Impairment Of Glucose-stimulated Insulin Release Research Articles

Role of nitric oxide synthase isoforms in glucose-stimulated insulin release.

The role of islet constitutive nitric oxide synthase (cNOS) in insulin-releasing mechanisms is controversial. By measuring enzyme activities and protein expression of NOS isoforms [i.e., cNOS and inducible NOS (iNOS)] in islets of Langerhans cells in relation to insulin secretion, we show that glucose dose-dependently stimulates islet activities of both cNOS and iNOS, that cNOS-derived nitric oxide (NO) strongly inhibits glucose-stimulated insulin release, and that short-term hyperglycemia in mice induces islet iNOS activity. Moreover, addition of NO gas or an NO donor inhibited glucose-stimulated insulin release, and different NOS inhibitors effected a potentiation. These effects were evident also in K+-depolarized islets in the presence of the ATP-sensitive K+ channel opener diazoxide. Furthermore, our results emphasize the necessity of measuring islet NOS activity when using NOS inhibitors, because certain concentrations of certain NOS inhibitors might unexpectedly stimulate islet NO production. This is shown by the observation that 0.5 mmol/l of the NOS inhibitor N(G)-monomethyl-L-arginine (L-NMMA) stimulated cNOS activity in parallel with an inhibition of the first phase of glucose-stimulated insulin release in perifused rats islets, whereas 5.0 mmol/l of L-NMMA markedly suppressed cNOS activity concomitant with a great potentiation of the insulin secretory response. The data strongly suggest, but do not definitely prove, that glucose indeed has the ability to stimulate both cNOS and iNOS in the islets and that NO might serve as a negative feedback inhibitor of glucose-stimulated insulin release. The results also suggest that hyperglycemia-evoked islet NOS activity might be one of multiple factors involved in the impairment of glucose-stimulated insulin release in type II diabetes mellitus.

TPN-evoked dysfunction of islet lysosomal activity mediates impairment of glucose-stimulated insulin release.

We examined the relation between nutrient-stimulated insulin secretion and the islet lysosome acid glucan-1,4-alpha-glucosidase system in rats undergoing total parenteral nutrition (TPN). During TPN treatment, serum glucose was normal, but free fatty acids, triglycerides, and cholesterol were elevated. Islets from TPN-infused rats showed increased basal insulin release, a normal insulin response to cholinergic stimulation but a greatly impaired response when stimulated by glucose or alpha-ketoisocaproic acid. This impairment of glucose-stimulated insulin release was only slightly ameliorated by the carnitine palmitoyltransferase 1 inhibitor etomoxir. However, in parallel with the impaired insulin response to glucose, islets from TPN-infused animals displayed reduced activities of islet lysosomal enzymes including the acid glucan-1,4-alpha-glucosidase, a putative key enzyme in nutrient-stimulated insulin release. By comparison, the same lysosomal enzymes were increased in liver tissue. Furthermore, in intact control islets, the pseudotetrasaccharide acarbose, a selective inhibitor of acid alpha-glucosidehydrolases, dose dependently suppressed islet acid glucan-1,4-alpha-glucosidase and acid alpha-glucosidase activities in parallel with an inhibitory action on glucose-stimulated insulin secretion. By contrast, when incubated with intact TPN islets, acarbose had no effect on either enzyme activity or glucose-induced insulin release. Moreover, when acarbose was added directly to TPN islet homogenates, the dose-response effect on the catalytic activity of the acid alpha-glucosidehydrolases was shifted to the right compared with control homogenates. We suggest that a general dysfunction of the islet lysosomal/vacuolar system and reduced catalytic activities of acid glucan-1,4-alpha-glucosidase and acid alpha-glucosidase may be important defects behind the impairment of the transduction mechanisms for nutrient-stimulated insulin release in islets from TPN-infused rats.

Cytokine-Induced Inhibition of Insulin Release from Mouse Pancreatic β-Cells Deficient in Inducible Nitric Oxide Synthase

