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Insulin and Exercise Decrease Glycogen Synthase Kinase-3 Activity by Different Mechanisms in Rat Skeletal Muscle

1999; Elsevier BV; Volume: 274; Issue: 35 Linguagem: Inglês

10.1074/jbc.274.35.24896

1083-351X

Jeffrey F. Markuns, Jørgen F. P. Wojtaszewski, Laurie J. Goodyear,

Adipose Tissue and Metabolism

Glycogen synthase activity is increased in response to insulin and exercise in skeletal muscle. Part of the mechanism by which insulin stimulates glycogen synthesis may involve phosphorylation and activation of Akt, serine phosphorylation and deactivation of glycogen synthase kinase-3 (GSK-3), leading to dephosphorylation and activation of glycogen synthase. To study Akt and GSK-3 regulation in muscle, time course experiments on the effects of insulin injection and treadmill running exercise were performed in hindlimb skeletal muscle from male rats. Both insulin and exercise increased glycogen synthase activity (%I-form) by 2–3-fold over basal. Insulin stimulation significantly increased Akt phosphorylation and activity, whereas exercise had no effect. The time course of the insulin-stimulated increase in Akt was closely matched by GSK-3α Ser21 phosphorylation and a 40–60% decrease in GSK-3α and GSK-3β activity. Exercise also deactivated GSK-3α and β activity by 40–60%. However, in contrast to the effects of insulin, there was no change in Ser21 phosphorylation in response to exercise. Tyrosine dephosphorylation of GSK-3, another putative mechanism for GSK-3 deactivation, did not occur with insulin or exercise. These data suggest the following: 1) GSK-3 is constitutively active and tyrosine phosphorylated under basal conditions in skeletal muscle, 2) both exercise and insulin are effective regulators of GSK-3 activity in vivo, 3) the insulin-induced deactivation of GSK-3 occurs in response to increased Akt activity and GSK-3 serine phosphorylation, and 4) there is an Akt-independent mechanism for deactivation of GSK-3 in skeletal muscle. Glycogen synthase activity is increased in response to insulin and exercise in skeletal muscle. Part of the mechanism by which insulin stimulates glycogen synthesis may involve phosphorylation and activation of Akt, serine phosphorylation and deactivation of glycogen synthase kinase-3 (GSK-3), leading to dephosphorylation and activation of glycogen synthase. To study Akt and GSK-3 regulation in muscle, time course experiments on the effects of insulin injection and treadmill running exercise were performed in hindlimb skeletal muscle from male rats. Both insulin and exercise increased glycogen synthase activity (%I-form) by 2–3-fold over basal. Insulin stimulation significantly increased Akt phosphorylation and activity, whereas exercise had no effect. The time course of the insulin-stimulated increase in Akt was closely matched by GSK-3α Ser21 phosphorylation and a 40–60% decrease in GSK-3α and GSK-3β activity. Exercise also deactivated GSK-3α and β activity by 40–60%. However, in contrast to the effects of insulin, there was no change in Ser21 phosphorylation in response to exercise. Tyrosine dephosphorylation of GSK-3, another putative mechanism for GSK-3 deactivation, did not occur with insulin or exercise. These data suggest the following: 1) GSK-3 is constitutively active and tyrosine phosphorylated under basal conditions in skeletal muscle, 2) both exercise and insulin are effective regulators of GSK-3 activity in vivo, 3) the insulin-induced deactivation of GSK-3 occurs in response to increased Akt activity and GSK-3 serine phosphorylation, and 4) there is an Akt-independent mechanism for deactivation of GSK-3 in skeletal muscle. glycogen synthase kinase-3 phosphatidylinositol phosphatidylinositol 4-morpholinepropanesulfonic acid Insulin and contractile activity are the most biologically relevant regulators of glycogen metabolism in skeletal muscle. In the postprandial state, insulin promotes muscle glucose uptake and the storage of glucose as glycogen (1Shulman G.I. Rothman D.L. Jue T. Stein P. DeFronzo R.A. Shulman R.G. N. Engl. J. Med. 1990; 322: 223-228Crossref PubMed Scopus (1056) Google Scholar). In contracting skeletal muscles glycogenolysis is activated, whereas the period following muscle contraction is characterized by a marked increase in glycogen synthesis (2Garetto L.P. Richter E.A. Goodman M.N. Ruderman N.B. Goodman M.N. Am. J. Physiol. 