Glucose Decreases Na+,K+-ATPase Activity in Pancreatic beta -Cells
AN EFFECT MEDIATED VIA Ca2+-INDEPENDENT PHOSPHOLIPASE A2 AND PROTEIN KINASE CDEPENDENT PHOSPHORYLATION OF THE alpha -SUBUNIT*

Shigeru Owada, Olof Larsson, Per ArkhammarDagger , Adrian I. Katz§, Alexander V. Chibalin, Per-Olof Berggren, and Alejandro M. Bertorello

From the Rolf Luft Center for Diabetes Research L6B:01, Department of Molecular Medicine, Karolinska Institutet, Karolinska Hospital, S-171 76 Stockholm, Sweden, the Dagger  NOVO NORDISK, BioImage, Moerkhoeg Bygade 28, 2860 Soeborg, Denmark, and the § Department of Medicine, University of Chicago, Chicago, Illinois 60637

    ABSTRACT
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Abstract
Introduction
References

In the pancreatic beta -cell, glucose-induced membrane depolarization promotes opening of voltage-gated L-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 beta -cell membrane depolarization and Ca2+ influx. Because glucose-induced beta -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 beta -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 alpha -subunit phosphorylation by a protein kinase C-dependent process.

These results demonstrate that glucose inhibits Na+,K+-ATPase activity in beta -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 beta -cell.

    INTRODUCTION
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Abstract
Introduction
References

Glucose metabolism increases the ATP:ADP ratio and initiates the beta -cell stimulus-secretion coupling by closing ATP-regulated potassium (K+ATP)1 channels (1). These channels are the main regulators of the beta -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) (2-5). 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 (6). The origin of this inward current is not known, but it has been suggested that the current is carried by Na+ or Ca2+ (6, 7). Thus, glucose may activate an as yet unidentified component affecting beta -cell membrane potential.

Further support for the proposition that glucose has effects on membrane potential distinct from closing K+ATP channels was provided by studies of action potential frequency in the presence of tolbutamide and glucose (8). 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 beta -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 beta -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 beta -cell membrane depolarization (7, 9) and Ca2+ influx (9, 10). It has therefore been postulated that a decrease in Na+,K+-ATPase-mediated ion gradients may be a contributing mechanism to insulin secretion (7-9). The precise role of the Na+,K+-ATPase in membrane depolarization and its possible regulation by glucose in beta -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 (11), others failed to demonstrate significant changes in enzyme activity (12). 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.

    EXPERIMENTAL PROCEDURES

Materials-- Immunoprecipitation of the Na+,K+-ATPase alpha -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.

Isolation of Pancreatic beta -Cells-- All experiments were performed in single isolated pancreatic islets and in beta -cells obtained from adult (12 months old) obese (ob/ob) mice from a local colony (13). 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 (14, 15). This preparation consists of 90-95% beta -cells (15). The medium used for islet and cell isolation and [Ca2+]i measurements contained the following: NaCl, 125 mM; KCl, 5.9 mM; CaCl2, 1.3 mM; MgCl2, 1.3 mM; HEPES, 25 mM, pH 7.4; and 1 mg/ml bovine serum albumin. For Na+,K+-ATPase activity determinations, bovine serum albumin was omitted. Primary cultures of beta -cells were kept in RPMI 1640 medium supplemented with 10% fetal calf serum, 100 µg/ml streptomycin, and 100 IU/ml penicillin. Cells were cultured on glass coverslips or kept in suspension by gentle agitation.

Determination of Na+,K+-ATPase Activity in Single Isolated Pancreatic Islets-- Single islets were isolated and transferred individually (in 5 µl of isolation medium) to the bottom of each well of a Nunclon-96 well plate (Nunc, Denmark). They were kept on ice until dissection was finished (this time never exceeded 60 min). After exposure to glucose under different protocols, the incubations were terminated by transferring the well plate to ice. An aliquot (50 µl) of Na+,K+-ATPase assay medium was added: NaCl, 50 mM; KCl, 5 mM; MgCl2, 10 mM; EGTA, 1 mM; Tris-HCl, 50 mM; Na2ATP, 10 mM (Sigma, St. Louis, MO); and [gamma -32P]ATP (NEN Life Science Products) (specific activity 3000 Ci/mmol) in tracer amounts (1.3 nCi/µl). Islets were transiently exposed to a thermic shock (10 min at -20 °C) in order to render the plasma membrane permeable to ATP. The plate was sealed in order to prevent evaporation and transferred to 37 °C for additional 15 min. The incubation was terminated by placing the well plate on ice and adding 175 µl of trichloroacetic acid/charcoal solution. Thereafter, the content of each well was transferred to Eppendorf tubes and centrifuged (12,000 × g for 5 min), and an aliquot from the supernatant (containing the liberated 32P) was counted in a liquid scintillation counter. In each experiment, equivalent to one animal, total and ouabain-insensitive Na+,K+-ATPase activity were determined in eight islets each, and the difference between means of these measurements represents one Na+,K+-ATPase data point. Na+,K+-ATPase activity was expressed as nmol of Pi/islet/h.

