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The Transcription Factors HIF-1 and HNF-4 and the Coactivator p300 Are Involved in Insulin-regulated Glucokinase Gene Expression via the Phosphatidylinositol 3-Kinase/Protein Kinase B Pathway*

  • Ulrike Roth
    Affiliations
    Institut für Biochemie und Molekulare Zellbiologie, Georg-August-Universität, Humboldtallee 23, D-37073 Göttingen, Germany
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  • Katja Curth
    Affiliations
    Institut für Biochemie und Molekulare Zellbiologie, Georg-August-Universität, Humboldtallee 23, D-37073 Göttingen, Germany
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  • Terry G. Unterman
    Affiliations
    Departments of Medicine and Physiology and Biophysics, University of Illinois at Chicago and Veterans Affair Chicago Healthcare System, Chicago, Illinois 60612
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  • Thomas Kietzmann
    Correspondence
    To whom correspondence should be addressed: Inst. für Biochemie und Molekulare Zellbiologie, Georg-August-Universität Göttingen, Humboldtallee 23, D-37073 Göttingen, Germany. Tel.: 49-551-395952; Fax: 49-551-395960
    Affiliations
    Institut für Biochemie und Molekulare Zellbiologie, Georg-August-Universität, Humboldtallee 23, D-37073 Göttingen, Germany
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  • Author Footnotes
    * This work was supported by the Deutsche Forschungsgemeinschaft Grants SFB 402, TP A1, and GRK335 (to T. K.), National Institutes of Health Grant DKK1430, and by the Department of Veterans Affairs Merrit Review program (to T. G. U.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.This study is dedicated to Kurt Jungermann who died on May 10, 2002.
Open AccessPublished:November 10, 2003DOI:https://doi.org/10.1074/jbc.M308391200
      Glucokinase plays a key role in the regulation of glucose utilization in liver and its expression is strongly enhanced by insulin and modulated by venous pO2. In primary rat hepatocytes, pO2 modulated insulin-dependent glucokinase (GK) gene expression was abolished by wortmannin an inhibitor of phosphatidylinositol 3-kinase (PI3K). Transfection of vectors encoding the p110 catalytic subunit of PI3K or constitutively active proteinkinase B (PKB) stimulated GK mRNA and protein expression. The transfection of GK promoter constructs together with expression vectors for p110 or constitutively active PKB revealed that the GK promoter region –87/–80 mediates the response to PI3K/PKB. Transfection experiments and gel shift assays show that this element is able to bind hypoxia-inducible factor-1 (HIF-1) in a hypoxia- and PKB-dependent manner. The ability of HIF-1α to activate the GK promoter was enhanced by hepatocyte nuclear factor-4α (HNF-4α), acting via the sequence –52/–39, and by the coactivator p300. Stimulation of the GK promoter by insulin was dependent on the intact –87/–80 region and maximal stimulation was achieved when HIF-1α, HNF-4, and p300 were cotransfected with the –1430 GK promoter Luc construct in primary hepatocytes. Maximal stimulation of GK promoter activity by insulin was inhibited when a p300 vector was used containing a mutation within a PKB phosphorylation site. Thus, a regulatory transcriptional complex consisting of HIF-1, HNF-4, and p300 appears to be involved in insulin-dependent GK gene activation.
      The mammalian enzyme glucokinase (GK),
      The abbreviations used are: GK, glucokinase; PI3K, phosphatidylinositol 3-kinase; PKB, proteinkinase B; myrPKB, myristoylated PKB; HNF, hepatocyte nuclear factor; HIF, hypoxia-inducible factor; SREBP, sterol-regulatory element-binding protein; USF, upstream stimulatory factor; HRE, hypoxia-responsive element; EMSA, electrophoretic mobility shift assay; EPO, erythropoietin; CREB, cAMP-responsive element-binding protein; CBP, CREB-binding protein.
      1The abbreviations used are: GK, glucokinase; PI3K, phosphatidylinositol 3-kinase; PKB, proteinkinase B; myrPKB, myristoylated PKB; HNF, hepatocyte nuclear factor; HIF, hypoxia-inducible factor; SREBP, sterol-regulatory element-binding protein; USF, upstream stimulatory factor; HRE, hypoxia-responsive element; EMSA, electrophoretic mobility shift assay; EPO, erythropoietin; CREB, cAMP-responsive element-binding protein; CBP, CREB-binding protein.
      also termed hexokinase IV, is a key enzyme of glucose utilization, playing a crucial role in maintaining blood glucose homeostasis. In contrast to the other hexokinase family members (hexokinase I–III), GK has a lower affinity for glucose with sigmoidal kinetics and is not inhibited by its reaction product glucose 6-phosphate. Compared with the other hexokinases with a molecular mass of about 100 kDa, GK has a molecular mass of only 50 kDa and binds to a glucokinase regulatory protein, which decreases its affinity for glucose. These attributes allow the GK enzyme to react adequately with glucose in concentrations reached in vivo (
      • Vandercammen A.
      • Van S.E.
      ,
      • Printz R.L.
      • Magnuson M.A.
      • Granner D.K.
      ,
      • Iynedjian P.B.
      ). GK expression is restricted to hepatocytes, the pancreatic β-cells, some neuroendocrine cells of the gastrointestinal tract, and the brain (
      • Jetton T.L.
      • Liang Y.
      • Pettepher C.C.
      • Zimmerman E.C.
      • Cox F.G.
      • Horvath K.
      • Matschinsky F.M.
      • Magnuson M.A.
      ).
      Insulin is a major regulator of GK gene expression in the liver. In primary hepatocytes, insulin concentrations from 10–10m up to 10–8m stimulate GK mRNA levels in a dose-dependent fashion, whereas glucagon and its second messenger cAMP inhibits GK expression (
      • Iynedjian P.B.
      • Jotterand D.
      • Nouspikel T.
      • Asfari M.
      • Pilot P.R.
      ). The ability of insulin to stimulate GK expression is positively modulated by perivenous pO2 (
      • Kietzmann T.
      • Roth U.
      • Freimann S.
      • Jungermann K.
      ), consistent with the predominant localization of GK in the less aerobic perivenous area of the liver acinus (
      • Jungermann K.
      • Kietzmann T.
      ).
      Following insulin binding to its receptor and receptor autophosphorylation, insulin signaling involves second messengers including members of the phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase cascades (
      • Saltiel A.R.
      • Kahn C.R.
      ). PI3K, which generates phosphatidylinositol 3,4,5-phosphate, plays a key role in the metabolic actions of insulin (
      • Shepherd P.R.
      • Nave B.T.
      • Rincon J.
      • Haigh R.J.
      • Foulstone E.
      • Proud C.
      • Zierath J.R.
      • Siddle K.
      • Wallberg-Henriksson H.
      ). Phosphatidylinositol 3,4,5-phosphate regulates the activity or subcellular localization of a variety of signaling molecules such as phosphatidylinositol-dependent kinase and protein kinase B (PKB, also known as Akt) (
      • Lietzke S.E.
      • Bose S.
      • Cronin T.
      • Klarlund J.
      • Chawla A.
      • Czech M.P.
      • Lambright D.G.
      ,
      • Alessi D.R.
      • James S.R.
      • Downes C.P.
      • Holmes A.B.
      • Gaffney P.R.
      • Reese C.B.
      • Cohen P.
      ), which in turn phosphorylates a number of target proteins including glycogen synthetase kinase-3, transcription factors of the foxoforkhead family, and coactivators such as p300.
