OBJECTIVE— To characterize the voltage-gated ion channels in human β-cells from nondiabetic donors and their role in glucose-stimulated insulin release. RESEARCH DESIGN AND METHODS— Insulin release was measured from intact islets. Whole-cell patch-clamp experiments and measurements of cell capacitance were performed on isolated β-cells. The ion channel complement was determined by quantitative PCR. RESULTS— Human β-cells express two types of voltage-gated K⁺ currents that flow through delayed rectifying (K_V2.1/2.2) and large-conductance Ca²⁺-activated K⁺ (BK) channels. Blockade of BK channels (using iberiotoxin) increased action potential amplitude and enhanced insulin secretion by 70%, whereas inhibition of K_V2.1/2.2 (with stromatoxin) was without stimulatory effect on electrical activity and secretion. Voltage-gated tetrodotoxin (TTX)-sensitive Na⁺ currents (Na_V1.6/1.7) contribute to the upstroke of action potentials. Inhibition of Na⁺ currents with TTX reduced glucose-stimulated (6–20 mmol/l) insulin secretion by 55–70%. Human β-cells are equipped with L- (Ca_V1.3), P/Q- (Ca_V2.1), and T- (Ca_V3.2), but not N- or R-type Ca²⁺ channels. Blockade of L-type channels abolished glucose-stimulated insulin release, while inhibition of T- and P/Q-type Ca²⁺ channels reduced glucose-induced (6 mmol/l) secretion by 60–70%. Membrane potential recordings suggest that L- and T-type Ca²⁺ channels participate in action potential generation. Blockade of P/Q-type Ca²⁺ channels suppressed exocytosis (measured as an increase in cell capacitance) by >80%, whereas inhibition of L-type Ca²⁺ channels only had a minor effect. CONCLUSIONS— Voltage-gated T-type and L-type Ca²⁺ channels as well as Na⁺ channels participate in glucose-stimulated electrical activity and insulin secretion. Ca²⁺-activated BK channels are required for rapid membrane repolarization. Exocytosis of insulin-containing granules is principally triggered by Ca²⁺ influx through P/Q-type Ca²⁺ channels.

Diabetes is characterized by hyperglycaemia due to impaired insulin secretion and aberrant glucagon secretion resulting from changes in pancreatic islet cell function and/or mass. The extent to which hyperglycaemia per se underlies these alterations remains poorly understood. Here we show that β-cell-specific expression of a human activating K_ATP channel mutation in adult mice leads to rapid diabetes and marked alterations in islet morphology, ultrastructure and gene expression. Chronic hyperglycaemia is associated with a dramatic reduction in insulin-positive cells and an increase in glucagon-positive cells in islets, without alterations in cell turnover. Furthermore, some β-cells begin expressing glucagon, whilst retaining many β-cell characteristics. Hyperglycaemia, rather than K_ATP channel activation, underlies these changes, as they are prevented by insulin therapy and fully reversed by sulphonylureas. Our data suggest that many changes in islet structure and function associated with diabetes are attributable to hyperglycaemia alone and are reversed when blood glucose is normalized.

Dysfunction and loss of insulin-producing pancreatic beta cells represent hallmarks of diabetes mellitus. Here, we show that mice lacking the mitogen-activated protein kinase (MAPK) p38delta display improved glucose tolerance due to enhanced insulin secretion from pancreatic beta cells. Deletion of p38delta results in pronounced activation of protein kinase D (PKD), the latter of which we have identified as a pivotal regulator of stimulated insulin exocytosis. p38delta catalyzes an inhibitory phosphorylation of PKD1, thereby attenuating stimulated insulin secretion. In addition, p38delta null mice are protected against high-fat-feeding-induced insulin resistance and oxidative stress-mediated beta cell failure. Inhibition of PKD1 reverses enhanced insulin secretion from p38delta-deficient islets and glucose tolerance in p38delta null mice as well as their susceptibility to oxidative stress. In conclusion, the p38delta-PKD pathway integrates regulation of the insulin secretory capacity and survival of pancreatic beta cells, pointing to a pivotal role for this pathway in the development of overt diabetes mellitus.

OBJECTIVE Paracrine signaling via γ-aminobutyric acid (GABA) and GABA_A receptors (GABA_ARs) 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 GABA_ARs 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 GABA_AR 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 GABA_AR α₂, β_3, γ₂, and π subunits in human islets. Patch-clamp experiments revealed expression of GABA_AR 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 GABA_AR 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 GABA_AR 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 GABA_AR constitutes an autocrine positive feedback loop in human β-cells. The presence of GABA_AR in non–β-cells suggests that GABA may also be involved in the regulation of somatostatin and glucagon secretion.

Glucagon, secreted from pancreatic islet α cells, stimulates gluconeogenesis and liver glycogen breakdown. The mechanism regulating glucagon release is debated, and variously attributed to neuronal control, paracrine control by neighbouring β cells, or to an intrinsic glucose sensing by the α cells themselves. We examined hormone secretion and Ca²⁺ responses of α and β cells within intact rodent and human islets. Glucose-dependent suppression of glucagon release persisted when paracrine GABA or Zn²⁺ signalling was blocked, but was reversed by low concentrations (1–20 μM) of the ATP-sensitive K⁺ (K_ATP) channel opener diazoxide, which had no effect on insulin release or β cell responses. This effect was prevented by the K_ATP channel blocker tolbutamide (100 μM). Higher diazoxide concentrations (≥30 μM) decreased glucagon and insulin secretion, and α- and β-cell Ca²⁺ responses, in parallel. In the absence of glucose, tolbutamide at low concentrations (10 μM) were inhibitory. In the presence of a maximally inhibitory concentration of tolbutamide (0.5 mM), glucose had no additional suppressive effect. Downstream of the K_ATP channel, inhibition of voltage-gated Na⁺ (TTX) and N-type Ca²⁺ channels (ω-conotoxin), but not L-type Ca²⁺ channels (nifedipine), prevented glucagon secretion. Both the N-type Ca²⁺ channels and α-cell exocytosis were inactivated at depolarised membrane potentials. Rodent and human glucagon secretion is regulated by an α-cell K_ATP channel-dependent mechanism. We propose that elevated glucose reduces electrical activity and exocytosis via depolarisation-induced inactivation of ion channels involved in action potential firing and secretion. Glucagon is a critical regulator of glucose homeostasis. Its major action is to mobilize glucose from the liver. Glucagon secretion from α cells of the pancreatic islets of Langerhans is suppressed by elevated blood sugar, a response that is often perturbed in diabetes. Much work has focused on the regulation of α-cell glucagon secretion by neuronal factors and by paracrine factors from neighbouring cells, including the important islet hormone insulin. In contrast, we provide evidence in support of a direct effect of glucose on α cells within intact rodent and human islets. Notably, our work implicates an α-cell glucose-sensing pathway similar to that found in insulin-secreting β cells, involving closure of ATP-dependent K⁺ channels in the presence of glucose. Furthermore, we find that membrane depolarisation results in inhibition of Na⁺ and Ca²⁺ channel activity and α-cell exocytosis. Thus, we propose that elevated blood glucose reduces α-cell electrical activity and glucagon secretion by inactivating the ion channels involved in action potential firing and secretion.