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.

OBJECTIVETo document the properties of the voltage-gated ion channels in human pancreatic α-cells and their role in glucagon release.RESEARCH DESIGN AND METHODSGlucagon 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.RESULTSGlucose 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 (<50 ms) component of exocytosis. Exocytosis was negligible at voltages below −20 mV and peaked at 0 mV. Blocking P/Q-type Ca2+-currents abolished depolarization-evoked exocytosis.CONCLUSIONSHuman 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.

Strategies aimed at mimicking or enhancing the action of the incretin hormone glucagon-like peptide 1 (GLP-1) therapeutically improve glucose-stimulated insulin secretion (GSIS); however, it is not clear whether GLP-1 directly drives insulin secretion in pancreatic islets. Here, we examined the mechanisms by which GLP-1 stimulates insulin secretion in mouse and human islets. We found that GLP-1 enhances GSIS at a half-maximal effective concentration of 0.4 pM. Moreover, we determined that GLP-1 activates PLC, which increases submembrane diacylglycerol and thereby activates PKC, resulting in membrane depolarization and increased action potential firing and subsequent stimulation of insulin secretion. The depolarizing effect of GLP-1 on electrical activity was mimicked by the PKC activator PMA, occurred without activation of PKA, and persisted in the presence of PKA inhibitors, the KATP channel blocker tolbutamide, and the L-type Ca(2+) channel blocker isradipine; however, depolarization was abolished by lowering extracellular Na(+). The PKC-dependent effect of GLP-1 on membrane potential and electrical activity was mediated by activation of Na(+)-permeable TRPM4 and TRPM5 channels by mobilization of intracellular Ca(2+) from thapsigargin-sensitive Ca(2+) stores. Concordantly, GLP-1 effects were negligible in Trpm4 or Trpm5 KO islets. These data provide important insight into the therapeutic action of GLP-1 and suggest that circulating levels of this hormone directly stimulate insulin secretion by β cells.

Glucagon is the body's main hyperglycemic hormone, and its secretion is dysregulated in type 2 diabetes mellitus (T2DM). The incretin hormone glucagon‐like peptide‐1 (GLP‐1) is released from the gut and is used in T2DM therapy. Uniquely, it both stimulates insulin and inhibits glucagon secretion and thereby lowers plasma glucose levels. In this study, we have investigated the action of GLP‐1 on glucagon release from human pancreatic islets. Immunocytochemistry revealed that only <0.5% of the α‐cells possess detectable GLP‐1R immunoreactivity. Despite this, GLP‐1 inhibited glucagon secretion by 50–70%. This was due to a direct effect on α‐cells, rather than paracrine signaling, because the inhibition was not reversed by the insulin receptor antagonist S961 or the somatostatin receptor‐2 antagonist CYN154806. The inhibitory effect of GLP‐1 on glucagon secretion was prevented by the PKA‐inhibitor Rp‐cAMPS and mimicked by the adenylate cyclase activator forskolin. Electrophysiological measurements revealed that GLP‐1 decreased action potential height and depolarized interspike membrane potential. Mathematical modeling suggests both effects could result from inhibition of P/Q‐type Ca2+ channels. In agreement with this, GLP‐1 and ω‐agatoxin (a blocker of P/Q‐type channels) inhibited glucagon secretion in islets depolarized by 70 mmol/L [K+]o, and these effects were not additive. Intracellular application of cAMP inhibited depolarization‐evoked exocytosis in individual α‐cells by a PKA‐dependent (Rp‐cAMPS‐sensitive) mechanism. We propose that inhibition of glucagon secretion by GLP‐1 involves activation of the few GLP‐1 receptors present in the α‐cell membrane. The resulting small elevation of cAMP leads to PKA‐dependent inhibition of P/Q‐type Ca2+ channels and suppression of glucagon exocytosis.

Glucagon-like peptide-1 (GLP-1) is an endogenous glucose-lowering hormone and GLP-1 receptor agonists are currently being used as antidiabetic drugs clinically. The canonical signalling pathway (including cAMP, Epac2, protein kinase A (PKA) and K channels) is almost universally accepted as the main mechanism of GLP-1-stimulated insulin secretion. This belief is based on in vitro studies that used nanomolar (1-100 nM) concentrations of GLP-1. Recently, it was found that the physiological concentrations (1-10 pM) of GLP-1 also stimulate insulin secretion from isolated islets, induce membrane depolarization and increase of intracellular [Ca] in isolated β cells/pancreatic islets. These responses were unaffected by PKA inhibitors and occurred without detectable increases in intracellular cAMP and PKA activity. These PKA-independent actions of GLP-1 depend on protein kinase C (PKC), involve activation of the standard GLP-1 receptor (GLP1R) and culminate in activation of phospholipase C (PLC), leading to an elevation of diacylglycerol (DAG), increased L-type Ca and TRPM4/TRPM5 channel activities. Here, we review these recent data and contrast them against the effects of nanomolar concentrations of GLP-1. The differential intracellular signalling activated by low and high concentrations of GLP-1 could provide a clue to explain how GLP-1 exerts different function in the central nervous system and peripheral organs.