1984; 246: E471-E475PubMed Google Scholar). The conversion of UDP-glucose to glycogen by glycogen synthase is the rate-limiting step in glycogen synthesis, and this enzyme is regulated by both allosteric and phosphorylation-dephosphorylation mechanisms (3Lawrence Jr., J.C. Roach P.J. Diabetes. 1997; 46: 541-547Crossref PubMed Google Scholar). Glycogen synthase is serine phosphorylated on multiple sites, and insulin treatment results in the hierarchal dephosphorylation of several of these sites, leading to activation of the enzyme and increased glycogen synthesis (3Lawrence Jr., J.C. Roach P.J. Diabetes. 1997; 46: 541-547Crossref PubMed Google Scholar). There is evidence for both protein phosphatase-1 activation and glycogen synthase kinase-3 (GSK-3)1 inhibition as regulators of glycogen synthase activity with insulin stimulation (3Lawrence Jr., J.C. Roach P.J. Diabetes. 1997; 46: 541-547Crossref PubMed Google Scholar). GSK-3 is a serine/threonine kinase which, in addition to phosphorylating glycogen synthase, has numerous other substrates including ATP-citrate lyase (4Hughes K. Ramakrishna S. Benjamin W.B. Woodgett J.R. Biochem. J. 1992; 288: 309-314Crossref PubMed Scopus (94) Google Scholar), type I protein Ser/Thr phosphatase (5Hemmings B.A. Resink T.J. Cohen P. FEBS Lett. 1982; 150: 319-324Crossref PubMed Scopus (136) Google Scholar), tau (6Mandelkow E.M. Drewes G. Biernat J. Gustke N. van Lint J. Vandenheede J.R. Mandelkow E. FEBS Lett. 1992; 314: 315-321Crossref PubMed Scopus (483) Google Scholar, 7Hong M. Chen D.C.R. Klein P.S. Lee V.M.-Y. J. Biol. Chem. 1997; 272: 25326-25332Abstract Full Text Full Text PDF PubMed Scopus (418) Google Scholar), and several transcription factors (8Boyle W.J. Smeal T. Defize L.H. Angel P. Woodgett J.R. Karin M. Hunter T. Cell. 1991; 64: 573-584Abstract Full Text PDF PubMed Scopus (856) Google Scholar, 9Saksela K. Makela T.P. Hughes K. Woodgett J.R. Alitalo K. Oncogene. 1992; 7: 347-353PubMed Google Scholar, 10Fiol C.J. Williams J.S. Chou C.H. Wang Q.M. Roach P.J. Andrisani O.M. J. Biol. Chem. 1994; 269: 32187-32193Abstract Full Text PDF PubMed Google Scholar). The two identified GSK-3 isoforms, GSK-3α and GSK-3β, contain serine and tyrosine phosphorylation sites that are critical in the regulation of enzyme activity (4Hughes K. Ramakrishna S. Benjamin W.B. Woodgett J.R. Biochem. J. 1992; 288: 309-314Crossref PubMed Scopus (94) Google Scholar, 11Woodgett J.R. EMBO J. 1990; 9: 2431-2438Crossref PubMed Scopus (1161) Google Scholar, 12Welsh G.I. Proud C.G. Biochem. J. 1993; 294: 625-629Crossref PubMed Scopus (351) Google Scholar). GSK-3 requires phosphorylation of a single tyrosine residue for full activity (13Hughes K. Nikolakaki E. Plyte S.E. Totty N.F. Woodgett J.R. EMBO J. 1993; 12: 803-808Crossref PubMed Scopus (525) Google Scholar, 14Wang Q.M. Fiol C.J. DePaoli-Roach A.A. Roach P.J. J. Biol. Chem. 1994; 269: 14566-14574Abstract Full Text PDF PubMed Google Scholar, 15Murai H. Okazaki M. Kikuchi A. FEBS Lett. 1996; 392: 153-160Crossref PubMed Scopus (63) Google Scholar), suggesting that dephosphorylation on tyrosine in vivo could be a mechanism for enzyme deactivation. Insulin results in phosphorylation on Ser21 and Ser9 in GSK-3α and β, respectively, leading to enzyme deactivation (16Sutherland C. Cohen P. FEBS Lett. 1994; 338: 37-42Crossref PubMed Scopus (192) Google Scholar, 17Sutherland C. Leighton I.A. Cohen P. Biochem. J. 1993; 296: 15-19Crossref PubMed Scopus (758) Google Scholar). GSK-3 phosphorylates glycogen synthase on two sites that are identical to those dephosphorylated by insulin (3Lawrence Jr., J.C. Roach P.J. Diabetes. 