©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Upstream Mechanisms of Glycogen Synthase Activation by Insulin and Insulin-like Growth Factor-I
GLYCOGEN SYNTHASE ACTIVATION IS ANTAGONIZED BY WORTMANNIN OR LY294002 BUT NOT BY RAPAMYCIN OR BY INHIBITING p21(*)

(Received for publication, July 21, 1994; and in revised form, November 1, 1994)

Ritsuko Yamamoto-Honda (1) Kazuyuki Tobe (1) Yasushi Kaburagi (1) Kohjiro Ueki (1) Shoji Asai (1) Makoto Yachi (1) Mikako Shirouzu (2) Junji Yodoi (3) Yasuo Akanuma (4) Shigeyuki Yokoyama (2) Yoshio Yazaki (1) Takashi Kadowaki (1)(§)

From the  (1)Third Department of Internal Medicine, Faculty of Medicine, and the (2)Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan, the (3)Department of Biological Responses, Laboratory of Infection and Prevention Institute for Virus Research, Kyoto University, Shogoin, Kawahara-Cho, Sakyo, Kyoto 606, Japan, and the (4)Institute for Diabetes Care and Research, Asahi Life Foundation, 1-6-1 Marunouchi, Chiyoda-ku, Tokyo 100, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

This study was undertaken to define intracellular signaling pathways upstream to glycogen synthase activation. First, we examined the role of the two pathways of insulin signaling, Ras-dependent and wortmannin/LY294002-sensitive, in glycogen synthase activation. Although negative dominant Ras (Ras17N) induction in PC12 cells markedly decreased activities of mitogen-activated protein kinase (MAP) and pp90 S6 kinase in response to insulin or insulin-like growth factor I (IGF-I), activation of glycogen synthase by these agents was unaffected by negative dominant Ras induction. In contrast, wortmannin and 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002), inhibitors of phosphatidylinositol 3-kinase, antagonized glycogen synthase activation in response to insulin or IGF-I. Next, we examined the contribution of pp70 S6 kinase, one of the wortmannin/LY294002-sensitive signaling molecules on glycogen synthase activation. Immunosuppressant rapamycin completely blocked activation of pp70 S6 kinase by insulin or IGF-I, but rapamycin alone or in combination with induction of negative dominant Ras failed to antagonize glycogen synthase activation by these hormones. These data suggest that 1) activation of Ras-MAP kinase is not necessary for stimulation of glycogen synthase and 2) activation of wortmannin/LY294002sensitive pathway, independent of pp70 S6 kinase, plays a key role in glycogen synthase regulation in PC12 cells.


INTRODUCTION

One of the major physiological roles of insulin is to regulate fuel metabolism. Insulin stimulates a number of processes that are central to the deposition of glucose into glycogen. Of these processes, activation of glycogen synthase is important in insulin's action. Defects of activating glycogen synthase by this hormone were observed in insulin resistance of type II diabetes mellitus, implicating defects in signaling upstream to the enzyme(1, 2, 3) . The stimulation of glycogen synthase by insulin and other polypeptide growth factors seems to possess a common intermediate element in the signaling pathways (4) and results in the dephosphorylation and activation of glycogen synthase. The precise mechanism by which insulin mediates signals to activate glycogen synthase, however, is still incompletely understood. Activation of tyrosine kinase of the insulin receptor might be the initial step for activating glycogen synthase(5, 6) , and this signal finally either activates protein phosphatase-1 (7) by increasing phosphorylation of site 1 of glycogen-associated subunit (G subunit) of protein phosphatase-1 (8) or inhibits glycogen synthase kinase-3 (9, 10) through the yet unidentified mechanisms. An insulin-stimulated protein kinase, a mammalian homologue of pp90 S6 kinase (10) has been a candidate molecule to link between the insulin receptor tyrosine kinase and dephosphorylation of glycogen synthase because it is shown to phosphorylate and activate the G subunit of phosphatase-1 and to phosphorylate and inactivate glycogen synthase kinase-3 in vitro (8, 12). (^1)These data suggested that cascade involving insulin-stimulated protein kinase, originating from p21 (Ras) (^2)signaling pathway leading to activation of mitogen-activated protein (MAP) kinase and pp90 S6 kinase, might play a pivotal role in glycogen synthesis.

