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Phosphatidylinositol 3-Kinase, but Not p70/p85 Ribosomal S6 Protein Kinase, Is Required for the Regulation of Phosphoenolpyruvate Carboxykinase (PEPCK) Gene Expression by Insulin

DISSOCIATION OF SIGNALING PATHWAYS FOR INSULIN AND PHORBOL ESTER REGULATION OF PEPCK GENE EXPRESSION (∗)
  • Calum Sutherland
    Affiliations
    Department of Molecular Physiology and Biophysics, Vanderbilt University Medical School, Nashville, Tennessee 37232-0615
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  • Richard M. O'Brien
    Affiliations
    Department of Molecular Physiology and Biophysics, Vanderbilt University Medical School, Nashville, Tennessee 37232-0615
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  • Daryl K. Granner
    Correspondence
    To whom correspondence should be addressed: Dept. of Molecular Physiology and Biophysics, 707 Light Hall, Vanderbilt University Medical School, Nashville, TN 37232-0615. Tel.: 615-322-7004; Fax: 615-322-7236
    Affiliations
    Department of Molecular Physiology and Biophysics, Vanderbilt University Medical School, Nashville, Tennessee 37232-0615
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  • Author Footnotes
    ∗ This work was supported by an American Diabetes Association Mentor-based fellowship (to C. S. and D. K. G.), by H. H. S. Grant DK35107, and by a Vanderbilt Diabetes Research and Training Center Grant DK20593 (to D. K. G.). 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:June 30, 1995DOI:https://doi.org/10.1074/jbc.270.26.15501
      Phosphoenolpyruvate carboxykinase (PEPCK) catalyzes the rate-limiting step in hepatic gluconeogenesis. Glucagon (via the second messenger cAMP) and glucocorticoids stimulate the transcription of the PEPCK gene, whereas insulin and phorbol esters inhibit, in a dominant fashion, these effects. Wortmannin, an inhibitor of phosphatidylinositol 3-kinase, prevents the stimulation of glycogen synthesis, glucose transport, mitogen-activated protein kinase, and p70/p85 ribosomal S6 protein kinase by insulin. We now show that wortmannin can also block the inhibition of glucocorticoid- and cAMP-stimulated PEPCK gene expression by insulin. PEPCK-chloramphenicol acetyltransferase fusion gene experiments demonstrate that wortmannin blocks an activity that is required for insulin signaling to elements within the PEPCK promoter. Phorbol esters mimic the action of insulin on the regulation of PEPCK gene expression, but wortmannin does not block the effect of these agents. Thus, phosphatidylinositol 3-kinase is required for the regulation of PEPCK gene expression by insulin, but not by phorbol esters. The immunosuppressant rapamycin, a potent inhibitor of insulin or phorbol ester stimulation of p70/p85 ribosomal S6 protein kinase, has no significant effect on the regulation of PEPCK gene expression by insulin or phorbol esters. Thus, p70/p85 ribosomal S6 protein kinase does not have a role in signaling to the PEPCK promoter by insulin or phorbol esters.

