Peter Thams

Phosphatidylinositol 4-kinase serves as a metabolic sensor and regulates priming of secretory granules in pancreatic beta cells.

Insulin secretion is controlled by the β cell′s metabolic state, and the ability of the secretory granules to undergo exocytosis increases during glucose stimulation in a membrane potential-independent fashion. Here, we demonstrate that exocytosis of insulin-containing secretory granules depends on phosphatidylinositol 4-kinase (PI 4-kinase) activity and that inhibition of this enzyme suppresses glucose-stimulated insulin secretion. Intracellular application of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] stimulated exocytosis by promoting the priming of secretory granules for release and increasing the number of granules residing in a readily releasable pool. Reducing the cytoplasmic ADP concentration in a way mimicking the effects of glucose stimulation activated PI 4-kinase and increased exocytosis whereas changes of the ATP concentration in the physiological range had little effect. The PI(4,5)P2-binding protein Ca2+-dependent activator protein for secretion (CAPS) is present in β cells, and neutralization of the protein abolished both Ca2+- and PI(4,5)P2-induced exocytosis. We conclude that ADP-induced changes in PI 4-kinase activity, via generation of PI(4,5)P2, represents a metabolic sensor in the β cell by virtue of its capacity to regulate the release competence of the secretory granules.

G protein-coupled receptor 39 deficiency is associated with pancreatic islet dysfunction.

G protein-coupled receptor (GPR)-39 is a seven-transmembrane receptor expressed mainly in endocrine and metabolic tissues that acts as a Zn(++) sensor signaling mainly through the G(q) and G(12/13) pathways. The expression of GPR39 is regulated by hepatocyte nuclear factor (HNF)-1alpha and HNF-4alpha, and in the present study, we addressed the importance of GPR39 for glucose homeostasis and pancreatic islets function. The expression and localization of GPR39 were characterized in the endocrine pancreas and pancreatic cell lines. Gpr39(-/-) mice were studied in vivo, especially in respect of glucose tolerance and insulin sensitivity, and in vitro in respect of islet architecture, gene expression, and insulin secretion. Gpr39 was down-regulated on differentiation of the pluripotent pancreatic cell line AR42J cells toward the exocrine phenotype but was along with Pdx-1 strongly up-regulated on differentiation toward the endocrine phenotype. Immunohistochemistry demonstrated that GRP39 is localized selectively in the insulin-storing cells of the pancreatic islets as well as in the duct cells of the exocrine pancreas. Gpr39(-/-) mice displayed normal insulin sensitivity but moderately impaired glucose tolerance both during oral and iv glucose tolerance tests, and Gpr39(-/-) mice had decreased plasma insulin response to oral glucose. Islet architecture was normal in the Gpr39 null mice, but expression of Pdx-1 and Hnf-1alpha was reduced. Isolated, perifused islets from Gpr39 null mice secreted less insulin in response to glucose stimulation than islets from wild-type littermates. It is concluded that GPR39 is involved in the control of endocrine pancreatic function, and it is suggested that this receptor could be a novel potential target for the treatment of diabetes.

Fat-induced changes in mouse pancreatic islet insulin secretion, insulin biosynthesis and glucose metabolism

Insulin secretion, insulin biosynthesis and islet glucose oxidation were studied in pancreatic islets isolated from fat-fed diabetic mice of both sexes. Insulin secretion from isolated islets was studied after consecutive stimulation with α-ketoisocaproic acid + glutamine, glucose, forskolin, and 12-O-tetradecanoylphorbol 13-acetate. Glucose-induced insulin secretion was impaired in islets from fat-fed mice. This was associated with a reduction of approximately 50% in islet glucose oxidation. Islet insulin secretion stimulated by the non-carbohydrate secretagogues tended to be higher in the fat-fed mice, but a statistically significant effect was not observed. Pancreatic insulin content was reduced by 50%, whereas the islet insulin and DNA content was unchanged after fat feeding. Proinsulin mRNA was reduced by 35% in islets from fat-fed mice, and was associated with a reduction of approximately 50% in glucose-stimulated (pro)insulin biosynthesis. It is concluded that the insulin secretory response of islets isolated from fat-fed mice is similar to the secretory pattern known from human type 2, non-insulin-dependent diabetics, and that a defect in islet glucose recognition, resulting in decreased glucose oxidation, may be responsible for the observed insulin secretory and biosynthetic defects seen after glucose stimulation.

