Streptozotocin,an O-GlcNAcase inhibitor,blunts insulin and growth hormone secretion

Type 2 diabetes mellitus results from a complex interaction between nutritional excess and multiple genes. Whereas pancreatic beta-cells normally respond to glucose challenge by rapid insulin release (first phase insulin secretion), there is a loss of this acute response in virtually all of the type 2 diabetes patients with significant fasting hyperglycemia. Our previous studies demonstrated that irreversible intracellular accumulation of a glucose metabolite, protein O-linked N-acetylglucosamine modification (O-GlcNAc), is associated with pancreatic beta-cell apoptosis. In the present study, we show that streptozotocin (STZ), a non-competitive chemical blocker of O-GlcNAcase, induces an insulin secretory defect in isolated rat islet cells. In contrast, transgenic mice with down-regulated glucose to glucosamine metabolism in beta-cells exhibited an enhanced insulin secretion capacity. Interestingly, the STZ blockade of O-GlcNAcase activity is also associated with a growth hormone secretory defect and impairment of intracellular secretory vesicle trafficking. These results provide evidence for the roles of O-GlcNAc in the insulin secretion and possible involvement of O-GlcNAc in general glucose-regulated hormone secretion pathways.     

Role of the M3 muscarinic acetylcholine receptor in beta-cell function and glucose homeostasis

The release of insufficient amounts of insulin in the presence of elevated blood glucose levels is one of the key features of type 2 diabetes. Various lines of evidence indicate that acetylcholine (ACh), the major neurotransmitter of the parasympathetic nervous system, can enhance glucose-stimulated insulin secretion from pancreatic beta-cells. Studies with isolated islets prepared from whole body M(3) muscarinic ACh receptor knockout mice showed that cholinergic amplification of glucose-dependent insulin secretion is exclusively mediated by the M(3) muscarinic receptor subtype. To investigate the physiological relevance of this muscarinic pathway, we used Cre/loxP technology to generate mutant mice that lack M(3) receptors only in pancreatic beta-cells. These mutant mice displayed impaired glucose tolerance and significantly reduced insulin secretion. In contrast, transgenic mice overexpressing M(3) receptors in pancreatic beta-cells showed a pronounced increase in glucose tolerance and insulin secretion and were resistant to diet-induced glucose intolerance and hyperglycaemia. These findings indicate that beta-cell M(3) muscarinic receptors are essential for maintaining proper insulin secretion and glucose homeostasis. Moreover, our data suggest that enhancing signalling through beta-cell M(3) muscarinic receptors may represent a new avenue in the treatment of glucose intolerance and type 2 diabetes.     

Expression of yeast hexokinase in pancreatic beta cells of transgenic mice reduces blood glucose, enhances insulin secretion, and decreases diabetes.

It has been proposed that endogenous hexokinases of the pancreatic beta cell control the rate of glucose-stimulated insulin secretion and that genetic defects that reduce beta-cell hexokinase activity may lead to diabetes. To test these hypotheses, we have produced transgenic mice that have a 2-fold increase in hexokinase activity specific to the pancreatic beta cell. This increase was sufficient to significantly augment glucose-stimulated insulin secretion of isolated pancreatic islets, increase serum insulin levels in vivo, and lower the blood glucose levels of transgenic mice by 20-50% below control levels. Elevation of hexokinase activity also significantly reduced blood glucose levels of diabetic mice. These results confirm the role of beta-cell hexokinase activity in the regulation of insulin secretion and glucose homeostasis. They also provide strong support for the proposal that reductions in beta-cell hexokinase activity can produce 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 -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.     

[Val12] HRAS downregulates GLUT2 in beta cells of transgenic mice without affecting glucose homeostasis.

Glucose-induced insulin release from pancreatic beta cells depends on the beta-cell metabolism of glucose, which generates intracellular signals for secretion. The beta-cell glucose transporter isotype GLUT2 and the glucose phosphorylating enzyme glucokinase have both been implicated in coupling insulin secretion to extracellular glucose levels. Here we present evidence that a pronounced decrease in beta-cell GLUT2 has no immediate effect on glucose homeostasis. Analysis of transgenic mice overexpressing human [Val12]HRAS oncoprotein under control of the insulin promoter reveals a great reduction in plasma-membrane GLUT2 levels. These mice are nonetheless able to maintain normal fed and fasting plasma glucose and insulin levels for a period of several months. Insulin secretion studied in isolated islets and the perfused pancreas is characterized by a normal incremental response to increasing glucose concentrations. Glucose metabolism, as measured by glucose phosphorylation and oxidation in isolated islets, shows a normal dose dependence on extracellular glucose concentrations. These findings suggest that normal GLUT2 expression in beta cells is not essential for glucose sensing. The transgenic mice provide an experimental system for studying the role of glucose phosphorylation in regulation of insulin release in the absence of GLUT2.     