In addition to the Ras-MAP kinase cascade, polypeptide growth factors, including insulin, activate PI 3-kinase cascade(13) . In insulin-sensitive cell lines, most of the PI 3-kinase activity is associated with insulin receptor substrate-1 by the interaction of the Src homology 2 domains of the 85-kDa subunit of PI 3-kinase with the YXXM motifs of insulin receptor substrate-1(14, 15) . Recently, wortmannin and 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002), inhibitors for PI 3-kinase(16, 17) , were reported to inhibit stimulation of 2-deoxyglucose uptake and inhibition of isoproterenol-induced lipolysis by insulin(16, 18) , suggesting that wortmannin/LY294002-sensitive mechanisms might lie upstream to these metabolic responses. These observations inspired us to examine whether wortmannin/LY294002-sensitive mechanism would contribute to glycogen synthase activation, another important metabolic response of insulin. Downstream to wortmannin/LY294002-sensitive signaling pathway lies pp70 S6 kinase(18, 19, 20) . pp70 S6 kinase phosphorylated and inactivated glycogen synthase kinase-3 in vitro(12) , raising the possibility that pp70 S6 kinase could contribute to stimulation of glycogen synthesis.

Prior studies implicating insulin-sensitive protein kinase and pp70 S6 kinase in glycogen synthase activation have relied exclusively on in vitro systems, and there were no reports examining the contribution of wortmannin/LY294002-sensitive pathway on insulin-induced glycogen synthase activation to date. Thus, we have investigated the role of Ras and wortmannin/LY294002-sensitive pathway, including pp70 S6 kinase, in the insulin-induced activation of glycogen synthase in PC12 cells. PC12 cells (21) were reported to possess receptors for insulin and IGF-I(22) , to stimulate the conversion of the inactive GDP-bound form of Ras to the active GTP-bound form(23) , to stimulate S6 kinase activity(24) , and to stimulate glycogen synthase (25) in response to insulin or IGF-I. In the present study, we found that glycogen synthase activation evoked by insulin or IGF-I was not affected by inhibiting Ras activation by induction of negative dominant Ras (Ras17N) but significantly affected by treatment with wortmannin or LY294002, indicating that glycogen synthase activation by insulin or IGF-I in PC12 cells is largely under the control of wortmannin/LY294002-sensitive pathway, not downstream to Ras-MAP kinase cascade. We also observed that treatment with rapamycin, alone or in combination with negative dominant Ras, failed to antagonize glycogen synthase activation by these hormones, indicating glycogen synthase activation was not downstream to pp70 S6 kinase.


EXPERIMENTAL PROCEDURES

Materials

Porcine insulin and recombinant human IGF-I were gifts from Eli Lilly Co. and Fujisawa Pharmaceuticals, respectively. Wortmannin, kindly provided by Dr. Yuzuru Matsuda (Tokyo Research Laboratories, Kyowa Hakko Kogyo Co.), was dissolved in dimethyl sulfoxide at 10 mM and stored at -20 °C in the dark just prior to use. Rapamycin, kindly provided by Fujisawa Pharmaceutical Co., was dissolved in ethanol at 100 µg/ml and stored at -20 °C. LY 294002 was synthesized as described(17) . When added, wortmannin or rapamycin was incubated for 30 min, and LY294002 was incubated for 5 min before the addition of insulin or IGF-I. All other materials were obtained from the same sources as previously described (26) .

Cells and Culture Conditions

PC12 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 5% equine serum. When necessary, cells were incubated with Dulbecco's modified Eagle's medium with 0.1% fetal bovine serum and 0.2% equine serum (low serum medium). The PC12Ras17N cells were obtained following transfection with plasmids expressing a dominant inhibitory mutant Ha-ras gene (containing an asparagine substitution at position 17, Ras17N) under the transcriptional control of murine mammary tumor virus promoter (27) . Induction of Ras17N was achieved by adding 1 µM dexamethasone to the medium and incubating for 24 h. The tyrosine phosphorylation of pp185 (presumably IRS-I) and pp95 (presumably the beta subunit of the insulin receptor or IGF-I receptor, respectively), evaluated by immunoblot with antiphosphotyrosine antibodies, PI 3-kinase activity in immunoprecipitates with antibodies against the epitopes of insulin receptor substrate-1, MAP kinase activity, pp90 S6 kinase activity, and pp70 S6 kinase activity in response to insulin or IGF-I in PC12Ras17N cells was almost equal to those in PC12 cells (data not shown).

