Defective pancreatic beta-cell glycolytic signaling in hepatocyte nuclear factor-1alpha-deficient mice.

Mutations in the hepatocyte nuclear factor-1alpha (HNF-1alpha) gene cause maturity onset diabetes of the young type 3, a form of type 2 diabetes mellitus. In mice lacking the HNF-1alpha gene, insulin secretion and intracellular calcium ([Ca2+]i) responses were impaired following stimulation with nutrient secretagogues such as glucose and glyceraldehyde but normal with non-nutrient stimuli such as potassium chloride. Patch clamp recordings revealed ATP-sensitive K+ currents (KATP) in beta-cells that were insensitive to suppression by glucose but normally sensitive to ATP. Exposure to mitochondrial substrates suppressed KATP, elevated [Ca2+]i, and corrected the insulin secretion defect. NAD(P)H responses to glucose were substantially reduced, and inhibitors of glycolytic NADH generation reproduced the mutant phenotype in normal islets. Flux of glucose through glycolysis in islets from mutant mice was reduced, as a result of which ATP generation in response to glucose was impaired. We conclude that hepatocyte nuclear factor-1alpha diabetes results from defective beta-cell glycolytic signaling, which is potentially correctable using substrates that bypass the defect.

Characterization of a Ca2+ release-activated nonselective cation current regulating membrane potential and [Ca2+]i oscillations in transgenically derived beta-cells.

Although stimulation of insulin secretion by glucose is regulated by coupled oscillations of membrane potential and intracellular Ca2+ ([Ca2+]i), the membrane events regulating these oscillations are incompletely understood. In the presence of glucose and tetraethylammonium, transgenically derived beta-cells (betaTC3-neo) exhibit coupled voltage and [Ca2+]i oscillations strikingly similar to those observed in normal islets in response to glucose. Using these cells as a model system, we investigated the membrane conductance underlying these oscillations. Alterations in delayed rectifier or Ca2+-activated K+ channels were excluded as a source of the conductance oscillations, as they are completely blocked by tetraethylammonium. ATP-sensitive K+ channels were also excluded, since the ATP-sensitive K+ channel blocker tolbutamide substituted for glucose in inducing [Ca2+]i oscillations. Thapsigargin, which depletes intracellular Ca2+ stores, and maitotoxin, an activator of nonselective cation channels, both converted the glucose-dependent [Ca2+]i oscillations into a sustained elevation. On the other hand, both SKF 96365, a blocker of Ca2+ store-operated channels, and external Na+ removal suppressed the glucose-stimulated [Ca2+]i oscillations. Maitotoxin activated a nonselective cation current in betaTC3 cells that was attenuated by removal of extracellular Na+ and by SKF 96365, in the same manner to a current activated in mouse beta-cells following depletion of intracellular Ca2+ stores. Currents similar to these are produced by the mammalian trp-related channels, a gene family that includes Ca2+ store-operated channels and inositol 1,4,5-trisphosphate-activated channels. We found several of the trp family genes were expressed in betaTC3 cells by reverse transcriptase polymerase chain reaction using specific primers, but by Northern blot analysis, mtrp-4 was the predominant message expressed. We conclude that a conductance underlying glucose-stimulated oscillations in beta-cells is provided by a Ca2+ store depletion-activated nonselective cation current, which is plausibly encoded by homologs of trp genes.

Genomic organization, chromosomal localization, tissue distribution, and biophysical characterization of a novel mammalian Shaker-related voltage-gated potassium channel, Kv1.7.

We report the isolation of a novel mouse voltage-gated Shaker-related K+ channel gene, Kv1.7 (Kcna7/KCNA7). Unlike other known Kv1 family genes that have intronless coding regions, the protein-coding region of Kv1.7 is interrupted by a 1.9-kilobase pair intron. The Kv1.7 gene and the related Kv3.3 (Kcnc3/KCNC3) gene map to mouse chromosome 7 and human chromosome 19q13.3, a region that has been suggested to contain a diabetic susceptibility locus. The mouse Kv1.7 channel is voltage-dependent and rapidly inactivating, exhibits cumulative inactivation, and has a single channel conductance of 21 pS. It is potently blocked by noxiustoxin and stichodactylatoxin, and is insensitive to tetraethylammonium, kaliotoxin, and charybdotoxin. Northern blot analysis reveals approximately 3-kilobase pair Kv1.7 transcripts in mouse heart and skeletal muscle. In situ hybridization demonstrates the presence of Kv1.7 in mouse pancreatic islet cells. Kv1.7 was also isolated from mouse brain and hamster insulinoma cells by polymerase chain reaction.