Determination of Na+,K+-ATPase Activity in Pancreatic beta -Cells-- Na+,K+-ATPase activity was measured essentially as described above (16). Freshly isolated cells in suspension (50 µl) were incubated at room temperature with the desired agonists. The incubation was terminated by rapid cooling of the samples to 4 °C. Aliquots (10 µl) of the cell suspension (protein concentration, 10-20 µg) were rapidly transferred to the Na+,K+-ATPase assay medium (final volume, 100 µl; see composition above). Cells were transiently exposed to a thermic shock (10 min at -20 °C), in order to render the plasma membrane permeable to [gamma -32P]ATP. The samples were then incubated at 37 °C for 15 min. The reaction was terminated by rapid cooling to 4 °C and addition of a mixture of trichloroacetic acid/charcoal (5%/10%). After separating the charcoal phase (12,000 × g for 5 min) containing the nonhydrolyzed nucleotide, the 32P liberated in the supernatant was counted. Na+,K+-ATPase activity was calculated as the difference between test samples (total ATPase activity) and samples assayed in a medium devoid of Na+ and K+ and in the presence of 2 mM ouabain (ouabain-insensitive ATPase activity). Protein determination was performed according to Bradford (17), using a conventional dye reagent (Bio-Rad, Richmond, CA).

Phosphorylation and Immunoprecipitation of Na+,K+-ATPase in Intact Cells-- beta -Cells in suspension were labeled for 3 h at 37 °C in a buffer containing the following: 120 mM NaCl, 5 mM KCl, 4 mM NaHCO3, 1 mM CaCl2, 1 mM MgSO4, 0.2 mM NaH2PO4, 0.15 mM Na2HPO4, 5 mM glucose, 10 mM lactate, 1 mM pyruvate, 20 mM HEPES, and 1% bovine serum albumin, pH 7.45, with the addition of 100 µCi/ml 32P-orthophosphate (NEN Life Science Products). Incubations with either 3 or 15 mM glucose, in the presence or absence of 1 µM bisindolylmaleimide, were performed at room temperature. The incubations were terminated by removing the medium and addition of cold immunoprecipitation buffer. Immunoprecipitation of the Na+,K+-ATPase alpha -subunit was performed as described (18). Briefly, aliquots were incubated overnight at 4 °C with 50 µl of rabbit polyclonal antibody and the simultaneous addition of excess protein A-Sepharose beads (Pharmacia Biotech, Uppsala, Sweden). Samples were analyzed by SDS-PAGE using the Laemmli buffer system (19). Proteins were transferred to polyvinylidene difluoride membranes (Immobilon-P, Millipore, Bedford, MA) and subjected to autoradiography.

Determination of [Ca2+]i-- Experiments were performed on primary cultures of beta -cells as described before (20, 21). Briefly, beta -cells cultured for 12-24 h on glass coverslips were incubated in basal medium (3 mM glucose) for 30-40 min with 1 µM Fura-2/acetoxymethyl ester (Sigma). After washing, the subsequent measurements were performed in a SPEX Fluorolog-2 CM1T11I system connected to an inverted Zeiss Axiovert 35 M epifluorescence microscope. During each experimental protocol (described in detail in the legend to Fig. 3), cells were superfused in a custom-built chamber (20, 21). The excitation and emission wavelengths were 340/380 and 510 nm, respectively. The results are presented as 340/380 ratios.

Electrophysiological Studies-- Inside-out, cell-attached, and perforated-patch configurations of the patch-clamp technique were used (22-24). Pipettes were pulled from borosilicate glass, fire-polished, and coated with Sylgard resin (Dow Corning) near the tips. Pipettes had resistances between 2 and 6 MOmega . Single-channel currents were recorded from inside-out or cell-attached membrane patches. Channel activity in excised patches was measured at 0 mV pipette potential (Vp), whereas single channel activity was recorded from intact cells (cell-attached) at Vp -70 mV. Membrane potential was monitored using the perforated-patch configuration, using amphotericin B as pore-forming agent. Current and voltage were recorded using an Axopatch 200 patch-clamp amplifier (Axon Instruments Inc., Foster City, CA). During experiments, the current and voltage signals were stored using a VR-100A digital recorder (Instrutech Corp., Elmont, NY) and a high-resolution video cassette recorder (JVC, Tokyo, Japan). Channel records are displayed according to the convention with upward deflections denoting outward currents.

