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Endocrinology Vol. 143, No. 12 4592-4598
Copyright © 2002 by The Endocrine Society

ARTICLE

Glucose Metabolites Inhibit Protein Phosphatases and Directly Promote Insulin Exocytosis in Pancreatic ß-Cells

Åke Sjöholm, Mikael Lehtihet, Alexandre M. Efanov, Sergei V. Zaitsev, Per-Olof Berggren and Richard E. Honkanen

Department of Internal Medicine (A.S., M.L.), Karolinska Institutet, Stockholm South Hospital, SE 118 83 Stockholm, Sweden; Department of Molecular Medicine (A.M.E., S.V.Z., P.-O.B.), Karolinska Hospital, SE-171 76 Stockholm, Sweden; Belozersky Institute of Physico-Chemical Biology (S.V.Z.), Moscow State University, 119899 Moscow, Russia; and Department of Biochemistry and Molecular Biology (R.E.H.), College of Medicine, University of South Alabama, Mobile, Alabama 36688

Address all correspondence and requests for reprints to: Ake Sjöholm, Karolinska Institutet, Stockholm Söder Hospital, SE 118 83 Stockholm, Sweden.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In human type 2 diabetes mellitus, loss of glucose-sensitive insulin secretion is an early pathogenetic event. Glucose is the cardinal physiological stimulator of insulin secretion from the pancreatic ß-cell, but the mechanisms involved in glucose sensing are not fully understood. Specific ser/thr protein phosphatase (PPase) inactivation by okadaic acid promotes Ca2+ entry and insulin exocytosis in the ß-cell. We now show that glycolytic and Krebs cycle intermediates, whose concentrations increase upon glucose stimulation, not only dose dependently inhibit ser/thr PPase enzymatic activities, but also directly promote insulin exocytosis from permeabilized ß-cells. Thus, fructose-1,6-bisphosphate, phosphoenolpyruvate, 3-phosphoglycerate, citrate, and oxaloacetate inhibit PPases and significantly enhance insulin exocytosis, nonadditive to that of okadaic acid, at micromolar Ca2+ concentrations. In contrast, the effect of GTP is potentiated by okadaic acid, suggesting that the action of GTP does not require PPase inactivation. We conclude that specific glucose metabolites and GTP inhibit ß-cell PPase activities and directly stimulate Ca2+-independent insulin exocytosis. The glucose metabolites, but not GTP, seem to require PPase inactivation for their stimulatory effect on exocytosis. Thus, an increase in phosphorylation state, through inhibition of protein dephosphorylation by metabolic intermediates, may be a novel regulatory mechanism linking glucose sensing to insulin exocytosis in the ß-cell.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
REVERSIBLE PHOSPHORYLATION OF specific intracellular proteins is believed to be an important and versatile mechanism for regulating their biological activity, which, in turn, controls a variety of cellular functions. For instance, significant changes in protein kinase activities and in protein phosphorylation patterns occur subsequent to stimulation of insulin release by glucose (1, 2, 3, 4). Therefore, the molecular mechanisms regulating phosphorylation of proteins involved in the insulin secretory process by the pancreatic ß-cell have been extensively investigated. However, far less is known about the role and regulation of protein dephosphorylation by various protein phosphatases (PPases).

Previous studies have established that 1) the ß-cell contains divalent-cation independent serine/threonine PPase activity, most of which is PPase-1 and PPase-2A (5, 6); 2) stimulation of protein phosphorylation by direct activation of protein kinase A (PKA) and protein kinase C with forskolin or phorbol ester results in a stimulated insulin secretion (7, 8, 9, 10); 3) physiological stimuli of insulin secretion increase ß-cell phosphorylation state (2); 4) short-term treatment of ß-cells or permeabilized rat pancreatic islets with the specific PPase inhibitor okadaic acid promotes Ca2+ entry and insulin exocytosis (6, 7, 11, 12).