Expression and function of pancreatic beta-cell delayed rectifier K+ channels. Role in stimulus-secretion coupling.

Voltage-dependent delayed rectifier K+ channels regulate aspects of both stimulus-secretion and excitation-contraction coupling, but assigning specific roles to these channels has proved problematic. Using transgenically derived insulinoma cells (betaTC3-neo) and beta-cells purified from rodent pancreatic islets of Langerhans, we studied the expression and role of delayed rectifiers in glucose-stimulated insulin secretion. Using reverse-transcription polymerase chain reaction methods to amplify all known candidate delayed rectifier transcripts, the expression of the K+ channel gene Kv2.1 in betaTC3-neo insulinoma cells and purified rodent pancreatic beta-cells was detected and confirmed by immunoblotting in the insulinoma cells. betaTC3-neo cells were also found to express a related K+ channel, Kv3.2. Whole-cell patch clamp demonstrated the presence of delayed rectifier K+ currents inhibited by tetraethylammonium (TEA) and 4-aminopyridine, with similar Kd values to that of Kv2.1, correlating delayed rectifier gene expression with the K+ currents. The effect of these blockers on intracellular Ca2+ concentration ([Ca2+]i) was studied with fura-2 microspectrofluorimetry and imaging techniques. In the absence of glucose, exposure to TEA (1-20 mM) had minimal effects on betaTC3-neo or rodent islet [Ca2+]i, but in the presence of glucose, TEA activated large amplitude [Ca2+]i oscillations. In the insulinoma cells the TEA-induced [Ca2+]i oscillations were driven by synchronous oscillations in membrane potential, resulting in a 4-fold potentiation of insulin secretion. Activation of specific delayed rectifier K+ channels can therefore suppress stimulus-secretion coupling by damping oscillations in membrane potential and [Ca2+]i and thereby regulate secretion. These studies implicate previously uncharacterized beta-cell delayed rectifier K+ channels in the regulation of membrane repolarization, [Ca2+]i, and insulin secretion.

K+ channels: generating excitement in pancreatic beta-cells.

K+ channels play a key role in cellular physiology by regulating the efflux of K+ ions. They are the most diverse group of ion channel proteins; more than 30 K+ channel genes have been characterized. Regulated by ATP, voltage, and calcium, multiple K+ channels coexist in the beta-cell to regulate membrane potential, cell excitability, and insulin secretion. Recent developments at the molecular level have greatly expanded our understanding of beta-cell K+ channel structure and function, especially in regard to the ATP-sensitive K+ channel, the target for sulfonylurea drugs. Mutations in K+ channel genes underlie diseases as diverse as persistent hyperinsulinemia of infancy, cardiac long QT syndrome, cerebellar degeneration, and certain ataxias. These discoveries have identified new pharmacological targets for possible therapeutic intervention in the treatment of diabetes.

Activation of stimulus-secretion coupling in pancreatic beta-cells by specific products of glucose metabolism. Evidence for privileged signaling by glycolysis.

The energy requirements of most cells supplied with glucose are fulfilled by glycolytic and oxidative metabolism, yielding ATP. In pancreatic beta-cells, a rise in cytosolic ATP is also a critical signaling event, coupling closure of ATP-sensitive K+ channels (KATP) to insulin secretion via depolarization-driven increases in intracellular Ca2+ ([Ca2+]i). We report that glycolytic but not Krebs cycle metabolism of glucose is critically involved in this signaling process. While inhibitors of glycolysis suppressed glucose-stimulated insulin secretion, blockers of pyruvate transport or Krebs cycle enzymes were without effect. While pyruvate was metabolized in islets to the same extent as glucose, it produced no stimulation of insulin secretion and did not block KATP. A membrane-permeant analog, methyl pyruvate, however, produced a block of KATP, a sustained rise in [Ca2+]i, and an increase in insulin secretion 6-fold the magnitude of that induced by glucose. These results indicate that ATP derived from mitochondrial pyruvate metabolism does not substantially contribute to the regulation of KATP responses to a glucose challenge, supporting the notion of subcompartmentation of ATP within the beta-cell. Supranormal stimulation of the Krebs cycle by methyl pyruvate can, however, overwhelm intracellular partitioning of ATP and thereby drive insulin secretion.

