Glucose Decreases Na+,K+-ATPase Activity in Pancreatic β-Cells

Abstract

In the pancreatic β-cell, glucose-induced membrane depolarization promotes opening of voltage-gatedl-type Ca2+ channels, an increase in cytoplasmic free Ca2+ concentration ([Ca2+]i), and exocytosis of insulin. Inhibition of Na+,K+-ATPase activity by ouabain leads to β-cell membrane depolarization and Ca2+ influx. Because glucose-induced β-cell membrane depolarization cannot be attributed solely to closure of ATP-regulated K+ channels, we investigated whether glucose regulates other transport proteins, such as the Na+,K+-ATPase. Glucose inhibited Na+,K+-ATPase activity in single pancreatic islets and intact β-cells. This effect was reversible and required glucose metabolism. The inhibitory action of glucose was blocked by pretreatment of the islets with a selective inhibitor of a Ca2+-independent phospholipase A2. Arachidonic acid, the hydrolytic product of this phospholipase A2, also inhibited Na+,K+-ATPase activity. This effect, like that of glucose, was blocked by nordihydroguaiaretic acid, a selective inhibitor of the lipooxygenase metabolic pathway, but not by inhibitors of the cyclooxygenase or cytochrome P450-monooxygenase pathways. The lipooxygenase product 12(S)-HETE (12-S-hydroxyeicosatetranoic acid) inhibited Na+,K+-ATPase activity, and this effect, as well as that of glucose, was blocked by bisindolylmaleimide, a specific protein kinase C inhibitor. Moreover, glucose increased the state of α-subunit phosphorylation by a protein kinase C-dependent process.These results demonstrate that glucose inhibits Na+,K+-ATPase activity in β-cells by activating a distinct intracellular signaling network. Inhibition of Na+,K+-ATPase activity may thus be part of the mechanisms whereby glucose promotes membrane depolarization, an increase in [Ca2+]i, and thereby insulin secretion in the pancreatic β-cell. In the pancreatic β-cell, glucose-induced membrane depolarization promotes opening of voltage-gatedl-type Ca2+ channels, an increase in cytoplasmic free Ca2+ concentration ([Ca2+]i), and exocytosis of insulin. Inhibition of Na+,K+-ATPase activity by ouabain leads to β-cell membrane depolarization and Ca2+ influx. Because glucose-induced β-cell membrane depolarization cannot be attributed solely to closure of ATP-regulated K+ channels, we investigated whether glucose regulates other transport proteins, such as the Na+,K+-ATPase. Glucose inhibited Na+,K+-ATPase activity in single pancreatic islets and intact β-cells. This effect was reversible and required glucose metabolism. The inhibitory action of glucose was blocked by pretreatment of the islets with a selective inhibitor of a Ca2+-independent phospholipase A2. Arachidonic acid, the hydrolytic product of this phospholipase A2, also inhibited Na+,K+-ATPase activity. This effect, like that of glucose, was blocked by nordihydroguaiaretic acid, a selective inhibitor of the lipooxygenase metabolic pathway, but not by inhibitors of the cyclooxygenase or cytochrome P450-monooxygenase pathways. The lipooxygenase product 12(S)-HETE (12-S-hydroxyeicosatetranoic acid) inhibited Na+,K+-ATPase activity, and this effect, as well as that of glucose, was blocked by bisindolylmaleimide, a specific protein kinase C inhibitor. Moreover, glucose increased the state of α-subunit phosphorylation by a protein kinase C-dependent process. These results demonstrate that glucose inhibits Na+,K+-ATPase activity in β-cells by activating a distinct intracellular signaling network. Inhibition of Na+,K+-ATPase activity may thus be part of the mechanisms whereby glucose promotes membrane depolarization, an increase in [Ca2+]i, and thereby insulin secretion in the pancreatic β-cell. ATP-regulated K+ channels protein kinase C bisindolylmaleimide haloenol lactone (E)-6-(bromomethylene)-3-(1-naphtalenyl)-2H-tetrahydropyran-2-one arachidonic acid indometacin ethoxyresorufin nordihydroguaiaretic acid Ca2+-independent phospholipase A2 analysis of variance 12-S-hydroxyeicosatetranoic acid. Glucose metabolism increases the ATP:ADP ratio and initiates the β-cell stimulus-secretion coupling by closing ATP-regulated potassium (K+ATP)1channels (1Ashcroft F.M. Harrison D.E. Ashcroft S.J.H. Nature. 