers LAMP2A and Hsc 70 but caused depletion of LC3B protein levels. P2X7 receptor gene deletion significantly decreased LAMP2A and inflammatory indicators while significantly increasing LC3B protein levels as compared to wild type mice treated with BDCM. P2X7 receptor deleted mice were also protected from NASH pathology as evidenced by decreased inflammation and fibrosis. Our studies establish that P2X7 receptor is a key regulator of autophagy induced by metabolic oxidative stress in NASH, thereby modulating hepatic inflammation. Further our findings presented here forms a basis for P2X7 receptor as a potential therapeutic target in the treatment for NASH.
For preterm neonates, the quality of the first milk is crucial for intestinal maturation and resistance to necrotizing enterocolitis (NEC). Among other factors, milk quality is determined by the stage of lactation and processing. We hypothesized that unprocessed mature bovine milk (BM, raw bovine milk) would have less bioactivity than corresponding bovine colostrum (BC) in a preterm pig model, but have improved bioactivity relative to its homogenized, pasteurized, spray-dried equivalent, whole milk powder (WMP), or a bovine milk protein based infant formula (IF). For five days, newborn preterm pigs received parenteral and enteral nutrition consisting of IF (n = 13), BM (n = 13), or BC (n = 14). In a second study, WMP (n = 15) was compared with the above-mentioned IF (n = 10) and BM (n = 9). Compared with IF pigs, BM pigs had significantly improved intestinal structure (mucosal weight, villus height) and function (increased nutrient absorption and enzyme activities, decreased gut permeability, nutrient fermentation and NEC severity). BC further improved these effects, relative to BM (lactase activity, lactose absorption, plasma citrulline, and tissue IL-8). WMP induced similar effects as BM, except for lactase activity and lactose absorption. In conclusion, the maturational and protective effects on the immature intestine decreased in the order BC>BM>WMP, but all three intact bovine milk diets were markedly better than IF. The stage of lactation (colostrum versus mature milk) and milk processing (e.g. homogenization, fractionation, pasteurization, spray-drying) are important factors determining milk quality during the early postnatal period of preterm neonates.
The outcome of liver injury is determined by the success of repair. Liver repair involves replacement of damaged liver tissue with healthy liver epithelial cells (including both hepatocytes and cholangiocytes), and reconstruction of normal liver structure and function. Current dogma posits that replication of surviving mature hepatocytes and cholangiocytes drives the regeneration of liver epithelium after injury, while failure of liver repair commonly leads to fibrosis, a scaring condition in which hepatic stellate cells, the main liver-resident mesenchymal cells, play the major role. The current review discusses other mechanisms that might be responsible for the regeneration of new liver epithelial cells and outgrowth of matrix-producing mesenchymal cells during hepatic injury. This theory proposes that during liver injury, some epithelial cells undergo epithelial-to-mesenchymal transition (EMT), acquire myofibroblastic phenotypes/features and contribute to fibrogenesis, while certain mesenchymal cells (namely hepatic stellate cells and stellate cell-derived myofibroblasts) undergo mesenchymal-to-epithelial transition (MET), revert to epithelial cells, and ultimately differentiate into either hepatocytes or cholangiocytes. Although this theory is highly controversial, it suggests that the balance between EMT and MET modulates the outcome of liver injury. This review summarizes recent advances that support or refute the concept that certain types of liver cells are capable of phenotype transition (i.e. EMT and MET) during both culture conditions and chronic liver injury.
Cytokines play important roles in all stages of steatohepatitis, including hepatocyte injury, the inflammatory response and the altered function of sinusoidal cells. This study examined the involvement of a major inflammatory cytokine, IFN-, in the progression of steatohepatitis. In a steatohepatitis model by feeding a methionine and choline-deficient high fat (MCDHF) diet to both wild-type and IFN--deficient mice, the liver histology, expression of genes encoding inflammatory cytokines and fibrosis-related markers were examined. To analyze the effects of IFN- on Kupffer cells in vitro, we examined the TNF-α production by a mouse macrophage cell line. 42 days of MCDHF diet resulted in weight loss, elevated aminotransferases, liver steatosis and inflammation in wild-type mice. However, the IFN--deficient mice exhibited less extensive changes. RT-PCR revealed that the expression of TNF-α, TGF-β1, iNOS, IL-4 and osteopontin were increased in wild-type mice, although they were suppressed in IFN--deficient mice. 70 days of MCDHF diet induced much more liver fibrosis in wild-type mice than in IFN--deficient mice. The expression levels of fibrosis-related genes, α-SMA, type-I collagen, TIMP-1 and MMP-2 were dramatically increased in wild-type mice, while they were significantly suppressed in IFN--deficient mice. Moreover, in vitro experiments showed that when RAW 264.7 macrophages were treated with IFN-, they produced TNF-α in a dose-dependent manner. The present study showed that IFN- deficiency might inhibit the inflammatory response of macrophages cells, subsequently suppress stellate cell activation and liver fibrosis. These findings highlight the critical role of IFN- in the progression of steatohepatitis.
Acetate, propionate and butyrate are the main short-chain fatty acids (SCFAs) that arise from the fermentation of fibers by the colonic microbiota. While many studies focus on the regulatory role of SCFAs, their quantitative role as a catabolic or anabolic substrate for the host has received relatively little attention. To investigate this aspect, we infused conscious mice with physiological quantities of stable isotopes [1-13C]acetate, [2-13C]propionate or [2,4-13C2]butyrate directly into the cecum, which is the natural production site in mice, and analyzed their interconversion by the microbiota as well as their metabolism by the host. Cecal interconversion - pointing to microbial cross-feeding - was high between acetate and butyrate, low between butyrate and propionate and almost absent between acetate and propionate. As much as 62% of infused propionate was used in whole-body glucose production, in line with its role as gluconeogenic substrate. Conversely, glucose synthesis from propionate accounted for 69% of total glucose production. The synthesis of palmitate and cholesterol in the liver was high from cecal acetate (2.8% and 0.7%, respectively) and butyrate (2.7% and 0.9%, respectively) as substrates, but low or absent from propionate (0.6% and 0.0%, respectively). Label incorporation due to chain elongation of stearate was approximately 8-fold higher than de novo synthesis of stearate. Microarray data suggested that SCFAs exert only a mild regulatory effect on the expression of genes involved in hepatic metabolic pathways during the 6h infusion period. Altogether, gut-derived acetate, propionate and butyrate play important roles as substrates for glucose, cholesterol and lipid metabolism.
The present study investigates the relative ability of α-Toc, -Toc and -Toc to modulate cell signaling events that are associated with inflammatory responses in fetal-derived (FHs 74 Int) intestinal cells. The secretion of pro-inflammatory IL8 cytokine secretion in FHs 74 Int cells was stimulated in the order α–Toc< – Toc < – Toc. A similar pro-inflammatory response was observed when inflammation was induced in FHs 74 Int cells. The effect of Toc to modulate IL8 expression corresponded to an isoform-specific modulation of NfB and Nrf-2 cell signaling pathways involved in the expression of pro-inflammatory cytokines and antioxidant enzymes, respectively. Delta-Toc and to a lesser extent -Toc activated both NfB and Nrf-2 signaling, as indicated by the greater nuclear translocation of transcription factors. Activation of NfB signaling by - and -Toc was accompanied by the up-regulation of NfB target genes, such as IL8 and PTGS2, occurring both with and without a prior IFN/PMA challenge. Nevertheless, - and -Tocs, particularly -Toc, concurrently down-regulated glutatamate cysteine ligase (GCLC), a Nrf-2 target gene that encodes for glutathione biosynthesis. Substantiation of this observation was made by confirming that both -Toc and -Toc were effective at decreasing GCLC protein expression and cellular glutathione content. Down-regulation of the glutathione content in fetal intestinal cells corresponded to the induction of apoptosis-mediated cytotoxicity. In conclusion, - and -Tocs are biologically active forms of vit E and show superior bioactivity to α-Toc in modulating cell signaling events that contribute to a pro-inflammatory response in fetal-derived intestinal cells.
Helicobacter pylori is the strongest risk factor for gastric cancer and strains harboring the cag pathogenicity island, which translocates the oncoprotein CagA into host cells, further augment cancer risk. We previously reported that in vivo adaptation of a non-carcinogenic H. pylori strain (B128) generated a derivative strain (7.13) with the ability to induce adenocarcinoma, providing a unique opportunity to define mechanisms that mediate gastric carcinogenesis. MicroRNAs (miRNAs) are small noncoding RNAs that regulate expression of oncogenes or tumor suppressors and are frequently dysregulated in carcinogenesis. To identify miRNAs and their targets involved in H. pylori-mediated carcinogenesis, miRNA microarrays were performed on RNA isolated from gastric epithelial cells co-cultured with H. pylori strains B128, 7.13, or a 7.13 cagA- isogenic mutant. Among 61 miRNAs differentially expressed in a cagA-dependent manner, the tumor suppressor miR-320 was significantly downregulated by strain 7.13. Since miR-320 negatively regulates the anti-apoptotic protein Mcl-1, we demonstrated that H. pylori significantly induced Mcl-1 expression in a cagA-dependent manner and that suppression of Mcl-1 results in increased apoptosis. To extend these results, mice were challenged with H. pylori strain 7.13 or its cagA- mutant; consistent with cell culture data, H. pylori induced Mcl-1 expression in a cagA-dependent manner. In human subjects, cag+ strains induced significantly higher levels of Mcl-1 than cag- strains, and Mcl-1 expression levels paralleled the severity of neoplastic lesions. Collectively, these results indicate that H. pylori suppresses miR-320, upregulates Mcl-1, and decreases apoptosis in a cagA-dependent manner, which likely confers an increased risk for gastric carcinogenesis.
The crural diaphragm (CD) is an essential component of the esophagogastric junction (EGJ) and inspiratory exercises may modify its function. This study's goal is to verify if inspiratory muscle training (IMT) improves EGJ motility and gastroesophageal reflux (GER). Twelve GERD (7 males, 20-47 years, 9 esophagitis, 3 NERD) and 7 healthy volunteers (3 males, 20-41 years) performed esophageal pH monitoring, manometry, and heart rate variability (HRV) studies. A 6-cm-sleeve catheter measured average EGJ pressure during resting, peak inspiratory EGJ pressures during sinus arrhythmia maneuver (SAM) and inhalations under 17-cmH2O, 35-cmH2O, and 70-cmH2O loads (TH maneuvers), and along 1 hour after a meal. GERD patients entered a 5-days-a-week IMT program. One author scored heartburn and regurgitation before and after IMT. IMT increased average EGJ pressure (19.7 ± 2.4 mmHg v 29.5 ± 2.1 mmHg, p < 0.001), inspiratory EGJ pressure during SAM (89.6 ± 7.6 mmHg v 125.6 ± 13.3 mmHg, p = 0.001), and during TH maneuvers. The EGJ-pressure gain across 35 cmH2O and 70 cmH2O loads was lower for GERD volunteers. The number and cumulative duration of the tLESR decreased after IMT. Proximal progression of GER decreased after IMT but not the distal acid exposure. LF power increased after IMT and the higher its increment the lower the increment of supine acid exposure. IMT decreased heartburn and regurgitation scores. In conclusion, IMT improved EGJ pressure, reduced GER proximal progression, and GERD symptoms. Some GERD patients have a CD failure and IMT may prove beneficial as a GERD add-on treatment.
