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• W2011132677 abstract "The current studies show FGF15 signaling decreases hepatic forkhead transcription factor 1 (FoxO1) activity through phosphatidylinositol (PI) 3-kinase-dependent phosphorylation. The bile acid receptor FXR (farnesoid X receptor) activates expression of fibroblast growth factor (FGF) 15 in the intestine, which acts through hepatic FGFR4 to suppress cholesterol-7α hydroxylase (CYP7A1) and limit bile acid production. Because FoxO1 activity and CYP7A1 gene expression are both increased by fasting, we hypothesized CYP7A1 might be a FoxO1 target gene. Consistent with recently reported results, we show CYP7A1 is a direct target of FoxO1. Additionally, we show that the PI 3-kinase pathway is key for both the induction of CYP7A1 by fasting and the suppression by FGF15. FGFR4 is the major hepatic FGF receptor isoform and is responsible for the hepatic effects of FGF15. We also show that expression of FGFR4 in liver was decreased by fasting, increased by insulin, and reduced by streptozotocin-induced diabetes, implicating FGFR4 as a primary target of insulin regulation. Because insulin and FGF both target the PI 3-kinase pathway, these observations suggest FoxO1 is a key node in the convergence of FGF and insulin signaling pathways and functions as a key integrator for the regulation of glucose and bile acid metabolism. The current studies show FGF15 signaling decreases hepatic forkhead transcription factor 1 (FoxO1) activity through phosphatidylinositol (PI) 3-kinase-dependent phosphorylation. The bile acid receptor FXR (farnesoid X receptor) activates expression of fibroblast growth factor (FGF) 15 in the intestine, which acts through hepatic FGFR4 to suppress cholesterol-7α hydroxylase (CYP7A1) and limit bile acid production. Because FoxO1 activity and CYP7A1 gene expression are both increased by fasting, we hypothesized CYP7A1 might be a FoxO1 target gene. Consistent with recently reported results, we show CYP7A1 is a direct target of FoxO1. Additionally, we show that the PI 3-kinase pathway is key for both the induction of CYP7A1 by fasting and the suppression by FGF15. FGFR4 is the major hepatic FGF receptor isoform and is responsible for the hepatic effects of FGF15. We also show that expression of FGFR4 in liver was decreased by fasting, increased by insulin, and reduced by streptozotocin-induced diabetes, implicating FGFR4 as a primary target of insulin regulation. Because insulin and FGF both target the PI 3-kinase pathway, these observations suggest FoxO1 is a key node in the convergence of FGF and insulin signaling pathways and functions as a key integrator for the regulation of glucose and bile acid metabolism. Hepatic cholesterol is converted to bile acids, secreted into the gallbladder, and during a meal is released into the small intestine to enhance digestion and absorption of dietary lipids and fat-soluble vitamins. The majority of the bile acid pool (95%) is recycled back to the liver, whereas the remaining 5% is eliminated through fecal excretion (1.Chiang J.Y. Endocr. Rev. 2002; 23: 443-463Crossref PubMed Scopus (389) Google Scholar, 2.Russell D.W. Annu. Rev. Biochem. 2003; 72: 137-174Crossref PubMed Scopus (1427) Google Scholar). This is an important route for the elimination of excess cholesterol and underscores the importance that bile acids play in regulating mammalian cholesterol metabolism. The initial and rate-controlling step in the classic pathway for cholesterol conversion into bile acids is catalyzed by cholesterol 7α-hydroxylase (CYP7A1). 