      INTRODUCTION

      Insulin regulates many metabolic responses in a variety of mammalian tissues, principally liver, muscle, and adipose(
      • Denton R.M.
      ,
      • White M.F.
      • Kahn C.R.
      ). The initial mechanism of insulin action involves its binding to a specific cell-surface receptor and the subsequent activation of the intrinsic receptor tyrosine kinase domain(
      • Denton R.M.
      ,
      • White M.F.
      • Kahn C.R.
      ). A major substrate for the insulin receptor tyrosine kinase is insulin receptor substrate-1 (IRS-1).1(
      The abbreviations used are: IRS-1
      insulin receptor substrate-1
      PI
      phosphatidylinositol
      p70S6k
      p70/p85 ribosomal S6 protein kinase
      PEPCK
      phosphoenolpyruvate carboxykinase
      8CPT-cAMP
      8-(4-chlorophenylthio)-cAMP
      PMA
      phorbol 12-myristate 13-acetate
      BSA
      bovine serum albumin
      CAT
      chloramphenicol acetyltransferase
      DMEM
      Dulbecco's modified Eagle's medium
      EGF
      epidermal growth factor.
      )Tyrosine phosphorylation of IRS-1 promotes the interaction of this protein with a number of other proteins via their src homology 2 domains, and this initiates divergent signaling cascades (for review, see Ref.
      • Myers M.G.
      • Sun X.J.
      • White M.F.
      ). For example, the activity of phosphatidylinositol 3-kinase (PI 3-kinase; EC 2.7.1.67) is stimulated upon binding to IRS-1(
      • Backer J.M.
      • Myers 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.
      ,
      • Carpenter C.L.
      • Auger K.R.
      • Chanudhuri M.
      • Yoakim M.
      • Schaffhausen B.
      • Shoelson S.
      • Cantley L.C.
      ,
      • Lavan B.E.
      • Kuhne M.R.
      • Garner C.W.
      • Anderson D.
      • Reedijk M.
      • Pawson T.
      • Lienhard G.E.
      ), and this leads to the accumulation of phosphatidylinositols phosphorylated at the 3-D position(
      • Whitman M.
      • Downes C.P.
      • Keeler M.
      • Keller T.
      • Cantley L.
      ,
      • Fry M.J.
      ). These phospholipids have been postulated to act as “second messenger” molecules(
      • Auger K.R.
      • Serunian L.A.
      • Soltoff S.P.
      • Libby P.
      • Cantley L.C.
      ), although their precise in vivo targets have yet to be identified(
      • Nakanishi H.
      • Brewer K.A.
      • Exton J.H.
      ,
      • Toker A.
      • Meyer M.
      • Reddy K.K.
      • Falck J.R.
      • Aneja R.
      • Aneja S.
      • Parra A.
      • Burns D.J.
      • Ballas L.M.
      • Cantley L.C.
      ). The activation of PI 3-kinase is important in insulin action since wortmannin (an inhibitor (IC50 = 1-10 nM) of PI 3-kinase(
      • Arcaro A.
      • Wymann M.P.
      )) blocks the antilipolytic action of insulin in adipocytes (
      • Rahn T.
      • Ridderstrale M.
      • Tornqvist H.
      • Manganiello V.
      • Fredrikson G.
      • Belfrage P.
      • Degerman E.
      ); the inhibition of glycogen synthase kinase-3 by insulin(
      • Cross D.A.E.
      • Alessi D.R.
      • Vandenheede J.R.
      • McDowell H.E.
      • Hundal H.S.
      • Cohen P.
      ,
      • Welsh G.I.
      • Foulstone E.J.
      • Young S.W.
      • Tavare J.M.
      • Proud C.G.
      ); the inhibition of apoptosis by insulin(
      • Yao R.
      • Cooper G.M.
      ); and insulin-stimulated membrane ruffling(
      • Kotani K.
      • Yonezawa K.
      • Hara K.
      • Ueda H.
      • Kitamura Y.
      • Sakaue H.
      • Ando A.
      • Chavanieu A.
      • Calas B.
      • Grigorescu F.
      ), glucose uptake(
      • Gould G.W.
      • Jess T.J.
      • Andrews G.C.
      • Herbst J.J.
      • Plevin R.J.
      • Gibbs E.M.
      ,
      • Clarke J.F.
      • Young P.W.
      • Yonezawa K.
      • Kasuga M.
      • Holman G.D.
      ,
      • Shimizu Y.
      • Shimazu T.
      ,
      • Yeh J.-I.
      • Gulve E.A.
      • Rameh L.
      • Birnbaum M.J.
      ), and glycogen synthesis (
      • Shepherd P.R.
      • Nave B.T.
      • Siddle K.
      ,
      • Yamamoto-Honda R.
      • Tobe K.
      • Kaburagi Y.
      • Ueki K.
      • Asai S.
      • Yachi M.
      • Shirouzu M.
      • Yodoi J.
      • Akanuma Y.
      • Yokoyama S.
      • Yazaki Y.
      • Kadowaki T.
      ).
      Wortmannin also blocks the activation of p70/p85 ribosomal S6 protein kinase (p70S6k) by insulin(
      • Yamamoto-Honda R.
      • Tobe K.
      • Kaburagi Y.
      • Ueki K.
      • Asai S.
      • Yachi M.
      • Shirouzu M.
      • Yodoi J.
      • Akanuma Y.
      • Yokoyama S.
      • Yazaki Y.
      • Kadowaki T.
      ,
      • Cheatham B.
      • Vlahos C.J.
      • Cheatham L.
      • Wang L.
      • Blenis J.
      • Khan C.R.
      ). p70S6k is the protein kinase responsible for the insulin- and growth factor-stimulated phosphorylation of ribosomal protein S6 in vivo(
      • Chung J.
      • Kuo C.J.
      • Crabtree G.R.
      • Blenis J.
      ,
      • Price D.J.
      • Grove J.R.
      • Calvo V.
      • Avruch J.
      • Bierer B.E.
      ), and this in turn may result in increased translation from specific polypyrimidine-containing mRNAs(
      • Terada N.
      • Patel H.R.
      • Takase K.
      • Kohno K.
      • Nairn A.C.
      • Gelfand E.W.
      ,
      • Jefferies H.B.J.
      • Reinhard C.
      • Kozma S.C.
      • Thomas G.
      ). The macrolide rapamycin is a potent inhibitor of insulin-stimulated p70S6k activity, ribosomal protein S6 phosphorylation(
      • Chung J.
      • Kuo C.J.
      • Crabtree G.R.
      • Blenis J.
      ), and glycogen synthesis(
      • Shepherd P.R.
      • Nave B.T.
      • Siddle K.
      ). The precise mechanism of p70S6k activation and the direct target of rapamycin remain unclear (for review, see Ref.
      • Ferrari S.
      • Thomas G.
      ); however, rapamycin has no effect on the stimulation of PI 3-kinase by insulin.
      Although insulin regulates the transcription of numerous genes in a variety of cell types(
      • O'Brien R.M.
      • Granner D.K.
      ), the regulation of phosphoenolpyruvate carboxykinase (PEPCK; GTP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.32) gene expression by insulin has been studied in most detail. Insulin and phorbol esters inhibit basal PEPCK gene expression in H4IIE cells, and both agents block the stimulatory action of cAMP and glucocorticoids(
      • O'Brien R.M.
      • Granner D.K.
      ). The signal transduction pathway(s) involved in these actions of insulin and phorbol ester have yet to be determined. Although these agents initiate their actions separately(
      • Chu D.T.W.
      • Stumpo D.J.
      • Blackshear P.J.
      • Granner D.K.
      ), it is known that these initially distinct signals converge on the same DNA sequence in the PEPCK gene promoter(
      • O'Brien R.M.
      • Lucas P.C.
      • Forest C.D.
      • Magnuson M.A.
      • Granner D.K.
      ,
      • O'Brien R.M.
      • Bonovich M.T.
      • Forest C.D.
      • Granner D.K.
      ). This paper describes the effects of wortmannin and rapamycin on the hormonal regulation of PEPCK gene transcription. Wortmannin blocks signaling to the PEPCK gene by insulin, but not by phorbol esters. Although p70S6k may lie downstream of PI 3-kinase, rapamycin does not affect signaling to the PEPCK gene by either insulin or phorbol ester. Thus, PI 3-kinase is required for insulin regulation of PEPCK gene expression, but p70S6k is apparently not involved. Moreover, the data confirm the existence of distinct signaling pathways to the PEPCK gene for phorbol ester and insulin.