MAP Kinase Activity in Immune Complex

This assay was performed as described (28) with modifications. PC12 cell lines were preincubated in low serum medium for 24 h with or without dexamethasone and then incubated with the indicated concentrations of insulin or IGF-I for 5 min. The cells were lysed with buffer A containing 20 mM Tris-HCl, pH 7.5, 25 mM beta-glycerophosphate, 100 mM NaCl, 1 mM sodium orthovanadate, 2 mM EGTA, and 1 mM phenylmethylsulfonyl fluoride. Lysates were incubated with protein A-Sepharose preadsorbed with alphaC92 (antibodies against the peptide corresponding to the residues 350-367 of extracellular signal-regulated kinase-1, described by Tobe et al.(29) ) for 1 h at 4 °C, and the immunoprecipitates were extensively washed. To 5 µl of the immunoprecipitates, 40 µl of kinase buffer (25 mM Tris-HCl, pH 7.4, 10 mM MgCl(2), 1 mM dithiothreitol, 40 µM ATP, 0.2 µCi of [-P]ATP, 2 µM protein kinase inhibitor (rabbit sequence, Sigma), 0.5 mM EGTA) and 5 µl of myelin basic protein (5 mg/ml) were added. After 10 min at 25 °C, the reaction was stopped, the aliquots were spotted on squares of P-81 paper, washed, and counted as described(29) .

pp90 S6 Kinase Activity in Immune Complex

This assay was performed as described(28) . alpharsk were antibodies prepared against the residues 699-715 of the amino acid sequence of mouse pp90 S6 kinase homologues(28, 30) . PC12 cell lines were preincubated in low serum medium for 24 h, incubated with the indicated concentrations of insulin or IGF-I for 10 min, and lysed with buffer B (25 mM Tris-HCl, pH 7.4, containing 1 mM sodium orthovanadate, 10 mM sodium pyrophosphate, 10 mM NaF, and 1 mM phenylmethylsulfonyl fluoride). Cell lysates were incubated with alpharsk preadsorbed to protein A-Sepharose for 1 h at 4 °C. The immunoprecipitates were washed and resuspended in 40 µl of kinase buffer and 50 µg of S6 peptide (KKKLSSLKA). After 15 min at 30 °C, the reactions were stopped and analyzed as described above.

Assay of PI 3-Kinase Activity in Immunoprecipitates with alphaPY

This assay was performed as described by Fukui and Hanafusa (31) with some modifications. Confluent PC12 cell lines were preincubated with low serum medium for 24 h and treated with or without 100 nM IGF-I for 1 min. The cells were lysed in buffer B containing 1 mM MgCl(2). The lysates were immunoprecipitated with alphaPY preadsorbed to protein A-Sepharose. The immunoprecipitates were washed once with buffer B containing 500 mM LiCl and once with PI 3-kinase reaction buffer (20 mM Tris-HCl, pH 7.5, 100 mM NaCl, and 0.5 mM EGTA). The reaction was started by adding 50 µl of PI 3-kinase reaction buffer containing 20 mM MgCl(2), 10 µM ATP, 3 µCi of [-P] ATP, and 0.2 mg/ml phosphatidylinositol dissolved in dimethy1 sulfoxide to the immunoprecipitates. After the incubation at 25 °C for 5 min, the reactions were terminated by addition of 150 µl of chloroform, methanol, 11.6 N HCl (100:20:2), 100 µl of chloroform was added, and the organic phase was separated and washed twice with methanol, 1 N HCl (1:1). The lipids were concentrated in vacuo, spotted onto a Silica Gel 60 plate, and developed in chloroform, methanol, 28% ammonium hydroxide, water (43:38:5:7) and were visualized by autoradiography.

pp70 S6 Kinase Activity in Immune Complex

This assay was performed according to the method of Chen and Blenis (32) with modifications. Antibodies against pp70 S6 kinase (C-18) were against the epitopes corresponding to residues 485-502 of rat pp70 S6 kinase and obtained from Santa Cruz Biotechnology (Santa Cruz, CA). PC12 cells were preincubated with low serum medium for 24 h, incubated with indicated concentrations of insulin or IGF-I for 30 min, and lysed with buffer C containing 75 mM beta-glycerophosphate, pH 7.5, 10 mM MgCl(2), 10 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 5 µg/ml aprotinin, 2 µM protein kinase inhibitor, and 5 µg/ml leupeptin. Cell lysates were immunoprecipitated, washed, and resuspended in 30 mM beta-glycerophosphate, pH 7.5, 15 mM MgCl(2), and 10 mM EGTA, and 50 ng of 32-mer peptides from the C-terminal sequence of ribosomal S6 protein (KEAKEKRQEQIAKRRRLSSLRASTSKSGGSQK, Life Technologies, Inc.). After 15 min at 30 °C, the reaction was stopped and analyzed as described above.

