Control of Glycogen Synthesis in Cultured Human Muscle Cells

Abstract

The regulation of glycogen synthesis and associated enzymes was studied in human myoblasts and myotubes maintained in culture. Both epidermal growth factor (EGF) and insulin stimulated glycogen synthesis approximately 2-fold, this stimulation being accompanied by a rapid and stable activation of the controlling enzyme glycogen synthase (GS). EGF also caused inhibition of glycogen synthase kinase 3 (GSK-3) and activation of the α isoform of protein kinase B (PKB) with the time-course and magnitude of its effects being similar to those induced by insulin. An inhibitor of the mitogen-activated protein (MAP) kinase pathway did not prevent stimulation of GS by EGF, suggesting that this pathway is not essential for the effect. A partial decrease in the fold activation of GS was, however, observed when p70S6k activation was blocked with rapamycin, suggesting a contribution of this pathway to the control of GS by either hormone. Wortmannin, a selective inhibitor of phosphatidylinositol 3′-kinase (PI-3 kinase) completely blocked the effects of both EGF and insulin in these cells. These results demonstrate that EGF, like insulin, activates glycogen synthesis in muscle, acting principally via the PKB/GSK-3 pathway but with a contribution from a rapamycin-sensitive component that lies downstream of PI-3 kinase. The regulation of glycogen synthesis and associated enzymes was studied in human myoblasts and myotubes maintained in culture. Both epidermal growth factor (EGF) and insulin stimulated glycogen synthesis approximately 2-fold, this stimulation being accompanied by a rapid and stable activation of the controlling enzyme glycogen synthase (GS). EGF also caused inhibition of glycogen synthase kinase 3 (GSK-3) and activation of the α isoform of protein kinase B (PKB) with the time-course and magnitude of its effects being similar to those induced by insulin. An inhibitor of the mitogen-activated protein (MAP) kinase pathway did not prevent stimulation of GS by EGF, suggesting that this pathway is not essential for the effect. A partial decrease in the fold activation of GS was, however, observed when p70S6k activation was blocked with rapamycin, suggesting a contribution of this pathway to the control of GS by either hormone. Wortmannin, a selective inhibitor of phosphatidylinositol 3′-kinase (PI-3 kinase) completely blocked the effects of both EGF and insulin in these cells. These results demonstrate that EGF, like insulin, activates glycogen synthesis in muscle, acting principally via the PKB/GSK-3 pathway but with a contribution from a rapamycin-sensitive component that lies downstream of PI-3 kinase. A key step in the lowering of blood glucose levels by insulin is the promotion of uptake of glucose into muscle and its subsequent storage as glycogen. This involves recruitment of additional glucose transporters to the plasma membrane and stimulation of the enzyme glycogen synthase (GS) 1The abbreviations used are: GS, glycogen synthase; GSK-3, glycogen synthase kinase 3; PP1G, glycogen-bound form of protein phosphatase 1; PKB, protein kinase B; PDK1, 3-phosphoinositide-dependent protein kinase 1; EGF, epidermal growth factor; MAP, mitogen-activated protein. 1The abbreviations used are: GS, glycogen synthase; GSK-3, glycogen synthase kinase 3; PP1G, glycogen-bound form of protein phosphatase 1; PKB, protein kinase B; PDK1, 3-phosphoinositide-dependent protein kinase 1; EGF, epidermal growth factor; MAP, mitogen-activated protein.(reviewed in Ref. 1Lawrence Jr., J.C. Roach P.J. Diabetes. 