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• W2002854251 abstract "Bile acid synthesis and pool size increases in diabetes, whereas insulin inhibits bile acid synthesis. The objective of this study is to elucidate the mechanism of insulin regulation of cholesterol 7α-hydroxylase gene expression in human hepatocytes. Real-time PCR assays showed that physiological concentrations of insulin rapidly stimulated cholesterol 7α-hydroxylase (CYP7A1) mRNA expression in primary human hepatocytes but inhibited CYP7A1 expression after extended treatment. The insulin-regulated forkhead box O1 (FoxO1) and steroid regulatory element-binding protein-1c (SREBP-1c) strongly inhibited hepatocyte nuclear factor 4α and peroxisome proliferator-activated receptor γ coactivator-1α trans-activation of the CYP7A1 gene. FoxO1 binds to an insulin response element in the rat CYP7A1 promoter, which is not present in the human CYP7A1 gene. Insulin rapidly phosphorylates and inactivates FoxO1, whereas insulin induces nuclear SREBP-1c expression in human primary hepatocytes. Chromatin immunoprecipitation assay shows that insulin reduced FoxO1 and peroxisome proliferators-activated receptor γ-coactivator-1α but increased SREBP-1c recruitment to CYP7A1 chromatin. We conclude that insulin has dual effects on human CYP7A1 gene transcription; physiological concentrations of insulin rapidly inhibit FoxO1 activity leading to stimulation of the human CYP7A1 gene, whereas prolonged insulin treatment induces SREBP-1c, which inhibits human CYP7A1 gene transcription. Insulin may play a major role in the regulation of bile acid synthesis and dyslipidemia in diabetes. Bile acid synthesis and pool size increases in diabetes, whereas insulin inhibits bile acid synthesis. The objective of this study is to elucidate the mechanism of insulin regulation of cholesterol 7α-hydroxylase gene expression in human hepatocytes. Real-time PCR assays showed that physiological concentrations of insulin rapidly stimulated cholesterol 7α-hydroxylase (CYP7A1) mRNA expression in primary human hepatocytes but inhibited CYP7A1 expression after extended treatment. The insulin-regulated forkhead box O1 (FoxO1) and steroid regulatory element-binding protein-1c (SREBP-1c) strongly inhibited hepatocyte nuclear factor 4α and peroxisome proliferator-activated receptor γ coactivator-1α trans-activation of the CYP7A1 gene. FoxO1 binds to an insulin response element in the rat CYP7A1 promoter, which is not present in the human CYP7A1 gene. Insulin rapidly phosphorylates and inactivates FoxO1, whereas insulin induces nuclear SREBP-1c expression in human primary hepatocytes. Chromatin immunoprecipitation assay shows that insulin reduced FoxO1 and peroxisome proliferators-activated receptor γ-coactivator-1α but increased SREBP-1c recruitment to CYP7A1 chromatin. We conclude that insulin has dual effects on human CYP7A1 gene transcription; physiological concentrations of insulin rapidly inhibit FoxO1 activity leading to stimulation of the human CYP7A1 gene, whereas prolonged insulin treatment induces SREBP-1c, which inhibits human CYP7A1 gene transcription. Insulin may play a major role in the regulation of bile acid synthesis and dyslipidemia in diabetes. The liver plays a central role in lipid metabolism and maintaining whole body lipid homeostasis, which is dysregulated in metabolic syndrome (syndrome X), obesity, and diabetes (1Reaven G. Abbasi F. McLaughlin T. Recent Prog. Horm. Res. 2004; 59: 207-223Crossref PubMed Scopus (270) Google Scholar). Bile acid synthesis in the liver is the predominant pathway for cholesterol catabolism and is regulated by cholesterol 7α-hydroxylase (CYP7A1) 2The abbreviations used are: CYP7A1, cholesterol 7α-hydroxylase; ChIP, chromatin immunoprecipitation; FoxO1, forkhead box O1; HNF4α, hepatocyte nuclear factor 4α; IRE, insulin response element; rIRE, rat IRE; PEPCK, phosphoenolpyruvate carboxykinase; PGC-1α, peroxisome proliferators-activated receptor γ-coactivator-1α; SRE, sterol response element; SREBP, SRE-binding protein; LXRα, liver orphan receptor α; EMSA, electrophoretic mobility shift assay; HA, hemagglutinin; Luc, luciferase. 