In all experiments, the extracellular solution contained the following: 138 mM NaCl, 5.6 mM KCl, 1.2 mM MgCl2, 2.6 mM CaCl2, and 5 mM HEPES (pH 7.4 with NaOH). For the excised-patch configuration, the "intracellular-like" solution consisted of the following: 125 mM KCl, 1 mM MgCl2, 10 mM EGTA, 30 mM KOH, and 5 mM HEPES (pH 7.15 with KOH). Patches were excised into nucleotide-free solution, and ATP was first added to test for channel inhibition. ATP was then removed, and patches were subsequently exposed to test substances. In the cell-attached recordings, the pipette solution consisted of the extracellular solution described above. Recordings of membrane potential were done using the following pipette solution: 10 mM KCl, 76 mM K2SO4, 10 mM NaCl, 1 mM MgCl2, 10 mM HEPES (pH 7.35 with KOH), and 240 µg/ml amphotericin B. All intact cell experiments (cell-attached and perforated-patch recordings) were done at 30-33 °C, whereas the inside-out patch experiments were performed at room temperature (20-22 °C). The bath had a volume of 0.4 ml, and cells were perifused at a rate of 4 ml/min. All test compounds were added to the perifusion medium. Each experimental condition was tested, with identical results, in at least five different cells.

Statistical Analysis-- Data are presented as means ± S.E. Statistical comparison of the data was performed using the paired Student's t test or ANOVA with Sheffe's F test when appropriate. Group differences with p < 0.05 were considered significant.

    RESULTS

In this study, we examined the extent to which changes in Na+,K+-ATPase activity contribute to beta -cell membrane depolarization and thereby to opening of L-type voltage-dependent Ca2+ channels and influx of Ca2+. Cells were exposed to 1 mM ouabain, a concentration that induced maximal inhibition of the pancreatic beta -cell Na+,K+-ATPase activity. The effect of ouabain on beta -cell membrane potential was determined using the perforated-patch configuration of the patch-clamp technique. Cells were perifused with a nonstimulatory concentration (4 mM) of glucose. Addition of ouabain to the medium resulted in prompt (within 60-100 s) depolarization of membrane potential (Fig. 1A), which increased from a resting potential of approximately -55 mV to a plateau potential of about -40 mV, upon which action potentials were superimposed.


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Fig. 1.   Effect of glucose and ouabain on beta -cell membrane potential and [Ca2+]i. A, recording of membrane potential in a single beta -cell using the perforated-patch configuration of the patch-clamp technique. The cell was perifused with a solution containing 4 mM glucose, which resulted in a membrane potential of approximately -50 mV. Upon inclusion of 1 mM ouabain, as indicated by the bar, the cell depolarized within 60 s to approximately -40 mV, which gave rise to a train of action potentials. B, effect of ouabain (1 mM) on [Ca2+]i in a Fura-2 loaded beta -cell cluster. Effect of ouabain (1 mM) in the presence or absence of external Ca2+. All protocols were terminated by addition of 15 mM glucose. The tracings are representative of four experiments, each performed in three separate cell preparations. C, ouabain and single K+ATP channel activity. i, lack of effect of ouabain on channel activity in inside-out patches. In the presence of ATP, channel activity was almost completely blocked, but it reappeared upon removal of the nucleotide. Addition of ouabain, as indicated by the bar, did not affect channel activity. This lack of effect was observed in five different cell preparations. ii, ouabain was also without effect on K+ATP channel activity in intact cells, as measured in the cell-attached mode. The lower tracing was obtained 2 min after the addition of ouabain and is representative of seven experiments from three separate cell preparations. iii, cell-attached recording from a beta -cell at 5 mM glucose exhibits a low action potential frequency. Inclusion of ouabain in the perifusion medium dramatically increased the frequency of action potentials. The recording was performed at 32 °C and is representative of five different cells.

The effect of ouabain on [Ca2+]i was determined in small clusters of 5-10 cells (Fig. 1B). Addition of 1 mM ouabain to cells incubated in the presence of 4 mM glucose induced a rapid and reversible increase (130 ± 17% above basal, n = 18, p < 0.001) in [Ca2+]i. A subsequent challenge with 15 mM glucose did not significantly increase [Ca2+]i further (153 ± 15% above basal, n = 18, p = 0.323 versus ouabain) when compared with the increase obtained with ouabain alone. The increase in [Ca2+]i elicited by ouabain was completely abolished in the absence of extracellular Ca2+ and rapidly restored upon addition of 1.3 mM Ca2+ to the perifusion medium. Moreover, the presence of 50 µM D-600, a voltage-dependent L-type Ca2+ channel blocker, abolished the increase in [Ca2+]i.

