Glucose-induced insulin mRNA accumulation is impaired in islets from neonatal streptozotocin-treated rats.

According to the glucose toxicity hypothesis, hyperglycemia contributes to defective beta-cell function in type 2, non-insulin-dependent diabetes mellitus. This concept is supported by substantial data in rodent models of diabetes. However, the ability of glucose to stimulate the accumulation of insulin mRNA, a critical feature of normal beta-cell physiology, has not been investigated in in vivo models of chronic hyperglycemia. The aim of this study was to determine whether glucose-induced insulin mRNA accumulation is impaired in the neonatal streptozotocin-treated rat (n0-STZ rat), a model of non-obese, non-insulin-dependent diabetes mellitus. Islets of Langerhans isolated from n0-STZ and control rats were cultured for 24 h in the presence of 2.8 or 16.7 mmol/L glucose, and insulin mRNA levels were measured by Northern analysis. Insulin mRNA levels were increased more than twofold by glucose in control islets. In contrast, no significant effect of glucose was found on insulin mRNA levels in n0-STZ islets. We conclude that insulin gene regulation by glucose is impaired in n0-STZ rat islets.     

Circadian and age-dependent expression patterns of GLUT2 and glucokinase in the pancreatic beta-cell of diabetic and nondiabetic rats.

Alterations in glucose sensing are well-known in both humans and animal models of non-insulin-dependent diabetes mellitus. However, the circadian- and age-dependent expression of glucose-sensing genes has not previously been investigated in vivo. In the present paper, we show a progressive loss of beta-cell GLUT2-mRNA and, by immunocytochemistry, a gain of soluble, cytoplasmic GLUT2-protein in Goto-Kakizaki rat islets. We report that GLUT2-mRNA shows significant diurnal variation, which is stronger in metabolically healthy rats. We also demonstrate the significant diurnal variation of glucokinase-mRNA, with higher levels in the pancreas of 6-week-old Goto-Kakizaki rats than in Wistar rats. This leads to a maximum glucose phosphorylation capacity in-phase with food intake, enhanced glucose-stimulated insulin secretion, and prevents postprandial hyperglycemia. Perfusion experiments showed a reduction in glucose-stimulated insulin secretion in Goto-Kakizaki rat islets with an impaired first phase. Hyperglycemia and hypoinsulinemia in newborn and up to 3-week-old Goto-Kakizaki rats are thus probably due to reduced pancreatic beta-cell content, reduced beta-cell insulin content and impaired glucose sensing. The de-compensation of the metabolic situation in 42-week-old Goto-Kakizaki rats is likely to be caused by beta-cell destruction accompanied by negligible accumulation of GLUT2 in the cell membrane and further reduction of glucokinase expression.     

Free fatty acids increase PGC-1alpha expression in isolated rat islets

PGC-1alpha mRNA and protein are elevated in islets from multiple animal models of diabetes. Overexpression of PGC-1alpha impairs glucose-stimulated insulin secretion (GSIS). However, it is not well known which metabolic events lead to upregulation of PGC-1alpha in the beta-cells under pathophysiological condition. In present study, we have investigated effects of chronic hyperlipidemia and hyperglycemia on PGC-1alpha mRNA expression in isolated rat islets. Isolated rat islets are chronically incubated with 0, 0.2 and 0.4 mM oleic acid/palmitic acid (free fatty acids, FFA) or 5.5 and 25 mM glucose for 72 h. FFA dose-dependently increases PGC-1alpha mRNA expression level in isolated islets. FFA also increases PGC-1alpha expression in mouse beta-cell-derived beta TC3 cell line. In contrast, 25 mM glucose decreases expression level of PGC-1alpha. Inhibition of PGC-1alpha by siRNA improves FFA-induced impairment of GSIS in islets. These data suggest that hyperlipidemia and hyperglycemia regulate PGC-1alpha expression in islets differently, and elevated PGC-1alpha by FFA plays an important role in chronic hyperlipidemia-induced beta-cell dysfunction.     

