Response Of Islets Research Articles

Soybean diet improves insulin secretion through activation of cAMP/PKA pathway in rats

Maternal malnutrition leads to permanent alterations in insulin secretion of offspring and the soybean diet contributes to improve insulin release. At least a soy component, genistein, seems to increase the insulin secretion by activating the cAMP/PKA and PLC/PKC pathways. Here, we investigated the effect of the soybean diet on the expression of PKAα and PKCα, and insulin secretion in response to glucose and activators of adenylate cyclase and PKC in adult pancreatic rat islets. Rats from mothers fed with 17% or 6% protein (casein) during pregnancy and lactation were maintained with 17% casein (CC and CR groups) or soybean (SC and SR groups) diet until 90 days of life. The soybean diet improved the insulin response to a physiological concentration of glucose in control islets, but only in the presence of supra-physiological concentrations of glucose in islets from CR and SR groups. PMA also improved the insulin response in islets of SC and SR groups. The expression of PKCα was similar in all groups. Forskolin increased the insulin secretion; however, the magnitude of the increment was lower in islets from CR and SR groups than in control animals and in those from rats maintained with soybean diet than in rats fed with casein diet. The PKAα expression was similar between SR and CR groups and lower in SC than in CC islets. Thus, soybean diet improved the secretory pattern of β cells, at least in part, by activating the cAMP/PKA-signaling cascade.

Involvement of chronic stresses in rat islet and INS-1 cell glucotoxicity induced by intermittent high glucose

In order to investigate the toxic effect of intermittent high glucose (IHG) and sustained high glucose (SHG) on rat pancreatic beta cell functions and the potential involved mechanisms, isolated rat islets and INS-1 beta cells were exposed to SHG (25 mmol/l) or IHG (11.1 and 25 mmol/l glucose alternating every 12 h) for 72 h. The results showed that IHG induced a more significant impairment of insulin release response in rat islets and INS-1 cell than SHG. Simultaneously, the intracellular levels of endoplasmic reticulum and oxidative stress were more markedly increased in islets and INS-1 cells exposed to IHG. However, there was no significant difference between reducing cell viability, insulin content and gene expression induced by SHG and IHG. Taken together, this study suggested the more serious toxic effect on rat pancreatic beta cell function induced by IHG treatment may be due to excessive activation of cellular stress.

Dual role of proapoptotic BAD in insulin secretion and beta cell survival

The proapoptotic BCL-2 family member BAD resides in a glucokinase-containing complex that regulates glucose-driven mitochondrial respiration. Here, we present genetic evidence of a physiologic role for BAD in glucose-stimulated insulin secretion by beta cells. This novel function of BAD is specifically dependent upon the phosphorylation of its BH3 sequence, previously defined as an essential death domain. We highlight the pharmacologic relevance of phosphorylated BAD BH3 by using cell-permeable, hydrocarbon-stapled BAD BH3 helices that target glucokinase, restore glucose-driven mitochondrial respiration and correct the insulin secretory response in Bad-deficient islets. Our studies uncover an alternative target and function for the BAD BH3 domain and emphasize the therapeutic potential of phosphorylated BAD BH3 mimetics in selectively restoring beta cell function. Furthermore, we show that BAD regulates the physiologic adaptation of beta cell mass during high-fat feeding. Our findings provide genetic proof of the bifunctional activities of BAD in both beta cell survival and insulin secretion.

Enhanced activation of phospholipase C and insulin secretion from islets incubated in fatty acid–free bovine serum albumin