Cytokines may participate in islet destruction during the development of type 1 diabetes. Expression of inducible nitric oxide synthase (iNOS) and subsequent NO formation induced by IL-1β or (IL-1β + IFN-γ) may impair islet function in rodent islets. Inhibition of iNOS or a deletion of the iNOS gene (iNOS −/− mice) protects against cytokine-induced β-cell suppression, although cytokines might also induce NO-independent impairment. Presently, we exposed wild-type (wt, C57BL/6 × 129SvEv) and iNOS −/− islets to IL-1β (25 U/ml) and (IL-1β (25 U/ml) + IFN-γ (1000 U/ml)) for 48 h. IL-1β and (IL-1β + IFN-γ) induced a significant increase in NO formation in wt but not in iNOS −/− islets. Both IL-1β and (IL-1β + IFN-γ) impaired glucose-stimulated insulin release and reduced the insulin content of wt islets, while (IL-1β + IFN-γ) reduced glucose oxidation rates and cell viability. IL-1β exposure to iNOS −/− islets impaired glucose-stimulated insulin release, increased insulin accumulation and reduced the insulin content, without any increase in cell death. Exposure to (IL-1β + IFN-γ) had no effect on iNOS −/− islets except reducing the insulin content. Our data suggest that IL-1β may inhibit glucose-stimulated insulin release by pathways that are not NO-dependent and not related to glucose metabolism or cell death.

Total Parenteral Nutrition Modulates Hormone Release by Stimulating Expression and Activity of Inducible Nitric Oxide Synthase in Rat Pancreatic Islets

The expression and activities of constitutive nitric oxide synthase (cNOS) and inducible nitric oxide synthase (iNOS) in relation to insulin and glucagon secretory mechanisms were investigated in islets isolated from rats subjected to total parenteral nutrition (TPN) for 10 d. TPN is known to result in significantly increased levels of plasma lipids during the infusion time. In comparison with islets from freely fed control rats, islets taken from TPN rats at d 10 displayed a marked decrease in glucose-stimulated insulin release (4.65 +/- 0.45 ng/[islet x h] vs 10.25 +/- 0.65 for controls) (p < 0.001) accompanied by a strong iNOS activity (18.3 +/- 1.1 pmol of NO/[min x mg of protein]) and a modestly reduced cNOS activity (11.3 +/- 3.2 pmol of NO/[min x mg of protein] vs 17.7 +/- 1.7 for controls) (p < 0.01). Similarly, Western blots showed the expression of iNOS protein as well as a significant reduction in cNOS protein in islets from TPN-treated rats. The enhanced NO production, which is known to inhibit glucose-stimulated insulin release, was manifested as a strong increase in the cyclic guanosine 5'-monophosphate content in the islets of TPN-treated rats (1586 +/- 40 amol/islet vs 695 +/- 64 [p < 0.001] for controls). Moreover, the content of cyclic adenosine monophosphate (cAMP) was greatly increased in the TPN islets (80.4 +/- 2.1 fmol/islet vs 42.6 +/- 2.6 [p < 0.001] for controls). The decrease in glucose-stimulated insulin release was associated with an increase in the activity of the secretory pathway regulated by the cAMP system in the islets of TPN-treated rats, since the release of insulin stimulated by the phosphodiesterase inhibitor isobutylmethylxanthine was greatly increased both in vivo after iv injection and after in vitro incubation of isolated islets. By contrast, the release of glucagon was clearly reduced in islets taken from TPN-treated rats (33.5 +/- 1.5 pg/[islet x h] vs 45.5 +/- 2.2 for controls) (p < 0.01) when islets were incubated at low glucose (1.0 mmol/L). The data show that long-term TPN treatment in rats brings about impairment of glucose-stimulated insulin release, that might be explained by iNOS expression and a marked iNOS-derived NO production in the beta-cells. The release of glucagon, on the other hand, is probably decreased by a direct "nutrient effect" of the enhanced plasma lipids. The results also suggest that the islets of TPN-treated rats have developed compensatory insulin secretory mechanisms by increasing the activity of their beta-cell cAMP system.

Impaired glucose-stimulated insulin release in islets from adult rats malnourished during foetal-neonatal life

Poor foetal and neonatal nutrition may impair normal pancreatic β-cell development and predispose to diabetes in later life. We investigate here the nature of the pancreatic β-cell dysfunction in sucrose-fed adult offspring malnourished during the foetal-neonatal period and examine glucose metabolism and the generation of signals involved in the secretory mechanism. In islets from sucrose-fed previously malnourished rats, rates of glucose utilisation (production of 3H 2O) and oxidation (production of 14CO 2), at 2, 6 and 10 mM glucose, were not lower than those of controls. ATP concentrations in islets from previously malnourished rats fed sucrose at 2 and 10 mM glucose were similar to those of controls. Glucose-stimulated insulin release was impaired (by 49–55%) in islets from these animals as was the response to keto-isocaproate (by 70%) and tolbutamide (by 70%). Under conditions in which ATP-sensitive K + channels were clamped open (40 mM K + and diazoxide), glucose-stimulated insulin release in islets from previously malnourished rats fed sucrose was reduced. These findings show that defects in insulin secretion in islets isolated from previously malnourished animals are located in both ATP-sensitive K + channel dependent and independent pathways. They do not involve alterations in the early steps of glucose handling in the β-cell, including glucose metabolism and ATP generation.