1984; 312: 446-448Crossref PubMed Scopus (920) Google Scholar). These channels are the main regulators of the β-cell resting membrane potential, and their closure will initiate membrane depolarization, opening of voltage-gated l-type Ca2+ channels, and thereby an increase in cytoplasmic free Ca2+ concentration ([Ca2+]i) (2Prentki M. Matschinsky F.M. Physiol. Rev. 1987; 67: 1185-1248Crossref PubMed Google Scholar, 3Zawalich W.S. Rasmussen H. Mol. Cell. Endocrinol. 1990; 70: 119-137Crossref PubMed Scopus (123) Google Scholar, 4Berggren P.-O. Larsson O. Biochem. Soc. Trans. 1994; 22: 12-18Crossref PubMed Scopus (80) Google Scholar, 5Ashcroft F.M. Ashcroft S.J.H. Harrison D.E. J. Physiol. 1988; 400: 501-527Crossref PubMed Scopus (130) Google Scholar). However, depolarization of the plasma membrane from resting potential to threshold potential of the voltage-dependent Ca2+ channels requires, in addition to closure of K+ATP channels, an inward current (6Ashcroft F.M. Rorsman P. Biophys. Mol. Biol. 1989; 54: 87-143Crossref PubMed Scopus (977) Google Scholar). The origin of this inward current is not known, but it has been suggested that the current is carried by Na+ or Ca2+ (6Ashcroft F.M. Rorsman P. Biophys. Mol. Biol. 1989; 54: 87-143Crossref PubMed Scopus (977) Google Scholar,7Henquin J.C. Meissner H.P. Experientia. 1984; 40: 1043-1052Crossref PubMed Scopus (292) Google Scholar). Thus, glucose may activate an as yet unidentified component affecting β-cell membrane potential. Further support for the proposition that glucose has effects on membrane potential distinct from closing K+ATPchannels was provided by studies of action potential frequency in the presence of tolbutamide and glucose (8Bozem M. Henquin J.-C. Pflügers Arch. 1988; 413: 147-152Crossref PubMed Scopus (16) Google Scholar). These studies show that stimulatory concentrations of glucose have the ability to induce an increased frequency of action potentials that is significantly higher than what is observed in the presence of maximal concentrations of the K+ATP channel blocker tolbutamide, thus suggesting the existence of an additional regulator of β-cell membrane potential that might be affected by glucose metabolism. The Na+,K+-ATPase is involved in maintaining the Na+ and K+ gradients across the β-cell plasma membrane. It extrudes three Na+ ions in exchange for two K+ ions, generating a net outward flow of cations through the cell membrane. This makes the pump electrogenic and results in a hyperpolarizing effect on membrane potential. Consequently, inhibition of Na+,K+-ATPase activity (for example, by ouabain) leads to β-cell membrane depolarization (7Henquin J.C. Meissner H.P. Experientia. 1984; 40: 1043-1052Crossref PubMed Scopus (292) Google Scholar, 9Nilsson T. Arkhammar P. Rorsman P. Berggren P.-O. J. Biol. Chem. 1988; 263: 1855-1860Abstract Full Text PDF PubMed Google Scholar) and Ca2+ influx (9Nilsson T. Arkhammar P. Rorsman P. Berggren P.-O. J. Biol. Chem. 1988; 263: 1855-1860Abstract Full Text PDF PubMed Google Scholar, 10Grapengiesser E. Berts A. Saha S. Lund P.-E. Gylfe E. Hellman B. Arch. Biochem. Biophys. 1993; 300: 372-377Crossref PubMed Scopus (29) Google Scholar). It has therefore been postulated that a decrease in Na+,K+-ATPase-mediated ion gradients may be a contributing mechanism to insulin secretion (7Henquin J.C. Meissner H.P. Experientia. 1984; 40: 1043-1052Crossref PubMed Scopus (292) Google Scholar, 8Bozem M. Henquin J.-C. Pflügers Arch. 1988; 413: 147-152Crossref PubMed Scopus (16) Google Scholar, 9Nilsson T. Arkhammar P. Rorsman P. Berggren P.