The Na+,K+-ATPase activity is inhibited by Ca2+ (25). Although this effect has not been reported to occur in intact cells but rather in a cell free system, the effect of glucose on Na+,K+-ATPase activity was examined after omitting Ca2+ from the extracellular medium. In the absence of Ca2+ and simultaneous presence of extracellular EGTA (0.5 mM), 10 mM glucose was still able to inhibit Na+,K+-ATPase activity (60 ± 8% of control, n = 6, p < 0.05).

Because the K+ATP channel is essential in regulating pancreatic beta -cell membrane potential (5, 6), one may question whether the depolarizing effect of ouabain is brought about via a direct and/or an indirect effect on this channel. To examine these possibilities, we studied K+ATP channel activity in excised patches as well as in intact cells. As seen in Fig. 1C, ouabain had no effect on channel activity in inside-out patches, suggesting that ouabain has no direct effect on the K+ATP channel complex. It has recently been reported that ouabain induces a reduction in the number of open K+ATP channels in intact beta -cells and that this might be the mechanism whereby ouabain depolarizes the beta -cell membrane and thereby increases [Ca2+]i (10). To correlate this finding with acute regulation of Na+,K+-ATPase activity, we studied channel activity using the cell-attached mode of the patch-clamp technique. To increase the number of active channels and thereby the resolution of channel events, we performed the experiments in the absence of glucose. As seen in Fig. 1C, ouabain did not affect channel activity within 2 min of exposure, the time period during which the cell depolarizes and an increase in [Ca2+]i is observed. Mean K+ATP channel currents were not altered (3 ± 4%, n = 7, not significant versus control prior exposure to ouabain). Ouabain was, however, still able to induce action potentials in the beta -cell when perifused with a threshold concentration of glucose (Fig. 1C).

We next studied the effect of glucose on Na+,K+-ATPase activity in single isolated pancreatic islets. Incubation of single medium size islets (2.4 ± 0.4 µg of protein, n = 5) with glucose resulted in a time-dependent (ANOVA p < 0.01) and dose-dependent (ANOVA p < 0.005) inhibition of Na+,K+-ATPase activity (Fig. 2, A and B). Ouabain-insensitive ATPase activity was not affected by glucose (control, 39.2 ± 2.3, n = versus 15 mM glucose, 38.2 ± 3.1, (nmol of Pi/islet/h) n = 8). The inhibitory effect of glucose was reversible (Fig. 2C). After exposure to 15 mM glucose for 10 min at 37 °C, the medium (5 µl) was removed and replaced with a medium containing 3 mM glucose for additional 10 min. After this washout period, Na+,K+-ATPase activity returned to control levels.


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Fig. 2.   Effect of glucose on Na+,K+-ATPase activity in single isolated pancreatic islets. A, Na+,K+-ATPase activity was determined in single islets preincubated with 15 mM glucose at 37 °C for different time intervals. Each point represents the mean ± S.E. of 5-8 experiments (animals) performed independently; ANOVA, p < 0.01. B, Na+,K+-ATPase activity was determined in single islets preincubated with different concentrations of glucose for 10 min at 37 °C. Each point represents the mean ± S.E. of 5-8 experiments (animals) performed independently; ANOVA, p < 0.005. C, glucose-induced inhibition of Na+,K+-ATPase activity is reversible and requires glucose metabolism. Na+,K+-ATPase activity was determined in single islets incubated with 15 mM glucose for 10 min at 37 °C and thereafter in a similar medium containing 3 mM glucose. Na+,K+-ATPase activity was also determined in single islets incubated with 15 mM glucose in the presence or absence of 15 mM mannoheptulose (M) at 37 °C for 10 min. Each column represents the mean + S.E. of five determinations (animals) performed independently. p values are as compared with control incubations (3 mM glucose).

In order for glucose to serve as a secretagogue, it has to be metabolized. Likewise, glucose needs to be metabolized in order to inhibit Na+,K+-ATPase activity. The presence of an equimolar concentration (15 mM) of the glycolytic inhibitor mannoheptulose substantially prevented the inhibitory effect of glucose (Fig. 2C). That glucose needs to be metabolized and the requirement of an intact intracellular signaling system were further supported by the lack of effect of glucose on Na+,K+-ATPase activity in a beta -cell-free system (not shown), similar to what has been previously reported (12).

