Search

Glucagon is a potent counterregulatory hormone that opposes the action of insulin in controlling glycemia. The cellular mechanisms by which pancreatic alpha-cell glucagon secretion occurs in response to hypoglycemia are poorly known. SUR1/K(IR)6.2-type ATP-sensitive K(+) (K(ATP)) channels have been implicated in the glucagon counterregulatory response at central and peripheral levels, but their role is not well understood. In this study, we examined hypoglycemia-induced glucagon secretion in vitro in isolated islets and in vivo using Sur1KO mice lacking neuroendocrine-type K(ATP) channels and paired wild-type (WT) controls. Sur1KO mice fed ad libitum have normal glucagon levels and mobilize hepatic glycogen in response to exogenous glucagon but exhibit a blunted glucagon response to insulin-induced hypoglycemia. Glucagon release from Sur1KO and WT islets is increased at 2.8 mmol/liter glucose and suppressed by increasing glucose concentrations. WT islets increase glucagon secretion approximately 20-fold when challenged with 0.1 mmol/liter glucose vs. approximately 2.7-fold for Sur1KO islets. Glucagon release requires Ca(2+) and is inhibited by nifedipine. Consistent with a regulatory interaction between K(ATP) channels and intra-islet zinc-insulin, WT islets exhibit an inverse correlation between beta-cell secretion and glucagon release. Glibenclamide stimulated insulin secretion and reduced glucagon release in WT islets but was without effect on secretion from Sur1KO islets. The results indicate that loss of alpha-cell K(ATP) channels uncouples glucagon release from inhibition by beta-cells and reveals a role for K(ATP) channels in the regulation of glucagon release by low glucose.

Ketosis-prone diabetes is heterogeneous. Its causes could include novel beta-cell functional defects. To characterize such defects, 103 patients with diabetic ketoacidosis were evaluated for beta-cell autoimmunity and human leukocyte antigen (HLA) class II alleles, with longitudinal measurements of beta-cell function and biochemical and clinical parameters. They were classified into four A beta groups, based on the presence of glutamic acid decarboxylase (GAD)65, GAD67, or IA-2 autoantibodies (A+ or A-) and beta-cell functional reserve (beta+ or beta-). The group distribution was: 18 A+beta-, 23 A-beta-, 11 A+beta+, and 51 A-beta+. Collectively, the two beta- groups differed from the two beta+ groups in earlier onset and longer duration of diabetes, lower body mass index, less glycemic improvement, and persistent insulin requirement. HLA class II genotyping showed that the A-beta- group differed from the A+beta- group in having lower frequencies of two alleles strongly associated with autoimmune type 1 diabetes susceptibility: DQA*03 and DQB1*02. Similarly, the A-beta+ group differed from the A+beta+ group in having a lower frequency of DQB1*02. Ketosis-prone diabetes comprises at least four etiologically distinct syndromes separable by autoantibody status, HLA genotype, and beta-cell functional reserve. Novel, nonautoimmune causes of beta-cell dysfunction are likely to underlie the A-beta+ and A-beta- syndromes.

We investigated the effect of nutrient intake on glucose metabolism in normal Mexican-Americans ( n = 6) and European-Americans ( n = 6). Subjects were studied after an 18-h fast and after 5–6 h of ingestion of hourly meals that supplied 6.35 or 12.75 μmol glucose ⋅ kg−1⋅ min−1. Endogenous glucose production (EGP), gluconeogenesis (GNG), and glycogenolysis (GLY) were estimated by mass isotopomer analysis with [U-13C]glucose infusions. Fasting EGP, GNG, and GLY did not differ between the groups. Food ingestion lowered the molar rate of GNG by only 31%. However, while consuming the lower quantity of nutrients, Mexican-Americans had higher plasma glucose ( P < 0.05), a 39% higher rate of EGP ( P< 0.05), and a 68% ( P < 0.025) higher rate of GLY than the European-Americans. At the higher intake, EGP and GLY were suppressed completely in both groups. There was a linear relationship between insulin concentrations, EGP, and GLY in both groups, but the slope of the line was significantly ( P < 0.05) greater in the European-Americans. We conclude that the sensitivity of GLY to nutrient intake differs between ethnic groups and that this may play a role in the increased predisposition of Mexican-Americans to type II diabetes.

