Expression levels of enzymes generating NO and CO in islets of murine and human diabetes.

The possible implication of the gasotransmitters NO and CO for the development of diabetes remains unresolved. Our previous investigations in rodents suggested NO being inhibitory, and CO stimulatory, to glucose-stimulated insulin secretion (GSIS). Here we studied the possible role of these gasotransmitters in both murine and human type 2 diabetes (T2D) by mapping the expression pattern of neural nitric oxide synthase (nNOS), inducible NOS (iNOS), constitutive heme oxygenase (HO-2), and inducible HO (HO-1) in isolated pancreatic islets. Two variants of obese murine diabetes with distinct phenotype, the db/db and the ob/ob mouse, were studied at the initiation of the diabetic condition. Plasma glucose and plasma insulin were recorded and ß-cell expression levels of the different enzymes were measured with confocal microscopy and fluorescence intensity recordings. In human islets taken from nondiabetic controls (ND) and type 2 diabetes (T2D) the expression of the enzymes was analyzed by RNA-sequencing and qPCR. At the initiation of murine diabetes plasma glucose was slightly increased, whereas plasma insulin was extremely enhanced in both db/db and ob/ob mice. The ß-cell expression of nNOS and iNOS was markedly increased over controls in db/db mice, known to develop severe diabetes, while it was very low in ob/ob mice, known to develop mild diabetes. HO-2 expression was unaffected in db/db and modestly decreased in ob/ob mice. HO-1 expression was slightly enhanced in ob/ob, but, in contrast, extremely enhanced in db/db mice, suggesting a counteracting, antidiabetic action by CO. Moreover, the diabetic pattern of highly increased nNOS, iNOS and HO-1 expression seen in db/db mice was also fully recognized in human T2D islets. These results suggest that increased expression of the NOS-enzymes, especially an early upregulation of nNOS, could be involved in the initial development of the severe diabetes of db/db mice as well as in human T2D. Hence, nNOS, iNOS and HO-1 might be regarded as interesting targets to take into consideration in the early treatment of a diabetic condition in different variants of T2D.

Metformin Ameliorates Dysfunctional Traits of Glibenclamide- and Glucose-Induced Insulin Secretion by Suppression of Imposed Overactivity of the Islet Nitric Oxide Synthase-NO System.

Metformin lowers diabetic blood glucose primarily by reducing hepatic gluconeogenesis and increasing peripheral glucose uptake. However, possible effects by metformin on beta-cell function are incompletely understood. We speculated that metformin might positively influence insulin secretion through impacting the beta-cell nitric oxide synthase (NOS)-NO system, a negative modulator of glucose-stimulated insulin release. In short-time incubations with isolated murine islets either glibenclamide or high glucose augmented insulin release associated with increased NO production from both neural and inducible NOS. Metformin addition suppressed the augmented NO generation coinciding with amplified insulin release. Islet culturing with glibenclamide or high glucose revealed pronounced fluorescence of inducible NOS in the beta-cells being abolished by metformin co-culturing. These findings were reflected in medium nitrite-nitrate levels. A glucose challenge following islet culturing with glibenclamide or high glucose revealed markedly impaired insulin response. Metformin co-culturing restored this response. Culturing murine islets and human islets from controls and type 2 diabetics with high glucose or high glucose + glibenclamide induced a pronounced decrease of cell viability being remarkably restored by metformin co-culturing. We show here, that imposed overactivity of the beta-cell NOS-NO system by glibenclamide or high glucose leads to insulin secretory dysfunction and reduced cell viability and also, importantly, that these effects are relieved by metformin inhibiting beta-cell NO overproduction from both neural and inducible NOS thus ameliorating a concealed negative influence by NO induced by sulfonylurea treatment and/or high glucose levels. This double-edged effect of glibenclamide on the beta-cellsuggests sulfonylurea monotherapy in type 2 diabetes being avoided.

GPR40 protein levels are crucial to the regulation of stimulated hormone secretion in pancreatic islets. Lessons from spontaneous obesity-prone and non-obese type 2 diabetes in rats.