Na+,K+-ATPase activity was also determined in beta -cells in suspension. Na+,K+-ATPase activity (nmol of Pi/mg of protein/min) in this preparation was 39 ± 1, n = 20, whereas the ouabain-insensitive ATPase was 37 ± 3 (n = 20). After isolation, beta -cells were suspended in 3.6 mM glucose for approximately 10 min at room temperature. A further increase in the glucose concentration induced a dose-dependent decrease in Na+,K+-ATPase catalytic activity, with a maximal effect at 7.5 mM of glucose (ANOVA, p < 0.01). Changes in enzyme activity were also time-dependent and occurred as early as 1 min after glucose administration and persisted for at least 5 min (ANOVA, p < 0.001). The ouabain-insensitive ATPase activity was not affected by glucose (control, 37.4 ± 4, n = 4 versus 15 mM glucose, 39.0 ± 2, n = 7, not significant). Also, the effect of glucose on Na+,K+-ATPase activity in beta -cell suspension was reversible (not shown).

The inhibitory action of glucose requires its metabolism and was absent when a broken cell preparation was used. The pancreatic beta -cell is endowed with a phospholipase A2 that is regulated in a Ca2+-independent fashion by the increase in ATP resulting from glucose metabolism (26, 27). This phospholipase is inhibited by HELSS. It was previously reported that pretreatment of pancreatic beta -cells with HELSS prevented the increase in [Ca2+]i and insulin secretion induced by glucose but not KCl or carbachol (28). Pancreatic beta -cells from ob/ob mice treated with 25 µM HELSS for 30 min at room temperature did not change islet Na+,K+-ATPase activity significantly, whereas HELSS abolished the inhibitory effect of glucose (Fig. 3).


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Fig. 3.   Effect of glucose on Na+,K+-ATPase activity in the presence or absence of the phospholipase A2 inhibitor HELSS. Islets were preincubated with HELSS (25 µM) for 30 min at 23 °C. Thereafter, the medium was replaced by a medium containing either 3 or 15 mM glucose as indicated, and the incubation proceeded for 10 min at 37 °C. Each column represents the mean + S.E. of six experiments (animals).

Activation of beta -cell phospholipase A2 by glucose results in hydrolysis of membrane phospholipids and the consequent increase in the production of arachidonic acid (27). Incubation of single islets with arachidonic acid (AA) resulted in a dose-dependent inhibition of Na+,K+-ATPase activity (Fig. 4A), with a half-maximal inhibitory concentration of approximately 4 nM. We evaluated the effect of glucose and AA on Na+,K+-ATPase activity in the presence and absence of relative selective inhibitors of the three main pathways of AA metabolism. The cyclooxygenase pathway was inhibited with indomethacin (Indo), the cytochrome P450-dependent monooxygenase with ethoxyresorufin (ETX) (29), and the lipooxygenase pathway with nordihydroguaiaretic acid (NDGA) (30). In previous studies, incubation with each pathway inhibitor alone for as long as 30 min at room temperature did not alter Na+,K+-ATPase activity in renal proximal tubules (31). Similarly, none of the inhibitors affected the enzyme activity in isolated pancreatic islets (not shown). However, the inhibitory effect of glucose (15 mM) or AA on Na+,K+-ATPase activity (10 nM) was completely blocked by 0.1 mM of the lipooxygenase inhibitor NDGA (Fig. 4B). In contrast, neither ETX nor Indo modified the inhibition of islet Na+,K+-ATPase by either glucose or AA. Because the effect of glucose was abolished by NDGA, we next examined whether 12(S)-HETE, the main lipooxygenase metabolite found in pancreatic beta -cells (32), inhibited Na+,K+-ATPase activity. 12(S)-HETE inhibited Na+,K+-ATPase activity in a dose-dependent manner, with a half-maximal inhibitory concentration of ~1 nM (Fig. 4C). We have also compared the effect of 12(S)-HETE with that of other eicosanoids on Na+,K+-ATPase activity (Table I). Whereas 1 nM 12(S)-HETE significantly inhibited Na+,K+-ATPase activity (as in Fig. 4) the same concentration of the 12(R)-HETE isomer did not. 20-HETE induced a ~20% inhibition of Na+,K+-ATPase activity as described previously in renal epithelial cells (31). The lipooxygenase metabolites 5- and 15-HETE did not significantly affect Na+,K+-ATPase activity.


