PKClambda regulates glucose-induced insulin secretion through modulation of gene expression in pancreatic beta cells

Altered regulation of insulin secretion by glucose is characteristic of individuals with type 2 diabetes mellitus, although the mechanisms that underlie this change remain unclear. We have now generated mice that lack the lambda isoform of PKC in pancreatic beta cells (betaPKClambda(-/-) mice) and show that these animals manifest impaired glucose tolerance and hypoinsulinemia. Furthermore, insulin secretion in response to high concentrations of glucose was impaired, whereas the basal rate of insulin release was increased, in islets isolated from betaPKClambda(-/-) mice. Neither the beta cell mass nor the islet insulin content of betaPKClambda(-/-) mice differed from that of control mice, however. The abundance of mRNAs for Glut2 and HNF3beta was reduced in islets of betaPKClambda(-/-) mice, and the expression of genes regulated by HNF3beta was also affected (that of Sur1 and Kir6.2 genes was reduced, whereas that of hexokinase 1 and hexokinase 2 genes was increased). Normalization of HNF3beta expression by infection of islets from betaPKClambda(-/-) mice with an adenoviral vector significantly reversed the defect in glucose-stimulated insulin secretion. These results indicate that PKClambda plays a prominent role in regulation of glucose-induced insulin secretion by modulating the expression of genes important for beta cell function.     

Increased islet apoptosis in Pdx1+/- mice

Mice with 50% Pdx1, a homeobox gene critical for pancreatic development, had worsening glucose tolerance with age and reduced insulin release in response to glucose, KCl, and arginine from the perfused pancreas. Surprisingly, insulin secretion in perifusion or static incubation experiments in response to glucose and other secretagogues was similar in islets isolated from Pdx1(+/-) mice compared with Pdx1(+/+) littermate controls. Glucose sensing and islet Ca(2+) responses were also normal. Depolarization-evoked exocytosis and Ca(2+) currents in single Pdx1(+/-) cells were not different from controls, arguing against a ubiquitous beta cell stimulus-secretion coupling defect. However, isolated Pdx1(+/-) islets and dispersed beta cells were significantly more susceptible to apoptosis at basal glucose concentrations than Pdx1(+/+) islets. Bcl(XL) and Bcl-2 expression were reduced in Pdx1(+/-) islets. In vivo, increased apoptosis was associated with abnormal islet architecture, positive TUNEL, active caspase-3, and lymphocyte infiltration. Although similar in young mice, both beta cell mass and islet number failed to increase with age and were approximately 50% less than controls by one year. These results suggest that an increase in apoptosis, with abnormal regulation of islet number and beta cell mass, represents a key mechanism whereby partial PDX1 deficiency leads to an organ-level defect in insulin secretion and diabetes.     

Gluco-incretins control insulin secretion at multiple levels as revealed in mice lacking GLP-1 and GIP receptors

The role of the gluco-incretin hormones GIP and GLP-1 in the control of beta cell function was studied by analyzing mice with inactivation of each of these hormone receptor genes, or both. Our results demonstrate that glucose intolerance was additively increased during oral glucose absorption when both receptors were inactivated. After intraperitoneal injections, glucose intolerance was more severe in double- as compared to single-receptor KO mice, and euglycemic clamps revealed normal insulin sensitivity, suggesting a defect in insulin secretion. When assessed in vivo or in perfused pancreas, insulin secretion showed a lack of first phase in Glp-1R(-/-) but not in Gipr(-/-) mice. In perifusion experiments, however, first-phase insulin secretion was present in both types of islets. In double-KO islets, kinetics of insulin secretion was normal, but its amplitude was reduced by about 50% because of a defect distal to plasma membrane depolarization. Thus, gluco-incretin hormones control insulin secretion (a) by an acute insulinotropic effect on beta cells after oral glucose absorption (b) through the regulation, by GLP-1, of in vivo first-phase insulin secretion, probably by an action on extra-islet glucose sensors, and (c) by preserving the function of the secretory pathway, as evidenced by a beta cell autonomous secretion defect when both receptors are inactivated.     

Virus-induced alterations in insulin release in hamster islets of Langerhans.