1997; 46: 541-547Crossref PubMed Google Scholar). The activity of GS is regulated by reversible phosphorylation at a number of sites. The key sites involved in the regulation of GS are collectively referred to as sites 3, phosphorylation of which by glycogen synthase kinase 3 (GSK-3) leads to inactivation of GS (2Cohen P. Biochem. Soc. Trans. 1993; 21: 555-567Crossref PubMed Scopus (84) Google Scholar). The dephosphorylation and re-activation of GS is catalyzed by a glycogen-bound form of protein phosphatase 1 (PP1G) (3Dent P. Lavoinne A. Nakielny S. Caudwell F.B. Watt P. Cohen P. Nature. 1990; 348: 302-308Crossref PubMed Scopus (403) Google Scholar). Insulin activates GS via dephosphorylation of the protein. There is evidence for insulin exerting this stimulatory effect on GS by both activating PP1G and by inhibiting GSK-3. Recently, attention has focused on the mechanism by which insulin inhibits GSK-3, with a plausible scheme emerging to link events at the plasma membrane with regulation of GSK-3. GSK-3 is inactivated by phosphorylation at a single serine residue close to its amino terminus (4Sutherland C. Cohen P. FEBS Lett. 1994; 338: 37-42Crossref PubMed Scopus (192) Google Scholar). At least three insulin-stimulated protein kinases, namely p70S6k, p90S6k, and protein kinase B (PKB) are capable of catalyzing this phosphorylation in vitro. Each of these kinases lies downstream of an insulin-stimulated cascade (5Cross D.A. Alessi D.R. Cohen P. Andjelkovich M. Hemmings B.A. Nature. 1995; 378: 785-789Crossref PubMed Scopus (4335) Google Scholar). However, current evidence indicates that PKB is responsible for this insulin-stimulated phosphorylation of GSK-3 in a variety of cell types (5Cross D.A. Alessi D.R. Cohen P. Andjelkovich M. Hemmings B.A. Nature. 1995; 378: 785-789Crossref PubMed Scopus (4335) Google Scholar, 6Hurel S.J. Rochford J.J. Borthwick A.C. Wells A.M. Vandenheede J.R. Turnbull D.M. Yeaman S.J. Biochem. J. 1996; 320: 871-877Crossref PubMed Scopus (83) Google Scholar, 7Moule 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). PKB is itself phosphorylated and activated in response to insulin, this being mediated, at least in part, by 3-phosphoinositide-dependent protein kinase 1 (PDK1) (8Alessi D.R. James S.R. Downes C.P. Holmes A.B. Gaffney P.R. Reese C.B. Cohen P. Curr. Biol. 1997; 7: 261-269Abstract Full Text Full Text PDF PubMed Google Scholar, 9Stokoe D. Stephens L.R. Copeland T. Gaffney P.R. Reese C.B. Painter G.F. Holmes A.B. McCormick F. Hawkins P.T. Science. 1997; 277: 567-570Crossref PubMed Scopus (1045) Google Scholar). The phosphorylation of PKB by PDK1 is dependent on the presence of phosphatidylinositol (3Dent P. Lavoinne A. Nakielny S. Caudwell F.B. Watt P. Cohen P. Nature. 1990; 348: 302-308Crossref PubMed Scopus (403) Google Scholar, 4Sutherland C. Cohen P. FEBS Lett. 1994; 338: 37-42Crossref PubMed Scopus (192) Google Scholar, 5Cross D.A. Alessi D.R. Cohen P. Andjelkovich M. Hemmings B.A. Nature. 1995; 378: 785-789Crossref PubMed Scopus (4335) Google Scholar) triphosphate (10Alessi D.R. Andjelkovic M. Caudwell B. Cron P. Morrice N. Cohen P. Hemmings B.A. EMBO J. 1996; 15: 6541-6551Crossref PubMed Scopus (2498) Google Scholar), the major lipid product of PI-3 kinase, an enzyme activated by insulin via binding to insulin receptor substrate 1 at the plasma membrane (reviewed in Ref. 11Shepherd P.R. Nave B.T. O' Rahilly S. J. Mol. Endocrinol. 