NIDDM is associated with loss of pancreatic beta-cell L-type Ca2+ channel activity.

Development of non-insulin-dependent diabetes mellitus (NIDDM) is associated with defects in glucose-stimulated insulin secretion. We have investigated Zucker diabetic fatty rats (ZDF), an animal model of NIDDM, and found that, compared with control islets, the expression of mRNA encoding C- and D-isoforms of alpha 1-subunits of beta-cell L-type voltage-dependent Ca2+ channels (VDCC) was significantly reduced in islets isolated from ZDF rats. This correlated with a substantial reduction of L-type Ca2+ currents (ICa) in ZDF beta-cells. Intracellular Ca2+ concentration responses in ZDF islets after glucose, KCI, or BAY K 8644 stimulation were markedly attenuated, whereas responses evoked by carbachol were unimpaired, consistent with a specific decrease in ICa in the diabetic islets. This reduction was accompanied by loss of pulsatile insulin secretion from ZDF islets treated with oscillatory increases of external glucose concentration. Our findings suggest that the attenuation of ICa in diabetic islets may contribute to the abnormal glucose-dependent insulin secretory responses associated with NIDDM and indicate that this defect is caused by decreased expression of genes encoding beta-cell VDCC alpha 1-subunits.

Depletion of intracellular Ca2+ stores activates a maitotoxin-sensitive nonselective cationic current in beta-cells.

Glucose stimulation of beta-cell insulin secretion is initiated by membrane depolarization coupled with an elevation in intracellular Ca2+ concentration ([Ca2+]i). Both depolarization-dependent Ca2+ entry and intracellular Ca2+ store release contribute to the sugar-induced rise in [Ca2+]i. Here we show that maneuvers depleting intracellular Ca2+ stores induce membrane depolarization and a sustained nitrendipine-sensitive Ca2+ influx, whereas interventions promoting Ca2+ store refilling produce a hyperpolarization and inhibit Ca2+ influx. Both intracellular Ca2+ store depletion and maitotoxin activated a depolarizing nonselective cation current carried principally by Na+ in the physiological range of membrane potentials. The activation of such a current may form the paradigm by which excitable cells refill depleted intracellular Ca2+ stores by depolarization-driven opening of voltage-activated Ca2+ channels.

Delayed rectifier K+ channel overexpression in transgenic islets and beta-cells associated with impaired glucose responsiveness.

Glucose stimulation of pancreatic beta-cell insulin secretion is closely coupled to alterations in ion channel conductances and intracellular Ca2+ ([Ca2+]i). To further examine this relationship after augmentation of voltage-dependent K+ channel expression, transgenic mice were produced which specifically overexpress a human insulinoma-derived, tetraethylammonium (TEA)-insensitive delayed rectifier K+ channel in their pancreatic beta-cells as shown by immunoblot of isolated islets and immunohistochemical analysis of pancreas sections. Whole-cell current recordings confirmed the presence of high amplitude TEA-resistant K+ currents in transgenic islet cells, whose expression correlated with hyperglycemia and hypoinsulinemia. Stable overexpression of the channel in insulinoma cells attenuated glucose-activated increases in [Ca2+]i and prevented the induction of TEA-dependent [Ca2+]i oscillations. These results, employing the first ion channel transgenic mouse, demonstrate the importance of membrane potential regulation in excitation-secretion coupling in the pancreatic beta-cell.

Defective glucose-dependent endoplasmic reticulum Ca2+ sequestration in diabetic mouse islets of Langerhans.