Chronological characterization of diabetes development in male Spontaneously Diabetic Torii rats

To characterize the underlying mechanisms of diabetes development in males of the Spontaneously Diabetic Torii (SDT) rat, a novel spontaneous model for diabetes, we chronologically examined them, focusing on their diabetic features and the pathological changes in the pancreatic islets. Male SDT rats exhibited glucose intolerance with impaired insulin secretion after 14 weeks and developed diabetes with remarkable hyperglycemia and marked hypoinsulinemia after 20 weeks. At prediabetic stage (10-20 weeks), they were normoglycemic, but had significantly lower insulin levels of plasma and pancreas than the normal rats. Their beta-cell volume was already smaller significantly at 10 weeks than that of normal rats. The primary changes of the pancreatic islets were microvascular events such as congestion and hemorrhage at 8-10 weeks. Thereafter, the SDT rat islets were affected with inflammation and progressive fibrosis (at 10-20 weeks), and eventually atrophied with a loss of beta-cells (at 38 weeks). These results indicate that the male SDT rats develop spontaneous diabetes with an absolute decrease in the insulin secretory capacity of the islets.     

Genetic regulation of metabolic pathways in beta-cells disrupted by hyperglycemia.

In models of type 2 diabetes the expression of beta-cell genes is altered, but these changes have not fully explained the impairment in beta-cell function. We hypothesized that changes in beta-cell phenotype and global alterations in both carbohydrate and lipid pathways are likely to contribute to secretory abnormalities. Therefore, expression of genes involved in carbohydrate and lipid metabolism were analyzed in islets 4 weeks after 85-95% partial pancreatectomy (Px) when beta-cells have impaired glucose-induced insulin secretion and ATP synthesis. Px rats after 1 week developed mild to severe hyperglycemia that was stable for the next 3 weeks, whereas neither plasma triglyceride, non-esterified fatty acid, or islet triglyceride levels were altered. Expression of peroxisome proliferator-activated receptors (PPARs), with several target genes, were reciprocally regulated; PPARalpha was markedly reduced even at low level hyperglycemia, whereas PPARgamma was progressively increased with increasing hyperglycemia. Uncoupling protein 2 (UCP-2) was increased as were other genes barely expressed in sham islets including lactate dehydrogenase-A (LDH-A), lactate (monocarboxylate) transporters, glucose-6-phosphatase, fructose-1,6-bisphosphatase, 12-lipoxygenase, and cyclooxygenase 2. On the other hand, the expression of beta-cell-associated genes, insulin, and GLUT2 were decreased. Treating Px rats with phlorizin normalized hyperglycemia without effecting plasma fatty acids and reversed the changes in gene expression implicating the importance of hyperglycemia per se in the loss of beta-cell phenotype. In addition, parallel changes were observed in beta-cell-enriched tissue dissected by laser capture microdissection from the central core of islets. In conclusion, chronic hyperglycemia leads to a critical loss of beta-cell differentiation with altered expression of genes involved in multiple metabolic pathways diversionary to normal beta-cell glucose metabolism. This global maladaptation in gene expression at the time of increased secretory demand may contribute to the beta-cell dysfunction found in diabetes.     

Islet amyloid polypeptide (amylin)-deficient mice develop a more severe form of alloxan-induced diabetes

To examine whether islet amyloid polypeptide (IAPP), other than through amyloid formation, may be of importance in diabetes pathogenesis, IAPP-deficient mice (IAPP(-/-)) were challenged with alloxan (day 0). Diabetes in IAPP(-/-) mice was more severe at day 35, indicated by greater weight loss; glucose levels were higher in alloxan-treated IAPP(-/-) mice, whereas insulin levels were lower, indicating a greater impairment of islet function. Accordingly, glucose levels upon intravenous glucose challenges at days 7 and 35 were consistently higher in alloxan-treated IAPP(-/-) mice. At day 35, insulin mRNA expression, but not beta-cell mass, was lower in untreated IAPP(-/-) mice. Yet, upon alloxan administration, beta-cell mass and numbers of beta-cell-containing islets were significantly more reduced in IAPP(-/-) mice. Furthermore, they displayed exaggerated beta-cell dysfunction, because in their remaining beta-cells, insulin mRNA expression was significantly more impaired and the localization of glucose transporter-2 was perturbed. Thus the lack of IAPP has allowed exaggerated beta-cell cytotoxic actions of alloxan, suggesting that there may be beneficial features of IAPP actions in situations of beta-cell damage.     