      Besides its major role in the transmission of the insulin signal (
      • Saltiel A.R.
      • Kahn C.R.
      ), the phosphatidylinositol 3-kinase/protein kinase B pathway appears also to be involved in the oxygen signaling cascade, regulating the activity of the transcription factor hypoxia-inducible factor-1 (HIF-1) (
      • Zhong H.
      • Chiles K.
      • Feldser D.
      • Laughner E.
      • Hanrahan C.
      • Georgescu M.M.
      • Simons J.W.
      • Semenza G.L.
      ,
      • Jiang B.H.
      • Jiang G.
      • Zheng J.Z.
      • Lu Z.
      • Hunter T.
      • Vogt P.K.
      ). HIF-1 is composed of an inducible subunit (HIF-1α) and a constitutively expressed subunit (HIF-1β, also known as ARNT), and both are members of the basic helix-loop-helix-PAS protein family. HIF-1 is involved in hypoxia-dependent induction of a variety of genes, including erythropoietin, glycolytic enzymes, but also plasminogen activator inhibitor-1 and vascular endothelial growth factor (
      • Samoylenko A.
      • Roth U.
      • Jungermann K.
      • Kietzmann T.
      ,
      • Bunn H.F.
      • Poyton R.O.
      ,
      • Semenza G.L.
      ,
      • Maxwell P.H.
      • Ratcliffe P.J.
      ,
      • Wenger R.H.
      ). Two other HIF α-subunits, HIF-2α (EPAS/HLF/HRF/MOP2) (
      • Wenger R.H.
      ,
      • Ema M.
      • Taya S.
      • Yokotani N.
      • Sogawa K.
      • Matsuda Y.
      • FujiiKuriyama Y.
      ,
      • Flamme I.
      • Frohlich T.
      • von-Reutern M.
      • Kappel A.
      • Damert A.
      • Risau W.
      ,
      • Hogenesch J.B.
      • Chan W.K.
      • Jackiw V.H.
      • Brown R.C.
      • Gu Y.Z.
      • Pray G.M.
      • Perdew G.H.
      • Bradfield C.A.
      ), and HIF-3α (
      • Gu Y.Z.
      • Moran S.M.
      • Hogenesch J.B.
      • Wartman L.
      • Bradfield C.A.
      ,
      • Kietzmann T.
      • Cornesse Y.
      • Brechtel K.
      • Modaressi S.
      • Jungermann K.
      ) have been cloned from human, mouse, and rat, and together with other ARNT isoforms (ARNT2 and ARNT3/BMAL-1/MOP-3), they give rise to the existence of several HIF dimers composed of different HIF α-subunits and ARNT isoforms (
      • Wenger R.H.
      ,
      • Semenza G.L.
      ). Interestingly HIF-α protein levels and transcriptional activity have been shown to be enhanced by insulin (
      • Zelzer E.
      • Levy Y.
      • Kahana C.
      • Shilo B.Z.
      • Rubinstein M.
      • Cohen B.
      ,
      • Stiehl D.P.
      • Jelkmann W.
      • Wenger R.H.
      • Hellwig-Burgel T.
      ,
      • Kietzmann T.
      • Samoylenko A.
      • Roth U.
      • Jungermann K.
      ).
      The transcription factors mediating the modulation of the insulin-dependent GK gene expression by perivenous pO2 in hepatocytes, and thus the zonated GK expression in liver, are not yet known. Transcription factors that could be involved include the HIF-1, sterol-regulatory element-binding protein-1 (SREBP-1) (
      • Foretz M.
      • Guichard C.
      • Ferre P.
      • Foufelle F.
      ), upstream stimulatory factors (USFs) (
      • Iynedjian P.B.
      ), and HNF-4 (
      • Roth U.
      • Jungermann K.
      • Kietzmann T.
      ), all of which can be regulated by oxygen or by insulin or by both. The transcriptional coactivator p300, which has been shown to contain a PKB phosphorylation site (
      • Guo S.
      • Cichy S.B.
      • He X.
      • Yang Q.
      • Ragland M.
      • Ghosh A.K.
      • Johnson P.F.
      • Unterman T.G.
      ) and to interact with HIF-1α and HNF-4 (
      • Madan A.
      • Curtin P.T.
      ,
      • Galson D.L.
      • Tsuchiya T.
      • Tendler D.S.
      • Huang L.E.
      • Ren Y.
      • Ogura T.
      • Bunn H.F.
      ,
      • Huang L.E.
      • Ho V.
      • Arany Z.
      • Krainc D.
      • Galson D.
      • Tendler D.
      • Livingston D.M.
      • Bunn H.F.
      ,
      • Arany Z.
      • Huang L.E.
      • Eckner R.
      • Bhattacharya S.
      • Jiang C.
      • Goldberg M.A.
      • Bunn H.F.
      • Livingston D.M.
      ), also might be involved. Thus, it was the aim of this study to investigate whether the PI3K/PKB pathway and the factors HIF-1, HNF-4, and p300 are involved in mediating the effect of insulin on GK gene expression.

      EXPERIMENTAL PROCEDURES

      All biochemicals and enzymes were of analytical grade and were purchased from commercial suppliers.
      Animals—Male Wistar rats (200–260 g) were kept on a 12 h day/night rhythm (light from 7 a.m. to 7 p.m.) with free access to water and food. Rats were anesthetized with pentobarbital (60 mg/kg body weight) prior to preparation of hepatocytes between 8 a.m. and 9 a.m.
      Plasmid Constructs—The reporter plasmid rat GK-1430 LUC was constructed by inserting a BglII-HindIII GK promoter fragment (–1430/+21) into pGL3 basic from Promega (Heidelberg, Germany). The reporter plasmid GK-101 LUC or GK-101HREm LUC which contains a mutation in the hypoxia-responsive element (HRE) (–87/–80) were constructed by inserting a SmaI-XhoI promoter fragment (–101/+79) (
      • Fleischmann M.
      • Iynedjian P.B.
      ) into pGL3 basic. The GK-1430HREm and GK-1430HNFm LUC construct was generated using the QuickChange mutagenesis kit (Promega) with GK-1430 LUC as template. All constructs were verified by sequencing in both directions.
      The p110 plasmid containing the p110α cDNA (
      • Khwaja A.
      • Lehmann K.
      • Marte B.M.
      • Downward J.
      ) was a kind gift from Dr. J. Downward. The vectors expressing PKB and the constitutively active form of PKB (myrPKB) were a kind gift from Dr. D. Stokoe and have already been described (
      • Franke T.F.
      • Yang S.I.
      • Chan T.O.
      • Datta K.
      • Kazlauskas A.
      • Morrison D.K.
      • Kaplan D.R.
      • Tsichlis P.N.
      ). The human SREBP-1a expression plasmid (
      • Shimano H.
      • Horton J.D.
      • Shimomura I.
      • Hammer R.E.
      • Brown M.S.
      • Goldstein J.L.
      ), the rat HNF-4α expression vector (
      • Sladek F.M.
      ), and the rat HIF-1α expression vector were already described (
      • Kietzmann T.
      • Roth U.
      • Jungermann K.
      ). The expression vectors encoding the human p300 as well as the p300mut containing a mutation replacing serine with alanine at position 1834 also have been described (
      • Guo S.
      • Cichy S.B.
      • He X.
      • Yang Q.
      • Ragland M.
      • Ghosh A.K.