-O. J. Biol. Chem. 1988; 263: 1855-1860Abstract Full Text PDF PubMed Google Scholar). The precise role of the Na+,K+-ATPase in membrane depolarization and its possible regulation by glucose in β-cells have been difficult to define, primarily because data reporting changes in enzyme activity have been obtained in cell homogenates or membrane preparations, lacking intact intracellular signaling pathways. Although it was reported that glucose had an inhibitory effect on Na+,K+-ATPase activity (11Levin S.R. Kasson B.G. Driessen J.F. J. Clin. Invest. 1978; 62: 692-701Crossref PubMed Scopus (55) Google Scholar), others failed to demonstrate significant changes in enzyme activity (12Gronda C.M. Rossi J.P. Gagliardino J.J. Methods Find. Exp. Clin. Pharmacol. 1989; 11: 341-344PubMed Google Scholar). Nevertheless, none of the studies mentioned above have examined short-term effects of glucose, i.e. within the time frame matching the initial cellular events in the insulin secretory process. Immunoprecipitation of the Na+,K+-ATPase α-subunit was performed using a polyclonal antibody raised against the whole enzyme (kindly provided by E. Féraille, University of Geneva, Switzerland). Fura-2/acetoxymethyl ester, ouabain, mannoheptulose, arachidonic acid, ethoxyresorufin, nordihydroguaiaretic acid, indometacin, bisindolylmaleimide, and 12,13-phorbol dibutyrate were purchased from Sigma. Haloenol lactone (E)-6-(bromomethylene)-3-(1-naphtalenyl)-2H-tetrahydropyran-2-one (HELSS) was obtained from Calbiochem (San Diego, CA). 12(S)-HETE, 12(R)-HETE, 20-HETE, 5-HETE, and 15-HETE were purchased from Cayman Chemicals (Ann Arbor, MI); all of these compounds were dissolved in ethanol (final concentration, <0.001%) except for HELSS, which was dissolved in Me2SO (final concentration, <0.01%). AA was dissolved in ethanol under N2 flow, and the stock solution was stored at −20 °C protected from light. Appropriate control with the diluents was exercised. All experiments were performed in single isolated pancreatic islets and in β-cells obtained from adult (12 months old) obese (ob/ob) mice from a local colony (13Hellman B. Ann. N. Y. Acad. Sci. 1965; 13: 541-558Crossref Scopus (306) Google Scholar). The mice were killed by decapitation. Pancreatic islets were isolated by collagenase digestion. Cells were obtained by dissociation of islets into single cells and small cell clusters (14Lernmark Å. Diabetologia. 1974; 10: 431-438Crossref PubMed Scopus (296) Google Scholar,15Nilsson T. Arkhammar P. Hallberg A. Hellman B. Berggren P.-O. Biochem. J. 1987; 248: 329-336Crossref PubMed Scopus (71) Google Scholar). This preparation consists of 90–95% β-cells (15Nilsson T. Arkhammar P. Hallberg A. Hellman B. Berggren P.-O. Biochem. J. 1987; 248: 329-336Crossref PubMed Scopus (71) Google Scholar). The medium used for and cell and the and Na+,K+-ATPase activity was of β-cells were in medium with and Cells were on or in by islets were isolated and of to the of well of a well were on was time to glucose under the were by the well to of Na+,K+-ATPase medium was and activity in were to a at −20 in to the plasma membrane to The was in to and to °C for additional The was by the well on and of the of well was to and for and an from the the was in a In to and Na+,K+-ATPase activity were in islets and the of these Na+,K+-ATPase data Na+,K+-ATPase activity was as of Na+,K+-ATPase activity was as above J.F. A. P. Nature. 1990; PubMed Scopus Google Scholar). isolated cells in were at with the The was by of the to of the cell concentration, were to the Na+,K+-ATPase medium (final Cells were to a at −20 in to the plasma membrane to The were at °C for The was by to °C and addition of a of the for the the in the was Na+,K+-ATPase activity was as the and in a medium of Na+ and K+ and in the presence of ouabain was performed to Biochem. 