These combined findings suggest an important functional role for protein (de)phosphorylation in regulation of the stimulus-secretion coupling in the ß-cell. Subsequent to stimulation of ß-cell insulin exocytosis with glucose, there is a rapid increase in cytosolic glucose metabolites and GTP levels through glycolytic and Krebs cycle enzymes (13). Additionally, GTP may be formed by interconversion of ATP, also generated during catabolism of glucose. In this study, we have investigated the effect of specific glucose metabolites on divalent-cation independent serine/threonine PPase activities and insulin exocytosis in permeabilized cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Okadaic acid was the kind gift of Dr. R. Dickey (U.S. Food and Drug Administration). cAMP, PKA, D-glucose, phosphoenolpyruvate, oxaloacetate, fructose-1,6-bisphosphate, 3-phosphoglycerate, citrate, fructose-6-phosphate, ß-glycerophosphate, 2-phosphoglycerate, isocitrate, {alpha}-ketoglutarate, succinate, fumarate, malate, GTP, NAD(H), NADP(H), L-glutamine, 12-O-tetradecanoylphorbol-13-acetate, and crude histone (type 2-AS) were from Sigma (St. Louis, MO). Ammonium molybdate was obtained from Mallinckrodt (Hazelwood, MO), whereas [{gamma}-32P]ATP (350 Ci/mol) was purchased from NEN Life Science Products (Boston, MA). KCl and CaCl2 were from Fisher Scientific (Pittsburgh, PA).

Preparation of phosphohistone
Histone type 2-AS was phosphorylated with rabbit muscle type I protein kinase A as described in (14). Incubation mixtures (4 ml) consisted of 20 mg histone, 1 mg PKA, 20 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 0.5 mM dithiothreitol (DTT), 0.7 µM cAMP and 150 µM [{gamma}-32P]ATP. The reaction mixture was incubated at 30 C for 3 h and terminated by addition of trichloroacetic acid (20% final concentration). The phosphorylated histone was recovered according to Ref. 15 .

Determination of phosphatase activity
Clonal rat insulinoma RINm5F cells, NIT-1 cells, or primary rodent pancreatic islet ß-cells (16, 17) were cultured for 2 d in 60-mm plastic Falcon culture dishes in medium basal medium Eagle supplemented with 10% fetal bovine serum, 100 U/ml benzylpenicillin, and 0.1 mg/ml streptomycin.

Cells were washed once in ice-cold PBS, scraped off plates in 1 ml ice-cold Tris buffer (20 mM Tris-HCl; 1 mM EDTA; and 2 mM DTT, pH 7.4), and disrupted in a Polytron homogenizer. After pelleting debris, homogenates were swiftly transferred to Eppendorf (Hamburg, Germany) tubes that were immediately plunged into liquid nitrogen and stored at -80 C pending analysis. Phosphatase activity against phosphohistone was determined by measuring the liberation of [32P]. Assays (80 µl total volume) containing 50 mM Tris-HCl (pH 7.4), 0.5 mM DTT, 1 mM EDTA, and [32P]histone (2 µM PO4) were conducted at 30 C for 10 min (14). Okadaic acid (final concentration 1 nM) or its solvent dimethyl formamide was also included. At a 1-nM concentration, okadaic acid will inhibit essentially all PPase-2A activity without appreciably affecting PPase-1 (5, 14).

When the effects of glucose metabolites or GTP were to be studied, these (or equal amounts of their solvents only) were added to the incubation mixtures as 10-fold concentrated stock solutions and pH adjusted if necessary. Reactions were stopped by addition of 100 µl 0.1 M H2SO4 containing 1 mM potassium phosphate. Substrate dephosphorylation was kept to less than 10% of total phosphorylated substrate, and the reaction was linear with respect to time and enzyme concentration. [32P] released was extracted as a phosphomolybdate complex and measured according to Ref. 14 .

Additionally, two different substrates (phosphohistone and phosphorylase a) were compared to exclude any substrate-directed artifacts. Protein phosphatase activity was assayed by the release of [32P]phosphate from phosphorylase a as described (14). The recombinant phosphatases were incubated with 10 µM phosphorylase a in 50 mM Tris-HCl (pH 7.0), 1 mg/ml BSA, 1 mM MnCl2, 0.3% (vol/vol) ß-mercaptoethanol (total volume 60 µl) at 37 C for 10 min. The reaction was terminated by the addition of 0.2 ml of 20% (wt/vol) trichloroacetic acid and 50 µl of BSA (6–10 mg/ml). Following centrifugation at 15,000 x g for 5 min, the supernatant (200 µl) was analyzed for 32P release by liquid scintillation counting. 32P release was restricted to 15–20% of the total counts present in the assay.