The role of islet GPR40 protein in the pathogenesis of diabetes is unclear. We explored the influence of GPR40 protein levels on hormone secretion in islets from two rat models of spontaneous type 2 diabetes displaying either hyperlipidaemia or hyperglycaemia. GPR40 expression was analysed by confocal microscopy, Western blot and qPCR in islets from preobese Zucker (fa/fa) rats, diabetic Goto-Kakizaki (GK) rats, and controls. Confocal microscopy of control islets showed expression of GPR40 protein in insulin, glucagon and somatostatin cells. GPR40 expression was strongly increased in islets of hyperlipidaemic fa/fa rats and coincided with a concentration-related increase in palmitate-induced release of insulin and glucagon and its inhibition of somatostatin release. Conversely, hyperglycaemic GK islets displayed an extremely faint expression of GPR40 as did high-glucose-cultured control islets. This was reflected in abolished palmitate-induced hormone response in GK islets and high-glucose-cultured control islets. The palmitate antagonist rosiglitazone promoted reappearance of GPR40 in high-glucose-cultured islets and served as partial agonist in glucose-stimulated insulin release. GPR40 protein is abundantly expressed in pancreatic islets and modulates stimulated hormone secretion. Mild hyperlipidaemia in obesity-prone diabetes creates increased GPR40 expression and increased risk for an exaggerated palmitate-induced insulin response and lipotoxicity, a metabolic situation suitable for GPR40 antagonist treatment. Chronic hyperglycaemia creates abrogated GPR40 expression and downregulated insulin release, a metabolic situation suitable for GPR40 agonist treatment to avoid glucotoxicity. GPR40 protein is interactively modulated by both free fatty acids and glucose and is a promising target for pharmacotherapy in different variants of type 2 diabetes.

Abnormally decreased NO and augmented CO production in islets of the leptin-deficient ob/ob mouse might contribute to explain hyperinsulinemia and islet survival in leptin-resistant type 2 obese diabetes.

The role of the gaseous messengers NO and CO for ß-cell function and survival is controversial. We examined this issue in the hyperglycemic-hyperinsulinemic ob/ob mouse, an animal model of type 2 obese diabetes, by studying islets from obese vs lean mice regarding glucose-stimulated insulin release in relation to islet NO and CO production and the influence of modulating peptide hormones. Glucose-stimulated increase in ncNOS-activity in incubated lean islets was converted to a decrease in ob/ob islets associated with markedly increased insulin release. Both types of islets displayed iNOS activity appearing after ~60 min in high-glucose. In ob/ob islets the insulinotropic peptides glucagon, GLP-1 and GIP suppressed NOS activities and amplified glucose-stimulated insulin release. The insulinostatic peptide leptin induced the opposite effects. Suppression of islet CO production inhibited, while stimulation amplified glucose-stimulated insulin release. Nonincubated isolated islets from young and adult obese mice displayed very low ncNOS and negligible iNOS activity. In contrast, production of CO, a NOS inhibitor, was impressively raised. Glucose injections induced strong activities of islet NOS isoforms in lean but not in obese mice and confocal microscopy revealed iNOS expression only in lean islets. Islets from ob/ob mice existing in a hyperglycemic in vivo milieu maintain elevated insulin secretion and protection from glucotoxicity through a general suppression of islet NOS activities achieved by leptin deficiency, high CO production and insulinotropic cyclic-AMP-generating hormones. Such a beneficial effect on islet function and survival might have its clinical counterpart in human leptin-resistant type 2 obese diabetes with hyperinsulinemia.

Insulinotropic and antidiabetic effects of 17ß-estradiol and the GPR30 agonist G-1 on human pancreatic islets.

