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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 γ (PPARγ) coactivator-1α-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 PPARγ1, fatty acid translocase, glucose-6-phospha...

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.

Nicotine obtained from tobacco can improve learning and memory on various tasks and has been linked to arousal, attention, rapid information processing, working memory, and long-term memories that can cause craving years after someone has stopped smoking. One likely target for these effects is the hippocampus, a centre for learning and memory that has rich cholinergic innervation and dense nicotinic acetylcholine receptor (nAChR) expression. During Alzheimer's dementia there are fewer nAChRs and the cholinergic inputs to the hippocampus degenerate. However, there is no evidence for fast synaptic transmission mediated by nAChRs in the hippocampus, and their role is not understood. Nicotine is known to act on presynaptic nAChRs within the habenula of chick to enhance glutamatergic transmission; here we report that a similar mechanism operates in the hippocampus. Measurements of intracellular Ca2+ in single mossy-fibre presynaptic terminals indicate that nAChRs containing the alpha7 subunit can mediate a Ca2+ influx that is sufficient to induce vesicular neurotransmitter release. We propose that nicotine from tobacco influences cognition by enhancing synaptic transmission. Conversely, a decreased efficacy of transmission may account for the deficits associated with the loss of cholinergic innervation during Alzheimer's disease.

We tested for the presence of sulfonylurea receptors in pancreatic alpha cells. Two high affinity sulfonylurea receptors were identified in clonal pancreatic alpha cells (alpha TC-6): a 140-kDa species observed previously in clonal pancreatic beta cells (HIT) and a second 150-kDa protein. The dissociation constant (Kd) for both receptors is approximately 3.5 nM for an iodinated glyburide analog, 5-iodo-2-hydroxyglyburide. The estimated number of receptors (Bmax) increases approximately 2-fold, from 3.1 to 6.8 pmol/mg of membrane protein as the pH of the binding buffer is reduced from 7.5 to 6. Consistent with the notion that high affinity sulfonylurea receptors are integral components of the ATP-sensitive K+ channel, we demonstrated the presence of ATP-sensitive K+ channels in inside-out patches of alpha TC-6 cells. Whole cell K+ currents that activated with time showed inward rectification at positive potentials (above 0 mV) and were almost completely suppressed by 5 nM glyburide. Likewise, glyburide blocked 86Rb+ efflux from ATP-depleted alpha TC-6 cells, an effect that was reversed by 400 microM diazoxide. The presence of sulfonylurea receptors provides a mechanism by which sulfonylureas can directly modulate alpha cell function. The properties of the 150-kDa receptor and the role of ATP-sensitive K+ channels in alpha cells remain to be elucidated, but as in beta cells, ATP-sensitive K+ channels may be involved in metabolic regulation of alpha cells by glucose.

Cell membranes isolated from hamster insulinoma (HIT T15) cells at passages 65-74 contain high and low affinity receptors for a sulfonylurea derivative, 5-[125I]iodo,2-hydroxyglyburide (KD values of approximately 7 nM and 16 microM). Between passages 75 and 85, the estimated B(max) for the high affinity receptor decreases approximately 10-fold from approximately 1.6 to 0.16 pmol/mg membrane protein. By contrast, the density of low affinity binding sites, 800-1000 pmol/mg, is unaltered. The drop in high affinity receptors is paralleled by a decrease in the density of KATP channels assessed using patch-clamp and 86Rb(+)-efflux techniques. These results strongly support the idea that the high affinity sulfonylurea receptor is an integral part of the KATP channel.

