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Activation of Mitogen-activated Protein Kinase and Phosphatidylinositol 3′-Kinase Is Not Sufficient for the Hormonal Stimulation of Glucose Uptake, Lipogenesis, or Glycogen Synthesis in 3T3-L1 Adipocytes (∗)

  • Russell J. Wiese
    Affiliations
    Department of Signal Transduction, Parke-Davis Pharmaceutical Research, Division of Warner-Lambert Company, Ann Arbor, Michigan 48105
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  • Cynthia Corley Mastick
    Affiliations
    Department of Signal Transduction, Parke-Davis Pharmaceutical Research, Division of Warner-Lambert Company, Ann Arbor, Michigan 48105
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  • Dan F. Lazar
    Affiliations
    Department of Signal Transduction, Parke-Davis Pharmaceutical Research, Division of Warner-Lambert Company, Ann Arbor, Michigan 48105
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  • Alan R. Saltiel
    Correspondence
    To whom correspondence should be addressed: Dept. of Signal Transduction, Parke-Davis Pharmaceutical Research, 2800 Plymouth Rd., Ann Arbor, MI 48105 . Tel.: 313-996-3960; Fax: 313-996-5668
    Affiliations
    Department of Signal Transduction, Parke-Davis Pharmaceutical Research, Division of Warner-Lambert Company, Ann Arbor, Michigan 48105
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  • Author Footnotes
    ∗ 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.
Open AccessPublished:February 17, 1995DOI:https://doi.org/10.1074/jbc.270.7.3442
      The precise mechanism by which insulin regulates glucose metabolism is not fully understood. However, it is known that insulin activates two enzymes, phosphatidylinositol 3′-kinase (PI 3′-K) and mitogen-activated protein kinase (MAPK), which may be involved in stimulating the metabolic effects of insulin. The role of these enzymes in glucose metabolism was examined by comparing the effects of insulin, platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) in 3T3-L1 adipocytes. Treatment of the cells with PDGF or EGF for 5 min increased the MAPK activity 3-5-fold, while insulin treatment produced a 2.5-fold increase. The MAPK activity remained elevated for 1 h after either PDGF or insulin treatment. PDGF and insulin, but not EGF, caused a transient increase in the amount PI 3′-K activity coprecipitated with tyrosine phosphorylated proteins. Although PDGF and insulin caused a similar increase in the activities of these two enzymes, only insulin caused substantial increases in glucose utilization. Insulin increased the transport of glucose and the synthesis of lipid 4- and 17-fold, respectively, while PDGF did not affect these processes significantly. Glycogen synthesis was increased 15-fold in response to insulin and only 3-fold in response to PDGF. Thus, the activation of MAPK and PI 3′-K are not sufficient for the complete stimulation of glucose transport, lipid synthesis, or glycogen synthesis by hormones in 3T3-L1 adipocytes, suggesting a requirement for other signaling mechanisms that may be uniquely responsive to insulin.