A key pathologic feature of the systemic inflammatory response of sepsis/endotoxemia is the accumulation of neutrophils within the microvasculature of organs such as the liver, where they cause tissue damage and vascular dysfunction. There is emerging evidence that the vascular endothelium is critical to the orchestration of inflammatory responses to blood-borne microbes and microbial products in sepsis/endotoxemia. In this study, we aimed to understand the role of endothelium, and specifically endothelial TLR4 activation, in the regulation of neutrophil recruitment to the liver during endotoxemia. Intravital microscopy of bone marrow chimeric mice revealed that TLR4 expression by non-bone marrow derived cells was required for neutrophil recruitment to the liver during endotoxemia. Furthermore, LPS-induced neutrophil adhesion in liver sinusoids was equivalent between wildtype mice and transgenic mice that express TLR4 only on endothelium (tlr4-/-Tie2tlr4), revealing that activation of endothelial TLR4 alone was sufficient to initiate neutrophil adhesion. Neutrophil arrest within sinusoids of endotoxemic mice requires adhesive interactions between neutrophil CD44 and endothelial hyaluronan. Intravital immunofluorescence imaging demonstrated that stimulation of endothelial TLR4 alone was sufficient to induce the deposition of serum-derived hyaluronan associated protein (SHAP) within sinusoids, which was required for CD44/hyaluronan-dependent neutrophil adhesion. In addition to endothelial TLR4 activation, Kupffer cells contribute to neutrophil recruitment via a distinct CD44/HA/SHAP-independent mechanism. This study sheds new light on the control of innate immune activation within the liver vasculature during endotoxemia, revealing a key role for endothelial cells as sentinels in the detection of intravascular infections and coordination of neutrophil recruitment to the liver.
Ethanol ingestion increases endogenous glucocorticoid levels in both humans and rodents. The present study aimed to define a mechanistic link between the increased glucocorticoids and alcoholic fatty liver in mice. Plasma corticosterone levels were not affected in mice on a 2-week ethanol diet regimen, but significantly increased upon four weeks of ethanol ingestion. Accordingly, hepatic triglyceride levels were not altered after two weeks of ethanol ingestion, but were elevated at four weeks. Based on the observation that two weeks of ethanol ingestion did not significantly increase endogenous corticosterone levels, we administered exogenous glucocorticoids along with the 2-week ethanol treatment to determine whether the elevated glucocorticoid contributes to the development of alcoholic fatty liver. Mice were subjected to ethanol feeding for two weeks with or without dexamethasone administration. Hepatic triglyceride contents were not affected by either ethanol or dexamethasone alone, but were significantly increased by administration of both. Microarray and protein level analyses revealed two distinct changes in hepatic lipid metabolism in mice administered with both ethanol and dexamethasone: accelerated triglyceride synthesis by diacylglycerol O-acyltransferase 2, and suppressed fatty acid β-oxidation by long chain acyl-CoA synthetase1, carnitine palmitoyltransferase 1a, and acyl-CoA oxidase 1. A reduction of hepatic peroxisome proliferation activator receptor α (PPARα) was associated with co-administration of ethanol and dexamethasone. These findings suggest that increased glucocorticoid levels may contribute to the development of alcoholic fatty liver, at least partially, through hepatic PPARα inactivation.
Gut microbiota alternations are associated with various disorders. In this study, gut microbiota changes were investigated in a methionine-choline deficient (MCD) diet-induced non-alcoholic steatohepatitis (NASH) rodent model, and the effects of administering Lactobacillus casei strain Shirota (LcS) on the development of NASH were also investigated. Mice were divided into three groups, given the normal chow diet (NCD), MCD diet, or the MCD diet plus daily oral administration of LcS for 6 weeks. Gut microbiota analyses for the three groups revealed that lactic acid bacteria such as Bifidobacterium and Lactobacillus in feces were markedly reduced by the MCD diet. Interestingly, oral administration of LcS to MCD diet-fed mice increased not only the L.casei subgroup but also other lactic acid bacteria. Subsequently, NASH development was evaluated based on hepatic histochemical findings, serum parameters and various mRNA and/or protein expression levels. LcS intervention markedly suppressed MCD-diet induced NASH development, with reduced serum lipopolysaccharide concentrations, suppression of inflammation and fibrosis in the liver, and reduced colon inflammation. Therefore, reduced populations of lactic acid bacteria in the colon may be involved in the pathogenesis of MCD diet-induced NASH, suggesting normalization of gut microbiota to be effective for treating NASH.
Endotoxemia is a causal factor in the development of alcoholic liver injury. The present study aimed at determining the interactions of ethanol with different fat sources at the gut-liver axis. Male Sprague Dawley rats were pair-fed control or ethanol liquid diets for 8 weeks. The liquid diets were based on the Lieber-DeCarli formula, with 30% total calories derived from corn oil (rich in polyunsaturated fatty acids). To test the effects of saturated fats, corn oil in the ethanol diet was replaced by either cocoa butter (CB, rich in long chain saturated fatty acids) or medium chain triglycerides (MCT, exclusively medium chain saturated fatty acids). Ethanol feeding increased hepatic lipid accumulation and inflammatory cell infiltration, and perturbed hepatic and serum metabolite profiles. Ethanol feeding with CB or MCT alleviated ethanol-induced liver injury and attenuated ethanol-induced metabolic perturbation. Both CB and MCT also normalized ethanol-induced hepatic macrophage activation, cytokine expression and neutrophil infiltration. Ethanol feeding elevated serum endotoxin level, which was normalized by MCT but not CB. In accordance, ethanol-induced downregulations of intestinal occludin and ZO-1 were normalized by MCT but not CB. However, CB normalized ethanol-increased hepatic endotoxin level in association with upregulation of the endotoxin detoxifying enzymes argininosuccinate synthase 1 (ASS1). Knockdown ASS1 in H4IIEC3 cells resulted in impaired endotoxin clearance and upregulated cytokine expression. These data demonstrate that the protection of saturated fats against alcohol-induced liver injury occur via different actions at the gut-liver axis and are chain length dependent.
Background: We have recently identified hexamethonium-resistant peristalsis in the guinea-pig colon. We showed that following acute blockade of nicotinic receptors, peristalsis recovers leading to normal propagation velocities of fecal pellets along the colon. This raises the fundamental question as to what mechanisms underlie hexamethonium-resistant peristalsis. Aims: In this study we investigated whether blockade of the major receptors that underlie excitatory neuro-muscular transmission are required for hexamethonium-resistant peristalsis to occur. Methods: Video imaging of colonic wall movements was used to make spatio-temporal maps and determine the velocity of peristalsis. Propagation of artificial fecal pellets in the guinea-pig distal colon was studied in hexamethonium, atropine, -conotoxin (GVIA), ibodutant (MEN15596) and tetrodotoxin (TTX). Results: Hexamethonium and ibodutant alone did not retard peristalsis. In contrast, -conotoxin abolished peristalsis in a proportion of preparations, and reduced the velocity of propagation in all remaining specimens. In fact, peristalsis could still occur, in a proportion of animals, in the combined presence of hexamethonium, atropine, ibodutant and -conotoxin. Peristalsis never occurred in the presence of tetrodotoxin. Discussion: The major finding of the current study is the unexpected observation that peristalsis can still occur after blockade of the major excitatory neuro-neuronal and neuro-muscular transmitters. Also, the colon retained an intrinsic polarity in the presence of these antagonists and was only able to expel pellets in an aboral direction. The nature of the mechanism(s)/ neurotransmitter(s) that generate peristalsis and facilitates natural fecal pellet propulsion, after blockade of major excitatory neurotransmitters, at the neuro-neuronal and neuro-muscular junction remains to be identified.
PN-Associated Liver Disease (PNALD) is a life-threatening complication of the administration of parenteral nutrition (PN). The development of PNALD may be partly due to the composition of the lipid emulsion administered with PN: soy-based lipid emulsions (SOLE) are associated with liver disease, while fish oil-based lipid emulsions (FOLE) are associated with prevention and improvement of liver disease. The objective of this study was to determine how the choice of lipid emulsion modified the production of bioactive lipid mediators. We utilized a mouse model of steatosis to study the differential effect of FOLE and SOLE. We subsequently validated these results in serum samples obtained from a small cohort of human infants transitioning from SOLE to FOLE. In mice, FOLE was associated with the production of anti-inflammatory, pro-resolving lipid mediators; SOLE was associated with increased production of inflammatory lipid mediators. In human infants, the transition from SOLE to FOLE was associated with a shift towards a pro-resolving lipidome. Together, these results demonstrate that the composition of the lipid emulsion directly modifies inflammatory homeostasis.
Hepatic stellate cells (HSC) and liver endothelial cells (LEC) migrate to sites of injury and perpetuate alcohol induced liver injury. High mobility group box 1 (HMGB1) is a protein released from the nucleus of injured cells that has been implicated as a proinflammatory mediator. We hypothesized that HMGB1 may be released from ethanol-stimulated liver parenchymal cells and contribute to HSC and LEC recruitment. Ethanol stimulation of rat hepatocytes and HepG2 cells resulted in translocation of HMGB1 from the nucleus as assessed by Western blot. HMGB1 protein levels were increased in the supernatant of ethanol-treated hepatocytes compared to vehicle-treated cells. Migration of both HSC and LEC was increased in response to conditioned media for ethanol-stimulated hepatocytes (CMEtOH) compared to vehicle-stimulated hepatocytes (CMVEH) (P<0.05). However, the effect of CMEtOH on migration was almost entirely reversed by treatment with HMGB1-neutralizing antibody or when HepG2 cells were pre-transfected with HMGB1-siRNA compared to control siRNA transfected HepG2 cells (P<0.05). Recombinant HMGB1 (100 ng/mL) also stimulated migration of HSC and LEC compared to vehicle stimulation (P<0.05 for both HSC and LEC). HMGB1 stimulation of HSC increased the phosphorylation of Src and Erk and HMGB1-induced HSC migration was blocked by the Src inhibitor PP2, and Erk inhibitor, U0126. Hepatocytes release HMGB1 in response to ethanol with subsequent recruitment of HSC and LEC. This pathway has implications for HSC and LEC recruitment to sites of ethanol induced liver injury.