We have recently shown that 17ß-estradiol (E2) and the synthetic G protein-coupled receptor 30 (GPR30) ligand G-1 have antiapoptotic actions in mouse pancreatic islets, raising the prospect that they might exert beneficial effects also in human islets. The objective of the present study was to identify the expression of GPR30 in human islets and clarify the role of GPR30 in islet hormone secretion and ß-cell survival. GPR30 expression was analyzed by confocal microscopy, Western blot, and quantitative PCR in islets from female and male donors. Hormone secretion, phosphatidylinositol hydrolysis, cAMP content, and caspase-3 activity in female islets were determined with conventional methods and apoptosis with the annexin-V method. Confocal microscopy revealed GPR30 expression in islet insulin, glucagon, and somatostatin cells. GPR30 mRNA and protein expression was markedly higher in female vs. male islets. An amplifying effect of G-1 or E2 on cAMP content and insulin secretion from isolated female islets was not influenced by the E2 genomic receptor (ERα and ERß) antagonists ICI 182,780 and EM-652. Cytokine-induced (IL-1ß plus TNFα plus interferon-γ) apoptosis in islets cultured for 24 h at 5 mmol/liter glucose was almost abolished by G-1 or E2 treatment and was not affected by the nuclear estrogen receptor antagonists. Concentration-response studies on female islets from healthy controls and type 2 diabetic subjects showed that both E2 and G-1 displayed important antidiabetic actions by improving glucose-stimulated insulin release while suppressing glucagon and somatostatin secretion. In view of these findings, we propose that small molecules activating GPR30 could be promising in the therapy of diabetes mellitus.

Activation of G protein-coupled receptor 30 modulates hormone secretion and counteracts cytokine-induced apoptosis in pancreatic islets of female mice.

The role of the newly discovered estrogen receptor GPR30 in islet physiology and pathophysiology is unclear. We examined GPR30 expression in relation to hormone secretion and possible anti-apoptotic effects in isolated mouse islets using the synthetic GPR30 ligand G-1. The mRNA and protein expression of GPR30 was analyzed by qPCR, Western blot and confocal microscopy. Hormone secretion and cAMP content were determined with RIA and apoptosis in islet cells with the Annexin-V method. GPR30 mRNA and protein expression was markedly higher in islets from females compared to male. This gender difference was not found for the genomic estrogen receptors ER alpha and ER beta, the ER alpha expression being 10-fold higher than ER beta in both genders. Confocal microscopy revealed abounden GPR30 expression in insulin, glucagon and somatostatin cells. Dose-response studies of G-1 vs 17beta-estradiol in isolated islets at 1 or 12 mM glucose showed an almost identical pattern in that both compounds increased insulin and inhibited glucagon and somatostatin secretion. ICI-182,780 and EM-652, potent antagonists of the 17beta-estradiol receptors (ER alpha and ER beta) did not influence the amplifying effect of G-1 or 17beta-estradiol on cAMP content or insulin secretion from isolated islets. Cytokine-induced (IL-1 beta+TNFalpha+INF gamma) apoptosis in islets, cultured for 24h at 5mM glucose, was almost abolished by G-1 or 17beta-estradiol treatment. Addition of ICI-182,780 or EM-652 did not affect this beneficial effect of G-1 or 17beta-estradiol. Taken together, our findings show that GPR30 is expressed in most islet endocrine cells. The synthetic GPR30 ligand G-1 mimics the non-genomic effects of 17beta-estradiol on islet hormone secretion, cAMP content in islets and its anti-apoptotic effects. G-1 or analogs thereof might be new potential candidates in the therapeutic strategy for type 2 diabetes in women.

Defective secretion of islet hormones in chromogranin-B deficient mice.

Granins are major constituents of dense-core secretory granules in neuroendocrine cells, but their function is still a matter of debate. Work in cell lines has suggested that the most abundant and ubiquitously expressed granins, chromogranin A and B (CgA and CgB), are involved in granulogenesis and protein sorting. Here we report the generation and characterization of mice lacking chromogranin B (CgB-ko), which were viable and fertile. Unlike neuroendocrine tissues, pancreatic islets of these animals lacked compensatory changes in other granins and were therefore analyzed in detail. Stimulated secretion of insulin, glucagon and somatostatin was reduced in CgB-ko islets, in parallel with somewhat impaired glucose clearance and reduced insulin release, but normal insulin sensitivity in vivo. CgB-ko islets lacked specifically the rapid initial phase of stimulated secretion, had elevated basal insulin release, and stored and released twice as much proinsulin as wildtype (wt) islets. Stimulated release of glucagon and somatostatin was reduced as well. Surprisingly, biogenesis, morphology and function of insulin granules were normal, and no differences were found with regard to beta-cell stimulus-secretion coupling. We conclude that CgB is not required for normal insulin granule biogenesis or maintenance in vivo, but is essential for adequate secretion of islet hormones. Consequentially CgB-ko animals display some, but not all, hallmarks of human type-2 diabetes. However, the molecular mechanisms underlying this defect remain to be determined.