1974; Scopus Google Scholar), using a CA). in were for at °C in a the glucose, and with the addition of with or glucose, in the presence or of bisindolylmaleimide, were performed at The were by the medium and addition of Immunoprecipitation of the Na+,K+-ATPase α-subunit was performed as Berggren P.-O. J. Physiol. PubMed Google Scholar). were at °C with of polyclonal antibody and the addition of protein were by using the Nature. PubMed Scopus Google Scholar). were to and to were performed on of β-cells as H. M. T. Arkhammar P. Rorsman P. S. Berggren P.-O. PubMed Scopus Google Scholar, P. Larsson O. M. E. Å. M. Berggren P.-O. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). β-cells for on were in medium for with Fura-2/acetoxymethyl the were performed in a to an in in the to cells were in a H. M. T. Arkhammar P. Rorsman P. S. Berggren P.-O. PubMed Scopus Google Scholar, P. Larsson O. M. E. Å. M. Berggren P.-O. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). The and were and The results are as and of the were used A. E. B. Pflügers Arch. PubMed Scopus Google Scholar, A. J. Physiol. 1988; PubMed Scopus Google Scholar, Rorsman P. T. Pflügers Arch. PubMed Scopus Google Scholar). were from and with the had and were from or membrane activity in was at potential single channel activity was from intact cells at potential was using the using as and were using an CA). the current and were stored using a and a are to the with outward In all the solution the and with the the solution of the and with were into and was to for channel was and were to In the the solution of the solution of membrane potential were using the with and B. All intact cell experiments and were at the experiments were performed at The had a of and cells were at a of All compounds were to the was with in at are as of the data was performed using the or with with were In this we examined the to which changes in Na+,K+-ATPase activity to β-cell membrane depolarization and thereby to opening of l-type voltage-dependent Ca2+ channels and influx of Cells were to ouabain, a concentration that maximal inhibition of the pancreatic β-cell Na+,K+-ATPase activity. The effect of ouabain on β-cell membrane potential was using the of the Cells were with a concentration of of ouabain to the medium in depolarization of membrane potential which increased from a resting potential of to a potential of which action potentials were The effect of ouabain on was in small clusters of cells of ouabain to cells in the presence of glucose a and reversible increase above in with glucose not significantly increase above ouabain) with the increase obtained with ouabain The increase in by ouabain was in the of Ca2+ and addition of Ca2+ to the Moreover, the presence of a voltage-dependent l-type the increase in The Na+,K+-ATPase activity is inhibited by Ca2+ Rev. Physiol. 1988; PubMed Scopus Google Scholar). Although this effect has not been reported to in intact cells but in a cell free the effect of glucose on Na+,K+-ATPase activity was examined Ca2+ from the In the of Ca2+ and presence of glucose was to Na+,K+-ATPase activity of Because the K+ATP channel is in pancreatic β-cell membrane potential F.M. Ashcroft S.J.H. Harrison D.E. J. Physiol. 1988; 400: 501-527Crossref PubMed Scopus (130) Google Scholar, F.M. Rorsman P. Biophys. Mol. Biol. 1989; 54: 87-143Crossref PubMed Scopus (977) Google Scholar), may whether the effect of ouabain is a an effect on this these we K+ATP channel activity in as well as in intact in ouabain had effect on channel activity in suggesting that ouabain has effect on the K+ATP channel It has been reported that ouabain a in the of K+ATP channels in intact β-cells and that this might be the mechanism whereby ouabain the β-cell membrane and thereby increases E. Berts A. Saha S. Lund P.-E. Gylfe E. Hellman B. Arch. Biochem. Biophys. 