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Fig. 4.   Effect of arachidonic acid and of inhibitors of its three main metabolic pathways on Na+,K+-ATPase activity. A, effect of different concentrations of AA on Na+,K+-ATPase activity. Islets were incubated with AA at 37 °C for 10 min. Each point represents the mean ± S.E. of 5 separate experiments (animals). B, the inhibitory effect of glucose and AA was examined in the presence of selective inhibitors of AA metabolic pathways. Isolated islets were preincubated at room temperature for 15 min with Indo, ETX, or NDGA (all 0.1 mM) or with vehicle. The islets were then incubated with 15 mM glucose for 10 min at 37 °C. Each column represents the mean + S.E. of six separate experiments (animals). *, p < 0.05; **, p < 0.01. p values are as compared with control incubations (3 mM glucose). C, effect of 12(S)-HETE on beta -cell Na+,K+-ATPase activity. Isolated islets were incubated with different concentrations of 12(S)-HETE at 37 °C for 10 min. Each data point represents the mean ± S.E. of five separate experiments (animals).

                              
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Table I
Effect of different eicosanoids on Na+, K+-ATPase activity from single isolated ob/ob islets
The concentration of all eicosanoids was 10-9 M. They were dissolved in ethanol and further diluted with isolation buffer to a final ethanol concentration of 0.001%.

It has been reported that the action of glucose on insulin secretion involves translocation of protein kinase C (PKC) (33). In the present study, the effect of glucose on Na+,K+-ATPase activity was blocked by the specific PKC inhibitor Bis (Fig. 5A). Similarly, Bis blocked the inhibitory effect of 12(S)-HETE. Additional experiments were performed in the presence of exogenous activators of PKC. Phorbol ester (phorbol 12,13-dibutyrate) inhibited Na+,K+-ATPase activity (vehicle, 14.6 ± 1.2, n = 5 versus 10 nM phorbol 12,13-dibutyrate, 7.1 ± 1.7, n = 5, p < 0.05), and this inhibition was not affected by HELSS (6.6 ± 1.9, n = 5, not significant). Accordingly, in beta -cells labeled with 32P-orthophosphate, glucose increased the state of phosphorylation of Na+,K+-ATPase alpha -subunit, and this effect was blocked by Bis (Fig. 5B).


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Fig. 5.   Effect of PKC inhibition on the ability of glucose and 12(S)-HETE to regulate islet Na+,K+-ATPase activity. A, isolated islets were incubated for 10 min at 37 °C with 15 mM glucose or 1 nM 12(S)-HETE, in the presence or absence of 1 mM Bis. Each column represents the mean ± S.E. of five separate experiments (animals). B, effect of glucose on Na+,K+-ATPase alpha -subunit phosphorylation. beta -Cells were incubated for 5 min at room temperature with either 3 or 15 mM glucose in the presence or absence of 1 µM Bis. In each protocol, equal amounts of immunoprecipitated material were confirmed by Western blot. The figure is representative of three experiments.

We examined the impact of PKC inhibition or 12(S)-HETE on glucose-regulated beta -cell membrane potential using the perforated patch configuration of the patch clamp technique (Fig. 6). A typical recording following an increase in the glucose concentration from 3 to 15 mM is shown in Fig. 6A. An increase in glucose concentration leads to depolarization of membrane potential, followed by a train of action potentials, which ceases as the concentration of glucose is lowered to 3 mM. Preincubation of beta -cells with 1 µM of the PKC inhibitor Bis for 30 min and its continuous presence throughout the experiment did not affect glucose-induced electrical activity (Fig. 6B). Inclusion of 12(S)-HETE was without effect on membrane potential or electrical activity at 3 or 15 mM glucose (Fig. 6, C and E, respectively). At 6 mM glucose, which is an intermediate glucose concentration at which the beta -cell membrane potential slowly fluctuates, 12(S)-HETE was also unable to affect electrical activity (Fig. 6D).


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Fig. 6.   Recordings of beta -cell membrane potential using the perforated patch configuration of the patch clamp technique. A, a typical recording of membrane potential following an increase in the glucose concentration from 3 to 15 mM. B, glucose-induced electrical activity in beta -cells preincubated with 1 µM PKC inhibitor Bis for 30 min. Bis was also present during the experiment. C-E, effect of 12(S)-HETE on beta -cell electrical activity at 3 (C), 6 (D), and 15 (E) mM glucose. Traces are representative experiments from series of at least three.