2The abbreviations used are: CYP7A1, cholesterol-7α hydroxylase; PEPCK, phosphoenolpyruvate carboxykinase; FGF, fibroblast growth factor; FGFR, FGF receptor; FoxO1, forkhead transcription factor 1; FXR, farnesoid X receptor; PGC-1, peroxisome proliferator-activated receptor-γ coactivator 1; SHP, small heterodimer partner; HA, hemagglutinin; JNK, c-Jun N-terminal kinase; PI, phosphatidylinositol; WT, wild type; GFP, green fluorescent protein; q-PCR, quantitative real time RT-PCR. CYP7A1 regulation is primarily transcriptional, and expression of its gene is dynamically regulated by hormones and metabolites (2.Russell D.W. Annu. Rev. Biochem. 2003; 72: 137-174Crossref PubMed Scopus (1427) Google Scholar, 3.Jelinek D.F. Andersson S. Slaughter C.A. Russell D.W. J. Biol. Chem. 1990; 265: 8190-8197Abstract Full Text PDF PubMed Google Scholar, 4.Ness G.C. Pendleton L.C. Zhao Z. Biochim. Biophys. Acta. 1994; 1214: 229-233Crossref PubMed Scopus (36) Google Scholar, 5.Shin D.J. Plateroti M. Samarut J. Osborne T.F. Nucleic Acids Res. 2006; 34: 3853-3861Crossref PubMed Scopus (36) Google Scholar, 6.Twisk J. Hoekman M.F. Lehmann E.M. Meijer P. Mager W.H. Princen H.M. Hepatology. 1995; 21: 501-510PubMed Google Scholar). Importantly, bile acids themselves regulate CYP7A1 gene expression through a multicomponent negative feedback pathway. One of the molecular pathways for bile acid regulation is initiated by the activation of the farnesoid X receptor (FXR) responding directly to bile acid agonists (7.Makishima M. Okamoto A.Y. Repa J.J. Tu H. Learned R.M. Luk A. Hull M.V. Lustig K.D. Mangelsdorf D.J. Shan B. Science. 1999; 284: 1362-1365Crossref PubMed Scopus (2182) Google Scholar). Ligand-activated FXR directly binds to a site in the promoter for the small heterodimer partner (SHP) gene and induces expression of SHP mRNA (8.Goodwin B. Jones S.A. Price R.R. Watson M.A. McKee D.D. Moore L.B. Galardi C. Wilson J.G. Lewis M.C. Roth M.E. Maloney P.R. Willson T.M. Kliewer S.A. Mol. Cell. 2000; 6: 517-526Abstract Full Text Full Text PDF PubMed Scopus (1531) Google Scholar, 9.Lu T.T. Makishima M. Repa J.J. Schoonjans K. Kerr T.A. Auwerx J. Mangelsdorf D.J. Mol. Cell. 2000; 6: 507-515Abstract Full Text Full Text PDF PubMed Scopus (1232) Google Scholar). The translated SHP protein lacks the signature nuclear receptor zinc finger DNA binding domain but uses its conserved dimerization motif to form protein-protein contacts with DNA bound activators, usually other nuclear receptors, to inhibit or interfere with their activation potential (10.Seol W. Choi H.-S. Moore D.D. Science. 1996; 272: 1336-1339Crossref PubMed Scopus (446) Google Scholar, 11.Fang S. Miao J. Xiang L. Ponugoti B. Treuter E. Kemper J.K. Mol. Cell. Biol. 2007; 27: 1407-1424Crossref PubMed Scopus (83) Google Scholar). The first identified target for SHP repression was the CYP7A1 promoter, and SHP was proposed to interfere with activation by the DNA-bound monomeric liver receptor homologue 1 (LRH-1) nuclear receptor (8.Goodwin B. Jones S.A. Price R.R. Watson M.A. McKee D.D. Moore L.B. Galardi C. Wilson J.G. Lewis M.C. Roth M.E. Maloney P.R. Willson T.M. Kliewer S.A. Mol. Cell. 2000; 6: 517-526Abstract Full Text Full Text PDF PubMed Scopus (1531) Google Scholar, 9.Lu T.T. Makishima M. Repa J.J. Schoonjans K. Kerr T.A. Auwerx J. Mangelsdorf D.J. Mol. Cell. 2000; 6: 507-515Abstract Full Text Full Text PDF PubMed Scopus (1232) Google Scholar). However, hepatic nuclear factor-4 (HNF-4), another nuclear receptor that stimulates CYP7A1 (12.Cooper A.D. Chen J. Botelho-Yetkinler M.J. Cao Y. Taniguchi T. Levy-Wilson B. J. Biol. Chem. 1997; 272: 3444-3452Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 13.De Fabiani E. Mitro N. Anzulovich A.C. Pinelli A. Galli G. Crestani M. J. Biol. Chem. 2001; 276: 30708-30716Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar), is also a target for SHP repression as well (14.Lee Y.