Glycogen Synthase

Glycogen synthase assays were performed according to the method of Thomas et al.(33) with modifications. PC12 cell lines were preincubated in low serum medium for 24 h. Cells were then washed twice with glucose-free Krebs-Ringer phosphate-HEPES buffer and incubated with various concentrations of insulin or IGF-I for 45 min. Cells were then lysed, the lysates were centrifuged, and the supernatant was used as enzyme preparation(3, 26) . Glycogen synthase activities were determined in the presence of 0.25 mM (for ligand-dependent activity) or 10 mM (for total activity) glucose 6-phosphate.


RESULTS

Effect of Ras17N Induction on Glycogen Synthase Activity

Insulin or IGF-I stimulated MAP kinase, pp90 S6 kinase, and pp70 S6 kinase activity in the alphaC92, alpharsk, and C-18 immunoprecipitates from PC12 cells. In a representative study, MAP kinase activity, pp90 S6 kinase activity, and pp70 S6 kinase activities were 0.097, 0.096, and 0.76 nmol/min/g in unstimulated PC12 cell lysates, increased to 0.87, 0.22, and 4.1 nmol/min/g in response to 1 µM insulin, and increased to 2.3, 0.24, and 4.2 nmol/min/g in response to 100 nM IGF-I, respectively. To evaluate the effect of inducing Ras17N, the MAP kinase activity and pp90 S6 kinase activities in PC12 cell lines were examined. Induction of Ras17N blocked an increase in those kinase activities in response to insulin or IGF-I stimulation (Fig. 1A). The -fold stimulation of myelin basic protein kinase activity in PC12-DEX and PC12Ras17N-DEX cells in response to insulin were 11.9 and 2.8, respectively. Insulin-stimulated pp90 S6 kinase activity was increased maximally to 255% in PC12-DEX cells and 140% in PC12Ras17N-DEX cells. IGF-I-stimulated pp90 S6 kinase activity was increased maximally to 282% in PC12-DEX cells and 130% in PC12Ras17N-DEX cells. Suppression of Ras-MAP kinase cascades in PC12Ras17N-DEX cells resulted in impaired cell growth in response to insulin or IGF-I as compared with PC12-DEX cells (data not shown). On the contrary, pp70 S6 kinase activity (Fig. 1A) and glycogen synthase activities (Fig. 1, B and C) were unaffected by Ras17N induction. pp70 S6 kinase activity was increased maximally to 3.9- and 3.6-fold in response to insulin and to 3.5- and 3.3-fold in response to IGF-I in PC12-DEX and PC12Ras17N-DEX cells, respectively, compared with unstimulated PC12-DEX cells. Glycogen synthase activity ratio was increased maximally to 0.11 and 0.11 in response to insulin and to 0.12 and 0.12 in response to IGF-I in PC12-DEX and PC12Ras17N-DEX cells, respectively. Half-maximal stimulation of glycogen synthase in PC12-DEX cells was at 40 nM insulin and 0.3 nM IGF-I, and that in PC12Ras17N-DEX cells was at 40 nM insulin and 0.4 nM IGF-I, respectively.


Figure 1: Effects of Ras17N induction on MAP kinase, pp90 S6 kinase, pp70 S6 kinase, and glycogen synthase. PanelA, PC12 cells were pretreated with low serum medium containing 1 µM dexamethasone for 24 h and stimulated with 1 µM insulin or 100 nM IGF-I. Immune complex kinase assays were performed as described under ``Experimental Procedures.'' The results were converted to the ratio against the unstimulated figure of PC12-DEX cells, and are the means of three separate experiments shown in triplicate (means ± S.D.). PanelB, PC12 cells were pretreated with low serum medium containing 1 µM dexamethasone for 24 h and stimulated with various concentrations of IGF-I. Glycogen synthase activity was measured as described under ``Experimental Procedures.'' The results (in dpm) were converted to the activity ratios determined by dividing the activity measured with 0.25 mM glucose 6-phosphate by the activity measured with 10 mM glucose 6-phosphate and are the means of three separate experiments shown in duplicate (means ± S.D.). bullet, PC12-DEX; circle, PC12Ras17N-DEX. PanelC, PC12 cells were pretreated with low serum medium containing 1 µM dexamethasone for 24 h and stimulated with various concentrations of insulin. Glycogen synthase activity was measured as described under ``Experimental Procedures.'' The results (in dpm) were converted to the activity ratios and are the means of three separate experiments shown in duplicate (means ± S.D.). bullet, PC12-DEX; circle, PC12Ras17N-DEX.