2The abbreviations used are: CYP7A1, cholesterol 7α-hydroxylase; ChIP, chromatin immunoprecipitation; FoxO1, forkhead box O1; HNF4α, hepatocyte nuclear factor 4α; IRE, insulin response element; rIRE, rat IRE; PEPCK, phosphoenolpyruvate carboxykinase; PGC-1α, peroxisome proliferators-activated receptor γ-coactivator-1α; SRE, sterol response element; SREBP, SRE-binding protein; LXRα, liver orphan receptor α; EMSA, electrophoretic mobility shift assay; HA, hemagglutinin; Luc, luciferase. (2Chiang J.Y.L. Am. J. Physiol. Gastrointest. Liver Physiol. 2003; 284: 349-356Crossref PubMed Scopus (146) Google Scholar). Bile acids are physiological agents that facilitate biliary cholesterol excretion, intestinal absorption of nutrients, and disposal of toxic metabolites. Bile acids are also signaling molecules that activate bile acid receptors to regulate bile acid synthesis and glucose metabolism (2Chiang J.Y.L. Am. J. Physiol. Gastrointest. Liver Physiol. 2003; 284: 349-356Crossref PubMed Scopus (146) Google Scholar). In vivo studies show that bile acid pool and excretion increase in diabetic human patients (3Bennion L.J. Grundy S.M. N. Engl. J. Med. 1977; 296: 1365-1371Crossref PubMed Scopus (181) Google Scholar, 4Andersen E. Karlaganis G. Sjovall J. Eur. J. Clin. Investig. 1988; 18: 166-172Crossref PubMed Scopus (50) Google Scholar) and in experimental diabetic animals (5Hassan A.S. Ravi Subbiah M.T. Thiebert P. Proc. Soc. Exp. Biol. Med. 1980; 164: 449-452Crossref PubMed Scopus (37) Google Scholar, 6Villanueva G.R. Herreros M. Perez-Barriocanal F. Bolanos J.P. Bravo P. Marin J.J. J. Lab. Clin. Med. 1990; 115: 441-448PubMed Google Scholar). Insulin treatment restores bile acid pool and synthesis to the normal levels. It has been reported that physiological concentrations of insulin (1.4–14 nm) inhibit bile acid synthesis by down-regulation of CYP7A1 (7Wang D.P. Stroup D. Marrapodi M. Crestani M. Galli G. Chiang J.Y. J. Lipid Res. 1996; 37: 1831-1841Abstract Full Text PDF PubMed Google Scholar, 8Crestani M. Sadeghpour A. Stroup D. Galli G. Chiang J.Y. J. Lipid Res. 1998; 39: 2192-2200Abstract Full Text Full Text PDF PubMed Google Scholar, 9Twisk J. Hoekman M.F. Lehmann E.M. Meijer P. Mager W.H. Princen H.M. Hepatology. 1995; 21: 501-510PubMed Google Scholar), sterol 12α-hydroxylase (CYP8B1) (10Ishida H. Yamashita C. Kuruta Y. Yoshida Y. Noshiro M. J. Biochem. (Tokyo). 2000; 127: 57-64Crossref PubMed Scopus (47) Google Scholar), and sterol 27-hydroxylase (CYP27A1) gene transcription (9Twisk J. Hoekman M.F. Lehmann E.M. Meijer P. Mager W.H. Princen H.M. Hepatology. 1995; 21: 501-510PubMed Google Scholar). We have previously reported that insulin plays a dominant role in the inhibition of CYP7A1 gene transcription (7Wang D.P. Stroup D. Marrapodi M. Crestani M. Galli G. Chiang J.Y. J. Lipid Res. 1996; 37: 1831-1841Abstract Full Text PDF PubMed Google Scholar, 8Crestani M. Sadeghpour A. Stroup D. Galli G. Chiang J.Y. J. Lipid Res. 1998; 39: 2192-2200Abstract Full Text Full Text PDF PubMed Google Scholar). How insulin regulates human CYP7A1 and what factors mediate the insulin effects remain unknown.Insulin is known to induce more than 100 genes but inhibit only a few hepatic genes involved in glucose metabolism (11Foufelle F. Ferre P. Biochem. J. 2002; 366: 377-391Crossref