      INTRODUCTION

      While the intracellular events that mediate insulin action are not fully understood, the regulation of both protein and lipid phosphorylation are thought to play a prominent role(
      • Saltiel A.R.
      ). Upon binding insulin, the insulin receptor undergoes autophosphorylation on tyrosine residues, resulting in increased kinase activity that leads to the phosphorylation of other intracellular proteins(
      • Kasuga M.
      • Karlsson F.A.
      • Kahn C.R.
      ,
      • Haring H.U.
      • White M.F.
      • Machicao F.
      • Ermel B.
      • Schleicher E.
      • Obermaier B.
      ,
      • Machicao F.
      • Haring H.
      • White M.F.
      • Carrascosa J.M.
      • Obermaier B.
      • Wieland O.H.
      ,
      • Rees-Jones R.
      • Taylor S.
      ,
      • Sedoul J.C.
      • Pegron J.F.
      • Ballotti R.
      • Debant A.
      • Fehlmann M.
      • Van Obberghen E.
      ,
      • Momomura K.
      • Tope K.
      • Seyama Y.
      • Takaku F.
      • Kasuga M.
      ,
      • Margolis R.N.
      • Taylor S.I.
      • Seminara D.
      • Hubbard A.L.
      ,
      • Bernier M.
      • Laird D.M.
      • Lane M.D.
      ,
      • Hoffmann R.D.
      • Flores-Riveros J.R.
      • Liao K.
      • Laird D.M.
      • Lane M.D.
      ,
      • Izumi T.
      • White M.F.
      • Kadowaki T.
      • Takaju F.
      • Akanuma Y.
      • Kasuga M.
      ). One of these phosphorylated proteins, the insulin receptor substrate-1 (IRS-1), (
      The abbreviations used are: IRS-1
      insulin receptor substrate-1
      PDGF
      platelet-derived growth factor
      EGF
      epidermal growth factor
      PI 3′-K
      phosphatidylinositol 3′-kinase
      MAPK
      mitogen activated protein kinase
      MAP2
      microtubule-associated protein 2
      SH2
      Src homology 2
      SH3
      Src homology 3
      PBS
      phosphate-buffered saline
      BSA
      bovine serum albumin.
      )is critical for the mitogenic effects of insulin in some cells(
      • Sun X.J.
      • Miralpeix M.
      • Myers Jr., M.G.
      • Glasheen E.M.
      • Backer J.M.
      • Kahn C.R.
      • White M.F.
      ,
      • Rose D.W.
      • Saltiel A.R.
      • Majumdar M.
      • Decker S.J.
      • Olefsky J.M.
      ,
      • Waters S.B.
      • Yamauchi K.
      • Pessin J.E.
      ). IRS-1 itself does not contain any catalytic activity, but may be one member of a family of docking proteins for signaling molecules. Tyrosine phosphorylation of IRS-1 induces its association with several proteins containing Src homology 2 (SH2) domains, including SHPTP2(
      • Kuhné M.R.
      • Pawson T.
      • Lienhard G.E.
      • Feng G.-S.
      ,
      • Sun X.J.
      • Crimmins D.L.
      • Myers Jr., M.G.
      • Miralpeix M.
      • White M.F.
      ), Nck (
      • Lee C.-H.
      • Li W.
      • Nishimura R.
      • Zhou M.
      • Batzer A.G.
      • Myers Jr., M.G.
      • White M.F.
      • Schlessinger J.
      • Skolnik E.Y.
      ), Grb2(

      Skolnik, E. Y., Lee, C.-H., Batzer, A., Vicentini, L. M., Zhou, M., Daly, R., Myers, M. G., Jr., Backer, J. M., Ullrich, A., White, M. F., Schlessinger, J., (1993)EMBO J., 12, 1929-1936