Overexpression of beta 2-microglobulin in transgenic mouse islet beta cells results in defective insulin secretion.

Overexpression of heavy chains of the class I major histocompatibility complex in islet beta cells of transgenic mice is known to induce nonimmune diabetes. We have now overexpressed the secretory protein beta 2-microglobulin in beta cells. Transgenic mice of one lineage had normal islets. Mice of another lineage did not become overtly diabetic but showed significant depletion of beta-cell insulin. When mice were made homozygous for the transgene locus, they developed diabetes. Introduction of the beta 2-microglobulin chain into class I heavy chain transgenic mice resulted in a significant improvement in their islet morphology and insulin content, and the female mice remained normoglycemic. These results suggest that different transgene molecules overexpressed in beta cells can cause islet dysfunction, though not necessarily overt diabetes, and that this effect is mediated by the level of transgene expression. Evidence is provided to show that beta-cell disruption by transgene overexpression occurs at the level of protein and involves a defect in insulin secretion.     

Inhibition of cholesterol biosynthesis impairs insulin secretion and voltage-gated calcium channel function in pancreatic beta-cells

Insulin secretion from pancreatic beta-cells is mediated by the opening of voltage-gated Ca2+ channels (CaV) and exocytosis of insulin dense core vesicles facilitated by the secretory soluble N-ethylmaleimide-sensitive factor attachment protein receptor protein machinery. We previously observed that beta-cell exocytosis is sensitive to the acute removal of membrane cholesterol. However, less is known about the chronic changes in endogenous cholesterol and its biosynthesis in regulating beta-cell stimulus-secretion coupling. We examined the effects of inhibiting endogenous beta-cell cholesterol biosynthesis by using the squalene epoxidase inhibitor, NB598. The expression of squalene epoxidase in primary and clonal beta-cells was confirmed by RT-PCR. Cholesterol reduction of 36-52% was observed in MIN6 cells, mouse and human pancreatic islets after a 48-h incubation with 10 mum NB598. A similar reduction in cholesterol was observed in the subcellular compartments of MIN6 cells. We found NB598 significantly inhibited both basal and glucose-stimulated insulin secretion from mouse pancreatic islets. CaV channels were markedly inhibited by NB598. Rapid photolytic release of intracellular caged Ca2+ and simultaneous measurements of the changes in membrane capacitance revealed that NB598 also inhibited exocytosis independently from CaV channels. These effects were reversed by cholesterol repletion. Our results indicate that endogenous cholesterol in pancreatic beta-cells plays a critical role in regulating insulin secretion. Moreover, chronic inhibition of cholesterol biosynthesis regulates the functional activity of CaV channels and insulin secretory granule mobilization and membrane fusion. Dysregulation of cellular cholesterol may cause impairment of beta-cell function, a possible pathogenesis leading to the development of type 2 diabetes.     

Age-dependent reduction in GLUT-2 levels is correlated with the impairment of the insulin secretory response in isolated islets of Sprague-Dawley rats

In this study we have investigated the insulin secretory response to glucose and other secretagogues (2-ketoisocaproate, 3-isobutyl-1-methyl-xanthine and arginine) of pancreatic islets isolated from Sprague-Dawley rats of various ages (from 2 to 28 months). Our results showed a significant decline in the glucose-stimulated insulin secretion, starting at 12 months of age. On the other hand, the response to non-glucose secretagogues (and mainly to 2-ketoisocaproate) was less impaired with advancing age than that to glucose. We also observed a progressive age-related decline of protein levels of the glucose transporter GLUT-2 in pancreatic islets, which was temporally concomitant and quantitatively comparable with the beta-cell alteration in glucose responsiveness (-40/50%). Finally, we observed a significant increase of the islets insulin content in older rats with respect to younger animals. We conclude that in the islet of older rats the impaired capability to respond to glucose could be dependent, at least in part, on the age-dependent reduction in GLUT-2 and could be compensated by mechanisms including a preserved responsiveness to non-glucose secretagogues and/or the development of islet hypertrophy.     

