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Insulin Stimulation of the Fatty Acid Synthase Promoter Is Mediated by the Phosphatidylinositol 3-Kinase Pathway

INVOLVEMENT OF PROTEIN KINASE B/Akt*
  • Dong Wang
    Affiliations
    Department of Nutritional Sciences, University of California, Berkeley, California 94720-3104
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  • Hei Sook Sul
    Correspondence
    To whom correspondence should be addressed. Tel.: 510-642-3978; Fax: 510-642-0535;
    Affiliations
    Department of Nutritional Sciences, University of California, Berkeley, California 94720-3104
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  • Author Footnotes
    * This work was supported in part by National Institutes of Health Grant DK-36264 (to H. S. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Open AccessPublished:September 25, 1998DOI:https://doi.org/10.1074/jbc.273.39.25420
      Fatty acid synthase (FAS) is a critical enzyme in de novo lipogenesis. It catalyzes the seven steps in the conversion of malonyl-CoA and acetyl-CoA to palmitate. We have shown that the rate of FAS transcription is induced dramatically when fasted animals are refed with a high carbohydrate, fat-free diet or when streptozotocin-diabetic mice are given insulin. The FAS promoter was up-regulated by insulin through the proximal insulin response sequence containing an E-box motif at the −65-base pair position. Binding of upstream stimulatory factors to the −65 E-box is functionally required for insulin regulation of the FAS promoter. In the present study, we characterized signaling pathways in the insulin stimulation of FAS transcription using specific inhibitors for various signaling molecules and transfecting engineered phosphatidylinositol (PI) 3-kinase subunits and protein kinase B (PKB)/Akt. PD98059 and rapamycin, which inhibit MAP kinase and P70 S6 kinase, respectively, had little effect on the insulin-stimulated FAS promoter activity in 3T3-L1 adipocytes. On the other hand, wortmannin and LY294002, which specifically inactivate PI 3-kinase, strongly inhibited the insulin-stimulated FAS promoter activity. As shown in RNase protection assays, LY294002 also inhibited insulin stimulation of the endogenous FAS mRNA levels in 3T3-L1 adipocytes. Cotransfection of expression vectors for the constitutively active P110 subunit of PI 3-kinase resulted in an elevated FAS promoter activity in the absence of insulin and a loss of further insulin stimulation. Transfecting a dominant negative P85 subunit of PI 3-kinase decreased FAS promoter activity and blocked insulin stimulation. Furthermore, cotransfected wild-type PKB/Akt increased FAS promoter activity in the absence of insulin and a loss of insulin responsiveness of the FAS promoter. On the other hand, kinase-dead PKB/Akt acted in a dominant negative manner to decrease the FAS promoter activity and abolished its insulin responsiveness. These results demonstrate that insulin stimulation of fatty acid synthase promoter is mediated by the PI 3-kinase pathway and that PKB/Akt is involved as a downstream effector.
      FAS
      fatty acid synthase
      IRS
      insulin receptor substrate
      PI
      phosphatidylinositol
      PKB
      protein kinase B
      USF
      upstream stimulatory factor
      MAP
      mitogen-activated protein
      LUC
      luciferase
      bp
      base pair(s)
      kb
      kilobase pair(s).
      Fatty acid synthase (FAS)1 plays an important role in de novo lipogenesis in mammals and birds. Using NADPH as reducing equivalents, FAS catalyzes the seven steps in the conversion of acetyl-CoA and malonyl-CoA to palmitate. FAS activity is exquisitely sensitive to nutritional, hormonal, and developmental status (
      • Volpe J.J.
      • Vagelos P.R.
      ,
      • Wakil S.J.
      • Stoops J.K.
      • Joshi V.C.
      ,
      • Sul H.S.
      • Wang D.
      ,
      • Hillgartner F.B.
      • Salati L.M.
      • Goodridge A.G.
      ). We previously reported that insulin increases FAS mRNA levels in streptozotocin-diabetic mice and cultured 3T3-L1 adipocytes and that the regulation is at the transcriptional level (
      • Paulauskis J.D.
      • Sul H.S.
      ,
      • Paulauskis J.D.
      • Sul H.S.
      ). Using chimeric constructs of serial 5′-deletions of the rat FAS promoter linked to the luciferase reporter gene and transfection into 3T3-L1 adipocytes, we mapped the insulin response sequence to the proximal promoter region from −68 to −52 (
      • Moustaid N.