      • Johnson P.F.
      • Unterman T.G.
      ).
      Cell Transfection and Luciferase Assay—Liver cells were isolated by collagenase perfusion and maintained under standard conditions in an atmosphere of periportal pO2 (16% O2/79% N2/5% CO2 (by volume)) in medium M199 containing 0.5 nm insulin added as a growth factor for culture maintenance, 100 nm dexamethasone required as a permissive hormone, and, until the first change of medium after 5 h, 4% newborn calf serum, as described previously (
      • Immenschuh S.
      • Hinke V.
      • Ohlmann A.
      • Gifhorn-Katz S.
      • Katz N.
      • Jungermann K.
      • Kietzmann T.
      ). Freshly isolated rat hepatocytes (about 1 × 106 cells per dish) were transfected as described (
      • Immenschuh S.
      • Hinke V.
      • Ohlmann A.
      • Gifhorn-Katz S.
      • Katz N.
      • Jungermann K.
      • Kietzmann T.
      ), thereby controlling transfection efficiency by cotransfection with 0.25 μg of Renilla luciferase expression vector (pRLSV40) (Promega). In brief, 2 μg of the respective LUC construct was transfected with 500 ng of p110, PKB-WT, myrPKB, SREBP-1a, HIF-1α, or HNF-4 and/or p300 expression vectors or with appropriate amounts of the respective empty expression vectors. After 5 h, the medium was changed and the cells were cultured under periportal pO2 (16% O2) for 19 h, then the medium was changed again and the cells were further cultured for 24 h under periportal pO2.
      Western Blot Analysis—Protein from primary cultured hepatocytes and transiently transfected hepatocytes were isolated as described previously (
      • Wiesener M.S.
      • Turley H.
      • Allen W.E.
      • Willam C.
      • Eckardt K.U.
      • Talks K.L.
      • Wood S.M.
      • Gatter K.C.
      • Harris A.L.
      • Pugh C.W.
      • Ratcliffe P.J.
      • Maxwell P.H.
      ). The protein content was determined using the Bradford method. 50 μg of protein dissolved in 27 μl of SDS sample buffer was loaded onto a 10% SDS-polyacrylamide gel and then transferred onto nitrocellulose membranes. Nonspecific binding was blocked with blocking buffer (10 mm Tris/HCl (pH 7.5), 100 mm NaCl, 0.1% Tween 20, 10% milk powder). Blots were incubated with primary goat antibody against GK (Santa Cruz Biotechnology, Heidelberg, Germany) in a 1:200 dilution or with 1:1000 dilution of the rabbit antibodies against phospho-PKB/Akt (Thr308), phospho-PKB/Akt (Ser473), or total PKB/Akt (New England Biolabs, Heidelberg, Germany) in blocking buffer overnight at 4 °C. Washing was performed with blocking buffer without milk powder. The secondary antibodies were anti-goat IgG (Dako, Hamburg, Germany) or anti-rabbit IgG (Santa Cruz Biotechnology) used in a 1:2000 dilution for 1 h. The primary rabbit antibody against golgi membrane (Bioscience, Göttingen, Germany) was used in a 1:8000 dilution. The secondary antibody was a goat anti-rabbit IgG horseradish peroxidase (Santa Cruz Biotechnology), used in a 1:2000 dilution. After washing for 30 min, the ECL Western blotting system (Amersham Biosciences, Freiburg, Germany) was used for detection. Under these conditions, GK was visible as a band of 53 kDa and PKB as a band of about 60 kDa.
      RNA Preparation and Northern Analysis—Isolation of total RNA and Northern analysis were performed as described previously (
      • Kietzmann T.
      • Roth U.
      • Freimann S.
      • Jungermann K.
      ). Digoxigenin-labeled antisense RNAs served as hybridization probes; they were generated by in vitro transcription from pBS-GK-1 using T3 RNA polymerase or from pBS-β-actin using T7 RNA polymerase and RNA labeling mixture containing 3.5 mm 11-digoxigenin-UTP, 6.5 mm UTP, 10 mm GTP, 10 mm CTP, and 10 mm ATP. Hybridizations and detections were carried out essentially as described previously (
      • Kietzmann T.
      • Roth U.
      • Freimann S.
      • Jungermann K.
      ). Blots were quantified with a videodensitometer (Biotech Fischer, Reiskirchen, Germany).
      Preparation of Nuclear Extracts—Nuclear extracts were prepared by modification of a standard protocol (
      • Kietzmann T.
      • Roth U.
      • Jungermann K.
      ,
      • Semenza G.L.
      • Wang G.L.
      ) with buffers A and C containing 0.5 mm dithioerythritol (Sigma, Taufkirchen, Germany), 0.4 mm phenylmethylsulfonyl fluoride (Serva, Heidelberg, Germany), 2 μg of leupeptin per ml (Roche Applied Science, Mannheim, Germany), 2 μg of pepstatin per ml (Roche Applied Science), 2 μg of aprotinin per ml (Bayer, Leverkusen, Germany), 1 mm sodium vanadate (Sigma) and the “complete” protease inhibitor mixture tablets (Roche Applied Science) essentially as described previously (
      • Kietzmann T.
      • Roth U.
      • Jungermann K.
      ,
      • Semenza G.L.
      • Wang G.L.
      ).
      Electrophoretic Mobility Shift Assay (EMSA)—The sequence of the GK-HRE oligonucleotide was 5′-ACCCCACGTGGTTCTTTGT-3′. Equal amounts of complementary oligonucleotides were annealed and labeled by 5′-end labeling with [γ-32P]ATP (Amersham Biosciences) and T4 polynucleotide kinase (MBI, St. Leon-Rot, Germany). They were purified with the Nucleotide Removal Kit (Qiagen, Hilden, Germany). Binding reactions were carried out in a total volume of 20 μl containing 50 mm KCl, 1 mm MgCl2, 1.1 mm EDTA, 5% glycerol, 10 μg of nuclear extract, 250 ng of poly(dI-dC), and 5 mm dithioerythritol. After preincubation for 5 min at room temperature, 1 μl of the labeled probe (104 cpm) was added, and the incubation was continued for an additional 10 min. For supershift analysis either 1 μl rat HIF-1α antibody (
      • Kietzmann T.
      • Roth U.
      • Jungermann K.
      ), USF-2 antibody (N18), or Myc (C33) antibody (Santa Cruz Biotechnology) was added to the EMSA reaction and then incubated at 4 °C for 2 h. The electrophoresis was then performed with a 5% non-denaturing polyacrylamide gel in TBE buffer (89 mm Tris, 89 mm boric acid, 5 mm EDTA) at 200 V. After electrophoresis the gels were dried and analyzed by phosphorimaging (
      • Kietzmann T.
      • Roth U.
      • Jungermann K.
      ,
      • Semenza G.L.
      • Wang G.L.
      ).

      RESULTS

      Venous pO2 Enhances Phosphorylation of PKB by Insulin in Primary Rat Hepatocytes—The signaling pathway(s) mediating the effects of insulin and pO2 on GK gene expression are unknown. Since insulin can exert effects on gene expression via PI3K and PKB (
      • Vanhaesebroeck B.
      • Alessi D.R.
      ), we considered the possibility that perivenous pO2 may modulate the activation of the PI3K/PKB pathway or its downstream targets in primary hepatocytes.