Publisher Summary Plasma membrane ion channels are protein tunnels on the surface of cells that permit cellular entry and exit of electrically charged ions. Depending on the signal that makes them open and close (“gating”), ion channels can be classified into ligand-gated and voltage-gated channels. Although these molecules are better known for their role in generating the spark of the heartbeat or in the lightning-like conduction of nerve action potentials, it is now evident that ion channels play a critical role in igniting the physiological process of insulin secretion in pancreatic β-cells. Recognition of a signal at the cell surface and its translation into an intracellular gesture defines the process of “signal transduction.” This chapter describes how signal transduction by specific ion channels mediates the stimulus-secretion coupling process in insulin-secreting β-cells. The primary physiological function of the pancreatic β-cell is to sense changes in circulating glucose levels and respond with an appropriate insulin secretory response. The significance of this apparently simple function is of profound importance in health because its failure disrupts normal blood glucose homeostasis and results in important insulin secretory abnormalities such as diabetes mellitus and familial hyperinsulinism. The β-cell is rather unique because its electrical activity changes in response to stimuli that are primarily metabolic in nature. Thus, examination of the electrical events in β-cell stimulus-secretion coupling is best understood by following the changes in response to its primary metabolic stimulus, glucose.

Activation of the sympathetic nervous system inhibits insulin secretion. We tested the hypothesis that activation of alpha 2-adrenergic receptors on the beta-cell by epinephrine or clonidine attenuates insulin release by an effect on the voltage-dependent Ca2+ channel (VDCC) and examined the role of G-proteins in this signal transduction pathway. Using a cultured SV40-transformed hamster beta-cell line (HIT cells) as a model system, we determined the effect of alpha 2-adrenergic agonists on insulin secretion, 86Rb+ efflux (a marker for K+ channel flux), and the free cytosolic Ca2+ level [( Ca2+]i) monitored in fura-2-loaded cells. In a dose-dependent manner, epinephrine and clonidine (10(-8)-10(-5)M) attenuated the increase in [Ca2+]i and insulin secretion induced by either K+ depolarization or stimulation of the VDCC with the agonist Bay K 8644. Epinephrine failed to affect the rise in [Ca2+]i induced by carbamylcholine, an agent that mobilizes intracellular Ca2+. Epinephrine also did not changes 86Rb+ efflux from HIT cells. The inhibitory effects of epinephrine were prevented by the alpha 2-adrenergic antagonist idazoxan, but were unaffected by the alpha 1-adrenergic antagonist phenoxybenzamine. Pretreatment of HIT cells with pertussis toxin (0.1 micrograms/ml) overnight abolished the inhibitory effects of epinephrine and clonidine on both [Ca2+]i and insulin secretion. These data suggest that one mechanism by which alpha 2-adrenergic agonists inhibit insulin secretion is by inhibiting Ca2+ influx through VDCC, an action that is mediated through a pertussis toxin-sensitive G-protein.

The orphan receptor small heterodimer partner (SHP; NROB2) is a transcriptional repressor that inhibits nuclear receptor signaling in diverse metabolic pathways. Here, we report that SHP(-/-) mice exhibited hypoinsulinemia with age, which was associated with increased peripheral insulin sensitivity and increased response of isolated islets to glucose stimulation, yet maintain normal levels of blood glucose. Deficiency in SHP function resulted in up-regulation of glucose transporter 4 mRNA and glucose uptake in muscles, and overexpression of SHP in C2C12 cells inhibited both basal and peroxisomal proliferator-activated receptor gamma (PPARgamma) coactivator-1alpha-stimulated glucose transporter 4 expression and glucose uptake. SHP(-/-) hepatocytes showed markedly decreased basal glucose production in cultures, and SHP(-/-) livers had increased glycogen stores and were more sensitive to insulin inhibition of glucose output, which were concomitant with decreased expression for PPARgamma1, fatty acid translocase, glucose-6-phosphatase, and phosphoenol/pyruvate carboxykinase, and increased mRNAs for glucokinase and pyruvate kinase. In white fat, SHP deficiency resulted in up-regulation of genes involved in insulin sensitizing, including PPARgamma2 and adiponectin. We show that, at the transcriptional level, SHP directly represses adiponectin promoter activity by PPARgamma/liver receptor homolog-1. The results suggest that the increases in insulin sensitivity through multiple signaling pathways in muscle, liver, and fat, with an increase in islet secretory function, represent the complex mechanism whereby SHP deficiency leads to improvement in insulin sensitivity, secretion, and diabetes.