-K. Dowhan D.H. Hadzopoulou-Cladaras M. Moore D.D. Mol. Cell. Biol. 2000; 20: 187-195Crossref PubMed Scopus (265) Google Scholar). Bile acids activate the JNK pathway, which may also play an important role in inhibition of CYP7A1 gene expression by a SHP-independent mechanism (15.Gupta S. Stravitz R.T. Dent P. Hylemon P.B. J. Biol. Chem. 2001; 276: 15816-15822Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar). Other kinase signaling pathways have been implicated in regulating CYP7A1, but mechanistic information is incomplete (16.Han S.I. Studer E. Gupta S. Fang Y. Qiao L. Li W. Grant S. Hylemon P.B. Dent P. Hepatology. 2004; 39: 456-463Crossref PubMed Scopus (55) Google Scholar, 17.Qiao D. Chen W. Stratagoules E.D. Martinez J.D. J. Biol. Chem. 2000; 275: 15090-15098Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 18.Stravitz R.T. Rao Y.P. Vlahcevic Z.R. Gurley E.C. Jarvis W.D. Hylemon P.B. Am. J. Physiol. 1996; 271: G293-G303PubMed Google Scholar). More recently, FXR has been shown to activate expression of both the human fibroblast growth factor (FGF) 19 in primary cultures of human hepatocytes (19.Holt J.A. Luo G. Billin A.N. Bisi J. McNeill Y.Y. Kozarsky K.F. Donahee M.Y.W.D. Mansfield T.A. Kliewer S.A. Goodwin B. Jones S.A. Genes Dev. 2003; 17: 1581-1591Crossref PubMed Scopus (543) Google Scholar) and its mouse orthologue, FGF15, in the intestine in response to bile acids (20.Inagaki T. Choi M. Moschetta A. Peng L. Cummins C.L. McDonald J.G. Luo G. Jones S.A. Goodwin B. Richardson J.A. Gerard R.D. Repa J.J. Mangelsdorf D.J. Kliewer S.A. Cell Metab. 2005; 2: 217-225Abstract Full Text Full Text PDF PubMed Scopus (1364) Google Scholar). FGF15 along with FGF21 and FGF23 compose a subfamily of FGFs that are regulated by nuclear receptors and function as metabolic hormones. Although FGF15 regulates bile acid metabolism, FGF21 has an important role in glucose metabolism, peroxisome proliferator-activated receptor-α-dependent activation of fatty acid oxidation, ketogenesis, and growth hormone function (21.Kharitonenkov A. Shiyanova T.L. Koester A. Ford A.M. Micanovic R. Galbreath E.J. Sandusky G.E. Hammond L.J. Moyers J.S. Owens R.A. Gromada J. Brozinick J.T. Hawkins E.D. Wroblewski V.J. Li D.S. Mehrbod F. Jaskunas S.R. Shanafelt A.B. J. Clin. Investig. 2005; 115: 1627-1635Crossref PubMed Scopus (1648) Google Scholar, 22.Inagaki T. Dutchak P. Zhao G. Ding X. Gautron L. Parameswara V. Li Y. Goetz R. Mohammadi M. Esser V. Elmquist J.K. Gerard R.D. Burgess S.C. Hammer R.E. Mangelsdorf D.J. Kliewer S.A. Cell Metab. 2007; 5: 415-425Abstract Full Text Full Text PDF PubMed Scopus (1207) Google Scholar, 23.Badman M.K. Pissios P. Kennedy A.R. Koukos G. Flier J.S. Maratos-Flier E. Cell Metab. 2007; 5: 426-437Abstract Full Text Full Text PDF PubMed Scopus (1187) Google Scholar, 24.Inagaki T. Lin V.Y. Goetz R. Mohammadi M. Mangelsdorf D.J. Kliewer S.A. Cell Metab. 2008; 8: 77-83Abstract Full Text Full Text PDF PubMed Scopus (330) Google Scholar), whereas FGF23 regulates phosphate metabolism (25.White K.E. Evans W.E. O'Riordan J.L.H. Speer M.C. Econs M.J. Lorenz-Depiereux B. Grabowski M. Meltinger T. Strom T.M. Nat. Genet. 