Search

The lactate dehydrogenase isoform A (LDHA) is a key metabolic enzyme that preferentially catalyzes the conversion of pyruvate to lactate. Whereas LDHA is highly expressed in many tissues, its expression is turned off in the differentiated adult β-cell within the pancreatic islets. The repression of LDHA under normal physiological condition and its inappropriate upregulation under a diabetogenic environment is well-documented in rodent islets/β-cells but little is known about LDHA expression in human islet cells and whether its abundance is altered under diabetic conditions. Analysis of public single-cell RNA-seq (sc-RNA seq) data as well as cell type-specific immunolabeling of human pancreatic islets showed that LDHA was mainly localized in human α-cells while it is expressed at a very low level in β-cells. Furthermore, LDHA, both at mRNA and protein, as well as lactate production is upregulated in human pancreatic islets exposed to chronic high glucose treatment. Microscopic analysis of stressed human islets and autopsy pancreases from individuals with type 2 diabetes (T2D) showed LDHA upregulation mainly in human α-cells. Pharmacological inhibition of LDHA in isolated human islets enhanced insulin secretion under physiological conditions but did not significantly correct the deregulated secretion of insulin or glucagon under diabetic conditions.

Objective Sodium-glucose cotransporter 2 (SGLT2) inhibitors (SGLT2i), or gliflozins, are anti-diabetic drugs that lower glycemia by promoting glucosuria, but they also stimulate endogenous glucose and ketone body production. The likely causes of these metabolic responses are increased blood glucagon levels, and decreased blood insulin levels, but the mechanisms involved are hotly debated. This study verified whether or not SGLT2i affect glucagon and insulin secretion by a direct action on islet cells in three species, using multiple approaches. Methods We tested the in vivo effects of two selective SGLT2i (dapagliflozin, empagliflozin) and a SGLT1/2i (sotagliflozin) on various biological parameters (glucosuria, glycemia, glucagonemia, insulinemia) in mice. mRNA expression of SGLT2 and other glucose transporters was assessed in rat, mouse, and human FACS-purified α- and β-cells, and by analysis of two human islet cell transcriptomic datasets. Immunodetection of SGLT2 in pancreatic tissues was performed with a validated antibody. The effects of dapagliflozin, empagliflozin, and sotagliflozin on glucagon and insulin secretion were assessed using isolated rat, mouse and human islets and the in situ perfused mouse pancreas. Finally, we tested the long-term effect of SGLT2i on glucagon gene expression. Results SGLT2 inhibition in mice increased the plasma glucagon/insulin ratio in the fasted state, an effect correlated with a decline in glycemia. Gene expression analyses and immunodetections showed no SGLT2 mRNA or protein expression in rodent and human islet cells, but moderate SGLT1 mRNA expression in human α-cells. However, functional experiments on rat, mouse, and human (29 donors) islets and the in situ perfused mouse pancreas did not identify any direct effect of dapagliflozin, empagliflozin or sotagliflozin on glucagon and insulin secretion. SGLT2i did not affect glucagon gene expression in rat and human islets. Conclusions The data indicate that the SGLT2i-induced increase of the plasma glucagon/insulin ratio in vivo does not result from a direct action of the gliflozins on islet cells., Highlights • Gliflozins (SGLT2 and SGLT1/2 inhibitors) increase plasma glucagon/insulin ratio. • SGLT2 is not expressed in rodent and human pancreatic α- and β-cells. • SGLT1 is however expressed in human α-cells. • SGLT2 and SGLT1/2 inhibitors do not directly affect glucagon and insulin secretion.

Store-operated Ca2+ entry (SOCE) is a dynamic process that leads to refilling of endoplasmic reticulum (ER) Ca2+ stores through reversible gating of plasma membrane Ca2+ channels by the ER Ca2+ sensor Stromal Interaction Molecule 1 (STIM1). Pathogenic reductions in β-cell ER Ca2+ have been observed in diabetes. However, a role for impaired SOCE in this phenotype has not been tested. We measured the expression of SOCE molecular components in human and rodent models of diabetes and found a specific reduction in STIM1 mRNA and protein levels in human islets from donors with type 2 diabetes (T2D), islets from hyperglycemic streptozotocin-treated mice, and INS-1 cells (rat insulinoma cells) treated with proinflammatory cytokines and palmitate. Pharmacologic SOCE inhibitors led to impaired islet Ca2+ oscillations and insulin secretion, and these effects were phenocopied by β-cell STIM1 deletion. STIM1 deletion also led to reduced ER Ca2+ storage and increased ER stress, whereas STIM1 gain of function rescued β-cell survival under proinflammatory conditions and improved insulin secretion in human islets from donors with T2D. Taken together, these data suggest that the loss of STIM1 and impaired SOCE contribute to ER Ca2+ dyshomeostasis under diabetic conditions, whereas efforts to restore SOCE-mediated Ca2+ transients may have the potential to improve β-cell health and function.

Objective Glucagon is secreted by pancreatic α-cells in response to hypoglycemia and its hyperglycemic effect helps to restore normal blood glucose. Insulin and somatostatin (SST) secretions from β- and δ-cells, respectively, are stimulated by glucose by mechanisms involving an inhibition of their ATP-sensitive K+ (KATP) channels, leading to an increase in [Ca2+]c that triggers exocytosis. Drugs that close KATP channels, such as sulfonylureas, are used to stimulate insulin release in type 2 diabetic patients. α-cells also express KATP channels. However, the mechanisms by which sulfonylureas control glucagon secretion are still largely debated and were addressed in the present study. In particular, we studied the effects of KATP channel blockers on α-cell [Ca2+]c and glucagon secretion in the presence of a low (1 mM) or a high (15 mM) glucose concentration and evaluated the role of SST in these effects. Methods Using a transgenic mouse model expressing the Ca2+-sensitive fluorescent protein, GCaMP6f, specifically in α-cells, we measured [Ca2+]c in α-cells either dispersed or within whole islets (by confocal microscopy). By measuring [Ca2+]c in α-cells within islets and glucagon secretion using the same perifusion protocols, we tested whether glucagon secretion correlated with changes in [Ca2+]c in response to sulfonylureas. We studied the role of SST in the effects of sulfonylureas using multiple approaches including genetic ablation of SST, or application of SST-14 and SST receptor antagonists. Results Application of the sulfonylureas, tolbutamide, or gliclazide, to a medium containing 1 mM or 15 mM glucose increased [Ca2+]c in α-cells by a direct effect as in β-cells. At low glucose, sulfonylureas inhibited glucagon secretion of islets despite the rise in α-cell [Ca2+]c that they triggered. This glucagonostatic effect was indirect and attributed to SST because, in the islets of SST-knockout mice, sulfonylureas induced a stimulation of glucagon secretion which correlated with an increase in α-cell [Ca2+]c. Experiments with exogenous SST-14 and SST receptor antagonists indicated that the glucagonostatic effect of sulfonylureas mainly resulted from an inhibition of the efficacy of cytosolic Ca2+ on exocytosis. Although SST-14 was also able to inhibit glucagon secretion by decreasing α-cell [Ca2+]c, no decrease in [Ca2+]c occurred during sulfonylurea application because it was largely counterbalanced by the direct stimulatory effect of these drugs on α-cell [Ca2+]c. At high glucose, i.e., in conditions where glucagon release was already low, sulfonylureas stimulated glucagon secretion because their direct stimulatory effect on α-cells exceeded the indirect effect by SST. Our results also indicated that, unexpectedly, SST-14 poorly decreased the efficacy of Ca2+ on exocytosis in β-cells. Conclusions Sulfonylureas exert two opposite actions on α-cells: a direct stimulation as in β-cells and an indirect inhibition by SST. This suggests that any alteration of SST paracrine influence, as described in diabetes, will modify the effect of sulfonylureas on glucagon release. In addition, we suggest that δ-cells inhibit α-cells more efficiently than β-cells., Highlights • KATP channel blockers control glucagon secretion by two mechanisms. • The first one is the direct stimulation of α-cell by a [Ca2+]c rise, as in β-cells. • The second one is an indirect inhibition mediated by δ-cells releasing somatostatin. • Somatostatin mainly reduces the efficacy of Ca2+ on exocytosis in α-cells. • Somatostatin more potently inhibits glucagon than insulin secretion.

Objective Non-coding RNAs constitute a major fraction of the β-cell transcriptome. While the involvement of microRNAs is well established, the contribution of long non-coding RNAs (lncRNAs) in the regulation of β-cell functions and in diabetes development remains poorly understood. The aim of this study was to identify novel islet lncRNAs differently expressed in type 2 diabetes models and to investigate their role in β-cell failure and in the development of the disease. Methods Novel transcripts dysregulated in the islets of diet-induced obese mice were identified by high throughput RNA-sequencing coupled with de novo annotation. Changes in the level of the lncRNAs were assessed by real-time PCR. The functional role of the selected lncRNAs was determined by modifying their expression in MIN6 cells and primary islet cells. Results We identified about 1500 novel lncRNAs, a number of which were differentially expressed in obese mice. The expression of two lncRNAs highly enriched in β-cells, βlinc2, and βlinc3, correlated to body weight gain and glycemia levels in obese mice and was also modified in diabetic db/db mice. The expression of both lncRNAs was also modulated in vitro in isolated islet cells by glucolipotoxic conditions. Moreover, the expression of the human orthologue of βlinc3 was altered in the islets of type 2 diabetic patients and was associated to the BMI of the donors. Modulation of the level of βlinc2 and βlinc3 by overexpression or downregulation in MIN6 and mouse islet cells did not affect insulin secretion but increased β-cell apoptosis. Conclusions Taken together, the data show that lncRNAs are modulated in a model of obesity-associated type 2 diabetes and that variations in the expression of some of them may contribute to β-cell failure during the development of the disease., Highlights • Mouse pancreatic islets express a large number of novel long non-coding RNAs. • Many long non-coding RNAs are differentially expressed in the islets of obese mice. • The level of two islet long non-coding RNAs correlates to body weight and glycemia. • The expression of these islet long non-coding RNAs is altered in Type 2 diabetes. • Altered expression of these long non-coding RNAs sensitise β-cells to apoptosis.