1996; 17: 175-184Crossref PubMed Scopus (62) Google Scholar). In addition to insulin, several other growth factors and hormones influence glycogen synthesis and the signaling pathways involved. Of particular interest is the action of epidermal growth factor (EGF) which apparently has different effects on glycogen metabolism in different cell types. In isolated rat adipocytes, EGF stimulates neither glycogen synthesis nor GS activity, despite stimulating p70S6k and p90S6k (12Lin T.A. Lawrence Jr., J.C. J. Biol. Chem. 1994; 269: 21255-21261Abstract Full Text PDF PubMed Google Scholar). Consistent with the failure to activate GS is the observation that, unlike insulin, EGF has only a small and transient effect on the activities of PKB and GSK-3 (13Cross D.A. Watt P.W. Shaw M. van der Kaay J. Downes C.P. Holder J.C. Cohen P. FEBS Lett. 1997; 406: 211-215Crossref PubMed Scopus (191) Google Scholar). In contrast, EGF inhibits GSK-3 in hepatocytes to the same extent as insulin (14Peak M. Rochford J.J. Borthwick A.C. Yeaman S.J. Agius L. Diabetologia. 1998; 41: 16-25Crossref PubMed Scopus (54) Google Scholar) and yet it does not stimulate glycogen synthesis, indeed it antagonizes the stimulatory effect of insulin on this metabolic effect (15Peak M. Agius L. Eur. J. Biochem. 1994; 221: 529-536Crossref PubMed Scopus (13) Google Scholar). This antagonistic effect is not however observed in adipocytes (12Lin T.A. Lawrence Jr., J.C. J. Biol. Chem. 1994; 269: 21255-21261Abstract Full Text PDF PubMed Google Scholar). It is well established that insulin inactivates GSK-3 and activates GS in a variety of muscle systems (5Cross D.A. Alessi D.R. Cohen P. Andjelkovich M. Hemmings B.A. Nature. 1995; 378: 785-789Crossref PubMed Scopus (4335) Google Scholar, 6Hurel S.J. Rochford J.J. Borthwick A.C. Wells A.M. Vandenheede J.R. Turnbull D.M. Yeaman S.J. Biochem. J. 1996; 320: 871-877Crossref PubMed Scopus (83) Google Scholar, 13Cross D.A. Watt P.W. Shaw M. van der Kaay J. Downes C.P. Holder J.C. Cohen P. FEBS Lett. 1997; 406: 211-215Crossref PubMed Scopus (191) Google Scholar, 16Cross D.A. Alessi D.R. Vandenheede J.R. McDowell H.E. Hundal H.S. Cohen P. Biochem. J. 1994; 303: 21-26Crossref PubMed Scopus (420) Google Scholar, 17Borthwick A.C. Wells A.M. Rochford J.J. Hurel S.J. Turnbull D.M. Yeaman S.J. Biochem. Biophys. Res. Commun. 1995; 210: 738-745Crossref PubMed Scopus (34) Google Scholar) but little work has been carried out using EGF, with the exception of one study in rat diaphragm which reported that EGF does not stimulate GS (18Azpiazu I. Saltiel A.R. DePaoli-Roach A.A. Lawrence Jr., J.C. J. Biol. Chem. 1996; 271: 5033-5039Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). This laboratory has utilized cultured human muscle cells to study the control of glycogen synthesis by insulin (6Hurel S.J. Rochford J.J. Borthwick A.C. Wells A.M. Vandenheede J.R. Turnbull D.M. Yeaman S.J. Biochem. J. 1996; 320: 871-877Crossref PubMed Scopus (83) Google Scholar, 17Borthwick A.C. Wells A.M. Rochford J.J. Hurel S.J. Turnbull D.M. Yeaman S.J. Biochem. Biophys. Res. Commun. 1995; 210: 738-745Crossref PubMed Scopus (34) Google Scholar). Here we report that EGF stimulates GS and glycogen synthesis in both human myoblasts and myotubes and that this is apparently mediated via the PKB/GSK-3 pathway. All tissue culture trays were from Costar (Cambridge, MA). Culture media, penicillin/streptomycin, and trypsin-EDTA were from Life Technologies, Inc. (Paisley, UK). Chick embryo extract was obtained from ICN (Costa Mesa, California). Anti-GSK-3α and anti-GSK-3β antibodies and antibodies to the PH-domain of PKBα were as described previously (16Cross D.A. Alessi D.R. Vandenheede J.R. McDowell H.E. Hundal H.S. Cohen P. Biochem. J. 1994; 303: 21-26Crossref PubMed Scopus (420) Google Scholar, 5Cross D.A. Alessi D.R. Cohen P. Andjelkovich M. Hemmings B.A. Nature. 