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Pancreatic islets are complex micro-organs consisting of at least three different cell types: glucagon-secreting α-, insulin-producing β- and somatostatin-releasing δ-cells1. Somatostatin is a powerful paracrine inhibitor of insulin and glucagon secretion2. In diabetes, increased somatostatinergic signalling leads to defective counter-regulatory glucagon secretion3. This increases the risk of severe hypoglycaemia, a dangerous complication of insulin therapy4. The regulation of somatostatin secretion involves both intrinsic and paracrine mechanisms5 but their relative contributions and whether they interact remains unclear. Here we show that dapagliflozin-sensitive glucose- and insulin-dependent sodium uptake stimulates somatostatin secretion by elevating the cytoplasmic Na+ concentration ([Na+]i) and promoting intracellular Ca2+-induced Ca2+ release (CICR). This mechanism also becomes activated when [Na+]i is elevated following the inhibition of the plasmalemmal Na+-K+ pump by reductions of the extracellular K+ concentration emulating those produced by exogenous insulin in vivo6. Islets from some donors with type-2 diabetes hypersecrete somatostatin, leading to suppression of glucagon secretion that can be alleviated by a somatostatin receptor antagonist. Our data highlight the role of Na+ as an intracellular second messenger, illustrate the significance of the intraislet paracrine network and provide a mechanistic framework for pharmacological correction of the hormone secretion defects associated with diabetes that selectively target the δ-cells.

OBJECTIVE To document the properties of the voltage-gated ion channels in human pancreatic α-cells and their role in glucagon release. RESEARCH DESIGN AND METHODS Glucagon release was measured from intact islets. [Ca2+]i was recorded in cells showing spontaneous activity at 1 mmol/l glucose. Membrane currents and potential were measured by whole-cell patch-clamping in isolated α-cells identified by immunocytochemistry. RESULTS Glucose inhibited glucagon secretion from human islets; maximal inhibition was observed at 6 mmol/l glucose. Glucagon secretion at 1 mmol/l glucose was inhibited by insulin but not by ZnCl2. Glucose remained inhibitory in the presence of ZnCl2 and after blockade of type-2 somatostatin receptors. Human α-cells are electrically active at 1 mmol/l glucose. Inhibition of KATP-channels with tolbutamide depolarized α-cells by 10 mV and reduced the action potential amplitude. Human α-cells contain heteropodatoxin-sensitive A-type K+-channels, stromatoxin-sensitive delayed rectifying K+-channels, tetrodotoxin-sensitive Na+-currents, and low-threshold T-type, isradipine-sensitive L-type, and ω-agatoxin-sensitive P/Q-type Ca2+-channels. Glucagon secretion at 1 mmol/l glucose was inhibited by 40–70% by tetrodotoxin, heteropodatoxin-2, stromatoxin, ω-agatoxin, and isradipine. The [Ca2+]i oscillations depend principally on Ca2+-influx via L-type Ca2+-channels. Capacitance measurements revealed a rapid ( CONCLUSIONS Human α-cells are electrically excitable, and blockade of any ion channel involved in action potential depolarization or repolarization results in inhibition of glucagon secretion. We propose that voltage-dependent inactivation of these channels underlies the inhibition of glucagon secretion by tolbutamide and glucose.

OBJECTIVE Paracrine signaling via γ-aminobutyric acid (GABA) and GABAA receptors (GABAARs) has been documented in rodent islets. Here we have studied the importance of GABAergic signaling in human pancreatic islets. RESEARCH DESIGN AND METHODS Expression of GABAARs in islet cells was investigated by quantitative PCR, immunohistochemistry, and patch-clamp experiments. Hormone release was measured from intact islets. GABA release was monitored by whole-cell patch-clamp measurements after adenoviral expression of α1β1 GABAAR subunits. The subcellular localization of GABA was explored by electron microscopy. The effects of GABA on electrical activity were determined by perforated patch whole-cell recordings. RESULTS PCR analysis detected relatively high levels of the mRNAs encoding GABAAR α2, β3, γ2, and π subunits in human islets. Patch-clamp experiments revealed expression of GABAAR Cl− channels in 52% of β-cells (current density 9 pA/pF), 91% of δ-cells (current density 148 pA/pF), and 6% of α-cells (current density 2 pA/pF). Expression of GABAAR subunits in islet cells was confirmed by immunohistochemistry. β-Cells secreted GABA both by glucose-dependent exocytosis of insulin-containing granules and by a glucose-independent mechanism. The GABAAR antagonist SR95531 inhibited insulin secretion elicited by 6 mmol/l glucose. Application of GABA depolarized β-cells and stimulated action potential firing in β-cells exposed to glucose. CONCLUSIONS Signaling via GABA and GABAAR constitutes an autocrine positive feedback loop in human β-cells. The presence of GABAAR in non–β-cells suggests that GABA may also be involved in the regulation of somatostatin and glucagon secretion.