Aims/hypothesis: The mechanisms responsible for beta cell compensation in obesity and for beta cell failure in type 2 diabetes are poorly defined. The mRNA levels of several metallothionein (MT) genes are upregulated in islets from individuals with type 2 diabetes, but their role in beta cells is not clear. Here we examined: 1) the temporal changes of islet Mt1 and Mt2 gene expression in mouse models of beta cell compensation and failure; and 2) the role of Mt1 and Mt2 in beta cell function and glucose homeostasis in mice. Methods: Mt1 and Mt2 expression was assessed in islets from: (1) control lean (chow diet-fed) and diet-induced obese (high-fat diet-fed for 6 weeks) mice; (2) mouse models of prediabetes (6-week-old db/db mice) and diabetes (16-week-old db/db mice) and age-matched db/+ (control) mice; and (3) obese non-diabetic ob/ob mice (16-week-old) and age-matched ob/+ (control) mice. MT1E, MT1X and MT2A expression was assessed in islets from humans with and without type 2 diabetes. Mt1-Mt2 double-knockout (KO) mice, transgenic mice overexpressing Mt1 under the control of its natural promoter (Tg-Mt1) and corresponding control mice were also studied. In MIN6 cells, MT1 and MT2 were inhibited by small interfering RNAs. mRNA levels were assessed by real-time RT-PCR, plasma insulin and islet MT levels by ELISA, glucose tolerance by i.p. glucose tolerance tests and fasting 1 h refeeding tests, insulin tolerance by i.p. insulin tolerance tests, insulin secretion by RIA, cytosolic free Ca2+ concentration with Fura-2 leakage resistant (Fura-2 LR), cytosolic free Zn2+ concentration with Fluozin-3, and NAD(P)H by autofluorescence. Results: Mt1 and Mt2 mRNA levels were reduced in islets of murine models of beta cell compensation, whereas they were increased in diabetic db/db mice. In humans, MT1X mRNA levels were significantly upregulated in islets from individuals with type 2 diabetes in comparison with non-diabetic donors, while MT1E and MT2A mRNA levels were unchanged. Ex vivo, islet Mt1 and Mt2 mRNA and MT1 and MT2 protein levels were downregulated after culture with glucose at 10–30 mmol/l vs 2–5 mmol/l, in association with increased insulin secretion. In human islets, mRNA levels of MT1E, MT1X and MT2A were downregulated by stimulation with physiological and supraphysiological levels of glucose. In comparison with wild-type (WT) mice, Mt1-Mt2 double-KO mice displayed improved glucose tolerance in association with increased insulin levels and enhanced insulin release from isolated islets. In contrast, isolated islets from Tg-Mt1 mice displayed impaired glucose-stimulated insulin secretion (GSIS). In both Mt1-Mt2 double-KO and Tg-Mt1 models, the changes in GSIS occurred despite similar islet insulin content, rises in cytosolic free Ca2+ concentration and NAD(P)H levels, or intracellular Zn2+ concentration vs WT mice. In MIN6 cells, knockdown of MT1 but not MT2 potentiated GSIS, suggesting that Mt1 rather than Mt2 affects beta cell function. Conclusions/interpretation: These findings implicate Mt1 as a negative regulator of insulin secretion. The downregulation of Mt1 is associated with beta cell compensation in obesity, whereas increased Mt1 accompanies beta cell failure and type 2 diabetes.

The sarcoendoplasmic reticulum (ER) Ca2+ ATPase 2 (SERCA2) pump is a P-type ATPase tasked with the maintenance of ER Ca2+ stores. Whereas β-cell SERCA2 expression is reduced in diabetes, the role of SERCA2 in the regulation of whole-body glucose homeostasis has remained uncharacterized. To this end, SERCA2 heterozygous mice (S2HET) were challenged with a high-fat diet (HFD) containing 45% of kilocalories from fat. After 16 weeks of the HFD, S2HET mice were hyperglycemic and glucose intolerant, but adiposity and insulin sensitivity were not different between HFD-fed S2HET mice and HFD-fed wild-type controls. Consistent with a defect in β-cell function, insulin secretion, glucose-induced cytosolic Ca2+ mobilization, and the onset of steady-state glucose-induced Ca2+ oscillations were impaired in HFD-fed S2HET islets. Moreover, HFD-fed S2HET mice exhibited reduced β-cell mass and proliferation, altered insulin production and proinsulin processing, and increased islet ER stress and death. In contrast, SERCA2 activation with a small molecule allosteric activator increased ER Ca2+ storage and rescued tunicamycin-induced β-cell death. In aggregate, these data suggest a critical role for SERCA2 and the regulation of ER Ca2+ homeostasis in the β-cell compensatory response to diet-induced obesity.

Type 2 diabetes is characterised by chronic hyperglycaemia and its incidence is highly increased by exaggerated food consumption. It results from a lack of insulin action/production, but growing evidence suggests that it might also involve hyperglucagonaemia and impaired control of glucose homeostasis by the brain. In recent years, the cocaine and amphetamine-regulated transcript (CART) peptides have generated a lot of interest in the battle against obesity because, via the brain, they exert anorexic effects and they increase energy expenditure. They are also localised, outside the brain, in discrete regions of the body and play a hormonal role in controlling various functions. In this issue of Diabetologia, the Wierup group (doi: 10.1007/s00125-016-4020-6 ) shows that CART peptides are expressed heterogeneously in islet cells of various species, including humans, and that their expression is upregulated in diabetes. The authors also shine a spotlight on some interesting effects of CART peptides on islet function, including stimulation of insulin secretion and inhibition of glucagon release. CART peptides would thus be at the centre of a cooperation between the brain and the endocrine pancreas to control glucose homeostasis. Although the mechanisms of action of CART peptides remain enigmatic because no specific receptor for these peptides has so far been discovered, their potential therapeutic use is evident and represents a new challenge for future research.

Glucose is the main physiological stimulus of pancreatic β-cells. However, chronic exposure of β-cells to elevated glucose concentrations induces glucotoxicity. In animal models of type 2 diabetes, it has been shown that several days of hyperglycemia impairs glucose-stimulated insulin secretion and increases β-cell apoptosis. In patients with type 2 diabetes, the multiple disorders caused by chronic hyperglycemia in β-cells include elevated basal insulin secretion, increased sensitivity to glucose, diminished response to insulinotropic stimuli and substantial depletion of insulin hoarding [1,2]. These defects associated with insulin resistance lead to a progressive loss of β-cell mass and function and to the onset of diabetes. It is crucial to study the mechanisms by which glucotoxicity induces β-cell failure to develop therapeutic strategies for protecting and recovering a functional β-cell mass. Several mechanisms might explain the glucotoxicity due to prolonged hyperglycemia, such as β-cell exhaustion, oxidative stress induced by free radical oxygen species, endoplasmic reticulum (ER) stress, inflammation caused by proinflammatory cytokines and chemokines, loss of neogenesis, proliferation of β-cells, and so on [3,4,5,6,7,8,9,10]. However, the precise mechanisms of glucotoxicity and its contribution to the pathology of type 2 diabetes mellitus (T2DM) are still not fully understood.

Steviol glycosides (SGs), such as stevioside and rebaudioside A, are natural, non-caloric sweet-tasting organic molecules, present in extracts of the scrub plant Stevia rebaudiana, which are widely used as sweeteners in consumer foods and beverages. TRPM5 is a Ca2+-activated cation channel expressed in type II taste receptor cells and pancreatic β-cells. Here we show that stevioside, rebaudioside A and their aglycon steviol potentiate the activity of TRPM5. We find that SGs potentiate perception of bitter, sweet and umami taste, and enhance glucose-induced insulin secretion in a Trpm5-dependent manner. Daily consumption of stevioside prevents development of high-fat-diet-induced diabetic hyperglycaemia in wild-type mice, but not in Trpm5−/− mice. These results elucidate a molecular mechanism of action of SGs and identify TRPM5 as a potential target to prevent and treat type 2 diabetes., Steviol glycosides are sweet-tasting compounds isolated from a South American shrub and are increasingly used as sweeteners in foods and beverages. Philippaert et al. demonstrate that steviol glycosides potentiate Ca2+-dependent TRPM5 activity and promote glucose-induced insulin secretion and glucose tolerance.

Glucagon secreted by pancreatic α-cells is the major hyperglycemic hormone correcting acute hypoglycaemia (glucose counterregulation). In diabetes the glucagon response to hypoglycaemia becomes compromised and chronic hyperglucagonemia appears. There is increasing awareness that glucagon excess may underlie important manifestations of diabetes. However opinions differ widely how glucose controls glucagon secretion. The autonomous nervous system plays an important role in the glucagon response to hypoglycaemia. But it is clear that glucose controls glucagon secretion also by mechanisms involving direct effects on α-cells or indirect effects via paracrine factors released from non-α-cells within the pancreatic islets. The present review discusses these mechanisms and argues that different regulatory processes are involved in a glucose concentration-dependent manner. Direct glucose effects on the α-cell and autocrine mechanisms are probably most significant for the glucagon response to hypoglycaemia. During hyperglycaemia, when secretion from β- and δ-cells is stimulated, paracrine inhibitory factors generate pulsatile glucagon release in opposite phase to pulsatile release of insulin and somatostatin. High concentrations of glucose have also stimulatory effects on glucagon secretion that tend to balance and even exceed the inhibitory influence. The latter actions might underlie the paradoxical hyperglucagonemia that aggravates hyperglycaemia in persons with diabetes.