1995; 378: 785-789Crossref PubMed Scopus (4335) Google Scholar). [γ-32P]ATP (148 TBq/mmol) was obtained from ICN.d-[U-14C]glucose (10 GBq/mmol) and UDP-[6-3H]glucose (252 GBq/mmol) were from Amersham Pharmacia Biotech (Buckinghamshire, UK). Wortmannin and rapamycin were from Sigma (Poole, UK), and PD98059 was from New England Biolabs (Massachusetts). Actrapid insulin was from Novo Nordisk (Copenhagen, Denmark). Mouse EGF was from Sigma. Human myoblasts were grown from needle biopsy samples taken from the gastrocnemius muscle of healthy subjects with no family history of Type 2 diabetes and with normal glucose tolerance and normal insulin sensitivity as assessed using the short insulin tolerance test. Myoblasts were maintained in growth medium consisting of Ham's F-10 nutrient mixture containing 20% fetal calf serum, 1% chick embryo extract, 10,000 units/ml penicillin, and 2 mg/ml streptomycin. Myoblasts were fused to form myotubes by incubation for up to 21 days in α-minimal essential medium containing 2% fetal calf serum, 2 mml-glutamine, 10,000 units/ml penicillin, and 2 mg/ml streptomycin. All experiments were performed using cells between the fifth and fifteenth passage at greater than 90% confluence. Prior to hormone treatment, cells were incubated for at least 2 h in serum-free medium. Glycogen synthesis was determined as 14C-glucose incorporation into glycogen. Cells were incubated for 1 h in culture medium containing [U-14C]glucose (5.5 mm glucose; 1.25 μCi/ml) and either insulin, EGF, or both. Medium was then removed, and the cells were rapidly washed five times in ice-cold phosphate-buffered saline. Cells were lysed by the addition of 20% w/v KOH that was neutralized after 1 h by the addition of 1m HCl. The wells were aspirated and washed with 400 μl of distilled water. The aspirates were boiled for 5 min, and glycogen was added to give a final concentration of 12 mg/ml. Glycogen was precipitated with ethanol at 0 °C for 1 h. The samples were centrifuged at 1700 × g for 10 min, pellets were redissolved in formic acid, and radioactivity was determined by scintillation counting. Results were as of glucose into glycogen of cell protein. the cells were rapidly washed three times with ice-cold and by into GS (10 mm mm mm mm 1 mm 10 1 mm 1 mm Cells were then by for using a were centrifuged at for 10 and the pellets Glycogen synthase activity was as incorporation of from into as described in J.J. A. J. FEBS Lett. PubMed Scopus Google Scholar). were incubated with mixture mm mm mm 1% activity containing either mm or 10 for at °C J.J. A. J. FEBS Lett. PubMed Scopus Google Scholar). Results were as activities incubation with growth or other cells were washed three times with ice-cold phosphate-buffered and was Cells were then into kinase containing mm 2 mm 1 mm 1 1 and 1 to and in were for 1 UK), and centrifuged at × g for 5 at GSK-3 or PKB were from of the containing approximately 10 of protein. In was carried out using antibodies that been to for 2 h at incubation of with the for h at the was by for 3 at × The was and the washed with kinase and with A mm containing 1 mm 1 mm 1 mm and 1 mg/ml of and GSK-3 activity in was in a final of μl containing mm mm substrate G.I. J.C. Proud C.G. Biochem. 1997; PubMed Scopus Google Scholar), 10 mm mm mm and mm of protein kinase (6Hurel S.J. Rochford J.J. Borthwick A.C. Wells A.M. Vandenheede J.R. Turnbull D.M. Yeaman S.J. Biochem. J. 1996; 320: 871-877Crossref PubMed Scopus (83) Google Scholar). incubation for at samples were centrifuged at × g for 3 min, and μl of the containing the product was in mm with the were and incorporation was determined by scintillation counting. PKB was in an that activity was the PKB substrate (5Cross D.A. Alessi D.R. Cohen P. Andjelkovich M. Hemmings B.A. Nature. 