PubMed Scopus (397) Google Scholar). The consensus sequence of negative insulin response element (IRE), T(G/A)TTT(T/G)(G/T), has been identified in promoters of phosphoenolpyruvate carboxykinase (PEPCK), glucose-6-phosphatase, insulin-like growth factor binding protein-1, tyrosine aminotransferase, and apolipoprotein CIII genes (12O'Brien R.M. Granner D.K. Physiol. Rev. 1996; 76: 1109-1161Crossref PubMed Scopus (435) Google Scholar). These IREs are known to bind hepatocyte nuclear factor 3α (HNF3α or FoxA1), but HNF3α does not mediate insulin action (12O'Brien R.M. Granner D.K. Physiol. Rev. 1996; 76: 1109-1161Crossref PubMed Scopus (435) Google Scholar). Recently forkhead box O1 (FoxO1), a mammalian homolog of Caenorhabditis elegans DAF-16 has been identified as an insulin-regulated transcription factor that plays a critical role in mediating insulin inhibition of the PEPCK, glucose-6-phosphatase, insulin-like growth factor binding protein-1, and apolipoprotein CIII genes (13Hall R.K. Yamasaki T. Kucera T. Waltner-Law M. O'Brien R. Granner D.K. J. Biol. Chem. 2000; 275: 30169-30175Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, 14Schmoll D. Walker K.S. Alessi D.R. Grempler R. Burchell A. Guo S. Walther R. Unterman T.G. J. Biol. Chem. 2000; 275: 36324-36333Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar, 15Puigserver P. Rhee J. Donovan J. Walkey C.J. Yoon J.C. Oriente F. Kitamura Y. Altomonte J. Dong H. Accili D. Spiegelman B.M. Nature. 2003; 423: 550-555Crossref PubMed Scopus (1155) Google Scholar, 16Nakae J. Kitamura T. Silver D.L. Accili D. J. Clin. Investig. 2001; 108: 1359-1367Crossref PubMed Scopus (498) Google Scholar, 17Altomonte J. Cong L. Harbaran S. Richter A. Xu J. Meseck M. Dong H.H. J. Clin. Investig. 2004; 114: 1493-1503Crossref PubMed Scopus (231) Google Scholar). Akt/protein kinase B, a down-stream serine kinase of the insulin-signaling pathway, phosphorylates FoxO1 (18Nakae J. Park B.C. Accili D. J. Biol. Chem. 1999; 274: 15982-15985Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar). The phosphorylated FoxO1 is excluded from the nucleus and degraded by the ubiquitin proteasome pathway, which results in inhibition of FoxO1 target genes (19Zhang X. Gan L. Pan H. Guo S. He X. Olson S.T. Mesecar A. Adam S. Unterman T.G. J. Biol. Chem. 2002; 277: 45276-45284Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar, 20Matsuzaki H. Daitoku H. Hatta M. Tanaka K. Fukamizu A. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 11285-11290Crossref PubMed Scopus (422) Google Scholar). FoxO1 exerts a positive effect by binding to its target gene promoters (15Puigserver P. Rhee J. Donovan J. Walkey C.J. Yoon J.C. Oriente F. Kitamura Y. Altomonte J. Dong H. Accili D. Spiegelman B.M. Nature. 2003; 423: 550-555Crossref PubMed Scopus (1155) Google Scholar). FoxO1 also regulates genes as a DNA binding-independent co-activator or co-repressor of many transcription factors (21Hirota K. Daitoku H. Matsuzaki H. Araya N. Yamagata K. Asada S. Sugaya T. Fukamizu A. J. Biol. Chem. 2003; 278: 13056-13060Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Dysregulation of FoxO1 expression and function leads to hyperglycemia and hypertriglyceridemia in the mouse models of type I and type II diabetes (17Altomonte J. Cong L. Harbaran S. Richter A. Xu J. Meseck M. Dong H.H. J. Clin. Investig. 2004; 114: 1493-1503Crossref PubMed Scopus (231) Google Scholar).Insulin stimulates the activity of sterol regulatory element-binding proteins (SREBPs), which are key regulators of lipid homeostasis and insulin actions (22Foretz M. Pacot C. Dugail I. Lemarchand P. Guichard C. Le Liepvre X. Berthelier-Lubrano C. Spiegelman B. Kim J.B. Ferre P. Foufelle F. Mol. Cell. Biol. 1999; 19: 3760-3768Crossref PubMed Scopus (451) Google Scholar). The predominant SREBP isoform, SREBP-1c in liver and adipose tissues, preferentially induces genes in fatty acid and triglyceride synthesis (23Horton J.D. Goldstein J.L. Brown M.S. J. Clin. Investig. 