      Therefore, we examined the activation of PKB in hepatocytes that were cultured under periportal and perivenous pO2 and stimulated with insulin. After insulin stimulation, the levels of serine 473-phosphorylated PKB were enhanced by about 7-fold under periportal pO2 and by about 10-fold under perivenous pO2. Threonine 308 phosphorylation of PKB protein was enhanced by about 2-fold under periportal pO2 and by about 4-fold under perivenous pO2. In contrast, the level of total PKB protein was not modulated by venous pO2 neither under control conditions or after insulin treatment (Fig. 1). The modulation by O2 of the insulin-induced PKB phosphorylation indicates a possible involvement of PKB mediating the effects of O2 on insulin-dependent GK expression.
      Figure thumbnail gr1
      Fig. 1Insulin-induced phosphorylation of PKB/Akt in primary rat hepatocytes: modulation by perivenous pO2. Hepatocytes were cultured for 24 h and stimulated for 10 min with 100 nm insulin under periportal and perivenous pO2. A total of 100 μg of protein from the hepatocytes were subjected to Western analyses with an antibody against phospho-PKB/Akt (Ser473), phospho-PKB/Akt (Thr308), or total PKB/Akt (see “Experimental Procedures”). Autoradiographic signals were obtained by chemiluminescence and scanned by videodensitometry.
      Induction of Glucokinase Expression by Insulin and by Overexpression of the PI3K Catalytic Subunit p110 or a Constitutively Active PKB in Primary Rat Hepatocytes: Inhibition by Wortmannin—To investigate whether the effects of insulin on GK mRNA expression is mediated via PI3K and/or PKB, rat hepatocytes were treated with insulin and/or the PI3K inhibitor wortmannin or transfected with an expression vector encoding a constitutively active form of PKB (myrPKB). Under periportal pO2 conditions, insulin treatment increased GK mRNA levels by about 6-fold (Fig. 2). Wortmannin completely inhibited the insulin-dependent GK mRNA induction. In the myrPKB-transfected cells, the GK mRNA level was enhanced by about 15-fold and was not increased further by treatment with insulin (Fig. 2). The PI3K- and PKB-dependent regulation of GK protein also was examined. The level of GK protein was increased by insulin treatment under venous pO2 by about 9-fold (Fig. 3). When the vectors encoding the catalytic PI3K subunit p110 or myrPKB were transfected into hepatocytes, GK protein levels were enhanced by about 10-fold and 14-fold, respectively (Fig. 3).
      Figure thumbnail gr2
      Fig. 2Induction of GK mRNA expression by insulin or overexpression of a constitutively active form of PKB: inhibition by wortmannin. Hepatocytes transfected with myrPKB expression vectors were cultured for 24 h under periportal pO2. At 24 h the medium was changed, and cells were further cultured for the next 24 h under periportal pO2. Where indicated the cells were treated with 100 nm insulin and 20 nm wortmannin for 3 h. A, the GK mRNA levels were measured by Northern blotting (see B). The mRNA level under periportal pO2 without insulin was set equal to 1. The values are from four independent culture experiments. B, Northern blot, 20 μg of total RNA prepared from the cultured hepatocytes were hybridized to digoxigenin-labeled GK and β-actin antisense RNA probes (see “Experimental Procedures”). Autoradiographic signals were obtained by chemiluminescence and scanned by videodensitometry. myrPKB, myristoylated PKB; Wo, wortmannin.
      Figure thumbnail gr3
      Fig. 3Induction of glucokinase expression by overexpression of p110 or PKB-WT in primary rat hepatocytes. Hepatocytes transfected either with p110 or PKB-WT expression vectors were cultured for 24 h under periportal pO2. At 24 h the medium was changed, and cells were further cultured for the next 24 h under periportal pO2. The untransfected cells were cultured under periportal and perivenous pO2. A total of 50 μg of protein of the cultured hepatocytes was subjected to Western analysis with an antibody against GK and as loading control against golgi membrane (GM) (see “Experimental Procedures”). Autoradiographic signals were obtained by chemiluminescence and scanned by videodensitometry.
      Stimulation of GK Promoter-controlled LUC Expression by Overexpression of p110 or PKB in Primary Rat Hepatocytes—To further investigate the role of PI3K and PKB in regulating GK expression we investigated whether GK promoter activity is regulated via the PI3K-PKB-pathway and cotransfected primary rat hepatocytes with liver-specific GK promoter LUC gene constructs and expression vectors encoding p110, PKB-WT, or myrPKB.
      Cotransfection of hepatocytes with the p110 vector together with GK-1430 LUC, containing the entire liver-specific GK promoter, enhanced Luc activity by about 2-fold (Fig. 4). Cotransfection with PKB-WT, increased luciferase activity by about 3-fold, while cotransfection with the myrPKB vector mediated an induction of Luc activity by about 4.3-fold. Similarly, cotransfection with the p110, PKB-WT, or myrPKB expression vectors stimulated promoter activity in a LUC gene construct containing the wild-type 101-bp rat GK promoter (GK-101 LUC) by about 2-fold. By contrast, the p110, PKB-WT, and myrPKB expression plasmids did not result in the activation of the GK-1430 HREm and GK-101 HREm constructs, where the P2 region (–87/–80) containing a putative binding site for HIF-1 has been mutated. These results indicate that factors binding to the putative HRE located in the proximal GK promoter could be targets of PKB signaling.
      Figure thumbnail gr4
      Fig. 4Activation of GK promoter-controlled LUC expression by overexpression of p110 and PKB in primary rat hepatocytes. A, luciferase gene constructs with rat liver-specific GK promoter regions: a wild-type 1430-bp rat GK promoter fragment (GK-1430 LUC), a 1430-bp GK promotor fragment mutated at the HRE, a 101-bp rat GK promoter fragment (GK-101 LUC), and a 101-bp promoter mutated at the HRE (GK-101 HREm LUC). The wild-type HRE is underlined, and mutated bases are shown in lowercase letters. B, hepatocytes were transiently cotransfected with the GK-1430 LUC, GK-1430 HREm LUC, GK-101 LUC, or GK-101 HREm LUC constructs and the p110, PKB-WT, and myrPKB expression vectors. After 24 h the transfected cells were cultured for another 24 h under periportal pO2. In each experiment the percentage of Luc activity was determined relative to the GK-1430 LUC control (–), which was set equal to 100%. For the determination of the fold stimulation of Luc activity the respective controls (–) were set to 1. The values represent means ± S.E. of four independent experiments. Statistics, Student's t test for paired values: *, significant differences control (–) versus +p110, control (–) versus +PKB-WT, control (–) versus +myrPKB; p ≤ 0.05.
      Stimulation of Rat GK Promoter Activity by SREBP-1a and HIF-1αThe putative GK-HRE 5′-CACGTGGT-3′ matches the HIF-1 hypoxia response element consensus sequence 5′-BACGTSSK-3′ (B = G/C/T; S = C/G; K = G/T) (
      • Kvietikova I.
      • Wenger R.H.
      • Marti H.H.
      • Gassmann M.
      ) with its core 5′-RCGTG-3′ (
      • Semenza G.L.
      • Jiang B.H.
      • Leung S.W.
      • Passantino R.
      • Concordet J.P.
      • Maire P.
      • Giallongo A.
      ) as well as the E-box consensus sequence 5′-CANNTG-3′ (n = A/G/C/T), which appears to be the common recognition site for bHLH transription factors such as SREBP-1a (
      • Foretz M.
      • Guichard C.
      • Ferre P.