After the inoculation of Golden Syrian hamsters with the TC-83 vaccine strain of Venezuelan encephalitis (VE) virus, a sustained diminution in glucose-stimulated insulin release and glucose intolerance of shorter duration develops. To understand better the mechanism of this defect in insulin release, we examined insulin secretion in response to several test agents in isolated perifused islets from control and 24-d post-VE virus-infected hamsters. 50 islets were used in all perifusion experiments, and data were expressed as total insulin released as well as peak response for each test agent during a 30-min perifusion period from control and VE-infected islets. After perifusion with 20 mM glucose, a 45% diminution of insulin release was noted in VE-infected islets in comparison with control islets, which in turn was similar to in vivo findings. However, following 1-mM tolbutamide stimulation, insulin release was similar in control and VE-infected islets. In separate studies, 1 mM tolbutamide, 10 mM theophilline, 1 mM dibutyryl cyclic (c)AMP, and 1 mM 8-bromo-cAMP resulted in statistically similar insulin-release curves in control and VE-infected islets. Additional experiments assessing [5-3H]glucose use in control and infected islets after 20 min of perifusion with 20 mM glucose revealed virtually identical values (239 +/- 30-control; and 222 +/- 27-VE-infected islets). Morphological and morphometric evaluation of VE-infected islets (21 d following virus inoculation) showed no changes in islet volume density, beta cell density, and beta cell granulation. Thus, VE virus induces a defect in glucose-stimulated insulin release from hamster beta cells that can be corrected by cAMP analogues and does not alter islet glucose use.     

Glucokinase and IRS-2 are required for compensatory beta cell hyperplasia in response to high-fat diet-induced insulin resistance

Glucokinase (Gck) functions as a glucose sensor for insulin secretion, and in mice fed standard chow, haploinsufficiency of beta cell-specific Gck (Gck(+/-)) causes impaired insulin secretion to glucose, although the animals have a normal beta cell mass. When fed a high-fat (HF) diet, wild-type mice showed marked beta cell hyperplasia, whereas Gck(+/-) mice demonstrated decreased beta cell replication and insufficient beta cell hyperplasia despite showing a similar degree of insulin resistance. DNA chip analysis revealed decreased insulin receptor substrate 2 (Irs2) expression in HF diet-fed Gck(+/-) mouse islets compared with wild-type islets. Western blot analyses confirmed upregulated Irs2 expression in the islets of HF diet-fed wild-type mice compared with those fed standard chow and reduced expression in HF diet-fed Gck(+/-) mice compared with those of HF diet-fed wild-type mice. HF diet-fed Irs2(+/-) mice failed to show a sufficient increase in beta cell mass, and overexpression of Irs2 in beta cells of HF diet-fed Gck(+/-) mice partially prevented diabetes by increasing beta cell mass. These results suggest that Gck and Irs2 are critical requirements for beta cell hyperplasia to occur in response to HF diet-induced insulin resistance.     

Stimulation of insulin secretion reveals heterogeneity of pancreatic B cells in vivo.

We examined the immunofluorescence and ultrastructural changes of insulin-producing B cells in the center and at the periphery of islets of Langerhans during in vivo stimulation by glucose and glibenclamide. A decreased insulin immunostaining was detected in islets from the splenic rat pancreas after 1.5 h of glucose stimulation. By contrast, immunofluorescence changes became apparent in islets from the duodenal pancreas only after greater than 3 h of hyperglycemia. In both cases, the immunolabeling of central B cells decreased before that of peripheral B cells. Similar changes were seen following in vivo stimulation of insulin secretion by glibenclamide. At the ultrastructural level, hyperglycemia decreased the volume density of B cell secretory granules and increased that of rough endoplasmic reticulum and Golgi apparatus. These changes were also detected earlier in central than in peripheral B cells and earlier in splenic than in duodenal islets. The data show that B cells form a heterogeneous population in vivo.     

The loss of GLUT2 expression by glucose-unresponsive beta cells of db/db mice is reversible and is induced by the diabetic environment.