Suppression of sulfonylurea- and glucose-induced insulin secretion in vitro and in vivo in mice lacking the chloride transport protein ClC-3.

Priming of insulin secretory granules for release requires intragranular acidification and depends on vesicular Cl(-)-fluxes, but the identity of the chloride transporter/ion channel involved is unknown. We tested the hypothesis that the chloride transport protein ClC-3 fulfills these actions in pancreatic beta cells. In ClC-3(-/-) mice, insulin secretion evoked by membrane depolarization (high extracellular K(+), sulfonylureas), or glucose was >60% reduced compared to WT animals. This effect was mirrored by a approximately 80% reduction in depolarization-evoked beta cell exocytosis (monitored as increases in cell capacitance) in single ClC-3(-/-) beta cells, as well as a 44% reduction in proton transport across the granule membrane. ClC-3 expression in the insulin granule was demonstrated by immunoblotting, immunostaining, and negative immuno-EM in a high-purification fraction of large dense-core vesicles (LDCVs) obtained by phogrin-EGFP labeling. The data establish the importance of granular Cl(-) fluxes in granule priming and provide direct evidence for the involvement of ClC-3 in the process.

Impaired insulin exocytosis in neural cell adhesion molecule-/- mice due to defective reorganization of the submembrane F-actin network.

The neural cell adhesion molecule (NCAM) is required for cell type segregation during pancreatic islet organogenesis. We have investigated the functional consequences of ablating NCAM on pancreatic beta-cell function. In vivo, NCAM(-/-) mice exhibit impaired glucose tolerance and basal hyperinsulinemia. Insulin secretion from isolated NCAM(-/-) islets is enhanced at glucose concentrations below 15 mM but inhibited at higher concentrations. Glucagon secretion from pancreatic alpha-cells evoked by low glucose was also severely impaired in NCAM(-/-) islets. The diminution of insulin secretion is not attributable to defective glucose metabolism or glucose sensing (documented as glucose-induced changes in intracellular Ca(2+) and K(ATP)-channel activity). Resting K(ATP) conductance was lower in NCAM(-/-) beta-cells than wild-type cells, and this difference was abolished when F-actin was disrupted by cytochalasin D (1 muM). In wild-type beta-cells, the submembrane actin network disassembles within 10 min during glucose stimulation (30 mM), an effect not seen in NCAM(-/-) beta-cells. Cytochalasin D eliminated this difference and normalized insulin and glucagon secretion in NCAM(-/-) islets. Capacitance measurements of exocytosis indicate that replenishment of the readily releasable granule pool is suppressed in NCAM(-/-) alpha- and beta-cells. Our data suggest that remodeling of the submembrane actin network is critical to normal glucose regulation of both insulin and glucagon secretion.

Deletion of the G protein-coupled receptor 30 impairs glucose tolerance, reduces bone growth, increases blood pressure, and eliminates estradiol-stimulated insulin release in female mice.

In vitro studies suggest that the G protein-coupled receptor (GPR) 30 is a functional estrogen receptor. However, the physiological role of GPR30 in vivo is unknown, and it remains to be determined whether GPR30 is an estrogen receptor also in vivo. To this end, we studied the effects of disrupting the GPR30 gene in female and male mice. Female GPR30((-/-)) mice had hyperglycemia and impaired glucose tolerance, reduced body growth, increased blood pressure, and reduced serum IGF-I levels. The reduced growth correlated with a proportional decrease in skeletal development. The elevated blood pressure was associated with an increased vascular resistance manifested as an increased media to lumen ratio of the resistance arteries. The hyperglycemia and impaired glucose tolerance in vivo were associated with decreased insulin expression and release in vivo and in vitro in isolated pancreatic islets. GPR30 is expressed in islets, and GPR30 deletion abolished estradiol-stimulated insulin release both in vivo in ovariectomized adult mice and in vitro in isolated islets. Our findings show that GPR30 is important for several metabolic functions in female mice, including estradiol-stimulated insulin release.

Impaired glucose-stimulated insulin secretion in the GK rat is associated with abnormalities in islet nitric oxide production.