      • Sakamoto K.
      • Clarke S.
      • Beyer R.S.
      • Sul H.S.
      ,
      • Moustaid N.
      • Beyer R.S.
      • Sul H.S.
      ), which contains an E-box (5′-CATGTG-3′) motif at −65. Three tandem copies of the −68/−52 FAS promoter region linked to the heterologous SV40 promoter were responsive to insulin (
      • Moustaid N.
      • Beyer R.S.
      • Sul H.S.
      ). We also reported that upstream stimulatory factors (USFs), members of basic helix-loop-helix leucine-zipper family of transcription factors, bind to the E-box at −65 in vitro (
      • Wang D.
      • Sul H.S.
      ). By correlating functional assays of mutated FAS promoter with USF binding activities and cotransfection of expression vectors of wild-type and dominant negative USFs, we demonstrated that USF binding to the E-box at −65 is functionally required for insulin regulation of the FAS promoter (
      • Wang D.
      • Sul H.S.
      ).
      Insulin regulates a wide variety of biological responses in coordination with other hormones, such as glucagon, to maintain glucose homeostasis. Insulin stimulates glucose transport by the peripheral tissues such as muscle and adipose tissue, inhibits glycogen synthesis and gluconeogenesis in the liver, and stimulates protein synthesis and lipogenesis. Some of these effects are exerted on the transcription level through a cascade of signaling events (
      • O'Brien R.M.
      • Granner D.K.
      ). Binding of insulin to the insulin receptor on cell membrane triggers tyrosine kinase activity of the insulin receptor and results in its autophosphorylation within the cytoplasmic domain (
      • Myers Jr., M.G.
      • White M.F.
      ,
      • Cheatham B.
      • Kahn C.R.
      ). Tyrosine-phosphorylated insulin receptor then interacts with insulin receptor substrates (IRSs). Phosphorylation of IRS-1 and IRS-2 on the tyrosine residues then results in the recruitment and activation of divergent signaling molecules, including those in the Ras/MAP kinase and phosphatidylinositol (PI) 3-kinase pathways. PI 3-kinase is composed of the regulatory subunit (P85) and the catalytic subunit (P110). P85 acts as an interface by interacting with the IRS-1 through its SH2 domain and thus recruits the P110 subunit to the cell membrane through its iSH2 domain (
      • Myers Jr., M.G.
      • White M.F.
      ). P110 then catalyzes the reaction to release phosphatidylinositol (3,4,5)-triphosphate as the second messenger using phosphatidylinositol (4,5)-bisphosphate as the substrate. Recently, the 3-phosphoinositide-dependent protein kinase (
      • Alessi D.R.
      • Deak M.
      • Casamayor A.
      • Caudwell F.B.
      • Morrice N.
      • Norman D.G.
      • Gaffney P.
      • Reese C.B.
      • MacDougall C.N.
      • Harbison D.
      • Ashworth A.
      • Bownes M.
      ,
      • Alessi D.R.
      • James S.R.
      • Downes C.P.
      • Holmes A.B.
      • Gaffney P.R.
      • Reese C.B.
      • Cohen P.
      ,
      • Cohen P.
      • Alessi D.R.
      • Cross D.A.
      ,
      • Stephens L.
      • Anderson K.
      • Stokoe D.
      • Erdjument-Bromage H.
      • Painter G.F.
      • Holmes A.B.
      • Gaffney P.R.
      • Reese C.B.
      • McCormick F.
      • Tempst P.
      • Coadwell J.
      • Hawkins P.T.
      ), a serine-threonine kinase, was shown to respond to phosphatidyl (3,4,5)-triphosphate and to lead to the phosphorylation and activation of PKB/Akt, which is suggested as one of the major downstream mediators of PI 3-kinase (
      • Kohn A.D.
      • Takeuchi F.
      • Roth R.A.
      ,
      • Klippel A.
      • Reinhard C.
      • Kavanaugh W.M.
      • Apell G.
      • Escobedo M.A.
      • Williams L.T.
      ,
      • Klippel A.
      • Kavanaugh W.M.
      • Pot D.
      • Williams L.T.
      ,
      • Burgering B.M.
      • Coffer P.J.
      ,
      • Franke T.F.
      • Yang S.I.
      • Chan T.O.
      • Datta K.
      • Kazlauskas A.
      • Morrison D.K.
      • Kaplan D.R.