1997; 46: 541-547Crossref PubMed Google Scholar). The mechanism leading to GSK-3 deactivation with insulin stimulation likely involves phosphatidylinositol (PI) 3-kinase, PtdIns 3,4,5-trisphosphate-dependent protein kinase-1, and Akt (also known as protein kinase B). The PtdIns 3,4-bisphosphate and PtdIns 3,4,5-trisphosphate lipid products of PI 3-kinase recruit and activate PtdIns 3,4,5-trisphosphate-dependent protein kinase-1, which in turn phosphorylates Akt on Thr308 (18Coffer P.J. Jin J. Woodgett J.R. Biochem. J. 1998; 335: 1-13Crossref PubMed Scopus (969) Google Scholar). Activation of Akt is also dependent upon Ser473phosphorylation, which presumably involves an additional PtdIns 3,4,5-trisphosphate-dependent protein kinase molecule (18Coffer P.J. Jin J. Woodgett J.R. Biochem. J. 1998; 335: 1-13Crossref PubMed Scopus (969) Google Scholar). PI 3-kinase as an upstream regulator of Akt is supported by studies demonstrating that wortmannin, dominant-negative PI 3-kinase mutants, and growth factor-receptor point mutations prevent the activation of Akt (18Coffer P.J. Jin J. Woodgett J.R. Biochem. J. 1998; 335: 1-13Crossref PubMed Scopus (969) Google Scholar, 19Burgering B.M.T. Coffer P.J. Nature. 1995; 376: 599-602Crossref PubMed Scopus (1884) Google Scholar, 20Franke T.F. Yang S. ChanT O. Datta K. Kazlauskas A. Morrison D.K. Kaplan D.R. Tsichlis P.N. Cell. 1995; 81: 727-736Abstract Full Text PDF PubMed Scopus (1829) Google Scholar, 21Kohn A.D. Kovacina K.S. Roth R.A. EMBO J. 1995; 14: 4288-4295Crossref PubMed Scopus (320) Google Scholar), and constitutively active mutants of PI 3-kinase are sufficient to stimulate Akt in cells (22Didichenko S.A. Tilton B. Hemmings B.A. Ballmer-Hofer K. Thelen M. Curr. Biol. 1996; 6: 1271-1278Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 23Klippel A. Reinhard C. Kavanaugh W.M. Apell G. Escobedo M.A. Williams L.T. Mol. Cell. Biol. 1996; 16: 4117-4127Crossref PubMed Scopus (418) Google Scholar). There is also strong evidence that Akt deactivates GSK-3 through serine phosphorylation (24Cross D.A.E. Alessi D.R. Cohen P. Andjelkovich M. Hemmings B.A. Nature. 1995; 378: 785-789Crossref PubMed Scopus (4397) Google Scholar, 25van Weeren P.C. de Bruyn K.M. de Vries-Smits A.M. van Lint J. Burgering B.M. J. Biol. Chem. 1998; 273: 13150-13156Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar). Although there has been considerable progress in elucidating signals regulating insulin activation of glycogen synthase in various cell types, there is little understanding of the molecular mechanisms leading to stimulation of glycogen synthase with exercise in skeletal muscle. In the current investigation we compared the effects of insulin and exercise on the regulation of glycogen synthesis in skeletal muscle and determined whether the activation of glycogen synthase was accompanied by changes in the phosphorylation state and activity of Akt and GSK-3. We show that both insulin and exercise increase glycogen synthase activity and that the time course of synthase activation is closely mirrored by deactivation of GSK-3. Insulin-induced GSK-3 deactivation is associated with GSK-3α serine phosphorylation and increased Akt activity, whereas exercise deactivates GSK-3 in the absence of changes in serine or tyrosine phosphorylation of GSK-3 or increased Akt activity. GSK-3α and Akt1 antibodies, Phospho-GS2 substrate peptide, and Akt/PKB-specific substrate peptide were purchased from Upstate Biotechnology Inc. (Lake Placid, NY). GSK-3β antibody was purchased from Transduction Laboratories (Lexington, KY). [γ-32P]ATP was from NEN Life ScienceProducts, and protein A- and G-Sepharose beads were from Amersham Pharmacia Biotech. Reagents for protein assays and electrophoresis were purchased from Bio-Rad (Rockville Center, NY). Chemiluminescence reagents were from Amersham Pharmacia Biotech, and all other standard chemicals were obtained from Sigma. Fed male Sprague-Dawley rats (190 g) were divided into three treatment groups: basal, exercise, and insulin. Insulin-stimulated animals were studied 5, 10, or 30 min after intraperitoneal insulin injection (maximal, 20 units/rat), and exercised animals were studied immediately after 5, 10, 30, or 60 min of treadmill running at 25 m/min, 10% grade. Serum glucose concentrations were determined by hexokinase assay (26Bondar R.J. Mead D.C. Clin. Chem. 1974; 20: 586-590Crossref PubMed Scopus (308) Google Scholar) and serum insulin concentrations by radioimmunoassay (27Soeldner J.S. Sloane D. Diabetes. 