OBJECTIVE Sarco-endoplasmic reticulum Ca2+-ATPase 2b (SERCA2b) and SERCA3 pump Ca2+ in the endoplasmic reticulum (ER) of pancreatic β-cells. We studied their role in the control of the free ER Ca2+ concentration ([Ca2+]ER) and the role of SERCA3 in the control of insulin secretion and ER stress. RESEARCH DESIGN AND METHODS β-Cell [Ca2+]ER of SERCA3+/+ and SERCA3−/− mice was monitored with an adenovirus encoding the low Ca2+-affinity sensor D4 addressed to the ER (D4ER) under the control of the insulin promoter. Free cytosolic Ca2+ concentration ([Ca2+]c) and [Ca2+]ER were simultaneously recorded. Insulin secretion and mRNA levels of ER stress genes were studied. RESULTS Glucose elicited synchronized [Ca2+]ER and [Ca2+]c oscillations. [Ca2+]ER oscillations were smaller in SERCA3−/− than in SERCA3+/+ β-cells. Stimulating cell metabolism with various [glucose] in the presence of diazoxide induced a similar dose-dependent [Ca2+]ER rise in SERCA3+/+ and SERCA3−/− β-cells. In a Ca2+-free medium, glucose moderately raised [Ca2+]ER from a highly buffered cytosolic Ca2+ pool. Increasing [Ca2+]c with high [K] elicited a [Ca2+]ER rise that was larger but more transient in SERCA3+/+ than SERCA3−/− β-cells because of the activation of a Ca2+ release from the ER in SERCA3+/+ β-cells. Glucose-induced insulin release was larger in SERCA3−/− than SERCA3+/+ islets. SERCA3 ablation did not induce ER stress. CONCLUSIONS [Ca2+]c and [Ca2+]ER oscillate in phase in response to glucose. Upon [Ca2+]c increase, Ca2+ is taken up by SERCA2b and SERCA3. Strong Ca2+ influx triggers a Ca2+ release from the ER that depends on SERCA3. SERCA3 deficiency neither impairs Ca2+ uptake by the ER upon cell metabolism acceleration and insulin release nor induces ER stress.

Glucose-induced insulin secretion is attributed to a rise of beta cell cytosolic free [Ca(2+)] ([Ca(2+)](c)) (triggering pathway) and amplification of the action of Ca(2+). This concept of amplification rests on observations that glucose can increase Ca(2+)-induced insulin secretion without further elevating an imposed already high [Ca(2+)](c). However, it remains possible that this amplification results from an increase in [Ca(2+)] just under the plasma membrane ([Ca(2+)](SM)), which escaped detection by previous measurements of global [Ca(2+)](c). This was the hypothesis that we tested here by measuring [Ca(2+)](SM).The genetically encoded Ca(2+) indicators D3-cpv (untargeted) and LynD3-cpv (targeted to plasma membrane) were expressed in clusters of mouse beta cells. LynD3-cpv was also expressed in beta cells within intact islets. [Ca(2+)](SM) changes were monitored using total internal reflection fluorescence microscopy. Insulin secretion was measured in parallel.Beta cells expressing D3cpv or LynD3cpv displayed normal [Ca(2+)] changes and insulin secretion in response to glucose. Distinct [Ca(2+)](SM) fluctuations were detected during repetitive variations of KCl between 30 and 32-35 mmol/l, attesting to the adequate sensitivity of our system. When the amplifying pathway was evaluated (high KCl + diazoxide), increasing glucose from 3 to 15 mmol/l consistently lowered [Ca(2+)](SM) while stimulating insulin secretion approximately two fold. Blocking Ca(2+) uptake by the endoplasmic reticulum largely attenuated the [Ca(2+)](SM) decrease produced by high glucose but did not unmask localised [Ca(2+)](SM) increases.Glucose can increase Ca(2+)-induced insulin secretion without causing further elevation of beta cell [Ca(2+)](SM). The phenomenon is therefore a true amplification of the triggering action of Ca(2+).

Zinc co-crystallizes with insulin in dense core secretory granules, but its role in insulin biosynthesis, storage and secretion is unknown. In this study we assessed the role of the zinc transporter ZnT8 using ZnT8-knockout ( ZnT8 −/− ) mice. Absence of ZnT8 expression caused loss of zinc release upon stimulation of exocytosis, but normal rates of insulin biosynthesis, normal insulin content and preserved glucose-induced insulin release. Ultrastructurally, mature dense core insulin granules were rare in ZnT8 −/− beta cells and were replaced by immature, pale insulin “progranules,” which were larger than in ZnT8 +/+ islets. When mice were fed a control diet, glucose tolerance and insulin sensitivity were normal. However, after high-fat diet feeding, the ZnT8 −/− mice became glucose intolerant or diabetic, and islets became less responsive to glucose. Our data show that the ZnT8 transporter is essential for the formation of insulin crystals in beta cells, contributing to the packaging efficiency of stored insulin. Interaction between the ZnT8 −/− genotype and diet to induce diabetes is a model for further studies of the mechanism of disease of human ZNT8 gene mutations.

OBJECTIVE: We studied how glucose and ATP-sensitive K(+) (K(ATP)) channel modulators affect alpha-cell [Ca(2+)](c). RESEARCH DESIGN AND METHODS: GYY mice (expressing enhanced yellow fluorescent protein in alpha-cells) and NMRI mice were used. [Ca(2+)](c), the K(ATP) current (I(KATP), perforated mode) and cell metabolism [NAD(P)H fluorescence] were monitored in single alpha-cells and, for comparison, in single beta-cells. RESULTS: In 0.5 mmol/l glucose, [Ca(2+)](c) oscillated in some alpha-cells and was basal in the others. Increasing glucose to 15 mmol/l decreased [Ca(2+)](c) by approximately 30% in oscillating cells and was ineffective in the others. alpha-Cell I(KATP) was inhibited by tolbutamide and activated by diazoxide or the mitochondrial poison azide, as in beta-cells. Tolbutamide increased alpha-cell [Ca(2+)](c), whereas diazoxide and azide abolished [Ca(2+)](c) oscillations. Increasing glucose from 0.5 to 15 mmol/l did not change I(KATP) and NAD(P)H fluorescence in alpha-cells in contrast to beta-cells. The use of nimodipine showed that L-type Ca(2+) channels are the main conduits for Ca(2+) influx in alpha-cells. gamma-Aminobutyric acid and zinc did not decrease alpha-cell [Ca(2+)](c), and insulin, although lowering [Ca(2+)](c) very modestly, did not affect glucagon secretion. CONCLUSIONS: alpha-Cells display similarities with beta-cells: K(ATP) channels control Ca(2+) influx mainly through L-type Ca(2+) channels. However, alpha-cells have distinct features from beta-cells: Most K(ATP) channels are already closed at low glucose, glucose does not affect cell metabolism and I(KATP), and it slightly decreases [Ca(2+)](c). Hence, glucose and K(ATP) channel modulators exert distinct effects on alpha-cell [Ca(2+)](c). The direct small glucose-induced drop in alpha-cell [Ca(2+)](c) contributes likely only partly to the strong glucose-induced inhibition of glucagon secretion in islets.

Glucose-induced insulin secretion requires a rise in beta-cell cytosolic Ca2+ ([Ca2+]c) that triggers exocytosis and a mechanistically unexplained amplification of the action of [Ca2+]c. Insulin granules are kept acidic by luminal pumping of protons with simultaneous Cl- uptake to maintain electroneutrality. Experiments using patched, dialyzed beta-cells prompted the suggestion that acute granule acidification by glucose underlies amplification of insulin secretion. However, others found glucose to increase granular pH in intact islets. In this study, we measured islet granular pH with Lysosensor DND-160, a fluorescent dye that permits ratiometric determination of pH methylamine (weak base) > Cl- omission. Concanamycin and methylamine did not alter glucose-induced [Ca2+]c increase in islets but strongly inhibited the two phases of insulin secretion. Omission of Cl- did not affect the first phase but decreased the second phase of both [Ca2+]c and insulin responses. Neither experimental condition affected the [Ca2+]c rise induced by 30 mM KCl, but the insulin responses were inhibited by concanamycin > methylamine and not affected by Cl- omission. The amplification of insulin secretion by glucose was not suppressed. We conclude that an acidic granular pH is important for insulin secretion but that the acute further acidification produced by glucose is not essential for the augmentation of secretion via the amplifying pathway.

The main function of pancreatic β cells is to synthesize and secrete insulin at appropriate rates to limit blood glucose fluctuations within a narrow range. Any alteration in β -cell functioning has a profound impact on glucose homeostasis: excessive secretion of insulin causes hypoglycaemia, and insufficient secretion leads to diabetes. It is therefore not surprising that insulin secretion is subject to very tight control. This control is primarily ensured by glucose itself but also involves an array of metabolic, neural, hormonal and sometimes pharmacological factors (Fig. 1). To integrate all these stimulatory and inhibitory influences, β cells rely on an astonishingly complex stimulus-secretion coupling. This review discusses how the hierarchy between two intracellular pathways, producing triggering and amplifying signals [1], optimizes adequate insulin secretion to changes in blood glucose concentration and enables the β cell to grade the numerous extracellular messages that it receives.