Non-insulin-dependent diabetes mellitus (NIDDM) is a metabolic disease associated with abnormal insulin secretion, the underlying mechanisms of which are unknown. Glucose-dependent signal transduction pathways were investigated in pancreatic islets derived from the db/db mouse, an animal model of NIDDM. After stimulation with glucose (4-12 mM), the changes in intracellular Ca2+ concentration ([Ca2+]i) were different; unlike control islets, db/db islets lacked an initial reduction of [Ca2+]i and the subsequent [Ca2+]i oscillations following stimulation with 12 mM glucose. The severity of these defects in Ca2+ signaling correlated with the age-dependent development of hyperglycemia. Similarly defective glucose-induced Ca2+ signaling were reproduced in control islets by pre-exposure to thapsigargin, a selective inhibitor of endoplasmic reticulum (ER) Ca(2+)-ATPase. Estimation of ATPase activities from rates of ATP hydrolysis and by immunoblot hybridization with an antiserum directed against the sarco/endoplasmic reticulum Ca(2+)-ATPase both demonstrated that the ER Ca(2+)-ATPase was almost entirely absent from db/db islets. The effects of inhibition of ER Ca(2+)-ATPase on insulin secretion were also examined; a 4-day exposure of control islets to 1 microM thapsigargin resulted in basal and glucose-stimulated insulin secretion levels similar to those found in db/db islets. These results suggest that aberrant ER Ca2+ sequestration underlies the impaired glucose responses in the db/db mouse and may play a role in defective insulin secretion associated with NIDDM.

Thapsigargin inhibits the glucose-induced decrease of intracellular Ca2+ in mouse islets of Langerhans.

Stimulation of pancreatic islets of Langerhans with glucose results in changes in intracellular Ca2+ concentration ([Ca2+]i). With the use of mouse islets loaded with fura 2, the earliest glucose-induced alteration of [Ca2+]i was a pronounced decline in [Ca2+]i. This effect (phase 0) was evident 1 min after increasing extracellular glucose from 2 to 12 mM and was sustained for 3-5 min. Phase 0 was also observed when glucose was increased from 5 to 12 mM, indicating that it was not an experimental artifact resulting from substrate depletion. The [Ca2+]i-lowering effect of glucose was mimicked by D-glyceraldehyde but not by 2-deoxyglucose, pyruvate, glyburide, or 30 mM extracellular KCl. Mannoheptulose inhibited phase 0, whereas diazoxide, sodium azide, calmidazolium, or increasing extracellular [Ca2+] to 10 mM were all without effect. After the elevation of islet [Ca2+]i with 5 microM glyburide, 12 mM glucose caused a considerable transient decrease in [Ca2+]i. Under similar conditions, 5 mM caffeine attenuated phase 0, whereas 1 microM thapsigargin, a specific inhibitor of the sarcoplasmic and endoplasmic reticulum family of Ca(2+)-adenosinetriphosphatases (SERCA), almost completely inhibited any glucose-induced reduction of [Ca2+]i. These observations suggest that glucose causes an elevation of beta-cell SERCA activity triggered by factors generated during the cytosolic stages of glycolysis.

Endoplasmic reticulum calcium store regulates membrane potential in mouse islet beta-cells.

Glucose stimulation of islet beta-cell insulin secretion is initiated by membrane depolarization and an elevation in intracellular free calcium concentration ([Ca2+]i) from a combination of influx through depolarization-activated Ca2+ channels and intracellular Ca2+ store release. Prevention of Ca2+ store refilling with thapsigargin produced a sustained depolarization, leading to enhanced Ca2+ influx and an elevation in [Ca2+]i in 12 mM glucose. Depletion of intracellular Ca2+ stores by external EGTA reduced [Ca2+]i and also caused a long-lasting depolarization. In single beta-cells, external EGTA activated an inward current, the voltage range and kinetic properties of which differed from those of voltage-dependent Ca2+ channels. A novel pathway thus exists in beta-cells by which depletion of endoplasmic reticulum Ca2+ stores results in the activation of an inward current that, by inducing depolarization, facilitates Ca2+ influx through voltage-gated Ca2+ channels. The physiological relevance of this pathway in the control of beta-cell function is indicated by the stimulation of insulin secretion by thapsigargin.

Dependence on NADH produced during glycolysis for beta-cell glucose signaling.

An increase in cytosolic ATP following glucose metabolism by pancreatic beta-cells is the key signal initiating insulin secretion by causing blockade of ATP-dependent K+ channels (KATP). This induces membrane depolarization, leading to an elevation in cytosolic Ca2+ ([Ca2+]i) and insulin secretion. In this report we identify the critical metabolic step by which glucose initiates changes in beta-cell KATP channel activity, membrane potential, and [Ca2+]i. The signal stems from the glycolytic production of NADH during the oxidation of glyceraldehyde 3-phosphate, which is subsequently processed into ATP by mitochondria via the operation of discrete shuttle systems.