      ,
      • Tobe K.
      • Matuoka K.
      • Tamemoto H.
      • Ueki K.
      • Kaburagi Y.
      • Asai S.
      • Noguchi T.
      • Matsuda M.
      • Tanaka S.
      • Hattori S.
      • Fukui Y.
      • Akanuma Y.
      • Yazaki Y.
      • Takenawa T.
      • Kadowaki T.
      ), and the p85 regulatory subunit of PI 3′-K(
      • Sun X.J.
      • Rothenberg P.
      • Kahn C.R.
      • Backer J.M.
      • Araki E.
      • Wilden P.A.
      • Cahill D.A.
      • Goldstein B.J.
      • White M.F.
      ). The association of this latter protein with IRS-1 confers an increase in the activity of the 110-kDa catalytic subunit of the enzyme (
      • Backer J.M.
      • Myers Jr., M.G.
      • Shoelson S.E.
      • Chin D.J.
      • Sun X.J.
      • Miralpeix M.
      • Hu P.
      • Margolis B.
      • Skolnik E.Y.
      • Schlessinger J.
      • White M.F.
      ,
      • Myers Jr., M.G.
      • Backer J.M.
      • Sun X.J.
      • Shoelson S.
      • Hu P.
      • Schlessinger J.
      • Yoakim M.
      • Schaffhausen B.
      • White M.F.
      ,
      • Folli F.
      • Saad M.J.A.
      • Backer J.M.
      • Kahn C.R.
      ), resulting in the production of phosphatidylinositol 3′,4′,5′-trisphosphate in cells(
      • Ruderman N.B.
      • Kapeller R.
      • White M.F.
      • Cantley L.C.
      ). Similarly, PI 3′-K activity is increased by virtually all tyrosine kinase receptors, although in most cases activation involves a direct interaction with the phosphorylated receptor(
      • Carpenter C.L.
      • Cantley L.C.
      ). While the exact role of this enzyme is not fully understood, its sequence similarity to the Saccharomyces cerevisiae protein VPS34 suggests that it may participate in membrane trafficking(
      • Hiles I.D.
      • Otsu M.
      • Volinia S.
      • Fry M.J.
      • Gout I.
      • Dhand R.
      • Panayotou G.
      • Ruiz-Larrea F.
      • Thompson A.
      • Totty N.F.
      • Hsuan J.J.
      • Courtneidge S.A.
      • Parker P.J.
      • Waterfield M.D.
      ). Compounds of unknown specificity that inhibit PI 3′-K activity can attenuate the stimulation of glucose transport and mitogenesis by insulin(
      • Kanai F.
      • Ito K.
      • Todaka M.
      • Hayashi H.
      • Kamohara S.
      • Ishii K.
      • Okada T.
      • Hazeki O.
      • Ui M.
      • Ebina Y.
      ,
      • Okada T.
      • Kawano Y.
      • Sakakibara T.
      • Hazeki O.
      • Ui M.
      ,
      • Cheatham B.
      • Vlahos C.J.
      • Cheatham L.
      • Wang L.
      • Blenis J.
      • Kahn C.R.
      ). Additionally, the stimulation of thymidine uptake by insulin can be inhibited by microinjection of the p85 SH2 domain or anti-p85 antibodies(
      • Jhun B.H.
      • Rose D.W.
      • Seely B.L.
      • Rameh L.
      • Cantley L.
      • Saltiel A.R.
      • Olefsky J.M.
      ). These data suggest that PI 3′-K activity may be necessary for certain actions of insulin.
      The Shc protooncogene product also appears to be phosphorylated on tyrosine in response to insulin or growth factor treatment(
      • Pronk G.J.
      • McGlade J.
      • Pelicci G.
      • Pawson T.
      • Bos J.L.
      ). The phosphorylation of Shc induces its association with Grb2(
      • Sasaoka T.
      • Rose D.W.
      • Jhun B.H.
      • Saltiel A.R.
      • Draznin B.
      • Olefsky J.M.
      ), which is an adapter protein containing both SH2 and SH3 domains(
      • Lowenstein E.J.
      • Daly R.J.
      • Batzer A.G.
      • Li W.
      • Margolis B.
      • Ullrich A.
      • Skolnik E.Y.
      • Bar-Sagi D.
      • Schlessinger J.
      ). Upon its activation, Grb2 can target the nucleotide exchange factor Sos, leading to the binding of GTP to p21ras(
      • Skolnik E.Y.
      • Batzer A.
      • Li W.
      • Lee C.-H.
      • Lowenstein E.
      • Mohammadi M.
      • Margolis B.
      • Schlessinger J.
      ,
      • Sasaoka T.
      • Draznin B.
      • Leitner J.W.
      • Langlois W.J.
      • Olefsky J.M.
      ). This activation of p21ras is required for the initiation of a phosphorylation cascade, leading ultimately to the stimulation of mitogen-activated protein kinase (MAPK)(
      • Skolnik E.Y.
      • Batzer A.
      • Li W.
      • Lee C.-H.
      • Lowenstein E.
      • Mohammadi M.
      • Margolis B.
      • Schlessinger J.
      ,
      • Wood K.W.
      • Sarnecki C.
      • Roberts T.M.
      • Blenis J.
      ). This enzyme is one of the major serine/threonine kinases known to be activated by insulin in tissue culture cells. Dent et al.(
      • Dent P.
      • Lavoinne A.
      • Nakielny S.
      • Caudwell F.B.
      • Watt P.
      • Cohen P.
      ) have suggested that MAPK is an important intermediate in the insulin-dependent activation of protein phosphatase-1, an enzyme that catalyzes the dephosphorylation and subsequent activation of glycogen synthetase and inhibition of phosphorylase kinase. However, numerous studies have shown that the activation of MAPK does not always correlate with the metabolic effects of insulin. For example, pharmacological agents that activate MAPK, such as phorbol esters and okadaic acid, can antagonize the metabolic effects of insulin(
      • Corvera S.
      • Jaspers S.
      • Pasceri M.
      ). Similarly, in cells expressing mutant insulin receptors, MAPK activation by insulin does not correlate with the metabolic activities of the hormone(
      • Pang L.
      • Lazar D.
      • Moller D.E.
      • Flier J.S.
      • Saltiel A.R.
      ,
      • Pang L.
      • Milarski K.L.
      • Ohmichi M.
      • Takata Y.
      • Olefsky J.M.
      • Saltiel A.R.
      ).
      3T3-L1 adipocytes represent one of the most sensitive tissue culture systems in which to elucidate insulin actions. These cells respond to insulin with increased glucose transport and incorporation of glucose into lipid and glycogen. Moreover, these cells also express receptors for other growth factors which have been shown to activate MAPK and PI 3′-K in other systems. We have exploited these cells to explore the role of MAPK and PI 3′-K in the metabolic effects of insulin.

