Stimulation of insulin secretion by the incretin hormones glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP) has been found to be diminished in type 2 diabetes. We hypothesized that this impairment is due to a defect at the receptor level induced by the diabetic state, particularly hyperglycemia. Gene expression of incretin receptors, GLP-1R and GIPR, were significantly decreased in islets of 90% pancreatectomized (Px) hyperglycemic rats, with recovery when glucose levels were normalized by phlorizin. Perifused islets isolated from hyperglycemic Px rats showed reduced insulin responses to GLP-1 and GIP. To examine the acute effect of hyperglycemia on incretin receptor expression, a hyperglycemic clamp study was performed for 96 h with reduction of GLP-1 receptor expression but increase in GIP receptor expression. Similar findings were found when islets were cultured at high glucose concentrations for 48 h. The reduction of GLP-1 receptor expression by high glucose was prevented by dominant-negative protein kinase C (PKC)␣ overexpression, whereas GLP-1 receptor expression was reduced with wild-type PKC␣ overexpression. Taken together, GLP-1 and GIP receptor expression is decreased with chronic hyperglycemia, and this decrease likely contributes to the impaired incretin effects found in diabetes. Diabetes

Although type 2 diabetes mellitus is associated with insulin resistance, many individuals compensate by increasing insulin secretion. Putative mechanisms underlying this compensation were assessed in the present study by use of 4-day glucose (GLC; 35% Glc, 2 ml/h) and lipid (LIH; 10% Intralipid + 20 U/ml heparin; 2 ml/h) infusions to rats. Within 2 days of beginning the infusion of either lipid or glucose, plasma glucose profiles were normalized (relative to saline-infused control rats; SAL; 0.45% 2 ml/h). During glucose infusion, plasma glucose was maintained in the normal range by an approximately twofold increase in plasma insulin and an approximately 80% increase in beta-cell mass. During LIH infusion, glucose profiles were also maintained in the normal range. Plasma insulin responses during feeding were doubled, and beta-cell mass increased 54%. For both groups, the increase in beta-cell mass was associated with increased beta-cell proliferation (98% increase during GLC and 125% increase during LIH). At the end of the 4-day infusions, no significant changes were observed in islet-specific gene transcription (i.e., the expression of islet hormone genes, glucose metabolism genes, and insulin transcription factors were unaffected). Two days after termination of the infusions, the glucose-stimulated plasma insulin response was increased approximately 67% in glucose-infused animals. No sustained effect on insulin secretory capacity was observed in the LIH animals. The increase in plasma insulin response after glucose infusion was achieved in the absence of any change in insulin clearance. We conclude that, in rats, an increase in insulin demand after an increase in glucose appearance or free fatty acid leads to an increase in beta-cell mass, mediated in part by an increase in beta-cell proliferation, and that these compensatory changes lead to increased insulin secretion, normal plasma glucose levels, and the maintenance of normal islet gene expression.