      EXPERIMENTAL PROCEDURES

      Materials

      Radioisotopes ([γ-32P]ATP and [3H]sodium acetate) were obtained from Amersham Corp. and ICN, respectively. The ribosomal S6 peptide (
      • Lavoinne A.
      • Erikson E.
      • Maller J.
      • Price D.J.
      • Avruch J.
      • Cohen P.
      ) related to the C-terminal 32 residues of ribosomal S6 protein (residues 218-249, KEAKEKRQEQIAKRRRLSSLRASTSKSGGSQK) was kindly provided by Prof. Philip Cohen (Dundee University, Dundee, Scotland). Insulin was purchased from Collaborative Bioproducts, and 8-(4-chlorophenylthio)-cAMP (8CPT-cAMP) was from Boehringer Mannheim. Phorbol 12-myristate 13-acetate (PMA), wortmannin, dexamethasone, BSA-Sepharose, and protein A-Sepharose were obtained from Sigma. Polyclonal antisera specific for p70/p85 ribosomal S6 protein kinase were obtained from Santa Cruz Biotechnologies Inc. All other chemicals were of the highest grade available.

      Cell Culture, Hormone Treatments, and CAT Assay

      The isolation of the H4IIE rat hepatoma-derived stable transfectant, HL1C, which contains the PEPCK promoter sequence from positions −2100 to +69 ligated to the CAT reporter gene, has been described previously(
      • Forest C.D.
      • O'Brien R.M.
      • Lucas P.C.
      • Magnuson M.A.
      • Granner D.K.
      ). HL1C cells were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 2.5% (v/v) fetal calf serum and 2.5% newborn calf serum as described previously(
      • Forest C.D.
      • O'Brien R.M.
      • Lucas P.C.
      • Magnuson M.A.
      • Granner D.K.
      ). Hormone and inhibitor treatments were carried out in serum-free medium for the times and at the concentrations indicated in the figure legends. Cells were harvested by tryptic digestion and sonicated in 400 μl of 250 mM Tris/HCl (pH 7.8) before CAT activity was determined(
      • Forest C.D.
      • O'Brien R.M.
      • Lucas P.C.
      • Magnuson M.A.
      • Granner D.K.
      ).

      Synthesis and Labeling of Oligonucleotides

      Two oligonucleotides, PC28 (5′-GGAGAGAGGCAGGGACTCTGGTGCCACC-3′) and ACT25 (5′-GGGTGTGGACCGGGACGGAGGAGCT-3′), were synthesized on a Cyclone Plus DNA synthesizer (Millipore Corp.). They were designed to be complementary to positions +102 to +129 and +42 to +67, relative to the transcription start site, in the PEPCK (
      • Beale E.G.
      • Chrapkiewicz N.B.
      • Scoble H.A.
      • Metz R.J.
      • Quick D.P.
      • Noble R.L.
      • Donelson J.E.
      • Biemann K.
      • Granner D.K.
      ) and β-actin (
      • Nudel U.
      • Zakut R.
      • Shani M.
      • Neumann S.
      • Levy Z.
      • Yaffe D.
      ) genes, respectively. The oligonucleotides were gel-purified and then 5′-end-labeled using [γ-32P]ATP (specific activity of ~109 cpm/μg) prior to their use in a primer extension assay(
      • Forest C.D.
      • O'Brien R.M.
      • Lucas P.C.
      • Magnuson M.A.
      • Granner D.K.
      ).

      RNA Isolation and Primer Extension Analysis

      HL1C cells were serum-starved overnight and treated with hormone/inhibitor for the times and at the concentrations indicated in the figure legends. Total cellular RNA (~100 μg/106 cells) was then isolated (
      • Forest C.D.
      • O'Brien R.M.
      • Lucas P.C.
      • Magnuson M.A.
      • Granner D.K.
      ). A primer extension assay was used to analyze PEPCK and β-actin mRNA accumulation as described previously(
      • Forest C.D.
      • O'Brien R.M.
      • Lucas P.C.
      • Magnuson M.A.
      • Granner D.K.
      ), and autoradiographs were quantified by PhosphorImager analysis (Molecular Dynamics, Inc.).