1993; 300: 372-377Crossref PubMed Scopus (29) Google Scholar). this with regulation of Na+,K+-ATPase activity, we channel activity using the of the increase the of channels and thereby the of channel we performed the experiments in the of in ouabain not channel activity within of the time which the cell and an increase in is K+ATP channel were not not control to to induce action potentials in the β-cell with a threshold concentration of glucose the effect of glucose on Na+,K+-ATPase activity in single isolated pancreatic of single medium islets of with glucose in a and inhibition of Na+,K+-ATPase activity and activity was not affected by glucose glucose, of The inhibitory effect of glucose was reversible to glucose for at the medium was and with a medium glucose for additional this Na+,K+-ATPase activity to control In for glucose to as a it has to be glucose to be in to Na+,K+-ATPase activity. The presence of an concentration of the inhibitor the inhibitory effect of glucose glucose to be and the of an intact intracellular signaling were by the of effect of glucose on Na+,K+-ATPase activity in a to what has been reported (12Gronda C.M. Rossi J.P. Gagliardino J.J. Methods Find. Exp. Clin. Pharmacol. 1989; 11: 341-344PubMed Google Scholar). Na+,K+-ATPase activity was also in β-cells in Na+,K+-ATPase activity of of in this preparation was the was β-cells were in glucose for at increase in the glucose concentration a decrease in Na+,K+-ATPase activity, with a maximal effect at of glucose in enzyme activity were also and as as glucose and for at The activity was not affected by glucose glucose, not the effect of glucose on Na+,K+-ATPase activity in β-cell was reversible The inhibitory action of glucose its metabolism and was a cell preparation was The pancreatic β-cell is with a phospholipase A2 that is in a Ca2+-independent by the increase in from glucose metabolism J. Biol. Chem. Full Text PDF PubMed Google Scholar, S. T. 1993; PubMed Scopus Google Scholar). This phospholipase is inhibited by It was reported that pretreatment of pancreatic β-cells with the increase in and insulin secretion by glucose but not or S. J. 1993; PubMed Scopus Google Scholar). Pancreatic β-cells from mice with for at not Na+,K+-ATPase activity the inhibitory effect of glucose of β-cell phospholipase A2 by glucose results in of membrane and the increase in the of arachidonic acid S. T. 1993; PubMed Scopus Google Scholar). of single islets with arachidonic acid in a inhibition of Na+,K+-ATPase activity with a inhibitory concentration of the effect of glucose and AA on Na+,K+-ATPase activity in the presence and of selective inhibitors of the three main of AA metabolism. The cyclooxygenase was inhibited with the cytochrome with ethoxyresorufin B. M. J. Physiol. Google Scholar), and the lipooxygenase with nordihydroguaiaretic acid J. Pharmacol. PubMed Scopus Google Scholar). In with inhibitor for as as at not Na+,K+-ATPase activity in M. T. J. Biol. PubMed Scopus Google Scholar). none of the inhibitors affected the enzyme activity in isolated pancreatic islets However, the inhibitory effect of glucose or AA on Na+,K+-ATPase activity was blocked by of the lipooxygenase inhibitor In the inhibition of Na+,K+-ATPase by glucose or Because the effect of glucose was by we examined whether 12(S)-HETE, the main lipooxygenase in pancreatic β-cells S. J. J. Physiol. 263: Google Scholar), inhibited Na+,K+-ATPase activity. 12(S)-HETE inhibited Na+,K+-ATPase activity in a with a inhibitory concentration of have also the effect of 12(S)-HETE with that of other on Na+,K+-ATPase activity significantly inhibited Na+,K+-ATPase activity in the concentration of the a inhibition of Na+,K+-ATPase activity as in cells M. T. J. Biol. PubMed Scopus Google Scholar). The lipooxygenase and 15-HETE not significantly Na+,K+-ATPase of on activity from single isolated of F.M. 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It that the signaling by glucose metabolism and increased cellular in cells is by of phospholipase A2 and of AA and 12(S)-HETE The 12(S)-HETE and the Na+,K+-ATPase may phosphorylation of Na+,K+-ATPase to what has been in Berggren P.