1965; 14: 771-779Crossref PubMed Scopus (590) Google Scholar). Rats were killed by decapitation, and the gastrocnemius muscles from both legs were quickly dissected, divided into red and white fractions, frozen in liquid N2, and stored at −80 °C until processed. Approximately 60 mg of muscle tissue was Polytron homogenized in 1 ml of buffer containing 50 mm Tris-HCl, 5 mm EDTA, 100 mmsodium fluoride, pH 7.8. Glycogen synthase activity in the absence or presence of 6.7 mm glucose 6-phosphate was determined as described previously (28Thomas J.A. Schlender K.K. Larner J. Anal. Biochem. 1968; 25: 486-499Crossref PubMed Scopus (949) Google Scholar) and is reported as the ratio of the glycogen synthase activity in the absence of glucose 6-phosphate to that in the presence of glucose 6-phosphate (%I form). To measure muscle glycogen concentrations, homogenates were hydrolyzed in 2 n HCl, neutralized, and assayed spectrophotometrically for glucose content as described previously using a hexokinase-dependent assay kit (26Bondar R.J. Mead D.C. Clin. Chem. 1974; 20: 586-590Crossref PubMed Scopus (308) Google Scholar). For studies of GSK-3 activity and phosphorylation, pulverized skeletal muscle was homogenized in GSK-3 buffer (1:5) containing 50 mm HEPES, 150 mm sodium chloride, 20 mm sodium pyrophosphate, 20 mm β-glycerophosphate, 10 mm sodium fluoride, 2 mm sodium vanadate, 2 mm EDTA, 1% Nonidet P-40, 10% glycerol, 2 mm phenylmethylsulfonyl fluoride, 1 mm magnesium chloride, 1 mm calcium chloride, 10 μg/ml leupeptin, and 10 μg/ml aprotinin, pH 7.6. For studies of Akt1 activity and phosphorylation, additional muscle was homogenized in Akt buffer (1:5) containing 20 mm HEPES, 2 mm EGTA, 50 mm β-glycerol phosphate, 1 mm dithiothreitol, 1 mm sodium vanadate, 1% Triton X-100, 10% glycerol, 10 μm leupeptin, 3 mm benzamidine, 5 μm pepstatin A, 10 μg/ml aprotinin, and 1 mm phenylmethylsulfonyl fluoride, pH 7.4. Samples were rotated end over end for 1 h at 4 °C and centrifuged at 15,500 × g for 1 h. Supernatants were retained and assayed for protein concentrations using the Bradford method (29Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217544) Google Scholar). Akt1 activity in the muscle lysates was measured as described previously (30Sherwood D.J. Dufresne S.D. Markuns J.F. Cheatham B. Moller D.E. Aronson D. Goodyear L.J. Am. J. Physiol. 1999; (in press)PubMed Google Scholar). Skeletal muscle lysates (100 μg) were preincubated with either anti-GSK-3α or anti-GSK-3β antibody in buffer A (25 mm HEPES, 10 mm β-glycerophosphate, 2 mm EDTA, 2 mm sodium vanadate, 1% Nonidet P-40, 10% glycerol, 10 μg/ml leupeptin, 5 mm sodium pyrophosphate, 2 mm benzamidine, 10 μg/ml aprotinin, 2 mmphenylmethylsulfonyl fluoride, and 0.1% β-mercaptoethanol, pH 7.6) for 2 h at 4 °C. Immune complexes were formed by incubating samples with protein G-Sepharose beads for 2 h at 4 °C. Pellets were washed once with buffer A, once with buffer containing 100 mm Tris-HCl, 500 mm lithium chloride, 100 μm sodium vanadate, and 1 mm dithiothreitol, pH 7.5, and twice with buffer containing 8 mm MOPS, 200 μm EDTA, 100 μm sodium vanadate, 1 mm dithiothreitol, and 10 mm magnesium acetate, pH 7.0. Beads were incubated in a reaction mixture containing 102 μm Phospho-GS2 substrate peptide (YRRAAVPPSPSLSRHSSPHQ pS EDEEE), 125 μm ATP, 8 mm MOPS, 10 nm microcystin, 200 μm EDTA, 500 μm sodium vanadate, 1 mm magnesium acetate, pH 7.0, and 1.5 μCi of [γ-32P]ATP for 15 min at 30 °C. The samples were spotted onto P81 phosphocellulose papers and extensively washed with 