STAT5 Activity in Pancreatic β-Cells Influences the Severity of Diabetes in Animal Models of Type 1 and 2 Diabetes

Pancreatic β-cell growth and survival and insulin production are stimulated by growth hormone and prolactin through activation of the transcription factor signal transducer and activator of transcription (STAT)5. To assess the role of STAT5 activity in β-cells in vivo, we generated transgenic mice that expressed a dominant-negative mutant of STAT5a (DNSTAT5) or constitutive active mutant of STAT5b (CASTAT5) under control of the rat insulin 1 promoter (RIP). When subjected to a high-fat diet, RIP-DNSTAT5 mice showed higher body weight, increased plasma glucose levels, and impairment of glucose tolerance, whereas RIP-CASTAT5 mice were more glucose tolerant and less hyperleptinemic than wild-type mice. Although the pancreatic insulin content and relative β-cell area were increased in high-fat diet–fed RIP-DNSTAT5 mice compared with wild-type or RIP-CASTAT5 mice, RIP-DNSTAT5 mice showed reduced β-cell proliferation at 6 months of age. The inhibitory effect of high-fat diet or leptin on insulin secretion was diminished in isolated islets from RIP-DNSTAT5 mice compared with wild-type islets. Upon multiple low-dose streptozotocin treatment, RIP-DNSTAT5 mice exhibited higher plasma glucose levels, lower plasma insulin levels, and lower pancreatic insulin content than wild-type mice, whereas RIP-CASTAT5 mice maintained higher levels of plasma insulin. In conclusion, our results indicate that STAT5 activity in β-cells influences the susceptibility to experimentally induced type 1 and type 2 diabetes.

Impact of fetal and neonatal environment on beta cell function and development of diabetes

The global epidemic of diabetes is a serious threat against health and healthcare expenses. Although genetics is important it does not explain the dramatic increase in incidence, which must involve environmental factors. Two decades ago the concept of the thrifty phenotype was introduced, stating that the intrauterine environment during pregnancy has an impact on the gene expression that may persist until adulthood and cause metabolic diseases like obesity and type 2 diabetes. As the pancreatic beta cells are crucial in the regulation of metabolism this article will describe the influence of normal pregnancy on the beta cells in both the mother and the fetus and how various conditions like diabetes, obesity, overnutrition and undernutrition during and after pregnancy may influence the ability of the offspring to adapt to changes in insulin demand later in life. The influence of environmental factors including nutrients and gut microbiota on appetite regulation, mitochondrial activity and the immune system that may affect beta cell growth and function directly and indirectly is discussed. The possible role of epigenetic changes in the transgenerational transmission of the adverse programming may be the most threatening aspect with regard to the global diabetes epidemics. Finally, some suggestions for intervention are presented.

The l-α-amino acid receptor GPRC6A is expressed in the islets of Langerhans but is not involved in l-arginine-induced insulin release

GPRC6A is a seven-transmembrane receptor activated by a wide range of l-α-amino acids, most potently by l-arginine and other basic amino acids. The receptor is broadly expressed, but its exact physiological role remains to be elucidated. It is well established that l-arginine stimulates insulin secretion; therefore, the receptor has been hypothesized to have a role in regulating glucose metabolism. In this study, we demonstrate that GPRC6A is expressed in islets of Langerhans, but activation of the receptor by l-arginine did not stimulate insulin secretion. We also investigated central metabolic parameters in GPRC6A knockout mice compared with wildtype littermates and found no difference in glucose metabolism or body fat percentage when mice were administered a standard chow diet. In conclusion, our data do not support a role for GPRC6A in l-arginine-induced insulin release and glucose metabolism under normal physiological conditions.

Pancreatic islet insulin secretion and metabolism in adult rats malnourished during neonatal life.