Glucose-induced insulin secretion by beta cells of diabetic db/db mice was studied by a pancreas perfusion technique, and the levels of GLUT2 protein in pancreatic islets were assessed by immunofluorescence microscopy and protein blot analysis. Beta cells from diabetic mice had a high basal rate of insulin secretion; they did not respond to glucose stimulation but displayed a normal secretory response to arginine. At the same time, GLUT2 expression by db/db islets was lost whereas beta cells from nondiabetic db/+ mice expressed high levels of this transporter. GLUT2 levels in liver or kidney of diabetic mice were, however, mostly unaltered. Transplanting islets from db/db mice under the kidney capsule of db/+ mice restored normal GLUT2 levels. Conversely, transplantation of db/+ islets into db/db mice induced the disappearance of GLUT2 expression. When islets from db/+ mice were transplanted under the kidney capsule of streptozocin-diabetic mice, the immunodetection of GLUT2 also disappeared. We conclude that: (a) GLUT2 expression is decreased in glucose-unresponsive beta cells from db/db mice; (b) the decreased expression of GLUT2 is reversible; (c) the loss of GLUT2 expression is induced by the diabetic environment of db/db and streptozocin-induced diabetic mice. These observations together with previously published data suggest that a factor different from glucose or insulin regulates the beta cell expression of GLUT2.     

Overexpression of monocarboxylate transporter and lactate dehydrogenase alters insulin secretory responses to pyruvate and lactate in beta cells

Previous investigations revealed low activities of lactate dehydrogenase (LDH) and plasma membrane monocarboxylate transporters (MCT) in the pancreatic beta cell. In this study the significance of these characteristics was explored by overexpressing type A LDH (LDH-A) and/or type 1 MCT (MCT-1) in the clonal INS-1 beta cells and isolated rat islets. Inducible overexpression of LDH-A resulted in an 87-fold increase in LDH activity in INS-1 cells. Adenovirus-mediated overexpression of MCT-1 increased lactate transport activity 3.7-fold in INS-1 cells. Although overexpression of LDH-A, and/or MCT-1 did not affect glucose-stimulated insulin secretion, LDH-A overexpression resulted in stimulation of insulin secretion even at a low lactate concentration with a concomitant increase in its oxidation in INS-1 cells regardless of MCT-1 co-overexpression. Adenovirus-mediated overexpression of MCT-1 caused an increase in pyruvate oxidation and conferred pyruvate-stimulated insulin release to isolated rat islets. Although lactate did not stimulate insulin secretion from control or MCT-1-overexpressing islets, co-overexpression of LDH-A and MCT-1 evoked lactate-stimulated insulin secretion with a concomitant increase in lactate oxidation in rat islets. These results suggest that low expression of MCT and LDH is requisite to the specificity of glucose in insulin secretion, protecting the organism from undesired hypoglycemic actions of pyruvate and lactate during exercise and other catabolic states.     

Enzyme-induced modifications of beta cell function. I. Effect of pronase on insulin secretion

Abstract. The effects upon immunoreactive insulin (IRI) release of beta cell membrane modifications induced by pronase, a mixture of proteolytic enzymes extracted from Streptomyces griseus, have been studied in isolated islets of Langerhans. Pretreatment of the islets for 90 min with 4 μg/ml pronase did not modify their IRI content; it slightly increased the basal rate of IRI release and potentiated the secretory response to glucose, leucine and tolbutamide during incubation. In perifused islets, the rapid phase of IRI secretion in response to glucose was more markedly enhanced than the late phase. After preincubation with 4 μg/ml pronase, glucose-induced IRI release was reversible and abolished in the absence of calcium. Pretreatment for 90 min with 500 μg/ml pronase decreased IRI content of the islets by approximately 25% and provoked a pronounced but transient rise of basal IRI release. This was considered to be a leakage of IRI from damaged beta cells since it persisted in a medium deprived of calcium. The rapid secretory phase in response to glucose was preserved in perifused islets whereas the late phase was markedly reduced. The secretory responses to leucine and tolbutamide were almost completely obliterated. When pretreatment with pronase was carried out in a calcium-depleted medium, basal IRI release from islets preincubated with 500 μg/ml pronase was only slightly increased whereas IRI secretion induced by glucose was inhibited by 40 and 65%, respectively in islets pretreated with 4 and 500 μg/ml pronase. These results indicate that pronase-induced modifications of the beta cell membrane influence IRI secretion in a way which depends on the concentration of the enzyme and the presence of extracellular calcium. They are considered to support the hypothesis that membrane systems are involved in IRI releasing mechanisms.     