      • Foufelle F.
      ,
      • Brown M.S.
      • Goldstein J.L.
      ), Myc/Max, or USF (
      • Iynedjian P.B.
      ). Since it has been shown that the expression of SREBP (
      • Foretz M.
      • Guichard C.
      • Ferre P.
      • Foufelle F.
      ,
      • Foretz M.
      • Pacot C.
      • Dugail I.
      • Lemarchand P.
      • Guichard C.
      • Le L.X.
      • Berthelier L.C.
      • Spiegelman B.
      • Kim J.B.
      • Ferre P.
      • Foufelle F.
      ) and HIF-1α (
      • Stiehl D.P.
      • Jelkmann W.
      • Wenger R.H.
      • Hellwig-Burgel T.
      ,
      • Kietzmann T.
      • Samoylenko A.
      • Roth U.
      • Jungermann K.
      ,
      • Treins C.
      • Giorgetti-Peraldi S.
      • Murdaca J.
      • Semenza G.L.
      • Van Obberghen E.
      ) can be stimulated by insulin, we examined whether SREBP and HIF-1α may regulate GK promoter activity via the putative HRE.
      Cotransfection of GK-1430 LUC with a plasmid encoding SREBP-1a enhanced the Luc activity by about 2.8-fold (Fig. 5). In contrast, the SREBP-1a vector did not stimulate promoter activity in cells transfected with either the GK-101 LUC or GK-101 HREm LUC gene constructs (Fig. 5).
      Figure thumbnail gr5
      Fig. 5Activation of GK promoter-controlled Luc expression by overexpression of SREBP-1a and HIF-1α in primary rat hepatocytes. Hepatocytes were transiently cotransfected with either the GK-1430 LUC, GK-101 LUC, or GK-101 HREm LUC constructs and SREBP-1a or HIF-1α expression vectors. After 24 h the transfected cells were cultured for another 24 h under periportal pO2. In each experiment the percentage of Luc activity was determined relative to the GK controls (–), which were set equal to 100% and for the determination of the fold stimulation of Luc activity to 1. The values represent means ± S.E. of six independent experiments. Statistics Student's t test for paired values: *, significant differences control (–) versus +SREBP-1a; control (–) versus +HIF-1α, p ≤ 0.05.
      When GK-1430 LUC was cotransfected with an expression vector for HIF-1α, luciferase activity was enhanced by about 3.3-fold. The HIF-1α mediated induction of Luc activity was also visible when the GK-101 LUC construct was cotransfected with the HIF-1α expression vector. By contrast, when GK-101 HREm LUC was cotransfected with the HIF-1α vector the HIF-1α-dependent increase in Luc activity was attenuated (Fig. 5).
      Together, these studies indicate that the activation of GK promoter LUC gene constructs by SREBP is not mediated by the GK-HRE. Instead, this site may mediate the activation of the GK promoter by HIF-1. These results also indicate that SREBP proteins also may contribute to the full effect of insulin on GK expression-just not through this element.
      Positive Modulation of Protein Binding to the HRE Sequence of the Rat GK Promoter by Hypoxia and Overexpression of PKB—To further substantiate that HIF-1 may be involved in the signaling cascade activating GK expression via the HRE downstream from PKB, we examined binding of nuclear proteins to an oligonucleotide probe containing the GK-HRE by EMSA. The GK-HRE oligonucleotide (–91/–71) was able to form two complexes with nuclear proteins prepared from primary hepatocytes. The formation of the slower migrating complex was enhanced when nuclear extracts were prepared from cells cultured under venous pO2 (mild hypoxia). By contrast, the ability of nuclear extracts to form the faster migrating complex was not altered by changes in pO2 (Fig. 6). Since the sequence of the GK-HRE was previously shown to be involved in binding of USF proteins, it was possible that the slower migrating complex might contain USF proteins. To determine whether HIF-1α and USF proteins are constituents of the observed complexes specific antibodies against HIF-1α and USF-2 were added to the EMSA reaction.
      Figure thumbnail gr6
      Fig. 6Venous pO2 and protein kinase B induce binding of HIF-1 to the rat GK-HRE. A, oligonucleotide. The HRE consensus (cons) sequence and the sense strand of the GK-HRE oligonucleotide are shown. Bases matching the consensus are underlined (B = G/C/T, S = G/C, K = G/T). B, EMSA: the 32P-labeled GK-HRE oligonucleotide was incubated with 10 μg of protein of nuclear extracts from periportal (16% O2) or perivenous (8% O2) cells or cells transfected with myrPKB vectors (see “Experimental Procedures”). For supershift analysis 1 μl of a HIF-1α antibody (a-HIF-1α), USF-2 antibody (a-USF-2), or Myc antibody (a-Myc) was added to the EMSA reaction, which was then incubated at 4 °C for 2 h. The DNA protein binding was analyzed by electrophoresis on 5% native polyacrylamide gels. Arrows indicate the HIF-1 and USF containing complexes. NE, nuclear extract; SS, supershift.
      Addition of the USF-2 antibody inhibited formation of the faster migrating (pO2-independent) complex whereas the hypoxia-inducible complex remained nearly intact. In contrast, when addition of the antibody against HIF-1α abolished the formation of the hypoxia-dependent complex and a slight supershift could now be detected, consistent with a previous study (
      • Samoylenko A.
      • Roth U.
      • Jungermann K.
      • Kietzmann T.
      ). The HIF-1α-containing nuclear protein complex was also detected by using nuclear extracts prepared from cells cultured under higher (periportal) pO2 and transfected with the PKB expression vector. The formation of this complex was again strongly suppressed in the presence of the antibody against HIF-1α and a supershift was formed. To ensure specificity of the observed results and to test whether members of the bHLH family such as Myc may participate in binding to the HRE, supershifts with Myc antibodies were performed. Addition of antibodies against Myc did not result in a supershift or inhibition of complex formation (Fig. 6). These findings indicate that the GK-HRE can be bound by HIF-1 and that this binding is positively modulated via the PI3K-PKB pathway.
      Insulin Stimulates Rat GK Promoter Activity in Hepatocytes via PI3K/PKB, HIF-1α, and HNF-4 with p300 Acting as Positive Modulator—Studies with the erythropoietin (EPO) HRE, enolase-1 HRE, and plasminogen activator inhibitor-1 HRE have shown that HIF-1 binding activity is stimulated by insulin (
      • Zelzer E.
      • Levy Y.
      • Kahana C.
      • Shilo B.Z.
      • Rubinstein M.
      • Cohen B.
      ,
      • Kietzmann T.
      • Samoylenko A.
      • Roth U.
      • Jungermann K.
      ). Furthermore, it was shown that downstream of the EPO-HRE, the binding of HNF-4 to an HNF-4-responsive element enhances HIF-1-mediated EPO gene activation (
      • Madan A.
      • Curtin P.T.
      ,
      • Galson D.L.
      • Tsuchiya T.
      • Tendler D.S.
      • Huang L.E.
      • Ren Y.
      • Ogura T.
      • Bunn H.F.
      ). HNF-4 is known to interact with the β-subunit of HIF-1 and both, the α- and β-subunits of HIF-1 are able to bind the transcriptional activator p300 (
      • Huang L.E.
      • Ho V.
      • Arany Z.
      • Krainc D.
      • Galson D.
      • Tendler D.
      • Livingston D.M.
      • Bunn H.F.
      ,
      • Arany Z.
      • Huang L.E.