Abstract. Glucagon-like peptide-1 (GLP-1) induces pancreatic insulin secretion via the cAMPdependent protein kinase (PKA) pathway. However, the GLP-1 concentration used in the previous in vitro experiments was far from the in vivo concentrations. Alteration of plasma GLP-1 concentration at pM order lowers blood glucose concentration. In this study, we examined the GLP-1 action mechanism at a physiological concentration on insulin secretion. A high concentration of GLP-1 (10 nM) stimulated intracellular cAMP accumulation and insulin secretion was significantly inhibited by KT5720, a selective inhibitor of PKA. Low GLP-1 concentrations (1 pM) also increased insulin secretion without significant accumulation of intracellular cAMP, and KT5720 did not affect insulin secretion. Insulin secretion stimulated by 1 pM GLP-1 was reduced by inhibitors of calcium action, including verapamil, dantrolene, and BAPTA. Thus, we concluded that relatively low GLP-1 concentrations-comparable to in vivo blood concentrations-promoted insulin secretion independent of the cAMP-PKA pathway. This effect was dependent on intracellular Ca 2+ concentration. The results of the present study may further the understanding of the dose-dependent response of GLP-1 signal transducing pathways and the complicated mechanism of insulin secretion. Studies of GLP-1 at physiologic concentrations may lead to new developments in studies of pancreatic β-cell function.

OBJECTIVE—The aim of this study was to investigate the effect of apolipoprotein (apo)E4 allele on plasma LDL cholesterol response to calorie-restricted diet therapy in type 2 diabetic patients. RESEARCH DESIGN AND METHODS—Twenty-four diabetic patients with the apoE3/3 genotype and 11 diabetic patients with the apoE4/3 genotype were recruited. Participants were hospitalized for calorie-restricted diet therapy (25.0 kcal · kg body wt−1 · day−1) for 14 days. Body weight, fasting plasma glucose (FPG) levels, and plasma lipid levels on hospital days 1 and 14 were compared between the two apoE genotype groups. RESULTS—There were no significant differences in baseline FPG levels, HbA1c levels, BMI, and plasma levels of total cholesterol, triglyceride, and HDL cholesterol between the two apoE genotype groups, but baseline plasma levels of LDL cholesterol were significantly higher in the apoE4/3 group than in the apoE3/3 group. Body weight decreased slightly and FPG levels decreased significantly after diet therapy in both apoE genotype groups. In the apoE3/3 group, only plasma levels of triglyceride decreased significantly after diet therapy, whereas in the apoE4/3 group, plasma levels of triglyceride, total cholesterol, and LDL cholesterol decreased significantly after diet therapy. The decrease (percentage of change) in total cholesterol (−16.3 vs. −6.6%) and LDL cholesterol (−15.6 vs. −0.7%) after diet therapy was significantly greater in the apoE4/3 group than in the apoE3/3 group. CONCLUSIONS—Calorie-restricted diet therapy is more effective in reducing plasma LDL cholesterol in type 2 diabetic patients with the apoE4 allele.

Abstract. To demonstrate an involvement of ATP-sensitive potassium (K ATP ) channelindependent pathways in the first phase of glucose-stimulated insulin secretion (GSIS) from pancreatic β cells, the time course of GSIS from MIN6 cells was analyzed at 30-s sample intervals. GSIS was biphasic with the first phase being observed 120 to 390 s after glucose addition, peaking at 180 s, and with a shoulder at 240 to 330 s. Both 10 µM diazoxide and 3 µM verapamil completely inhibited tolbutamide-or glibenclamide-induced insulin secretion and suppressed the peak of the first phase of GSIS, but did not result in complete suppression. The shoulder following the peak was suppressed by 1 µM dantrolene. The peak, but not shoulder, disappeared under the extracellular Ca 2+ -free condition. A significant amount of insulin secretion remained even in the combined presence of verapamil and dantrolene. The Na + channel blocker tetrodotoxin (30 nM) nearly completely inhibited the first phase release. These results suggest that the first phase of GSIS from MIN6 cells depends on both Ca 2+-dependent and -independent mechanisms. The former mechanism includes the extracellular Ca 2+ influx via L-type voltagedependent calcium channel and intracellular Ca 2+ release from endoplasmic reticulum via ryanodine receptors, and the latter mechanism involves the pathways associated with Na + channels.