Glucose-induced insulin secretion from hyperglycemic 90% pancreatectomized rats is markedly impaired, possibly because of loss of ␤ cell differentiation. Association of these changes with ␤ cell hypertrophy, increased mRNA levels of the transcription factor c-Myc, and their complete normalization by phlorizin treatment suggested a link between chronic hyperglycemia, increased c-Myc expression, and altered ␤ cell function.In this study, we tested the effect of hyperglycemia on rat pancreatic islet c-Myc expression both in vivo and in vitro. Elevation of plasma glucose for 1-4 days (glucose infusion/clamp) was followed by parallel increases in islet mRNA levels (relative to TATA-binding protein) of c-Myc and two of its target genes, ornithine decarboxylase and lactate dehydrogenase A. Similar changes were observed in vitro upon stimulation of cultured islets or purified ␤ cells with 20 and 30 mmol⅐liter ؊1 glucose for 18 h. These effects of high glucose were reproduced by high potassium-induced depolarization or dibutyrylcAMP and were inhibited by agents decreasing cytosolic Ca 2؉ or cAMP concentrations. In conclusion, the expression of the early response gene c-Myc in rat pancreatic ␤ cells is stimulated by high glucose in a Ca 2؉ -dependent manner and by cAMP. c-Myc could therefore participate to the regulation of ␤ cell growth, apoptosis, and differentiation under physiological or pathophysiological conditions.Besides being the major stimulus of insulin secretion, glucose exerts pleiotropic effects in pancreatic ␤ cells, including stimulation of protein synthesis (1) and cell proliferation and cell growth (2). Stimulus-secretion coupling is largely understood (3), but how glucose exerts its other effects is unclear. Moreover, the influence of glucose is not always beneficial, because chronic hyperglycemia impairs ␤ cell gene expression, function and survival (4 -6). Several mechanisms have been proposed to explain this so called "glucose toxicity," including ␤ cell overstimulation, increased oxidative stress, protein glycation, and islet amyloid pancreatic polypeptide deposition. We have recently shown that 2-4 weeks after a 90% pancreatectomy, chronic hyperglycemia induces a loss of ␤ cell differentiation that could account for the marked alteration of glucoseinduced insulin secretion in this animal model of diabetes (7,8). Thus, the expression of several transcription factors involved in ␤ cell differentiation (transcription factor Pdx1, hepatocyte nuclear factors, etc.) and of a panel of genes involved in glucose recognition and stimulation of secretion (insulin, glucose transporter 2, mitochondrial glycerol-P dehydrogenase, etc.) was decreased in islets from hyperglycemic 90% pancrea-

Differentiation and maturation of porcine neonatal pancreatic cell clusters (NPCCs) microencapsulated in barium alginate were assessed after transplantation into immunocompetent mice. Microencapsulated NPCCs were transplanted into the peritoneal cavity of streptozocin-induced diabetic B6AF1 mice (n ‫؍‬ 32). The microcapsules were removed at 2, 6, and 20 weeks and examined for cellular overgrowth, insulin content, and insulin secretory responses to glucose and glucose with theophylline. The differentiation, maturation, and proliferation of the ␤-cells in the NPCCs were assessed by immunohistochemistry. Blood glucose levels were normalized in 81% of the animals that received a transplant and remained normal until termination of the experiments at 20 weeks. Hyperglycemic blood glucose levels after explantation of the capsules confirmed the function of the encapsulated NPCCs. Insulin content of the encapsulated NPCCs was increased 10-fold at 20 weeks after transplantation compared with pretransplantation levels. A 3.2-fold increase of the ratio of the ␤-cell area to the total cellular area was observed at 20 weeks, demonstrating the maturation of NPCCs into ␤-cells. A s a result of recent progress (1), there is increased interest in islet transplantation as a potential therapy for type 1 diabetes. However, two major barriers must be overcome before islet transplantation can be provided for more patients: 1) the limited availability of human pancreatic tissue and 2) the need for permanent immunosuppression to prevent graft rejection and autoimmunity (2). Xenogeneic islets from pigs and cows (3-5) have been considered as potential sources of islets for transplantation. Many factors favor the use of pigs: the similar structure of porcine and human insulin, the comparable glucose levels, and that both pigs and humans are omnivores. Islet cells can be isolated in large numbers from adult (6 -9) or neonatal pigs (10,11). However, adult pig islets have proved to be difficult to isolate and tend to fare poorly in tissue culture, which has limited their use. Neonatal pancreatic cell clusters (NPCCs) contain a high proportion of islet precursor cells, can be maintained in culture, and differentiate into ␤-cells after transplantation (11). Naked (10,12) or microencapsulated NPCCs (13) have been shown to restore normoglycemia after transplantation into streptozotocin (STZ)-diabetic nude mice.The concept of a bioartificial pancreas, consisting of islets enclosed within immunobarrier membranes, provides a potential way to overcome the need for immunosuppression. Our group recently developed a promising encapsulation method that uses highly purified alginate cross-linked with BaCl 2 , without a separate permselective barrier, which protects islets against allorejection and autoimmunity (14). The aims of this study were to assess the protective capacity of simple barium-alginate capsules in a xenotransplantation model of NPCCs transplanted into STZ-induced diabetic immunocompetent mice and then to evaluate the growth, maturation, a...