Amylin modulates beta-cell glucose sensing via effects on stimulus-secretion coupling.

The release of insulin from the pancreatic beta cell is dependent upon a complex interplay between stimulators and inhibitors. Recently, amylin, a peptide secreted by pancreatic beta cells, has been implicated in the development of type II (noninsulin dependent) diabetes through its modulation of the peripheral effects of insulin. However, the effect of amylin on insulin secretion from the beta cell has remained controversial. It is reported here that in single beta cells exhibiting normal glucose sensing, amylin causes membrane hyperpolarization, increases in net outward current, and reductions in insulin secretion. In contrast, in cells with abnormal glucose sensing (e.g., from db/db diabetic mice), amylin has no effect on electrical activity or secretion. Thus, amylin's effects on excitation-secretion coupling in the beta cell of the pancreas appear to be linked to the cell's capacity for normal glucose sensing.

Voltage-dependent intracellular calcium release from mouse islets stimulated by glucose.

Glucose-activated beta-cell insulin secretion depends upon elevation of intracellular calcium concentration, [Ca2+]i, which is thought to arise from Ca2+ influx through voltage-dependent calcium channels. Using fura-2-loaded mouse islets, we demonstrate, in fact, that the major component of the glucose-activated [Ca2+]i rise represents voltage-dependent intracellular Ca2+ release. Furthermore, the Ca2+ release pool possesses a novel pharmacology in that it is caffeine-sensitive but ryanodine-insensitive. In the absence of external Ca2+, glucose still caused intracellular Ca2+ release, an effect blockable by tetrodotoxin. However, depolarization of the islet with KCl in low Ca(2+)-containing solutions induced intracellular Ca2+ release, which was resistant to tetrodotoxin. We conclude that glucose release of intracellular Ca2+ is dependent upon depolarization alone, possibly through increasing inositol 1,4,5-trisphosphate production.

Calcium current regulation of depolarization-evoked calcium transients in beta-cells (HIT-T15).

Glucose-induced insulin secretion by beta-cells is linked to phasic increases in intracellular Ca2+ concentration ([Ca2+]i) arising from membrane depolarization. We examined the source of this Ca2+ in cultured beta-cells using rapid dual-wavelength spectroscopy of fura-2 under voltage-clamp conditions. Depolarization of the beta-cell initiated a sustained rise in [Ca2+]i that was dependent on the activation of L-type Ca2+ current that exhibited very slow inactivation. Neither release of internally stored Ca2+ nor Na(+)-Ca2+ exchange contributed significantly to this calcium rise, as evidenced by the suppressive effect of rapid application of Cd2+ and the lack of effect of elevations of intracellular Na+ concentration. Restoration of control Ca2+ levels was primarily dependent on Ca2+ channel closure, but both a voltage-dependent and voltage-independent Ca2+ efflux system also contributed. Both the fluorescence-based and charge-based estimates of the rise in [Ca2+]i were in broad agreement, indicating that Ca current activation was the primary source of the Ca2+ transient. The results suggest that nutrient-induced changes in beta-cell membrane potential tightly regulate [Ca2+]i, and thereby insulin release, primarily via alterations in the conductive state of slowly inactivating Ca2+ channels.

Divalent cations modulate the transient outward current in rat ventricular myocytes.

The modulation of the transient outward K+ current (Ito) by divalent cations was studied in enzymatically isolated rat ventricular myocytes with the whole cell patch-clamp technique. At holding potentials negative to -70 mV, 1 mM Cd2+ suppressed Ito, whereas, at potentials positive to -50 mV, the current was augmented. These effects were caused by shifts in the voltage dependence of both activation and inactivation of Ito toward more positive potentials. Cd2+ also slowed the activation kinetics of Ito by shifting the voltage dependence of its rate of activation, but the rate of inactivation was unaffected. Other divalent cations produced similar shifts but at markedly different concentrations. Thus, in the millimolar range, a rightward shift of approximately 20 mV was produced by 3 Co2+, 5 Ni2+, and 10 Ca2+, whereas 10 microM concentrations of Cu2+ and Zn2+ produced equivalent shifts. Similar effects were seen in hippocampal neurons with micromolar concentrations of Zn2+. Thus divalent cations have marked and specific effects on the kinetics and voltage dependence of Ito and may serve as a regulatory mechanism in its activation, particularly in cells with resting potentials positive to -60 mV.