We review the role of ion channels in regulating insulin secretion from pancreatic beta-cells. By controlling ion permeability, ion channels at the membrane play a major role in regulating both electrical activity and signal transduction in the beta-cell. A proximal step in the cascade of events required for stimulus-secretion coupling is the closure of ATP-sensitive K+ channels, resulting in cell depolarization. Of particular relevance is the finding that this channel is directly regulated by a metabolite of glucose, which is the primary insulin secretagogue. In addition, this channel, or a closely associated protein, contains the sulfonylurea-binding site. Another K+ channel, the Ca2(+)-activated K+ channel, may be involved in cell repolarization to create homeostasis. Voltage-dependent Ca2+ channels are activated by cell depolarization and regulate Ca2+ influx into the cell. By controlling cytosolic free-Ca2+ levels ([Ca2+]i), these channels play an important role in transducing the initial stimulus to the effector systems that modulate insulin secretion. The link between a rise in [Ca2+]i and the terminal event of exocytosis is the least-understood aspect of stimulus-secretion coupling. However, phosphorylation studies have identified substrate proteins that may correspond to those involved in smooth muscle contraction, suggesting an analogy in the processes of stimulus secretion and excitation contraction. The advent of new methodology, particularly the patch-clamp technique, has fostered a more detailed characterization of the beta-cell ion channels. Furthermore, biochemical and molecular approaches developed for the structural analysis of ion channels in other tissues can now be applied to the isolation and characterization of the beta-cell ion channels. This is of particular significance because there appear to be tissue-specific variations in the different types of ion channels. Given the importance of ion channels in cell physiology, a knowledge of the structure and properties of these channels in the beta-cell is required for understanding the abnormalities of insulin secretion that occur in non-insulin-dependent diabetes mellitus. Ultimately, these studies should also provide new therapeutic approaches to the treatment of this disease.

Although there is compelling pharmacological evidence based on Ca2+-channel antagonist studies suggesting that the voltage-dependent Ca2+ channels regulate insulin release, no direct comparison with Ca2+ currents exists. This is particularly important because of the recent demonstration in other cell types of one and possibly two Ca2+ channels that are insensitive to Ca2+-channel antagonists, the dihydropyridines and the phenylalkylamines. Using an SV40-transformed pancreatic β-cell line (HIT cells), we determined how voltage-dependent Ca2+ channels are involved in stimulus-secretion coupling. Ca2+ currents were measured with the tight-seal technique for wholecell recording. The cytosolic free-Ca2+ concentration ([Ca2+]1) was followed with the fluorescent probe Fura 2, and the measurements were compared with insulin secretion stimulated by depolarizing the cells with K+. The Ca2+ current contained two components: arapidly decaying current activated at –50 to –40 mV that decayed with a time constant of 25 ms anda very slowly decaying component activated at –40 mV. Both components were sensitive to the Ca2+-channel antagonist nimodipine. There is excellent agreement in the concentration of nimodipine that inhibited Ca2+ current and the increase in [Ca2+1], in response to K+ depolarization (IC50 of 15 and 6 nM, respectively). Nimodipine inhibited insulin release over a similar dose-response range with an IC50of 1.5 × 10 9 M. These studies indicate that the increase in [Ca2+1], in response to β-cell depolarization can be accounted for by the influx of this ion through a single class of dihydropyridine-sensitive Ca2+ channels in the cell membrane.

With a glucose-responsive (β-cell line (HIT cells), we tested the hypothesis that the cytosolic free Ca2+ level ([Ca2+],) is an intracellular signal through which a rise in cyclic AMP (cAMP) levels is transmitted to potentiate glucose-stimulated insulin secretion. In these cells, glucose stimulates the acute release of insulin without increasing [Ca2+], or altering cAMP content. Either forskolin or 3-isobutylmethylxanthine (IBMX) potentiated glucose-stimulated insulin secretion and increased cAMP levels. At either a submaximal glucose concentration or maximally stimulatory glucose concentration, both IBMX and forskolin triggered a rapid rise in [Ca2+], (1.9- and 1.5-fold increase over basal levels, respectively). Similarly, glucagon stimulated a 1.3-fold increase in [Ca2+]1 over basal levels. The effect on [Ca2+]1 required glucose and was secondary to Ca2+ influx through voltage-dependent Ca2+ channels because it was blocked by either chelation of extracellular Ca2+ with EGTA or by the Ca2+-channel blockers verapamil and nimodipine. Verapamil also inhibited IBMX potentiation of glucose-stimulated insulin secretion and the IBMX-induced rise in [ca2+]1 in a dose-dependent manner with IC50s of 2 × 10−5 and 4 × 10−6 M, respectively. We conclude that in the β-cell, a rise in cAMP levels increases Ca2+ influx through voltage-dependent Ca2+channels and that this represents a mechanism by which cAMP potentiates glucose-stimulated insulin secretion in β-cells.