75 mm H3PO4. Papers were dried, placed in vials with scintillation fluid, and counted with a Beckman scintillation counter. Aliquots of muscle lysates (90 μg) were separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The nitrocellulose membranes were blocked with Tris-buffered saline containing 100 mm Tris, 1.5m NaCl, and 0.01% sodium azide (TNA) + 5% nonfat dry milk + 0.05% Tween 20. Membranes were incubated overnight with phosphospecific anti-Akt Ser473 antibody (1:1000), anti-GSK-3α Ser21 antibody (2 μg/ml), or anti-GSK-3β Tyr216 antibody (1 μg/ml) in TNA + 5% nonfat dry milk + 0.05% Tween 20 at 4 °C. To study tyrosine phosphorylation, immunoprecipitation/immunoblotting experiments were done by incubating muscle lysates (1 mg) with either anti-phosphotyrosine antibody (1 μg/ml), anti-GSK-3α (2 μg/ml), or GSK-3β (2 μg/ml), followed by immunoblotting with anti-GSK-3α (2 μg/ml), anti-GSK-3β antibody (1:2500), or anti-phosphotyrosine antibody (1 μg/ml). Primary antibodies were bridged with horseradish peroxidase-conjugated secondary antibody, and immune complexes were visualized with enhanced chemiluminescence. Specific bands were quantitated by densitometry. All data are expressed as the means ± S.E. Statistical analysis was performed using SAS General Linear model. Differences were considered significant when p ≤ 0.05. Fig.1 shows the time course for the activation of the %I-form of glycogen synthase in red gastrocnemius muscle in response to treadmill running exercise and maximal insulin injection. Only 5 min of exercise was necessary to produce a significant activation of glycogen synthase, but the highest levels of activity were not observed until 30 or 60 min of running exercise (3-fold above basal). Insulin was slightly less effective in increasing glycogen synthase activity (2-fold above basal), but maximal activation occurred more rapidly (10 min). Similar results were observed in white gastrocnemius muscle (data not shown). Muscle glycogen content was decreased by exercise at all time points, whereas glycogen levels were unaffected by insulin injection (data not shown). To determine whether the magnitude and time course of glycogen synthase activation with insulin and exercise corresponded to changes in Akt, we measured both Akt activity and serine phosphorylation in red and white gastrocnemius muscle. Similar to the activation of glycogen synthase, Akt activity (Fig.2 A) and serine phosphorylation (Fig. 2, B and C) were significantly increased with insulin in the red gastrocnemius muscle. Maximal insulin-stimulated Akt activation occurred earlier than peak glycogen synthase activation (5 min versus 10 min), and Akt phosphorylation/activity had returned to base-line levels by 30 min after insulin injection. Insulin also increased Akt activity in white gastrocnemius with a similar time course of activation, although peak stimulation was somewhat less than in the red muscle (2.5-fold above basal; data not shown). In contrast to the consistent stimulation of Akt with insulin, exercise did not alter Akt activity or serine phosphorylation in the red (Fig. 2) or white skeletal muscle (data not shown). The temporal relationship between Akt and glycogen synthase activation is consistent with the hypothesis that part of the mechanism by which insulin stimulates glycogen synthesis is through stimulation of Akt. In contrast, exercise must utilize a different mechanism to increase glycogen synthase activity. Consistent with the stimulation of Akt, insulin rapidly decreased GSK-3α and β activities in red gastrocnemius muscle (Fig.3). With 5 min of insulin stimulation, both GSK-3α and β were decreased by approximately 40% and remained significantly lower than basal after 30 min of insulin treatment. In contrast with the lack of effect of exercise on Akt activity and phosphorylation, exercise decreased GSK-3α and β activities in the muscle (Fig. 3). Activities of both isoforms were significantly decreased within 10 min of exercise and remained depressed with 30 and 60 