BACKGROUND & AIMS: Farnesoid X receptor (FXR, Nr1h4) is a ligand-activated transcription factor belonging to the nuclear receptor superfamily. FXR is essential in maintaining bile acid (BA) homeostasis and FXR-/- mice develop cholestasis, inflammation, and spontaneous liver tumors. The signal transducer and activator of transcription 3 (STAT3) is well-known ro regulate liver growth, and STAT3 is feedback inhibited by its target gene, the suppressor of cytokine signaling 3 (SOCS3). Strong activation of STAT3 was detected in FXR-/- mouse livers. However, the mechanism of STAT3 activation with FXR deficiency remains elusive. METHODS: Wild type and FXR-/- mice were used to detect STAT3 pathway activation in the liver. In vivo BA feeding or deprivation was used to determine the role of BAs in STAT3 activation and in vitro molecular approaches were used to determine the direct transcriptional regulation of SOCS3 by FXR. RESULTS: STAT3 was activated in FXR-/- but not WT mice. BA feeding increased, but deprivation by cholestyramine reduced, serum inflammatory markers and STAT3 activation. Furthermore, the Socs3 gene was determined as a direct FXR target gene. CONCLUSIONS: The elevated BAs and inflammation, along with reduced SOCS3, collectively contribute to the activation of the STAT3 signaling pathway in the liver of FXR-/- mice. This study suggests that the constitutive activation of STAT3 maybe a mechanism of liver carcinogenesis in FXR-/- mice.
Changes in substrate utilization and reduced mitochondrial function following exposure to energy dense, high fat diets are putatively key components in the development of obesity related metabolic disease. Here we examined the effect of a 3-day high-fat diet (HFD) on isolated liver mitochondrial respiration and whole-body energy utilization in obesity prone (OP) rats. We also examined if hepatic overexpression (o/e) of peroxisomal proliferator-activated receptor co-activator-1α (PGC-1α), a master regulator of mitochondrial function and biogenesis would modify liver and whole body responses to the high fat diet. Acute, 3-day HFD (45% kcals) in OP rats resulted in increased daily energy intake, energy balance, weight gain, and adiposity; without producing increased liver triglyceride (TAG) accumulation. HFD fed OP rats also displayed decreased whole body substrate switching from the dark to light cycle which was paired with reductions in hepatic mitochondrial respiration of multiple substrates under multiple respiratory states. Hepatic PGC-1α o/e was observed to protect whole body substrate switching, as well as, maintaining mitochondrial respiration following the acute HFD. Additionally, liver PGC-1α o/e did not alter whole-body dietary fatty acid oxidation, but resulted in greater storage of dietary FFA in liver lipid, primarily as TAG. Together, these data demonstrate that short-term HFD can result in decreased metabolic flexibility and hepatic mitochondrial function in OP rats, which is completely prevented by hepatic overexpression of PGC-1α.
Multidrug resistance-associated protein 2 (MRP2)/ATP-binding cassette protein C2 (ABCC2) and multidrug resistance protein 1 (MDR1)/ABCB1 are well-known efflux transporters located on the brush border membrane of the small intestinal epithelia, where they limit the absorption of a broad range of substrates. The expression patterns of MRP2/ABCC2 and MDR1/ABCB1 along the small intestinal tract are tightly regulated. Several reports have demonstrated the participation of ERM (ezrin/radixin/moesin) proteins in the post-translational modulation of MRP2/ABCC2 and MDR1/ABCB1, especially with regard to their membrane localization. The present study focused on the in vivo expression profiles of MRP2/ABCC2, MDR1/ABCB1, ezrin, and phosphorylated ezrin to further elucidate the relationship between the efflux transporters and the ERM proteins. The current results showed good correlation between the phosphorylation status of ezrin and Mrp2/Abcc2 expression along the gastrointestinal tract of rats, and between the expression profiles of both ezrin and Mdr1/Abcb1 in the small intestine. We also demonstrated the involvement of conventional protein kinase C isoforms in the regulation of ezrin phosphorylation. Furthermore, experiments conducted with wild-type (WT) ezrin and a T567A (Ala substituted Thr) dephosphorylated mutant showed a decrease in membrane surface-localized and total expressed MRP2/ABCC2 in T567A-expressing vs. WT ezrin-expressing Caco-2 cells. In contrast, T567A- and WT-expressing cells both showed an increase in membrane surface-localized and total expressed MDR1/ABCB1. These findings suggest that the phosphorylation status and the expression profile of ezrin differentially direct MRP2/ABCC2 and MDR1/ABCB1 expression, respectively, along the small intestinal tract.
Glucose-dependent insulinotropic polypeptide (GIP), which is released into the circulation from jejunal mucosal K-cells. Circulating GIP augments pancreatic insulin secretion and hyperinsulinemia plays critical roles in the pathogenesis of obesity and type-2 diabetes mellitus. In recent studies, we have shown GIP directly activates Na-glucose co-transporter-1 (SGLT1) and enhances glucose absorption in mouse jejunum. It is not known whether GIP would also regulates other intestinal nutrient absorptive process(es). Present study investigated the effect of GIP on H+-peptide cotransporter-1 (PepT1) that mediates di- and tripeptides as well as peptidomimetic drugs. The activity of PepT1 is driven by a proton (H+)-gradient that is, in part, maintained by apical Na:H exchange is form 3 (NHE3). Immunofluorescence studies revealed GIP-receptor and PepT1 like proteins are localized in the villus cells of normal mouse jejunum. Gly-Sar (a relatively non-digestible peptide) fluxes were measured under voltage clamp condition in Ussing type chambers. Mucosal to cytosolic acid pH-gradient (i.e., H+ gradient) significantly enhanced the mucosal to serosal Gly-Sar absorption as an evidence for the presence of PepT1 (i.e., H+-Gly-Sar cotransports) on the apical membranes of mouse jejunum. The H+-gradient driven Gly-Sar absorption was: 1) completely inhibited by cephalexin (a competitive inhibitor of Pept1); 2 significantly activated by GIP; and 3) not affected by GLP-1. The GIP-activated Gly-Sar absorption was completely inhibited by RP-cAMP (a cAMP-antagonist). These observations indicate that GIP directly activates the Pept1 activity by a cAMP-dependent signaling pathway in jejunum.
Changes in the intestinal microbiota have been linked to diabetes, obesity, IBD, and Clostridium difficile-associated disease. Despite this, it remains unclear how the intestinal environment, set by ion transport, affects luminal and mucosa-associated bacterial composition. Na+/H+-Exchanger isoform 3 (NHE3), a target of C. difficile toxin B, plays an integral role in intestinal Na+ absorption. Thus, the NHE3-deficient mouse model was chosen to examine the effect of pH and ion composition on bacterial growth. We hypothesized that ion transport-induced change in the intestinal environment would lead to alteration of the microbiota. Region-specific changes in ion composition and pH correlated with region-specific alteration of luminal and mucosal-associated bacteria with general decreases in Firmicutes and increases in Bacteroidetes members. Bacteroides thetaiotaomicron increased in NHE3-/- terminal ileum and was examined in vitro to determine if altered Na+ was sufficient to affect growth. Increased in vitro growth of B. thetaiotaomicron occurred in 43 mM Na+ correlating with the NHE3-/- mouse terminal ileum [Na+]. NHE3-/- terminal ileum displayed increased fut2 mRNA and fucosylation correlating with B. thetaiotaomicron growth. Inoculation of B. thetaiotaomicron in WT and NHE3-/- terminal ileum organoids displayed increased fut2 and fucosylation, indicating that B. thetaiotaomicron alone is sufficient for the increased fucosylation seen in vivo. These data demonstrate that loss of NHE3 alters the intestinal environment leading to region-specific changes in bacteria and shed light on the growth requirements of some gut microbiota members, which is vital for creating better treatments of complex diseases with an altered gut microbiota.
The enteric microbiota contributes to the pathogenesis of inflammatory bowel disease but the pathways involved and bacterial participants may vary in different hosts. We previously reported that some components of the human commensal microbiota, particularly Clostridium perfringens, have the proteolytic capacity for host matrix degradation and reduce transepithelial resistance. Here, we examined the C. perfringens-derived proteolytic activity against epithelial tight junction proteins using human intestinal epithelial cell lines. We showed that the protein levels of E-cadherin, occludin and JAM-1 decrease in colonic cells treated with C. perfringens culture supernatant. E-cadherin ectodomain shedding in C. perfringens-stimulated intestinal epithelial cells was detected with antibodies against the extracellular domain of E-cadherin and we demonstrate that this process occurs in a time- and dose-dependent manner. In addition, we showed that the filtered sterile culture supernatant of C. perfringens has no cytotoxic activity on the human intestinal cells at the concentrations used in this study. The direct cleavage of E-cadherin by the proteases from the C. perfringens culture supernatant was confirmed by C. perfringens supernatant-induced in vitro degradation of the human recombinant E-cadherin. We conclude that C. perfringens culture supernatant mediates digestion of epithelial cell junctional proteins which is likely to enable access to the extracellular matrix components by the paracellular pathway.
Background: Gastrointestinal motility involves interactions between myogenic and neurogenic processes intrinsic to the gut wall. We have compared the presence of propagating myogenic contractions of the isolated colon in four experimental animals (guinea-pig, mouse, rabbit and rat), following blockade of enteric neural activity. Methods: Isolated colonic preparations were distended with fluid, with the anal end either closed or open. Spatio-temporal maps of changes in diameter were constructed from video recordings. Results: Distension-induced peristaltic contractions were abolished by tetrodotoxin (TTX; 0.6 µM) in all animal species. Subsequent addition of carbachol (0.1-1 µM) did not evoke myogenic motor patterns in the mouse or guinea-pig, although some activity was observed in rabbit and rat colon. These myogenic contractions propagated both orally and anally and differed from neurogenic propagating contractions in their frequency, extent of propagation and polarity. Niflumic acid (300µM) used to block myogenic activity also blocked neural peristalsis and thus cannot be used to discriminate between these mechanisms. In all species, except the mouse colon, small myogenic "ripple" contractions were revealed in TTX, but in both rat and rabbit an additional, higher frequency ripple-type contraction was superimposed. Conclusion: Following blockade of enteric nerve function, a muscarinic agonist can evoke propulsive myogenic peristaltic contractions in isolated rabbit and rat colon, but not in guinea-pig or mouse colon. Marked differences between species exist in the ability of myogenic mechanisms to propel luminal content, but in all species there is normally a complex interplay between neurogenic and myogenic processes.