The transient K+ current in rat ventricular myocytes: evaluation of its Ca2+ and Na+ dependence.

1. The transient outward K+ current (Ito) was studied in enzymatically isolated rat ventricular myocytes using the whole-cell patch clamp technique. 2. At holding potentials between -100 and -60 mV, depolarizing pulses activated outward current which was composed of transient and maintained components. These components differed from each other in their activation voltage range as well as in their kinetics of inactivation. 3. The transient component, in turn, appeared to be composed of rapidly and slowly inactivating components. Subtraction of ICa from the total current, or nifedipine pre-treatment, eliminated the slowly inactivating component of Ito indicating that the time course of inactivation of Ito may be contaminated by ICa. 4. Reduction of the holding potential from -100 mV to less negative holding potentials reduced all components of Ito, such that at holding potentials of -40 mV, very little or no Ito could be measured. 5. Elevation of [Ca2+]o activated Ito at holding potentials of -40 mV, and substitution of external Ca2+ by Sr2+ suppressed Ito, consistent with findings from other preparations and in support of a Ca(2+)-activated component of Ito. 6. Elevations of [Ca2+]o, however, also shifted the steady-state activation and inactivation parameters of the transient K+ current, such that a greater proportion of Ito channels were activated at the less negative holding potentials. 7. The shifts in the activation and inactivation parameters of the transient outward current were not mimicked by equivalent changes in external Mg2+. 8. Modulators of Ca2+ release from the sarcoplasmic reticulum (SR) such as caffeine and ryanodine suppressed Ito regardless of whether the myocytes were dialysed with low or high concentrations of Ca2+ buffers (EGTA or BAPTA, 0.5-14 mM) or whether nifedipine was used to block ICa. 9. 4-Aminopyridine (4-AP) blocked Ito in a dose-dependent manner, completely suppressing it at 10 mM. Similarly, tedisamil, a new K+ channel blocker, completely and reversibly blocked Ito at 5-20 microM concentrations. 10. TTX (10 microM) or removal of external Na+ decreased Ito, consistent with the idea that a component of Ito was Na+ activated. Both interventions, however, also shifted the voltage dependence of the activation and inactivation of Ito to more negative potentials, such that at -100 mV neither intervention had a significant effect on Ito. Alterations in [Na+]i had no effect on Ito.(ABSTRACT TRUNCATED AT 400 WORDS)

Tedisamil blocks the transient and delayed rectifier K+ currents in mammalian cardiac and glial cells.

The potassium currents in rat and guinea pig ventricular myocytes and mouse astrocytes were studied using tedisamil, a novel antiarrhythmic agent. A 1 to 20 microM dosage of tedisamil caused marked prolongation of the action potential in isolated rat ventricular myocytes, mimicking its reported effects on multicellular rat heart preparations. Under voltage clamp conditions, tedisamil caused a dose-dependent increase in the speed of inactivation of the transient outward K+ current (Ito), the predominant outward current in rat ventricular myocytes. In cardiac myocytes, the tedisamil block was neither use- nor voltage-dependent. The slow reversibility of drug action when applied from the outside, and its effectiveness when applied intracellularly, suggested an internal site of drug action. In guinea pig ventricular myocytes, tedisamil blocked the slowly developing time-dependent delayed rectifier K+ current (IK) over the same concentration range as that found for Ito in the rat myocytes. Tedisamil reduced this current without changing the characteristics of its slow (tau approximately 1 sec) activation. The effects of tedisamil on Ito and IK were independent of the phosphorylation state of the channel, as assessed by the equal effectiveness of the drug in the presence or absence of isoproterenol. Tedisamil also blocked the transient K+ current and the delayed rectifier current (IK) in mouse astrocytes over the same concentration range as that found in the cardiac myocytes and by a process that accelerated (transient K+ current) or mimicked (IK) inactivation. At concentrations of up to 50 microM, tedisamil had little effect on the time-dependent inward rectifier K+ current, or inward calcium current in rat or guinea pig ventricular myocytes.(ABSTRACT TRUNCATED AT 250 WORDS)