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.

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.

CaV2.3 calcium channels control second-phase insulin release.

Concerted activation of different voltage-gated Ca( (2+) ) channel isoforms may determine the kinetics of insulin release from pancreatic islets. Here we have elucidated the role of R-type Ca(V)2.3 channels in that process. A 20% reduction in glucose-evoked insulin secretion was observed in Ca(V)2.3-knockout (Ca(V)2.3(-/-)) islets, close to the 17% inhibition by the R-type blocker SNX482 but much less than the 77% inhibition produced by the L-type Ca(2+) channel antagonist isradipine. Dynamic insulin-release measurements revealed that genetic or pharmacological Ca(V)2.3 ablation strongly suppressed second-phase secretion, whereas first-phase secretion was unaffected, a result also observed in vivo. Suppression of the second phase coincided with an 18% reduction in oscillatory Ca(2+) signaling and a 25% reduction in granule recruitment after completion of the initial exocytotic burst in single Ca(V)2.3(-/-) beta cells. Ca(V)2.3 ablation also impaired glucose-mediated suppression of glucagon secretion in isolated islets (27% versus 58% in WT), an effect associated with coexpression of insulin and glucagon in a fraction of the islet cells in the Ca(V)2.3(-/-) mouse. We propose a specific role for Ca(V)2.3 Ca(2+) channels in second-phase insulin release, that of mediating the Ca(2+) entry needed for replenishment of the releasable pool of granules as well as islet cell differentiation.

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.

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.

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.

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.

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.

Defective glucose-stimulated insulin release in the diabetic Goto-Kakizaki (GK) rat coincides with reduced activity of the islet carbon monoxide signaling pathway.

The Goto-Kakizaki (GK) rat displays a markedly reduced insulin response to glucose, a defect that is thought to be coupled to an impaired glucose signaling in the beta-cell. We have examined whether carbon monoxide (CO), derived from beta-cell heme oxygenase (HO), might be involved in the secretory dysfunction. Immunocytochemical labeling of constitutive HO (HO-2) showed no overt difference in fluorescence pattern in islets from GK vs. Wistar controls. However, isolated islets from GK rats displayed a markedly impaired HO activity measured as CO production (-50%), and immunoblotting revealed an approximately 50% reduction of HO-2 protein expression compared with Wistar controls. Furthermore, there was a prominent expression of inducible HO (HO-1) in GK islets. Incubation of isolated islets showed that the glucose-stimulated CO production and the glucose-stimulated insulin response were considerably reduced in GK islets compared with Wistar islets. Addition of the HO activator hemin or gaseous CO to the incubation media brought about a similar amplification of glucose-stimulated insulin release in GK and Wistar islets, suggesting that distal steps in the HO-CO signaling pathway were not appreciably affected. We conclude that the defective insulin response to glucose in the GK rat can be explained, at least in part, by a marked impairment of the glucose-HO-CO signaling pathway as manifested by a prominent decrease in glucose stimulation of islet CO production and a reduced expression of HO-2. A possible role of HO-1 expression as a compensatory mechanism in the GK islets is presently unclear.

SUR1 regulates PKA-independent cAMP-induced granule priming in mouse pancreatic B-cells.

Measurements of membrane capacitance were applied to dissect the cellular mechanisms underlying PKA-dependent and -independent stimulation of insulin secretion by cyclic AMP. Whereas the PKA-independent (Rp-cAMPS-insensitive) component correlated with a rapid increase in membrane capacitance of approximately 80 fF that plateaued within approximately 200 ms, the PKA-dependent component became prominent during depolarizations >450 ms. The PKA-dependent and -independent components of cAMP-stimulated exocytosis differed with regard to cAMP concentration dependence; the K(d) values were 6 and 29 micro M for the PKA-dependent and -independent mechanisms, respectively. The ability of cAMP to elicit exocytosis independently of PKA activation was mimicked by the selective cAMP-GEFII agonist 8CPT-2Me-cAMP. Moreover, treatment of B-cells with antisense oligodeoxynucleotides against cAMP-GEFII resulted in partial (50%) suppression of PKA-independent exocytosis. Surprisingly, B-cells in islets isolated from SUR1-deficient mice (SUR1(-/-) mice) lacked the PKA-independent component of exocytosis. Measurements of insulin release in response to GLP-1 stimulation in isolated islets from SUR1(-/-) mice confirmed the complete loss of the PKA-independent component. This was not attributable to a reduced capacity of GLP-1 to elevate intracellular cAMP but instead associated with the inability of cAMP to stimulate influx of Cl(-) into the granules, a step important for granule priming. We conclude that the role of SUR1 in the B cell extends beyond being a subunit of the plasma membrane K(ATP)-channel and that it also plays an unexpected but important role in the cAMP-dependent regulation of Ca(2+)-induced exocytosis.