2002; 109: 1125-1131Crossref PubMed Scopus (3685) Google Scholar). A SREBP precursor (125 kDa) forms a complex with SREBP cleavage-activating protein in the endoplasmic reticulum. Insulin inducing gene-1 and -2a (Insig) anchor the SREBP·SREBP cleavage-activating protein complex to the endoplasmic reticulum membrane (24Engelking L.J. Kuriyama H. Hammer R.E. Horton J.D. Brown M.S. Goldstein J.L. Liang G. J. Clin. Investig. 2004; 113: 1168-1175Crossref PubMed Scopus (217) Google Scholar). When cellular sterol levels are low, Insigs release the SREBP cleavage-activating protein (SCAP)·SREBP complex and allow SCAP to escort SREBPs to the Golgi apparatus. Two sterol-regulated proteases act sequentially to release the N-terminal basic-helix-loop-helix leucine zipper domain. The mature SREBP (68 kDa) enters the nucleus, binds to the sterol regulatory element (SRE), and induces target gene transcription. Insulin stimulates liver orphan receptor α (LXRα) which induces SREBP-1c gene expression (25Tobin K.A. Ulven S.M. Schuster G.U. Steineger H.H. Andresen S.M. Gustafsson J.A. Nebb H.I. J. Biol. Chem. 2002; 277: 10691-10697Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 26Repa J.J. Liang G. Ou J. Bashmakov Y. Lobaccaro J.M. Shimomura I. Shan B. Brown M.S. Goldstein J.L. Mangelsdorf D.J. Genes Dev. 2000; 14: 2819-2830Crossref PubMed Scopus (1401) Google Scholar). Insulin also promotes SREBP-1c cleavage and increases insulin-stimulated lipogenesis by inhibition of Insig-2a expression in livers (11Foufelle F. Ferre P. Biochem. J. 2002; 366: 377-391Crossref PubMed Scopus (397) Google Scholar, 24Engelking L.J. Kuriyama H. Hammer R.E. Horton J.D. Brown M.S. Goldstein J.L. Liang G. J. Clin. Investig. 2004; 113: 1168-1175Crossref PubMed Scopus (217) Google Scholar). Studies have shown that insulin-dependent proteolytic cleavage is crucial and sufficient for SREBP-1c activation (27Hegarty B.D. Bobard A. Hainault I. Ferre P. Bossard P. Foufelle F. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 791-796Crossref PubMed Scopus (160) Google Scholar).In this study we investigated the insulin regulation of CYP7A1 gene transcription in human hepatocytes. Results indicate that short-term treatment of physiological concentrations of insulin rapidly induces, whereas prolonged treatment of insulin represses human CYP7A1 gene expression. This dual effect of insulin action may be mediated by two insulin sensitive transcription factors, FoxO1 and SREBP-1c. This study provides new insights into how bile acid synthesis is regulated by insulin under normal physiological conditions and how dysregulation of bile acid homeostasis may contribute to glucose and lipid abnormalities in diabetes.EXPERIMENTAL PROCEDURESCell Culture—The human hepatoblastoma cell line, HepG2, was purchased from American Type Culture Collection (Manassas, VA). The cells were cultured in Dulbecco's modified Eagle's medium and F-12 (Sigma) supplemented with 100 units/ml penicillin G/streptomycin sulfate (Mediatech, Herndon, VA) and 10% (v/v) heat inactivated fetal bovine serum (Irvine Scientific, Santa Ana, CA). Primary human hepatocytes HH1247 (3 y, M), HH1248 (42 y, F), HH1249 (59 y, M), HH1251 (29 y, M), HH1274 (55 y, F), HH1281 (16 y, M), HH1286 (50 y, M), and HH1308 (64 y, M) were isolated from human donors and were obtained through the Liver Tissue Procurement and Distribution System of the National Institutes of Health. For the first 36 h after isolation, cells were maintained in