1995; 378: 785-789Crossref PubMed Scopus (4335) Google Scholar). enzyme of activity was as that which the incorporation of 1 of into substrate in 1 In the effect of of EGF on glycogen synthesis in myoblasts was of cultured myoblasts with EGF caused a in the incorporation of glucose into glycogen from to with This was similar to that by insulin which stimulated the of glycogen synthesis to experiments in cells from three with with EGF also caused a in the activity of GS, with stimulation being observed 5 and a of approximately after 1 The magnitude and are similar to those observed previously in response to insulin (6Hurel S.J. Rochford J.J. Borthwick A.C. Wells A.M. Vandenheede J.R. Turnbull D.M. Yeaman S.J. Biochem. J. 1996; 320: 871-877Crossref PubMed Scopus (83) Google Scholar) the in the work are than reported effect was observed when the were added effects were observed in EGF caused a in the activity of GS from to experiments in cells from three with work from several has that the activation of GS by insulin is mediated via inactivation by GSK-3. The effect of EGF and insulin on the activity of GSK-3 is in EGF a rapid decrease in the activity of GSK-3 2 and both the magnitude and (6Hurel S.J. Rochford J.J. Borthwick A.C. Wells A.M. Vandenheede J.R. Turnbull D.M. Yeaman S.J. Biochem. J. 1996; 320: 871-877Crossref PubMed Scopus (83) Google Scholar) of this effect are similar to those observed with insulin. the events of GSK-3, selective of p70S6k activation p90S6k activation and PI-3 kinase were that inactivation of GSK-3 by EGF is not by rapamycin or PD98059 but is blocked completely by These are with those for insulin (5Cross D.A. Alessi D.R. Cohen P. Andjelkovich M. Hemmings B.A. Nature. 1995; 378: 785-789Crossref PubMed Scopus (4335) Google Scholar, 6Hurel S.J. Rochford J.J. Borthwick A.C. Wells A.M. Vandenheede J.R. Turnbull D.M. Yeaman S.J. Biochem. J. 1996; 320: 871-877Crossref PubMed Scopus (83) Google Scholar), PKB as the kinase responsible for phosphorylation and inactivation of GSK-3 in response to This is by the in which that PKBα is rapidly activated by EGF, the activation being rapid to for the of GSK-3 inactivation the magnitude of activation in response to EGF was similar to that by insulin of PKB by EGF and insulin. cells were with EGF for the times to was determined in as described Results are the of (EGF) or three PKB activity was determined after of the enzyme from of myoblasts incubated for 10 in the or presence of EGF or insulin. are the 5 experiments in three with is by The effect of these on the activity of GS was also rapamycin little effect on the activity of GS, both PD98059 and caused a decrease in the activity in the of addition of EGF, activation was observed in the presence of that the kinase p90S6k pathway is not essential for the activation of GS by EGF, despite EGF being a of that pathway (12Lin T.A. Lawrence Jr., J.C. J. Biol. Chem. 1994; 269: 21255-21261Abstract Full Text PDF PubMed Google Scholar). Wortmannin completely blocked activation of GS by The stimulation of GS activity was, however, in the presence of rapamycin at at previously reported a similar effect of rapamycin on the activation of GS insulin of these cells (6Hurel S.J. Rochford J.J. Borthwick A.C. Wells A.M. Vandenheede J.R. Turnbull D.M. Yeaman S.J. Biochem. J. 1996; 320: 871-877Crossref