Reduced beta-cell mass and altered glucose sensing impair insulin-secretory function in betaIRKO mice

Pancreatic beta-cell-restricted knockout of the insulin receptor results in hyperglycemia due to impaired insulin secretion, suggesting that this cell is an important target of insulin action. The present studies were undertaken in beta-cell insulin receptor knockout (betaIRKO) mice to define the mechanisms underlying the defect in insulin secretion. On the basis of responses to intraperitoneal glucose, approximately 7-mo-old betaIRKO mice were either diabetic (25%) or normally glucose tolerant (75%). Total insulin content was profoundly reduced in pancreata of mutant mice compared with controls. Both groups also exhibited reduced beta-cell mass and islet number. However, insulin mRNA and protein were similar in islets of diabetic and normoglycemic betaIRKO mice compared with controls. Insulin secretion in response to insulin secretagogues from the isolated perfused pancreas was markedly reduced in the diabetic betaIRKOs and to a lesser degree in the nondiabetic betaIRKO group. Pancreatic islets of nondiabetic betaIRKO animals also exhibited defects in glyceraldehyde- and KCl-stimulated insulin release that were milder than in the diabetic animals. Gene expression analysis of islets revealed a modest reduction of GLUT2 and glucokinase gene expression in both the nondiabetic and diabetic mutants. Taken together, these data indicate that loss of functional receptors for insulin in beta-cells leads primarily to profound defects in postnatal beta-cell growth. In addition, altered glucose sensing may also contribute to defective insulin secretion in mutant animals that develop diabetes.     

UCP-2 and UCP-3 proteins are differentially regulated in pancreatic beta-cells

Background

Increased uncoupling protein-2 (UCP-2) expression has been associated with impaired insulin secretion, whereas UCP-3 protein levels are decreased in the skeleton muscle of type-2 diabetic subjects. In the present studies we hypothesize an opposing effect of glucose on the regulation of UCP-2 and UCP-3 in pancreatic islets.

Methodology

Dominant negative UCP-2 and wild type UCP-3 adenoviruses were generated, and insulin release by transduced human islets was measured. UCP-2 and UCP-3 mRNA levels were determined using quantitative PCR. UCP-2 and UCP-3 protein expression was investigated in human islets cultured in the presence of different glucose concentrations. Human pancreatic sections were analyzed for subcellular localization of UCP-3 using immunohistochemistry.

Principal Findings

Dominant negative UCP-2 expression in human islets increased insulin secretion compared to control islets (p<0.05). UCP-3 mRNA is expressed in human islets, but the relative abundance of UCP-2 mRNA was 8.1-fold higher (p<0.05). Immunohistochemical analysis confirmed co-localization of UCP-3 protein with mitochondria in human beta-cells. UCP-2 protein expression in human islets was increased ∼2-fold after high glucose exposure, whereas UCP-3 protein expression was decreased by ∼40% (p<0.05). UCP-3 overexpression improved glucose-stimulated insulin secretion.

Conclusions

UCP-2 and UCP-3 may have distinct roles in regulating beta-cell function. Increased expression of UCP-2 and decreased expression of UCP-3 in humans with chronic hyperglycemia may contribute to impaired glucose-stimulated insulin secretion. These data imply that mechanisms that suppress UCP-2 or mechanisms that increase UCP-3 expression and/or function are potential therapeutic targets to offset defects of insulin secretion in humans with type-2 diabetes.     