hepatocyte maintenance medium supplemented with 10–7 m insulin and dexamethasone (Cambrex, NJ).Reporters and Expression Plasmids—Rat and human CYP7A1/luciferase (Luc) reporter were constructed as previously described (Crestani (44Crestani M. Stroup D. Chiang J.Y.L. J. Lipid Res. 1995; 36: 2419-2432Abstract Full Text PDF PubMed Google Scholar); Wang et al. (76)). Expression plasmid for human peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) (pcDNA3/HA-PGC-1α) was obtained from A. Kralli (The Scripps Research Institute, La Jolla, CA). Expression plasmid for pCMV-rHNF3α was kindly provided by Robert H. Costa (University of Illinois, Chicago, IL). pCMV5-FoxO1 and pBluescript-FoxO1 were kindly provided by D. Accili (Columbia University, New York). SREBP-1c expression plasmid (pCMV-SREBP-1c-436) was purchased from the ATCC. pCMX-hLXRα and pCMX-RXRα were provided by D. Mangelsdorf (University of Texas Southwestern) and R. Evans (The Salk Institute for Biological Studies, La Jolla, CA), respectively. The construction of the expression plasmid for HNF4α (pCMV-HNF4α) was previously described (8Crestani M. Sadeghpour A. Stroup D. Galli G. Chiang J.Y. J. Lipid Res. 1998; 39: 2192-2200Abstract Full Text Full Text PDF PubMed Google Scholar). The β-galactosidase expression plasmid (pCMV-β-gal) and the mammalian expression vector pcDNA3 were obtained from Clontech (Palo Alto, CA). The reporter vector (pGL3-Basic) was purchased from Promega (Madison, WI). The PEPCK reporter plasmid, p2000Luc, was obtained from R. Hanson (Case Western Reserve University). The synthetic reporter 5XUAS-TK-Luc, which contains 5 copies of Gal4 binding site UAS located upstream of the thymidine kinase promoter and luciferase gene, was provided by A. Takeshita (Toranomon Hospital, Tokyo, Japan) (28Li T. Chiang J.Y. Am. J. Physiol. Gastrointest. Liver Physiol. 2004; 288: 74-84Crossref PubMed Scopus (175) Google Scholar). The Gal4-HNF4α fusion plasmid pBx-HNF4-LBD was obtained from I. Talianidis (Institute of Molecular Biology and Biotechnology Foundation for Research and Technology, Hellas, Herakleion Crete, Greece).Transient Transfection Assay—HepG2 cells were grown to ∼80% confluence in 24-well tissue culture plates. Luciferase reporters and expression plasmids were transfected using Lipofectamine 2000 reagent (Invitrogen) following the manufacturer's instructions. The pcDNA3 empty vector was added to normalize the amounts of DNA transfected in each assay. Luciferase and β-galactosidase activities were assayed and expressed in relative luciferase units as described previously (28Li T. Chiang J.Y. Am. J. Physiol. Gastrointest. Liver Physiol. 2004; 288: 74-84Crossref PubMed Scopus (175) Google Scholar).RNA Isolation and Quantitative Real-time PCR—Primary human hepatocytes were cultured in serum-free and insulin-free media for 24 h and treated with insulin (Sigma) as indicated. RNA isolation, reverse transcription reactions, and real-time PCR were performed as described previously (29Song K.H. Li T. Chiang J.Y. J. Biol. Chem. 2006; 281: 10081-10088Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). All TaqMan Probes, CYP7A1 (HS00167982), FoxO1 (HS00231106), SREBP-1 (HS00231674), HNF4α (HS00230853), and UBC (HS00824723), were ordered from Gene Expression Assays (Applied Biosystems Inc., Foster City, CA). PEPCK probe was custom made from Custom Gene Expression Assays (Applied Biosystems, Inc.). Ubiquitin C was used as an internal control for all PCR amplification reactions. Relative mRNA expression was quantified using the comparative Ct (ΔCt) method and expressed as 2–ΔΔCt. Each assay was done in triplicate and expressed as the mean ± S.D.Electrophoretic Mobility Shift Assay (EMSA)—FoxO1 and FoxA1 were synthesized in vitro using the transcription/translation (TNT) system programmed with the FoxO1 and FoxA1 expression plasmids according to the manufacturer's instruction (Promega). [α-32P]dCTP (3000 Ci/mol) was obtained from PerkinElmer Life Sciences. Synthetic oligonucleotides of complimentary strands were labeled with [α-32P]dCTP and incubated with in vitro translated proteins (5 μl) as described previously (28Li T. Chiang J.Y. Am. J. Physiol. Gastrointest. Liver Physiol. 