-O. J. Physiol. PubMed Google Scholar, A. P. Acad. Sci. S. A. PubMed Scopus Google Scholar, J.P. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar, E. Berggren P.-O. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The that 12(S)-HETE A. J. M. B. J. Biochem. Biophys. PubMed Scopus Google Scholar, 1984; PubMed Scopus Google Scholar, Y. 1989; PubMed Scopus Google Scholar, S. O. P. A. J. Clin. Invest. PubMed Scopus Google Scholar) is by the that the inhibitory effect of glucose or 12(S)-HETE on Na+,K+-ATPase activity was by the presence of a inhibitor and that in the state of phosphorylation of the Na+,K+-ATPase α-subunit was increased by glucose in a that was The that the and are in to glucose S. S. Arkhammar P. Berggren P.-O. Acad. Sci. S. A. PubMed Scopus Google Scholar) may that or of these are involved in the regulation of Na+,K+-ATPase activity. The electrogenic of the Na+,K+-ATPase has been well in E. T. of Scholar). In pancreatic inhibition or of the pump is by changes in membrane potential J.C. Meissner H.P. J. Physiol. PubMed Scopus (55) Google Scholar, Meissner H.P. J. Physiol. Scholar), that the pump is electrogenic also in this Thus, as the Na+,K+-ATPase is it depolarization of the β-cell plasma membrane J.C. Meissner H.P. J. Physiol. PubMed Scopus (55) Google Scholar). The that glucose a inhibitory effect of the Na+,K+-ATPase that part of the effect of glucose is to a decrease in the This the Na+,K+-ATPase to to the resting membrane potential, as well as to as a of activity to glucose have that of activity not the effect of glucose on cytoplasmic free Ca2+ concentration, effects on membrane potential or insulin secretion P. Larsson O. M. E. Å. M. Berggren P.-O. J. Biol. 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However, in activity was by with Because this will the action of that are three of we performed experiments in the presence of a specific inhibitor of such as The data and that the role of in glucose-induced insulin secretion be to be of a than of a In the we that glucose-induced inhibition of the Na+,K+-ATPase is by However, it is that the effect of this inhibition in β-cell membrane depolarization be and may therefore not be the 12(S)-HETE not membrane potential at resting threshold and stimulatory concentrations of we that inhibition of Na+,K+-ATPase activity by but not ouabain, is not to the plasma membrane and that a action of glucose on channels and Na+,K+-ATPase is to such an This is also in with the of effect of 12(S)-HETE on insulin with the that not membrane potential, these results support to the that inhibition of Na+,K+-ATPase activity not have a effect but a effect on insulin results not support the that the effect of ouabain is to a K+ATP channels activity, as suggested E. Berts A. Saha S. Lund P.-E. Gylfe E. Hellman B. Arch. Biochem. Biophys. 1993; 300: 372-377Crossref PubMed Scopus (29) Google Scholar). It be that E. Berts A. Saha S. Lund P.-E. Gylfe E. Hellman B. Arch. Biochem. Biophys. 1993; 300: 372-377Crossref PubMed Scopus (29) Google Scholar) reported an effect on K+ATP activity of to were to demonstrate an effect of ouabain on K+ATP activity within which is the time frame for glucose-induced inhibition of Na+,K+-ATPase activity as well as for membrane depolarization and increase in and J.C. Meissner H.P. J. Physiol. PubMed Scopus (55) Google and Meissner H.P. J. Physiol. Scholar). In this for a distinct intracellular signaling by glucose that results in inhibition of β-cell Na+,K+-ATPase. Although this is by of a Ca2+-independent phospholipase A2 and of a lipooxygenase it is of and thereby increased phosphorylation of the α-subunit that is for Na+,K+-ATPase Further studies have to this inhibitory effect on Na+,K+-ATPase activity is in to the effects of glucose in the β-cell stimulus-secretion for