Palmitate modulates the early steps of insulin signalling pathway in pancreatic islets

Insulin stimulates its own secretion and synthesis by pancreatic beta-cells. Although the exact molecular mechanism involved is unknown, changes in beta-cell insulin signalling have been recognized as a potential link between insulin resistance and its impaired release, as observed in non-insulin-dependent diabetes. However, insulin resistance is also associated with elevated plasma levels of free fatty acids (FFA) that are well known modulators of insulin secretion by pancreatic islets. This information led us to investigate the effect of FFA on insulin receptor signalling in pancreatic islets. Exposure of pancreatic islets to palmitate caused up-regulation of several insulin-induced activities including tyrosine phosphorylation of insulin receptor and pp185. This is the first evidence that short exposure of these cells to 100 microM palmitate activates the early steps of insulin receptor signalling. 2-Bromopalmitate, a carnitine palmitoyl-CoA transferase-1 inhibitor, did not affect the effect of the fatty acid. Cerulenin, an acylation inhibitor, abolished the palmitate effect on protein levels and phosphorylation of insulin receptor. This result supports the proposition that protein acylation may be an important mechanism by which palmitate exerts its modulating effect on the intracellular insulin signalling pathway in rat pancreatic islets.     

Islet amyloid polypeptide (IAPP) transgenic rodents as models for type 2 diabetes

Blood glucose concentrations are maintained by insulin secreted from beta-cells located in the islets of Langerhans. There are approximately 2000 beta-cells per islet, and approximately one million islets of Langerhans scattered throughout the pancreas. The islet in type 2 diabetes mellitus (T2D) has deficient beta-cell mass due to increased beta-cell apoptosis and islet amyloid derived from islet amyloid polypeptide (IAPP). Accumulating evidence implicates toxic IAPP oligomers in the mediation of beta-cell apoptosis in T2D. Humans, monkeys, and cats express an amyloidogenic toxic form of IAPP and spontaneously develop diabetes characterized by islet amyloid deposits. However, longitudinal studies of islet pathology in humans are impossible, and studies in nonhuman primates and cats are costly and impractical. Rodent IAPP is not amyloidogenic, thus commonly used rodent models of diabetes do not recapitulate islet pathology in humans. To investigate the diabetogenic role of human IAPP (h-IAPP), several mouse models and, more recently, a rat model transgenic for h-IAPP have been developed. Studies in these models have revealed that the toxic effect of h-IAPP on beta-cell apoptosis demonstrates a threshold-dependent effect. Specifically, increasing h-IAPP transgene expression by breeding or induction of insulin resistance leads to increased beta-cell apoptosis and diabetes. These transgenic rodent models for h-IAPP provide an opportunity to elucidate the mechanisms responsible for h-IAPP-induced beta-cell apoptosis further and to test novel approaches to the prevention and treatment of T2D.     

A new mouse model of spontaneous diabetes derived from ddY strain.

By the selective breeding of obese male mice of the ddY strain and using indices of the heavy body weight and appearance of urinary glucose, we established two inbred strains in 1992: one with obesity and urinary glucose (Tsumura, Suzuki, Obese Diabetes: TSOD) and the other without them (Tsumura, Suzuki, Non Obesity: TSNO). The male TSOD mice constantly showed signs of obesity and urinary glucose with increases in food and water intake, body weight and some fat weight. The body mass index (BMI) clearly showed moderate obesity. Increases in the levels of diabetic blood parameters (glucose, insulin and lipids) were also found in males, in which the levels of blood glucose and insulin were high to the ages past the growth peak. In the histological studies, pancreatic islets of the TSOD males were found hypertrophic without any signs of insulitis or fibrous formation. Among these diabetic characteristics, some of which were similar to the reported models of non-insulin-dependent diabetes mellitus (NIDDM), the stable appearances of the hyperglycemia, the hyperinsulinemia and the hypertrophy of pancreatic islets to the ages past the growth peak were the prominent features. In these respect the TSOD mouse may be a useful model for researching the mechanisms of human diabetes and its complications.     

Insulin secretion and islet endocrine cell content at onset and during the early stages of diabetes in the BB rat: effect of the level of glycemic control.

