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.

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.

Cloning, expression, and distribution of a Ca(2+)-activated K+ channel beta-subunit from human brain.

We have cloned and expressed a Ca(2+)-activated K+ channel beta-subunit from human brain. The open reading frame encodes a 191-amino acid protein possessing significant homology to a previously described subunit cloned from bovine muscle. The gene for this subunit is located on chromosome 5 at band q34 (hslo-beta). There is no evidence for alternative RNA splicing of this gene product. hslo-beta mRNA is abundantly expressed in smooth muscle, but expression levels are low in most other tissues, including brain. Brain subregions in which beta-subunit mRNA expression is relatively high are the hippocampus and corpus callosum. The coexpression of hslo-beta mRNA together with hslo-alpha subunits in either Xenopus oocytes or stably transfected HEK 293 cells give rise to Ca(2+)-activated potassium currents with a much increased calcium and/or voltage sensitivity. These data indicate that the beta-subunit shows a tissue distribution different to that of the alpha-subunit, and in many tissues there may be no association of alpha-subunits with beta-subunits. These beta-subunits can play a functional role in the regulation of neuronal excitability by tuning the Ca2+ and/or the voltage dependence of alpha-subunits.

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.

Cloning, expression, and distribution of functionally distinct Ca(2+)-activated K+ channel isoforms from human brain.

We have cloned and expressed nine Ca(2+)-activated K+ channel isoforms from human brain. The open reading frames encode proteins ranging from 1154 to 1195 amino acids, and all possess significant identity with the slowpoke gene products in Drosophila and mouse. All isoforms are generated by alternative RNA splicing of a single gene on chromosome 10 at band q22.3 (hslo). RNA splicing occurs at four sites located in the carboxy-terminal portion of the protein and gives rise to at least nine ion channel constructs (hbr1-hbr9). hslo mRNA is expressed abundantly in human brain, and individual isoforms show unique expression patterns. Expression of hslo mRNA in Xenopus oocytes produces robust voltage and Ca(2+)-activated K+ currents. Splice variants differ significantly in their Ca2+ sensitivity, suggesting a broad functional role for these channels in the regulation of neuronal excitability.

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.

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.

High activity of mitochondrial glycerol phosphate dehydrogenase in insulinomas and carcinoid and other tumors of the amine precursor uptake decarboxylation system.

The activity of the mitochondrial glycerol phosphate dehydrogenase (EC 1.1.99.5), the enzyme unique to the glycerol phosphate hydrogen shuttle, was measured in normal human tissues and tumors and compared with the activity of succinate dehydrogenase, another enzyme that transfers electrons to ubiquinone at site II of the electron transport chain. Six of 7 insulinomas and 10 of 12 carcinoid tumors showed high glycerol phosphate dehydrogenase activity. The activity was also increased in 3 of 4 gastrinomas, 2 paraganglionomas, 1 of 4 thyroid nodules, and 1 parathyroid tumor. These tissues belong to the amine precursor uptake decarboxylation system. The activity of glycerol phosphate dehydrogenase was generally unremarkable in non-amine precursor uptake decarboxylation system tumors and in normal tissues studied. However, 1 of 2 breast carcinomas, 1 submandibular tumor, and 2 of 3 melanomas were enriched in glycerol phosphate dehydrogenase activity. In general, succinate dehydrogenase activity exceeded that of glycerol phosphate dehydrogenase in all tissues except some of the tissues in which glycerol phosphate dehydrogenase activity was high. Normal tissues, such as the pancreatic beta-cell, which aerobically metabolize glucose rapidly utilize the glycerol phosphate shuttle to oxidize the large amount of NADH formed from glucose metabolism in the cytosol. Whether this is the reason for the enriched activity of the glycerol phosphate dehydrogenase in certain amine precursor uptake decarboxylation system tumors is unknown.

Purification, renaturation, and reconstituted protein kinase activity of the Sendai virus large (L) protein: L protein phosphorylates the NP and P proteins in vitro.