Pancreatic islets were isolated from rats that had been nursed by dams fed with a control or an 8.7% protein diet during the first 12 d of the lactation period. Glucose-induced insulin secretion from islets in the 8.7% protein group was reduced 50%. The islet insulin and DNA content were similar, whereas the pancreatic insulin content was reduced by 30 % in the rats fed 8.7 % protein. In order to elucidate the mechanism responsible for the attenuation of insulin secretion, measurements were performed of the activity of several islet enzymes that had previously been supposed to be involved in the coupling of glucose stimulation to insulin secretion. Islet glucose oxidation was unaffected, but glucose-stimulated hydrolysis of phosphatidylinositol was reduced by one-third in the islets of rats fed 8.7% protein. The activity of mitochondrial glycerophosphate dehydrogenase was similar in islets of rats fed the 8.7% protein diet and those fed the control diet. The activity of Ca-independent phospholipase A2 was increased fourfold in the islets of rats fed 8.7% protein. It is concluded that impairment of glucose-induced insulin secretion in rats fed a low-protein diet may be caused by attenuation of islet phosphatidylinositol hydrolysis, and it is tentatively suggested that the increased activity of Ca-independent phospholipase A2 in islets of rats fed a low-protein diet may participate in the stimulation of apoptosis.

Differential mechanisms of glucose and palmitate in augmentation of insulin secretion in mouse pancreatic islets

Aims/hypothesis. To assess the possible importance of saturated fatty acids in glucose amplification of K+ATP channel-independent insulin secretion. Methods. Insulin release from perifused pancreatic islets of NMRI mice was determined by radioimmunoassay. Results. In the presence of K+ (20 mmol/l) and diazoxide (250 μmol/l), which stimulates Ca2+ influx and opens K+ATP channels, palmitate (165 μmol/l total; 1.2 μmol/l free) increased insulin secretion at 3.3, 10 and 16.7 mmol/l glucose while glucose (10; 16.7 mmol/l) did not increase insulin secretion. In the presence of K+ (60 mmol/l) and diazoxide (250 μmol/l), glucose (10; 16.7 mmol/l) stimulation of K+ATP channel-independent insulin secretion increased, whereas the effectiveness of palmitate (165 μmol/l total; 1.2 μmol/l free) on insulin secretion at both 3.3, 10 or 16.7 mmol/l glucose was reduced. Palmitate thereby mimicked the stimulatory pattern of the protein kinase C activator, 12-O-tetradecanoylphorbol 13-acetate (0.16 μmol/l), which also failed to increase insulin secretion at maximum depolarising concentrations of K+ (60 mmol/l). Furthermore, the protein kinase C inhibitor calphostin C (1 μmol/l), led to a complete suppression of the effects of both palmitate (165 μmol/l total; 1.2 μmol/l free) and myristate (165 μmol/l total; 2.4 μmol/l free) stimulation of glucose (16.7 mmol/l)-induced insulin secretion. Calphostin C (1 μmol/l), however, failed to affect insulin secretion induced by glucose (16.7 mmol/l). Conclusion/interpretation. These data suggest that glucose could increase insulin secretion independently of saturated fatty acids like palmitate and myristate, which amplify glucose-induced insulin secretion by activation of protein kinase C. [Diabetologia (2001) 44: 738–746]

Inhibition of glucose-induced insulin secretion by the diacylglycerol lipase inhibitor RHC 80267 and the phospholipase A2 inhibitor ACA through stimulation of K+ permeability without diminution by exogenous arachidonic acid