Although it is agreed that autoimmune destruction of pancreatic islets in diabetic BB rats is rapid, reports of endocrine cell content of islets from BB diabetic rats at the time of onset of diabetes vary considerably. Because of the rapid onset of the disease (hours) and the attendant changes in islet morphology and insulin secretion, it was the aim of this study to compare islet beta-cell numbers to other islet endocrine cells as close to the time of onset of hyperglycemia as possible (within 12 h). As it has been reported that hyperglycemia renders the beta cell insensitive to glucose, the early effects of different levels of insulin therapy (well-controlled vs. poorly controlled glycemia) on islet morphology and insulin secretion were examined. When measured within 12 h of onset, insulin content of BB diabetic islets, measured by morphometric analysis or pancreatic extraction, was 60% of insulin content of control islets. Despite significant amounts of insulin remaining in the pancreas, 1-day diabetic rats exhibited fasting hyperglycemia and were glucose intolerant. The insulin response from the isolated perfused pancreas to glucose and the glucose-dependent insulinotropic hormone, gastric inhibitory polypeptide (GIP), was reduced by 95%. Islet content of other endocrine peptides, glucagon, somatostatin, and pancreatic polypeptide, was normal at onset and at 2 weeks post onset. A group of diabetic animals, maintained in a hyperglycemic state for 7 days with low doses of insulin, were compared with a group kept normoglycemic by appropriate insulin therapy. No insulin could be detected in islets of poorly controlled diabetics, while well-controlled animals had 30% of the normal islet insulin content.(ABSTRACT TRUNCATED AT 250 WORDS)     

Visualizing superoxide production in normal and diabetic rat islets of Langerhans

Oxygen free radicals have been implicated in beta-cell dysfunction and apoptosis associated with type 1 and type 2 diabetes mellitus. The roles of free radicals in diabetes have thus far been defined indirectly by monitoring oxidative tissue damage and the effects of antioxidants, free radical scavengers, and overexpression of superoxide dismutase. We employed the superoxide-mediated oxidation of hydroethidine to ethidium to dynamically and directly assess the relative rates of mitochondrial superoxide anion generation in isolated islets in response to glucose stimulation. Superoxide content of isolated islets increased in response to glucose stimulation. We next compared the oxyradical levels in Zucker lean control and Zucker diabetic fatty rat islets by digital imaging microfluorometry. The superoxide content of Zucker diabetic fatty islets was significantly higher than Zucker lean control islets under resting conditions, relatively insensitive to elevated glucose concentrations, and correlated temporally with a decrease in glucose-induced hyperpolarization of the mitochondrial membrane. Importantly, superoxide levels were elevated in islets from young, pre-diabetic Zucker diabetic fatty animals. Overproduction of superoxide was associated with perturbed mitochondrial morphology and may contribute to abnormal glucose signaling found in the Zucker diabetic fatty model of type 2 diabetes mellitus.     

Genetic deficiency of glycogen synthase kinase-3beta corrects diabetes in mouse models of insulin resistance

Despite treatment with agents that enhance beta-cell function and insulin action, reduction in beta-cell mass is relentless in patients with insulin resistance and type 2 diabetes mellitus. Insulin resistance is characterized by impaired signaling through the insulin/insulin receptor/insulin receptor substrate/PI-3K/Akt pathway, leading to elevation of negatively regulated substrates such as glycogen synthase kinase-3beta (Gsk-3beta). When elevated, this enzyme has antiproliferative and proapoptotic properties. In these studies, we designed experiments to determine the contribution of Gsk-3beta to regulation of beta-cell mass in two mouse models of insulin resistance. Mice lacking one allele of the insulin receptor (Ir+/-) exhibit insulin resistance and a doubling of beta-cell mass. Crossing these mice with those having haploinsufficiency for Gsk-3beta (Gsk-3beta+/-) reduced insulin resistance by augmenting whole-body glucose disposal, and significantly reduced beta-cell mass. In the second model, mice missing two alleles of the insulin receptor substrate 2 (Irs2-/-), like the Ir+/- mice, are insulin resistant, but develop profound beta-cell loss, resulting in early diabetes. We found that islets from these mice had a 4-fold elevation of Gsk-3beta activity associated with a marked reduction of beta-cell proliferation and increased apoptosis. Irs2-/- mice crossed with Gsk-3beta+/- mice preserved beta-cell mass by reversing the negative effects on proliferation and apoptosis, preventing onset of diabetes. Previous studies had shown that islets of Irs2-/- mice had increased cyclin-dependent kinase inhibitor p27(kip1) that was limiting for beta-cell replication, and reduced Pdx1 levels associated with increased cell death. Preservation of beta-cell mass in Gsk-3beta+/- Irs2-/- mice was accompanied by suppressed p27(kip1) levels and increased Pdx1 levels. To separate peripheral versus beta-cell-specific effects of reduction of Gsk3beta activity on preservation of beta-cell mass, mice homozygous for a floxed Gsk-3beta allele (Gsk-3(F/F)) were then crossed with rat insulin promoter-Cre (RIP-Cre) mice to produce beta-cell-specific knockout of Gsk-3beta (betaGsk-3beta-/-). Like Gsk-3beta+/- mice, betaGsk-3beta-/- mice also prevented the diabetes of the Irs2-/- mice. The results of these studies now define a new, negatively regulated substrate of the insulin signaling pathway specifically within beta-cells that when elevated, can impair replication and increase apoptosis, resulting in loss of beta-cells and diabetes. These results thus form the rationale for developing agents to inhibit this enzyme in obese insulin-resistant individuals to preserve beta-cells and prevent diabetes onset.     