      EXPERIMENTAL PROCEDURES

      Materials

      Cell culture reagents were purchased from Life Technologies, Inc. [14C-(U)]2-Deoxyglucose (323 mCi/mmol), [14C-(U)]glucose (298 mCi/mmol), and [γ-32P]ATP (3000 Ci/mmol) were purchased from DuPont NEN. The phosphatidylinositol was purchased from Avanti (Birmingham, AL). The mouse anti-phosphotyrosine monoclonal antibody and anti-p85 antiserum were obtained from Upstate Biotechnology Inc. (Lake Placid, NY). The ECL detection system was purchased from Amersham Corp.. The insulin was obtained from Eli Lilly, while the PDGF and EGF were from Harlan (Indianapolis, IN). Other reagents were from Sigma.

      Cell Culture

      3T3-L1 fibroblasts were maintained in Dulbecco's modified Eagle's medium (4500 g/liter glucose) supplemented with 10% calf serum in an atmosphere of 5% CO2/air. The cells were differentiated into adipocytes as described (
      • Rubin C.S.
      • Lai E.
      • Rosen O.M.
      ) and experiments conducted 1-2 days after removing the insulin medium.

      MAPK Assay

      Adipocytes (24-well culture dishes) were serum-deprived for 3 h prior to treatment with insulin, EGF, or PDGF. The cells were washed twice with phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4) and harvested in lysis buffer (50 mM β-glycerol phosphate, 10 mM HEPES, pH 8.0, 70 mM NaCl, 1 mM Na3VO4, 10 μg/ml leupeptin, 10 μg/ml aprotinin). The MAP kinase assay was performed as described(
      • Miyasaka T.
      • Chao M.V.
      • Sherline P.
      • Saltiel A.R.
      ).

      Phosphatidylinositol 3′-Kinase Assay

      After serum starvation and hormone stimulation, the cells (10-cm culture dishes) were harvested in Nonidet P-40 buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 5 mM EDTA, 1 mM Na3VO4, 10 μg/ml leupeptin, 10 μg/ml aprotinin). Insoluble material was removed from cell lysates by centrifugation, and the supernatants were precleared by incubating 15 min at 4°C with Pansorbin cells and agarose-coupled rabbit IgG. The resulting supernatants were immunoprecipitated overnight at 4°C with anti-phosphotyrosine antibodies. The precipitated pellets were washed and the PI 3′-K activity assayed as described previously(
      • Ohmichi M.
      • Decker S.J.
      • Saltiel A.R.
      ).

      Immunoprecipitation of p85

      Adipocytes (10-cm culture dishes) were serum-starved for 3 h and treated with insulin, PDGF, or EGF for 5 min. The cells were harvested in HNTG buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl2, 1 mM EDTA, 1 mM Na3VO4, 30 mMp-nitrophenyl phosphate, 10 μg/ml leupeptin, 10 μg/ml aprotinin) and the lysates cleared by centrifugation. The supernatants were immunoprecipitated with anti-p85 antisera and protein A-Sepharose. The pellets were washed three times with HNTG buffer and the proteins separated on a 7.5% polyacrylamide gel. After transfer to nitrocellulose, the membranes were immunoblotted with anti-phosphotyrosine antibodies as described(
      • Mastick C.C.
      • Kato H.
      • Roberts Jr., C.T.
      • LeRoith D.
      • Saltiel A.R.
      ).

      Glucose Uptake Assay

      Adipocytes (12-well culture dishes) were incubated for 3 h prior to assay in Krebs-Ringer bicarbonate buffer supplemented with 30 mM HEPES, pH 7.4, 0.5% BSA (KRBH/BSA) and 2.5 mM glucose. The cells were washed once with PBS and incubated for another 15 min in KRBH/BSA without glucose. Insulin or PDGF were added, and the cells incubated 15 min. The assay was initiated by the addition of [14C-(U)]2-deoxyglucose (0.4 μCi/sample) and glucose (5 mM final concentration). The assay was terminated after 15 min by washing cells three times in ice-cold PBS with 10 mM glucose. Cells were solubilized in 0.5 M NaOH, the extracts were neutralized by addition of glacial acetic acid, and cell-associated radioactivity determined by scintillation counting.