Incubation in 100 μmol/L fatty acid–free bovine serum albumin (FAF-BSA) significantly amplifies insulin secretion from isolated, perifused rat islets. When compared with the responses of control islets incubated in 100 μmol/L radioimmunoassay-grade BSA, insulin secretion rates were increased 2- to 3-fold when these islets were stimulated with 10 mmol/L glucose alone or with the combination of 10 mmol/L glucose, 15 mmol/L KCl, and 100 μmol/L diazoxide. These amplified secretory responses were paralleled by significant increases in the phospholipase C (PLC) activation monitored by fractional increases in 3H-inositol efflux from these same islets. Amplified PLC responses were also observed with the cholinergic agonist carbachol (50 μmol/L). No differences in the secretory responses to the protein kinase C activator phorbol 12-myristate 13-acetate (200 nmol/L) could be detected between control and FAF-BSA–pretreated rat islets. Mouse islets were also immune to the amplifying impact of this treatment protocol. These findings demonstrate that short-term incubation in FAF-BSA significantly augments the activation of PLC in rat islets by a number of agonists. This proximal event provides the impetus for the distal activation of protein kinase C. If applicable to human islets, this manipulation may provide a mechanism to enhance the secretory responses from islets destined for transplantation, thus improving their in vivo secretory capacity.

Glucose Toxicity and Pancreatic Beta Cell Dysfunction in Type 2 Diabetes

The adverse effects of prolonged exposure of pancreatic islets to supraphysiologic glucose concentrations(i.e. glucose toxicity) is mediated at least in part by glucose oxidation and the subsequent generation of reactive oxygen species(ROS) that can impair insulin gene expression and β cell function. Multiple biochemical pathways and mechanisms of action for glucose toxicity have been suggested. These include glucose autoxidation, protein kinase C activation, methylglyoxal formation and glycation, hexosamine metabolism, sorbitol formation, and oxidative phosphorylation. There are many potential mechanisms whereby excess glucose metabolites traveling along these pathways might cause β cell damage. However, all these pathways have in common the formation of reactive oxygen species that, in excess and over time, cause chronic oxidative stress, which in turn causes defective insulin gene expression and insulin secretion as well as increased apoptosis. The intracellular peroxide levels of the pancreatic islets(INS-1 cells, rat islets) by flow cytometry were increased in the high glucose media compared to 5.6 mM glucose media. The insulin, MafA, PDX-1 mRNA levels and glucose stimulated insulin secretion(GSIS) were decreased in high glucose media compared to 5.6 mM glucose media. The HO-1 seems to mediate the protective response of pancreatic islets against the oxidative stress that is due to high glucose conditions. Also, we observed decreased glutathione level, γ-GCS expression and increased oxidized LDL, malondialdehyde level at leukocytes and mesothelial cells from patients with Korean Type 2 Diabetes(esp, poorly controlled patients). In conclusion, this pathophysiologic sequence sets the scene for considering antioxidant therapy as an adjunct in the management of diabetes, especially type 2 Diabetes.(KOREAN DIABETES J 32:175-181, 2008)

Different secretory response of pancreatic islets and insulin secreting cell lines INS-1 and INS-1E to osmotic stimuli

Objective of this study was to characterize osmotically-induced insulin secretion in two tumor cell lines. We compared response of freshly isolated rat pancreatic islets and INS-1 and INS-1E tumor cell lines to high glucose, 30 % hypotonic medium and 20 % hypertonic medium. In Ca(2+)-containing medium glucose induced insulin release in all three cell types. Hypotonicity induced insulin secretion from islets and INS-1 cells but not from INS-1E cells, in which secretion was inhibited despite similar increase in cell volume in both cell types. GdCl(3) (100 micromol/l) did not affect insulin response from INS-1E cells to hypotonic challenge. Hypertonic medium inhibited glucose-induced insulin secretion from islets but not from tumor cells. Noradrenaline (1 micromol/l) inhibited glucose-induced but not swelling-induced insulin secretion from INS-1 cells. Surprisingly, perifusion with Ca(2+)-depleted medium showed distinct secretory response of INS-1E cells to hypotonicity while that of INS-1 cells was partially inhibited. Functioning glucose-induced insulin secretion is not sufficient prerequisite for hypotonicity-induced response in INS-1E cells suggesting that swelling-induced exocytosis is not essential step in the mechanism mediating glucose-induced insulin secretion. Both cell lines are resistant to inhibitory effect of hyperosmolarity on glucose-induced insulin secretion. Response of INS-1E cells to hypotonicity is inhibited by the presence of Ca(2+) in medium.