min of exercise (Fig. 3). This time course of GSK-3 deactivation (Fig. 3) followed closely with the time course of activation of glycogen synthase (Fig. 2). The pattern and magnitude of decrease in GSK-3α activity with insulin and exercise was similar in the red and white gastrocnemius muscle. However, decreases in GSK-3β activity in response to insulin and exercise were highly variable in white muscle (data not shown), suggesting that GSK-3β may be less sensitive to metabolic alterations in the more glycolytic fibers. Phosphorylation of the GSK-3 isoforms on Ser21 or Ser9 results in a decrease in enzyme activity. To determine whether the insulin- and exercised-induced decreases in GSK-3 activity were associated with increased serine phosphorylation, we immunoblotted muscle lysates with an antibody that recognizes GSK-3α only when phosphorylated on Ser21. Fig. 4 shows that insulin increased GSK-3 Ser21 phosphorylation in both red and white skeletal muscle in a time-dependent manner, strikingly similar to the deactivation of the enzyme and the activation and phosphorylation of Akt. On the other hand, the deactivation of GSK-3 activity with exercise was not accompanied by an increase in Ser21 phosphorylation of GSK-3α. These data provide compelling evidence that exercise regulates GSK-3 activity in skeletal muscle by a mechanism other than Ser21 phosphorylation. GSK-3α and β may be constitutively active in resting cells, and this activated state may be maintained by phosphorylation on Tyr279 and Tyr216, respectively. Because some studies have suggested that tyrosine dephosphorylation may regulate kinase activity in vivo (14Wang Q.M. Fiol C.J. DePaoli-Roach A.A. Roach P.J. J. Biol. Chem. 1994; 269: 14566-14574Abstract Full Text PDF PubMed Google Scholar, 15Murai H. Okazaki M. Kikuchi A. FEBS Lett. 1996; 392: 153-160Crossref PubMed Scopus (63) Google Scholar, 31Shaw M. Cohen P. Alessi D.R. FEBS Lett. 1997; 416: 307-311Crossref PubMed Scopus (216) Google Scholar), we determined whether insulin or exercise altered GSK-3 tyrosine phosphorylation in the muscle. Immunoblotting muscle lysates with a phosphospecific antibody that recognizes GSK-3β when phosphorylated on Tyr216failed to demonstrate any effect of insulin or exercise (Fig.5). Furthermore, immunoprecipitation/immunoblotting with antibodies to phosphotyrosine, GSK-3α, and GSK-3β showed significant phosphorylation in the basal state but no regulation with insulin or exercise (data not shown). The current results demonstrate that physical exercise decreases GSK-3 activity in skeletal muscle and that the magnitude of GSK-3 deactivation is similar to that observed with maximal insulin stimulation in vivo. In addition to the novel finding that exercise decreases GSK-3 activity in muscle, our data also suggest that there are distinct mechanisms leading to GSK-3 deactivation in response to exercise and insulin. For the insulin-induced deactivation of GSK-3, the findings are consistent with an Akt-dependent phosphorylation of GSK-3 on Ser21. Previous studies have provided direct evidence that Akt can phosphorylate GSK-3 on Ser9 or Ser21 (24Cross D.A.E. Alessi D.R. Cohen P. Andjelkovich M. Hemmings B.A. Nature. 1995; 378: 785-789Crossref PubMed Scopus (4397) Google Scholar, 31Shaw M. Cohen P. Alessi D.R. FEBS Lett. 1997; 416: 307-311Crossref PubMed Scopus (216) Google Scholar). Although we could not make this type of assessment in the intact animal, it is compelling that the time course of Akt activation and GSK-3 phosphorylation with in vivo insulin stimulation was so similar (Figs.2 A and 4 B). The magnitude and time course of GSK-3α serine phosphorylation also strikingly resembles the deactivation of GSK-3α (Figs. 3 A and 4 B). Furthermore, the return of Akt activity to near basal activity occurred more rapidly than the dephosphorylation of GSK-3. Thus, the pattern of regulation of muscle Akt and GSK-3 in the intact animal supports the hypothesis that insulin results in the sequential phosphorylation and activation of Akt and GSK-3 in skeletal muscle. In contrast to the effects of insulin to cause serine phosphorylation of GSK-3, exercise did not result in Ser21 phosphorylation of GSK-3α. The inability of exercise to elicit GSK-3 serine phosphorylation is consistent with experiments demonstrating that Akt activity (current study and Refs. 30Sherwood D.J. Dufresne S.D. Markuns J.F. Cheatham B. Moller D.E. Aronson D. Goodyear L.J. Am. J. Physiol. 