TThe mechanism involved in the sorting and accumulation of secretory cargo proteins, such as amylase, into secretory granules of exocrine cells remains to be solved. To clarify that sorting mechanism, we expressed a reporter protein HaloTag fused with partial sequences of salivary amylase protein in primary cultured parotid acinar cells. We found that a HaloTag protein fused with only the signal peptide sequence (Met1-Ala25) of amylase, termed SS25H, co-localized well with endogenous amylase, which was confirmed by immunofluorescence microscopy. Percoll-density gradient centrifugation of secretory granule fractions shows that the distributions of amylase and SS25H were similar. These results suggest that SS25H is transported to secretory granules and is not discriminated from endogenous amylase by the machinery that functions to remove proteins other than granule cargo from immature granules. Another reporter protein, DsRed2, that has the same signal peptide sequence also co-localized with amylase, suggesting that the sorting to secretory granules is not dependent on a characteristic of the HaloTag protein. While Blue Native PAGE demonstrates that endogenous amylase forms a high molecular weight complex, SS25H does not participate in the complex and does not form self-aggregates. Nevertheless, SS25H was released from cells by the addition of a β-adrenergic agonist, isoproterenol, which also induces amylase secretion. These results indicate that addition of the signal peptide sequence, which is necessary for the translocation in the ER, is sufficient for the transportation and storage of cargo proteins in secretory granules of exocrine cells.
Chronic inflammation and enteric infections are frequently associated with epithelial Na+/H+ exchange (NHE) inhibition. Alterations in electrolyte transport and in mucosal pH associated with inflammation may represent a key mechanism leading to changes in the intestinal microbial composition. NHE3 expression is essential for the maintenance of the epithelial barrier function. NHE3-/- mice develop spontaneous distal chronic colitis and are highly susceptible to dextran sulfate (DSS)-induced mucosal injury. Spontaneous colitis is reduced with broad-spectrum antibiotics treatment, thus highlighting the importance of the microbiota composition in NHE3-deficiency mediated colitis. We herein characterized the colonic microbiome of wildtype (WT) and NHE3-/- mice housed in a conventional environment using 454 pyrosequencing. We demonstrated a significant decrease in the phylogenetic diversity of the luminal and mucosal microbiota of conventional NHE3-/- mice as compared to WT. Rederivation of NHE3-/- mice from conventional to an ultraclean barrier facility eliminated the signs of colitis and decreased DSS susceptibility. Reintroduction of the conventional microflora into WT and NHE3-/- mice from the barrier facility resulted in the restoration of the symptoms initially described in the conventional environment. Interestingly, qPCR analysis of the microbiota composition in mice kept in the barrier facility compared to reconventionalized mice showed a significant reduction of Clostridia classes IV and XIVa. Therefore, the gut microbiome plays a prominent role in the pathogenesis of colitis in NHE3-/- mice and reciprocally, NHE3 also plays a critical role in shaping the gut microbiota. Therefore, NHE3 deficiency may be a critical contributor to dysbiosis observed in IBD patients.
Aims: We tested the hypothesis that the sensory-motor characteristics of aerodigestive reflexes are dependent on stimulus type and volumes, sleep or awake states and maturation. Methods: Thirteen neonates were studied at 33.6 ± 0.5 (time-1) and 37.3 ± 0.5 weeks (time-2) postmenstrual age using multi-modal provocative esophageal manometry concurrent with video-polysomnography. Effects of graded volumes (399 infusions at time-1; 430 infusions at time-2) of mid-esophageal stimulation with air, water and apple juice on the sensory thresholds and recruitment frequency of upper esophageal sphincter (UES), esophageal body and lower esophageal sphincter (LES) reflexes were investigated during sleep and awake states. Results: Sensory thresholds for aerodigestive reflexes between maturational stages were similar. Increased frequency recruitment of UES contractile reflex, LES relaxation reflex and peristaltic reflexes were noted at time-2 (all, P < 0.05). Graded stimulus-response relationships were evident at time-1 and time-2 during awake and sleep states (P < 0.05). Secondary peristalsis vs. esophago-deglutition response proportions during sleep at time-1 vs. time-2 (P = 0.001), and awake vs. sleep at time-2 (P = 0.02) were distinct. Conclusions: Sensory-motor effects of esophageal mechanosensitivity, osmosensitivity and chemosensitivity are advanced in sleep during maturation. Sleep further modulates the recruitment frequency and type of aerodigestive reflexes.
Hydrogen sulfide (H2S) has been reported to be involved in the signaling of the inflammatory response; however there are differing views as to whether it is pro- or anti-inflammatory. In this study, we sought to determine if endogenously synthesized H2S via cystathionine--lyase (CSE) plays a pro- or anti-inflammatory role in caerulein-induced pancreatitis. To investigate this, we used mice genetically deficient in CSE to elucidate the function of CSE in caerulein induced acute pancreatitis. We compared the inflammatory response and tissue damage of wild type (WT) and CSE KO mice following ten hourly administrations of 50 μg/kg caerulein or saline control. From this we found that the CSE KO mice showed significantly less local pancreatic damage as well as acute pancreatitis-associated lung injury in comparison to the WT mice. There were also lower levels of pancreatic eicosanoid and cytokines as well as reduced acinar cell NF-B activation in the CSE KO mice when compared with WT mice. Additionally, in WT mice, there was a greater level of pancreatic CSE expression and sulfide synthesizing activity in caerulein-induced when compared to the saline control. When comparing the two saline treated control groups, the CSE KO mice showed significantly less pancreatic H2S synthesizing activity relative to the WT mice. These results indicate that endogenous H2S generated by CSE plays a key pro-inflammatory role via NF-B activation in caerulein-induced pancreatitis and its genetic deletion affords significant protection against acute pancreatitis and associated lung injury.
The sodium taurocholate cotransporting polypeptide (Ntcp) is the major uptake transporter for bile salts into liver parenchymal cells and PKC-mediated endocytosis was shown to regulate the number of Ntcp molecules at the plasma membrane. In this study, mechanisms of Ntcp internalization were analyzed by flow cytometry, immunofluorescence and Western blot analyses in HepG2 cells. PKC activation induced endocytosis of Ntcp from the plasma membrane by about 30%. Endocytosis of Ntcp was clathrin-dependent and was followed by lysosomal degradation. A dileucine motif located in the third intracellular loop of Ntcp was essential for endocytosis but also for processing and plasma membrane targeting, suggesting a dual function of this motif for intracellular trafficking of Ntcp. Mutation of two from five potential phosphorylation sites surrounding the dileucine motif (Thr225 and Ser226) inhibited PKC-mediated endocytosis. In conclusion, we could identify a motif, which is critical for Ntcp plasma membrane localization. Endocytic retrieval protects hepatocytes from elevated bile salt concentrations and is of special interest, because NTCP has been identified as a receptor for the hepatitis B and D virus.
Hepatocyte apoptosis is a hallmark of nonalcoholic steatohepatitis (NASH). We have previously observed that the saturated free fatty acids (FFAs) induce hepatocyte apoptosis in part, via a death receptor 5 (DR5)-mediated signaling pathway. Cellular inhibitor of apoptosis 1 and 2 (cIAP-1 and cIAP-2) proteins are potent inhibitors of death receptor-mediated apoptosis. However, role of the cIAPs in FFA-mediated hepatocyte apoptosis is unexplored. Our aim was to determine if cIAPs are dysregulated during hepatocyte lipoapoptosis. cIAP proteins underwent rapid cellular elimination following treatment with the saturated FFAs palmitate (PA) and stearate (SA). In contrast, palmitate did not decrease cIAP-1 and cIAP-2 mRNA expression in the cells. Degradation of cIAPs was dependent on their E3-ligase activity, suggesting that cIAPs undergo auto-ubiquitination which leads to proteasomal degradation. Huh-7 cells stably expressing shRNA targeting cIAP-1, but not cIAP-2, displayed enhanced sensitivity to PA-mediated apoptosis. Incubation with the SMAC mimetic JP1584, which induces rapid degradation of cIAPs, also enhanced PA-mediated apoptosis. Hepatocytes isolated from DR5 knockout mice exhibited reduced apoptosis following treatment with PA plus JP1584, implying that degradation of cIAPs sensitizes to DR5 mediated cell death pathways. A decrease of cIAP-1 was also observed in tissue from NASH patients compared to normal obese subjects. Collectively, these results implicate proteasomal degradation of cIAPs by FFA as a mechanism contributing to hepatocyte lipoapoptosis.
Intestinal fat absorption is known to be, overall, a highly efficient process, but much less is known about the efficiency with which individual dietary fatty acids (FA) are absorbed by the adult small intestine. We therefore measured the absorption efficiency of the major dietary FA using sucrose polybehenate (SPB) as a non-absorbable marker, and analyzed how it is modulated by acyl chain physicochemical properties and polymorphisms of proteins involved in chylomicron assembly. Dietary FA absorption efficiency was measured in 44 healthy subjects fed a standard diet containing 35% fat and 5% SPB. FA and behenic acid (BA) were measured in homogenized diets and stool samples by GC-mass spectroscopy, and coefficients of absorption for each FA were calculated as 1-[(FA/BA)feces/(FA/BA)diet]. Absorption coefficients for saturated FA decreased with increasing chain length and hydrophobicity (mean ± SE), and ranged from 0.95 ± 0.02 for myristate (14:0), 0.80 ± 0.03 for stearate (18:0), to 0.26 ± 0.02 for arachidate (20:0). Absorption coefficients for unsaturated FA increased with increasing desaturation from 0.79 ± 0.03 for elaidic acid (18:1t), 0.96 ± 0.01 for linoleate (18:2), to near complete absorption for eicosapentaenoic (20:5) and docosahexaenoic (22:6) acids. Of several common genetic polymorphisms in key proteins involved in the chylomicron assembly pathway, only the intestinal fatty acid binding protein-2 A54T allele (rs1799883) had any impact on FA absorption. We conclude that acyl chain length, saturation, and hydrophobicity are the major determinants of the efficiency with which dietary FA are absorbed by the adult small intestine.
Colorectal cancer is the third most prevalent form of cancer worldwide and fourth-leading cause of cancer-related mortality, leading to approximately 600,000 deaths annually, and predominantly affecting the developed world. Lysyl oxidase is a secreted, extracellular matrix-modifying enzyme previously suggested to act as a tumor suppressor in colorectal cancer. However, emerging evidence has rapidly implicated lysyl oxidase in promoting metastasis of solid tumors and in particular colorectal cancer at multiple stages, affecting tumor cell proliferation, invasion and angiogenesis. This emerging research has stimulated significant interest in lysyl oxidase as a strong candidate for developing and deploying inhibitors as functional efficacious cancer therapeutics. In this review, we discuss the rapidly expanding body of knowledge concerning lysyl oxidase in solid tumor progression, highlighting recent advancements in the field of colorectal cancer.
During the last decade, biological therapies have an increasing share in the modern therapeutics of various diseases including inflammatory bowel diseases (IBD). Animal models of IBD have been often used to identify the targets of biologic therapies, to test their relevance to disease pathogenesis, to assess their therapeutic efficacy in vivo, and to check for drug toxicity. In the field of inflammatory diseases the majority of biologics under development have failed to reach the clinic. This review examines the ability of pre-clinical data from animal models of IBD to predict success or failure of biologics in human IBD. Specifically, it describes the murine models of IBD, the mechanism of disease induction, the phenotype of the disease, its relevance to human IBD, and the specific immunological features of disease pathogenesis in each model and mainly compares the results of the phase II and III trials of biologics in IBD with pre-clinical data obtained from studies in animal models. Finally, it examines the possible reasons for low success in translation from bench to bedside and offers some suggestions in order to improve translation rates.