Summary Glucagon, secreted by pancreatic islet α cells, is the principal hyperglycemic hormone. In diabetes, glucagon secretion is not suppressed at high glucose, exacerbating the consequences of insufficient insulin secretion, and is inadequate at low glucose, potentially leading to fatal hypoglycemia. The causal mechanisms remain unknown. Here we show that α cell KATP-channel activity is very low under hypoglycemic conditions and that hyperglycemia, via elevated intracellular ATP/ADP, leads to complete inhibition. This produces membrane depolarization and voltage-dependent inactivation of the Na+ channels involved in action potential firing that, via reduced action potential height and Ca2+ entry, suppresses glucagon secretion. Maneuvers that increase KATP channel activity, such as metabolic inhibition, mimic the glucagon secretory defects associated with diabetes. Low concentrations of the KATP channel blocker tolbutamide partially restore glucose-regulated glucagon secretion in islets from type 2 diabetic organ donors. These data suggest that impaired metabolic control of the KATP channels underlies the defective glucose regulation of glucagon secretion in type 2 diabetes., Graphical Abstract, Highlights • KATP channel closure stimulates insulin secretion but inhibits glucagon release • α cell depolarization reduces voltage-gated Ca2+ entry and glucagon release • An activating KATP channel mutation impairs glucagon release in mice • KATP channel closure corrects glucagon secretion defect in type 2 diabetic islets

The majority of genetic risk variants for type 2 diabetes (T2D) affect insulin secretion, but the mechanisms through which they influence pancreatic islet function remain largely unknown. We functionally characterized human islets to determine secretory, biophysical, and ultrastructural features in relation to genetic risk profiles in diabetic and nondiabetic donors. Islets from donors with T2D exhibited impaired insulin secretion, which was more pronounced in lean than obese diabetic donors. We assessed the impact of 14 disease susceptibility variants on measures of glucose sensing, exocytosis, and structure. Variants near TCF7L2 and ADRA2A were associated with reduced glucose-induced insulin secretion, whereas susceptibility variants near ADRA2A, KCNJ11, KCNQ1, and TCF7L2 were associated with reduced depolarization-evoked insulin exocytosis. KCNQ1, ADRA2A, KCNJ11, HHEX/IDE, and SLC2A2 variants affected granule docking. We combined our results to create a novel genetic risk score for β-cell dysfunction that includes aberrant granule docking, decreased Ca2+ sensitivity of exocytosis, and reduced insulin release. Individuals with a high risk score displayed an impaired response to intravenous glucose and deteriorating insulin secretion over time. Our results underscore the importance of defects in β-cell exocytosis in T2D and demonstrate the potential of cellular phenotypic characterization in the elucidation of complex genetic disorders.

Glucagon secretion is regulated by glucose but the mechanisms involved remain hotly debated. Both intrinsic (within the α-cell itself) and paracrine (mediated by factors released β- and/or δ-cells) have been postulated. Glucagon secretion is maximally suppressed by glucose concentrations that do not affect insulin and somatostatin secretion, a finding that highlights the significance of intrinsic regulation of glucagon secretion. Experiments on islets from mice lacking functional ATP-sensitive potassium channels (K(ATP)-channels) indicate that these channels are critical to the α-cell's capacity to sense changes in extracellular glucose. Here, we review recent data on the intrinsic and paracrine regulation of glucagon secretion in human pancreatic islets. We propose that glucose-induced closure of the K(ATP)-channels, via membrane depolarization, culminates in reduced electrical activity and glucagon secretion by voltage-dependent inactivation of the ion channels involved in action potential firing. We further demonstrate that glucagon secretion measured in islets isolated from donors with type-2 diabetes is reduced at low glucose and that glucose stimulates rather than inhibits secretion in these islets. We finally discuss the relative significance of paracrine and intrinsic regulation in the fed and fasted states and propose a unifying model for the regulation of glucagon secretion that incorporates both modes of control.