Sodium dodecyl sulfate-solubilized Sendai virus large (L) protein was highly purified by a one-step procedure, using hydroxylapatite column chromatography. Monoclonal antibodies addressed to the carboxyl-terminal amino acid sequence of the L protein were used for monitoring L protein during purification. By removing sodium dodecyl sulfate from purified L protein, a protein kinase activity was successfully renatured. P and NP proteins served as its substrates. After immunoprecipitation with anti-L antibodies, the immunocomplex already showed protein kinase activity. In the presence of P protein, the NP protein was more highly phosphorylated. The results show that Sendai virus L protein possesses a protein kinase activity phosphorylating the other proteins of the viral nucleocapsid in vitro.

Interleukin-1 beta inhibits glucokinase activity in clonal HIT-T15 beta-cells.

Interleukin-1 beta (IL-1 beta) has been implicated in the pathogenesis of insulin-dependent diabetes mellitus. In the present study we have investigated the effects of IL-1 beta on glucose metabolism in clonal HIT-T15 beta cells. In the short-term (1 h), 25 U/ml IL-1 beta significantly increased the rates of insulin release and glucose utilisation, but not glucose oxidation. In contrast, after 48 h, IL-1 beta inhibited insulin release and glucose utilisation and oxidation. By assaying enzymes (hexokinase, glucokinase, pyruvate dehydrogenase, glucose 6-phosphatase) and nucleotides (ATP, ADP) associated with the regulation of glycolysis and glucose oxidation, we conclude that the inhibitory effects of IL-1 beta may be due to impaired glucokinase activity.

Insulin-secreting beta-cells possess specific receptors for interleukin-1 beta.

The effect of the cytokine interleukin-1 beta on the insulin secretory responsiveness of single beta-cells (HIT-T15) was investigated. In the short-term, IL-1 beta induced a dosage-dependent stimulation of insulin release. In contrast, in the long-term, IL-1 beta, inhibited both basal and secretagogue-stimulated insulin secretion. We also demonstrate the simultaneous presence of specific high and low affinity binding sites for IL-1 beta on beta-cells. IL-1 beta, which has been implicated in the pathogenesis of insulin-dependent diabetes, may therefore mediate its opposing effects on beta-cells through a specific plasma membrane receptor.

Nuclear factor E2F mediates basic transcription and trans-activation by E1a of the human MYC promoter.

Transcription from one of the two initiation sites, P1 and P2, of the dual human MYC promoter seems to be essential in all proliferating cells. To identify proteins and target structures for MYC regulation, a DNA region was analyzed that is critical for P2 promoter activity. Here, we show that a nuclear factor binds to a DNA element within P2, which is conserved perfectly between mouse and man and displays a striking homology to the E1a-inducible E2 promoter of adenovirus type 5 (Ad5). We demonstrate that the same transcription factor, defined recently as E2F, which plays an essential role in the activation of adenovirus early promoters and enhancers, also interacts as a dominant nuclear factor with the MYC promoter. The presence of an intact E2F binding site is required for basic expression and for trans-activation of the P2 promoter by E1a proteins. The human MYC promoter is the first cellular target described for E2F. The results suggest that expression of MYC might be regulated via modulation of E2F by cellular 'E1a-like' factors.

Effect of esters of succinic acid and other citric acid cycle intermediates on insulin release and inositol phosphate formation by pancreatic islets.

Esters of carboxylic acids are permeable to cells and once inside the cell are hydrolyzed to carboxylic acids. Methyl and ethyl esters of succinate and other citric acid cycle intermediates were tested to find out whether they are insulin secretagogues. Monomethyl succinate stimulated insulin release from pancreatic islets in a concentration-dependent manner with maximal release attained at a concentration of 10 mM. Dimethyl succinate (10 mM) was as effective as monomethyl succinate, but pyruvate methyl ester, monoethyl succinate, and dimethyl fumarate were ineffective as primary secretagogues. However, dimethyl fumarate potentiated both leucine- and leucine-plus-glutamine-induced insulin release. Glucose, leucine, leucine plus glutamine, and monomethyl succinate increased inositol tris-, bis- and monophosphate formation in pancreatic islets and antimycin A inhibited this formation. Since mitochondrial metabolism is probably essential for glucose-induced insulin release and the metabolism of succinate and leucine (without or with glutamine) involves mitochondrial respiration exclusively, these results might indicate that mitochondrial metabolism generates conditions or factors that are transmitted to the cytosol to increase inositol trisphosphate formation and thus calcium mobilization and insulin release. Since succinate is believed to enter metabolism at site II of the mitochondrial respiratory chain, it is interesting that rotenone, an inhibitor of NADH dehydrogenase and site I of the respiratory chain, was a potent inhibitor of monomethyl succinate-induced insulin released. Rotenone also inhibited leucine (plus or minus glutamine)-induced insulin release. These results indicate that beta cell metabolism of monomethyl succinate and leucine, like glucose, influences dehydrogenases that produce NADH.