      Preparation of Cell Extract for Kinase Assays

      HL1C cells (2-4 X 106) were incubated in serum-free medium with hormones and inhibitor for 3 h at the concentrations indicated in the legend to Table I. Cells were then scraped into 0.5 ml of ice-cold lysis buffer (25 mM Tris/HCl (pH 7.4), 50 mM NaF, 100 mM NaCl, 0.5 mM sodium vanadate, 5 mM EGTA, 1 mM EDTA, 1% (v/v) Nonidet P-40, 10 mM sodium pyrophosphate, 1 mM benzamidine, 0.1 mM phenylmethylsulfonyl fluoride, 5% (v/v) glycerol, and 0.1% (v/v) 2-mercaptoethanol). Cell debris was removed by centrifugation at 13,000 X g for 5 min, and the protein concentration was determined, using BSA as an internal standard, by the method of Bradford(
      • Bradford M.M.
      ).
      Table I:Rapamycin blocks insulin-stimulated p70/p85 ribosomal S6 protein kinase activity in HL1C cells

      Immunoprecipitation of p70/p85 Ribosomal S6 Protein Kinase

      Cell extracts, prepared as described above, were incubated with BSA-Sepharose and centrifuged at 13,000 X g for 5 min. Protein A-Sepharose was used to precipitate the p70S6k in the supernatants following incubation with 1 μg of a specific p70S6k antibody (Santa Cruz Biotechnologies Inc.) that does not cross-react with members of the p90 ribosomal S6 protein kinase family.

      Assay of p70/p85 Ribosomal S6 Protein Kinase

      p70S6k assays were performed as described previously using a peptide based on the C terminus of ribosomal protein S6 as substrate (see “Materials” and Ref.
      • Lavoinne A.
      • Erikson E.
      • Maller J.
      • Price D.J.
      • Avruch J.
      • Cohen P.
      ). One unit of p70S6k activity catalyzes the phosphorylation of 1.0 nmol of substrate/min.