2004; 288: 74-84Crossref PubMed Scopus (175) Google Scholar). Gels were dried and autoradiographed using a PhosphorImager 445Si (GE Healthcare). Sequences of the probes (tagged with GATC at the 5′ end for labeling) used in the experiments are listed in the supplemental data. Oligonucleotides were synthesized by MWG Biotech (High Point, NC).Site-directed Mutagenesis—A PCR-based QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) was used for mutation of the reporter constructs; mutant rIRE2 sequence was introduced into rat p-344/luc plasmid, and the HNF4α binding site mutation was introduced into ph-1887/Luc plasmid as described previously (28Li T. Chiang J.Y. Am. J. Physiol. Gastrointest. Liver Physiol. 2004; 288: 74-84Crossref PubMed Scopus (175) Google Scholar).Chromatin Immunoprecipitation (ChIP) Assay—Human primary hepatocytes were plated in T75 tissue culture flasks. HepG2 cells overexpressing HA-PGC-1α were cultured in serum-free and insulin-free media for 24 h. ChIP assays were performed as described previously (28Li T. Chiang J.Y. Am. J. Physiol. Gastrointest. Liver Physiol. 2004; 288: 74-84Crossref PubMed Scopus (175) Google Scholar). Antibodies against HNF4α (sc-8987), SREBP-1 (sc-366), and HA tag (sc-805, Lot#K0303) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and FoxO1 (#9462) was from Cell Signaling Technology, Inc. (Danvers, MA). Antibodies were used to immunoprecipitate chromatin. Non-immuno IgG (Santa Cruz) was used as negative control for immunoprecipitation. A 391-bp fragment containing the HNF4α binding site was amplified by PCR and analyzed on a 1.5% agarose gel.Isolation of Cell Nuclei—Approximately 107 cells were washed 3 times with cold 1× phosphate-buffered saline. Cells were collected by centrifugation at 4 °C and re-suspended in 1 ml of buffer A (10 mm Tris-Cl, pH 7.5, 10 mm NaCl, 3 mm MgCl2) containing protease inhibitors (Sigma) and incubated on ice for 10 min. 100 μl of 10% Nonidet P-40 was then added, and cells were incubated for another 10 min on ice. Cells were lysed by passing through a 22-gauge needle 20 times. Cell lysates were centrifuged at 3000 rpm at 4 °C for 10 min to precipitate the nuclei. Nuclei were washed 3 times in ice-cold buffer A and lysed in SDS lysis buffer for immunoblotting analysis.Immunoblotting Analysis—Primary human hepatocytes were treated with 10 nm insulin as described above. Total cell lysates or nuclei fractions were analyzed by SDS-polyacrylamide gel electrophoresis. An antibody against phospho-FoxO1 (Ser-256) (#9461; Cell Signaling Technology, Inc.) and antibodies against FoxO1 (sc-9462), HNF4α (sc-8987), and actin (sc-1615) (Santa Cruz Biotechnology) were used for Western blotting and detected by ECL Western blotting detection kit (Amersham Biosciences).RESULTSEffect of Insulin on CYP7A1 and Transcription Factor mRNA Expression in Human Primary Hepatocytes—We first studied the effect of insulin on CYP7A1 mRNA expression in human primary hepatocytes using quantitative real-time PCR. Fig. 1A shows that increasing doses of insulin (0.1 nm to 100 nm) rapidly induced CYP7A1 mRNA levels in 2 h, with a maximum induction of ∼22-fold at the physiological concentration of 10 nm in this donor liver. Higher concentrations of insulin had less stimulatory effect