Biphasic insulin secretion from freshly isolated or cultured, perifused rodent islets: comparative studies with rats and mice

In the present report, we compared the insulin secretory responses of freshly isolated, perifused rat and mouse islets to glucose. Prestimulatory glucose levels were changed to assess their influence on the subsequent secretory responses. Additional studies included experiments with the incretin factor glucagon-like peptide-1 (GLP-1), the cholinergic agonist carbachol, and the α2 agonist epinephrine. Our findings demonstrate that under conditions where glucose (8.5-11.1 mmol/L) evokes a dramatic biphasic insulin secretory response from perifused rat islets, mouse islets exhibit little response. Increasing the prestimulatory glucose level to 8.5 mmol/L dramatically distorts subsequently measured glucose-induced insulin secretion from rat islets but allows the evocation of a modest but clear biphasic response from mouse islets in response to 30 mmol/L, but not 11.1 or 16.7 mmol/L, glucose. In the presence of a minimally effective glucose level (10 mmol/L), mouse islets remain exquisitely sensitive to the combined stimulatory effects of GLP-1 (2.5 nmol/L) plus carbachol (0.5 μmol/L) and to the inhibitory influence of epinephrine (10 nmol/L). Short-term culture of rat islets in CMRL 1066 containing 5.6 mmol/L glucose resulted in a significant decrease in the secretory response to 11.1 mmol/L glucose, whereas the same manipulation improved mouse islet responses. It is concluded that the process of collagenase isolating islets does not alter mouse islet sensitivity in any adverse way and that increasing the prestimulatory glucose level can indeed alter the pattern of insulin secretion in either a positive or negative manner depending upon the species being investigated. Prior short-term culture of rodent islets differentially affects secretion from these 2 species.

Diminished Phosphodiesterase-8B Potentiates Biphasic Insulin Response to Glucose

cAMP activates multiple signal pathways, crucial for the pancreatic beta-cells function and survival and is a major potentiator of insulin release. A family of phosphodiesterases (PDEs) terminate the cAMP signals. We examined the expression of PDEs in rat beta-cells and their role in the regulation of insulin response. Using RT-PCR and Western blot analyses, we identified PDE3A, PDE3B, PDE4B, PDE4D, and PDE8B in rat islets and in INS-1E cells and several possible splice variants of these PDEs. Specific depletion of PDE3A with small interfering (si) RNA (siPDE3A) led to a small (67%) increase in the insulin response to glucose in INS-1E cells but not rat islets. siPDE3A had no effect on the glucagon-like peptide-1 (10 nmol/liter) potentiated insulin response in rat islets. Depletion in PDE8B levels in rat islets using similar technology (siPDE8B) increased insulin response to glucose by 70%, the potentiation being of similar magnitude during the first and second phase insulin release. The siPDE8B-potentiated insulin response was further increased by 23% when glucagon-like peptide-1 was included during the glucose stimulus. In conclusion, PDE8B is expressed in a small number of tissues unrelated to glucose or fat metabolism. We propose that PDE8B, an 3-isobutyl-1-methylxanthine-insensitive cAMP-specific phosphodiesterase, could prove a novel target for enhanced insulin response, affecting a specific pool of cAMP involved in the control of insulin granule trafficking and exocytosis. Finally, we discuss evidence for functional compartmentation of cAMP in pancreatic beta-cells.

Interferons induce an antiviral state in human pancreatic islet cells

Enterovirus infections, in particular those with Coxsackieviruses, have been linked to the development of type 1 diabetes (T1D). Although animal models have demonstrated that interferons (IFNs) regulate virus-induced T1D by acting directly on the beta cell, little is known on the human pancreatic islet response to IFNs. Here we show that human islet cells respond to IFNs by expressing signature genes of antiviral defense. We also demonstrate that they express three intracellular sensors for viral RNA, the toll like receptor 3 (TLR3) gene, the retinoic acid-inducible gene I (RIG-I) and the melanoma differentiation-associated gene-5 (MDA-5), which induce type I IFN production in infected cells. Finally, we show for the first time that the IFN-induced antiviral state provides human islets with a powerful protection from the replication of Coxsackievirus. This may be critical for beta cell survival and protection from virus-induced T1D in humans.