Insulin Secretory Defect in Zucker fa/fa Rats Is Improved by Ameliorating Insulin Resistance

The role of insulin resistance in the impaired glucose-stimulated insulin release of Zucker fatty rats was investigated using the insulin-sensitizing thiazolidinedione drug pioglitazone. Fatty rats had fasting hyperinsulinemia yet a blunted secretory response to intravenous glucose compared with lean age-matched controls. Islets from fatty rats secreted less insulin (based on islet DNA) in response to high glucose than islets from lean rats but secreted normal amounts of insulin when tolbutamide or alpha-ketoisocaproic acid (alpha-KIC) was the stimulus. Administering pioglitazone for 9 days diminished basal hyperinsulinemia and increased the insulin response to high glucose by fatty rats but not by lean controls. Pioglitazone pretreatment augmented the secretory response by isolated islets to high glucose, alpha-KIC, and tolbutamide. Augmentation of islet insulin release was not associated with reduced plasma glucose concentration, suggesting that altered glycemia was not involved. Pancreas and islet insulin content was greater in fatty rats than in lean controls and was decreased by pioglitazone; hence, insulin stores and glucose-stimulated insulin release did not correlate. Pioglitazone treatment did not affect the rate of islet glucose usage or ATP/ADP in the presence of 2.75 or 16 mmol/l glucose. These data indicate that ameliorating insulin resistance reverses defective glucose-stimulated insulin release by Zucker fa/fa rats. After pioglitazone administration, insulin secretion may be augmented by increased generation of a metabolic coupling factor from glucose or at a later step in the secretory process that is common to both glucose and nonglucose secretagogues.

SAM prevents impairment of glucose-stimulated insulin secretion caused by hexose deprivation or starvation

Succinic acid monomethyl ester (SAM) was recently proposed as an insulinotropic tool in non-insulin-dependent diabetes mellitus. Three models were now used to investigate whether SAM protects the B-cell against the impairment of glucose-stimulated insulin release caused by either glucose deprivation or starvation. In the first model, preincubation of the islets for 180 min at low glucose concentration in the presence of SAM prevented the decrease in the secretory response to D-glucose otherwise observed during a subsequent incubation. In the second model, an impaired secretory response to D-glucose was observed after 3-day culture at low (2.8 or 5.6 mM) as distinct from high (11.1 mM) hexose concentration and the presence of SAM in the culture medium again protected against this anomaly. In the third model, the infusion of SAM for 3 days to starved rats restored the secretory potential of isolated islets to a level comparable to that otherwise found in fed rats. Thus, during glucose deprivation or starvation, SAM is indeed able to maintain B-cell responsiveness to D-glucose.

Protective Action of Succinic Acid Monomethyl Ester Against the Impairment of Glucose-Stimulated Insulin Release Caused by Glucopenia or Starvation: Metabolic Determinants

The monomethyl ester of succinic acid (SME) was recently found to protect pancreatic islet B-cells against the impairment of glucose-stimulated insulin release caused by either glucopenia or starvation. The possible metabolic determinants of such a protective action are now scrutinized. After 180 min preincubation at 2.8 mM D-glucose in the presence of SME (10 mM), the oxidation of D-[U-14C]glucose, relative to either the utilization of D-[5-3H]glucose or the generation of 14C-labeled acidic metabolites, was higher than that after preincubation in the absence of SME and became close to that otherwise found after preincubation at 16.7 mM D-glucose. Likewise, after 3 days of culture at a low concentration of D-glucose (2.8 mM), the presence of SME in the culture medium tended to increase the subsequent oxidation of D-[6-14C]glucose and utilization of D-[5-3H]glucose. These two variables increased as a function of the concentration of D-glucose in the culture medium, this coinciding with a modest increase in hexokinase activity and a more pronounced increase in glucokinase activity. The presence of SME in the culture medium failed, however, to exert any obvious effect upon the respiration of the islets, suggesting that the protective action of the ester against glucopenia may also involve variables distinct from the metabolism of either endogenous or exogenous nutrients. Likewise, the fact that SME infusion to starved rats prevents the impairment of glucose-induced insulin release otherwise attributable to starvation may involve enzymatic determinants, such as a less severe decrease in glucokinase activity, metabolic variables, such as a greater relative increase in D-[U-14C]glucose oxidation relative to D-[5-3H]glucose utilization in response to a rise in extracellular D-glucose concentration, and other factors yet to be identified that participate in the secretory sequence at a site distal to those metabolic events triggered by D-glucose in the islet cells.