Enzyme-Induced Modifications of Beta Cell Function

Abstract. The effects upon immunoreaetive insulin (IRI) release of beta cell membrane modifications induced by pronase, a mixture of proteolytic enzymes extracted from Streptomyces griseus , have been studied in isolated islets of Langerbans. Pretreatment of the islets for 90 min with 4 μg/ml pronase did not modify their IRI content; it slightly increased the basal rate of IRI release and potentiated the secretory response to glucose, leucine and tolbutamide during incubation. In perifused islets, the rapid phase of IRI secretion in response to glucose was more markedly enhanced than the late phase. After preincubation with 4 μg/ml pronase, glucose-induced IRI release was reversible and abolished in the absence of calcium. Pretreatment for 90 min with 500 μg/ml pronase decreased IRI content of the islets by approximately 25% and provoked a pronounced but transient rise of basal IRI release. This was considered to be a leakage of IRI from damaged beta cells since it persisted in a medium deprived of calcium. The rapid secretory phase in response to glucose was preserved in perifused islets whereas the late phase was markedly reduced. The secretory responses to leucine and tolbutamide were almost completely obliterated. When pretreatment with pronase was carried out in a calcium-depleted medium, basal IRI release from islets preincubated with 500 μg/ml pronase was only slightly increased whereas IRI secretion induced by glucose was inhibited by 40 and 65%, respectively in islets pre treated with 4 and 500 μg/ml pronase. These results indicate that pronase-induced modifications of the beta cell membrane influence IRI secretion in a way which depends on the concentration of the enzyme and the presence of extracellular calcium. They are considered to support the hypothesis that membrane systems are involved in IRI releasing mechanisms.     

Effect of clonidine on insulin secretion: a case report

We have recently seen a case of non-insulin-dependent diabetes mellitus with hypertension in which chronic treatment with oral clonidine gave rise to elevation of blood glucose and decreased insulin secretion. When the response of insulin secretion to glucose administration during clonidine therapy was compared with that after 12 days of wash-out for clonidine in this patient (who was then receiving phentolamine mesylate), there was a marked suppression of insulin secretion to stimulation by intravenous glucose during oral clonidine therapy. This result indicates that the decreased insulin secretion associated with oral clonidine therapy is very unlikely to be due to any direct action of clonidine on beta cells of the pancreatic islets and may be due to suppression of catecholamine release via central alpha-adrenergic receptor stimulation.     

Direct glucocorticoid inhibition of insulin secretion. An in vitro study of dexamethasone effects in mouse islets.

The direct effects of glucocorticoids on pancreatic beta cell function were studied with normal mouse islets. Dexamethasone inhibited insulin secretion from cultured islets in a concentration-dependent manner: maximum of approximately 75% at 250 nM and IC50 at approximately 20 nM dexamethasone. This inhibition was of slow onset (0, 20, and 40% after 1, 2, and 3 h) and only slowly reversible. It was prevented by a blocker of nuclear glucocorticoid receptors, by pertussis toxin, by a phorbol ester, and by dibutyryl cAMP, but was unaffected by an increase in the fuel content of the culture medium. Dexamethasone treatment did not affect islet cAMP levels but slightly reduced inositol phosphate formation. After 18 h of culture with or without 1 microM dexamethasone, the islets were perifused and stimulated by a rise in the glucose concentration from 3 to 15 mM. Both phases of insulin secretion were similarly decreased in dexamethasone-treated islets as compared with control islets. This inhibition could not be ascribed to a lowering of insulin stores (higher in dexamethasone-treated islets), to an alteration of glucose metabolism (glucose oxidation and NAD(P)H changes were unaffected), or to a lesser rise of cytoplasmic Ca2+ in beta cells (only the frequency of the oscillations was modified). Dexamethasone also inhibited insulin secretion induced by arginine, tolbutamide, or high K+. In this case also the inhibition was observed despite a normal rise of cytoplasmic Ca2+. In conclusion, dexamethasone inhibits insulin secretion through a genomic action in beta cells that leads to a decrease in the efficacy of cytoplasmic Ca2+ on the exocytotic process.     