      • Eckner R.
      • Bhattacharya S.
      • Jiang C.
      • Goldberg M.A.
      • Bunn H.F.
      • Livingston D.M.
      ,
      • Kallio P.J.
      • Okamoto K.
      • O'Brien S.
      • Carrero P.
      • Makino Y.
      • Tanaka H.
      • Poellinger L.
      ). We recently identified an HNF-4-responsive element located downstream to the GK-HRE, implicating a similar mechanism of transcriptional regulation as with the EPO gene (
      • Roth U.
      • Jungermann K.
      • Kietzmann T.
      ).
      To investigate the role of HIF-1α, HNF-4α, and p300 in insulin-regulated GK expression, we transfected primary rat hepatocytes with either the wild-type GK-1430 LUC construct, the GK construct lacking the HRE (GK-1430HREm), or the construct lacking the HNF-4 binding site (GK-1430HNFm) together with the expression vectors for HIF-1α, HNF-4α, and p300 alone or in combination.
      In hepatocytes transfected with GK-1430 LUC, treatment with insulin resulted in an about 2-fold enhancement of Luc activity (Fig. 7). Cotransfecion of GK-1430 with HIF-1α increased Luc activity by about 2-fold and insulin increased Luc activity further to about 4-fold. The enhancement of Luc activity by HNF-4 was about 4.5-fold, consistent with a previous study (
      • Jiang B.H.
      • Jiang G.
      • Zheng J.Z.
      • Lu Z.
      • Hunter T.
      • Vogt P.K.
      ), and insulin now increased Luc activity by about 7-fold. Transfection of GK-1430 with HIF-1α and HNF-4α together increased Luc activity by about 6-fold, and insulin treatment elicited a 10-fold increase in Luc activity under these conditions (Fig. 7).
      Figure thumbnail gr7
      Fig. 7Activation of GK promoter-controlled LUC expression by overexpression of HIF-1, HNF-4α, and p300 in primary rat hepatocytes: regulation by insulin. A, luciferase gene constructs with 1430-bp rat liver-specific GK promoter regions: a wild-type 1430-bp rat GK promoter fragment (GK-1430 LUC) and constructs with mutated HRE (GK-1430-HREm) or mutated HNF-4 binding site (GK-1430-HNFm) are shown. The wild-type HRE and HNF-4 element are underlined; mutated bases in the respective constructs are indicated by lowercase letters. B, hepatocytes were transiently cotransfected with either the GK-1430 LUC, GK-1430-HREm LUC, and GK-1430-HNFm LUC constructs and HIF-1α, HNF-4α, p300WT, and p300mut (containing the Ser1834 → Ala mutation in the PKB phosphorylation site) expression vectors alone or in combination. After 24 h the transfected cells were cultured for another 24 h under periportal pO2 and as indicated the cells were treated for 24 h with 100 nm insulin (black bars). In each experiment the Luc activity was determined relative to the GK-1430 LUC, GK-1430-HREm LUC, or GK-1430-HNFm LUC control (–), which was set equal to 1. The values represent means ± S.E. of six independent experiments. Statistics Student's t test for paired values: *, significant differences, –insulin versus +insulin; p ≤ 0.05.
      Cotransfection of GK-1430 with p300WT mediated an increase in Luc activity by about 4-fold, which was enhanced upon insulin treatment to about 7-fold. The transfection of GK-1430 with HIF-1α, HNF-4α, and p300WT increased Luc activity by about 8-fold. Treatment with insulin now maximally increased Luc activity by about 20-fold. Since cooperative effects of p300 with HIF-1 involve the C/H1 domain of p300, we also sought to interrupt the cooperativity by cotransfection of a p300 mutant lacking the C/H1 domain (p300Δ C/H1). When GK-1430 LUC was transfected together with HIF-1α, HNF4α, and the vector encoding p300Δ C/H1, the cooperative induction of GK promoter activity was abolished (data not shown). This result indicates that interactions with this region of p300 are required for cooperative activation of the GK-1430 promoter by HIF and HNF4α, similar to previous studies with the EPO gene (
      • Huang L.E.
      • Ho V.
      • Arany Z.
      • Krainc D.
      • Galson D.
      • Tendler D.
      • Livingston D.M.
      • Bunn H.F.
      ,
      • Arany Z.
      • Huang L.E.
      • Eckner R.
      • Bhattacharya S.
      • Jiang C.
      • Goldberg M.A.
      • Bunn H.F.
      • Livingston D.M.
      ).
      While insulin has been shown to enhance HIF-1α protein levels, it is so far unknown whether PKB might modulate cooperative actions of HIF and HNF-4. Since neither HIF-1α or HNF-4 contain a predicted PKB phosphorylation sequence we considered the possibility that PKB might promote cooperativity between HIF-1 and HNF-4 via the coactivator p300, which contains a PKB phosphorylation site at Ser1834 (
      • Guo S.
      • Cichy S.B.
      • He X.
      • Yang Q.
      • Ragland M.
      • Ghosh A.K.
      • Johnson P.F.
      • Unterman T.G.
      ). To determine whether phosphorylation of serine 1834 may be critical for the ability of insulin to regulate GK promoter activity, we performed cotransfection studies with the –1430 GK promoter LUC constructs together with vectors allowing expression of HIF-1α, HNF4, and p300mut, in which serine 1834 is replaced with alanine, a neutral amino acid that is not susceptible to phosphorylation.
      When GK-1430 LUC was transfected together with HIF-1α, HNF4α, and the vector encoding the mutated unphosphorylatable p300, Luc activity was enhanced by about 7-fold and treatment with insulin had only a limited effect, thus resulting in about the same values as with HIF-1α and HNF-4α alone (Fig. 7).
      Mutation of the HRE abolished the ability of insulin to stimulate GK promoter activity, and neither HIF-1α, nor p300WT alone or in combination could enhance Luc activity significantly, whereas the HNF-4-mediated induction persisted similarly as with the wild type GK construct (Fig. 7).
      In contrast, mutation of the HNF-4 binding site diminished the ability of HIF-1α and p300 to stimulate promoter activity but did not disrupt the effect of insulin. No increase in Luc activity was measurable by using the HNF-4α expression vector, and the cooperative induction of Luc activity by HIF-1α, HNF-4α, and p300 as observed with the wild-type GK promoter was completely abolished by using GK-1430HNFm (Fig. 7).
      Together, these results indicate that the ability of insulin to stimulate GK promoter activity involves the effects of HIF-1 at the HRE, while the cooperative interactions between HIF-1, HNF-4, and p300 phosphorylated at serine 1834 appears to be important for a robust induction.

      DISCUSSION

      In this study, we found that the expression of the GK gene can be enhanced by insulin via the PI3K/PKB pathway in primary rat hepatocytes. To our knowledge these studies provide the first report that insulin is able to activate the liver-specific GK promoter through the hypoxia response element in the proximal GK promoter and that interactions between HIF-1, HNF-4, and p300 contribute to this effect.
      Involvement of the PI3K/PKB Pathway in the Regulation of Gene Expression—The results of this study which show that insulin can activate GK expression via PI3K/PKB are in line with another study in rat hepatocytes showing that the insulin-induced increase in GK mRNA abundance was completely abolished by PI3K inhibitors such as wortmannin and LY294002 (
      • Iynedjian P.B.
      • Roth R.A.
      • Fleischmann M.
      • Gjinovci A.