Carrier-bound synthetic peptides. Use as antigen in HIV-1 ELISA tests and in antiserum production.

Chemically synthesize carrier-bound peptides have been used as antigens in diagnostic test systems (ELISA) and for raising antipeptide-specific antisera. The method does not require prior cleavage of the peptides from the support used for the solid-phase synthesis. Using the same resin for both the synthesis and the subsequent applications it was possible to avoid expensive and time-consuming purification procedures and artificial recoupling to solid supports. A quick and specific ELISA-based diagnostic test system for HIV-specific antipeptide antibodies in human sera was established. In addition the carrier-bound peptides were shown to be potent antigens for raising antibodies in animals.

Glyceraldehyde phosphate: an insulin secretagogue with possible effects on inositol phosphate formation in pancreatic islets.

The insulinotropic action of glucose, the most potent physiologic insulin secretagogue, involves its metabolism. However, no glucose metabolite has ever been identified as a key intermediate. We tested the abilities of a number of glucose metabolites to stimulate insulin release from pancreatic islets. Of all of these metabolites, glyceraldehyde 3-phosphate was the most potent insulin secretagogue. In numerous experiments over 3 years, insulin release by 4 mM glyceraldehyde phosphate ranged from 50 to 200% of that initiated by 16.7 mM glucose--a near-maximal insulin stimulus. At concentrations of 1 and 4 mM, glyceraldehyde phosphate was even more potent than the known secretagogues glucose and glyceraldehyde. Glucose metabolites were also tested for their ability to stimulate inositol tris-, bis-, and monophosphate formation by permeabilized islets. Only glyceraldehyde phosphate stimulated inositol phosphate formation and this stimulation occurred at concentrations of glyceraldehyde phosphate which could be present in the beta cell under physiologic conditions (K0.5 = 25 microM). The current results are consistent with the idea that glyceraldehyde phosphate is a key insulinotropic glucose metabolite that might act directly (or rather directly via a receptor) on the phospholipase C that forms inositol trisphosphate in the plasma membrane.

[Ethically chosen studies with early-weaned piglets during their raising in pens with different applications of straw. 1. The effects of different applications of straw and different floor conditions in areas of uniform size]. / Ethologische Wahlversuche mit frühabgesetzten Ferkeln während der Haltung in Buchten mit unterschiedlicher Anwendung von Stroh. 1. Mitteilung: Auswirkungen verschiedener Anwendungen des Strohes und unterschiedlicher Bodenbeschaffenheit bei einheitlicher Flächengrösse.

In extension of earlier experiments in housing systems with perforated floors, utilization rates of solid floor with litter, straw in a rack and deep litter, as well as the behaviour and physical condition, especially of claws, were investigated. Each pen had an area of 0.45 m2/piglet. In further experiments the influences of race (DL instead of DL x Piétrain), kind of rearing (without straw instead of rearing with straw), lowering temperature and the effect of a preferred perforated floor in comparison with solid floor were investigated. Deep litter was preferred for activity and lying behaviour only in the case of low temperature (14-18 degrees C) whereas solid floor with litter was preferred in the case of higher temperature (19-25 degrees C). The perforated floor was equivalent to solid floor on the condition that the area dimension was sufficient, temperature 3 degrees C higher than in the other experiments and straw in a rack was offered. However, the utilization rates of solid floor increased in the case of lowering temperature by 4 degrees C. There was no significant influences of the different race or kind of rearing. In the experiments there were significant signs of a more harmonious condition of the animals and a shorter, respectively no period where the animals had to adapt to the housing conditions. These findings were in contrast to those of earlier investigations. The possibility of acting with straw led to an undisturbed circadian rhythm and to a higher rate of standing within the total activity which was as high as in earlier studies. Further social companions and environmental objects lost attractiveness.(ABSTRACT TRUNCATED AT 250 WORDS)