Protein Kinase Cδ Plays a Non-redundant Role in Insulin Secretion in Pancreatic β Cells

Protein kinase C (PKC) is considered to modulate glucose-stimulated insulin secretion. Pancreatic beta cells express multiple isoforms of PKCs; however, the role of each isoform in glucose-stimulated insulin secretion remains controversial. In this study we investigated the role of PKCdelta, a major isoform expressed in pancreatic beta cells on beta cell function. Here, we showed that PKCdelta null mice manifested glucose intolerance with impaired insulin secretion. Insulin tolerance test showed no decrease in insulin sensitivity in PKCdelta null mice. Studies using islets isolated from these mice demonstrated decreased glucose- and KCl-stimulated insulin secretion. Perifusion studies indicated that mainly the second phase of insulin secretion was decreased. On the other hand, glucose-induced influx of Ca2+ into beta cells was not altered. Immunohistochemistry using total internal reflection fluorescence microscopy and electron microscopic analysis showed an increased number of insulin granules close to the plasma membrane in beta cells of PKCdelta null mice. Although PKC is thought to phosphorylate Munc18-1 and facilitate soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors complex formation, the phosphorylation of Munc18-1 by glucose stimulation was decreased in islets of PKCdelta null mice. We conclude that PKCdelta plays a non-redundant role in glucose-stimulated insulin secretion. The impaired insulin secretion in PKCdelta null mice is associated with reduced phosphorylation of Munc18-1.

Intramuscular Injection of Islet Neogenesis-Associated Protein Peptide Stimulates Pancreatic Islet Neogenesis in Healthy Dogs

Diabetes is a serious health problem. It has been proposed that islet neogenesis from pancreatic progenitor cells may restore insulin secretion in diabetic mammals. Islet neogenesis- associated protein (INGAP) stimulates islet neogenesis; therefore, we hypothesized that it would stimulate islet neogenesis in dogs. Forty nondiabetic beagle dogs were randomly divided into 4 groups. Group 1 received daily intramuscular injections of vehicle, whereas the other 3 groups received daily INGAP injections of 0.5, 1.5, or 10 mg/kg. After 30 days, pancreatic tissues were collected, and RNA and histological sections were analyzed. In dogs treated with 1.5 mg/kg INGAP, there was a significant (P < 0.001) increase in the percentage of insulin-positive cells (P < 0.001) and insulin gene expression. There was a trend to increased insulin-positive cells and gene expression with treatments of 0.5 and 10 mg/kg peptide. Protein gene product 9.5-positive cells were increased with treatment. These results indicate that INGAP stimulates cells in the pancreatic duct epithelium of healthy dogs (putative islet progenitor cells) to develop along a neuroendocrine pathway and form new islets in response to INGAP peptide. The INGAP might be an effective therapy for diabetes.

Glucose Stimulates Ca2+ Influx and Insulin Secretion in 2-Week-old β-Cells Lacking ATP-sensitive K+ Channels