1999; (in press)PubMed Google Scholar, 32Brozinick Jr., J.T. Birnbaum M.J. J. Biol. Chem. 1998; 273: 14679-14682Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, and 33Lund S. Pryor P.R. Ostergaard S. Schmitz O. Pedersen O. Holman G.D. FEBS Lett. 1998; 425: 472-474Crossref PubMed Scopus (54) Google Scholar) and PI 3-kinase activity (34Goodyear L.J. Giorgino F. Balon T.W. Condorelli G. Smith R.J. Am. J. Physiol. 1995; 268: E987-E995Crossref PubMed Google Scholar) are not increased in response to treadmill running exercise or muscle contractions induced by electrical stimulation. There are other kinases that have been suggested to serine phosphorylate GSK-3 (35Skoglund G. Hansson A. Ingelman-Sundberg M. Eur. J. Biochem. 1985; 148: 407-412Crossref PubMed Scopus (48) Google Scholar, 36Moxham C.M. Tabrizchi A. Davis R.J. Malbon C.C. J. Biol. Chem. 1996; 271: 30765-30773Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar), and we have shown that two of these molecules, the p90 ribosomal S6 kinase 2 (RSK2) and the c-Jun NH2-terminal kinase (37Goodyear L.J. Chung P.-Y. Sherwood D. Dufresne S.D. Moller D.E. Am. J. Physiol. 1996; 271: E403-E408PubMed Google Scholar), are highly activated by the same exercise protocol used in the current study. This suggests that high levels of RSK2 and c-Jun NH2-terminal kinase activity in skeletal muscle do not necessarily lead to increased Ser21phosphorylation of GSK-3 in vivo. There are other examples in the literature of an Akt-independent mechanism for deactivation of GSK-3. In mouse fibroblasts, Wingless inactivation of GSK-3 is wortmannin-insensitive (38Cook D. Fry M.J. Hughes K. Sumathipala R. Woodgett J.R. Dale T.C. EMBO J. 1996; 15: 4526-4536Crossref PubMed Scopus (344) Google Scholar). Evidence that GSK-3β Ser9 phosphorylation is not required for a decrease in enzyme activity also comes from a study showing that mutation of Ser9 to alanine does not diminish lithium effects on GSK-3-mediated tau phosphorylation (7Hong M. Chen D.C.R. Klein P.S. Lee V.M.-Y. J. Biol. Chem. 1997; 272: 25326-25332Abstract Full Text Full Text PDF PubMed Scopus (418) Google Scholar). Interestingly, a recent study has demonstrated that lithium increases glycogen synthase activity by a wortmannin-independent mechanism in rat skeletal muscle (39Furnsinn C. Noe C. Herdlicka R. Roden M. Nowotny P. Leighton B. Waldhausl W. Am. J. Physiol. 1997; 273: E514-E520PubMed Google Scholar). Thus, exercise and lithium may work by a similar, serine phosphorylation-independent mechanism for deactivation of GSK-3. In the activated state, GSK-3α and β are tyrosine phosphorylated (Tyr279 and Tyr216, respectively) (13Hughes K. Nikolakaki E. Plyte S.E. Totty N.F. Woodgett J.R. EMBO J. 1993; 12: 803-808Crossref PubMed Scopus (525) Google Scholar, 14Wang Q.M. Fiol C.J. DePaoli-Roach A.A. Roach P.J. J. Biol. Chem. 1994; 269: 14566-14574Abstract Full Text PDF PubMed Google Scholar, 15Murai H. Okazaki M. Kikuchi A. FEBS Lett. 1996; 392: 153-160Crossref PubMed Scopus (63) Google Scholar), making it plausible that dephosphorylation of these residues could facilitate enzyme deactivation. Although one study has demonstrated tyrosine autophosphorylation of GSK-3 (14Wang Q.M. Fiol C.J. DePaoli-Roach A.A. Roach P.J. J. Biol. Chem. 1994; 269: 14566-14574Abstract Full Text PDF PubMed Google Scholar), a clear mechanism for tyrosine dephosphorylation in vivo has not been established, and data are conflicting regarding tyrosine dephosphorylation of GSK-3 as a result of insulin stimulation (15Murai H. Okazaki M. Kikuchi A. FEBS Lett. 1996; 392: 153-160Crossref PubMed Scopus (63) Google Scholar, 31Shaw M. Cohen P. Alessi D.R. FEBS Lett. 1997; 416: 307-311Crossref PubMed Scopus (216) Google Scholar). Because we did not observe Ser21 phosphorylation of GSK-3α with exercise, we hypothesized that the enzyme may be regulated by tyrosine dephosphorylation. However, we found no evidence for exercise regulation of GSK-3 tyrosine phosphorylation in the current study and found stable levels of tyrosine phosphorylation under all conditions. This raises the possibility that exercise deactivates GSK-3 by phosphorylation at an alternative site. Indeed, some isoforms of protein kinase C can phosphorylate and inactivate GSK-3, and the exact sites of phosphorylation have not been determined (15Murai H. Okazaki M. Kikuchi A. FEBS Lett. 1996; 392: 153-160Crossref PubMed Scopus (63) Google Scholar, 38Cook D. Fry M.J. Hughes K. Sumathipala R. Woodgett J.R. Dale T.C. EMBO J. 1996; 15: 4526-4536Crossref PubMed Scopus (344) Google Scholar, 40Goode N. Hughes K. Woodgett J.R. Parker P.J. J. Biol. Chem. 1992; 267: 16878-16882Abstract Full Text PDF PubMed Google Scholar). We could also speculate that glycogen or glycogen-bound molecules function as allosteric regulators of GSK-3 activity in skeletal muscle. If glycogen itself could act as an allosteric activator of GSK-3, this would limit glycogen synthesis under conditions where glycogen is abundant. Following exercise, decreased glycogen content within the muscle fibers could reduce the allosteric activation of GSK-3, leading to increased glycogen synthesis. Our data showing a close correlation between the exercise- and insulin-induced decrease in GSK-3 activity and increase in glycogen synthase activity are consistent with the hypothesis that GSK-3 functions to regulate glycogen synthesis in skeletal muscle. However, recent studies in adipocytes have suggested that activation of phosphatases is the primary factor regulating glycogen synthase activity (41Moule S.K. Welsh G.I. Edgell N.J. Foulstone E.J. Proud C.G. Denton R.M. J. Biol. Chem. 1997; 272: 7713-7719Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar, 42Brady M.J. Bourbonais F.J. Saltiel A.R. J. Biol. Chem. 1998; 273: 14063-14066Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). In adipocytes isoproterenol-induced deactivation of GSK-3 is not accompanied by glycogen synthase activation (41Moule S.K. Welsh G.I. Edgell N.J. Foulstone E.J. Proud C.G. Denton R.M. J. Biol. Chem. 1997; 272: 7713-7719Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar). In 3T3L1 fibroblasts glycogen synthase activity may be regulated by GSK-3 deactivation, but when these cells differentiate into adipocytes, control of synthase with insulin stimulation appears to shift to protein phosphatase-1 activation (42Brady M.J. Bourbonais F.J. Saltiel A.R. J. Biol. Chem. 1998; 273: 14063-14066Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). On the other hand, in cultured human myoblasts (43Halse R. 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Zangen D. Fielding R.A. Goodyear L.J. J. Clin. Invest. 1997; 99: 1251-1257Crossref PubMed Scopus (162) Google Scholar) and c-Jun NH2-terminal kinase signaling cascades (52Aronson D. Dufresne S.D. Goodyear L.J. J. Biol. Chem. 1997; 272: 25636-25640Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar) are also increased by exercise and muscle contraction in skeletal muscle. Thus, there is likely to be convergence of multiple signals at the nucleus in response to contractile activity, with integration of these signals critical in controlling transcription and ultimately determining muscle phenotype. We are beginning to develop an understanding of molecular signaling mechanisms in contracting skeletal muscle, and the current observation that exercise decreases GSK-3 activity should be an important step toward elucidating the molecular mechanisms that regulate glycogen synthesis, protein synthesis, and gene transcription in working muscle. We thank Drs. Lawrence Mandarino and Katsumi Maezono for assistance with the GSK-3 assay.