      Lipogenesis Assay

      Adipocytes (six-well culture dishes) were serum- and glucose-deprived as described for the glucose uptake assay. The cells were incubated for 15 min with insulin or PDGF, and the reaction initiated by the addition of [14C-(U)]glucose (0.125 μCi/sample) and glucose (5 mM final concentration). The assay was terminated after 1 h by washing the cells three times in ice-cold PBS, the cells were harvested using a rubber policeman in 1 ml of PBS, and 5 ml of Betafluor scintillant (National Diagnostics, Manville, NJ) were added. The samples were allowed to settle overnight, and radioactivity in 4 ml of the organic phase was determined by scintillation counting.

      Glycogen Synthesis Assay

      Adipocytes (six-well culture dishes) were serum- and glucose-deprived as described for the glucose uptake assay. The cells were incubated for 15 min with insulin or PDGF, and the reaction initiated by the addition of [14C-(U)]glucose (2 μCi/sample) and glucose (5 mM final concentration). The assay was terminated after 1 h by washing with ice-cold PBS, and the cells were solubilized in 30% KOH. The glucose incorporation into glycogen was determined as described previously(
      • Hess S.L.
      • Suchin C.R.
      • Saltiel A.R.
      ).

      RESULTS

      Growth Factors Activate MAP Kinase in 3T3-L1 Cells

      Numerous growth factors such as PDGF and EGF have been shown to activate the MAPK pathway. To determine whether this enzyme plays a unique role in insulin action, we compared the ability of insulin, PDGF, and EGF to stimulate MAPK in 3T3-L1 adipocytes. After a 5-min treatment with insulin, PDGF, or EGF, cell lysates were prepared and the MAPK activity in these lysates was measured by 32P incorporation into microtubule-associated protein 2 (MAP2) in vitro (Fig. 1A). Insulin (100 nM) treatment caused a 2.5-fold increase in MAP kinase activity, while PDGF (10 or 100 ng/ml) and EGF (100 ng/ml) treatments produced a 3-5-fold increase.
      Figure thumbnail gr1
      Figure 1:MAP kinase is activated in response to insulin, PDGF, or EGF. A, 3T3-L1 adipocytes were either untreated or exposed to insulin (INS; 100 nM), PDGF (1, 10 or 100 ng/ml), or EGF (100 ng/ml) for 5 min prior to preparing extracts. The MAP kinase activity after each treatment was measured as 32P incorporated into MAP2 in vitro. The phosphorylated MAP2 was excised from polyacrylamide gels and counted. The results shown are the mean ± standard error for three experiments each done in duplicate. B, the cells were exposed to 100 nM insulin (squares; solidline) or 10 ng/ml PDGF (circles; brokenline) for the indicated periods prior to preparing extracts and measuring the MAP kinase activity in those extracts. The results shown are the mean ± standard error for two experiments each done in duplicate.
      To compare the kinetics of MAPK activation by insulin and PDGF, the time courses were evaluated. Cells were incubated with PDGF (10 ng/ml) or insulin (100 nM) for various times, and the MAPK activity was then measured (Fig. 1B). The MAPK activity reached a 3-fold stimulation 5 min after the addition of insulin, and declined thereafter, although after 1 h of exposure to insulin the enzyme was still modestly elevated. PDGF caused a 5-fold stimulation in enzyme activity that peaked after 10 min and was still elevated 1 h after treatment.