    DISCUSSION

Although it has been suggested that the activity of the Na+,K+-ATPase in the pancreatic beta -cell affects electrical properties of the plasma membrane (34), its regulation by glucose has remained controversial (11, 12). One reason for this may be that previous investigations were performed on broken-cell preparations in which glucose metabolism was not preserved. In this study, we present evidence that glucose indeed causes a rapid, reversible, and dose-dependent inhibition of the Na+,K+-ATPase, and we suggest possible mechanisms involved in this effect. The experiments were performed in intact, single islets or in beta -cells in suspension, under conditions that preserve signal-transduction pathways, including those necessary for glucose metabolism. The requirement for glucose metabolism is supported by the lack of effect of glucose on Na+,K+-ATPase activity in the presence of mannoheptulose, which inhibits glucokinase (Ref. 35 and references therein) and thereby blocks further glucose metabolism.

Several intracellular signaling messengers have been associated with the insulin secretory process induced by glucose. Among the earliest events in the signaling cascade is the activation of phospholipase A2, which is Ca2+-independent and is regulated by glucose and ATP (27). Similarly, the inhibitory effect of glucose on Na+,K+-ATPase activity is Ca2+-independent and is blocked by HELSS, a selective blocker of certain PLA2 isoforms (36). Phospholipase A2 has been localized both in the plasma membrane and in the cytosol, but only in the latter location is it regulated by ATP (27), suggesting that its translocation to the plasma membrane could be a possible mechanism of activation and targeting to the specific substrate. Despite the specificity of HELSS for the Ca2+-independent PLA2, we cannot exclude the contribution of other HELSS-insensitive PLA2 isoforms, which release arachidonic acid within the beta -cells.

Among the PLA2, the preferred substrates are the plasmalogens, rather than diacyl substrates (27). The enzyme catalyzes the hydrolysis of arachidonate from the sn-2 position of choline and ethanolamine phospholipids. In the present study, AA decreased Na+,K+-ATPase activity in a dose-dependent manner, similar to results reported earlier in kidney tubules (31). This observation could explain previous reports that AA induces beta -cell membrane depolarization and thereby facilitates Ca2+ entry and insulin secretion (37). Inhibition of Na+,K+-ATPase activity by glucose occurred independently of [Ca2+]i. Moreover, glucose-induced AA metabolism and eicosanoid release do not require Ca2+ influx (37).

Arachidonate 12-lipooxygenase products are involved in the secretory process of insulin (38, 39). Pancreatic beta -cells (but not alpha -cells) are endowed with a specific 12-lipooxygenase responsible for the production of 12(S)-HETE (32). The 12-lipooxygenase generates 12(S)-hydroperoxyeicosatetraenoic acid (12(S)-HPETE) from AA, and 12-HPETE is further reduced by peroxidase to 12-HETE. This compound can also be formed by the action of monooxygenases, but apparently not in pancreatic islets, in which 12-HETE is produced by the action of 12-lipooxygenase (40). In our studies, blocking the 12-lipooxygenase pathway by means of the selective inhibitor NDGA abolished the effect of both AA and glucose on Na+,K+-ATPase activity. Furthermore, the 12-lipooxygenase product 12(S)-HETE decreased islet Na+,K+-ATPase activity in a dose-dependent manner. Considering that upon addition of glucose there is an increased production of HETEs (41) and that decreased 12-lipooxygenase activity is associated with inhibition of insulin secretion, these results suggest that glucose is likely to modulate Na+,K+-ATPase activity by increasing the cellular levels of 12(S)-HETE. This hypothesis is further supported by the findings that neither ETX nor Indo affects islet Na+,K+-ATPase activity in response to glucose or AA.

Receptor-mediated regulation of Na+,K+-ATPase activity in epithelial cells is a complex phenomenon that involves the activation and integration of diverse intracellular signals (42, 43). It appears that the signaling cascade initiated by glucose metabolism and increased cellular ATP in islet cells is followed by activation of phospholipase A2 and subsequent stimulation of AA and 12(S)-HETE formation (Fig. 7). The molecular links between 12(S)-HETE and the Na+,K+-ATPase may include phosphorylation and/or internalization of Na+,K+-ATPase subunits, similar to what has been described in transporting epithelia (18, 44-46). The possibility that 12(S)-HETE directly activates PKC (47-50) is supported by the fact that the inhibitory effect of glucose or 12(S)-HETE on Na+,K+-ATPase activity was abolished by the presence of a PKC inhibitor (Fig. 4) and that in intact, metabolically labeled beta -cells, the state of phosphorylation of the Na+,K+-ATPase alpha -subunit was increased by glucose in a process that was PKC-dependent (Fig. 5). The fact that only the PKC alpha  and epsilon  isoforms are translocated in response to glucose (33) may suggest that either one or both of these isoforms are involved in the regulation of Na+,K+-ATPase activity.


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Fig. 7.   Schematic representation of the intracellular signals activated by glucose that eventually bring about the decrease in Na+,K+-ATPase activity and stimulation of insulin exocytosis. For explanation, see under "Discussion."