Two sarcoendoplasmic reticulum Ca(2+)-ATPases, SERCA3 and SERCA2b, are expressed in pancreatic islets. Immunocytochemistry showed that SERCA3 is restricted to beta-cells in the mouse pancreas. Control and SERCA3-deficient mice were used to evaluate the role of SERCA3 in beta-cell cytosolic-free Ca(2+) concentration ([Ca(2+)](c)) regulation, insulin secretion, and glucose homeostasis. Basal [Ca(2+)](c) was not increased by SERCA3 ablation. Stimulation with glucose induced a transient drop in basal [Ca(2+)](c) that was suppressed by inhibition of all SERCAs with thapsigargin (TG) but unaffected by selective SERCA3 ablation. Ca(2+) mobilization by acetylcholine was normal in SERCA3-deficient beta-cells. In contrast, [Ca(2+)](c) oscillations resulting from intermittent glucose-stimulated Ca(2+) influx and [Ca(2+)](c) transients induced by pulses of high K(+) were similarly affected by SERCA3 ablation or TG pretreatment of control islets; their amplitude was increased and their slow descending phase suppressed. This suggests that, during the decay of each oscillation, the endoplasmic reticulum releases Ca(2+) that was pumped by SERCA3 during the upstroke phase. SERCA3 ablation increased the insulin response of islets to 15 mmol/l glucose. However, basal and postprandial plasma glucose and insulin concentrations in SERCA3-deficient mice were normal. In conclusion, SERCA2b, but not SERCA3, is involved in basal [Ca(2+)](c) regulation in beta-cells. SERCA3 becomes operative when [Ca(2+)](c) rises and is required for normal [Ca(2+)](c) oscillations in response to glucose. However, a lack of SERCA3 is insufficient in itself to alter glucose homeostasis or impair insulin secretion in mice.

The specific phenotype of mature differentiated beta cells not only depends on the specific presence of genes that allow beta cell function but also on the selective absence of housekeeping genes (“disallowed genes”) that would interfere with this function. Recent studies have shown that both histone modifications and DNA methylation via the de novo methyltransferase DNMT3A are involved in repression of disallowed genes in neonatal beta cells when these cells acquire their mature phenotype. It is unknown, however, if the environmental influence of advanced age, pregnancy and the metabolic stress of high fat diet or diabetes could alter the repression of disallowed genes in beta cells. In the present study, we show that islet disallowed genes—which are also deeply repressed in FACS-purified beta cells—remain deeply repressed in animals of advanced age and in pregnant females. Moreover, the stability of this repression was correlated with strong and stable histone repression marks that persisted in islets isolated from 2 year old mice and with overall high expression of Dnmt3a in islets. Furthermore, repression of disallowed genes was unaffected by the metabolic stress of high fat diet. However, repression of about half of the disallowed genes was weakened in 16 week-old diabetic db/db mice. In conclusion, we show that the disallowed status of islet genes is stable under physiological challenging conditions (advanced age, pregnancy, high fat diet) but partially lost in islets from diabetic animals.

Changes in cytosolic free Ca(2+) concentration ([Ca(2+)]c) play a crucial role in the control of insulin secretion from the electrically excitable pancreatic β-cell. Secretion is controlled by the finely tuned balance between Ca(2+) influx (mainly through voltage-dependent Ca(2+) channels, but also through voltage-independent Ca(2+) channels like store-operated channels) and efflux pathways. Changes in [Ca(2+)]c directly affect [Ca(2+)] in various organelles including the endoplasmic reticulum (ER), mitochondria, the Golgi apparatus, secretory granules and lysosomes, as imaged using recombinant targeted probes. Because most of these organelles have specific Ca(2+) influx and efflux pathways, they mutually influence free [Ca(2+)] in the others. In this article, we review the mechanisms of control of [Ca(2+)] in various compartments and particularly the cytosol, the endoplasmic reticulum ([Ca(2+)]ER), acidic stores and mitochondrial matrix ([Ca(2+)]mito), focusing chiefly on the most important physiological stimulus of β-cells, glucose. We also briefly review some alterations of β-cell Ca(2+) homeostasis in Type 2 diabetes.

Proteins with a short half-life are potential sites of pancreatic ss cell dysfunction under pathophysiological conditions. In this study, mouse islets were used to establish which step in the regulation of insulin secretion is most sensitive to inhibition of protein synthesis by 10 microM cycloheximide (CHX). Although islet protein synthesis was inhibited approximately 95% after 1 h, the inhibition of insulin secretion was delayed and progressive. After long (18-20 h) CHX-treatment, the strong (80%) inhibition of glucose-, tolbutamide-, and K(+)-induced insulin secretion was not due to lower insulin stores, to any marked impairment of glucose metabolism or to altered function of K(+)-ATP channels (total K(+)-ATP currents were however decreased). It was partly caused by a decreased Ca(2+) influx (whole-cell Ca(2+) current) resulting in a smaller rise in cytosolic Ca(2+) ([Ca(2+)](i)). The situation was very different after short (2-5 h) CHX-treatment. Insulin secretion was 50-60% inhibited although islet glucose metabolism was unaffected and stimulus-induced [Ca(2+)](i) rise was not (2 h) or only marginally (5 h) decreased. The efficiency of Ca(2+) on secretion was thus impaired. The inhibition of insulin secretion by 15 h of CHX treatment was more slowly reversible (>4 h) than that of protein synthesis. This reversibility of secretion was largely attributable to recovery of a normal Ca(2+) efficiency. In conclusion, inhibition of protein synthesis in islets inhibits insulin secretion in two stages: a rapid decrease in the efficiency of Ca(2+) on exocytosis, followed by a decrease in the Ca(2+) signal mediated by a slower loss of functional Ca(2+) channels. Glucose metabolism and the regulation of K(+)-ATP channels are more resistant. Proteins with a short half-life appear to be important to ensure optimal Ca(2+) effects on exocytosis, and are the potential Achille's heel of stimulus-secretion coupling.

1. To determine how pretreatment with sulphonylureas alters the beta cell function, mouse islets were cultured (18 - 20 h) without (controls) or with (test) 0.01 microM glibenclamide. Acute responses to glucose were then determined in the absence of glibenclamide. 2. Test islets were insensitive to drugs (sulphonylureas and diazoxide) acting on K+-ATP channels, and their [Ca2+]i was already elevated in the absence of stimulation. 3. Insulin secretion was increased in the absence of glucose, and mainly stimulated between 0 - 10 instead of 7 - 20 mM glucose in controls. The maximum response was halved, but this difference disappeared after correction for the 45% decrease in the islet insulin content. 4. The first phase of glucose-induced insulin secretion was abrogated because of a paradoxical decrease of the high basal [Ca2+]i in beta cells. The second phase was preserved but occurred with little rise of [Ca2+]i. These abnormalities did not result from alterations of glucose metabolism (NADPH fluorescence). 5. In islets cultured with 50 microM tolbutamide, glucose induced biphasic increases in [Ca2+]i and insulin secretion. The decrease in the secretory response was matched by the decrease in insulin content (45%) except at maximal glucose concentrations. Islets pretreated with tolbutamide, however, behaved like those cultured with glibenclamide if tolbutamide was also present during the acute functional tests. 6. In conclusion, treatment with a low glibenclamide concentration causes long-lasting blockade of K+-ATP channels and rise of [Ca2+]i in beta cells. Glucose-induced insulin secretion occurs at lower concentrations, is delayed and is largely mediated by a modulation of Ca2+ action on exocytosis. It is suggested that glucose regulation of insulin secretion mainly depends on a K+-ATP channel-independent pathway during in vivo sulphonylurea treatment.

We studied whether reverse Na(+)-Ca2+ exchange can increase cytoplasmic Ca2+ ([Ca2+]i) in mouse islets and contribute to insulin release. The exchange was stimulated by replacing Na+ with choline, sucrose, or lithium in a medium containing 15 mM glucose. Na+ omission increased electrical activity in B cells, [Ca2+]i, and insulin release. When voltage-dependent Ca2+ channels were blocked by nimodipine or closed by holding the membrane polarized with diazoxide, Na+ omission caused a slight hyperpolarization, a small rise in [Ca2+]i, and a marginal increase in insulin release (the latter only with choline). This small rise in [Ca2+]i was dependent on extracellular Ca2+ but was hardly augmented when intracellular Na+ was raised with alanine. When B cells were depolarized by 30 mM K+, Na+ omission did not affect the membrane potential but increased [Ca2+]i and insulin release. If Ca2+ channels were blocked by nimodipine, only marginal increases in Ca2+ and insulin release persisted, which were not different from those observed when the cells were not depolarized. This indicates that Ca2+ influx through voltage-dependent Ca2+ channels rather than via reverse Na(+)-Ca2+ exchange underlies the rise in [Ca2+]i and in insulin release produced by Na+ removal. No decisive support for Ca2+ influx by reverse Na(+)-Ca2+ exchange could be found.