Polymyxin-B suppresses endotoxin-induced insulin hypersecretion in pancreatic islets.

Endotoxin induces insulin hypersecretion in vivo, which results in hyperinsulinemia and glucose dyshomeostasis. Polymyxin-B (PMX-B), an inhibitor of protein kinase C (PKC), has been shown to ameliorate the consequences of endotoxin-induced hyperinsulinemia in vivo. To explore the mechanism for this effect in vitro, this study determined whether PMX-B could alter endotoxin-induced insulin hypersecretion in isolated pancreatic islets of Langerhans. Pancreases were obtained from fasted, male, Sprague-Dawley rats treated with either saline (control) or endotoxin (S. enteritidis B, 16.7 mg/kg, i.v.). Three hours after the experimental treatment, islets were isolated by collagenase digestion and then incubated for 1 hr in Krebs Ringer bicarbonate buffer containing 0.5% bovine albumin, 10 mM HEPES, 300 mg/dl D-glucose, phorbol 12-myristate 13-acetate (PMA, 1 microM when present), and PMX-B (1 or 10 mM when present). In the absence of PMA and PMX-B, "endotoxic" islets hypersecreted immunoreactive insulin (IRI) relative to control islets. PMA, the prototypical PKC activator, significantly increased IRI secretion from both control and "endotoxic" islets. The additional inclusion of PMX-B (1 mM or 10 mM) in the incubation media significantly reduced insulin secretion from both control and "endotoxic" islets and suppressed the insulin hypersecretion observed in "endotoxic" islets. Since insulin secretion occurs at least partially through mechanisms dependent on PKC activation, the ability of PMX-B to suppress insulin hypersecretion in "endotoxic" islets suggests that activation of PKC within pancreatic beta-cells may play a role in the excess insulin secretion and hyperinsulinemia associated with endotoxicosis.     

Insulin release in aging: dynamic response of isolated islets of Langerhans of the rat to D-glucose and D-glyceraldehyde

Glucose-stimulated insulin release is diminished in islets of Langerhans from older rats compared to that in islets from young controls. The causes of this age-related decrease in hormone release and its relationship to the hyperglycemia seen in aging populations have not been fully elucidated. In attempts to define this secretory defect, we demonstrated in static studies that the insulin secretion to D-glyceraldehyde is not diminished in aging. To gain further insight into the effects of D-glyceraldehyde vs. D-glucose in aging and to understand the dynamics of insulin release from islets of older rats, dynamic insulin release from isolated islets of 2.5- and 13-month-old rats was studied by the technique of perifusion to 2.8 mM and 16.7 mM D-glucose or 2.8 mM D-glucose with 5, 10, or 14 mM D-glyceraldehyde. Insulin secretion at nonstimulatory glucose concentrations (2.8 mM) was similar in the two groups of islets. Insulin release was reduced by 36% from islets of older rats incubated in the presence of 16.7 mM D-glucose, and the first phase of insulin release was largely blunted compared with that in islets from young controls. In the presence of 5.0, 10.0, or 14.0 mM D-glyceraldehyde (plus 2.8 mM D-glucose), total insulin secretion was similar from islets of older and young rats, and normal biphasic release was restored to islets from older rats. Response to the secretagogues was delayed by 1 min in studies on islets from older rats. These findings demonstrate that while the aging process leads to a profound defect in glucose-stimulated insulin release from the pancreatic beta-cell, this defect is not present with every secretagogue, since the normal secretory response is restored in the presence of D-glyceraldehyde. The differences in the insulin secretory responses to D-glucose and D-glyceraldehyde in islets from older rats support the hypothesis that the major rate-limiting step in stimulus-secretion coupling in aging is before the metabolism of the trioses.     

Beta-cell lines derived from transgenic mice expressing a hybrid insulin gene-oncogene.