      ). The involvement of PKB in regulating GK expression also has been shown by transducing primary rat hepatocytes with an adenovirus vector allowing an 4-hydroxytamoxifen-activable expression of PKB. The 4-hydroxytamoxifen treatment of the transduced cells resulted in a GK mRNA induction similar to the insulin-induced mRNA accumulation (
      • Iynedjian P.B.
      • Roth R.A.
      • Fleischmann M.
      • Gjinovci A.
      ). This regulation does not appear to be hepatocyte-specific, since GK gene expression also is stimulated by insulin in pancreatic β cells via involvement of the insulin receptor type B, PI3K, and PKB (
      • Leibiger B.
      • Leibiger I.B.
      • Moede T.
      • Kemper S.
      • Kulkarni R.N.
      • Kahn C.R.
      • de-Vargas L.M.
      • Berggren P.O.
      ). However, those studies did not provide information about the transcription factors involved in this regulation of GK expression.
      In addition to GK gene, the PI3K/PKB pathway has been shown to be involved in the insulin-dependent regulation of several other genes such as SREBP-1 (
      • Fleischmann M.
      • Iynedjian P.B.
      ), GLUT-1 (
      • Barthel A.
      • Okino S.T.
      • Liao J.
      • Nakatani K.
      • Li J.
      • Whitlock J.-P.J.
      • Roth R.A.
      ), IG-FBP-1 (
      • Yeagley D.
      • Guo S.
      • Unterman T.
      • Quinn P.G.
      ), G-6-Pase (
      • Schmoll D.
      • Walker K.S.
      • Alessi D.R.
      • Grempler R.
      • Burchell A.
      • Guo S.
      • Walther R.
      • Unterman T.G.
      ), and FAS (
      • Wang D.
      • Sul H.S.
      ). As with the GK gene, the transcription factors mediating the effects of insulin via PKB on the expression of SREBP and GLUT-1 are not yet known, whereas USF has been implicated in mediating the effects of insulin on FAS gene expression (
      • Wang D.
      • Sul H.S.
      ). FOXO forkhead proteins are thought to mediate negative effects of insulin on the expression of IGFBP-1 and G-6-Pase (
      • Guo S.
      • Cichy S.B.
      • He X.
      • Yang Q.
      • Ragland M.
      • Ghosh A.K.
      • Johnson P.F.
      • Unterman T.G.
      ,
      • Schmoll D.
      • Walker K.S.
      • Alessi D.R.
      • Grempler R.
      • Burchell A.
      • Guo S.
      • Walther R.
      • Unterman T.G.
      ,
      • Nakae J.
      • Biggs III, W.H.
      • Kitamura T.
      • Cavenee W.K.
      • Wright C.V.
      • Arden K.C.
      • Accili D.
      ).
      Involvement of Hypoxia-inducible Factor 1 in the Insulin-mediated Gene Expression—In the present study, cotransfection experiments with plasmids allowing the expression of the p110 catalytic subunit of PI3K, the wild-type PKB, or constitutively active myristoylated PKB (myrPKB) showed that the HRE located in the proximal GK promoter participates in PI3K/PKB-dependent GK gene expression (Fig. 4). Enhanced interaction of HIF-1α proteins at the GK-HRE was demonstrated by EMSA using nuclear extracts from cells cultured under perivenous pO2 (mild hypoxia) or under periportal pO2 but transfected with PKB expression plasmids.
      The above mentioned findings are in line with studies showing that insulin enhances the binding of HIF-1 to HREs in HepG2 hepatoma cells, L6 rat skeletal muscle myoblasts, and primary hepatocytes (
      • Zelzer E.
      • Levy Y.
      • Kahana C.
      • Shilo B.Z.
      • Rubinstein M.
      • Cohen B.
      ,
      • Stiehl D.P.
      • Jelkmann W.
      • Wenger R.H.
      • Hellwig-Burgel T.
      ,
      • Kietzmann T.
      • Samoylenko A.
      • Roth U.
      • Jungermann K.
      ). Furthermore, transfection studies with an HIF-1-dependent Luc reporter gene containing HRE sequences demonstrate an insulin-dependent enhancement of Luc activity, which also is in line with the present findings. In addition, the insulin-induced enhancement of HIF-1α protein levels is inhibited in a dose-dependent manner by specific PI3K inhibitors such as LY294002 and wortmannin in prostate carcinoma-derived cell lines (
      • Jiang B.H.
      • Jiang G.
      • Zheng J.Z.
      • Lu Z.
      • Hunter T.
      • Vogt P.K.
      ) and hepatocytes (
      • Kietzmann T.
      • Samoylenko A.
      • Roth U.
      • Jungermann K.
      ), implicating the involvement of a PI3K-dependent pathway. Insulin appeared to act via a translational-dependent pathway, since cycloheximide inhibited the insulin-dependent enhancement of HIF-1α protein levels (
      • Treins C.
      • Giorgetti-Peraldi S.
      • Murdaca J.
      • Semenza G.L.
      • Van Obberghen E.
      ). Additionally, the HIF-1-dependent gene transcription was inhibited by overexpression of a dominant negative PI3K or PKB, whereas these inhibitory actions could be counteracted by a constitutively active PKB (
      • Zhong H.
      • Chiles K.
      • Feldser D.
      • Laughner E.
      • Hanrahan C.
      • Georgescu M.M.
      • Simons J.W.
      • Semenza G.L.
      ,
      • Jiang B.H.
      • Jiang G.
      • Zheng J.Z.
      • Lu Z.
      • Hunter T.
      • Vogt P.K.
      ). Together, these studies indicate that signaling via the PI3K/PKB pathway may contribute to the induction of HIF-1α at both the translational and post-translational level.
      Besides insulin, other hormones, growth factors, and clotting factors, which are able to activate the PI3K/PKB pathway such as insulin-like growth factor (
      • Zelzer E.
      • Levy Y.
      • Kahana C.
      • Shilo B.Z.
      • Rubinstein M.
      • Cohen B.
      ,
      • Feldser D.
      • Agani F.
      • Iyer N.V.
      • Pak B.
      • Ferreira G.
      • Semenza G.L.
      ), angiotensin II (
      • Richard D.E.
      • Berra E.
      • Pouyssegur J.
      ), platelet-derived growth factor (
      • Richard D.E.
      • Berra E.
      • Pouyssegur J.
      ,
      • Gorlach A.
      • Diebold I.
      • Schini-Kerth V.B.
      • Berchner-Pfannschmidt U.
      • Roth U.
      • Brandes R.P.
      • Kietzmann T.
      • Busse R.
      ), thrombin (
      • Gorlach A.
      • Diebold I.
      • Schini-Kerth V.B.
      • Berchner-Pfannschmidt U.
      • Roth U.
      • Brandes R.P.
      • Kietzmann T.
      • Busse R.
      ), and tumor necrosis factor α (
      • Hellwig-Burgel T.
      • Rutkowski K.
      • Metzen E.
      • Fandrey J.
      • Jelkmann W.
      ), have been shown to enhance the HIF-1α levels and HIF-1 activity independent from the oxygen tension in different cells.
      While increased binding of USF to the –65/–60 E-box in the FAS promoter contributed to the ability of insulin to stimulate FAS expression in 3T3-L1 fat cells through a PI3K and PKB-dependent mechanism (
      • Wang D.
      • Sul H.S.