The effects of nonglucose nutrient insulin secretagogues on cytoplasmic pH (pHi) in pancreatic B cells are unclear. These were studied with intact mouse islets loaded with BCECF and stimulated with ketoisocaproic acid (KIC) or leucine, which, unlike glucose, are exclusively metabolized in mitochondria. The changes in pHi were compared to those in cytoplasmic Ca2+ ([Ca2+]i; islets loaded with fura-2), metabolism [NAD(P)H fluorescence], and insulin release. In HCO3- buffer containing 3 mM glucose, 10 mM KIC produced a rapid and sustained increase in metabolism, [Ca2+]i, pHi, and insulin release. In HEPES buffer, the increase in metabolism, [Ca2+]i, and release were also rapid but not as sustained, whereas the alkalinization was delayed. The changes in release thus follow a time course more similar to that of [Ca2+]i and metabolism than to that of pHi. The role of [Ca2+]i in pHi changes was next examined. A similar rapid rise in pHi was produced by KIC in both HCO3- and HEPES buffers when its effects on [Ca2+]i were prevented, whether [Ca2+]i was kept low (4.8 mM KCl plus diazoxide) or high (30 mM KCl plus diazoxide). When the Na(+):H+ exchanger was blocked by dimethylamiloride, the alkalinizing effect of KIC was unaffected in HCO3- buffer, indicating that it does not result from an activation of this exchanger. In HEPES buffer, however, KIC strongly decreased pHi unless the rise in [Ca2+]i was prevented, in which case KIC increased pHi. When the HCO3-/Cl-exchanger was blocked by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), the effect of KIC in HCO3- buffer became similar to that in HEPES buffer without or with DIDS. The effects of leucine on pHi were similar to those of KIC. In conclusion, the effect of KIC and leucine on islet cell pHi, like that of glucose, is the complex result of an alkalinizing action of their metabolism and an acidifying action of the [Ca2+]i rise that they also produce. Compensation of this acidification is achieved by operation of the pHi-regulating exchangers, of which the HCO3-/Cl- exchanger plays a predominant role.

There exists a good temporal correlation between the oscillations of cytoplasmic Ca2+ ([Ca2+]i) in pancreatic B cells and insulin release. Here, single mouse islets loaded with fura-2 were used to investigate whether there also exists a quantitative correlation between both events during stimulation by different glucose concentrations. The frequency of [Ca2+]i oscillations was decreased by raising the CaCl2 concentration to 10 mM in the perifusion medium (to ensure adequate resolution of insulin oscillations by the immunoassay), and the glucose concentration was increased from 10 to 20 or from 15 to 30 mM. Raising the glucose level was followed by changes in [Ca2+]i oscillations. Their duration increased slightly, and that of the intervals decreased, whereas the nadir between the oscillations became less deep. On the other hand, the peak of [Ca2+]i oscillations did not change. As a result, the rhythm was accelerated, and the average [Ca2+]i was increased. The concomitant increase in insulin release resulted from similar changes, with, in addition, a marked increase in the peak of insulin oscillations. In two thirds of the islets, variations in the amplitude of successive [Ca2+]i oscillations occurred during stimulation with 10 mM glucose and disappeared at higher glucose levels. This was due to temporal variations in the responsiveness of all regions of the islet, rather than to the existence of nonresponsive regions that would be recruited into an active state by high glucose. In conclusion, there exists a good temporal correlation between insulin and [Ca2+]i oscillations in islets stimulated by various glucose concentrations. The quantitative correlation is not as close, indicating that the relationship between the two phenomena is nonlinear and supporting previous evidence that glucose also increases the efficacy of Ca2+ on secretion. This mechanism, rather than the development of a Ca2+ rise in nonresponsive cells, might underlie B cell recruitment in intact islets.

Pancreatic β cells respond to increases in glucose concentration with enhanced metabolism, the closure of ATP-sensitive K+ channels and electrical spiking. The latter results in oscillatory Ca2+ influx through voltage-gated Ca2+ channels and the activation of insulin release. The relationship between changes in cytosolic and mitochondrial free calcium concentration ([Ca2+]cyt and [Ca2+]mit, respectively) during these cycles is poorly understood. Importantly, the activation of Ca2+-sensitive intramitochondrial dehydrogenases, occurring alongside the stimulation of ATP consumption required for Ca2+ pumping and other processes, may exert complex effects on cytosolic ATP/ADP ratios and hence insulin secretion. To explore the relationship between these parameters in single primary β cells, we have deployed cytosolic (Fura red, Indo1) or green fluorescent protein-based recombinant-targeted (Pericam, 2mt8RP for mitochondria; D4ER for the ER) probes for Ca2+ and cytosolic ATP/ADP (Perceval) alongside patch-clamp electrophysiology. We demonstrate that: (1) blockade of mitochondrial Ca2+ uptake by shRNA-mediated silencing of the uniporter MCU attenuates glucose- and essentially blocks tolbutamide-stimulated, insulin secretion; (2) during electrical stimulation, mitochondria decode cytosolic Ca2+ oscillation frequency as stable increases in [Ca2+]mit and cytosolic ATP/ADP; (3) mitochondrial Ca2+ uptake rates remained constant between individual spikes, arguing against activity-dependent regulation (“plasticity”) and (4) the relationship between [Ca2+]cyt and [Ca2+]mit is essentially unaffected by changes in endoplasmic reticulum Ca2+ ([Ca2+]ER). Our findings thus highlight new aspects of Ca2+ signalling in β cells of relevance to the actions of both glucose and sulphonylureas. Electronic supplementary material The online version of this article (doi:10.1007/s00424-012-1177-9) contains supplementary material, which is available to authorized users.

Glucose induces insulin release from pancreatic β-cells by stimulating ATP synthesis, membrane depolarisation and Ca(2+) influx. As well as activating ATP-consuming processes, cytosolic Ca(2+) increases may also potentiate mitochondrial ATP synthesis. Until recently, the ability to study the role of mitochondrial Ca(2+) transport in glucose-stimulated insulin secretion has been hindered by the absence of suitable approaches either to suppress Ca(2+) uptake into these organelles, or to examine the impact on β-cell excitability. Here, we have combined patch-clamp electrophysiology with simultaneous real-time imaging of compartmentalised changes in Ca(2+) and ATP/ADP ratio in single primary mouse β-cells, using recombinant targeted (Pericam or Perceval, respectively) as well as entrapped intracellular (Fura-Red), probes. Through shRNA-mediated silencing we show that the recently-identified mitochondrial Ca(2+) uniporter, MCU, is required for depolarisation-induced mitochondrial Ca(2+) increases, and for a sustained increase in cytosolic ATP/ADP ratio. By contrast, silencing of the mitochondrial Na(+)-Ca(2+) exchanger NCLX affected the kinetics of glucose-induced changes in, but not steady state values of, cytosolic ATP/ADP. Exposure to gluco-lipotoxic conditions delayed both mitochondrial Ca(2+) uptake and cytosolic ATP/ADP ratio increases without affecting the expression of either gene. Mitochondrial Ca(2+) accumulation, mediated by MCU and modulated by NCLX, is thus required for normal glucose sensing by pancreatic β-cells, and becomes defective in conditions mimicking the diabetic milieu.

It is often assumed, but has not been demonstrated, that the oscillations of cytoplasmic calcium (Ca2+i) that occur in various secretory cells induce oscillations in secretion. Here, we have used single pancreatic islets from normal mice to monitor simultaneously insulin release and Ca2+i in beta-cells for periods up to 25 min. Ca2+i and insulin secretion were found to oscillate in synchrony during stimulation by glucose. This synchrony persisted when the frequency of both events changed spontaneously or upon addition of the hypoglycaemic sulfonylurea tolbutamide, or when their relative amplitudes varied. Repolarizing the beta-cell membrane with diazoxide abolished both Ca2+i and insulin oscillations. In contrast, epinephrine suppressed insulin oscillations in spite of the persistence of Ca2+i oscillations. This study is the first direct demonstration that, in an intact organ, sustained oscillations of Ca2+i induced by a physiological stimulus entrain synchronous oscillations of the functional response, and that both events can vary simultaneously or be dissociated depending on the experimental conditions.

Glucose homeostasis is critically dependent on insulin release from pancreatic β-cells, which is strictly regulated by glucose-induced oscillations in membrane potential (V m ) and the cytosolic calcium level ([Ca 2+ ] cyt ). We propose that TRPM5, a Ca 2+ -activated monovalent cation channel, is a positive regulator of glucose-induced insulin release. Immunofluorescence revealed expression of TRPM5 in pancreatic islets. A Ca 2+ -activated nonselective cation current with TRPM5-like properties is significantly reduced in Trpm 5 −/− cells. Ca 2+ -imaging and electrophysiological analysis show that glucose-induced oscillations of V m and [Ca 2+ ] cyt have on average a reduced frequency in Trpm5 −/− islets, specifically due to a lack of fast oscillations. As a consequence, glucose-induced insulin release from Trpm5 −/− pancreatic islets is significantly reduced, resulting in an impaired glucose tolerance in Trpm5 −/− mice.

Glucose homeostasis is critically dependent on insulin release from pancreatic beta cells, which is strictly regulated by glucose-induced simultaneously oscillations in membrane potential (Vm) and cytosolic calcium concentrations [Ca2+]cyt. We propose that TRPM5, a Ca2+-activated monovalent cation channel, is a positive regulator of glucose-induced insulin release. Micro-array screening and immunostaining reveal high and selective expression of TRPM5 in pancreatic islets. Whole cell current measurements in WT pancreatic islet cells demonstrate a Ca2+-activated non-selective cation current with properties comparable to TRPM5 measured in over-expression, including the bell-shaped dependency on intracellular Ca2+, time constant of activation and permeation properties. This current is significantly reduced in Trpm5-/- cells. Ca2+-imaging and electrophysiological analysis show that glucose-induced oscillations of Vm and [Ca2+]cyt have a reduced frequency in Trpm5-/- islets. WT islets display either slow or fast oscillations, or mixed oscillations, consisting of fast oscillations superimposed on slow ones. In contrast, Trpm5-/- islets never show a fast oscillation pattern. Fast oscillations in Vm show a shorter burst interval, due to a higher slope of depolarization towards the threshold potential for burst initiation. Our results indicate that TRPM5 accelerates the depolarization during the interburst interval, initiating rapid oscillations and higher insulin release. As a consequence, glucose-induced insulin release from Trpm5-/- pancreatic isletsis significantly reduced, resulting in an impaired glucose tolerance in these mice. Pharmacological modulation of TRPM5 activity may represent a novel means to adjust insulin release in diabetic patients.