We investigated implications of nitric oxide (NO) derived from islet neuronal constitutive NO synthase (ncNOS) and inducible NOS (iNOS) on insulin secretory mechanisms in the mildly diabetic GK rat. Islets from GK rats and Wistar controls were analysed for ncNOS and iNOS by HPLC, immunoblotting and immunocytochemistry in relation to insulin secretion stimulated by glucose or l-arginine in vitro and in vivo. No obvious difference in ncNOS fluorescence in GK vs control islets was seen but freshly isolated GK islets displayed a marked iNOS expression and activity. After incubation at low glucose GK islets showed an abnormal increase in both iNOS and ncNOS activities. At high glucose the impaired glucose-stimulated insulin release was associated with an increased iNOS expression and activity and NOS inhibition dose-dependently amplified insulin secretion in both GK and control islets. This effect by NOS inhibition was also evident in depolarized islets at low glucose, where forskolin had a further amplifying effect in GK but not in control islets. NOS inhibition increased basal insulin release in perfused GK pancreata and amplified insulin release after glucose stimulation in both GK and control pancreata, almost abrogating the nadir separating first and second phase in controls. A defective insulin response to l-arginine was seen in GK rats in vitro and in vivo, being partially restored by NOS inhibition. The results suggest that increased islet NOS activities might contribute to the defective insulin response to glucose and l-arginine in the GK rat. Excessive iNOS expression and activity might be deleterious for the beta-cells over time.

Palmitate-induced beta-cell dysfunction is associated with excessive NO production and is reversed by thiazolidinedione-mediated inhibition of GPR40 transduction mechanisms.

BACKGROUND: Type 2 diabetes often displays hyperlipidemia. We examined palmitate effects on pancreatic islet function in relation to FFA receptor GPR40, NO generation, insulin release, and the PPARgamma agonistic thiazolidinedione, rosiglitazone. PRINCIPAL FINDINGS: Rosiglitazone suppressed acute palmitate-stimulated GPR40-transduced PI hydrolysis in HEK293 cells and insulin release from MIN6c cells and mouse islets. Culturing islets 24 h with palmitate at 5 mmol/l glucose induced beta-cell iNOS expression as revealed by confocal microscopy and increased the activities of ncNOS and iNOS associated with suppression of glucose-stimulated insulin response. Rosiglitazone reversed these effects. The expression of iNOS after high-glucose culturing was unaffected by rosiglitazone. Downregulation of GPR40 by antisense treatment abrogated GPR40 expression and suppressed palmitate-induced iNOS activity and insulin release. CONCLUSION: We conclude that, in addition to mediating acute FFA-stimulated insulin release, GPR40 is an important regulator of iNOS expression and dysfunctional insulin release during long-term exposure to FFA. The adverse effects of palmitate were counteracted by rosiglitazone at GPR40, suggesting that thiazolidinediones are beneficial for beta-cell function in hyperlipidemic type 2 diabetes.

Excessive islet NO generation in type 2 diabetic GK rats coincides with abnormal hormone secretion and is counteracted by GLP-1.

BACKGROUND: A distinctive feature of type 2 diabetes is inability of insulin-secreting beta-cells to properly respond to elevated glucose eventually leading to beta-cell failure. We have hypothesized that an abnormally increased NO production in the pancreatic islets might be an important factor in the pathogenesis of beta-cell dysfunction. PRINCIPAL FINDINGS: We show now that islets of type 2 spontaneous diabetes in GK rats display excessive NO generation associated with abnormal iNOS expression in insulin and glucagon cells, increased ncNOS activity, impaired glucose-stimulated insulin release, glucagon hypersecretion, and impaired glucose-induced glucagon suppression. Pharmacological blockade of islet NO production by the NOS inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) greatly improved hormone secretion from GK islets suggesting islet NOS activity being an important target to inactivate for amelioration of islet cell function. The incretin hormone GLP-1, which is used in clinical practice suppressed iNOS and ncNOS expression and activity with almost full restoration of insulin release and partial restoration of glucagon release. GLP-1 suppression of iNOS expression was reversed by PKA inhibition but unaffected by the proteasome inhibitor MG132. Injection of glucose plus GLP-1 in the diabetic rats showed that GLP-1 amplified the insulin response but induced a transient increase and then a poor depression of glucagon. CONCLUSION: The results suggest that abnormally increased NO production within islet cells is a significant player in the pathogenesis of type 2 diabetes being counteracted by GLP-1 through PKA-dependent, nonproteasomal mechanisms.

