Neurons in the supraoptic nuclei (SON) produce oxytocin and vasopressin and express insulin receptors (InsR) and glucokinase. Since oxytocin is an anorexigenic agent and glucokinase and InsR are hallmarks of cells that function as glucose and/or metabolic sensors, we evaluated the effect of glucose, insulin, and their downstream effector, K_ATP channels on calcium signaling in SON neurons and on oxytocin and vasopressin release from explants of the rat hypothalamo-neurohypophyseal system. We also evaluated the effect of blocking glucokinase and phosphatidylinositol 3 kinase (PI3K; mediates insulin-induced mobilization of glucose transporter, GLUT4) on responses to glucose and insulin. Glucose and insulin increased intracellular calcium ([Ca⁺⁺]_i ). The responses were glucokinase and PI3K dependent, respectively. Insulin and glucose alone increased vasopressin release (p<0.002). Oxytocin release was increased by glucose in the presence of insulin. The oxytocin and vasopressin responses to insulin+glucose were blocked by the glucokinase inhibitor, alloxan (4mM; p≤0.002), and the PI3K inhibitor, wortmannin (50nM; OT: p=0.03; VP: p≤0.002). Inactivating KATP channels with 200nM glibenclamide increased oxytocin and vasopressin release (OT: p<0.003; VP: p<0.05). These results suggest that insulin activation of PI3K increases glucokinase-mediated ATP production inducing closure of K_ATP channels, opening of voltage sensitive calcium channels, and stimulation of oxytocin and vasopressin release. The findings are consistent with SON oxytocin and vasopressin neurons functioning as glucose and 'metabolic' sensors to participate in appetite regulation.