The catalytic subunits of PPases types 1 and 2A were purified from rabbit muscle, demonstrating a single band upon SDS-PAGE and intense silver staining. PPase-1 was purified (specific activity 28 ± 1.5 nmol/min·mg protein using 2 µM [32P]phosphohistone as a substrate), essentially as described in (17). The catalytic subunit of PPase-2A was purified (specific activity 221 ± 5 nmol/min·mg protein against 2 µM [32P]phosphohistone), essentially as described (14), using G-75 Sephadex in the place of Ultragel-AcA44 as previously reported (14). Protein measurements were done with a Bio-Rad Laboratories, Inc. (Hercules, CA) DC assay kit using BSA as standard.

Insulin exocytosis from permeabilized ß-cells
Cells, cultured overnight, were washed three times in a cold permeabilization buffer consisting of (in mM): KAc 140, NaCl 5, MgCl2 1, BSA 0.025% (wt/vol) and HEPES 25 at pH 7.0 (adjusted with KOH). Cells were subsequently electropermeabilized in this buffer by six pulses of 3 kV/cm electric field. Then groups of permeabilized cells were transferred to tubes with 0.3 ml of a modified permeabilization buffer containing 2 mM Mg-ATP, an ATP-regenerating system (2 mM creatine phosphate and 10 U/ml creatine kinase), pCa 8 and 5 (pH and pCa adjusted with KOH and CaCl2, respectively). The actual pCa values in the incubation buffers were adjusted using a Ca2+-sensitive electrode (Orion Research, Inc., Boston, MA) and Ca2+ standard solutions (World Precision Instruments, Inc., Sarasota, FL). Additions of metabolites and okadaic acid were made to the buffer with pCa 5 or pCa8 and pH was readjusted when necessary. Cells were incubated at 37 C for 20 min, pelleted by centrifugation and supernatants frozen at -20 C pending analysis of insulin concentration in incubation media by RIA (18).

Statistical analysis
ANOVA was performed with a Statistica for Windows (version 5.0) software package (StatSoft, Inc., Tulsa, OK).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Glucose metabolites and GTP inactivate PPases
In the first set of experiments, the actions of specific glucose metabolites and GTP, generated during breakdown in glycolysis and the Krebs cycle, on the enzymatic activities of divalent-cation independent PPases were investigated in dilute ß-cell homogenates (Fig. 1Go, A–D). When insulin secretion is stimulated by glucose, these metabolites rapidly accumulate inside the ß-cell (19, 20). When added to cell homogenates, they inhibited ß-cell divalent-cation independent PPase activities in a dose-dependent manner (Fig. 1Go, A–D). The IC50 values for PPase activity were approximately 0.25 mM for phosphoenolpyruvate and approximately 5 mM for oxaloacetate. Additionally, fructose-1,6-bisphosphate, 3-phosphoglycerate and citrate were potent inhibitors of ß-cell PPase activities. In contrast, D-glucose, fructose-6-phosphate, ß-glycerophosphate, 2-phosphoglycerate, isocitrate, {alpha}-ketoglutarate, succinate, fumarate, malate, NAD(H), and NADP(H) had little or no effects on PPase type 1 or 2A activities when added to ß-cell homogenates. The values shown in Fig. 1Go, A and B (screening), are representative of three separate experiments. The data in Fig. 1Go, C and D (dose response), represent the means of four sets of experiments, each set conducted with a different passage of cells and the assays performed in triplicate. Qualitatively identical results in terms of PPase activity were obtained irrespective of using RINm5F, NIT-1, or primary pancreatic islet ß-cells. Additionally, two different substrates (phosphohistone and phosphorylase a) were used and yielded the same results, thus excluding any substrate-directed artifacts. To exclude the possibility that residual in vitro metabolism of the intermediates—when added to cell homogenates—would confound the results, comparisons were made with purified enzymes. Qualitatively similar results were obtained (not shown), thus validating the use of dilute cell homogenates.



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  		Figure 1. A–D, Concentration-dependent changes in divalent-cation-independent PPase activities after addition of glucose metabolite and GTP to ß-cell homogenates. Dilute cell homogenates or purified PPases were incubated for 10 min at 30 C with [32P]histone as a substrate and the desired test substance (10 mM, A and B). In C and D, dose-response curves are plotted. PPase activity was assayed as detailed in the Materials and Methods section. Values are mean percent of controls ± SEM for four separate experiments. F-6-P, Fructose-6-phosphate; BFP, fructose-1,6-bisphosphate; ß-glycero-P; ß-glycerophosphate; 3-PG, 3-phosphoglycerate; 2-PG, 2-phosphoglycerate; PEP, phosphoenolpyruvate; Citr, citrate; Isocitr, isocitrate.