on CYP7A1 mRNA expression. In contrast, insulin treatment for 6 h inhibited CYP7A1 mRNA by ∼40% at 100 nm (Fig. 1B). As a positive control, insulin strongly reduced PEPCK mRNA expression in primary hepatocytes at both 2 and 6 h (Fig. 1, A and B). To further study the insulin effect on CYP7A1 mRNA expression, we treated primary hepatocytes with insulin (10 nm) for a period of time from 2 to 24 h. Quantitative real-time PCR analysis of primary hepatocytes from five donors showed that insulin induced CYP7A1 mRNA expression levels at 2 h by an average of ∼9-fold and inhibited CYP7A1 mRNA at 6 and 24 h by ∼36 and ∼39%, respectively (supplemental Table 1). As a positive control, insulin strongly inhibited PEPCK mRNA in a time-dependent manner.We also measured the mRNA expression levels of HNF4α, the key regulator of human CYP7A1 gene transcription, and SREBP-1c and FoxO1, two transcription factors that are known to mediate the effects of insulin (Table 1, supplemental data). Insulin significantly induced HNF4α mRNA levels at 2 h by ∼30% but inhibited it at 6 and 24 h. On the other hand, insulin induced SREBP-1c by about 2-fold, consistent with a previous report (30Cagen L.M. Deng X. Wilcox H.G. Park E.A. Raghow R. Elam M.B. Biochem. J. 2004; 385: 207-216Crossref Scopus (125) Google Scholar). Insulin had no effect on FoxO1 mRNA expression, consistent with previous studies that insulin regulates FoxO1 mainly at post-translational levels (15Puigserver P. Rhee J. Donovan J. Walkey C.J. Yoon J.C. Oriente F. Kitamura Y. Altomonte J. Dong H. Accili D. Spiegelman B.M. Nature. 2003; 423: 550-555Crossref PubMed Scopus (1155) Google Scholar, 18Nakae J. Park B.C. Accili D. J. Biol. Chem. 1999; 274: 15982-15985Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar, 19Zhang X. Gan L. Pan H. Guo S. He X. Olson S.T. Mesecar A. Adam S. Unterman T.G. J. Biol. Chem. 2002; 277: 45276-45284Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar).Identification of the IREs in the CYP7A1 Gene—FoxO1 is known to bind to the consensus IRE, T(G/A)TTT(T/G)(G/T), in the promoters of glucose-6-phosphatase, PEPCK, insulin-like growth factor binding protein-1, and tyrosine aminotransferase and mediates the inhibitory effect of insulin on these genes (31O'Brien R.M. Noisin E.L. Suwanichkul A. Yamasaki T. Lucas P.C. Wang J.C. Powell D.R. Granner D.K. Mol. Cell. Biol. 1995; 15: 1747-1758Crossref PubMed Google Scholar). Analysis of the nucleotide sequences in the CYP7A1 promoter identified four putative IREs in the rat gene and two putative IREs in the human gene (Fig. 2A). Only the IRE located in the bile acid response element-I (–81 to –75) is conserved in rat and human CYP7A1 genes. We then performed an EMSA to test if FoxO1 bound to these IREs in the CYP7A1 gene. Fig. 2B shows that FoxO1 bound strongly to the rat IRE2 (rIRE2) but did not bind to rIRE1, rIRE3, rIRE4, human (h) IRE1 and hIRE2 despite the similarity in nucleotide sequences (Fig. 2A). FoxO1 binding to rIRE2 was competed out by a large excess of unlabeled rIRE2 probe and a glucose-6-phosphatase probe that contained three FoxO1 binding sites (Fig. 2C) (16Nakae J. Kitamura T. Silver D.L. Accili D. J. Clin. Investig. 