To the Editor: The heterogeneous nature of the autoimmune process in type 1 diabetes mellitus can, on occasion, result in the preservation of beta cells in patients with longstanding disease [1]. The detection of low levels of basal C-peptide secretion in these patients suggests some beta cell secretory capacity [2]. Whether these residual beta cells are functionally intact, however, remains unanswered. Here we report in vitro observations on islets isolated from a cadaveric donor who had had type 1 diabetes mellitus for 13 years. The objectives were to isolate islets from the pancreas to determine islet hormone secretion profiles and cellular electrophysiological responses, and to compare these variables with data from islets from non-diabetic donors. In addition, we compared the pancreatic histology of this donor pancreas with that of other type 1 diabetic mellitus donors with established or newly diagnosed disease. The donor, a white female, was 19 years old at diagnosis, weighed 73.8 kg (BMI 28 kg/m) and had no family history of diabetes. She was treated with gliclazide and metformin. Seven weeks after diagnosis, despite maximal doses of oral therapy, she developed ketonuria and had a fasting blood glucose of 24 mmol/l. She therefore started insulin treatment (0.27 U/kg insulin twice daily). This was increased to 0.6 U/kg using a basal bolus regimen over a 6 month period. She underwent four admissions for severe diabetic ketoacidosis. Unfortunately no serum samples were taken for determination of C-peptide or antibody status at any time. Samples taken at the time of death showed HLA-DR3 and -4 positivity. The donor’s pancreas was retrieved for research with the informed consent of the family and with the approval of the local ethics committee. Islets were isolated using established protocols [3]. An islet count was omitted as poor dithizone uptake was predicted. Islets that were visibly structurally intact were hand-picked from the purified digest for the secretion experiments and the residual islets were retained for morphological analysis. The islets used for the functional experiments were therefore not a true representation of the whole isolated population. Insulin and glucagon secretion was measured from batches of 15 islets (in quadruplicates) pre-incubated in 1 mmol/l glucose for 1 h followed by incubation for 1 h at 1, 6 or 20 mmol/l glucose. The hormone contents of islets and supernatant fractions were determined by radioimmunoassay [4]. Membrane capacitance, as a measure of insulin granule exocytosis, was recorded from isolated alpha and beta cells using the patch-clamp technique [5]. Pancreatic tissue samples taken from the body and tail regions of this donor pancreas and from the donor pancreases of three additional type 1 diabetic patients (diabetes duration >10 years, n=2; diabetes duration

OBJECTIVE The aim of the study was to elucidate the cellular mechanism underlying the suppression of glucose-induced insulin secretion in mice fed a high-fat diet (HFD) for 15 weeks. RESEARCH DESIGN AND METHODS C57BL6J mice were fed a HFD or a normal diet (ND) for 3 or 15 weeks. Plasma insulin and glucose levels in vivo were assessed by intraperitoneal glucose tolerance test. Insulin secretion in vitro was studied using static incubations and a perfused pancreas preparation. Membrane currents, electrical activity, and exocytosis were examined by patch-clamp technique measurements. Intracellular calcium concentration ([Ca2+]i) was measured by microfluorimetry. Total internal reflection fluorescence microscope (TIRFM) was used for optical imaging of exocytosis and submembrane depolarization-evoked [Ca2+]i. The functional data were complemented by analyses of histology and gene transcription. RESULTS After 15 weeks, but not 3 weeks, mice on HFD exhibited hyperglycemia and hypoinsulinemia. Pancreatic islet content and β-cell area increased 2- and 1.5-fold, respectively. These changes correlated with a 20–50% reduction of glucose-induced insulin secretion (normalized to insulin content). The latter effect was not associated with impaired electrical activity or [Ca2+]i signaling. Single-cell capacitance and TIRFM measurements of exocytosis revealed a selective suppression (>70%) of exocytosis elicited by short (50 ms) depolarization, whereas the responses to longer depolarizations were (500 ms) less affected. The loss of rapid exocytosis correlated with dispersion of Ca2+ entry in HFD β-cells. No changes in gene transcription of key exocytotic protein were observed. CONCLUSIONS HFD results in reduced insulin secretion by causing the functional dissociation of voltage-gated Ca2+ entry from exocytosis. These observations suggest a novel explanation to the well-established link between obesity and diabetes.