Effect of Wortmannin on Glycogen Synthase Activity

Treatment of wortmannin inhibited alphaPY-associated PI 3-kinase activity in response to IGF-I (Fig. 2A). The IGF-I-induced activation of pp70 S6 kinase and glycogen synthase were antagonized by addition of wortmannin. The inhibitory effect was observed as low as at 1 nM and was almost complete at 100 nM (Fig. 2, B and C). Similar inhibitory effects of wortmannin on insulin-induced activation of pp70 S6 kinase and glycogen synthase were observed (data not shown). The total glycogen synthase activity, assayed in the presence of 10 mM glucose 6-phosphate, was unaffected by treatment with 30 nM wortmannin, indicating that the inhibitory effect of wortmannin was due to modification of phosphorylation state of glycogen synthase rather than direct inhibition of the enzyme. Wortmannin did not inhibit glycogen synthase in vitro at concentrations up to 100 nM. The activation of MAP kinase and pp90 S6 kinase was not significantly inhibited by 30 nM wortmannin treatment (Fig. 2D).


Figure 2: Effects of wortmannin on PI 3-kinase, MAP kinase, pp90 S6 kinase, pp70 S6 kinase, and glycogen synthase. PanelA, PI 3-kinase activity in alphaPY immunoprecipitates from cells pretreated without (vehicle, 0.01% dimethyl sulfoxide) or with various concentrations of wortmannin and stimulated with or without IGF-I was analyzed as described under ``Experimental Procedures.'' Autoradiograms were exposed at -70 °C for 12 h. PanelB, PC12 cells were preincubated for 30 min without (vehicle, 0.01% dimethyl sulfoxide) or with various concentrations of wortmannin and incubated for a further 45 min with or without 100 nM IGF-I. Glycogen synthase activity was measured as described under ``Experimental Procedures.'' The results are expressed as the (means ± S.D.) percentages of maximal stimulation by IGF-I and are the means of three separate experiments in duplicate. PanelC, PC12 cells were preincubated for 30 min without (vehicle, 0.01% dimethyl sulfoxide) or with various concentrations of wortmannin and incubated for a further 30 min with or without 100 nM IGF-I. pp70 S6 kinase activity was measured as described under ``Experimental Procedures.'' The results are expressed as the (means ± S.D.) percentages of maximal stimulation by IGF-I and are the means of two separate experiments in triplicate. PanelD, PC12 cells were pretreated with low serum medium for 24 h, preincubated for 30 min without (vehicle, 0.01% dimethyl sulfoxide) or with 30 nM wortmannin, and incubated with 1 µM insulin or 100 nM IGF-I for a further 5 min (MAP kinase) or 10 min (pp90 S6 kinase). Immune complex kinase assays were performed as described under ``Experimental Procedures.'' The results were converted to the ratio against the unstimulated figure of PC12 cells and are the means of two separate experiments shown in triplicate (means ± S.D.).



Effect of LY294002 on Glycogen Synthase Activity and Kinase Activation

Treatment of LY294002 inhibited alphaPY-associated PI 3-kinase activity in response to IGF-I (Fig. 3A). LY294002 also antagonized insulin-induced activation of pp70 S6 kinase and glycogen synthase (Fig. 3, B and C). IC of pp70 S6 kinase inhibition occurred at 2.0 µM, and that of glycogen synthase inhibition occurred at 13 µM. Total glycogen synthase activity was unaffected by treatment with 30 µM LY294002. LY294002 did not affect glycogen synthase activity in vitro at concentrations up to 30 µM. The activation of MAP kinase and pp90 S6 kinase was not significantly inhibited by 30 µM LY294002 treatment (Fig. 3D).