In adult beta-cells glucose-induced insulin secretion involves two mechanisms (a) a K(ATP) channel-dependent Ca(2+) influx and rise of cytosolic [Ca(2+)](c) and (b) a K(ATP) channel-independent amplification of secretion without further increase of [Ca(2+)](c). Mice lacking the high affinity sulfonylurea receptor (Sur1KO), and thus K(ATP) channels, have been developed as a model of congenital hyperinsulinism. Here, we compared [Ca(2+)](c) and insulin secretion in overnight cultured islets from 2-week-old normal and Sur1KO mice. Control islets proved functionally mature: the magnitude and biphasic kinetics of [Ca(2+)](c) and insulin secretion changes induced by glucose, and operation of the amplifying pathway, were similar to adult islets. Sur1KO islets perifused with 1 mm glucose showed elevation of both basal [Ca(2+)](c) and insulin secretion. Stimulation with 15 mm glucose produced a transient drop of [Ca(2+)](c) followed by an overshoot and a sustained elevation, accompanied by a monophasic, 6-fold increase in insulin secretion. Glucose also increased insulin secretion when [Ca(2+)](c) was clamped by KCl. When Sur1KO islets were cultured in 5 instead of 10 mm glucose, [Ca(2+)](c) and insulin secretion were unexpectedly low in 1 mm glucose and increased following a biphasic time course upon stimulation by 15 mm glucose. This K(ATP) channel-independent first phase [Ca(2+)](c) rise was attributed to a Na(+)-, Cl(-)-, and Na(+)-pump-independent depolarization of beta-cells, leading to Ca(2+) influx through voltage-dependent calcium channels. Glucose indeed depolarized Sur1KO islets under these conditions. It is suggested that unidentified potassium channels are sensitive to glucose and subserve the acute and long-term metabolic control of [Ca(2+)](c) in beta-cells without functional K(ATP) channels.

The Role of Arachidonic Acid and Its Metabolites in Insulin Secretion From Human Islets of Langerhans

The roles played by arachidonic acid and its cyclooxygenase (COX)-generated and lipoxygenase (LOX)-generated metabolites have been studied using rodent islets and insulin-secreting cell lines, but very little is known about COX and LOX isoform expression and the effects of modulation of arachidonic acid generation and metabolism in human islets. We have used RT-PCR to identify mRNAs for cytosolic phospholipase A(2) (cPLA(2)), COX-1, COX-2, 5-LOX, and 12-LOX in isolated human islets. COX-3 and 15-LOX were not expressed by human islets. Perifusion experiments with human islets indicated that PLA(2) inhibition inhibited glucose-stimulated insulin secretion, whereas inhibitors of COX-2 and 12-LOX enzymes enhanced basal insulin secretion and also secretory responses induced by 20 mmol/l glucose or by 50 mumol/l arachidonic acid. Inhibition of COX-1 with 100 mumol/l acetaminophen did not significantly affect glucose-stimulated insulin secretion. These data indicate that the stimulation of insulin secretion from human islets in response to arachidonic acid does not require its metabolism through COX-2 and 5-/12-LOX pathways. The products of COX-2 and LOX activities have been implicated in cytokine-mediated damage of beta-cells, so selective inhibitors of these enzymes would be expected to have a dual protective role in diabetes: they would minimize beta-cell dysfunction while maintaining insulin secretion through enhancing endogenous arachidonic acid levels.

Palmitic acid increase levels of pancreatic duodenal homeobox-1 and p38/stress-activated protein kinase in islets from rats maintained on a low protein diet

A severe reduction in insulin release in response to glucose is consistently noticed in protein-deprived rats and is attributed partly to the chronic exposure to elevated levels of NEFA. Since the pancreatic and duodenal transcription factor homeobox 1 (PDX-1) is important for the maintenance of beta-cell physiology, and since PDX-1 expression is altered in the islets of rats fed a low protein (LP) diet and that rats show high NEFA levels, we assessed PDX-1 and insulin mRNA expression, as well as PDX-1 and p38/stress activated protein kinase 2 (SAPK2) protein expression, in islets from young rats fed low (6%) or normal (17%; control) protein diets and maintained for 48 h in culture medium containing 5.6 mmol/l glucose, with or without 0.6 mmol/l palmitic acid. We also measured glucose-induced insulin secretion and glucose metabolism. Insulin secretion by isolated islets in response to 16.7 mmol/l glucose was reduced in LP compared with control rats. In the presence of NEFA, there was an increase in insulin secretion in both groups. At 2.8 mmol/l glucose, the metabolism of this sugar was reduced in LP islets, regardless of the presence of this fatty acid. However, when challenged with 16.7 mmol/l glucose, LP and control islets showed a severe reduction in glucose oxidation in the presence of NEFA. The PDX-1 and insulin mRNA were significantly higher when NEFA was added to the culture medium in both groups of islets. The effect of palmitic acid on PDX-1 and p38/SAPK2 protein levels was similar in LP and control islets, but the increase was much more evident in LP islets. These results demonstrate the complex interrelationship between nutrients in the control of insulin release and support the view that fatty acids play an important role in glucose homeostasis by affecting molecular mechanisms and stimulus/secretion coupling pathways.