Ghrelin activates neuronal constitutive nitric oxide synthase in pancreatic islet cells while inhibiting insulin release and stimulating glucagon release.

In view of our previous data, showing that ghrelin and nitric oxide (NO) display apparently parallel effects on insulin secretion (inhibitory) and glucagon secretion (stimulatory), we have now investigated the effect of ghrelin on islet hormone secretion in relation to its effect on NO synthase (NOS) isoenzymes in isolated rat pancreatic islets. Dose-response studies revealed that ghrelin at concentrations of 0.01-1 micromol l-1 inhibited insulin secretion stimulated by 8.3 mmol l-1 glucose, while ghrelin at concentrations lower than the physiological range (0.01 pmol l-1 to 1 nmol l-1) were without effect. In contrast, glucagon secretion was stimulated by 1.0 nmol l-1 to 1 micromol l-1 ghrelin. These effects of ghrelin on insulin and glucagon secretion were accompanied by increased NO production through activation of neuronal constitutive NOS (ncNOS). Ghrelin had no appreciable effect on the activity of inducible NOS (iNOS) in the islets. Addition of an NO scavenger (cPTIO) or the NOS inhibitor L-NAME to the incubation medium prevented the effects of ghrelin on hormone secretion from isolated islets. The present results confirm our previous data showing that ghrelin inhibits insulin and stimulates glucagon secretion from pancreatic islets of the mouse and we now show similar effects in rat islets. The effects of ghrelin were accompanied by an increased rate of NO production. Conceivably, ncNOS activation partly accounts for to the inhibitory effect of ghrelin on insulin secretion and the stimulatory effect of ghrelin on glucagon secretion.

Long-term infusion of nutrients (total parenteral nutrition) suppresses circulating ghrelin in food-deprived rats.

BACKGROUND: Ghrelin derives from endocrine cells (A-like cells) in the stomach (mainly the oxyntic mucosa). Its concentration in the circulation increases during fasting and decreases upon re-feeding. This has fostered the notion that the absence of food in the upper gastrointestinal (GI) tract stimulates the secretion of ghrelin. The purpose of the present study was to determine the concentration of ghrelin in serum and oxyntic mucosa after replacing food with intravenous (iv) infusion of nutrients for 8 days using the technique known as total parenteral nutrition (TPN) MATERIALS AND METHODS: Male Sprague-Dawley rats (200-250 g) were given nutrients (lipids, glucose, amino acids, minerals and vitamins) by iv infusion for 8 days during which time they were deprived of food and water; another group was deprived of food for 24-48 h (fasted controls), while fed controls had free access to food and water. Serum ghrelin, gastrin and pancreastatin concentrations were measured together with the ghrelin content of the oxyntic mucosa. Plasma insulin and glucose as well as serum lipid concentrations were also determined. RESULTS: Fasted rats had higher serum ghrelin than TPN rats and fed controls. The oxyntic mucosal ghrelin concentration (and content) was lower in TPN rats than in fasted rats or fed controls. The serum gastrin and pancreastatin concentrations were lower in TPN rats and fasted rats than in fed controls. The plasma insulin concentration was 87 pmol/l+/-8 (SEM) in TPN rats compared to 101+/-16 pmol/l in fed controls; it was 26+/-14 pmol/l in fasted rats. The basal plasma glucose level was 11+/-0.6 mmol/l in TPN rats and 12+/-0.8 mmol/l in fed controls; it was 7+/-0.3 mmol/l in fasted rats. In TPN rats, the serum concentrations of free fatty acids, triglycerides and cholesterol were increased by 100%, 50% and 25%, respectively, compared to fed controls. Fasted rats had higher circulating concentrations of free fatty acids (20%) and lower concentrations of triglycerides (-40%) than fed controls; fasted rats did not differ from fed controls with respect to serum cholesterol. CONCLUSION: The circulating ghrelin concentration is high in situations of nutritional deficiency (starvation) and low in situations of nutritional plenty (free access to food or TPN). The actual presence or absence of food in the GI tract seems irrelevant. Circulating insulin and glucose concentrations did not differ much between TPN rats and fed controls; serum lipids, however, were elevated in the TPN rats. We suggest that elevated blood lipid levels contribute to the suppression of circulating ghrelin in rats subjected to TPN for 8 days.

Islet amyloid polypeptide inhibits glucagon release and exerts a dual action on insulin release from isolated islets.