CAPS1 and CAPS2 regulate stability and recruitment of insulin granules in mouse pancreatic beta cells.

CAPS1 and CAPS2 regulate dense-core vesicle release of transmitters and hormones in neuroendocrine cells, but their precise roles in the secretory process remain enigmatic. Here we show that CAPS2(-/-) and CAPS1(+/-);CAPS2(-/-) mice, despite having increased insulin sensitivity, are glucose intolerant and that this effect is attributable to a marked reduction of glucose-induced insulin secretion. This correlates with diminished Ca(2+)-dependent exocytosis, a reduction in the size of the morphologically docked pool, a decrease in the readily releasable pool of secretory vesicles, slowed granule priming, and suppression of second-phase (but not first-phase) insulin secretion. In beta cells of CAPS1(+/-);CAPS2(-/-) mice, the lowered insulin content and granule numbers were associated with an increase in lysosome numbers and lysosomal enzyme activity. We conclude that although CAPS proteins are not required for Ca(2+)-dependent exocytosis to proceed, they exert a modulatory effect on insulin granule priming, exocytosis, and stability.

Proghrelin-derived peptides influence the secretion of insulin, glucagon, pancreatic polypeptide and somatostatin: a study on isolated islets from mouse and rat pancreas.

Proghrelin, the precursor of the orexigenic and adipogenic peptide hormone ghrelin, is synthetized in endocrine (A-like) cells in the gastric mucosa. During its cellular processing, proghrelin gives rise to the 28-amino acid peptide desacyl ghrelin, which after octanoylation becomes active acyl ghrelin, and to the 23-amino acid peptide obestatin, claimed to be a physiological opponent of acyl ghrelin. This study examines the effects of the proghrelin products, alone and in combinations, on the secretion of insulin, glucagon, pancreatic polypeptide (PP) and somatostatin from isolated islets of mice and rats. Surprisingly, acyl ghrelin and obestatin had almost identical effects in that they stimulated the secretion of glucagon and inhibited that of PP and somatostatin from both mouse and rat islets. Obestatin inhibited insulin secretion more effectively than acyl ghrelin. In mouse islets, acyl ghrelin inhibited insulin secretion at low doses and stimulated at high. In rat islets, acyl ghrelin inhibited insulin secretion in a dose-dependent manner but the IC(50) for the acyl ghrelin-induced inhibition of insulin release was 7.5 x 10(-8) M, while the EC(50) and IC(50) values, with respect to stimulation of glucagon release and to inhibition of PP and somatostatin release, were in the 3 x 10(-12)-15 x 10(-12) M range. The corresponding EC(50) and IC(50) values for obestatin ranged from 5 x 10(-12) to 20 x 10(-12) M. Desacyl ghrelin per se did not affect islet hormone secretion. However, at a ten times higher concentration than acyl ghrelin (corresponding to the ratio of the two peptides in circulation), desacyl ghrelin abolished the effects of acyl ghrelin but not those of obestatin. Acyl ghrelin and obestatin affected the secretion of glucagon, PP and somatostatin at physiologically relevant concentrations; with obestatin this was the case also for insulin secretion. The combination of obestatin, acyl ghrelin and desacyl ghrelin in concentrations and proportions similar to those found in plasma resulted in effects that were indistinguishable from those induced by obestatin alone. From the data it seems that the effects of endogenous, circulating acyl ghrelin may be overshadowed by obestatin or blunted by desacyl ghrelin.

A1 receptor deficiency causes increased insulin and glucagon secretion in mice.