Figure 3: Effects of LY294002 on PI 3-kinase, MAP kinase, pp90 S6 kinase, pp70 S6 kinase, and glycogen synthase. PanelA, PI 3-kinase activity in alphaPY immunoprecipitates from cells pretreated without (vehicle, 1% dimethyl sulfoxide) or with various concentrations of LY294002 and stimulated with or without IGF-I was analyzed as described under ``Experimental Procedures.'' Autoradiograms were exposed at -70 °C for 12 h. PanelB, PC12 cells were preincubated for 5 min without (vehicle, 1% dimethyl sulfoxide) or with various concentrations of LY294002 and incubated for a further 45 min with or without 100 nM IGF-I. Glycogen synthase activity was measured as described under ``Experimental Procedures.'' The results are expressed as the (means ± S.D.) percentages of maximal stimulation by IGF-I and are the means of three separate experiments in duplicate. PanelC, PC12 cells were preincubated for 5 min without (vehicle, 1% dimethyl sulfoxide) or with various concentrations of LY294002 and incubated for a further 30 min with or without 100 nM IGF-I. pp70 S6 kinase activity was measured as described under ``Experimental Procedures.'' The results are expressed as the (means ± S.D.) percentages of maximal stimulation by IGF-I and are the means of two separate experiments in triplicate. PanelD, PC12 cells were pretreated with low serum medium for 24 h, preincubated for 5 min without (vehicle, 1% dimethyl sulfoxide) or with 30 µM LY294002, and incubated for a further 5 min (MAP kinase) or 10 min (pp90 S6 kinase) with 1 µM insulin or 100 nM IGF-I. Immune complex kinase assays were performed as described under `` Experimental Procedures.'' The results were converted to the ratio against the unstimulated figure of PC12 cells and the means of two separate experiments in triplicate (means ± S.D.).



Effect of Rapamycin on Glycogen Synthase Activity

The immunosuppressant rapamycin has been shown to inhibit pp70 S6 kinase activity by insulin(34) . Treatment of 25 ng/ml rapamycin completely blocked basal, insulin-stimulated, and IGF-I-stimulated pp70 S6 kinase activity while MAP kinase activity and pp90 S6 kinase activities were unaffected by rapamycin (Fig. 4A). Glycogen synthase activity stimulated by insulin or IGF-I was not affected by rapamycin in PC12 cells (Fig. 4B) nor in PC12Ras17N-DEX cells (Fig. 4C). Glycogen synthase activity ratio in PC12 cells was increased maximally to 0.12 and 0.12 in response to insulin and to 0.16 and 0.15 in response to IGF-I with or without rapamycin treatment, respectively. Glycogen synthase activity ratio in PC12-DEX cells was increased maximally to 0.13 and 0.12 in response to insulin and to 0.15 and 0.15 in response to IGF-I with or without rapamycin treatment, respectively. Half-maximal stimulation of glycogen synthase in PC12 cells was at 0.25 and 0.25 nM IGF-I with and without rapamycin treatment, and that in PC12-DEX cells was at 0.30 and 0.35 nM IGF-I with and without rapamycin treatment, respectively.


Figure 4: Effects of rapamycin on MAP kinase, pp90 S6 kinase, pp70 S6 kinase, and glycogen synthase. PanelA, PC12 cells were pretreated with low serum medium for 24 h, preincubated for 30 min without (vehicle, 0.25% ethanol) or with 25 ng/ml rapamycin, and incubated for a further 5 min (MAP kinase), 10 min (pp90 S6 kinase), or 30 min (pp70 S6 kinase) with 1 µM insulin or 100 nM IGF-I. Immune complex kinase assays were performed as described under ``Experimental Procedures.'' The results were converted to the ratio against the unstimulated figure of PC12 cells and are the means of two separate experiments in triplicate (means ± S.D.). PanelB, PC12 cells were preincubated for 30 min without (vehicle, 0.25% ethanol) or with 25 ng/ml rapamycin and incubated for further a 45 min with various concentrations of IGF-I. Glycogen synthase activity was measured as described under ``Experimental Procedures.'' The results (in dpm) were converted to the activity ratios determined by dividing the activity measured with 0.25 mM glucose 6-phosphate by the activity measured with 10 mM glucose 6-phosphate and are the means of three separate experiments in duplicate (means ± S.D.). bullet, with rapamycin; circle, without rapamycin. PanelC, PC12Ras17N cells were pretreated with low serum medium containing 1 µM dexamethasone for 24 h, preincubated for 30 min without (vehicle, 0.25% ethanol) or with 25 µg/ml rapamycin, and incubated for a further 45 min with various concentrations of IGF-I. Glycogen synthase activity was measured as described under ``Experimental Procedures.'' The results (in dpm) were converted to the activity ratios and are the means of three separate experiments in duplicate (means ± S.D.). bullet, with rapamycin; circle, without rapamycin.