 
Glucose metabolites and GTP promote insulin exocytosis in a Ca2+-dependent manner
In the second set of experiments, the effect of the metabolites and GTP on insulin exocytosis from permeabilized ß-cells was investigated (Fig. 2Go, A–C). As shown in Fig. 2CGo, GTP significantly stimulated insulin exocytosis, an effect that was potentiated by okadaic acid. In contrast, okadaic acid (in 10 µM Ca2+) did not augment the secretagogic action of fructose-1,6-bisphosphate, phosphoenolpyruvate, 3-phosphoglycerate or citrate (Fig. 2BGo). Interestingly, however, when the Ca2+ concentration was lowered to 30 nM, okadaic acid potentiated the stimulatory actions of these glucose metabolites on insulin exocytosis (Fig. 2AGo). Qualitatively identical results on insulin exocytosis were obtained irrespective of using RINm5F, NIT-1, or primary pancreatic islet ß-cells.



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  		Figure 2. Calcium- and okadaic acid-dependent stimulation of insulin release from permeabilized ß-cells by glucose metabolites and GTP. Permeabilized ß-cells were incubated for 20 min with the indicated substances (10 mM) and in the presence or absence of 1 µM okadaic acid at low (30 nM, A) or high (10 µM, B) free Ca2+ concentrations. Values are means ± SEM for at least 12 observations. *, **, and *** denote P < 0.05, P < 0.01, and P < 0.001, respectively, for chance differences vs. insulin secretion at corresponding Ca2+ concentration. #, ##, and ### denote P < 0.05, P < 0.01, and P < 0.001, respectively, for chance differences vs. insulin secretion in the presence of corresponding glucose metabolite and without okadaic acid. PEP, Phosphoenolpyruvate; Citr, citrate; BFP, fructose-1,6-bisphosphate; PG, 3-phosphoglycerate; OA, okadaic acid.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this paper, a novel mechanism of linking glucose sensing to promotion of insulin exocytosis is described. In human type 2 diabetes mellitus, loss of glucose-sensitive insulin secretion is an early pathogenetic event (1). The pancreatic islet ß-cell serves as a unique fuel-sensing organ and is the sole producer of insulin, the key hormone in maintenance of normoglycemia, whose exocytosis is tightly controlled by the ambient plasma glucose concentrations (2, 3, 4). In contrast to most other cells in which signaling is controlled through plasma membrane receptor-operated processes, the ß-cell senses subtle changes in glucose concentration by intracellular levels of glucose metabolites, which translates into finely tuned changes in insulin exocytosis (20). Glucose is the cardinal physiological stimulator of insulin secretion and is capable of potentiating the action of other secretagogues. Metabolism of the sugar is necessary for its insulin-releasing effect because application of mannoheptulose, which inhibits glycolysis, leads to an attenuated secretory response to glucose, and insulin release does not occur in the presence of nonmetabolizable sugars (19). When islets are exposed to high glucose, the plasma membrane GLUT-2 system ensures that the extracellular and intracellular glucose concentrations are rapidly equilibrated (19). Through a mass effect and other mechanisms that favor oxidative metabolism, high (millimolar or submillimolar) concentrations of the metabolic intermediates are attained (13, 20, 21, 22, 23, 24, 25, 26, 27, 28). The major route of glucose catabolism in ß-cells involves its conversion into pyruvate and acetyl-CoA by the glycolytic pathway and funneling into mitochondria for subsequent oxidative metabolism in the Krebs cycle (3, 19). Findings have revealed the existence in the ß-cell plasma membrane of ATP-regulated K+ channels (KATP channels) that control transmembrane K+ fluxes (29). With regard to the regulation of the ATP-regulated K+ channels, ATP is the link between glucose metabolism and changes in ß-cell electrical activity elicited by the sugar. More specifically, ATP generated by glucose metabolism may close K+ channels resulting in depolarization and subsequent influx of Ca2+ through voltage-activated Ca2+ channels located in the plasma membrane, an event setting in motion secretory granule translocation and exocytotic discharge of insulin (29). However, experiments by the Henquin and Aizawa laboratories (21, 30, 31, 32, 33, 34) indicate that glucose retains an excellent ability to secrete insulin even in the presence of maximally effective concentrations of K+ and diazoxide, an opener of K+ channels. Thus, signaling molecules other than ATP and Ca2+ are involved in glucose sensing in the ß-cell. Previous studies have suggested that the NADH shuttle and unidentified factors are important in coupling glucose sensing to insulin exocytosis (35, 36), partly by stimulating mitochondrial oxidation or anaplerosis, but the precise nature by which these mechanisms promote secretion and the KATP-independent signaling pathways activated by glucose has remained elusive.