We have studied the influence of a wide concentration range of islet amyloid polypeptide (IAPP) on both glucagon and insulin release stimulated by various types of secretagogues. In an islet incubation medium devoid of glucose, the rate of glucagon release being high, we observed a marked suppressive action by low concentrations of IAPP, 10(-10) and 10(-8) M, on glucagon release. Similarly, glucagon release stimulated by L-arginine, the cholinergic agonist carbachol, or the phosphodiesterase inhibitor isobutylmethyl xanthine (IBMX), an activator of the cyclic AMP system, was inhibited by IAPP in the 10(-10) and 10(-8) M concentration range. Moreover, basal glucagon release at 7 and 10 mM glucose was suppressed by IAPP. In contrast, IAPP exerted a dual action on insulin release. Hence, low concentrations of IAPP brought about a modest increase of basal insulin secretion at 7 mM glucose and also of insulin release stimulated by carbachol. High concentrations of IAPP, however, inhibited insulin release stimulated by glucose (10 and 16.7 mM), IBMX, carbachol and L-arginine. In conclusion, our data suggest that IAPP has complex effects on islet hormone secretion serving as an inhibitor of glucagon release and having a dual action on insulin secretion exerting mainly a negative feedback on stimulated and a positive feedback on basal insulin release.

Insulin feedback actions: complex effects involving isoforms of islet nitric oxide synthase.

The present study examined the effects of exogenous insulin on C-peptide release in relation to islet activities of neural constitutive nitric oxide synthase (ncNOS) and inducible NOS (iNOS). The dose-response curves for glucose-stimulated insulin and C-peptide release from isolated islets were practically identical: 0.05-0.1 nmol/l insulin stimulated, 1-100 nmol/l had no effect, whereas concentrations >/=250 nmol/l ("high insulin"), inhibited C-peptide release. Both the stimulatory and inhibitory effects were abolished by the phosphatidylinositol 3'-kinase inhibitor wortmannin. Addition of a NOS inhibitor partially reversed the inhibitory action of high insulin, but had no effect on the stimulatory action of low insulin (0.1 nmol/l). Moreover, high insulin markedly increased islet ncNOS activity and induced a strong iNOS activity. As shown biochemically and with confocal microscopy, the stimulatory action of high insulin on NOS activities and the associated inhibition of C-peptide release were reversed by raising cyclic AMP through addition of either glucagon-like peptide 1 (GLP-1) or dibutyryl cyclic AMP (Bt(2)cAMP) to the incubated islets. We conclude that the positive feedback mechanisms of action of insulin are independent of islet NOS activities and remain unclear. The negative feedback action of insulin, however, can be explained by its ability to stimulate both islet ncNOS activity and the expression and activity of iNOS. The effects on iNOS are most likely transduced through phosphatidylinositol 3'-kinase and are counteracted by raising islet cyclic AMP levels.

Effects of ghrelin on insulin and glucagon secretion: a study of isolated pancreatic islets and intact mice.

We combined in vitro and in vivo methods to investigate the effects of ghrelin, a novel gastric hormone, on insulin and glucagon release. Studies of isolated mouse islets showed that ghrelin concentrations in the physiological range (0.5-3 nmol l(-1)) had no effect on glucose-stimulated insulin release, while low ghrelin concentrations (1-100 pmol l(-1)) inhibited and high (0.1 and 1 micromol l(-1)) stimulated. The insulin response to glucose was enhanced in the presence of a high ghrelin concentration (100 nmol l(-1)). Glucagon release was stimulated by ghrelin (0.1 pmol l(-1) to 1 micromol l(-1)); this effect was maintained in the presence of glucose (0-20 mmol l(-1)). In intact mice, basal plasma insulin was suppressed by 1 and 10 nmol kg(-1) of ghrelin, 2 and 6 min after i.v. injection. Ghrelin (0.2-10 nmol kg(-1) i.v.) suppressed also the glucose-stimulated insulin response and impaired the glucose tolerance (at a ghrelin dose of 3.3 nmol kg(-1)). Ghrelin (1 or 10 nmol kg(-1) i.v.) inhibited the insulin response to the phospholipase C stimulating agent carbachol and enhanced the insulin response to the phosphodiesterase inhibitor isobutyl-methylxanthine (IBMX) but did not affect the response to the membrane-depolarizing amino acid l-arginine. These observations suggest that the inhibitory effect of ghrelin on glucose-induced insulin release is in part exerted on phospholipase C pathways (and not on Ca(2+)entry), while the stimulatory effect of high doses of ghrelin depends on cyclic AMP. In contrast to the spectacular glucagon-releasing effect of ghrelin in vitro, ghrelin did not raise plasma glucagon. Carbachol, IBMX and l-arginine stimulated glucagon release. These responses were impaired by ghrelin, suggesting that it suppresses the various intracellular pathways (phospholipase C, cyclic AMP and Ca(2+)), that are activated by the glucagon secretagogues. Together these observations highlight (but do not explain) the different effects of ghrelin on glucagon release in vitro and in vivo. The results show that ghrelin has powerful effects on islet cells, suggesting that endogenous ghrelin may contribute to the physiological control of insulin and glucagon release. However, the narrow "window" of circulating ghrelin concentrations makes this doubtful.

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.