PubMed Scopus (83) Google of selective on the activation of GS by human myoblasts were incubated in the or presence of EGF (EGF) for 10 cells were with rapamycin for PD98059 for 1 h or for to incubation in the or presence of were and for GS are of in cells from different with is by in a human myoblasts were incubated in the or presence of EGF (EGF) for 10 cells were with rapamycin for PD98059 for 1 h or for to incubation in the or presence of were and for GS are of in cells from different with is by Human muscle cells in culture an for the study of cell Myoblasts and myotubes in culture of muscle in of to insulin and other but are cells and several from muscle (6Hurel S.J. Rochford J.J. Borthwick A.C. Wells A.M. Vandenheede J.R. Turnbull D.M. Yeaman S.J. Biochem. J. 1996; 320: 871-877Crossref PubMed Scopus (83) Google A.C. Wells A.M. Rochford J.J. Hurel S.J. Turnbull D.M. Yeaman S.J. Biochem. Biophys. Res. Commun. 1995; 210: 738-745Crossref PubMed Scopus (34) Google Scholar). is that myoblasts not levels of the glucose and little in glucose uptake in response to insulin L. A. J. PubMed Scopus Google Scholar). this is a of the it does the that observed effects on glycogen synthesis are of effects on glucose uptake and of in the activity of the cells a for study of events in human muscle in of the with when to the in these when using cell taken from a variety of The reported that, in myoblasts obtained from healthy human EGF an stimulation of the of incorporation of glucose into glycogen. This is associated with a rapid and activation of glycogen In both the extent of stimulation and the of the effects were similar to these observed in response to insulin (6Hurel S.J. Rochford J.J. Borthwick A.C. Wells A.M. Vandenheede J.R. Turnbull D.M. Yeaman S.J. Biochem. J. 1996; 320: 871-877Crossref PubMed Scopus (83) Google Scholar). that pathways are involved in the stimulation of GS by both EGF and insulin. both the and extent of the effects of the were similar for the the stimulation of GS activity by EGF and insulin was not both were rapamycin was to prevent the stimulation of GS by both EGF and insulin. the that the stimulation of glycogen synthesis in response to both EGF and insulin is mediated principally via the PKB/GSK-3 to the dephosphorylation of GS and stimulation of its activity, but with an additional contribution via a rapamycin-sensitive pathway that does not inhibition of GSK-3. work on adipocytes has of a rapamycin-sensitive component in the stimulation of glycogen synthesis and GS by insulin P.R. B.T. Biochem. J. 1995; PubMed Scopus Google Scholar). It has been reported using that the phosphorylation and inactivation of GSK-3 in response to EGF is not blocked by rapamycin, out a for p70S6k in that Vandenheede J.R. Cohen P. Biochem. J. 1994; 303: PubMed Scopus Google Scholar). This is by the work which that rapamycin does not the inhibition of GSK-3. PD98059 is effect on this inhibition that the kinase signaling pathway is not involved. previously that rapamycin and PD98059 no effect on the inhibition of GSK-3 in response to insulin (6Hurel S.J. Rochford J.J. Borthwick A.C. Wells A.M. Vandenheede J.R. Turnbull D.M. Yeaman S.J. Biochem. J. 1996; 320: 871-877Crossref PubMed Scopus (83) Google Scholar). The of different effects of EGF on glycogen synthesis in different cell systems (12Lin T.A. Lawrence Jr., J.C. J. Biol. Chem. 1994; 269: 21255-21261Abstract Full Text PDF PubMed Google Scholar, 13Cross D.A. Watt P.W. Shaw M. van der Kaay J. Downes C.P. Holder J.C. Cohen P. FEBS Lett. 1997; 406: 211-215Crossref PubMed Scopus (191) Google Scholar, M. Rochford J.J. Borthwick A.C. Yeaman S.J. Agius L. Diabetologia. 