A great deal of interest has been focused on how reversible phosphorylation of proteins is involved in regulation of cellular functions (37, 38, 39). Reversible changes in levels of phosphoserine and phosphothreonine at specific residues are a means by which the activity of many key proteins is regulated and a way through which cells convey extracellular signals into such diverse biological responses as mitogenesis, ion channel activity, substrate uptake and metabolism, and hormone secretion (37, 38, 39).

Compared with protein kinases, relatively little attention has been paid to the role of PPases in the ß-cell. However, divalent-cation independent serine/threonine PPases have been identified in ß-cells by Western blotting and by enzymatic assay (5, 6). Additionally, the specific PPase inhibitor, okadaic acid (14, 28), was shown to promote Ca2+ entry and insulin exocytosis, possibly through hyperphosphorylation (and thereby activation) of voltage-activated L-type Ca2+ channels (6, 7, 11, 12). Likewise, inhibition of insulin secretion by neurotransmitters was recently reported to occur through activation of PPase-2B (40). Furthermore, in intact ß-cells several insulin secretagogues evoked a rapid and transient inhibition of PPase activity, presumably contributing to a hyperphosphorylated state of ß-cell regulatory proteins (41). Consistent with this model is the finding in ß-cells that the 36-kDa catalytic subunit of PPase-2Ac undergoes carboxyl methylation, an effect accompanied by increased PPase-2A activity and suppressed insulin secretion (42). Likewise, PPase-2A can be activated in the ß-cell by ceramide (43), a second messenger that inhibits ß-cell function and insulin secretion (44).

We now report that specific glucose metabolites, generated in glycolysis and the Krebs cycle, inhibit the activities of divalent-cation independent PPases in a dose-dependent fashion in ß-cell homogenates and at physiological concentrations that promote insulin exocytosis. Interestingly, fructose-2,6-bisphosphate and glucose-1,6-bisphosphate, which are known to allosterically activate phosphofructokinase, one of the rate-limiting enzymes in the glycolytic pathway, have been reported to have inhibitory effects on porcine heart PPase-2A (45). It can thus be envisaged that glucose stimulation suppresses a tide of PPase activity, i.e. the metabolites may not be acting on a particular point in a signaling cascade. Rather they may act to suppress a lot of PPase activity. The net effect may be that many phosphorylation-stimulated events are augmented when PPase activity is suppressed. Thus, these glucose metabolites, in addition to Ca2+ and ATP, may sustain glucose-sensitive insulin exocytosis in ß-cells by contributing to a hyperphosphorylated state of ß-cell key regulatory proteins. One such key protein, with a critical regulatory role in glucose-stimulated insulin exocytosis, is the voltage-activated Ca2+ channel whose activity in the ß-cell is modulated through PPase inhibition by okadaic acid (11, 12). Consistent with this model, previous studies have shown that 3-phosphoglycerate and phosphoenolpyruvate increase protein phosphorylation in permeabilized ß-cells (46).