The effects of the diacylglycerol lipase inhibitor 1,6-bis-(cyclohexyloximinocarbonyl-amino)-hexane (RHC 80267) and the phospholipase A2 inhibitor N-(p-amylcinnamoyl)anthranilic acid (ACA) on insulin secretion and 86Rb+ efflux in mouse pancreatic islets were studied. RHC 80267 (35 microM) and ACA (100 microM) inhibited glucose (16.7 mM)-induced insulin secretion, but did not inhibit insulin secretion induced by K+ (40 mM) or the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA; 0.16 microM). K+ (40 mM) or TPA (0.16 microM) potentiated glucose (16.7 mM)-induced insulin secretion, and prevented inhibition of glucose (16.7 mM)-induced insulin secretion by RHC 80267 and ACA. In comparison, potentiation of glucose-induced insulin secretion by albumin-bound arachidonic acid (AA; 200 microM total; 10 microM free unbound) failed to counteract inhibition of glucose-induced insulin secretion by RHC 80267 or ACA, suggesting that inhibition of insulin secretion by these agents was not mediated by a decrease in AA accumulation in islets. Determination of 86Rb+ efflux, a marker of K+ channel activity, revealed that both RHC 80267 and ACA stimulated K+ efflux from islets. These effects of RHC 80267 and ACA were observed at both 3.3 and 16.7 mM glucose and persisted in Ca2+-free medium, suggesting that they may represent an opening of ATP-sensitive K+ channels. RHC 80267-mediated stimulation of 86Rb+ efflux was not mimicked by the diacylglycerol analog TPA (0.16 microM) and was not prevented by the diacylglycerol kinase inhibitor R 59022 (50 microM), suggesting that stimulation of 86Rb+ efflux did not reflect a conditional increase in diacylglycerol or in phosphatidic acid upon inhibition of diacylglycerol lipase. In contrast, TPA (0.16 microM) attenuated RHC 80267 and ACA stimulation of 86Rb+ efflux. Addition of AA (200 microM total; 10 microM free unbound) stimulated 86Rb+ efflux, suggesting that stimulation of 86Rb+ efflux by RHC 80267 and ACA was not due to a decrease in AA accumulation. This stimulation by AA was not dependent on AA metabolism because it persisted in the presence of the lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA; 50 microM) or the cyclooxygenase inhibitor indomethacin (50 microM). In contrast to RHC 80267 and ACA, AA stimulation of 86Rb+ efflux was attenuated in Ca2+-free medium, probably implicating Ca2+-sensitive K+ channels in AA regulation of 86Rb+ efflux. Parallel experiments with diazoxide (100 microM) revealed that RHC 80267 and ACA mimicked the effects of diazoxide, a specific activator of ATP-sensitive K+ channels in islets, on both insulin secretion and 86Rb+ efflux. In conclusion, it is suggested that RHC 80267 and ACA, independently of their action on AA release, may inhibit glucose-induced insulin secretion by the opening of ATP-sensitive K+ channels in islets.

Transgenic Rescue of Adipocyte Glucose-dependent Insulinotropic Polypeptide Receptor Expression Restores High Fat Diet-induced Body Weight Gain

Background: Glucose-dependent insulinotropic polypeptide (GIP) is pursued as an anti-obesity target. Results: The adipocyte GIP receptor (GIPr) promotes high fat diet (HFD)-induced body weight gain by an increase in lean mass rather than fat mass in mice. Conclusion: The adipocyte GIPr regulates both body weight and body composition. Significance: Targeting the GIPr may have effects beyond lipid storage and acute glucose metabolism. The glucose-dependent insulinotropic polypeptide receptor (GIPr) has been implicated in high fat diet-induced obesity and is proposed as an anti-obesity target despite an uncertainty regarding the mechanism of action. To independently investigate the contribution of the insulinotropic effects and the direct effects on adipose tissue, we generated transgenic mice with targeted expression of the human GIPr to white adipose tissue or beta-cells, respectively. These mice were then cross-bred with the GIPr knock-out strain. The central findings of the study are that mice with GIPr expression targeted to adipose tissue have a similar high fat diet -induced body weight gain as control mice, significantly greater than the weight gain in mice with a general ablation of the receptor. Surprisingly, this difference was due to an increase in total lean body mass rather than a gain in total fat mass that was similar between the groups. In contrast, glucose-dependent insulinotropic polypeptide-mediated insulin secretion does not seem to be important for regulation of body weight after high fat feeding. The study supports a role of the adipocyte GIPr in nutrient-dependent regulation of body weight and lean mass, but it does not support a direct and independent role for the adipocyte or beta-cell GIPr in promoting adipogenesis.