Improved Outcomes in Islet Isolation and Transplantation by the Use of a Novel Hemoglobin-based O2 Carrier

During isolation, islets are exposed to warm ischemia. In this study, intraductal administration of oxygenated polymerized, stroma-free hemoglobin-pyridoxalated (Poly SFH-P) was performed to improve O2 delivery. Rat pancreata subjected to 30-min warm ischemia were perfused intraductally with collagenase in oxygenated Poly SFH-P/RPMI or RPMI (control). PO2 was increased by Poly SFH-P (381.7 +/- 35.3 mmHg vs. 202.3 +/- 28.2, p = 0.01) and pH maintained within physiological range (7.4-7.2 vs. 7.1-6.6, p = 0.009). Islet viability (77% +/- 4.6 vs. 63% +/- 4.7, p = 0.04) was improved and apoptosis lower with Poly SFH-P (caspase-3: 34,714 +/- 2167 vs. 45,985 +/- 1382, respectively, p = 0.01). Poly SFH-P improved islet responsiveness to glucose as determined by increased intracellular Ca2+ levels and improved insulin secretion (SI 5.4 +/- 0.1 vs. 3.1 +/- 0.2, p = 0.03). Mitochondrial integrity was improved in Poly SFH-P-treated islets, which showed higher percentage change in membrane potential after glucose stimulation (14.7% +/- 1.8 vs. 9.8 +/- 1.4, respectively, p < 0.05). O2 delivery by Poly SFH-P did not increase oxidative stress (GSH 7.1 +/- 2.9 nm/mg protein for Poly SFH-P vs. 6.8 +/- 2.4 control, p = 0.9) or oxidative injury (MDA 1.8 +/- 0.9 nmol/mg protein vs. 6.2 +/- 2.4, p = 0.19). Time to reach normoglycemia in transplanted diabetic nude mice was shorter (1.8 +/- 0.4 vs. 7 +/- 2.5 days, p = 0.02), and glucose tolerance improved in the Poly SFH-P group (AUC 8106 +/- 590 vs. 10,863 +/- 946, p = 0.03). Oxygenated Poly SFH-P improves islet isolation and transplantation outcomes by preserving mitochondrial integrity.

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.

Muscarinic M 2 receptor is active on pancreatic islets from hypothalamic obese rat

Hypothalamic obese rats, obtained by neonatal treatment with monosodium l-glutamate (MSG), are hyperinsulinemic, and secrete more insulin than lean ones do when stimulated by glucose, while acetylcholine insulinotropic effect decreases. The effect of acetylcholine on glucose-induced insulin secretion is attributed to muscarinic receptors of pancreatic beta cells, mainly to M 3 subtype. However, it has been observed that activation of M 2 or M 4 subtypes causes inhibition of glucose-induced insulin secretion in insulin secreting cell line. Insulin secretion was measured, stimulated by glucose in the presence of acetylcholine plus methoctramine, a muscarinic M 2 antagonist, on pancreatic islets isolated from MSG-obese and lean rats to investigate whether impairment of acetylcholine insulinotropic effect on pancreatic islets from MSG-obese rats has any relationship with muscarinic M 2 receptor function in beta cells. Insulin secretion stimulated by 8.3 mM glucose was higher in islets from obese rats than from lean ones. Insulinotropic effect of acetylcholine was reported in islets of both animals, albeit less than in obese ones. Blockage of muscarinic M 2 receptor, using methoctramine at 1; 5 and 10 μM, increased acetylcholine secretory response in islets of obese rats, while no effect has been observed in lean ones. Results demonstrate that muscarinic M 2 receptors are functioning in pancreatic islets of MSG-obese rats. The inhibitory action of muscarinic M 2 receptor may be a mechanism by which acetylcholine discloses weak insulinotropic effect in MSG-obese rats.