Previous investigations have shown that GTP and other guanine nucleotides stimulate insulin exocytosis from permeabilized ß-cells (47), that nutrient insulin secretagogues rapidly increase ß-cell ATP and GTP levels through oxidative metabolism before initiation of insulin exocytosis (13), and that islet GTP is required (and can be rate limiting) for insulin secretion (48). Furthermore, small elevations in glucose concentrations redirect and amplify GTP synthesis, which may potentiate Ca2+-induced insulin exocytosis in intact islets (49, 50). However, the molecular mechanisms by which these nucleotides stimulate insulin secretion are not fully understood. Previous investigators have shown that guanine nucleotide-induced insulin secretion cannot fully be explained by phosphatidylinositol bisphosphate breakdown, Ca2+, cAMP or pertussis toxin-sensitive GTP-binding proteins (47). Concordant with this, we have shown that physiological concentrations of natural adenine and guanine nucleotides (ATP>ADP, GTP>GDP) were found to inhibit PPase activity in ß-cell homogenates or purified PPases in a dose-dependent fashion (41). Hence, adenine and guanine nucleotides cause concentration-dependent inhibitory effects on ß-cell PPase activities that may contribute to the increase in phosphorylation state that occurs during stimulation of insulin exocytosis. Thus, PPase inhibition may be one important mechanism, complimentary to effects on protein kinases and ion channels, by which these nucleotides promote insulin exocytosis. It should be noted that the concentrations of GTP and glucose metabolites are in the (sub)millimolar range in the living ß-cell and elevated by glucose (13, 20, 21, 22, 23, 24, 25, 26, 27, 28, 51), making our findings potentially physiologically significant. We tested some of the metabolites that were inactive on PPase activity also for their effects on insulin release (data not shown). They had no effect on insulin release, thus confirming the findings of previous reports (52, 53). The high expression of pyruvate carboxylase noted in the ß-cell, and its rapid induction by glucose (20), ensures a large supply of oxaloacetate when islets are exposed to hyperglycemia. Additionally, glucose is known to cause a dose-dependent rise in the cellular contents of several metabolites, which closely correlates to the dose dependence of glucose-induced insulin exocytosis (23, 24).

In contrast to GTP, the glucose metabolites stimulate insulin exocytosis in a mode nonadditive with that of okadaic acid, suggesting that PPase inactivation is the mechanism by which the glucose metabolites—but not GTP—promote secretion. Additionally, the potentiating effect of okadaic acid on insulin exocytosis by the glucose metabolites was strictly dependent on the level of Ca2+ (Fig. 2Go, A and B), the effect vanishing in high Ca2+. This phenomenon clearly warrants further investigation, but it is conceivable that the insulin secretory process becomes less dependent on dephosphorylation cascades when high Ca2+ concentrations prevail, which directly may promote distal events in exocytosis (31, 32, 33, 34).

In this context, it is of interest that mice genetically deficient in hepatocyte nuclear factor-1{alpha} not only display impaired insulin secretion (54) but also defect glycolytic signaling (55). Additionally, a recent paper suggested that mitochondrial glutamate might act as a messenger in glucose-sensitive insulin exocytosis (56). Our present findings complement the latter study, but also extend it inasmuch as they show that several glucose-derived metabolites other than glutamate may also act as metabolic coupling factors in the insulin stimulus-secretion coupling and offer a potential target for their action distal in the exocytosis process.

It is concluded that specific glucose metabolites and GTP inhibit ß-cell divalent-cation-independent ser/thr PPase activities and directly stimulate insulin exocytosis. An increase in ß-cell phosphorylation state, through the inhibition of protein dephosphorylation by metabolic intermediates, may thus represent a novel and important regulatory mechanism in linking glucose sensing to insulin exocytosis.


   Acknowledgments
 


   Footnotes
 
Financial support was received from funds of Karolinska Institutet, the Swedish Medical Research Council (72X-12550, 72X-00034, 72X-09890, 72XS-12708, and 12P-10151), GlaxoSmithKline Pharma AB, Petrus and Augusta Hedlund’s Foundation, the European Foundation for the Study of Diabetes, the Swedish Society of Medicine, the Swedish Diabetes Association, the Swedish Society of Medicine, the Sigurd and Elsa Golje Memorial Foundation, Svenska Försäkringsföreningen, the Nordic Insulin Foundation Committee, Swedish Match, Svenska Diabetesstiftelsen, Barndiabetesfonden, Magnus Bergvalls Foundation, Torsten and Ragnar Söderberg’s Foundations, Novo-Nordisk Sweden Pharma AB, Harald Jeansson’s and Harald and Greta Jeansson’s Foundations, Tore Nilsson’s Foundation for Medical Research, Åke Wiberg’s Foundation, Syskonen Svensson’s Fund, Berth von Kantzows Foundation, and Fredrik and Inger Thuring’s Foundation.

Abbreviations: DTT, Dithiothreitol; PKA, protein kinase A; PPase, protein phosphatase.

Received July 1, 2002.

Accepted for publication August 29, 2002.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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