Inhibition of insulin secretion, but normal peripheral insulin sensitivity, in a patient with a malignant endocrine pancreatic tumour producing high amounts of an islet amyloid polypeptide-like molecule

Islet amyloid polypeptide or amylin is a polypeptide secreted mainly from the pancreatic beta cells together with insulin upon stimulation. High levels of islet amyloid polypeptide have also been shown to increase the peripheral insulin resistance and consequently a role for islet amyloid polypeptide in the glucose homeostasis has been suggested. We have studied the glucose homeostasis in a patient with a malignant endocrine pancreatic tumour producing large amounts of an islet amyloid polypeptide-like molecule (about 400 times the upper reference level for islet amyloid polypeptide). This patient developed insulin-requiring diabetes mellitus shortly after the tumour diagnosis. Both intravenous and oral glucose tolerance tests revealed inhibited early responses in insulin and C-peptide release, but the insulin and C-peptide response to glucagon stimulation was less affected. Aneuglycaemic insulin clamp showed normal insulin-mediated glucose disposal. In vitro experiments, where isolated rat pancreatic islets were cultured with serum from the patient, showed a moderately decreased islet glucose oxidation rate and glucose-stimulated insulin release compared to islets cultured with serum from healthy subjects. However, culture of rat islets with normal human serum supplemented with synthetic rat islet amyloid polypeptide did not affect the glucose-stimulated insulin release. In conclusion, the observed effects show that the diabetic state in this patient was associated with an impaired glucose-stimulated insulin release but not with an increased peripheral insulin resistance.(ABSTRACT TRUNCATED AT 250 WORDS)

Survival and B-Cell Function of Neonatal Pig Pancreatic Islet-Like Cell Clusters in an Extracellular Matrix

Islet-like cell clusters (ICCs), formed from single cells of pig pancreas in suspension culture, were embedded in an extracellular matrix. It was recently reported that nicotinamide prevented dissolution of the extracellular matrix by the ICCs. In this experiment, various conditions for embedded culture of ICCs in an extracellular matrix were studied, in an attempt to maintain the function as well as the extracted insulin content of culture specimens. The ICCs in the matrix were refed with RPMI 1640, containing 10 mM nicotinamide, 10% fetal bovine serum (FBS), 11 mM D-glucose and with or without 0.1 mM 3-isobutyl-1-methylxanthine (IBMX) or 1.0 micrograms/ml caerulein. A comparison between the different culture media showed that embedded ICCs, maintained in RPMI 1640 with caerulein, in the presence of nicotinamide, had higher insulin content accumulation than when maintained in medium containing nicotinamide alone, but had impaired glucose-stimulated insulin secretion. In the medium containing IBMX and nicotinamide, embedded ICCs showed higher insulin accumulation but lower insulin content, compared to ICCs maintained in the presence of caerulein, and also showed impaired glucose-stimulated insulin release. Thus, the effect of nicotinamide on the survival and function of B-cells is amplified by the presence of caerulein or IBMX.

Heat shock protein induction in rat pancreatic islets by recombinant human interleukin 1?

Interleukin 1 beta, potentiated by tumour necrosis factor alpha, is cytotoxic to pancreatic Beta cells in vitro. We have hypothesized that interleukin 1 beta induces oxygen free radicals in Beta cells. Since cytotoxicity induced by free radicals and by heat may activate the same cellular repair mechanism (the heat shock response), the aim of this study was to investigate the pattern of protein synthesis in isolated islets after exposure to interleukin 1 beta (150 pg/ml, 24 h), tumour necrosis factor alpha (50 ng/ml, 24 h) heat shock (43 degrees C, 30 min) and H2O2 (0.1 mmol/l, 20 min). By polyacrylamide gel electrophoresis, autoradiography, Western-blot analysis and partial peptide mapping of 35S-methionine labelled islets, interleukin 1 beta was found to induce a 73 kilodalton protein belonging to the heat shock protein family heat shock protein 70, a heat shock protein 90, and haem oxygenase. A minor induction of heat shock protein 73 and haem oxygenase was seen after H2O2. Interleukin 1 beta did not induce heat shock proteins in rat thyroid cells, rat mesangial cells or in human monocytes. Tumour necrosis factor alpha did not induce selective protein synthesis. Pre-exposure of islets to heat, tumour necrosis factor alpha, or H2O2 did not prevent the impairment of glucose-stimulated insulin release seen after 24 h of interleukin 1 beta exposure. The data are compatible with free radical induction by interleukin 1 beta. However, the heat shock response is not specific for oxidative injury, and previous studies have shown discrepant effects as to a protective effect of free radical scavengers against interleukin 1 beta-mediated beta-cytotoxicity.(ABSTRACT TRUNCATED AT 250 WORDS)

Impaired insulin biosynthetic capacity in a rat model for non-insulin-dependent diabetes. Studies with dexamethasone.