Obestatin is a hormone released from the stomach deriving from the same peptide precursor as ghrelin. It is known to act as an anorectic hormone decreasing food intake but contrasting results have been reported about the effects of obestatin on gastrointestinal motility. The aim of the present study was to investigate whether this peptide may act on the gastric longitudinal smooth muscle by using a combined mechanical and electrophysiological approach. Gastric fundal strips from mice were mounted in organ baths for isometric recording of the mechanical activity. Obestatin caused a TTX-insensitive decrease of the basal tension and a reduction in amplitude of the neurally-induced cholinergic contractile responses even in the presence of the nitric oxide synthesis inhibitor L-N-nitro-arginine. Obestatin reduced the amplitude of the response to the ganglionic stimulating agent dimethylphenyl piperazinium iodide (DMPP) but did not influence that to methacholine. In non-adrenergic, non-cholinergic conditions, obestatin still decreased the basal tension of the preparations without influencing the neurally-induced relaxant responses. Electrophysiological experiments, performed by a single microelectrode inserted in a gastric longitudinal smooth muscle cell, showed that obestatin increased the membrane resistance, caused the inhibition of Ca2+ currents and positively shifted their voltage threshold of activation. In conclusion, the present results indicate that obestatin, other than exerting a neuromodulatory action at the postganglionic level, directly influences the gastric longitudinal smooth muscle. The reduction of the contractile responses might contribute to the distension of the gastric wall, one of the major signal involved in the regulation of food intake.
Crohn's disease (CD) is a chronic, immune-mediated, inflammatory disorder of the intestine that has been linked to numerous susceptibility genes, including the Immunity Related GTPase (IRG) M. IRGs are a family of proteins known to confer resistance to intracellular infections through various mechanisms, including the regulation of phagosome processing, cell motility, and autophagy. However, despite its association with CD, the role of IRGM and other IRGs in regulating intestinal inflammation is unclear. In the present study, we investigated the involvement of Irgm1, an orthologue of IRGM, in the genesis of murine intestinal inflammation. After DSS exposure, Irgm1-/- mice showed increased acute inflammation in the colon and ileum, with worsened clinical responses. Marked alterations of Paneth cell location and granule morphology were present in Irgm1-/- mice, even without DSS exposure, and were associated with impaired mitophagy and autophagy in Irgm1-/- intestinal cells (including Paneth cells). This was manifested by frequent tubular and swollen mitochondria, and increased LC3-positive autophagic structures. Interestingly, these LC3-positive structures often contained Paneth cell granules. These results suggest that Irgm1 modulates acute inflammatory responses in the mouse intestine, putatively through the regulation of gut autophagic processes that may be pivotal for proper Paneth cell functioning.
The ligand-gated channels TRPV1 and P2X3 have been reported to facilitate colorectal afferent neuron sensitization, thus contributing to organ hypersensitivity and pain. In the present study, we hypothesized that TRPV1 and P2X3 cooperate to modulate colorectal nociception and afferent sensitivity. To test this hypothesis, we employed TRPV1-P2X3 double knockout (TPDKO) mice and channel-selective pharmacological antagonists and evaluated combined channel contributions to behavioral responses to colorectal distension (CRD) and afferent fiber responses to colorectal stretch. Baseline responses to CRD were unexpectedly greater in TPDKO compared with control mice, but zymosan-produced CRD hypersensitivity was absent in TPDKO mice. Relative to control mice, proportions of mechano-sensitive and -insensitive pelvic nerve afferent classes were not different in TPDKO mice. Responses of mucosal and serosal class afferents to mechanical probing were unaffected, whereas responses of muscular (but not muscular/mucosal) afferents to stretch were significantly attenuated in TPDKO mice; sensitization of both muscular and muscular/mucosal afferents by inflammatory soup was also significantly attenuated. In pharmacological studies, the TRPV1 antagonist A889425 and P2X3 antagonist TNP-ATP, alone and in combination, applied onto stretch-sensitive afferent endings attenuated responses to stretch; combined antagonism produced greater attenuation. In the aggregate, these observations suggest that: (1) genetic manipulation of TRPV1 and P2X3 leads to reduction in colorectal mechanosensation peripherally and compensatory changes and/or disinhibition of other channels centrally; (2) combined pharmacological antagonism produces more robust attenuation of mechanosensation peripherally than does antagonism of either channel alone; and (3) the relative importance of these channels appears to be enhanced in colorectal hypersensitivity.
Gastric infection by H. pylori is the most common cause of ulcer disease and gastric cancer. The mechanism of progression from gastritis and inflammation to, in a fraction of those infected, ulcers and cancer, is not definitively known. Significant acidity is unique to the gastric environment and is required for ulcer development. The interplay between gastric acidity and H. pylori pathogenesis is important in progression to advanced disease. The aim of this study was to characterize the impact of acid on gastric epithelial integrity and cytokine release and how H. pylori infection alters these responses. HGE-20 cells were grown on porous inserts, and survival, barrier function, and cytokine release were studied at various apical pHs in the presence and absence of H. pylori. With apical acidity, gastric epithelial cells demonstrate increased barrier function, as evidenced by increased transepithelial electrical resistance (TEER) and decreased paracellular permeability. This effect is reduced in the presence of wildtype, but not urease knockout H. pylori. Epithelial inflammatory response is also modulated by both acidity and H. pylori infection. Without H. pylori, epithelial IL-8 release decreases in acid while IL-6 release increases. In the presence of H. pylori, acidic pH diminishes the magnitude of the previously reported increase in IL-8 and IL-6 release. H. pylori interferes with the gastric epithelial response to acid, contributing to altered barrier function and inflammatory response. H. pylori diminishes acid-induced tightening of cell junctions in a urease dependent manner, suggesting local pH elevation promotes barrier compromise and progression to mucosal damage.
This review focuses on recent advances in our understanding of the mechanisms and regulation of water-soluble vitamins (WSV) transport in the large intestine and the pancreas, two important organs of the digestive system that have not gotten their fair share of progress until recently. WSV, a group of structurally unrelated compounds, are essential for normal cellular functions and development, and thus, for overall health and survival of the organism. Humans cannot synthesize WSV endogenoursly, rather they obtain them from exogenous sources via intestinal absorption. The intestine is exposed to two sources of WSV: a dietary source, and a bacterial source (i. e., the WSV that are generated by the large intestinal microbiota). Contribution of the latter source toward human nutrition/health has been a subject of debate, and doubt, mostly on the basis that the large intestine does not have specialized systems for efficient uptake of WSV. However, recent studies utilizing a variety of human and animal colonic preparations have clearly demonstrated that such systems do exist in the large intestine. This has provided strong support that the microbiota-generated WSV are of nutritonal value to the host, and especially to the nutritional needs of the local colonocytes and their helath. With regards to the pancreas, WSV are essential for normal metabolic activities of all its cell types, and for its exocrine and endocrine functions. Again significant progress has also been made regarding the mechanisms involved in the uptake of WSV, and the effect of chronic alcohol exposure on the uptake processes.
The intestinal thiamine uptake process is adaptively regulated by level of vitamin in the diet, but the molecular mechanism involved is not fully understood. Here we used the human intestinal epithelial Caco-2 cells exposed to different levels of extracellular thiamine to delineate the molecular mechanism involved. Our results showed that maintaining Caco-2 cells in a thiamine-deficient medium resulted in a specific and significant increase of 3H-thiamine uptake compared with cell exposure to high level of thiamine (1 mM). This adaptive regulation was also associated with a higher level of mRNA expression of thiamine transporter-2 (THTR-2), but not thiamine transporter-1 (THTR-1), in the deficient condition and a higher level of promoter activity of gene encoding THTR-2 (SLC19A3). Using 5'-truncated promoter-luciferase constructs, we identified the thiamine level-responsive region in the SLC19A3 promoter to be between -77 and -29 (using transcriptional start site as +1). By means of mutational analysis, a key role for a SP1/GC-box in mediating the effect of extracellular thiamine level on SLC19A3 promoter was established. Furthermore, extracellular level of thiamine was found to affect SP1 protein expression and binding pattern to the thiamine level-responsive region of SLC19A3 promoter in Caco-2 cells as shown by Western blotting and EMSA analysis, respectively. These studies demonstrate that the human intestinal thiamine uptake is adaptively regulated by the extracellular substrate level via transcriptional regulation of the THTR-2 system, and report that SP1 transcriptional factor is involved in this regulation.
Enterohepatic helicobacter species (EHS) infect the intestinal tract and biliary tree triggering intestinal and hepatic disorders. Helicobacter hepaticus, the prototypic murine EHS, is also associated with inflammation. Necrotizing enterocolitis (NEC) is a devastating disease of premature infants. The cause of NEC is not fully understood, but anomalies of bacterial colonization (dysbiosis) are thought to play an important role in disease onset. To evaluate the effect of H. hepaticus infection on the development of NEC, premature formula-fed rats were kept either in H. hepaticus-free conditions (FF) or colonized with H. hepaticus (FF+H.hep); both groups were exposed to asphyxia and cold stress. The incidence of NEC, expression of Toll-like receptors (TLRs), production of cytokines and mucins, and presence of autophagy regulators were evaluated at the site of injury. H. hepaticus infection increased the incidence of NEC from 39% to 71% and significantly increased levels of TLR4 receptor, expression of pro-inflammatory cytokines CXCL1, IL-1β, IL-12, and IL-23, and altered activation of autophagy. H. hepaticus induces inflammation and increases the incidence and severity of experimental NEC; this is consistent with observations in neonates of blooms of pro-inflammatory microbes just prior to the onset of NEC. Future studies using rodent NEC models should include testing for H. hepaticus infection. Further studies in neonates of early identification and/or diminution of pro-inflammatory microbes may be beneficial in decreasing the incidence of NEC.