Due to their high glucose permeability, insulin-secreting pancreatic β-cells likely undergo strong intracellular protein glycation at high glucose concentrations. They may, however, be partly protected from the glucotoxic alterations of their survival and function by fructosamine-3-kinase (FN3K), a ubiquitous enzyme that initiates deglycation of intracellular proteins. To test that hypothesis, we cultured pancreatic islets from Fn3k-knockout ( Fn3k−/−) mice and their wild-type (WT) littermates for 1–3 wk in the presence of 10 or 30 mmol/l glucose (G10 or G30, respectively) and measured protein glycation, apoptosis, preproinsulin gene expression, and Ca2+and insulin secretory responses to acute glucose stimulation. The more potent glycating agent d-ribose (25 mmol/l) was used as positive control for protein glycation. In WT islets, a 1-wk culture in G30 significantly increased the amount of soluble intracellular protein-bound fructose-ε-lysines and the glucose sensitivity of β-cells for changes in Ca2+and insulin secretion, whereas it decreased the islet insulin content. After 3 wk, culture in G30 also strongly decreased β-cell glucose responsiveness and preproinsulin mRNA levels, whereas it increased islet cell apoptosis. Although protein-bound fructose-ε-lysines were more abundant in Fn3k−/−vs. WT islets, islet cell survival and function and their glucotoxic alterations were almost identical in both types of islets, except for a lower level of apoptosis in Fn3k−/−islets cultured for 3 wk in G30. In comparison, d-ribose (1 wk) similarly decreased preproinsulin expression and β-cell glucose responsiveness in both types of islets, whereas it increased apoptosis to a larger extent in Fn3k−/−vs. WT islets. We conclude that, despite its ability to reduce the glycation of intracellular islet proteins, FN3K is neither required for the maintenance of β-cell survival and function under control conditions nor involved in protection against β-cell glucotoxicity. The latter, therefore, occurs independently from the associated increase in the level of intracellular fructose-ε-lysines.

Glucose-induced insulin secretion requires a rise in beta-cell cytosolic Ca2+ ([Ca2+]c) that triggers exocytosis and a mechanistically unexplained amplification of the action of [Ca2+]c. Insulin granules are kept acidic by luminal pumping of protons with simultaneous Cl- uptake to maintain electroneutrality. Experiments using patched, dialyzed beta-cells prompted the suggestion that acute granule acidification by glucose underlies amplification of insulin secretion. However, others found glucose to increase granular pH in intact islets. In this study, we measured islet granular pH with Lysosensor DND-160, a fluorescent dye that permits ratiometric determination of pH6 in acidic compartments. Stimulation of mouse islets with glucose reversibly decreased granular pH by mechanisms that are dependent on metabolism and Cl- ions but independent of changes in [Ca2+]c and protein kinase A or C activity. Granular pH was increased by concanamycin (blocker of the vesicular type H+-ATPase)methylamine (weak base)Cl- omission. Concanamycin and methylamine did not alter glucose-induced [Ca2+]c increase in islets but strongly inhibited the two phases of insulin secretion. Omission of Cl- did not affect the first phase but decreased the second phase of both [Ca2+]c and insulin responses. Neither experimental condition affected the [Ca2+]c rise induced by 30 mM KCl, but the insulin responses were inhibited by concanamycinmethylamine and not affected by Cl- omission. The amplification of insulin secretion by glucose was not suppressed. We conclude that an acidic granular pH is important for insulin secretion but that the acute further acidification produced by glucose is not essential for the augmentation of secretion via the amplifying pathway.

Stimulatory concentrations of glucose induce two patterns of cytosolic Ca2+concentration ([Ca2+]c) oscillations in mouse islets: simple or mixed. In the mixed pattern, rapid oscillations are superimposed on slow ones. In the present study, we examined the role of the membrane potential in the mixed pattern and the impact of this pattern on insulin release. Simultaneous measurement of [Ca2+]cand insulin release from single islets revealed that mixed [Ca2+]coscillations triggered synchronous oscillations of insulin secretion. Simultaneous recordings of membrane potential in a single β-cell within an islet and of [Ca2+]cin the whole islet demonstrated that the mixed pattern resulted from compound bursting (i.e., clusters of membrane potential oscillations separated by prolonged silent intervals) that was synchronized in most β-cells of the islet. Each slow [Ca2+]cincrease during mixed oscillations was due to a progressive summation of rapid oscillations. Digital image analysis confirmed the good synchrony between subregions of an islet. By contrast, islets from sarco(endo)plasmic reticulum Ca2+-ATPase isoform 3 (SERCA3)-knockout mice did not display typical mixed [Ca2+]coscillations in response to glucose. This results from a lack of progressive summation of rapid oscillations and from altered spontaneous electrical activity, i.e., lack of compound bursting, and membrane potential oscillations characterized by lower-frequency but larger-depolarization phases than observed in SERCA3+/+β-cells. We conclude that glucose-induced mixed [Ca2+]coscillations result from compound bursting in all β-cells of the islet. Disruption of SERCA3 abolishes mixed [Ca2+]coscillations and augments β-cell depolarization. This latter observation indicates that the endoplasmic reticulum participates in the control of the β-cell membrane potential during glucose stimulation.

Chronic hyperglycemia has been shown to induce either a lack of response or an increased sensitivity to glucose in pancreatic β-cells. We reinvestigated this controversial issue in a single experimental model by culturing rat islets for 1 wk in 10 or 30 mmol/l glucose (G10, Controls; or G30, High-glucose islets) before testing the effect of stepwise glucose stimulation from G0.5 to G20 on key β-cell stimulus-secretion coupling events. Compared with Controls, the glucose sensitivity of High-glucose islets was markedly increased, leading to maximal stimulation of oxidative metabolism and both triggering and amplifying pathways of insulin secretion in G6 rather than G20, hence to loss of glucose effect above G6. This enhanced glucose sensitivity occurred despite an approximately twofold increase in islet uncoupling protein 2 mRNA expression. Besides this increased glucose sensitivity, the maximal glucose stimulation of insulin secretion in High-glucose islets was reduced by ∼50%, proportionally to the reduction of insulin content. In High-glucose islets, changes in45Ca2+influx induced by glucose and diazoxide were qualitatively similar but quantitatively smaller than in Control islets and, paradoxically, did not lead to detectable changes in the intracellular Ca2+concentration measured by microspectrofluorimetry (fura PE 3). In conclusion, after 1 wk of culture in G30, the loss of glucose stimulation of insulin secretion in the physiological range of glucose concentrations (G5–G10) results from the combination of an increased sensitivity to glucose of both triggering and amplifying pathways of insulin secretion and an ∼50% reduction in the maximal glucose stimulation of insulin secretion.

Acetylcholine (ACh), the major parasympathetic neurotransmitter, is released by intrapancreatic nerve endings during the preabsorptive and absorptive phases of feeding. In beta-cells, ACh binds to muscarinic M(3) receptors and exerts complex effects, which culminate in an increase of glucose (nutrient)-induced insulin secretion. Activation of PLC generates diacylglycerol. Activation of PLA(2) produces arachidonic acid and lysophosphatidylcholine. These phospholipid-derived messengers, particularly diacylglycerol, activate PKC, thereby increasing the efficiency of free cytosolic Ca(2+) concentration ([Ca(2+)](c)) on exocytosis of insulin granules. IP3, also produced by PLC, causes a rapid elevation of [Ca(2+)](c) by mobilizing Ca(2+) from the endoplasmic reticulum; the resulting fall in Ca(2+) in the organelle produces a small capacitative Ca(2+) entry. ACh also depolarizes the plasma membrane of beta-cells by a Na(+)- dependent mechanism. When the plasma membrane is already depolarized by secretagogues such as glucose, this additional depolarization induces a sustained increase in [Ca(2+)](c). Surprisingly, ACh can also inhibit voltage-dependent Ca(2+) channels and stimulate Ca(2+) efflux when [Ca(2+)](c) is elevated. However, under physiological conditions, the net effect of ACh on [Ca(2+)](c) is always positive. The insulinotropic effect of ACh results from two mechanisms: one involves a rise in [Ca(2+)](c) and the other involves a marked, PKC-mediated increase in the efficiency of Ca(2+) on exocytosis. The paper also discusses the mechanisms explaining the glucose dependence of the effects of ACh on insulin release.