      • Tsichlis P.N.
      ). PI 3-kinase has also been suggested to activate P70 S6 kinase (
      • Chung J.
      • Grammer T.C.
      • Lemon K.P.
      • Kazlauskas A.
      • Blenis J.
      ), which is thought to be important for stimulation of protein synthesis by insulin. While the Ras/MAP kinase pathway is believed to play an important role in mitogenic effects of insulin (
      • Myers Jr., M.G.
      • White M.F.
      , ), PI 3-kinase is being demonstrated as an important mediator in metabolic regulation including GLUT4 translocation (
      • Cheatham B.
      • Vlahos C.J.
      • Cheatham L.
      • Wang L.
      • Blenis J.
      • Kahn C.R.
      ,
      • Kotani K.
      • Carozzi A.J.
      • Sakaue H.
      • Hara K.
      • Robinson L.J.
      • Clark S.F.
      • Yonezawa K.
      • James D.E.
      • Kasuga M.
      ,
      • Quon M.J.
      • Chen H.
      • Ing B.L.
      • Liu M.L.
      • Zarnowski M.J.
      • Yonezawa K.
      • Kasuga M.
      • Cushman S.W.
      • Taylor S.I.
      ,
      • Sakaue H.
      • Ogawa W.
      • Takata M.
      • Kuroda S.
      • Kotani K.
      • Matsumoto M.
      • Sakaue M.
      • Nishio S.
      • Ueno H.
      • Kasuga M.
      ,
      • Martin S.S.
      • Haruta T.
      • Morris A.J.
      • Klippel A.
      • Williams L.T.
      • Olefsky J.M.
      ) and activation of glycogen synthase (
      • Cross D.A.
      • Alessi D.R.
      • Cohen P.
      • Andjelkovich M.
      • Hemmings B.A.
      ). Recently, PI 3-kinase has been shown to mediate insulin inhibition of the transcription of the PEPCK gene, which encodes the rate-limiting enzyme in gluconeogenesis (
      • Gabbay R.A.
      • Sutherland C.
      • Gnudi L.
      • Kahn B.B.
      • O'Brien R.M.
      • Granner D.K.
      • Flier J.S.
      ,
      • Sutherland C.
      • O'Brien R.M.
      • Granner D.K.
      ,
      • Sutherland C.
      • Waltner-Law M.
      • Gnudi L.
      • Kahn B.B.
      • Granner D.K.
      ).
      Molecular mechanisms mediating insulin regulation of lipogenesis, especially the signaling pathways involved, is largely unknown. Since FAS is a critical enzyme involved in lipogenesis, we set out to investigate the signaling pathways involved in the regulation of FAS transcription by insulin. In this report, we provide experimental evidence to demonstrate that the PI 3-kinase signaling pathway mediates insulin regulation of FAS transcription. While inhibition of MAP kinase and S6 kinase activity by PD98059 and rapamycin, respectively, had little effect on the insulin stimulation of FAS promoter activity, wortmannin and LY294002, which inhibit PI 3-kinase, blocked the insulin stimulation of FAS promoter activity. Regulation of the endogenous FAS mRNA by insulin was also blocked by treating 3T3-L1 adipocytes with LY294002. Cotransfection of expression vectors encoding a constitutively active P110 subunit of PI 3-kinase resulted in elevated FAS promoter activity in the absence of insulin and a loss of insulin response of the FAS promoter. On the other hand, a dominant negative P85 subunit of PI 3-kinase inhibited FAS promoter activity and abolished insulin stimulation of the FAS promoter. Moreover, cotransfection of PKB/Akt stimulated FAS promoter activity in the absence of insulin to that comparable to the insulin-stimulated level. Acting in a dominant-inhibitory fashion, kinase-dead PKB/Akt inhibited FAS promoter activity in the presence and the absence of insulin. These data suggest that insulin regulation of FAS transcription is mediated by the PI 3-kinase signaling pathway and that PKB/Akt is involved as a downstream effector.

      DISCUSSION

      When circulating insulin is high, there is an increase in lipogenesis in liver and adipose tissue. These processes occur in coordination with the increase in glucose uptake by peripheral tissues such as muscle and adipose tissue, with inhibition of hepatic gluconeogenesis and glycogenesis, and so forth. Increase in lipogenesis is impaired when insulin is low, and administration of insulin restores the rate to its normal level. FAS is a key lipogenic enzyme, and insulin increases its activity dramatically, not through allosteric effectors or covalent modification but through changes in transcription (
      • Paulauskis J.D.