Adenosine influences metabolism and the adenosine receptor antagonist caffeine decreases the risk of type 2 diabetes. In this study the metabolic role of one adenosine receptor subtype, the adenosine A(1)R, was evaluated in mice lacking this receptor [A(1)R (-/-)]. The HbA1c levels and body weight were not significantly different between wild type [A(1)R (+/+)] and A(1)R (-/-) mice (3-4 months) fed normal lab chow. At rest, plasma levels of glucose, insulin and glucagon were similar in both genotypes. Following glucose injection, glucose tolerance was not appreciably altered in A(1)R (-/-) mice. Glucose injection induced sustained increases in plasma insulin and glucagon levels in A(1)R (-/-) mice, whereas A(1)R (+/+) control mice reacted with the expected transient increase in insulin and decrease in glucagon levels. Pancreas perfusion experiments showed that A(1)R (-/-) mice had a slightly higher basal insulin secretion than A(1)R (+/+) mice. The first phase insulin secretion (initiated with 16.7 mM glucose) was of the same magnitude in both genotypes, but the second phase was significantly enhanced in the A(1)R (-/-) pancreata compared with A(1)R (+/+). Insulin- and contraction-mediated glucose uptake in skeletal muscle were not significantly different between in A(1)R (-/-) and A(1)R (+/+) mice. All adenosine receptors were expressed at mRNA level in skeletal muscle in A(1)R (+/+) mice and the mRNA A(2A)R, A(2B)R and A(3)R levels were similar in A(1)R (-/-) and A(1)R (+/+) mice. In conclusion, the A(1)R minimally affects muscle glucose uptake, but is important in regulating pancreatic islet function.

Expression of islet inducible nitric oxide synthase and inhibition of glucose-stimulated insulin release after long-term lipid infusion in the rat is counteracted by PACAP27.

Chronic exposure of pancreatic islets to elevated plasma lipids (lipotoxicity) can lead to beta-cell dysfunction, with overtime becoming irreversible. We examined, by confocal microscopy and biochemistry, whether the expression of islet inducible nitric oxide synthase (iNOS) and the concomitant inhibition of glucose-stimulated insulin release seen after lipid infusion in rats was modulated by the islet neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP)27. Lipid infusion for 8 days induced a strong expression of islet iNOS, which was mainly confined to beta-cells and was still evident after incubating islets at 8.3 mmol/l glucose. This was accompanied by a high iNOS-derived NO generation, a decreased insulin release, and increased cyclic GMP accumulation. No iNOS expression was found in control islets. Addition of PACAP27 to incubated islets from lipid-infused rats resulted in loss of iNOS protein expression, increased cyclic AMP, decreased cyclic GMP, and suppression of the activities of neuronal constitutive (nc)NOS and iNOS and increased glucose-stimulated insulin response. These effects were reversed by the PKA inhibitor H-89. The suppression of islet iNOS expression induced by PACAP27 was not affected by the proteasome inhibitor MG-132, which by itself induced the loss of iNOS protein, making a direct proteasomal involvement less likely. Our results suggest that PACAP27 through its cyclic AMP- and PKA-stimulating capacity strongly suppresses not only ncNOS but, importantly, also the lipid-induced stimulation of iNOS expression, possibly by a nonproteasomal mechanism. Thus PACAP27 restores the impairment of glucose-stimulated insulin release and additionally might induce cytoprotection against deleterious actions of iNOS-derived NO in beta-cells.

Secretory and electrophysiological characteristics of insulin cells from gastrectomized mice: evidence for the existence of insulinotropic agents in the stomach.

Mice were subjected to gastrectomy (GX) or sham operation (controls). Four to six weeks later the pancreatic islets were isolated and analysed for cAMP or alternatively incubated in a Krebs-Ringer based medium in an effort to study insulin secretion and cAMP accumulation in response to glucose or the adenylate cyclase activator forskolin. Freshly isolated islets from GX mice had higher cAMP content than islets from control mice, a difference that persisted after incubation for 1 h at a glucose concentration of 4 mmol/l. Addition of forskolin to this medium induced much greater cAMP and insulin responses in islets from GX mice than in islets from control mice. In contrast, the insulin response to high glucose (16.7 mmol/l) was much weaker in GX islets than in control islets. Glucose-induced insulin release was associated with a 2-fold rise in the cAMP content in control islets. Surprisingly no rise in cAMP was noted in GX islets incubated at high glucose. Capacitance measurements conducted on isolated insulin cells from GX mice revealed a much lower exocytotic response to a single 500 ms depolarisation (from -70 mV to zero) than in control insulin cells. Addition of cAMP to the cytosol enhanced the exocytotic response in insulin cells from control mice but not from GX mice. The depolarisation-triggered inward Ca(2+) current in insulin cells from GX mice did not differ from that in control mice, and hence the reduced exocytotic response following GX cannot be ascribed to a decreased Ca(2+) influx. Experiments involving a train of ten 500 ms depolarisations revealed that the exocytotic response was prominent in control insulin cells but modest in GX insulin cells. It seems that cAMP is capable of eliciting insulin release from insulin cells of GX mice only when cAMP is generated in a specific microdomain conceivably through the intervention of membrane-associated adenylate cyclases that can be activated by forskolin. The GX-evoked impairment of depolarisation-induced exocytosis and glucose-stimulated insulin release may reflect the lack of a gastric agent that serves to maintain an appropriate insulin response to glucose and an appropriate exocytotic response to depolarisation by raising cAMP in a special glucose-sensitive compartment possibly regulated by a soluble adenylate cyclase.