Fluorescence-activated cell sorting (FACS) is an essential tool for studies requiring isolation of distinct intestinal epithelial cell populations. Inconsistent or lack of reporting the critical parameters associated with FACS methodologies have complicated interpretation, comparison, and reproduction of findings. To address this, a comprehensive multi-center study was designed to develop guidelines that limit experimental and data reporting variability, providing a foundation for accurate comparison of data between studies. Common methodologies and data reporting protocols for tissue dissociation, cell yield, cell viability, FACS, and post-sort purity were established. Seven centers tested the standardized methods by FACS-isolating a specific crypt-based epithelial population (EpCAM+/CD44+) from murine small intestine. Genetic biomarkers for stem/progenitor (Lgr5 and Atoh1) and differentiated cell lineages (Lysozyme, Mucin2, ChromograninA and Sucrase Iso-maltase) were interrogated in target and control populations to assess intra- and inter-center variability. Wilcoxon rank sum test on gene expression levels showed limited intra-center variability between biological replicates. Principle component analysis demonstrated significant inter-center reproducibility among four centers. Analysis of data collected using standardized cell isolation methods and data reporting requirements readily identified methodological problems, indicating that standard reporting parameters facilitate post-hoc error identification. These results indicate the complexity of FACS isolation of target intestinal epithelial populations can be highly reproducible between biological replicates and different institutions by adherence to common cell isolation methods and FACS gating strategies. This study can be considered a foundation for continued method development and a starting point for investigators developing cell isolation expertise to study physiology and pathophysiology of the intestinal epithelium.
Gastrointestinal myofibroblasts are contractile, electrically non-excitable, transitional cells, which play role in extracellular matrix production, in ulcer healing and in pathophysiological conditions they contribute to chronic inflammation and tumor development. Sodium-calcium exchangers (NCX) are known to have a crucial role in Ca2+ homeostasis of contractile cells, however, no information is available concerning the role of NCX in the proliferation and migration of gastrointestinal myofibroblasts. In this study, our aim was to investigate the role of NCX in the Ca2+ homeostasis, migration and proliferation of human gastroinstestinal myofibroblasts, focusing on human gastric myofibroblasts (HGMs). We used microfluorometric measurements to investigate the intracellular Ca2+ and Na+ concentrations, PCR analysis and immunostaining to show the presence of the NCX, patch clamp for measuring NCX activity, and proliferation and migration assays to investigate the functional role of the exchanger. We showed that 53.0±8.1% of the HGMs present Ca2+ oscillations, which depend on extracellular Ca2+ and Na+, and can be inhibited by NCX inhibitors. NCX1, NCX2 and NCX3 were expressed at both mRNA and protein levels in HGMs and they contribute to the intracellular calcium and sodium homeostasis as well - regardless of the oscillatory activity. NCX inhibitors significantly blocked the basal and IGF-II stimulated migration and proliferation rates of HGMs. In conclusion, we showed that NCX plays a pivotal role in regulating the Ca2+ homeostasis, migration and proliferation of HGMs. The inhibition of NCX activity may be a potential therapeutic target in hyperproliferative gastric diseases.
Shortcomings of previously reported preclinical models of non-alcoholic steatohepatitis (NASH) include inadequate methods used to induce disease and assess liver pathology. We have developed a dietary model of NASH displaying features observed clinically, and methods for objectively assessing disease progression. Mice fed a diet containing 40% fat (of which ~18% was trans-fat), 22% fructose, and 2% cholesterol developed three stages of non-alcoholic fatty liver disease (steatosis, steatohepatitis with fibrosis, and cirrhosis) as assessed by histological and biochemical methods. Using digital pathology to reconstruct the left lateral and right medial lobes of the liver, comparisons between and within lobes were made to determine the uniformity of collagen deposition, and in turn informed experimental sampling methods for histological, biochemical, and gene expression analyses. Gene-expression analyses conducted using animals stratified by disease severity led to the identification of several genes for which expression highly correlated with the histological assessment of fibrosis. Importantly, we have established a biopsy method allowing assessment of disease progression. Mice subjected to liver biopsy recovered well from the procedure when compared to sham-operated controls with no apparent effect on liver function. Tissue obtained by biopsy was sufficient for gene and protein expression analyses, providing the opportunity to establish an objective method of assessing liver pathology before subjecting animals to treatment. The improved assessment techniques, and the observation that mice fed the high fat diet exhibit many clinically relevant characteristics of NASH establishes a preclinical model for identifying pharmacological interventions with greater likelihood of translating to the clinic.
The sodium-taurocholate (TC) cotransporting polypeptide Ntcp/NTCP mediates TC uptake across the sinusoidal membrane of hepatocytes. Previously, we demonstrated that nitric oxide (NO) inhibits TC uptake through S-nitrosylation of a cysteine residue. Our current aim was to determine which of the 8 cysteine residues of Ntcp is responsible for NO-mediated S-nitrosylation and inhibition of TC uptake. Thus, we tested the effect of NO on TC uptake in HuH7 cells transiently transfected with cysteine to alanine mutant Ntcp constructs. Of the eight mutants tested, only C44A Ntcp displayed decreased total and plasma membrane (PM) expression levels which were also reflected in decreased TC uptake. C266A Ntcp showed a decrease in TC uptake that was not explained by a decrease in PM expression or localization indicating that C266 is required for optimal uptake. We speculated that NO would target C266 since a previous report had shown the thiol reactive compound MTSET inhibits TC uptake by wild type NTCP but not by C266A NTCP. We confirmed that MTSET targets C266 of Ntcp but surprisingly, we found that C266 was not responsible for NO-mediated inhibition of TC uptake. Instead we found that C96 was targeted by NO since C96A Ntcp was insensitive to NO-mediated inhibition of TC uptake. We also found that WT but not C96A Ntcp is S-nitrosylated by NO suggesting that C96 is important in regulating Ntcp function in response to elevated levels of NO.
Epidemiological studies show that subsets of adult and pediatric IBS patients have prior exposures to psychological or inflammatory stress. We investigated the cellular mechanisms of colonic smooth muscle dysfunction in adult rats subjected to neonatal inflammation. Ten-day old male rat pups received 2,4,6-Trinitrobenzene sulfonic acid to induce colonic inflammation. Colonic circular smooth muscle strips were obtained 6 to 8 weeks later. We found that only about half of the neonate pups subjected to inflammatory insult showed a significant increase in expression of the pore-forming α1C-subunit of Cav1.2b channels in adult life. These were the same rats in whom Vip mRNA increased in the colon muscularis externae. Additional experiments showed reduced interaction of histone deacetylase (HDAC) 3 with α1C1b promoter that increased the acetylation of histone H3 lysine 9 (H3K9) in the core promoter region. VIP-treatment of naïve muscularis externae swiftly recruited CREB binding protein (CBP) to the α1C1b promoter and dissociated HDAC3 from this region to initiate transcription. The CBP interaction with the α1C1b promoter was transient, but the dissociation of HDAC3 persisted to sustain H3K9 hyperacetylation and increase in transcription. Intraperitoneal treatment of adult naïve rats with butyrate mimicked the effects of neonatal colon inflammation. We conclude neonatal inflammation upregulates VIP in the colon muscularis externae, which modulates epigenetic events at the α1C1b promoter to activate α1C1b gene transcription. Inflammatory insult in early-life may be one of the etiologies of smooth muscle dysfunction in adult life, which contribute to the altered motility function in diarrhea-predominant IBS patients.
Active, transcellular oxalate transport in the mammalian intestine contributes to the homeostasis of this important lithogenic anion. Several members of the Slc26a gene family of anion exchangers have a measurable oxalate affinity and are expressed along the gut, apically and basolaterally. Mouse Slc26a6 (PAT1) targets to the apical membrane of enterocytes in the small intestine and its deletion results in net oxalate absorption and hyperoxaluria. Apical exchangers of the Slc26a family that mediate oxalate absorption have not been established, yet Slc26a3 (DRA) protein is a candidate mediator of oxalate uptake. To test this hypothesis we evaluated the role of DRA in intestinal oxalate (Ox2-) and chloride (Cl-) transport by comparing unidirectional and net ion fluxes across short-circuited segments of small (ileum) and large (cecum and distal colon) intestine from wild type (WT) and DRA knockout (DRA KO) mice. In WT mice all segments studied exhibited net Ox2- and Cl- absorption to varying degrees. In KO mice, however, all segments exhibited net anion secretion which was consistently, and solely, due to a significant reduction in the absorptive unidirectional components. In KO mice, daily urinary oxalate excretion was reduced 66% compared to WT, while urinary creatinine excretion was unchanged. We conclude that DRA mediates a predominance of the apical uptake of oxalate and chloride absorbed in the small and large intestine of mice under short-circuit conditions. The large reductions in urinary oxalate excretion underscores the importance of transcellular intestinal oxalate absorption in general, and more specifically the DRA exchanger, to oxalate homeostasis.
The pathophysiology of irritable bowel syndrome (IBS) is believed to involve alterations in the brain-gut axis; however, the mechanisms by which these changes lead to symptoms of IBS remain poorly understood. While often considered a condition without an identified "organic" etiology, emerging evidence suggests that alterations in the gastrointestinal (GI) microbiota and altered GI immune function may play a role in the pathophysiology of IBS. These recent data suggest a plausible model in which changes in the GI microbiota and activation of the GI immune system may impinge upon the brain-gut axis, causing the alterations in GI motility and functional GI symptoms observed in patients with IBS. This review summarizes the current evidence for altered intestinal microbiota and immune function in IBS. It discusses the potential etiologic role of these factors and suggests an updated conceptual model for the pathogenesis of the disorder.
Increased hepatic vascular resistance mainly due to elevated vascular tone and to fibrosis is the primary factor in the development of portal hypertension in cirrhosis. Leptin, a hormone associated with reduction in nitric oxide bioavailability, vascular dysfunction and liver fibrosis, is increased in patients with cirrhosis. We aimed at evaluating whether leptin influences the increased hepatic resistance in portal hypertension. CCl4-cirrhotic rats received the leptin receptor-blocker ObR antibody, or its vehicle, every other day for 1 week. Hepatic and systemic hemodynamics were measured in both groups. Hepatic nitric oxide production and bioavalability, together with oxidative stress, nitrotyrosinated proteins, and liver fibrosis were evaluated. In cirrhotic rats, leptin-receptor blockade significantly reduced portal pressure without modifying portal blood flow, suggesting a reduction in the intrahepatic resistance. Portal pressure reduction was associated with increased nitric oxide bioavailability, and decreased O2- levels and nitrotyrosinated proteins. No changes in systemic hemodynamics and liver fibrosis were observed. Conclusion: The present study shows that blockade of the leptin signalling pathway in cirrhosis significantly reduces portal pressure. This effect is probably due to a nitric oxide-mediated reduction in the hepatic vascular tone.