      RESULTS

      We have utilized the HL1C cell line to begin to investigate the signal transduction pathways involved in insulin- and phorbol ester-regulated PEPCK gene expression. HL1C cells were generated by stably transfecting a PEPCK-CAT fusion gene (with promoter sequence from positions −2100 to +69, relative to the transcription start site of the PEPCK gene) into H4IIE rat hepatoma cells(
      • Forest C.D.
      • O'Brien R.M.
      • Lucas P.C.
      • Magnuson M.A.
      • Granner D.K.
      ). Individually, dexamethasone (3-5-fold) and cAMP (~1.2-fold) stimulate CAT expression in these cells, while together they act synergistically to promote a 20-50-fold increase in CAT expression after 18 h(
      • O'Brien R.M.
      • Lucas P.C.
      • Forest C.D.
      • Magnuson M.A.
      • Granner D.K.
      ,
      • O'Brien R.M.
      • Bonovich M.T.
      • Forest C.D.
      • Granner D.K.
      ,
      • Forest C.D.
      • O'Brien R.M.
      • Lucas P.C.
      • Magnuson M.A.
      • Granner D.K.
      ,
      • O'Brien R.M.
      • Noisin E.L.
      • Granner D.K.
      ). Insulin and phorbol esters act, in a dominant fashion, to block these stimulatory effects (Fig. 1-4)(
      • O'Brien R.M.
      • Lucas P.C.
      • Forest C.D.
      • Magnuson M.A.
      • Granner D.K.
      ,
      • O'Brien R.M.
      • Bonovich M.T.
      • Forest C.D.
      • Granner D.K.
      ,
      • Forest C.D.
      • O'Brien R.M.
      • Lucas P.C.
      • Magnuson M.A.
      • Granner D.K.
      ,
      • O'Brien R.M.
      • Noisin E.L.
      • Granner D.K.
      ).
      Figure thumbnail gr1
      Figure 1:Rapamycin does not affect insulin-regulated PEPCK gene transcription. HL1C cells (5-10 X 106) were incubated in serum-free DMEM in the absence of hormone (control); in 500 nM dexamethasone and 0.1 mM 8CPT-cAMP (Dex/cAMP); or in 500 nM dexamethasone, 0.1 mM 8CPT-cAMP, and 10 nM insulin (Dex/cAMP/insulin). A, effect of rapamycin on endogenous PEPCK gene transcription. Total RNA was prepared from HL1C cells treated for 3 h as described above in the presence or absence of 400 nM rapamycin. In the absence of drug, an equivalent amount of Me2SO carrier was added. Radiolabeled oligonucleotides PC28 (PEPCK) and ACT25 (β-actin) were annealed to 50 μg of total cellular RNA that was isolated from HL1C cells after each hormone/inhibitor treatment. A primer extension reaction was performed, and the products were separated by urea-acrylamide gel electrophoresis and visualized by autoradiography as described under “Experimental Procedures.” Similar results were obtained from two different experiments. The measurement of β-actin mRNA was used to establish equal loading of mRNA in each lane, and although it has previously been reported that insulin stimulates β-actin expression (
      • Messina J.L.
      ,
      • Buchou T.
      • Gaben A.-M.
      • Phan-Dhin-Tuy F.
      • Mester J.
      ), this was not apparent under our experimental conditions (see “Results”). B, effect of rapamycin (rapa) on PEPCK-CAT gene transcription. Rapamycin at 100 or 400 nM or Me2SO (DMSO) carrier was included in the hormone treatments, and the cells were harvested after 16 h as described under “Experimental Procedures.” Extracts were assayed for CAT activity (as described under “Experimental Procedures”), and results are expressed as percent CAT activity relative to dexamethasone/cAMP-stimulated CAT activity in the absence of rapamycin. Results are the means ± S.E. of three different experiments.
      The activity of the protein kinase p70S6k is stimulated by treatment of several cell types with a number of factors, including insulin(
      • Chung J.
      • Kuo C.J.
      • Crabtree G.R.
      • Blenis J.
      ,
      • Price D.J.
      • Grove J.R.
      • Calvo V.
      • Avruch J.
      • Bierer B.E.
      ). The immunosuppressant rapamycin blocks the stimulation of p70S6k (and the in vivo phosphorylation of ribosomal protein S6) in a number of cell types and by a variety of agents, including insulin(
      • Cross D.A.E.
      • Alessi D.R.
      • Vandenheede J.R.
      • McDowell H.E.
      • Hundal H.S.
      • Cohen P.
      ,
      • Welsh G.I.
      • Foulstone E.J.
      • Young S.W.
      • Tavare J.M.
      • Proud C.G.
      ,
      • Chung J.
      • Kuo C.J.
      • Crabtree G.R.
      • Blenis J.
      ,
      • Price D.J.
      • Grove J.R.
      • Calvo V.
      • Avruch J.
      • Bierer B.E.
      ). Table I shows that 200 nM rapamycin completely blocked insulin-stimulated p70S6k activity in the HL1C cell line; however, even at 400 nM, rapamycin had very little effect on the ability of insulin to inhibit endogenous PEPCK gene expression (Fig. 1A). The effect of insulin on PEPCK mRNA was assessed relative to β-actin mRNA. Although β-actin gene expression has been reported to be stimulated by insulin(
      • Messina J.L.
      ,
      • Buchou T.
      • Gaben A.-M.
      • Phan-Dhin-Tuy F.
      • Mester J.
      ), under our experimental conditions, no significant effect on β-actin mRNA accumulation was observed (1.1 ± 0.2-fold, as determined by PhosphorImager analysis; see Figs. 1A and 2A). Consistent with this lack of effect on endogenous PEPCK gene expression, rapamycin (at concentrations up to 400 nM) did not affect the ability of insulin to block the stimulation of PEPCK-CAT expression by dexamethasone and cAMP (Fig. 1B). This suggests that the stimulation of p70S6k by insulin is not required for the regulation of PEPCK gene transcription by this hormone.
      The antibiotic wortmannin, a relatively specific PI 3-kinase inhibitor in vitro(
      • Arcaro A.
      • Wymann M.P.
      ), blocks the stimulation of PI 3-kinase by insulin in vivo(
      • Clarke J.F.
      • Young P.W.
      • Yonezawa K.
      • Kasuga M.
      • Holman G.D.
      ,
      • Yeh J.-I.
      • Gulve E.A.
      • Rameh L.
      • Birnbaum M.J.
      ,
      • Okada T.
      • Kawano Y.
      • Sakakibara T.
      • Hazeki O.
      • Ui M.
      ,
      • Lam K.
      • Carpenter C.L.
      • Ruderman N.B.
      • Friel J.C.
      • Kelly K.L.
      ). Unlike rapamycin, wortmannin completely blocked the action of insulin on dexamethasone- and cAMP-stimulated PEPCK gene expression. The same result was obtained with both the endogenous PEPCK gene (Fig. 2, A and B) and the PEPCK-CAT reporter gene (Fig. 3A). The effect of wortmannin was concentration-dependent, with an IC50 between 50 and 75 nM (Fig. 3A). The concentration of wortmannin required to completely block the action of insulin on PEPCK gene expression (500 nM) was higher than that generally reported to be required for complete inhibition of PI 3-kinase. This may be due to the reported instability of wortmannin in buffered pH 7.4 solution (