      Insulin and PDGF, but Not EGF, Stimulate PI 3′-K Activity in 3T3-L1 Cells

      Numerous studies have shown that PI 3′-K activity may be increased by several tyrosine kinase receptors. To explore the potential role of this enzyme in fat cell metabolism, we evaluated its activity in cells treated with PDGF, EGF, and insulin. After a 5-min incubation with insulin, PDGF, or EGF, cell lysates were prepared and then immunoprecipitated with anti-phosphotyrosine antibodies. The PI 3′-K activity was measured in the anti-phosphotyrosine immunoprecipitates by the in vitro phosphorylation of phosphatidylinositol (Fig. 2). Insulin produced a marked increase in PI 3′-K activity associating with tyrosine-phosphorylated proteins. Treatment of the cells with PDGF for 5 min produced even a greater increase in phosphotyrosine-associated PI 3′-K activity (Fig. 2A). In contrast, EGF was without effect. These results were compared with the tyrosine-phosphorylated proteins, which were coprecipitated with antisera against the p85 subunit of PI 3′-K. After a 5-min incubation with insulin, PDGF or EGF, cell lysates were prepared and immunoprecipitated with anti-p85 antisera. The immunoprecipitated complexes were separated by SDS-polyacrylamide gel electrophoresis and the phosphotyrosine-containing proteins visualized by immunoblotting. Insulin produced a modest increase in the amount of IRS-1 complexed with PI 3′-K, while PDGF stimulated a marked increase in PI 3′-K associated with the PDGF receptor (Fig. 2B). As described above, EGF was without effect.
      Figure thumbnail gr2
      Figure 2:Phosphatidylinositol 3′-kinase activation in response to insulin, PDGF, or EGF. A, the cells were treated as in A. The cell extracts were immunoprecipitated with anti-phosphotyrosine antibodies. The PI 3′-K activity in anti-phosphotyrosine immunoprecipitates was assayed in vitro by 32P incorporated into phosphatidylinositol. The resulting phosphatidylinositol 3′-phosphate (PIP) was resolved by thin layer chromatography. The results shown are representative of four individual experiments. B, the cells were treated with insulin (INS; 100 nM), EGF (100 ng/ml), or PDGF (10 ng/ml) for 5 min. Cell extracts were prepared and immunoprecipitated with antisera against the p85 subunit of PI 3′-K. The precipitated proteins were separated by SDS-polyacrylamide gel electrophoresis and visualized by immunoblotting with anti-phosphotyrosine antibodies. C, the cells were treated with 100 nM insulin or 10 ng/ml PDGF for the indicated times prior harvesting the cells and measuring the PI 3′-K activity. The results shown are representative of three individual experiments.
      Since the phosphoproteins responsible for PI 3′-K activation are different for PDGF and insulin, the time course of activation after addition of the ligands was compared. Both hormones produced a marked increase in activity after only a 5-min exposure, which began to decline by 30 min. After exposure of cells to insulin for 60 min, a fraction of PI 3′-K activity remained associated with anti-phosphotyrosine immunoprecipitates, although PDGF-dependent activity returned to basal (Fig. 2C).

      PDGF Does Not Mimic the Actions of Insulin on Glucose Utilization

      The similarities in the activation of both MAP kinase and PI 3′-K activity by insulin and PDGF in 3T3-L1 adipocytes allowed us to evaluate the role of these pathways in glucose metabolism. 3T3-L1 adipocytes were treated for 15 min with PDGF or insulin prior to the addition of [14C-(U)]2-deoxyglucose. After 15 min, the [14C]2-deoxyglucose uptake was determined by scintillation counting. The treatment of the cells with insulin caused a 4-fold increase in [14C]2-deoxyglucose accumulation, while exposure to PDGF produced only a modest 1.4-fold increase (Fig. 3A). Thus, the effect of PDGF on glucose uptake was less than 10% of that seen with insulin in marked contrast to the similar increases observed for these two hormones in MAPK and PI 3′-K activities.
      Figure thumbnail gr3
      Figure 3:Stimulation glucose transport, lipid synthesis or glycogen synthesis in response to insulin or PDGF. A, glucose transport. After a 15-min incubation with insulin (INS; 100 nM) or PDGF (1, 10, or 100 ng/ml), [14C]2-deoxyglucose was added and the incubation continued an additional 15 min. After extensive washing, the [14C]2-deoxyglucose that had been internalized by the cells was determined by scintillation counting. The results are shown as the mean ± standard error for two experiments each done in triplicate. B, lipid synthesis. The cells were treated as in A and incubated with [14C]glucose for 1 h. After harvesting the cells, the lipids were extracted and the [14C]glucose converted to lipid determined by scintillation counting. The results are the mean ± standard error for two experiments each done in triplicate. C, glycogen synthesis. The cells were treated with insulin (INS; 100 nM) or PDGF (100 ng/ml) for 15 min. [14C]Glucose was added and the incubation continued for an additional hour. Cell lysates were prepared, and the 14C incorporated into glycogen was determined by scintillation counting. The results are shown as the mean ± standard error for two experiments each done in triplicate. ∗, p ≤ 0.05;∗∗, p ≤ 0.025;∗∗∗, p ≤ 0.005.
      Insulin can also increase the rate of lipid synthesis in 3T3-L1 adipocytes. The cells were incubated with either PDGF or insulin 15 min prior to the addition of [14C-(U)]glucose (5 mM), conditions under which lipid synthesis is not rate-limited by glucose uptake. After a 1-h exposure, the lipid synthesis from glucose was determined by scintillation counting. As observed for 2-deoxyglucose transport, PDGF caused only a 1.5-fold increase in lipogenesis, whereas insulin produced a 17-fold increase (Fig. 3B).
      Insulin also increases the conversion of glucose into glycogen in the 3T3-L1 adipocytes. Cells were treated with either PDGF or insulin for 15 min prior to the addition of [14C-(U)]glucose. Following a 60-min incubation, glycogen synthesis was determined by scintillation counting. Insulin produced a 15-fold increase in glycogen synthesis. In contrast, glycogen synthesis was increased only 3-fold in response to PDGF treatment (Fig. 3C).