Interactions between the epithelium and surrounding mesenchyme/stroma play an important role in normal gut morphogenesis, the epithelial response to injury, and epithelial carcinogenesis. The tumor microenvironment, composed of stromal cells including myofibroblasts and immune cells, regulates tumor growth and the cancer stem cell niche. Deletion of epimorphin (Epim), a syntaxin family member expressed in myofibroblasts and macrophages, results in partial protection from colitis and from inflammation-induced colon cancer in mice. We sought to determine whether epimorphin deletion protects from polyposis in the Apcmin/+ mouse model of intestinal carcinogenesis. Methods: Epim-/- mice were crossed to Apcmin/+ mice; Apcmin/+ and Apcmin/+ /Epim-/- mice were sacrificed at 3 months of age. Polyp numbers and sizes were quantified in small intestine and colon, and gene expression analyses for pathways relevant to epithelial carcinogenesis were performed. Primary myofibroblast cultures were isolated, and expression and secretion of selected growth factors from Apcmin/+ and Apcmin/+ /Epim-/- myofibroblasts was examined by ELISA. Results: Small bowel polyposis was significantly inhibited in Apcmin/+ /Epim-/- compared to Apcmin/+ mice. Apcmin/+ /Epim-/- compared to Apcmin/+ polyps and adjacent uninvolved intestinal mucosa had increased TGFβ expression and signaling with increased P-Smad2/3 expression. Myofibroblasts isolated from Apcmin/+ /Epim-/- vs. Apcmin/+ mice had markedly decreased hepatocyte growth factor (HGF) expression and secretion. Conclusions: Epim deletion inhibits polyposis in Apcmin/+ mice, associated with increased mucosal TGFβ signaling and decreased myofibroblast HGF expression and secretion. Our data suggest that Epim deletion reduces tumorigenicity of the stromal microenvironment.
Activated pancreatic stellate cells (PSCs) are responsible for the fibrotic matrix of chronic pancreatitis and pancreatic cancer. In vitro protocols examining PSC biology have usually involved PSCs cultured on plastic, a non-physiological surface. However, PSCs cultured on physiological matrices e.g. Matrigel™ (normal basement membrane) and collagen (fibrotic pancreas), may have distinctly different behaviours compared to cells cultured on plastic. Therefore, we aimed to i) compare PSC gene expression after culture on plastic, Matrigel™ and collagen I; ii) validate the gene array data for transgelin, the most highly dysregulated gene in PSCs grown on activating versus non-activating matrices, at mRNA and protein levels; iii) examine the role of transgelin in PSC function; and iv) assess transgelin expression in human chronic pancreatitis sections. Culture of PSCs on different matrices significantly affected their gene expression pattern. 146, 619 and 432 genes respectively were differentially expressed (p<0.001) in PSCs cultured on collagen I vs Matrigel™, Matrigel™ vs plastic and collagen I vs plastic. The highest fold change (12.5 fold upregulation) in gene expression in cells on collagen I vs Matrigel™, was observed for transgelin (an actin stress fibre associated protein). Transgelin was significantly increased in activated PSCs versus quiescent PSCs. Silencing transgelin expression decreased PSC proliferation and also reduced PDGF-induced PSC migration. Notably, transgelin was highly expressed in chronic pancreatitis in stromal areas and peri-acinar spaces but was absent in acinar cells. These findings suggest that transgelin is a potentially useful target protein to modulate PSC function so as to ameliorate pancreatic fibrosis.
The CFTR High Expresser (CHE) cells express 8-fold higher levels of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel compared to neighboring enterocytes, and were first identified by our laboratory (Ameen et al 1995, Gastroenterology 108:1016). We used double label immunofluorescence microscopy to further study these enigmatic epithelial cells in rat intestine in vivo or ex vivo. CHE cells were found in duodenum, most frequent in proximal jejunum, and absent in ileum and colon. CFTR abundance increased in CHE cells along the crypt-villus axis. The basolateral Na+K+Cl- co-transporter NKCC1, a key transporter involved in Cl- secretion was detected at similar levels in CHE cells and neighboring enterocytes at steady state. Microvilli appeared shorter in CHE cells, with low levels of Myosin1A - a villus enterocyte specific motor that retains sucrase/isomaltase in the brush border membrane (BBM). CHE cells lacked alkaline phosphatase and absorptive villus enterocyte BBM proteins including: Na+H+ exchanger NHE3, Cl-/HCO3- exchanger SLC26A6 (PAT1), and sucrase/isomaltase. High levels of the vacuolar-ATPase proton pump were observed in the apical domain of CHE cells. Levels of the NHE regulatory factor NHERF1, Na-K-ATPase and Syntaxin 3 were similar to that of neighboring enterocytes. cAMP or acetylcholine stimulation robustly increased apical CFTR and basolateral NKCC1 disproportionately in CHE cells relative to neighboring enterocytes. These data strongly argue for a specialized role of CHE cells in Cl--mediated "high-volume" fluid secretion on the villi of the proximal small intestine.
Primary biliary cirrhosis (PBC) is a chronic liver disease characterised by cholestasis. Recent MRI studies have confirmed the presence of cardiac abnormalities in non-cirrhotic PBC patients. However cardiorespiratory consequences of these abnormalities have not been explored. Thoracic fluid content (TFC) is a non-invasive bio-electrical impedance measure of the electrical conductivity of the chest cavity. We explored TFC and its relationship with cardiac contractility parameters in early stage PBC patients, compared to chronic liver disease and community controls. Methods: TFC was measured in early stage PBC (non-cirrhotic; n=78), non-alcoholic fatty liver disease (NAFLD; n=23) and primary sclerosing cholangitis (PSC; n=18) and a community control population (n=78). Myocardial contractility was measured as index of contractility (IC), acceleration index (ACI), cardiac index (CI), stroke index (SI), Left Ventricular Ejection Time (LVET) and left ventricular work index (LVWI). We also measured total arterial compliance (TAC) and the Heather Index (HI; cardiac inotropy). Results: The PBC group had significantly lower TFC compared to controls and the chronic liver disease groups (p<0.0001). There was an association between increasing TFC and markers of cardiac function (CI, SI, EDI , IC and ACI), together with indicators of cardiac inotropy (HI) and total arterial compliance (TAC). Multivariate analysis confirmed that the only parameter that independently associated with TFC was the marker of cardiac inotropy HI (p=0.037; Beta 0.5). Conclusion: This study has confirmed that TFC is reduced in those with PBC, that this is specific to PBC and that it associates independently with markers of cardiac inotropy.
Zymogen granule (ZG) formation in acinar cells involves zymogen-cargo sorting from trans-Golgi into immature secretory granules (ISGs). ISG maturation progresses by removal of lysosomal membrane and select-content proteins which enter endosomal intermediates prior to their apical exocytosis. Constitutive and stimulated secretion through this mechanism is termed the constitutive-like (CLP) and minor-regulated (MRP) pathways, respectfully. However, the molecular components that control membrane trafficking within these endosomal compartments are unknown. We show that tumor protein D52 is highly expressed in endosomal compartments following pancreatic acinar stimulation and regulates apical exocytosis of an apically directed endolysosomal compartment. Secretion from the endolysosomal compartment was detected by cell-surface labeling of lysosome associated membrane protein LAMP1, which is absent from ZGs, and had incomplete overlap with surface labeling of synaptotagmin, a marker of ZG exocytosis. Although culturing (16-18 h) of isolated acinar cells is accompanied by a loss of secretory responsiveness, levels of SNARE proteins necessary for ZG exocytosis were preserved. However, levels of endolysosomal proteins D52, EEA1, Rab5 and LAMP1 markedly decreased. When D52 levels were restored by adenoviral delivery, the levels of these regulatory proteins and secretion of both LAMP1 (endolysosomal) and amylase was strongly enhanced. These secretory effects were absent in alanine and aspartate substitutions of serine 136, the major D52 phosphorylation site, and were inhibited by brefeldin A, which does not directly affect the ZG compartment. Results indicate D52 directly regulates apical endolysosomal secretion and are consistent with previous studies suggesting this pathway indirectly regulates ZG secretion of digestive enzymes.
Injury to the intestinal mucosa is a life-threatening problem in a variety of clinical disorders, including hemorrhagic shock, trauma, burn, pancreatitis and heat stroke. However, the susceptibility to injury of different regions of intestine in these disorders is not well understood. We compared histological injury across the small intestine in two in vivo mouse models of injury, hemorrhagic shock (30% loss of blood volume) and heat stroke (peak core temperature,42.4°C). In both, areas near the duodenum showed significantly greater mucosal injury and reductions in villi height. To determine if these effects were dependent on circulating factors, additional experiments were performed on isolated intestinal segments to test for permeability to 4 kDa FITC-dextran. The segments were exposed to hyperthermia (42°C for 90 min) moderate simulated ischemia (PO230; PCO2 60 Torr; pH = 7.1), severe ischemia: (PO220; PCO280 Torr; pH6.9), or severe hypoxia (PO20, PCO235 Torr) for 90 min, and each group was compared to sham controls. All treatments resulted in marked elevations in permeability within segments near the duodenum. In severe hypoxia or hyperthermia, permeability was also moderately elevated in the jejunum and ileum, but in moderate or severe ischemia, permeability was unaffected in these regions. The results demonstrate increased susceptibility of proximal regions of the small intestine to acute stress-induced damage, irrespective of circulating factors. The predominant injury in the duodenum may impact the pattern of acute inflammatory responses arising from breach of the intestinal barrier and may be useful knowledge for designing therapeutic strategies.
Although ursodeoxycholic acid (UDCA) has long been used for patients with chronic cholestatic liver diseases, particularly primary biliary cirrhosis, it may modulate the host immune response. This study investigated the effect of UDCA feeding on experimental hepatitis, endotoxin shock, and bacterial infection in mice. C57BL/6 mice were fed a diet supplemented with or without 0.3% (w/v) UDCA for four weeks. UDCA improved hepatocyte injury and survival in concanavalin-A (Con-A)-induced hepatitis by suppressing IFN- production by liver mononuclear cells (MNC), especially NK and NKT cells. UDCA also increased survival after lipopolysaccharide (LPS)-challenge; however, it decreased survival of mice following Escherichia coli (E. coli) infection due to the worsening of infection. UDCA-fed mice showed suppressed serum IL-18 levels and production of IL-18 from liver Kupffer cells, which together with IL-12 potently induces IFN- production. However, unlike normal mice, exogenous IL-18 pretreatment did not increase the serum IFN- levels after E. coli, LPS or Con-A challenge in the UDCA-fed mice. Interestingly, however, glucocorticoid receptor (GR) expression was significantly upregulated in the liver MNC of the UDCA-fed mice but not in their whole liver tissue homogenates. Silencing GR in the liver MNC abrogated the suppressive effect of UDCA on LPS- or Con-A-induced IFN- production. Furthermore, RU486, a GR antagonist, restored the serum IFN- level in UDCA-fed mice after E. coli, LPS or Con-A challenge. Taken together, these results suggest that IFN--reducing immunomodulatory property of UDCA is mediated by elevated GR in the liver lymphocytes in an IL-12/18 independent manner.