      • Woscholski R.
      • Kodaki T.
      • McKinnon M.
      • Waterfield M.D.
      • Parker P.J.
      ) since the incubations carried out in this study were significantly longer (≥3 h) than those employed in most previous studies (<30 min)(
      • Rahn T.
      • Ridderstrale M.
      • Tornqvist H.
      • Manganiello V.
      • Fredrikson G.
      • Belfrage P.
      • Degerman E.
      ,
      • Cross D.A.E.
      • Alessi D.R.
      • Vandenheede J.R.
      • McDowell H.E.
      • Hundal H.S.
      • Cohen P.
      ,
      • Welsh G.I.
      • Foulstone E.J.
      • Young S.W.
      • Tavare J.M.
      • Proud C.G.
      ,
      • Kotani K.
      • Yonezawa K.
      • Hara K.
      • Ueda H.
      • Kitamura Y.
      • Sakaue H.
      • Ando A.
      • Chavanieu A.
      • Calas B.
      • Grigorescu F.
      ,
      • Gould G.W.
      • Jess T.J.
      • Andrews G.C.
      • Herbst J.J.
      • Plevin R.J.
      • Gibbs E.M.
      ,
      • Clarke J.F.
      • Young P.W.
      • Yonezawa K.
      • Kasuga M.
      • Holman G.D.
      ,
      • Shimizu Y.
      • Shimazu T.
      ,
      • Shepherd P.R.
      • Nave B.T.
      • Siddle K.
      ,
      • Lam K.
      • Carpenter C.L.
      • Ruderman N.B.
      • Friel J.C.
      • Kelly K.L.
      ). More pronounced effects of wortmannin were seen at shorter hormone/inhibitor treatments (Fig. 3B, compare 4 h with 16 h), which is consistent with a loss of drug potency during prolonged incubation.
      Figure thumbnail gr2
      Figure 2:Wortmannin blocks insulin signaling to the endogenous PEPCK gene. Total RNA was prepared from HL1C cells treated for 3 h with hormones (as described in the legend to Fig. 1) in the presence or absence of 500 nM wortmannin. Radiolabeled oligonucleotides PC28 (PEPCK) and ACT25 (β-actin) were annealed to 50 μg of total RNA that was isolated from HL1C cells after each hormone/inhibitor treatment. A primer extension reaction was performed, and the products were separated by urea-acrylamide gel electrophoresis, visualized by autoradiography, and quantified by PhosphorImager analysis. Panel A provides an indication of the relative PEPCK and β-actin mRNA levels of a representative experiment. The measurement of β-actin mRNA was used to establish equal loading of mRNA in each lane. Panel B provides quantitation of three different experiments by PhosphorImager analysis. Results are presented as percent PEPCK mRNA relative to that obtained in the presence of dexamethasone (Dex)/cAMP alone and represent the means ± S.E. of three different experiments. DMSO, Me2SO.
      Figure thumbnail gr3
      Figure 3:Wortmannin blocks insulin signaling to the stably transfected PEPCK-CAT fusion gene. HL1C cells were incubated in serum-free medium in the absence of hormone (control); in 500 nM dexamethasone and 0.1 mM 8CPT-cAMP (Dex/cAMP); or in 500 nM dexamethasone, 0.1 mM 8CPT-cAMP, and 10 nM insulin (Dex/cAMP/insulin). A, wortmannin (at the indicated concentrations) or Me2SO carrier (an equivalent amount of carrier in all treatments) was included with each hormone treatment, and the cells were harvested after 3 h as described under “Experimental Procedures.” B, wortmannin (500 nM; closed symbols) or Me2SO carrier (open symbols) was included with each hormone treatment, and the cells were harvested at the indicated times. Squares indicate dexamethasone/cAMP-treated cells, circles indicate dexamethasone/cAMP/insulin treatment, and the dashed line represents the control. Extracts were assayed for CAT activity (as described under “Experimental Procedures”), and results are expressed as percent CAT activity relative to that obtained with dexamethasone/cAMP alone. Results represent the means ± S.E. of five (A) or three (B) different experiments.
      The role of protein kinase C in insulin signaling remains controversial (
      • Blackshear P.J.
      • Haupt D.M.
      • Stumpo D.J.
      ,
      • Messina J.L.
      • Standaert M.L.
      • Ishizuka T.
      • Weinstock R.S.
      • Farese R.V.
      ,
      • Stumpo D.J.
      • Haupt D.M.
      • Blackshear P.J.
      ), but phorbol esters, like insulin, inhibit dexamethasone- and cAMP-stimulated PEPCK gene transcription. However, in a previous study, the down-regulation of protein kinase C (by prolonged exposure to PMA) did not affect insulin signaling to the PEPCK promoter(
      • Chu D.T.W.
      • Stumpo D.J.
      • Blackshear P.J.
      • Granner D.K.
      ). This result suggested that protein kinase C is not required for the inhibition of PEPCK gene expression by insulin and that distinct pathways must exist for insulin and phorbol ester signaling to this promoter(
      • O'Brien R.M.
      • Bonovich M.T.
      • Forest C.D.
      • Granner D.K.
      ). This interpretation has recently been challenged by the recognition that not all forms of protein kinase C are down-regulated by this method(
      • Messina J.L.
      • Standaert M.L.
      • Ishizuka T.
      • Weinstock R.S.
      • Farese R.V.
      ). However, Fig. 4 demonstrates that the inhibition of dexamethasone- and cAMP-stimulated PEPCK-CAT gene transcription by phorbol ester was not significantly affected by wortmannin in HL1C cells. In addition, the ability of wortmannin to block the regulation of PEPCK-CAT by insulin was lost in the presence of PMA (Fig. 4). This result independently demonstrates that distinct signaling pathways are utilized by these agents.
      Figure thumbnail gr4
      Figure 4:Wortmannin does not affect phorbol ester signaling to the stably transfected PEPCK-CAT fusion gene. HL1C cells were incubated in serum-free DMEM in the absence of hormone (control); in 500 nM dexamethasone and 0.1 mM 8CPT-cAMP (Dex/cAMP); or in 500 nM dexamethasone, 0.1 mM 8CPT-cAMP, and 10 nM insulin (Dex/cAMP/insulin). Wortmannin (500 nM), PMA (1 μM), wortmannin (Wort) plus PMA, or Me2SO (DMSO) carrier (as a control) was included in each hormone treatment, and the cells were harvested after a 3-h incubation. Extracts were assayed for CAT activity (as described under “Experimental Procedures”), and the results are expressed as the percent CAT activity relative to that obtained with Dex/cAMP alone. Results represent the means ± S.E. of three different experiments.