1998; 41: 16-25Crossref PubMed Scopus (54) Google Scholar, M. Agius L. Eur. J. Biochem. 1994; 221: 529-536Crossref PubMed Scopus (13) Google Scholar). is that cells a by EGF which to the stimulatory effects on GS and glycogen Of particular interest is the of the in muscle with with isolated rat hepatocytes EGF inactivates GSK-3 to the same extent as insulin and also a transient activation of PKBα (14Peak M. Rochford J.J. Borthwick A.C. Yeaman S.J. Agius L. Diabetologia. 1998; 41: 16-25Crossref PubMed Scopus (54) Google Scholar) and yet does not stimulate glycogen synthesis and indeed antagonizes the stimulatory effect of insulin on that metabolic (15Peak M. Agius L. Eur. J. Biochem. 1994; 221: 529-536Crossref PubMed Scopus (13) Google Scholar). It is however, that EGF has a of effects in which both and of the EGF receptor M. T. PubMed Scopus Google Scholar). The effects of EGF on glycogen synthesis in hepatocytes are in that are dependent on of cell culture cell and the of the cells. to a component to the action of EGF (15Peak M. Agius L. Eur. J. Biochem. 1994; 221: 529-536Crossref PubMed Scopus (13) Google Scholar). The of EGF to activate in hepatocytes L. A.M. S. D.R. J.R. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar) to in the levels of also a which the stimulatory effects of EGF on glycogen In contrast, the major not effect of EGF in muscle is to stimulate glycogen synthesis, via activation of The stimulatory effects of EGF are observed in the of insulin and are not by insulin, a pathway and no evidence for an additional pathway. It has been previously reported that EGF to stimulate the activity of GS in isolated rat diaphragm (18Azpiazu I. Saltiel A.R. DePaoli-Roach A.A. Lawrence Jr., J.C. J. Biol. Chem. 1996; 271: 5033-5039Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). The that EGF also to stimulate p70S6k in that is with the that a rapamycin-sensitive pathway is the major pathway controlling GS by EGF, the studied in that The of the signaling pathways in human muscle in to The observation that the kinase inhibitor PD98059 a decrease in the activity of GS that the kinase pathway is involved in the of GS activity in these cells. This is with observation that p90S6k activity is in human myoblasts in the of (6Hurel S.J. Rochford J.J. Borthwick A.C. Wells A.M. Vandenheede J.R. Turnbull D.M. Yeaman S.J. Biochem. J. 1996; 320: 871-877Crossref PubMed Scopus (83) Google Scholar). However, addition of EGF, the activity of GS and the in the presence of PD98059 is fold to the induced by EGF in the of the It is that the effect of PD98059 on the of GS is of the of the kinase pathway in the regulation of a GS PP1G (3Dent P. Lavoinne A. Nakielny S. Caudwell F.B. Watt P. Cohen P. Nature. 1990; 348: 302-308Crossref PubMed Scopus (403) Google Scholar). levels of kinase activity in the activate the GS inhibition of kinase activity with PD98059 the GS This for subsequent stimulation of GS via inhibition of GSK-3 a which is by lowering of the activity of GS by a for PI-3 kinase and the kinase pathway (16Cross D.A. Alessi D.R. Vandenheede J.R. McDowell H.E. Hundal H.S. Cohen P. Biochem. J. 1994; 303: 21-26Crossref PubMed Scopus (420) Google Scholar) in the of In the reported demonstrate that EGF stimulates both glycogen synthesis and GS in human muscle cells in culture. This is mediated via the but is also a contribution to the activation of GS from a component which is rapamycin-sensitive and lies downstream of PI-3 kinase. for major contribution to the tissue culture work and D. M. Turnbull for