DISCUSSION

The defect in insulin action observed in type II diabetic patients affects insulin-stimulated glycogen synthesis as well as glucose uptake (1, 2) , suggesting defects in signaling upstream to glycogen synthase. The longstanding paradox that insulin promotes the phosphorylation of some proteins while this hormone also promotes the dephosphorylation of glycogen synthase was recently explained that the latter mechanism was due to activation of the G subunit of type 1 protein phosphatase and inactivation of glycogen synthase kinase-3 by phosphorylation(8, 12) . To date, pp90 S6 kinase and/or pp70 S6 kinase have been believed to be involved in the process because pp90 S6 kinase phosphorylated both G subunits of protein phosphatase-1 and glycogen synthase kinase-3 and pp70 S6 kinase phosphorylated glycogen synthase kinase-3 in vitro(8, 12) . In addition, wortmannin and LY294002 were recently reported to inhibit stimulation of 2-deoxyglucose uptake(16, 18) , raising the possibility that wortmannin/LY294002-sensitive mechanisms might lie upstream to metabolic responses of insulin in common. Thus, the present experiments were designed to investigate whether the upstream mechanism of glycogen synthase activation would be Ras-MAP kinase-dependent or wortmannin/LY294002-sensitive and to evaluate the hypothesis that activation of pp90 S6 kinase and/or pp70 S6 kinase mediates glycogen synthase activation in intact cell system.

Several insulin-stimulated signal transduction pathways have been shown to be mediated by Ras. Insulin, as well as other polypeptide growth factors, activates Ras by stimulating the conversion of the inactive GDP-bound form of Ras to the active GTP-bound form(35, 36) . Ras activates the downstream kinases (37, 38, 39) such as MAP kinase and pp90 S6 kinase (or an insulin-sensitive protein kinase)(28, 40, 41) , and the latter is presumably involved in glycogen synthesis. As shown in Fig. 1, we found that inhibition of Ras by Ras17N did not affect glycogen synthase activity stimulated by insulin or IGF-I, although the activation of MAP kinase and pp90 S6 kinase and cellular growth by these hormones were impaired. This observation in vivo differed from those observed in vitro(8, 11) and provides first direct evidence that activation of Ras, MAP kinases, and pp90 S6 kinase is not required to activate glycogen synthase in response to insulin or IGF-I. We also observed no significant increase in glycogen synthase activity in response to insulin in Chinese hamster ovary cells overexpressing human insulin receptor and MAP kinase, although in these cell lines, pp90 S6 kinase activity was more sensitive to insulin than the parental cell lines overexpressing human insulin receptor. (^3)These data, together with the recent reports that epidermal growth factor activates Ras signaling but fails to activate glycogen synthase in 3T3-L1 adipocytes and rat adipocytes(42, 43) , strongly suggest that activation of Ras-MAP kinase pathway is neither necessary nor sufficient for activation of glycogen synthase by polypeptide growth factors in these cell lines.

Wortmannin/LY294002-sensitive pathway is another important cascade in insulin signaling(13) . PI 3-kinase was initially characterized as a phosphatidylinositol kinase that associated with activated tyrosine kinases or tyrosine-phosphorylated proteins such as IRS-I(14, 15) . Recently, distinct forms of PI 3-kinase activated by beta subunits of GTP-binding proteins (44) as well as mammalian target of rapamycin (also called RAFT1 and FRAP) with strong sequence similarity to the lipid kinase domain of p110alpha and beta of PI 3-kinase (45, 46, 47) were characterized. These ``PI 3-kinase family'' molecules also seem to be targets of LY294002, inhibitors originally designed as specific inhibitors of PI 3-kinase associated with activated tyrosine kinase (25) , and low concentrations of wortmannin(16, 44) . Wortmannin and/or LY294002 antagonized insulin-induced stimulation of 2-deoxyglucose uptake(16, 18) , inhibition of isoproterenol-induced lipolysis by insulin(18) , and activation of pp70 S6 kinase(18, 19, 20) . As molecules of the PI 3-kinase family responsible for activating the above processes have not yet been specified, these processes are now defined as wortmannin/LY294002-sensitive(48) . It is interesting that activation of glycogen synthase by insulin or IGF-I was strongly inhibited by low concentrations of wortmannin (Fig. 2B). Under these conditions, wortmannin did not affect MAP kinase nor pp90 S6 kinase activities significantly (Fig. 2D). In addition, the inhibitory effect of LY294002 was observed on glycogen synthase activation with an IC of 13 µM (Fig. 3B). This value is comparable with that to prevent proliferation of smooth muscle cells in cultured rabbit aortic segments (IC, 32 µM(17) ). These observations have strongly suggested that the wortmannin/LY294002-sensitive pathway might be a point of divergence in stimulating glycogen synthesis in PC12 cells.