Objective Polyethylene glycol (PEG) is a frequently used osmotic laxative that accelerates gastrointestinal transit. It has remained unclear however, if PEG affects intestinal functions. We aimed to determine the effect of PEG treatment on intestinal sterol metabolism. Design Rats were treated with PEG in drinking water (7%) for two weeks or left untreated (controls). We studied the enterohepatic circulation of the major bile salt (BS) cholate with a plasma stable isotope dilution technique and determined BS profiles and concentrations in bile, intestinal lumen contents and feces. We determined the fecal excretion of cholesterol plus its intestinally formed metabolites. Finally, we determined the cytolytic activity of fecal water (a surrogate marker of colorectal cancer risk) and the amount and composition of fecal microbiota. Results Compared with control rats, PEG treatment increased the pool size (+51%; p<0.01) and decreased the fractional turnover of cholate (-32%; p<0.01). PEG did not affect the cholate synthesis rate, corresponding with an unaffected fecal primary BS excretion. PEG reduced fecal excretion of secondary BS and of cholesterol metabolites (each p<0.01). PEG decreased the cytolytic activity of fecal water (54 [46-62] vs. 87 [85-92] % erythrocyte potassium release in PEG treated and control rats, respectively; p<0.01). PEG treatment increased the contribution of Verrucomicrobia (p<0.01) and decreased that of Firmicutes (p<0.01) in fecal flora. Conclusion PEG treatment changes the intestinal bacterial composition, decreases the bacterial dehydroxylation of primary bile salts and the metabolism of cholesterol and increases the pool size of the primary BS cholate in rats.
Clinical studies have linked the increased consumption of fructose to the development of obesity, dyslipidemia and impaired glucose tolerance, and a role in hepatosteatosis development is presumed. Fructose can undergo a non-enzymatic reaction from which advanced glycation endproducts (AGEs) are derived, leading to the formation of dysfunctional, fructosylated proteins, however the in vivo formation of AGEs from fructose is still less known than that from glucose. In the present study C57Bl/6J mice received 15% (w/v) fructose (FRT) or 15% (w/v) glucose (GLC) in water to drink for 30 weeks, resembling human habit to consume sugary drinks. At the end of protocol both FRT and GLC drinking mice had increased fasting glycaemia, glucose intolerance, altered plasma lipid profile, and marked hepatosteatosis. FRT mice had higher hepatic triglycerides deposition than GLC, paralleled by a greater increased expression and activity of the sterol regulatory element-binding protein 1 (SREBP1), the transcription factor responsible for the de novo lipogenesis, and of its activating protein SCAP. LC-MS analysis showed a different pattern of AGEs production in liver tissue between FRT and GLC mice, with larger amount of carboxymethyl lysine (CML) generated by FRT. Double immunofluorescence and coimmunoprecipitation analysis revealed an interaction between CML and SCAP that could lead to prolonged activation of SREBP1. Overall, the high levels of CML and activation of SCAP/SREBP pathway associated to high fructose exposure here reported may suggest a key role of this signaling pathway in mediating fructose-induced lipogenesis.
The mucus that protects the surface of the gastrointestinal tract is rich in specialized O-glycoproteins called mucins, but little is known about other mucus proteins or their variability along the GI tract. To ensure that only mucus was analyzed, we combined collection from explant tissues mounted in perfusion chambers, liquid sample preparation, single-shot mass spectrometry, and specific bioinformatics tools, to characterize the proteome of the murine mucus from stomach to distal colon. With our approach, we identified ~1,300 proteins in the mucus. We found no differences in the protein composition or abundance between genders, but there were clear differences in mucus along the tract. Noticeably, mucus from duodenum showed similarities to the stomach, probably reflecting the normal distal transport. Qualitatively, there were however fewer differences than might had been anticipated, suggesting a relatively stable core proteome (~80% of the total identified). Quantitatively, we found significant differences (~40% of the proteins) that could reflect mucus specialization throughout the gastrointestinal tract. Hierarchical clustering pinpointed a number of such proteins that correlated with Muc2 (e.g. Clca1, Zg16, Klk1). This study provides a deeper knowledge of the gastrointestinal mucus proteome that will be important in further understanding this poorly studied mucosal protection system.
The mouse intestinal mucus is mainly made up by the gel-forming Muc2 mucin and the stomach surface mucus Muc5ac, both extensively O-glycosylated. The oligosaccharide diversity provides a vast library of potential recognition sites for both commensal and pathogenic organisms. The mucin glycans are thus likely very important for the selection and maintenance of a stable intestinal flora. Here we have explored the O-glycan patterns of the mouse gastrointestinal tract mucins. The mucins from the mucus of the distal and proximal colon, ileum, jejunum, duodenum and stomach of conventionally raised wild-type (C57BL/6) mice were separated by composite gel electrophoresis. The O-linked glycans were released by reductive elimination and structurally characterized by liquid chromatography-mass spectrometry. The mucins glycans were mostly core 2 type (Galβ1-3(GlcNAcβ1-6)GalNAcol), but also core 1 (Galβ1-3GalNAcol). In the stomach Muc5ac about half of the mucin O-glycans were neutral and many monosulfated, but with a low grade of sialylation and fucosylation. Mouse ileum, jejunum and duodenum had similar glycan patterns dominated by sialylated and sulfated core 2 glycans, but few fucosylated. Colon was on the other hand dominated by highly charged fucosylated glycans. The distal colon is different from the proximal colon as different biosynthetic pathways are utilized, although sialylated and sulfated glycans were highly abundant in both parts. The sulfation was higher in the distal colon, while sialic acid was more common in the proximal colon. Many fucosylated glycans were found in both the proximal and distal colon. Thus the mucin O-glycans vary along the mouse gastrointestinal tract.
Colon has been shown to have a two-layered mucus system where the inner is devoid of bacteria. However, a complete overview of the mouse gastrointestinal mucus system is lacking. We now describe the mucus system from stomach to distal colon, including Peyer's patches, as mucus thickness, growth over time, adhesive properties and penetrability to fluorescent beads. Colon displayed spontaneous mucus release and all parts of the alimentary tract responded to carbachol and PGE2 stimulation, except the distal colon and domes of Peyer's patches. Stomach and colon had an inner mucus layer that was adherent to the epithelium. In contrast, the small intestine and Peyer's patches had a single mucus layer that was easily aspirated. The inner mucus layer of the distal colon was not penetrable to beads the size of bacteria and the inner layer of the proximal colon was only partly penetrable. In contrast, the inner mucus layer of stomach was fully penetrable, as was the small intestinal mucus. This suggest a functional organization of the intestinal mucus system where the small intestine has loose and penetrable mucus that allow easy penetration of nutrients in contrast to the stomach where the mucus provides physical protection and the colon where the mucus separates bacteria from the epithelium. This knowledge of the mucus system and its organization improves our understanding of the gastrointestinal tract physiology.
The molecular mechanisms behind human liver disease progression to cirrhosis remain elusive. Nuclear receptor small heterodimer partner (SHP/Nr0b2) is a critical regulator of liver function. SHP expression is diminished in human cirrhotic livers, suggesting a regulatory role in human liver diseases. We conducted the first comprehensive RNA sequencing analysis of SHP-/- mice, compared the results to human hepatitis C cirrhosis RNA-seq and non-alcoholic steatohepatitis (NASH) microarray datasets and verified novel results in human liver biospecimens. This approach revealed novel gene signatures associated with chronic liver disease and regulated by SHP. The physiological significance of markedly upregulated genes with minimally characterized functions in liver, such as peptidoglycan recognition protein 2 (PGLYRP2), dual specific phosphatase-4 (DUSP4), tetraspanin 4 (TSPAN4), thrombospondin 1 (THBS1), and SPARC-related modular calcium binding protein-2 (SMOC-2), was validated by q-PCR analysis of 126 human liver specimens including steatosis, fibrosis and NASH, alcohol and hepatitis C cirrhosis and in mouse models of liver inflammation and injury. This RNA-seq analysis identifies novel genes both regulated by the nuclear receptor SHP and implicated in the molecular pathogenesis of human chronic liver diseases. The results provide valuable information at the transcriptome level for characterizing mechanisms of these diseases.
Liver fibrosis, with subsequent development of cirrhosis and ultimately portal hypertension, results in the death of patients with end stage liver disease if liver transplantation is not performed. Hepatic stellate cells (HSCs), central mediators of liver fibrosis, resemble tissue pericytes, and regulate intrahepatic blood flow by modulating pericapillary resistance. Therefore, HSCs can contribute to portal hypertension in patients with chronic liver disease (CLD). We have previously demonstrated that activated HSCs express functional chemokine receptor, CXCR4, and that receptor engagement by its ligand, CXCL12, which is increased in patients with CLD, leads to further stellate cell activation in a CXCR4 specific manner. We therefore hypothesized that CXCL12 promotes HSC contraction in a CXCR4 dependent manner. Stimulation of HSCs on collagen gel lattices with CXCL12 led to gel contraction and myosin light-chain (MLC) phosphorylation which was blocked by addition of AMD3100, a CXCR4 small molecule inhibitor. These effects were further mediated by the Rho-kinase pathway as both Rho-kinase knockdown or Y-27632, a Rho-kinase inhibitor, blocked CXCL12 induced phophorylation of MLC and gel contraction. BAPTA-AM, a calcium chelator, had no effect indicating that this pathway is Ca2+-sensitive but not Ca2+-dependent. In conclusion, CXCL12 promotes stellate cell contractility in a predominantly Ca2+-independent fashion. Our data demonstrates a novel role of CXCL12 in stellate cell contraction and the availability of small molecule inhibitors of the CXCL12/CXCR4 axis justifies further investigation into its potential as therapeutic target for portal hypertension.
Nutritional stimulation of the cholecystokinin-1 receptor (CCK-1R) and nicotinic acetylcholine receptor (nAChR)-mediated vagal reflex was shown to reduce inflammation and preserve intestinal integrity. Mast cells are important early effectors of the innate immune response, therefore modulation of mucosal mast cells is a potential therapeutic target to control the acute inflammatory response in the intestine. The present study investigates intestinal mast cell responsiveness upon nutritional activation of the vagal anti-inflammatory reflex during acute inflammation. Mucosal mast cell degranulation was induced in C57/Bl6 mice by administration of Salmonella enterica LPS. Lipid-rich enteral feeding prior to LPS significantly decreased circulatory levels of mouse mast cell protease at 30 minutes post-LPS compared with isocaloric low-lipid nutrition or fasting. CCK-1R blockage reversed the inhibitory effects of lipid-rich feeding, whereas stimulation of the peripheral CCK-1R mimicked nutritional mast cell inhibition. The effects of lipid-rich nutrition were negated by nAChR blockers chlorisondamine and α-bungarotoxin and vagal intestinal denervation. In line, release of β-hexosaminidase by MC/9 mast cells following LPS or IgE-OVA complexes was dose-dependently inhibited by acetylcholine and nicotine. Application of GSK1345038A, a specific agonist of the nAChR α7, in bone marrow derived mast cells from nAChR β2-/- and wild types indicated that cholinergic inhibition of mast cells is mediated by the nAChR α7 and is independent of the nAChR β2. Together, the current study reveals mucosal mast cells as a previously unknown target of the nutritional anti-inflammatory vagal reflex.