      DISCUSSION

      Insulin affects the activity and/or amount of many proteins in a variety of cell types. The precise molecular mechanisms by which insulin elicits these changes are the subject of continued interest(
      • Denton R.M.
      ,
      • White M.F.
      • Kahn C.R.
      ). The potential physiological role(s) of p70S6k(
      • Cross D.A.E.
      • Alessi D.R.
      • Vandenheede J.R.
      • McDowell H.E.
      • Hundal H.S.
      • Cohen P.
      ,
      • Welsh G.I.
      • Foulstone E.J.
      • Young S.W.
      • Tavare J.M.
      • Proud C.G.
      ,
      • Chung J.
      • Kuo C.J.
      • Crabtree G.R.
      • Blenis J.
      ,
      • Price D.J.
      • Grove J.R.
      • Calvo V.
      • Avruch J.
      • Bierer B.E.
      ) and PI 3-kinase(
      • Cross D.A.E.
      • Alessi D.R.
      • Vandenheede J.R.
      • McDowell H.E.
      • Hundal H.S.
      • Cohen P.
      ,
      • Welsh G.I.
      • Foulstone E.J.
      • Young S.W.
      • Tavare J.M.
      • Proud C.G.
      ,
      • Clarke J.F.
      • Young P.W.
      • Yonezawa K.
      • Kasuga M.
      • Holman G.D.
      ,
      • Okada T.
      • Kawano Y.
      • Sakakibara T.
      • Hazeki O.
      • Ui M.
      ), two well described insulin-stimulated enzymes, in the regulation of a variety of metabolic processes have been analyzed using the inhibitors rapamycin and wortmannin, respectively. In this report, the effects of rapamycin and wortmannin on PEPCK gene expression were examined in order to study insulin signaling to the nucleus. The inhibition of insulin-stimulated p70S6k activity in HL1C cells by rapamycin had no significant effect on PEPCK gene expression (Fig. 1). In contrast, insulin stimulation of glyceraldehyde-3-phosphate dehydrogenase gene expression is inhibited by nanomolar concentrations of rapamycin,2(
      M. Alexander-Bridges, personal communication.
      )providing further evidence for the existence of multiple pathways of insulin-regulated gene expression (
      • O'Brien R.M.
      • Granner D.K.
      ).
      Unlike rapamycin, wortmannin inhibited the action of insulin on the expression of both the endogenous PEPCK gene and the PEPCK-CAT fusion gene in the HL1C stable cell line. Thus, PI 3-kinase is absolutely required for the regulation of PEPCK gene expression by insulin. Interestingly, wortmannin enhanced glucocorticoid- and cAMP-stimulated PEPCK-CAT expression (1.3-3-fold; Figure 3:, Figure 4:). Whether this is due to effects on constitutive PI 3-kinase activity, a direct effect on dexamethasone and/or cAMP signaling, or some other action is unclear. In a recent study, wortmannin reduced basal levels of glucose transport in rat skeletal muscle, an effect possibly due to the inhibition of a constitutive PI 3-kinase activity(
      • Yeh J.-I.
      • Gulve E.A.
      • Rameh L.
      • Birnbaum M.J.
      ). Similarly, Welsh et al.(
      • Welsh G.I.
      • Foulstone E.J.
      • Young S.W.
      • Tavare J.M.
      • Proud C.G.
      ) demonstrated that basal glycogen synthase kinase-3 activity (an activity inhibited by insulin) was increased by wortmannin in Chinese hamster ovary cells. However, the enhancement of glucocorticoid/cAMP-stimulated PEPCK-CAT expression was not observed in assays that measured endogenous PEPCK mRNA (Fig. 2), which suggests a selective action on reporter gene expression. When added in vitro, wortmannin had no direct effect on CAT activity3(
      C. Sutherland, unpublished data.
      ); thus, a simple activation of reporter protein cannot explain this phenomenon.
      In contrast to its effect on insulin action, wortmannin did not block the inhibition of dexamethasone- and cAMP-stimulated PEPCK-CAT gene expression by PMA (Fig. 4). This result is analogous to the effect of wortmannin on the regulation of glycogen synthase kinase-3 by insulin and PMA(
      • Welsh G.I.
      • Foulstone E.J.
      • Young S.W.
      • Tavare J.M.
      • Proud C.G.
      ). Surprisingly, we have found that the same cis-acting DNA element mediates part of the negative effect of both insulin and phorbol esters on PEPCK gene transcription(
      • O'Brien R.M.
      • Bonovich M.T.
      • Forest C.D.
      • Granner D.K.
      ). Thus, even though insulin and phorbol esters initially activate different pathways, their actions converge at a point downstream of PI 3-kinase. p70S6k does not appear to be this point of convergence. Although this enzyme can be activated by both insulin and phorbol esters (effects blocked by rapamycin)(
      • Chung J.
      • Kuo C.J.
      • Crabtree G.R.
      • Blenis J.
      ,
      • Susa M.
      • Olivier A.R.
      • Fabbro D.
      • Thomas G.
      ), rapamycin had no effect on insulin (Fig. 1) or PMA3 signaling to the PEPCK promoter in HL1C cells. Thus, neither PI 3-kinase nor p70S6k is required for the inhibition of glucocorticoid- and cAMP-stimulated PEPCK gene expression by PMA.
      PI 3-kinase can be stimulated by a wide range of cellular activators and growth factors (e.g. EGF, platelet-derived growth factor, and hepatic growth factor). EGF mimics the action of insulin on the inhibition of cAMP-stimulated PEPCK expression in primary hepatocyte cultures(