pp70 S6 kinase is another kinase that phosphorylates glycogen synthase kinase-3 in vitro(12) . This kinase is known to be activated by polypeptide growth factors including insulin(49, 50) , the upstream mechanism of which is independent on Ras-signaling cascades (18) (Fig. 1A) and dependent on wortmannin/LY294002-sensitive mechanisms(18, 19, 20) . In the present study, rapamycin failed to antagonize glycogen synthase activation by insulin or IGF-I, although pp70 S6 kinase activity was abolished by rapamycin treatment. In addition, inhibitory profiles of LY294002 on glycogen synthase activation was somewhat different from those of pp70 S6 kinase activation, supporting the conclusion drawn from experiments on rapamycin treatment. Thus, we demonstrated that pp70 S6 kinase is not involved in the activation of glycogen synthase by these hormones in PC12 cells. Similar results were recently reported in rat adipocytes that epidermal growth factor activates pp70 S6 kinase but fails to activate glycogen synthase and that activation of glycogen synthase was not antagonized by rapamycin(43) . Moreover, it is also unlikely that pp90 S6 kinase and pp70 S6 kinase are complementary; that is, activation of either S6 kinase is sufficient for glycogen synthase activation because rapamycin, in combination with Ras17N induction, also failed to antagonize glycogen synthase activation by these hormones (Fig. 2C).

In summary, we propose that wortmannin/LY294002-sensitive mechanism, not pp70 S6 kinase nor Ras-MAP kinase-signaling cascade, might be a point of divergence in glycogen synthase activation in PC12 cells. This novel finding might provide a clue to pathogenesis of impaired activation of glycogen synthase observed in type II diabetes mellitus.


FOOTNOTES

*
This work was supported by Grant 192125 from the Juvenile Diabetes Foundation International (to T. K.) and by a grant for diabetes research from the Ohtsuka Pharmaceutical Co., Ltd. (to T. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom all correspondence should be addressed. Tel.: 81-3-3815-5411 (ext. 3111); Fax: 81-3-3815-2087.

(^1)
In this paper, the term in vivo refers to intact cells, whereas the term in vitro refers to a cell-free system.

(^2)
The abbreviations used are: Ras, p21; MAP, mitogen-activated protein; PI 3-kinase, phosphatidylinositol 3-kinase; IGF-I, insulin-like growth factor I; alphaPY, antibodies specific for phosphotyrosine; pp, phosphoprotein; Ras17N, mutant Ha-ras with Asn substitution at position 17; PC12Ras17N, PC12 cells transfected with plasmids expressing Ras17N gene under the transcriptional control of murine mammary tumor virus promoter; PC12-DEX, PC12 cells pretreated with dexamethasone; PC12Ras17N-DEX, PC12Ras17N cells pretreated with dexamethasone.

(^3)
K. Ueki and T. Kadowaki, unpublished observation.


ACKNOWLEDGEMENTS

We thank Dr. Kinori Kosaka, Dr. Ryoko Hagura, Dr. Hiroko Kadowaki, Dr. Osamu Koshio, Hajime Kawashima (Institute for Diabetes Care and Research, Asahi Life Foundation), Dr. Ingvar M. Ferby, Dr. Takao Shimizu (Department of Biochemistry, Faculty of Medicine, University of Tokyo), and Dr. Zen-ichiro Honda (Department of Internal Medicine and Physical Therapy, Faculty of Medicine, University of Tokyo) for helpful suggestions and support, Dr. Yuzuru Matsuda for reagents, and Dr. Masato Kasuga (Second Department of Internal Medicine, Faculty of Medicine, Kobe University) for discussions. Kimiko Toyoshima and Asako Nishida are acknowledged for skillful typing of this manuscript.


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