The overall objectives of this proposal are to understand why glucagon responses to hypoglycemia from native pancreatic islets in type 1 diabetes and from islets transplanted intrahepatically are absent and to establish whether the omental or intraperitoneal transplant sites provide more optimal support of alpha and beta cell function than the intrahepatic site. Specific Aim #1. TO EVALUATE THE ALTERNATE HYPOTHESIS THAT THE RISE IN GLUCAGON LEVELS DURING HYPOGLYCEMIA IN RESPONSE TO SWITCHING OFF ZnCI2 INFUSIONS IS CAUSED BY DECREASED CLEARANCE OF GLUCAGON FROM THE CIRCULATION RATHER THAN INCREASED GLUCAGON SECRETION AS WE HAVE SUGGESTED. TO DETERMINE WHETHER THE MECHANISM OF THE RESTORATIVE EFFECT ON ALPHA CELL RESPONSES CAUSED BY SWITCHING OFF ZINC CHLORIDE INFUSIONS IS MEDIATED BY CLASSICAL INSULIN RECEPTOR PATHWAYS. WE HYPOTHESIZE THAT REGULATORY EFFECTS OF INSULIN, BUT NOT ZINC, ON ALPHA CELL FUNCTION DURING HYPOGLYCEMIA ARE MEDIATED BY CLASSICAL INSULIN RECEPTOR PATHWAYS. SpecfJC Aim #2. To determine whether depletion of liver glycogen by starvation and treatment with a blocker of gluconeogenesis will restore intrahepatic islet glucagon responses to hypoglycemia. We hypothesize that the mechanism of action for defective glucagon responses of islets transplanted intrahepatically is intrahepatic glucose flux, which blinds the transplanted islet to systemic hypoglycemia. Specific Aim #3. To determine whether the intrahepatic, intraperitoneal, or the omental sac transplantation sites is most optimal for beta and alpha cell function. We hypothesize that the non-hepatic sites will be more protective against beta cell toxicity caused by immunosuppressive drugs and will be more supportive of normal alpha cell responses to hypoglycemia. These experiments will be carried out using diabetes-prone BB (DP-BB) rats and inbred Lewis rats. The methods involve establishment of an intra-pancreaticoduodenal arterial insulin infusion that can be switched off when diabetic animals become hypoglycemic; prolonged fasting and AICAR infusion to deplete hepatic glycogen; and transplantation of donor islets into the hepatic, omental, and peritoneal sites to examine consequences on alpha and beta cell function as well as protection against the beta cell toxic effects of a calcineurin inhibitor. At the conclusion of these studies we will have established whether the alpha cell defect in DP-BB rats is metabolic or autoimmune in nature; whether intrahepatic glucose flux is responsible for defective glucagon secretion from intrahepatic islets; and whether non-hepatic sites are more efficacious than the hepatic site for islet transplantation in terms of alpha and beta cell function as well as protection against adverse effects of calcineruin inhibitors. [unreadable] [unreadable] [unreadable]