      • Sul H.S.
      ,
      • Paulauskis J.D.
      • Sul H.S.
      ). Rapid and high level induction of the FAS gene by insulin makes FAS an excellent model for studying the transcriptional activation of lipogenic genes by insulin. Previously, we had defined the FAS insulin response sequence to the proximal promoter region at −68/−52 and had shown that USF binding to the E-box motif within this region is functionally required for insulin stimulation of FAS transcription. However, the signaling pathway(s) that leads to the increase in lipogenesis or the activation of lipogenic genes by insulin is not known. In this report, we provide evidence that the PI 3-kinase signaling pathway, but not the P70 S6 kinase nor the MAP kinase pathways, mediates the insulin effect on FAS transcription. Inhibitors of PI 3-kinase (wortmannin and LY294002) abolished the insulin stimulation of endogenous FAS mRNA (Fig. 2) as well as the transfected FAS promoter-reporter construct in 3T3-L1 adipocytes (Fig. 1). Expression of the constitutively active P110 catalytic subunit of PI 3-kinase resulted in the loss of insulin responsiveness of the FAS promoter at an elevated activity level (Fig. 3). Expression of the dominant negative P85 regulatory subunit also resulted in loss of insulin responsiveness of the FAS promoter, but at a repressed activity level (Fig. 4). Similar effects on the insulin responsiveness of FAS promoter were also observed when wild-type and kinase-dead (dominant negative) PKB/Akt were expressed, suggesting PKB/Akt is a downstream mediator of the insulin stimulation of the FAS transcription (Fig. 5). This is the first report, to our knowledge, that demonstrates the involvement of the PI 3-kinase and PKB/Akt signaling pathway in the transcriptional regulation of lipogenic genes. Our data, therefore, suggest that PI 3-kinase is not only involved in the stimulation of glucose transportation, glycogen synthesis, and inhibition of gluconeogenesis, but also in the stimulation of lipogenesis. It also suggests that the effect of PI 3-kinase is a major branching point of the insulin signaling pathways that exert the insulin effect at multiple levels of gene expression, including control of transcription (e.g. stimulation of FAS and inhibition of PEPCK), phosphorylation of glycogen synthase, and translocation of the GLUT4 transporter. Since inhibiting MAP kinase activity by PD98059 had no effect on insulin stimulation of the FAS promoter, it appears that the MAP kinase pathway, which plays an important role in mediating the mitogenic-effect of insulin, is not likely to be involved in insulin stimulation of lipogenesis.
      From the results presented in Fig. 5, it is suggested that PKB/Akt is the downstream mediator of the insulin stimulation of FAS transcription. However, further targets of this signaling pathway remain unknown. We previously reported that binding of the helix-loop-helix leucine zipper transcription factor USF to the insulin response sequence of the FAS gene is required for insulin regulation (
      • Wang D.
      • Sul H.S.
      ), but thus far there has been no conclusive report on the regulation of USF by a phosphorylation/dephosphorylation mechanism. It is possible that either USF itself is regulated by phosphorylation status, or likely a USF-interacting protein is regulated by phosphorylation. In this regard, four proteins including Ets-1 (
      • Sieweke M.H.
      • Tekotte H.
      • Jarosch U.
      • Graf T.
      ), Fra-1 (
      • Pognonec P.
      • Boulukos K.E.
      • Aperlo C.
      • Fujimoto M.
      • Ariga H.
      • Nomoto A.
      • Kato H.
      ), c-Maf (
      • Kurschner C.
      • Morgan J.I.
      ), and Fos (
      • Blanar M.A.
      • Rutter W.J.
      ), which are known to be phosphorylated, were reported to be able to interact with USF and are likely the candidates for regulation by phosphorylation. However, whether phosphorylation of any of these USF interacting proteins plays a role in affecting stimulation of transcription by USF requires further investigation. Whether any of these USF interacting proteins is required for insulin regulation of FAS promoter remains to be elucidated.

      ACKNOWLEDGEMENTS

      We thank Dr. M. J. Quon and Dr. M. Kasuga for the P85 expression vectors, Dr. L. T. Williams and Dr. A. Klippel for the P110 and PKB/Akt expression vectors. We also thank Dr. L. T. Williams for his suggestion on using the PKB/Akt expression vectors in our studies.

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