Three pancreatic beta-cell lines have been established from insulinomas derived from transgenic mice carrying a hybrid insulin-promoted simian virus 40 tumor antigen gene. The beta tumor cell (beta TC) lines maintain the features of differentiated beta cells for about 50 passages in culture. The cells produce both proinsulin I and II and efficiently process each into mature insulin, in a manner comparable to normal beta cells in isolated islets. Electron microscopy reveals typical beta-cell type secretory granules, in which insulin is stored. Insulin secretion is inducible up to 30-fold by glucose, although with a lower threshold for maximal stimulation than that for normal beta cells. beta TC lines can be repeatedly derived from primary beta-cell tumors that heritably arise in the transgenic mice. Thus, targeted expression of an oncogene with a cell-specific regulatory element can be used both to immortalize a rare cell type and to provide a selection for the maintenance of its differentiated phenotype.     

Glucagon-like peptide-1 induces cell proliferation and pancreatic-duodenum homeobox-1 expression and increases endocrine cell mass in the pancreas of old, glucose-intolerant rats

Glucose homeostasis in mammals is maintained by insulin secretion from the beta-cells of the islets of Langerhans. Type 2 diabetes results either from primary beta-cell failure alone and/or a failure to secrete enough insulin to overcome insulin resistance. Here, we show that continuous infusion of glucagon-like peptide-1 (7-36) (GLP-1; an insulinotropic agent), to young and old animals, had effects on the beta-cell of the pancreas other than simply on the insulin secretory apparatus. Our previous studies on a rodent model of glucose intolerance, the aging Wistar rat, show that a plateau in islet size, insulin content, and beta-cell mass is reached at 13 months, despite a continuing increase in body weight. Continuous sc infusion of GLP-1 (1.5 pM/kg x min), over 5 days, resulted in normal glucose tolerance. Our current results in both young and old rats demonstrate that treatment caused an up-regulation of pancreatic-duodenum homeobox-1 (PDX-1) expression in islets and total pancreas, induced pancreatic cell proliferation, and beta-cell neogenesis. The effects on levels of PDX-1 messenger RNA were abrogated by simultaneous infusion of Exendin (9-39), a specific antagonist of GLP-1. PDX-1 protein levels increased 4-fold in whole pancreata and 6-fold in islets in response to treatment. Beta-cell mass increased to 7.2 +/- 0.58 from 4.88 +/- 0.38 mg, treated vs. control, respectively, P < 0.02. Total pancreatic insulin content also increased from 0.55 +/- 0.02 to 1.32 +/- 0.11 microg/mg total pancreatic protein. Therefore, GLP-1 would seem to be a unique therapy that can stimulate pancreatic cell proliferation and beta-cell differentiation in the pancreas of rodents.     

Increased and persistent circulating insulin-like growth factor II in neonatal transgenic mice suppresses developmental apoptosis in the pancreatic islets

In rats, a proportion of pancreatic beta-cells are deleted by apoptosis in the second week of postnatal life and replaced by endocrine cell neogenesis from pancreatic ductal epithelium. This coincides with a reduction in pancreatic insulin-like growth factor II (IGF-II) expression, and IGF-II has been shown to act as a beta-cell survival factor in vitro. To examine whether IGF-II regulates beta-cell apoptosis in vivo, an IGF-II transgenic mouse model was used in which mouse IGF-II is overexpressed in skin, gut, and uterus driven by a keratin promoter, so that circulating IGF-II is retained postnatally. Mice were killed between postnatal days 7 and 26, and the pancreas was examined histologically. Apoptotic cells were visualized by the terminal deoxynucleotidyltransferase-mediated deoxy-UTP nick end labeling method, and proliferating cells were examined by immunohistochemistry for proliferating cell nuclear antigen. In nontransgenic mice, serum IGF-II was absent by 26 days, but mean (+/-SEM) values were 45+/-9 ng/ml (n = 5) in transgenic animals. A 2- to 3-fold rise in islet cell apoptosis was seen in normal animals between days 11 and 16, but this was substantially decreased in IGF-II transgenic mice (day 11; control, 12+/-1%; transgenic, 6+/-1%; P < 0.01; n = 5). Consequently, islets from IGF-II transgenic mice had a significantly greater mean area from days 11-16, but the proportions of beta- and alpha-cells and circulating insulin levels were not changed. Islet cell DNA synthesis was increased in transgenic mice on days 13 and 16. The total islet number per section did not alter. The results show that a persistent presence of circulating IGF-II postnatally alters endocrine pancreatic ontogeny in the mouse and largely prevents the wave of developmental apoptosis that precipitates beta-cell turnover in neonatal life.     