Thea3 isoform of V-ATPase regulates insulin secretion from pancreatic β-cells

Vacuolar-type H(+)-ATPase (V-ATPase) is a multi-subunit enzyme that has important roles in the acidification of a variety of intracellular compartments and some extracellular milieus. Four isoforms for the membrane-intrinsic subunit (subunit a) of the V-ATPase have been identified in mammals, and they confer distinct cellular localizations and activities on the proton pump. We found that V-ATPase with the a3 isoform is highly expressed in pancreatic islets, and is localized to membranes of insulin-containing secretory granules in beta-cells. oc/oc mice, which have a null mutation at the a3 locus, exhibited a reduced level of insulin in the blood, even with high glucose administration. However, islet lysates contained mature insulin, and the ratio of the amount of insulin to proinsulin in oc/oc islets was similar to that of wild-type islets, indicating that processing of insulin was normal even in the absence of the a3 function. The insulin contents of oc/oc islets were reduced slightly, but this was not significant enough to explain the reduced levels of the blood insulin. The secretion of insulin from isolated islets in response to glucose or depolarizing stimulation was impaired. These results suggest that the a3 isoform of V-ATPase has a regulatory function in the exocytosis of insulin secretion.

Calcineurin/NFAT signalling regulates pancreatic β-cell growth and function

The growth and function of organs such as pancreatic islets adapt to meet physiological challenges and maintain metabolic balance, but the mechanisms controlling these facultative responses are unclear. Diabetes in patients treated with calcineurin inhibitors such as cyclosporin A indicates that calcineurin/nuclear factor of activated T-cells (NFAT) signalling might control adaptive islet responses, but the roles of this pathway in beta-cells in vivo are not understood. Here we show that mice with a beta-cell-specific deletion of the calcineurin phosphatase regulatory subunit, calcineurin b1 (Cnb1), develop age-dependent diabetes characterized by decreased beta-cell proliferation and mass, reduced pancreatic insulin content and hypoinsulinaemia. Moreover, beta-cells lacking Cnb1 have a reduced expression of established regulators of beta-cell proliferation. Conditional expression of active NFATc1 in Cnb1-deficient beta-cells rescues these defects and prevents diabetes. In normal adult beta-cells, conditional NFAT activation promotes the expression of cell-cycle regulators and increases beta-cell proliferation and mass, resulting in hyperinsulinaemia. Conditional NFAT activation also induces the expression of genes critical for beta-cell endocrine function, including all six genes mutated in hereditary forms of monogenic type 2 diabetes. Thus, calcineurin/NFAT signalling regulates multiple factors that control growth and hallmark beta-cell functions, revealing unique models for the pathogenesis and therapy of diabetes.

Desensitization of the pancreatic β-cell: effects of sustained physiological hyperglycemia and potassium

The impact of modest but prolonged (3 h) exposure to high physiological glucose concentrations and hyperkalemia on the insulin secretory and phospholipase C (PLC) responses of rat pancreatic islets was determined. In acute studies, glucose (5-20 mM) caused a dose-dependent increase in secretion with maximal release rates 25-fold above basal secretion. When measured after 3 h of exposure to 5-10 mM glucose, subsequent stimulation of islets with 10-20 mM glucose during a dynamic perifusion resulted in dose-dependent decrements in secretion and PLC activation. Acute hyperkalemia (15-30 mM) stimulated calcium-dependent increases in both insulin secretion and PLC activation; however, prolonged hyperkalemia resulted in a biochemical and secretory lesion similar to that induced by sustained modest hyperglycemia. Glucose- (8 mM) desensitized islets retained significant sensitivity to stimulation by either carbachol or glucagon-like peptide-1. These findings emphasize the vulnerability of the beta-cell to even moderate sustained hyperglycemia and provide a biochemical rationale for achieving tight glucose control in diabetic patients. They also suggest that PLC activation plays a critically important role in the physiological regulation of glucose-induced secretion and in the desensitization of release that follows chronic hyperglycemia or hyperkalemia.