2001; 108: 1359-1367Crossref PubMed Scopus (498) Google Scholar). This rIRE2 also bound FoxA1 (HNF3α) as expected. A single mutation of G to T in the rIRE2 abolished Foxo1 binding (Fig. 2C). To test the effect of FoxO1 on rat CYP7A1 gene transcription, reporter assays were performed in HepG2 cells with a rat CYP7A1 promoter/luciferase construct. Co-transfection of a FoxO1 expression plasmid stimulated rat CYP7A1 reporter activity by ∼4-fold (Fig. 2D, left panel). When a single mutation that abolished FoxO1 binding was introduced into the rat CYP7A1 reporter, the basal reporter activity was reduced by more than 80%. Co-transfection of FoxO1 failed to stimulate the mutant reporter activity (Fig. 2D, left panel). This mutant reporter did respond to HNF4α and LXRα because it retained the HNF4α and LXRα binding sites as in the wild type plasmid (Fig. 2D, right panel).FIGURE 2EMSA of FoxO1 and FoxA1 interaction with the putative rat and human IREs. A, putative IREs in rat (r) and human (h) CYP7A1 promoter. B, EMSA of in vitro synthesized FoxO1 and FoxA1 binding to rat and human IRE probes. A glucose-6-phosphatase IRE probe was used as a positive control. Unprogrammed TNT lysates (T3 and T7) were used as negative controls for nonspecific bindings. Sequences of IRE probes used are described in the supplemental data. C, EMSA of in vitro synthesized FoxO1 and FoxA1 with 32P-labeled rat CYP7A1 IRE2 and mutant IRE2 probes. Competition assays were done with a 100-fold excess of unlabeled probes. Mutations in rIRE2 probes is shown. G6Pase, glucose 6-phosphatase. D, effects of FoxO1 on rat CYP7A1 reporter activities. WT, wild type rat CYP7A1 reporter (p-344/luc). RLU, relative light units. Mutant, rat CYP7A1 reporter (p-344/luc) containing single nucleotide mutation in IRE2 sequence. Reporter (0.2 μg) and the indicated expression plasmids (0.1 μg) were transfected into HepG2 cells. Transient transfection and luciferase assays were performed as described “Experimental Procedures.” A mutation in rat IRE2 is shown. Statistical analysis was performed by Student's t test; *, significant, p < 0.05.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FoxO1 Inhibited Human CYP7A1 Reporter Activity by Blocking HNF4α Trans-activation Activity—The effect of FoxO1 on human CYP7A1 reporter activity was studied using transfection assays. In contrast to the stimulatory effect on the rat CYP7A1 reporter, co-transfection of FoxO1 inhibited human CYP7A1 reporter activities of ph-1887/Luc, ph-371/Luc, and ph-150/Luc by ∼70% (Fig. 3A). Further deletion of the sequence downstream of –150 abolished the inhibitory effect of FoxO1. Thus, the inhibitory effect of FoxO1 required the region from –150 to –135, which was previously mapped as the bile acid response element-II that contained an HNF4α binding site (32Stroup D. Chiang J.Y. J. Lipid Res. 2000; 41: 1-11Abstract Full Text Full Text PDF PubMed Google Scholar). It has been reported that FoxO1 interacts with the DNA binding domain of HNF4α and inhibits HNF4α trans-activation activity and its DNA binding (21Hirota K. Daitoku H. Matsuzaki H. Araya N. Yamagata K. Asada S. Sugaya T. Fukamizu A. J. Biol. Chem. 2003; 278: 13056-13060Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). To test if the HNF4α binding site was involved in FoxO1 regulation of the human CYP7A1 gene, the HNF4α binding site in the ph-1887/Luc reporter was mutated. Fig. 3B shows that the HNF4α binding site mutant reporter (mHNF4α-ph-1887/luc) had a much lower basal activity, and FoxO1 did not inhibit the mutant reporter activity. These results suggest that FoxO1 might inhibit human CYP7A1 gene by inhibiting HNF4α trans-activation activity. A mammalian one-hybrid assay was used to study the effect of FoxO1 on HNF4α trans-activation of a Gal4 (5×UAS)/TK/Luc reporter. Fig. 3C shows that Gal4-HNF4α activation of the GAL4 reporter activity was drastically increased by PGC-1α, and FoxO1 dose-dependently inhibited reporter activity (Fig. 3C). These results suggest that in addition to interfering with HNF4α bindi" @default.
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• W2002854251 title "Insulin Regulation of Cholesterol 7α-Hydroxylase Expression in Human Hepatocytes" @default.
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