The electrogenic nature of the Na+,K+-ATPase has been well documented in excitable tissues (51). In pancreatic beta -cells, inhibition or activation of the pump is followed by rapid changes in membrane potential (34, 52), indicating that the pump is electrogenic also in this tissue. Thus, as the Na+,K+-ATPase is inhibited, it induces depolarization of the beta -cell plasma membrane (34). The present finding that glucose induces a pronounced inhibitory effect of the Na+,K+-ATPase suggests that part of the depolarizing effect of glucose is due to a decrease in the Na+,K+-ATPase-generated current. This would allow the Na+,K+-ATPase to contribute to the resting membrane potential, as well as to serve as a modulator of electrical activity subsequent to glucose stimulation.

We have previously demonstrated that down-regulation of PKC activity does not dramatically affect the effect of glucose on cytoplasmic free Ca2+ concentration, reflecting effects on membrane potential or insulin secretion (21). However, in those experiments, PKC activity was down-regulated by prolonged (overnight) incubation with phorbol esters. Because this treatment will only prevent the action of PKCs that are phorbol ester-sensitive, three isoforms out of eight, we performed experiments in the presence of a more specific inhibitor of PKCs, such as Bis. The present data confirm our previous observations and clearly suggest that the role of PKC in glucose-induced insulin secretion should be considered to be of a modulatory rather than of a direct regulatory nature. In the present study, we demonstrated that glucose-induced inhibition of the Na+,K+-ATPase is mediated by PKC. However, it is clear that the effect of this inhibition in beta -cell membrane depolarization should only be minor and may therefore not be resolved. Similarly, the 12(S)-HETE isomer did not affect membrane potential at resting (3 mM), threshold (6 mM), and stimulatory (15 mM) concentrations of glucose. Hence, we believe that inhibition of Na+,K+-ATPase activity by second messengers, but not ouabain, is not sufficient to depolarize the plasma membrane and that a combined action of glucose on K+-ATP channels and Na+,K+-ATPase is needed to achieve such an effect. This is also in agreement with the lack of effect of 12(S)-HETE on insulin secretion. Together with the observation that PKC blockade does not affect membrane potential, these results add further support to the hypothesis that inhibition of Na+,K+-ATPase activity does not have a direct regulatory effect but rather a modulatory effect on insulin release.

Our results do not support the concept that the depolarizing effect of ouabain is due to a decreased K+ATP channels activity, as previously suggested (10). It should be noted that Grapengiesser et al. (10) reported an effect on K+ATP activity following 15 min of exposure to ouabain. We were unable to demonstrate an effect of ouabain on K+ATP activity within 2 min, which is the time frame for glucose-induced inhibition of Na+,K+-ATPase activity as well as for ouabain-induced membrane depolarization and increase in [Ca2+]i (this study and Refs. 34 and 52).

In conclusion, this study presents compelling evidence for a distinct intracellular signaling pathway activated by glucose that results in inhibition of beta -cell Na+,K+-ATPase. Although this process is initiated by activation of a Ca2+-independent phospholipase A2 and activation of a lipooxygenase metabolite (2(S)-HETE), it is activation of PKC and thereby increased phosphorylation of the alpha -subunit that is responsible for Na+,K+-ATPase inhibition. Further studies have to clarify how important this inhibitory effect on Na+,K+-ATPase activity is in relation to the overall effects of glucose in the beta -cell stimulus-secretion coupling.

    ACKNOWLEDGEMENT

We thank Martin Wahl for useful discussions.

    FOOTNOTES

* This study was supported by the Swedish Medical Research Council (Grants 19X-10860, 03X-09890, 03XS-12708, and 03X-09891), funds of the Karolinska Institutet, the Swedish Diabetes Association, the Nordic Insulin Foundation Committee, the Berth von Kantzows Foundation, the Åke Wibergs Foundation, and the Emil and Vera Cornells Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 46-8-517-75727; Fax: 46-8-517-73658; E-mail: alejan{at}enk.ks.se.

The abbreviations used are: K+ATP, ATP-regulated K+ channels; PKC, protein kinase C; Bis, bisindolylmaleimide; HELSS, haloenol lactone (E)-6-(bromomethylene)-3-(1-naphtalenyl)-2H-tetrahydropyran-2-one; AA, arachidonic acid; Indo, indometacin; ETX, ethoxyresorufin; NDGA, nordihydroguaiaretic acid; PLA2, Ca2+ -independent phospholipase A2; ANOVA, analysis of variance; 12(S)-HETE, 12-S-hydroxyeicosatetranoic acid.
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