Effect on insulin production sorting and secretion by major histocompatibility complex class II gene expression in the pancreatic beta-cell of transgenic mice.

Expression of major histocompatibility complex (MHC) class II protein in islet beta-cells of transgenic mice causes severe diabetes without an attendant autoimmune component. Little is known of the aberrant beta-cell function and site of biological lesions responsible for the diabetic state. Therefore, changes in (pro)insulin production, processing, sorting, storage, and secretion were evaluated using the in vitro perfused pancreas from male hyperglycemic BALB/cBYJ Tg (O pinsproA alpha d pinsproA beta d) mice and a RIA capable of detecting mouse insulin or proinsulin with quantitative equivalency. Results were compared to control pancreases from normal BALB/cBYJ mice. Extractable pancreatic insulin plus proinsulin content in the transgenics was 4% of normal. Normal pancreases responded characteristically with a diphasic insulin release during 30-min stimulation by glucose, a response that was enhanced by subsequent forskolin. In contrast, hormone release from transgenic pancreases was undetectable; based on the sensitivity of the immunoassay, fractional secretion of the residual pancreatic hormone content from the transgenic pancreases was less than 25% of normal. Proinsulin or insulin constitutive release was also not detected in the absence or presence of glucose-containing stimuli even when experiments were extended to 3 h. In contrast, fractional secretion in response to nonglucose stimuli (carbachol-leucine and arginine-leucine) was greater than normal from the transgenic diabetic pancreases. Responses to glucose stimuli did not normalize even after 90 min in the absence of glucose. In other experiments, pancreases were stimulated with carbachol/leucine/forskolin for 90 min, and the proportion of proinsulin to insulin released by the regulated pathway was determined after Sep-Pak and HPLC separation of combined eluates. Proinsulin was undetectable (and, therefore, accounted for less than 10% of the total hormone secretion). It is concluded from the observations of hyperglycemia, low pancreatic insulin content, and impaired release that insulin production in the pancreas of the MHC diabetic transgenic is severely depressed. The limited insulin production and chronic hyperglycemia do not (as speculated) cause missorting to a constitutive pathway or impaired conversion of proinsulin to insulin, since a proportionately increased proinsulin release does not occur. Although the response of the secretory process to glucose-containing stimuli is almost completely destroyed, fractional secretion in response to nonglucose stimuli is enhanced. The possible contribution of hyperglycemia-induced beta-cell desensitization or specific lesions in the glucose recognition signals induced by MHC expression are discussed. Results suggest that expression of MHC class II protein causes highly specific beta-cell lesions which, in themselves, could be a contributing factor in human insulin-dependent diabetes.     

Lysosomes and pancreatic islet function: adaptation of beta-cell lysosomes to various metabolic demands

The effect of various functional demands on the lysosomes of pancreatic islet beta cells was studied in vivo. To expose pancreatic islets to different metabolic situations, normal syngeneic mouse islets were transplanted to either lean mice, alloxan-diabetic mice, or obese hyperglycemic mice. Two weeks after transplantation, primary and secondary beta-cell lysosomes of the islet grafts were analyzed by morphometry. The beta-cell lysosomes and secretory granules of the endogenous islets of lean and obese hyperglycemic mice were also measured. The beta cells of the islets transplanted to lean normoglycemic mice showed only a moderately developed synthetic apparatus and a great number of secretory granules. They had mainly secondary lysosomes, frequently containing secretory granule material, indicating a high crinophagic activity. The islet beta cells exposed to the high serum glucose concentration of alloxan-diabetic and obese hyperglycemic mice had an extensive synthetic apparatus, but a very small content of secretory granules. In these beta cells, there was a predominance of small primary lysosomes, indicating a low crinophagic activity. It is concluded that crinophagy may provide a mechanism for the pancreatic beta cell to moderate its content of insulin. When its secretory granule stores are diminished due to increased demands on insulin secretion, the beta cell seems able to drastically decrease the crinophagic activity. The detailed morphometric analysis of the endogenous islets of the lean and obese hyperglycemic mice showed that the beta cells of the obese hyperglycemic mice had a smaller number and size of the secretory granules.(ABSTRACT TRUNCATED AT 250 WORDS)