We tested the hypothesis that somatostatin (SRIF) inhibits insulin secretion from an SV40 transformed hamster beta cell line (HIT cells) by an effect on the voltage-dependent Ca2+ channels and examined whether G-proteins were involved in the process. Ca2+ currents were recorded by the whole cell patch-clamp method, the free cytosolic calcium, [Ca2+]i, was monitored in HIT cells by fura-2, and cAMP and insulin secretion were measured by radioimmunoassay. SRIF decreased Ca2+ currents, [Ca2+]i, and basal insulin secretion in a dose-dependent manner over the range of 10(-12)-10(-7)M. The increase in [Ca2+]i and insulin secretion induced by either depolarization with K+ (15 mM) or by the Ca2+ channel agonist, Bay K 8644 (1 microM) was attenuated by SRIF in a dose-dependent manner over the same range of 10(-12)-10(-7) M. the half-maximal inhibitory concentrations (IC50) for SRIF inhibition of insulin secretion were 8.6 X 10(-12) M and 8.3 X 10(-11) M for K+ and Bay K 8644-stimulated secretion and 1 X 10(-10) M and 2.9 X 10(-10) M for the SRIF inhibition of the K+ and Bay K 8644-induced rise in [Ca2+]i, respectively. SRIF also attenuated the rise in [Ca2+]i induced by the cAMP-elevating agent, isobutylmethylxanthine (1 mM) in the presence of glucose. Bay K 8644, K+ and SRIF had no significant effects on cAMP levels and SRIF had no effects on adenylyl cyclase activity at concentrations lower than 1 microM. SRIF (100 nM) did not change K+ efflux (measured by 86Rb+) through ATP-sensitive K+ channels in HIT cells. SRIF (up to 1 microM) had no significant effect on membrane potential measured by bisoxonol fluorescence. Pretreatment of the HIT cells with pertussis toxin (0.1 microgram/ml) overnight abolished the effects of SRIF on Ca2+ currents, [Ca2+]i and insulin secretion implying a G-protein dependence in SRIF's actions. Thus, one mechanism by which SRIF decreases insulin secretion is by inhibiting Ca2+ influx through voltage-dependent Ca2+ channels, an action mediated through a pertussis toxin-sensitive G-protein.

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.

Sulfonylureas interact with specific, high affinity receptors on the pancreatic beta-cell to close ATP-sensitive K+ channels, depolarize the cell, activate Ca2+ influx through voltage-dependent Ca2+ channels, and trigger insulin secretion. We tested the hypothesis that sulfonylureas promote glucose uptake into 3T3-L1 cells or isolated rat adipocytes by similar mechanisms. Using 125I-labeled 5-iodo-2-hydroxyglyburide and either equilibrium binding or photoaffinity labeling, a high affinity sulfonylurea receptor was not found on plasma membranes of either the 3T3-L1 cells or rat adipocytes. Furthermore, glyburide did not inhibit 86Rb+ efflux (a marker for ATP-sensitive K+ channel conductance), increase free cytosolic calcium in adipocytes or 3T3-L1 cells, or increase basal or insulin-stimulated glucose uptake into 3T3-L1 cells or rat adipocytes. Parallel studies using a hamster insulin-secreting tumor cell line (HIT cells) easily demonstrated both the receptor and biological effects of glyburide on free cytosolic calcium and insulin secretion. Thus, rat adipocytes and 3T3-L1 cells do not possess the high affinity sulfonylurea receptor or respond to glyburide alone. We conclude that the antidiabetogenic effects of sulfonylureas are not mediated by a direct action of sulfonylureas to increase glucose uptake into adipose tissue and suggest that the major locus for sulfonylurea action is the beta-cell.

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.

In the pancreatic beta cells the proximal step in sulfonylurea signal transduction is the binding of these clinically important drugs to high-affinity receptors in the beta cell membrane. Using HIT cells as a model system, we have established an extremely close correlation between the affinity of binding of glyburide and its analog, iodoglyburide, and the activation of various steps in stimulus-secretion coupling--inhibition of 86Rb+ efflux, increase in [Ca2+]i resulting from gating of voltage-gated calcium channels by cell depolarization, and the exocytosis of insulin. Two different L-type channel cDNAs have been identified in an HIT cell library, one neuroendocrine in type and one more cardiac-like. A HIT cell membrane protein of Mr 140,000, which we believe to be the high-affinity sulfonylurea receptor, can be covalently linked to 5(125)-iodo-2-hydroxyglyburide by ultraviolet irradiation. The receptor has been solubilized and retains binding activity and the same rank order of displacement of the 5(125)-iodo-2-hydroxyglyburide as observed with the native receptor. The Mr 140,000 protein has been partially purified and the amino acid sequences of three proteolytic fragments have been used to design oligonucleotides to screen HIT cell cDNA libraries. Since the binding constant of glyburide or iodoglyburide is closely correlated with the ability of these compounds to inhibit the ATP-sensitive K+ channel, increase [Ca2+]i, and elicit insulin secretion, we have identified the Mr 140,000 protein as the sulfonylurea receptor. Expression of the cloned cDNA should allow us to test this hypothesis directly.

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.