      DISCUSSION

      Although the molecular mechanisms by which insulin regulates glucose metabolism are not fully understood, changes in protein and lipid phosphorylation are likely to be involved. While the tyrosine kinase activity of the insulin receptor initiates the intracellular signaling cascades responsible for the action of the hormone, the regulation of key events downstream from the receptor are generally controlled by changes in serine or threonine phosphorylation. Although the precise phosphorylation events that regulate these pathways remain elusive, insulin can stimulate phosphorylation of some proteins and the dephosphorylation of others(
      • Saltiel A.R.
      ). The many cellular responses to insulin can be easily distinguished by virtue of the dose, the length of exposure, and the cell type examined(
      • Saltiel A.R.
      ,
      • Rosen O.M.
      ), suggesting that insulin activates several signaling pathways. It is likely that the divergence of insulin signaling may occur at the receptor itself, since differences in insulin action have been observed in cells expressing mutant receptors. For example, mutation of the two C-terminal tyrosines of the receptor results in a phenotype, which is normal with respect to glucose uptake and glycogen synthesis(
      • Takata Y.
      • Webster N.J.G.
      • Olefsky J.M.
      ), but increased in its sensitivity with respect to mitogenesis(
      • Takata Y.
      • Webster N.J.G.
      • Olefsky J.M.
      ), S6 kinase(
      • Takata Y.
      • Webster N.J.G.
      • Olefsky J.M.
      ), and MAPK activation(
      • Pang L.
      • Milarski K.L.
      • Ohmichi M.
      • Takata Y.
      • Olefsky J.M.
      • Saltiel A.R.
      ). Also, overexpression of a mutant receptor with serine substituted at Trp1200 confers normal glucose transport and glycogen synthesis, but deficient autophosphorylation, DNA synthesis (
      • Moller D.E.
      • Benecke H.
      • Flier J.S.
      ), p21ras(
      • Osterop A.P.R.M.
      • Medema R.H.
      • Bos J.L.
      • v. d. Zon G.C.M.
      • Moller D.E.
      • Flier J.S.
      • Moller W.
      • Maassen J.A.
      ), and MAPK activation(
      • Pang L.
      • Lazar D.
      • Moller D.E.
      • Flier J.S.
      • Saltiel A.R.
      ).
      The ability of insulin to activate both distinct signaling pathways and diverse metabolic responses in a single cell has complicated the identification of relevant transduction mechanisms. Much attention has been focussed on two enzymes, MAPK and PI 3′-K, which are activated by hormone treatment and lie in distinct signaling pathways. Dent et al.(
      • Dent P.
      • Lavoinne A.
      • Nakielny S.
      • Caudwell F.B.
      • Watt P.
      • Cohen P.
      ) have suggested that the insulin-dependent activation of pp90rsk, via phosphorylation by MAPK, leads to the phosphorylation of site-1 on the regulatory G subunit of glycogen-associated protein phosphatase-1. This results in both the dephosphorylation and stimulation of glycogen synthetase and the inhibition of phosphorylase kinase. Although MAPK is activated in response to insulin, it is not generally correlated with the metabolic responsiveness to the hormone in tissue culture models. Several recent reports have demonstrated that EGF, serum, or thrombin treatments can activate MAPK in the absence of increased glucose transport and glycogen synthesis(
      • Robinson L.J.
      • Razzack Z.F.
      • Lawrence Jr., J.C.
      • James D.E.
      ,
      • Fingar D.C.
      • Birnbaum M.J.
      ,
      • van den Berghe N.
      • Ouwens D.M.
      • Maassen J.A.
      • van Mackelenbergh M.G.H.
      • Sips H.C.M.
      • Krans H.M.J.
      ,
      • Lin T.-A.
      • Lawrence Jr., J.C.