      • Molero C.
      • Valverde A.M.
      • Benito M.
      • Lorenzo M.
      ,
      • Fillat C.
      • Valera A.
      • Bosch F.
      ). It would thus be of interest to determine whether wortmannin blocks this action of EGF and whether the inhibitory effects of EGF and insulin or of EGF and PMA on PEPCK gene transcription are mediated by a common signal transduction pathway. The inhibition of cAMP-stimulated PEPCK gene expression by EGF was partially attenuated by prolonged exposure to phorbol esters; thus, protein kinase C may play a role in this action of EGF(
      • Fillat C.
      • Valera A.
      • Bosch F.
      ).
      At least part of the effect of insulin on PEPCK gene transcription is mediated by a cis-acting element, located between positions −413 and −407 relative to the transcription start site of the PEPCK gene, that has been designated the insulin response sequence(
      • O'Brien R.M.
      • Lucas P.C.
      • Forest C.D.
      • Magnuson M.A.
      • Granner D.K.
      ,
      • O'Brien R.M.
      • Noisin E.L.
      • Suwanichkul A.
      • Yamasaki T.
      • Lucas P.L.
      • Wang J.-C.
      • Powell D.R.
      • Granner D.K.
      ). This has the core sequence T(G/A)TTTTG(
      • O'Brien R.M.
      • Noisin E.L.
      • Suwanichkul A.
      • Yamasaki T.
      • Lucas P.L.
      • Wang J.-C.
      • Powell D.R.
      • Granner D.K.
      ). Although a number of proteins bind to the insulin response sequence in vitro (
      • O'Brien R.M.
      • Noisin E.L.
      • Suwanichkul A.
      • Yamasaki T.
      • Lucas P.L.
      • Wang J.-C.
      • Powell D.R.
      • Granner D.K.
      ,
      • O'Brien R.M.
      • Lucas P.L.
      • Yamasaki T.
      • Noisin E.L.
      • Granner D.K.
      ), it remains unclear which, if any, of these DNA-binding proteins actually mediates the action of insulin on PEPCK gene transcription. The signal transduction molecules that lie between PI 3-kinase and potential PEPCK insulin response sequence-binding proteins remain to be identified. The phosphorylated lipids produced by the action of PI 3-kinase may act as second messengers. Indeed, in vitro, one such lipid, PI 3,4,5-trisphosphate, may directly activate the phorbol ester-insensitive isoform of protein kinase C termed protein kinase C-ζ(
      • Nakanishi H.
      • Brewer K.A.
      • Exton J.H.
      ). Toker et al.(
      • Toker A.
      • Meyer M.
      • Reddy K.K.
      • Falck J.R.
      • Aneja R.
      • Aneja S.
      • Parra A.
      • Burns D.J.
      • Ballas L.M.
      • Cantley L.C.
      ) recently reported that other phorbol ester-insensitive protein kinase C isoforms are activated by PI 3,4,5-trisphosphate to a greater extent than protein kinase C-ζ. Thus, a role for one or more of these phorbol ester-insensitive forms of protein kinase C in insulin signaling cannot be excluded. PI 3-kinase appears to be tightly associated with, or contains, a protein kinase activity(
      • Carpenter C.L.
      • Auger K.R.
      • Duckworth B.C.
      • Hou W.-M.
      • Schaffhausen B.
      • Cantley L.C.
      ,
      • Dhand R.
      • Hiles I.
      • Panayotou G.
      • Roche S.
      • Fry M.J.
      • Gout I.
      • Totty N.F.
      • Truong O.
      • Vicendo P.
      • Yonezawa K.
      • Kasuga M.
      • Courtneidge S.A.
      • Waterfield M.D.
      ); thus, it is conceivable that novel protein substrates may be found for this enzyme, one or more of which may mediate insulin's action on PEPCK gene expression. There is evidence that, in certain cell types, the activation of PI 3-kinase may result in the activation of the mitogen-activated protein kinase pathway(
      • Cross D.A.E.
      • Alessi D.R.
      • Vandenheede J.R.
      • McDowell H.E.
      • Hundal H.S.
      • Cohen P.
      ,
      • Welsh G.I.
      • Foulstone E.J.
      • Young S.W.
      • Tavare J.M.
      • Proud C.G.
      ,
      • Ferby I.M.
      • Wago I.
      • Sakanaka C.
      • Kume K.
      • Shimizu T.
      ). Therefore, it will be of interest to determine whether the stimulation of mitogen-activated protein kinase activity by insulin is blocked by wortmannin in H4IIE cells and whether inhibitors of this well described protein kinase cascade influence the regulation of PEPCK gene transcription by insulin.
      In conclusion, PI 3-kinase is required for the regulation of PEPCK gene expression by insulin, whereas p70S6k is apparently not involved. Thus, although p70S6k lies downstream of PI 3-kinase, a bifurcation of this signaling pathway must exist such that PI 3-kinase activation leads to the regulation of other, as yet unidentified, factors. These data provide a direct link between PI 3-kinase activation and the regulation of expression of a specific gene. The identification of the transcription factor(s) responsible for the regulation of PEPCK gene expression by insulin should aid in the clarification of the signaling pathway between PI 3-kinase and this gene.

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