Role of endogenous ROS production in impaired metabolism-secretion coupling of diabetic pancreatic β cells

One of the characteristics of type 2 diabetes is that the insulin secretory response of β cells is selectively impaired to glucose. In the Goto-Kakizaki (GK) rat, a genetic model of type 2 diabetes mellitus, glucose-induced insulin secretion is selectively impaired due to deficient ATP production derived from impaired glucose metabolism. In addition, islets in GK rat and human type 2 diabetes are oxidatively stressed. In this issue, role of endogenous reactive oxygen species (ROS) production in impaired metabolism-secretion coupling of diabetic pancreatic β cells is reviewed. In β cells, ROS is endogenously produced by activation of Src, a non-receptor tyrosine kinase. Src inhibitors restore the impaired insulin release and impaired ATP elevation by reduction in ROS production in diabetic islets. Src is endogenously activated in diabetic islets, since the level of Src pY416 in GK islets is higher than that in control islets. In addition, exendin-4, a glucagon-like peptide-1 (GLP-1) receptor agonist, decreases Src pY416 and glucose-induced ROS production and ameliorates impaired ATP production dependently on Epac in GK islets. These results indicate that GLP-1 signaling regulates endogenous ROS production due to Src activation and that incretin has unique therapeutic effects on impaired glucose metabolism in diabetic β cells.     

Overexpression of Bcl-x(L) in beta-cells prevents cell death but impairs mitochondrial signal for insulin secretion

To study effects of Bcl-x(L) in the pancreatic beta-cell, two transgenic lines were produced using different forms of the rat insulin promoter. Bcl-x(L) expression in beta-cells was increased 2- to 3-fold in founder (Fd) 1 and over 10-fold in Fd 2 compared with littermate controls. After exposure to thapsigargin (10 microM for 48 h), losses of cell viability in islets of Fd 1 and Fd 2 Bcl-x(L) transgenic mice were significantly lower than in islets of wild-type mice. Unexpectedly, severe glucose intolerance was observed in Fd 2 but not Fd 1 Bcl-x(L) mice. Pancreatic insulin content and islet morphology were not different from control in either transgenic line. However, Fd 2 Bcl-x(L) islets had impaired insulin secretory and intracellular free Ca(2+) ([Ca(2+)](i)) responses to glucose and KCl. Furthermore, insulin and [Ca(2+)](i) responses to pyruvate methyl ester (PME) were similarly reduced as glucose in Fd 2 Bcl-x(L) islets. Consistent with a mitochondrial defect, glucose oxidation, but not glycolysis, was significantly lower in Fd 2 Bcl-x(L) islets than in wild-type islets. Glucose-, PME-, and alpha-ketoisocaproate-induced hyperpolarization of mitochondrial membrane potential, NAD(P)H, and ATP production were also significantly reduced in Fd 2 Bcl-x(L) islets. Thus, although Bcl-x(L) promotes beta-cell survival, high levels of expression of Bcl-x(L) result in reduced glucose-induced insulin secretion and hyperglycemia due to a defect in mitochondrial nutrient metabolism and signaling for insulin secretion.