      ), we did not observe any increase in the binding of USF to the GK-HRE with the nuclear extracts from hypoxic cells or from PKB-transfected cells. This argues against a dominant role of USF in the insulin- and PI3K/PKB-mediated GK promoter activation (Fig. 6).
      Another possible binding partner at the GK-HRE could be SREBP. SREBP-1 is induced by insulin and can bind to E-box motifs (
      • Foretz M.
      • Guichard C.
      • Ferre P.
      • Foufelle F.
      ,
      • Brown M.S.
      • Goldstein J.L.
      ,
      • Kim J.B.
      • Sarraf P.
      • Wright M.
      • Yao K.M.
      • Mueller E.
      • Solanes G.
      • Lowell B.B.
      • Spiegelman B.M.
      ). In the liver, the SREBP proteins are represented mainly by SREBP-1a and -1c, respectively (
      • Shimano H.
      • Horton J.D.
      • Shimomura I.
      • Hammer R.E.
      • Brown M.S.
      • Goldstein J.L.
      ,
      • Shimomura I.
      • Shimano H.
      • Horton J.D.
      • Goldstein J.L.
      • Brown M.S.
      ). It was shown that in primary rat hepatocytes the expression of SREBP-1c is positively controlled by insulin and negatively controlled by glucagon and cAMP. The adenovirus-mediated transduction of a dominant negative form of SREBP-1c into rat hepatocytes has shown that SREBP can be involved in glucose-dependent stimulation of the type L pyruvate kinase, FAS, Spot 14, and acetyl-coenzyme A carboxylase gene expression (
      • Foretz M.
      • Guichard C.
      • Ferre P.
      • Foufelle F.
      ). Additionally, this dominant negative form of SREBP inhibited the insulin-dependent GK expression (
      • Foretz M.
      • Guichard C.
      • Ferre P.
      • Foufelle F.
      ). This suggested that SREBP may contribute to insulin-regulated GK gene expression. Nevertheless, we observed that SREBP does not regulate GK expression via an element within the first 101 bp of the GK promoter as shown in cotransfection studies (Fig. 5). However, this does not exclude the possibility that SREBP proteins may contribute to GK regulation through other elements under different conditions.
      Interestingly, PKB and related PI3K-dependent kinases have been shown to phosphorylate transcription factors of the Foxo forkhead family such as FKHR, AFX, and FKHRL at specific serine and threonine residues in vitro and in intact cells. This phosphorylation in turn results in inhibition and in loss of transcriptional activation of target genes, including IG-FBP-1 (
      • Yeagley D.
      • Guo S.
      • Unterman T.
      • Quinn P.G.
      ) and G-6-Pase (
      • Schmoll D.
      • Walker K.S.
      • Alessi D.R.
      • Grempler R.
      • Burchell A.
      • Guo S.
      • Walther R.
      • Unterman T.G.
      ). The liver-specific GK promoter does not contain putative FKHR binding sites, supporting the concept that other mechanisms, such as HRE binding of HIF-1, are involved in the insulin- and PI3K/PKB-regulated GK expression.
      Cooperation of HIF-1, HNF-4, and CBP/p300 at the GK Promoter—This study shows that HIF-1α, HNF-4, and p300 function cooperatively to enhance the activity of the liver-specific GK-1430 promoter (Fig. 7). Interestingly, HIF-1α, HNF-4, and p300WT cotransfected cells show the most robust induction of Luc activity upon treatment with insulin (Fig. 7). This positive effect of insulin could be abolished by wortmannin (data not shown), which is in line with our finding that an expression vector encoding a mutant form of p300 in which a PKB phosphorylation site was mutated, abrogated this insulin-dependent activation of Luc activity (Fig. 7). The cooperative effects of HIF-1α, HNF-4, and p300 appear to be similar as proposed for the EPO gene where, under hypoxia, heterodimeric HIF-1 binds to an HRE, while HNF-4 binds to a direct repeat down-stream of the HRE. The binding of HNF-4 is crucial for an enhanced hypoxic response, and recruitment of the transcriptional coactivator p300 by HIF-1 and HNF-4 further enhanced the EPO expression (
      • Huang L.E.
      • Ho V.
      • Arany Z.
      • Krainc D.
      • Galson D.
      • Tendler D.
      • Livingston D.M.
      • Bunn H.F.
      ,
      • Arany Z.
      • Huang L.E.
      • Eckner R.
      • Bhattacharya S.
      • Jiang C.
      • Goldberg M.A.
      • Bunn H.F.
      • Livingston D.M.
      ).
      The specific interaction of these three factors in the regulation of GK and EPO expression also is supported by results from yeast two-hybrid studies and pull-down assays with glutathione S-transferase fusion proteins containing the first cysteine/histidine-rich p300 region (CH1) and radioactively labeled HIF-1α (
      • Arany Z.
      • Huang L.E.
      • Eckner R.
      • Bhattacharya S.
      • Jiang C.
      • Goldberg M.A.
      • Bunn H.F.
      • Livingston D.M.
      ). The p300 CH1 domain serves as a scaffold for folding of the HIF-1α C-terminal transactivation domain, which forms a vise-like clamp on the CH1 domain (
      • Freedman S.J.
      • Sun Z.Y.
      • Poy F.
      • Kung A.L.
      • Livingston D.M.
      • Wagner G.
      • Eck M.J.
      ). Upon binding to the CH1 domain of p300, three short helices are formed in the HIF-1α C-terminal transactivation domain which are stabilized by intermolecular interactions (
      • Dames S.A.
      • Martinez-Yamout M.
      • De Guzman R.N.
      • Dyson H.J.
      • Wright P.E.
      ). Several hydrophobic residues, Leu795, Cys800, Ile802, Leu808, Leu814, Leu815, Leu818, Leu822, within the HIF-1α C-terminal transactivation domain are thereby critical for binding (
      • Gu J.
      • Milligan J.
      • Huang L.E.
      ,
      • Ruas J.L.
      • Poellinger L.
      • Pereira T.
      ). Likewise, Leu344, Leu345, Cys388, and Cys393 in the C/H1 domain of p300 have also been shown to be essential for functional interaction (
      • Gu J.
      • Milligan J.
      • Huang L.E.
      ). Whether phosphorylation of serine 1834 within the Q domain of p300 is also critical for direct interaction with HIF-1 and/or HNF-4, or whether other factors also required for effective interaction between these proteins and cooperative function, was not addressed in the present investigation. Additional studies will be required to address this question.
      The interaction between HIF-1, cAMP-responsive element-binding protein (CREB) together with CBP/p300 also was found to be necessary for the robust hypoxia-dependent activation of the lactate dehydrogenase A gene (
      • Ebert B.L.
      • Bunn H.F.
      ). Thus, besides the EPO gene and the lactate dehydrogenase A gene, this study with the GK gene represents another example in which a multifactorial HIF-containing transcriptional complex requires p300 for optimal activation.
      Taken together, the results of the present study indicated that insulin-stimulated GK gene expression is mediated through the PI3K/PKB pathway. PKB activation results in the enhancement of HIF-1α protein levels and thus formation of active HIF-1 dimers. In addition, phosphorylation of p300, which interacts with HIF-1 and HNF-4, also may contribute to the stimulation of GK gene expression.

      Acknowledgments

      We thank Professor P. B. Iynedjian (Division of Clinical and Diabetes Research, University of Geneva School of Medicine, Geneva, Switzerland) and Professor F. Sladek (Department of Cell Biology and Neuroscience, University of California, Riverside, CA) for the gifts of plasmids.

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