The literature on ion channels is extensive; searching PubMed with the term ‘ion channel’ results in close to 120,000 hits. Few of us have the energy—let alone the time—to keep up. However, should you wish to know everything about one functionally important channel, then read on; here might be the channel just for you! The Na+ leak channel, non‐selective (NALCN) was first cloned in 1999 (Lee et al , 1999) but its true nature was not unveiled until 2007. To date, the entire literature on NALCN comprises fewer than ten papers, even when one considers its orthologues in Drosophila melanogaster and Caenorhabditis elegans . NALCN is a member of the 4 × 6TM family of ion channels, which comprise four homologous domains, each with six transmembrane (TM) segments. The family is encoded by 21 genes and includes voltage‐gated Na+‐selective and Ca2+‐selective ion channels (Lu et al , 2007) that have crucial roles in almost every excitable cell of the body. Nalcn encodes a protein that has fewer positive charges in the voltage‐sensing S4 segment than other members of the 4 × 6TM family and that is responsible for a voltage‐independent and tetrodotoxin (TTX)‐resistant non‐selective Na+ ‘leak’ current. The physiological significance of NALCN was highlighted by the finding that mice lacking the channel have abnormal respiratory rhythm and die within 24 h of birth. In addition, hippocampal neurons isolated from Nalcn knockout mice have reduced excitability. It seems that NALCN contributes to—but is almost certainly not the only channel responsible for—the elusive background leakage current often referred to as I L, which was first postulated by Hodgkin & Huxley (1952) but has been difficult to identify due to the lack of selective antagonists. A recent study revealed that …

Glucose-induced insulin secretion depends on an acceleration of glucose metabolism, requires a rise in the cytoplasmic free Ca2+ concentration ([Ca2+]i), and is modulated by activation of protein kinases in beta-cells. Normal mouse islets were used to determine whether oscillations of these three signals are able and necessary to trigger oscillations of insulin secretion. The approach was to minimize or abolish spontaneous oscillations and to compare the impact of forced oscillations of each signal on insulin secretion. In a control medium, repetitive increases in the glucose concentration triggered oscillations in metabolism [NAD(P)H fluorescence], [Ca2+]i (fura-PE3 method), and insulin secretion. In the presence of diazoxide, metabolic oscillations persisted, but [Ca2+]i and insulin oscillations were abolished. When the islets were depolarized with high K+ with or without diazoxide, [Ca2+]i was elevated, and insulin secretion was stimulated. Forced metabolic oscillations transiently decreased or did not affect [Ca2+]i and potentiated insulin secretion with oscillations of small amplitude. These oscillations of secretion followed metabolic oscillations only when [Ca2+]i did not change. When [Ca2+]i fluctuated, these changes prevailed over those of metabolism for timing secretion. Repetitive depolarizations with high K+ in the presence of stable glucose (10 mmol/l) induced synchronous pulses of [Ca2+]i and insulin secretion with only small oscillations of metabolism. Continuous stimulation of protein kinase A (PKA) and protein kinase C (PKC) did not dissociate the [Ca2+]i and insulin pulses from the high K+ pulses. However, the amplitude of the insulin pulses was consistently increased, whereas that of the [Ca2+]i pulses was either increased (PKA) or decreased (PKC). In conclusion, metabolic oscillations can induce oscillations of insulin secretion independently of but with a lesser effectiveness than [Ca2+]i oscillations. Although oscillations in metabolism may cyclically influence secretion through an ATP-sensitive K+ channel (K+-ATP channel)-independent pathway, their regulatory effects are characterized by a hysteresis that makes them unlikely drivers of fast oscillations, unless they also involve [Ca2+]i changes through the K+-ATP channel-dependent pathway.

In mouse pancreatic beta-cells, extracellular ATP (0.1 mmol/l) effectively reduced glucose-induced insulin secretion. This inhibitory action resulted from a direct interference with the secretory machinery, and ATP suppressed depolarization-induced exocytosis by 60% as revealed by high-resolution capacitance measurements. Suppression of Ca2+-dependent exocytosis was mediated via binding to P2Y1 purinoceptors but was not associated with inhibition of the voltage-dependent Ca2+ currents or adenylate cyclase activity. Inhibition of exocytosis by ATP resulted from G-protein-dependent activation of the serine/threonine protein phosphatase calcineurin and was abolished by cyclosporin A and deltamethrin. In contrast to the direct inhibitory action on exocytosis, ATP reduced the whole-cell ATP-sensitive K+ (K(ATP)) current by 30% (via activation of cytosolic phospholipase A2), leading to membrane depolarization and stimulation of electrical activity. The stimulatory effect of ATP also involved mobilization of Ca2+ from thapsigargin-sensitive intracellular stores. We propose that the inhibitory action of ATP, by interacting with the secretory machinery at a level downstream to an elevation in [Ca2+]i, is important for autocrine regulation of insulin secretion in mouse beta-cells.

Defects in the regulation of insulin secretion from pancreatic β-cells largely contribute to the perturbations of glucose homeostasis in noninsulin-dependent diabetes (Type 2 diabetes). Under physiological conditions, the rate of insulin release is controlled by variations in the concentration of circulating nutrients and by various hormonal and neural signals. Hormones and neurotransmitters modulate β-cell function by binding to membrane receptors and activating transduction pathways essentially similar to those existing in other cell types. In contrast, glucose, the major physiological stimulus, does not bind to a receptor but must be metabolized to induce insulin secretion. The acceleration of β-cell metabolism results in the production of a number of potential second messengers that might trigger or modulate exocytosis of storage granules containing insulin. Another peculiarity of β-cells is not so much that they are electrically excitable, but that their membrane potential is controlled by metabolism. The electrical activity induced by glucose involves marked changes in the flux of many ions across the plasma membrane. One of these ions, Ca2+, plays a pre-eminent role in the regulation of insulin secretion. The first indication that Ca2+ ions are essential players in stimulus-secretion coupling in β-cells dates back to the observation that the stimulation of insulin secretion by glucose and other agents was abrogated by omission of extracellular Ca2+.

1-Cells from rodents and humans express different receptors recognizing hormones of the secretin-glucagon family, which--when activated--synergize with glucose in the control of insulin release. We have recently reported that isolated islets from mice homozygous for a GLP-1 receptor null mutation (GLP-1R(-/-)) exhibit a well-preserved insulin-secretory response to glucose. This observation can be interpreted in two different ways: 1) the presence of GLP-1R is not essential for the secretory response of isolated islets to glucose alone; 2) beta-cells in GLP-1R(-/-) pancreases underwent compensatory changes in response to the null mutation. To explore these possibilities, we studied islets from control GLP-IR(+/+) mice in the absence or presence of 1 pmol/l exendin (9-39)amide, a specific and potent GLP-1R antagonist. Exendin (9-39)amide (15-min exposure) reduced glucose-induced insulin secretion from both perifused and statically incubated GLP-1R(+/+) islets by 50% (P < 0.05), and reduced islet cAMP production in parallel (P < 0.001). Furthermore, GLP-1R(-/-) islets exhibited: 1) reduced cAMP accumulation in the presence of 20 mmol/l glucose (knockout islets versus control islets, 12 +/- 1 vs. 27 +/- 3 fmol x islet(-1) x 15 min(-1); P < 0.001) and exaggerated acceleration of cAMP production by 10 nmol/l glucose-dependent insulinotropic peptide (GIP) (increase over 20 mmol/l glucose by GIP in knockout islets versus control islets: 66 +/- 5 vs. 14 +/- 3 fmol x islet(-1) x 15 min(-1); P < 0.001); 2) increased mean cytosolic [Ca2+] ([Ca2+]c) at 7, 10, and 15 mmol/l glucose in knockout islets versus control islets; and 3) signs of asynchrony of [Ca2+]c oscillations between different islet subregions. In conclusion, disruption of GLP-1R signaling is associated with reduced basal but enhanced GIP-stimulated cAMP production and abnormalities in basal and glucose-stimulated [Ca2+]c. These abnormalities suggest that GLP-1R signaling is an essential upstream component of multiple beta-cell signaling pathways.

In pancreatic β cells, the increase in the ATP/ADP ratio that follows a stimulation by glucose is thought to play an important role in the Ca2+-dependent increase in insulin secretion. Here we have investigated the possible interactions between Ca2+ and adenine nucleotides in mouse islets. Measurements of both parameters in the same single islet showed that the rise in the ATP/ADP ratio precedes any rise in the cytoplasmic free-Ca2+ concentration ([Ca2+]i) and is already present during the initial transient lowering of [Ca2+]i produced by the sugar. Blockade of Ca2+ influx with nimodipine did not prevent the concentration-dependent increase in the ATP/ADP ratio produced by glucose and even augmented the ratio at all glucose concentrations which normally stimulate Ca2+ influx. In contrast, stimulation of Ca2+ influx by 30 mM K+ or 100 µM tolbutamide lowered the ATP/ADP ratio. This lowering was of rapid onset and reversibility, sustained and prevented by nimodipine or omission of extracellular Ca2+. It was, however, not attenuated after blockade of secretion by activation of α2-adrenoceptors. The difference in islet ATP/ADP ratio during blockade and stimulation of Ca2+ influx was similar to that observed between threshold and submaximal glucose concentrations. The results suggest that the following feedback loop could control the oscillations of membrane potential and [Ca2+]i in β cells. Glucose metabolism increases the ATP/ADP ratio in a Ca2+-independent manner, which leads to closure of ATP-sensitive K+ channels, depolarization and stimulation of Ca2+ influx. The resulting increase in [Ca2+]i causes a larger consumption than production of ATP, which induces reopening of ATP-sensitive K+ channels and arrest of Ca2+ influx. Upon lowering of [Ca2+]i the ATP/ADP ratio increases again and a new cycle may start.

Sulfonylureas stimulate insulin secretion by blocking ATP-sensitive K+ channels (K+-ATP channels) of the beta-cell membrane, thereby causing depolarization, Ca2+ influx, and rise in cytoplasmic Ca2+ concentration ([Ca2+]i), whereas diazoxide inhibits insulin secretion by opening K+-ATP channels. It has been suggested recently that these drugs also respectively increase and decrease the efficacy of Ca2+ on exocytosis. This hypothesis was tested here with intact islets or single beta-cells from normal mice. Depolarizing islet cells by raising extracellular K+ from 4.8 to 15, 30, and 60 mmol/l progressively raised [Ca2+]i and stimulated insulin secretion. The magnitude of the [Ca2+]i rise produced by a subsequent addition of 100 micromol/l tolbutamide decreased as the concentration of K+ was increased. The effect on insulin secretion paralleled that on [Ca2+]i. Similarly, the magnitudes of the [Ca2+]i drop and of the inhibition of insulin secretion produced by 250 micromol/l diazoxide were inversely related to the concentration of K+. Either drug was effective on secretion only when it increased or decreased [Ca2+]i. Exocytosis of insulin granules from single, voltage-clamped beta-cells was also studied by measuring cell capacitance changes. In the perforated patch configuration, exocytosis was evoked by depolarizing pulses. Addition of tolbutamide to the extracellular medium did not affect the Ca2+ current and the resulting change in cell capacitance. In the whole-cell configuration, cell capacitance increased with the concentration of free Ca2+ in the solution diffusing from the pipette into the cell. It was markedly potentiated by cAMP, was inhibited by activation of alpha2-adrenoceptors with clonidine, and was strongly augmented by acetylcholine. In contrast, tolbutamide was ineffective whether applied intra- or extracellularly, at low or high free Ca2+, and with or without cAMP. Diazoxide also failed to interfere directly with exocytosis. These results indicate that tolbutamide and diazoxide affect insulin secretion by changing the concentration, not the action, of Ca2+ in beta-cells.