Hypoplasia of pancreatic islets in transgenic mice expressing activin receptor mutants.

Activin, a member of the TGF-beta superfamily, regulates the growth and differentiation of a variety of cell types. Based on the expression of activin in pancreatic rudiments of rat embryos and stimulation of insulin secretion from adult rat pancreatic islets by activin, activin is implicated in the development and function of islets. To examine the significance of activin signaling in the fetal and postnatal development of islets, transgenic mice expressing a dominant negative form of activin receptor (dn-ActR) or a constitutively active form of activin receptor (ActR-T206D) in islets were generated together with the transgenic mice expressing intact activin receptor (intact ActR) as a negative control. Transgenic mice with both dn-ActR and ActR-T206D showed lower survival rates, smaller islet area, and lower insulin content in the whole pancreas with impaired glucose tolerance when compared with transgenic mice with intact ActR or littermates, but they showed the same alpha cell/beta cell ratios as their littermates. In addition to islet hypoplasia, the insulin response to glucose was severely impaired in dn-ActR transgenic mice. It is suggested that a precisely regulated intensity of activin signaling is necessary for the normal development of islets at the stage before differentiation into alpha and beta cells, and that activin plays a role in the postnatal functional maturation of islet beta cells.     

Probing cell type-specific functions of Gi in vivo identifies GPCR regulators of insulin secretion

The in vivo roles of the hundreds of mammalian G protein-coupled receptors (GPCRs) are incompletely understood. To explore these roles, we generated mice expressing the S1 subunit of pertussis toxin, a known inhibitor of G(i/o) signaling, under the control of the ROSA26 locus in a Cre recombinase-dependent manner (ROSA26(PTX)). Crossing ROSA26(PTX) mice to mice expressing Cre in pancreatic beta cells produced offspring with constitutive hyperinsulinemia, increased insulin secretion in response to glucose, and resistance to diet-induced hyperglycemia. This phenotype underscored the known importance of G(i/o) and hence of GPCRs for regulating insulin secretion. Accordingly, we quantified mRNA for each of the approximately 373 nonodorant GPCRs in mouse to identify receptors highly expressed in islets and examined the role of several. We report that 3-iodothyronamine, a thyroid hormone metabolite, could negatively and positively regulate insulin secretion via the G(i)-coupled alpha(2A)-adrenergic receptor and the G(s)-coupled receptor Taar1, respectively, and protease-activated receptor-2 could negatively regulate insulin secretion and may contribute to physiological regulation of glucose metabolism. The ROSA26(PTX) system used in this study represents a new genetic tool to achieve tissue-specific signaling pathway modulation in vivo that can be applied to investigate the role of G(i/o)-coupled GPCRs in multiple cell types and processes.     

Involvement of the autonomic nervous system in the in vivo memory to glucose of pancreatic beta cell in rats.

The fact that the potentiating effect of prolonged hyperglycemia on the subsequent insulin secretion is observed in vivo but not in vitro suggests the involvement of extrapancreatic factors in the in vivo memory of pancreatic beta cells to glucose. We have investigated the possible role of the autonomic nervous system. Rats were made hyperglycemic by a 48-h infusion with glucose (HG rats). At the end of glucose infusion as well as 6 h postinfusion, both parasympathetic and sympathetic nerve activities were profoundly altered: parasympathetic and sympathetic activities, assessed by the firing rate either of the thoracic vagus nerve or the superior cervical ganglion, were dramatically increased and decreased, respectively. Moreover, 6 h after the end of glucose infusion, insulin secretion in response to a glucose load was dramatically increased in HG rats compared to controls. To determine whether these changes could be responsible for the increased sensitivity of the beta cell to glucose, insulin release in response to glucose was measured in HG and control rats, either under subdiaphragmatic vagotomy or after administration of the alpha 2A-adrenergic agonist oxymetazoline. Both treatments partially abolished the hyperresponsiveness of the beta cell to glucose in HG rats. Therefore chronic hyperglycemia brings about changes in the activity of the autonomic nervous system, which in turn are responsible, at least in part, for the generation of enhanced beta cell responsiveness to glucose in vivo.