Nitric oxide inhibits, and carbon monoxide activates, islet acid alpha-glucoside hydrolase activities in parallel with glucose-stimulated insulin secretion.

We have studied the influence of nitric oxide (NO) and carbon monoxide (CO), putative messenger molecules in the brain as well as in the islets of Langerhans, on glucose-stimulated insulin secretion and on the activities of the acid alpha-glucoside hydrolases, enzymes which we previously have shown to be implicated in the insulin release process. We have shown here that exogenous NO gas inhibits, while CO gas amplifies glucose-stimulated insulin secretion in intact mouse islets concomitant with a marked inhibition (NO) and a marked activation (CO) of the activities of the lysosomal/vacuolar enzymes acid glucan-1,4-alpha-glucosidase and acid alpha-glucosidase (acid alpha-glucoside hydrolases). Furthermore, CO dose-dependently potentiated glucose-stimulated insulin secretion in the range 0.1-1000 microM. In intact islets, the heme oxygenase substrate hemin markedly amplified glucose-stimulated insulin release, an effect which was accompanied by an increased activity of the acid alpha-glucoside hydrolases. These effects were partially suppressed by the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one. Hemin also inhibited inducible NO synthase (iNOS)-derived NO production probably through a direct effect of CO on the NOS enzyme. Further, exogenous CO raised the content of both cGMP and cAMP in parallel with a marked amplification of glucose-stimulated insulin release, while exogenous NO suppressed insulin release and cAMP, leaving cGMP unaffected. Emiglitate, a selective inhibitor of alpha-glucoside hydrolase activities, was able to markedly inhibit the stimulatory effect of exogenous CO on both glucose-stimulated insulin secretion and the activityof acid glucan-1,4-alpha-glucosidase and acid alpha-glucosidase, while no appreciable effect on the activities of other lysosomal enzyme activities measured was found. We propose that CO and NO, both produced in significant quantities in the islets of Langerhans, have interacting regulatory roles on glucose-stimulated insulin secretion. This regulation is, at least in part, transduced through the activity of cGMP and the lysosomal/vacuolar system and the associated acid alpha-glucoside hydrolases, but probably also through a direct effect on the cAMP system.

Does epidermal growth factor participate in the regulation of glucose, insulin and glucagon levels?

BACKGROUND/AIMS: The presence of receptors for epidermal growth factor (EGF) on beta cells in the rat pancreatic islets has been established, but the physiological role remains to be settled. The aim of the present study was to evaluate the effect of EGF on glucose homeostasis. METHODS: Fasted rats were treated with intraperitoneal injections of 10, 40 or 80 microg/kg body weight, either with EGF or 1% bovine serum albumin (controls). In a second experiment, fasted rats received an intraperitoneal injection of 1 mg glucose/kg body weight, followed by an injection of EGF or bovine serum albumin. Blood was drawn before the injections and at different time points afterwards. The plasma concentrations of glucose, insulin and glucagon were measured. RESULTS: A modest elevation of the concentrations of glucose and insulin in plasma during the study was found in fasted rats in experiment 1. The increase in insulin concentration was attenuated by EGF, but after glucose injection this effect was reversed. Plasma glucagon levels were dose-dependently elevated by EGF and increased instead of decreased after glucose injection. CONCLUSION: Our data suggest that EGF might play an important role in the regulation of glucagon secretion by preventing the lowering effect of glucose on plasma glucagon levels.