1. Phosphorylation by kinases and dephosphorylation by phosphatases markedly affect the biological activity of proteins involved in stimulus-response coupling. In this study, we have characterized the effects of okadaic acid, an inhibitor of protein phosphatases 1 and 2A, on insulin secretion. Mouse pancreatic islets were preincubated for 60 min in the presence of okadaic acid before their function was studied. 2. Okadaic acid dose-dependently (IC50 approximately 200 nM) inhibited insulin secretion induced by 15 mM glucose. At 0.5 microM, okadaic acid also inhibited insulin secretion induced by tolbutamide, ketoisocaproate and high K+, and its effects were not reversed by activation of protein kinases A or C. 3. The inhibition of insulin secretion did not result from an alteration of glucose metabolism (estimated by the fluorescence of endogenous pyridine nucleotides) or a lowering of the ATP/ADP ratio in the islets. 4. Okadaic acid treatment slightly inhibited voltage-dependent Ca2+ currents in beta cells (perforated patch technique), which diminished the rise in cytoplasmic Ca2+ (fura-2 method) that glucose and high K+ produce in islets. However, this decrease (25%), was insufficient to explain the corresponding inhibition of insulin secretion (90%). Moreover, mobilization of intracellular Ca2+ by acetylcholine was barely affected by okadaic acid, whereas the concomitant insulin response was decreased by 85%. 5. Calyculin A, another inhibitor of protein phosphatases 1 and 2A largely mimicked the effects of okadaic acid, whereas 1-norokadaone, an inactive analogue of okadaic acid on phosphatases, did not alter beta cell function. 6. In conclusion, okadaic acid inhibits insulin secretion by decreasing the magnitude of the Ca2+ signal in beta cells and its efficacy on exocytosis. The results suggest that, contrary to current concepts, both phosphorylation and dephosphorylation of certain beta cell proteins may be involved in the regulation of insulin secretion.

The direct effects of glucocorticoids on pancreatic beta cell function were studied with normal mouse islets. Dexamethasone inhibited insulin secretion from cultured islets in a concentration-dependent manner: maximum of approximately 75% at 250 nM and IC50 at approximately 20 nM dexamethasone. This inhibition was of slow onset (0, 20, and 40% after 1, 2, and 3 h) and only slowly reversible. It was prevented by a blocker of nuclear glucocorticoid receptors, by pertussis toxin, by a phorbol ester, and by dibutyryl cAMP, but was unaffected by an increase in the fuel content of the culture medium. Dexamethasone treatment did not affect islet cAMP levels but slightly reduced inositol phosphate formation. After 18 h of culture with or without 1 microM dexamethasone, the islets were perifused and stimulated by a rise in the glucose concentration from 3 to 15 mM. Both phases of insulin secretion were similarly decreased in dexamethasone-treated islets as compared with control islets. This inhibition could not be ascribed to a lowering of insulin stores (higher in dexamethasone-treated islets), to an alteration of glucose metabolism (glucose oxidation and NAD(P)H changes were unaffected), or to a lesser rise of cytoplasmic Ca2+ in beta cells (only the frequency of the oscillations was modified). Dexamethasone also inhibited insulin secretion induced by arginine, tolbutamide, or high K+. In this case also the inhibition was observed despite a normal rise of cytoplasmic Ca2+. In conclusion, dexamethasone inhibits insulin secretion through a genomic action in beta cells that leads to a decrease in the efficacy of cytoplasmic Ca2+ on the exocytotic process.

The following sequence of events is thought to underlie the stimulation of insulin release by hypoglycaemic sulphonylureas. Interaction of the drugs with a high-affinity binding site (sulphonylurea receptor) in the B-cell membrane leads to closure of ATP-sensitive K+ channels, depolarization, opening of voltage-dependent Ca2+ channels, Ca2+ influx and rise in cytoplasmic [Ca2+]i. Recent experiments using permeabilized islet cells or measuring changes in B-cell membrane capacitance have suggested that sulphonylureas can increase insulin release by a mechanism independent of a change in [Ca2+]i. This provocative hypothesis was tested here with intact mouse islets. When B-cells were strongly depolarized by 60 mM K+, [Ca2+]i was increased and insulin secretion stimulated. Under these conditions, tolbutamide did not further increase [Ca2+]i or insulin release, whether it was applied before or after high K+, and whether the concentration of glucose was 3 or 15 mM. This contrasts with the ability of forskolin and phorbol 12-myristate 13-acetate (PMA) to increase release in the presence of high K+. Tolbutamide also failed to increase insulin release from islets depolarized with barium (substituted for extracellular Ca2+) or with arginine in the presence of high glucose. Glibenclamide and its non-sulphonylurea moiety meglitinide were also without effect on insulin release from already depolarized B-cells. In the absence of extracellular Ca2+, acetylcholine induced monophasic peaks of [Ca2+]i and insulin secretion which were both unaffected by tolbutamide. Insulin release from permeabilized islet cells was stimulated by raising free Ca2+ (between 0.1 and 23 microM). This effect was not affected by tolbutamide and inconsistently increased by glibenclamide. In conclusion, the present study does not support the proposal that hypoglycaemic sulphonylureas can increase insulin release even when they do not also raise [Ca2+]i in B-cells.

The role of protein kinase-C (PKC) in the potentiation of insulin release by arginine vasopressin (AVP) and acetylcholine (ACh) was investigated with normal mouse islets. The islets were submitted to a short term (30-min) or long term (22-h) treatment with phorbol 12-myristate 13-acetate (PMA) to stimulate acutely or down-regulate PKC before being stimulated by AVP or ACh. Control islets were treated with the inactive 4 alpha-phorbol 12,13-didecanoate. In the presence of 15 mM glucose and 2.5 mM Ca2+, AVP and ACh stimulated inositol phosphate (IP) formation, increased cytoplasmic Ca2+ (Cai2+), and potentiated insulin release. These effects were greater with ACh than with AVP, in particular on Cai2+, which was scarcely affected by AVP. In the absence of extracellular Ca2+, only ACh induced a short-lived increase in Cai2+ and insulin. Acute treatment with PMA in the presence of extracellular Ca2+ strongly increased insulin release in spite of a marked lowering of Cai2+. Under these conditions, the effects of AVP and ACh on IP production and Cai2+ were practically abolished, and only ACh transiently increased insulin release. In the absence of Ca2+, the small mobilization of Cai2+ by ACh triggered a peak of insulin, whereas a similar mobilization of Cai2+ by AVP was ineffective on insulin. After long term treatment of the islets with PMA, AVP normally increased IP formation, but did not affect insulin release. The effect of ACh on IP was still inhibited. However, ACh produced a marked transient increase in Cai2+, with a small transient release of insulin. The releasing effect of ACh was also inhibited in the absence of Ca2+. In conclusion, PKC plays a dual role in the B-cell responses to ACh and AVP. Its activation is necessary for the sustained potentiation of insulin release that both agents produce. This effect probably results from a sensitization of the secretory machinery to Cai2+, the triggering signal. PKC also exerts a negative feedback control on the signal transduction mechanisms involving phospholipase-C, but the ACh and AVP responses are not equally affected by this feedback.

The mechanisms by which fatty acids increase insulin release are not known. In this study, mouse islets were used as a model and palmitate as a reference compound to study in vitro how saturated fatty acids influence pancreatic β-cells. Palmitate (625 μM) was bound to albumin. It did not affect basal insulin release (3 mM glucose) but increased the release induced by 10–15 mM glucose. This effect was dependent on the concentration of free rather than total palmitate. It was reversible and abolished by epinephrine, diazoxide, nimodipine, or omission of extracellular Ca. Bromopalmitate and methyl palmoxirate, two inhibitors of fatty acid oxidation, were ineffective alone, and only bromopalmitate partially inhibited the effects of palmitate on insulin release. The increase in insulin release produced by palmitate could not be ascribed to a blockade of ATP-sensitive K+-channels because the fatty acid only barely decreased 86Rb efflux and did not depolarize β-cells in 3 mM glucose. The small effect on 86Rb efflux might be attributed to a slight rise in the ATP/ADP ratio. No such rise occurred when palmitate was tested in 15 mM glucose, and the fatty acid consistently accelerated 86Rb efflux under these conditions. Measurements of β-cell membrane potential (intracellular microelectrodes) and of free cytoplasmic calcium (Cai 2+) in β-cells (Fura 2 technique) showed that palmitate increases Ca2+ influx; it also caused a very small mobilization of intracellular Ca. The persistence of this stimulation of Ca2+ influx in the presence of diazoxide and high K+ suggests that palmitate might act on Ca2+ channels. The rise in Ca12+ produced by palmitate was accompanied by an increase in insulin release only if the concentration of glucose was sufficiently high. The β-cell response to palmitate thus differs from the responses to glucose and other metabolized nutrients in several respects. Saturated fatty acids appear to potentiate insulin release through an increase in Ca12+ and another, yet unidentified, fuel-dependent mechanism.