      ). The studies presented here demonstrate that the profound activation of MAPK by PDGF in a highly responsive cell line does not result in significant stimulation of glucose transport, lipid synthesis, or glycogen synthesis, indicating that MAPK activation is not sufficient for stimulating glucose metabolism. Additional studies have suggested that MAPK may not be necessary for the regulation of glucose utilization by insulin. Insulin fails to activate MAPK in PC-12 cells, but nevertheless stimulates the synthesis of glycogen, lipid and protein(
      • Ohmichi M.
      • Pang L.
      • Ribon V.
      • Saltiel A.R.
      ). In addition, a low molecular weight inhibitor that specifically blocks the stimulation of MAPK by inhibiting its upstream activator, MEK (MAP kinase kinase), has no effect on the metabolic actions of insulin in 3T3-L1 cells. (
      D. Lazar, R. J. Wiese, C. C. Mastick, and A. R. Saltiel, submitted for publication.
      )
      Although MAPK appears to play a limited role in the metabolic actions of insulin, there is evidence that the enzyme PI 3′-K may be important. This enzyme is activated upon the association of its regulatory p85 subunit with insulin receptor substrates such as IRS-1 (
      • Backer J.M.
      • Myers Jr., M.G.
      • Shoelson S.E.
      • Chin D.J.
      • Sun X.J.
      • Miralpeix M.
      • Hu P.
      • Margolis B.
      • Skolnik E.Y.
      • Schlessinger J.
      • White M.F.
      ,
      • Myers Jr., M.G.
      • Backer J.M.
      • Sun X.J.
      • Shoelson S.
      • Hu P.
      • Schlessinger J.
      • Yoakim M.
      • Schaffhausen B.
      • White M.F.
      ,
      • Folli F.
      • Saad M.J.A.
      • Backer J.M.
      • Kahn C.R.
      ) or pp60 (
      • Lavan B.E.
      • Lienhard G.E.
      ) or the insulin receptor itself(
      • Ruderman N.B.
      • Kapeller R.
      • White M.F.
      • Cantley L.C.
      ). PI 3′-K inhibitors, such as wortmannin or LY294002, can block the stimulation of glucose transport and lipogenesis by insulin, suggesting that PI3′-kinase may be necessary for these actions of the hormone(
      • Kanai F.
      • Ito K.
      • Todaka M.
      • Hayashi H.
      • Kamohara S.
      • Ishii K.
      • Okada T.
      • Hazeki O.
      • Ui M.
      • Ebina Y.
      ,
      • Okada T.
      • Kawano Y.
      • Sakakibara T.
      • Hazeki O.
      • Ui M.
      ,
      • Cheatham B.
      • Vlahos C.J.
      • Cheatham L.
      • Wang L.
      • Blenis J.
      • Kahn C.R.
      ). Interestingly, PDGF is also a potent activator of the enzyme in 3T3-L1 adipocytes, presumably through the direct interaction of p85 with the PDGF receptor. However, glucose transport, lipogenesis, and glycogen synthesis remain largely unaffected by exposure to PDGF, suggesting that increased association of PI 3′-K with tyrosine-phosphorylated proteins alone is not sufficient for the stimulation of glucose metabolism. The possibility remains that the subcellular redistribution of PI 3′-K activity in response to PDGF is different than that produced by insulin. Recent reports suggest that in rat adipocytes PI 3′-K activity is localized to low density microsomes in response to insulin (
      • Kelly K.L.
      • Ruderman N.B.
      ). However, whether PDGF causes a similar redistribution is not yet known. Thus, compartmentalization of the enzyme may be the key determinant in signal generation. On the other hand, it is possible that while PI 3′-K activity is involved in membrane vesicle trafficking necessary for metabolic regulation, other intracellular biochemical events that are unique to insulin may provide the important signals regulating glucose metabolism. Moreover, it is likely that the regulation of protein serine/threonine dephosphorylation, which is generally not observed with other growth factors, will provide the critical clue toward understanding metabolic signaling for insulin.

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