These studies of a rat model for non-insulin-dependent diabetes mellitus (NIDDM) were performed to determine whether hyperglycemia occurs when capacity to synthesize insulin is exceeded. The neonatal streptozocin (STZ)-treated rat has acute hyperglycemia with marked destruction of pancreatic beta-cells, followed by gradual regeneration to 50-70% normal beta-cell number. At age 4 wk, fed serum glucose concentration is only mildly elevated relative to controls. With age, the rats become progressively hyperglycemic, and by 12 wk they have marked impairment of glucose-stimulated insulin release. In these studies, dexamethasone (0.125 mg/kg/day for 4 days) was administered to control and to STZ-treated animals to produce insulin resistance. The relationship between insulin biosynthesis and serum glucose concentrations was assessed. In control rats, response to dexamethasone was similar at both 4 and 12 wk. Serum glucose levels and pancreatic insulin concentration remained unchanged. Both insulin biosynthetic rates (as measured by 3H-leucine incorporation into proinsulin) and proinsulin mRNA levels increased twofold. STZ-treated rats at age 4 wk demonstrated mild hyperglycemia. Dexamethasone injection resulted in an increase in insulin biosynthesis and proinsulin mRNA in these animals, while serum glucose did not increase. STZ-treated rats at 12 wk showed more profound hyperglycemia (serum glucose 315 +/- 38 mg/dl versus control, 187 +/- 12 mg/dl). A marked rise in serum glucose (to 519 +/- 42 mg/dl) was observed after 4 days of dexamethasone injection. Pancreatic insulin content became severely depleted relative to saline-injected, STZ-treated animals, and there was no response of levels of proinsulin mRNA.

Insulin secretion by fetal human pancreas in organ culture.

Whole fetal human pancreases of 12-22 weeks gestation, showed histological growth and differentiation in vitro over 3 weeks. At glucose concentrations of 1-4 g/l, there was no difference in insulin secretion into culture medium over 1 h. There was no stimulation of insulin release by D-glyceraldehyde, thus defective glucose-stimulated insulin release was probably not due to impairment of an early step in glycolysis. In the presence of 0.5 mmol/l dibutyryl cyclic AMP, insulin secretion was enhanced (0.188 +/- 0.030 versus 0.100 +/- 0.012 mU x mg tissue-1 x h-1, p less than 0.001) independently of glucose concentrations. It thus appears that impairment of glucose-stimulated insulin release was unlikely to be due to insufficient intracellular cyclic AMP. Insulin release increased in response to tolbutamide and theophylline. Insulin secretion was stimulated in the presence of a fivefold increase in amino acid concentration (0.118 +/- 0.018 versus 0.031 +/- 0.008 mU x mg tissue-1 x h-1, p less than 0.001). There was a fourfold increase in basal insulin secretion from islets previously grown in high concentration of amino acids compared with standard culture medium, (0.284 +/- 0.052 versus 0.067 +/- 0.011 mU x mg tissue-1 x h-1, p less than 0.001), emphasizing the important role of amino acids as substrates for B cell metabolism and development.

Effects of starvation on oxidative metabolism and insulin release by isolated mouse pancreatic islets.

Abstract. Pancreatic islets were isolated from either 60-h-starved or fed mice and subsequently incubated in order to determine the insulin release in response to various secretagogues, rates of glucose, leucine or glutamine oxidation or the acetoacetate production from leucine. It was found that in contrast to findings with islets isolated from fed mice 16.7 mm glucose, 10 mM leucine and 10 mm α-ketoisocaproic acid did not stimulate the insulin release of islets isolated from starved mice. Moreover, the insulin release in response to leucine plus glutamine or glucose plus glutamine was decreased after starvation although these values were higher than those obtained for glutamine addition alone. Theophylline, however, restored partly the impaired insulin response to glucose and completely that to leucine or α-ketoisocaproic acid. Starvation was found to inhibit the islet glucose oxidation rate but the addition of theophylline was without effect irrespective of whether the islets were prepared from fed or starved mice. On the contrary, islet leucine oxidation was increased after starvation and again theophylline did not affect the islet leucine oxidation rate. Likewise, the islet acetoacetate production was increased after starvation. The glutamine oxidation rates were not affected by starvation, either when tested alone or together with glucose or leucine. It is concluded that although the starvation-induced impairment of glucose-stimulated insulin release may well be explained by an influence on the oxidative metabolism other factors are also involved as regards leucine-stimulated insulin release.