OBJECTIVE-To characterize the voltage-gated ion channels in human -cells from nondiabetic donors and their role in glucosestimulated 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 -cells. The ion channel complement was determined by quantitative PCR.RESULTS-Human -cells express two types of voltage-gated K ϩ currents that flow through delayed rectifying (K V 2.1/2.2) and large-conductance Ca 2ϩ -activated K ϩ (BK) channels. Blockade of BK channels (using iberiotoxin) increased action potential amplitude and enhanced insulin secretion by 70%, whereas inhibition of K V 2.1/2.2 (with stromatoxin) was without stimulatory effect on electrical activity and secretion. Voltage-gated tetrodotoxin (TTX)-sensitive Na ϩ currents (Na V 1.6/1.7) contribute to the upstroke of action potentials. Inhibition of Na ϩ currents with TTX reduced glucose-stimulated (6 -20 mmol/l) insulin secretion by 55-70%. Human -cells are equipped with L-(Ca V 1.3), P/Q-(Ca V 2.1), and T-(Ca V 3.2), but not N-or R-type Ca 2ϩ channels. Blockade of L-type channels abolished glucosestimulated insulin release, while inhibition of T-and P/Q-type Ca 2ϩ channels reduced glucose-induced (6 mmol/l) secretion by 60 -70%. Membrane potential recordings suggest that L-and T-type Ca 2ϩ channels participate in action potential generation. Blockade of P/Q-type Ca 2ϩ channels suppressed exocytosis (measured as an increase in cell capacitance) by Ͼ80%, whereas inhibition of L-type Ca 2ϩ channels only had a minor effect.CONCLUSIONS-Voltage-gated T-type and L-type Ca 2ϩ channels as well as Na ϩ channels participate in glucose-stimulated electrical activity and insulin secretion. Ca 2ϩ -activated BK channels are required for rapid membrane repolarization. Exocytosis of insulin-containing granules is principally triggered by Ca 2ϩ influx through P/Q-type Ca 2ϩ channels. Diabetes 57:1618-1628, 2008
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+ (KATP) channel opener diazoxide, which had no effect on insulin release or β cell responses. This effect was prevented by the KATP 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 (<1 μM) stimulated glucagon secretion, whereas high 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 KATP 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 KATP 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.
SummaryGlucagon, 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.
Glucagon secretion is inhibited by glucagon-like peptide-1 (GLP-1) and stimulated by adrenaline. These opposing effects on glucagon secretion are mimicked by low (1-10 nM) and high (10 muM) concentrations of forskolin, respectively. The expression of GLP-1 receptors in alpha cells is <0.2% of that in beta cells. The GLP-1-induced suppression of glucagon secretion is PKA dependent, is glucose independent, and does not involve paracrine effects mediated by insulin or somatostatin. GLP-1 is without much effect on alpha cell electrical activity but selectively inhibits N-type Ca(2+) channels and exocytosis. Adrenaline stimulates alpha cell electrical activity, increases [Ca(2+)](i), enhances L-type Ca(2+) channel activity, and accelerates exocytosis. The stimulatory effect is partially PKA independent and reduced in Epac2-deficient islets. We propose that GLP-1 inhibits glucagon secretion by PKA-dependent inhibition of the N-type Ca(2+) channels via a small increase in intracellular cAMP ([cAMP](i)). Adrenaline stimulates L-type Ca(2+) channel-dependent exocytosis by activation of the low-affinity cAMP sensor Epac2 via a large increase in [cAMP](i).
OBJECTIVEParacrine 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 METHODSExpression 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.RESULTSPCR 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.CONCLUSIONSSignaling 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.
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 KATP 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 KATP 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.
SummaryDysfunction and loss of insulin-producing pancreatic β cells represent hallmarks of diabetes mellitus. Here, we show that mice lacking the mitogen-activated protein kinase (MAPK) p38δ display improved glucose tolerance due to enhanced insulin secretion from pancreatic β cells. Deletion of p38δ results in pronounced activation of protein kinase D (PKD), the latter of which we have identified as a pivotal regulator of stimulated insulin exocytosis. p38δ catalyzes an inhibitory phosphorylation of PKD1, thereby attenuating stimulated insulin secretion. In addition, p38δ null mice are protected against high-fat-feeding-induced insulin resistance and oxidative stress-mediated β cell failure. Inhibition of PKD1 reverses enhanced insulin secretion from p38δ-deficient islets and glucose tolerance in p38δ null mice as well as their susceptibility to oxidative stress. In conclusion, the p38δ-PKD pathway integrates regulation of the insulin secretory capacity and survival of pancreatic β cells, pointing to a pivotal role for this pathway in the development of overt diabetes mellitus.
Melatonin is known to inhibit insulin secretion from rodent beta-cells through interactions with cell-surface MT1 and/or MT2 receptors, but the function of this hormone in human islets of Langerhans is not known. In the current study, melatonin receptor expression by human islets was examined by reverse transcription-polymerase chain reaction (RT-PCR) and the effects of exogenous melatonin on intracellular calcium ([Ca2+]i) levels and islet hormone secretion were determined by single cell microfluorimetry and radioimmunoassay, respectively. RT-PCR amplifications indicated that human islets express mRNAs coding for MT1 and MT2 melatonin receptors, although MT2 mRNA expression was very low. Analysis of MT1 receptor mRNA expression at the single cell level indicated that it was expressed by human islet alpha-cells, but not by beta-cells. Exogenous melatonin stimulated increases in intracellular calcium ([Ca2+]i) in dissociated human islet cells, and stimulated glucagon secretion from perifused human islets. It also stimulated insulin secretion and this was most probably a consequence of glucagon acting in a paracrine fashion to stimulate beta-cells as the MT1 receptor was absent in beta-cells. Melatonin did not decrease 3', 5'-cyclic adenosine monophosphate (cyclic AMP) levels in human islets, but it inhibited cyclic AMP in the mouse insulinoma (MIN6) beta-cell line and it also inhibited glucose-stimulated insulin secretion from MIN6 cells. These data suggest that melatonin has direct stimulatory effects at human islet alpha-cells and that it stimulates insulin secretion as a consequence of elevated glucagon release. This study also indicates that the effects of melatonin are species-specific with primarily an inhibitory role in rodent beta-cells and a stimulatory effect in human islets.
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