2000; 26: 345-348Crossref PubMed Scopus (1297) Google Scholar). Most FGFs require strong binding to cell surface heparin sulfate proteoglycans to stabilize binding to their cognate FGF receptor. However, these three metabolic FGF hormones do not bind tightly to heparin sulfate; rather, they utilize one of two distinct but related cell surface co-receptors called klotho or β-klotho, apparently as co-receptors (26.Goetz R. Beenken A. Ibrahimi O.A. Kalinina J. Olsen S.K. Eliseenkova A.V. Xu C. Neubert T.A. Zhang F. Linhardt R.J. Yu X. White K.E. Inagaki T. Kliewer S.A. Yamamoto M. Kurosu H. Ogawa Y. Kuro-o M. Lanske B. Razzaque M.S. Mohammadi M. Mol. Cell. Biol. 2007; 27: 3417-3428Crossref PubMed Scopus (434) Google Scholar, 27.Kurosu H. Choi M. Ogawa Y. Dickson A.S. Goetz R. Eliseenkova A.V. Mohammadi M. Rosenblatt K.P. Kliewer S.A. Kuro O.M. J. Biol. Chem. 2007; 282: 26687-26695Abstract Full Text Full Text PDF PubMed Scopus (604) Google Scholar, 28.Kurosu H. Ogawa Y. Miyoshi M. Yamamoto M. Nandi A. Rosenblatt K.P. Baum M.G. Schiavi S. Hu M.C. Moe O.W. Kuro-o M. J. Biol. Chem. 2006; 281: 6120-6123Abstract Full Text Full Text PDF PubMed Scopus (1077) Google Scholar, 29.Ogawa Y. Kurosu H. Yamamoto M. Nandi A. Rosenblatt K.P. Goetz R. Eliseenkova A.V. Mohammadi M. Kuro-o M. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 7432-7437Crossref PubMed Scopus (488) Google Scholar). FGF15 produced in the distal small intestine signals through FGFR4 in hepatocytes to inhibit expression of the liver CYP7A1 gene, providing an intriguing example of tissue communication in metabolic regulation (24.Inagaki T. Lin V.Y. Goetz R. Mohammadi M. Mangelsdorf D.J. Kliewer S.A. Cell Metab. 2008; 8: 77-83Abstract Full Text Full Text PDF PubMed Scopus (330) Google Scholar). Another report demonstrated that serum levels of FGF19 in humans are increased by bile acid feeding and decreased by a bile acid sequestrant (30.Lundasen T. Galman C. Angelin B. Rudling M. J. Intern. Med. 2006; 260: 530-536Crossref PubMed Scopus (327) Google Scholar). Thus, it is likely that FGF-dependent bile acid regulation is conserved between rodents and humans. FXR and bile acids have been implicated as integrated regulators of bile acid and glucose metabolism (31.Houten S.M. Watanabe M. Auwerx J. EMBO J. 2006; 25: 1419-1425Crossref PubMed Scopus (441) Google Scholar, 32.Lee F.Y. Lee H. Hubbert M.L. Edwards P.A. Zhang Y. Trends Biochem. Sci. 2006; 31: 572-580Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar). In support of this connection, we found that CYP7A1 was induced by fasting and during streptozotocin induced diabetes (33.Shin D.-J. Campos J.A. Gil G. Osborne T.F. J. Biol. Chem. 2003; 278: 50047-50052Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar), two stressful metabolic conditions where insulin signaling and glucose metabolism are compromised. More recently, other reports have also revealed an important role for bile acids in glucose metabolism (34.Ma K. Saha P.K. Chan L. Moore D.D. J. Clin. Investig. 2006; 116: 1102-1109Crossref PubMed Scopus (660) Google Scholar, 35.Zhang Y. Lee F.Y. Barrera G. Lee H. Vales C. Gonzalez F.J. Willson T.M. Edwards P.A. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 1006-1011Crossref PubMed Scopus (729) Google Scholar). Insulin has long been known to inhibit CYP7A1 (6.Twisk J. Hoekman M.F. Lehmann E.M. Meijer P. Mager W.H. Princen H.M. Hepatology. 1995; 21: 501-510PubMed Google Scholar, 36.Wang 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), but the mechanism has not been fully revealed. Insulin binding to its cell surface receptor initiates a signaling cascade through the PI 3-kinase pathway, resulting in phosphorylation and activation of the serine/threonine protein kinase Akt. Akt in turn phosphorylates forkhead transcription factor 1 (FoxO1) on three key residues, Thr-24, Ser-253, and Ser-316, converting FoxO1 from a predominantly nuclear to a predominantly cytoplasmic location, rendering it unable to activate its nuclear target genes (37.Accili D. Arden K.C. Cell. 2004; 117: 421-426Abstract Full Text Full Text PDF PubMed Scopus (1115) Google Scholar, 38.Brunet A. Bonni A. Zigmond M.J. Lin M.Z. Juo P. Hu L.S. Anderson M.J. Arden K.C. Blenis J. Greenberg M.E. Cell. 1999; 96: 857-868Abstract Full Text Full Text PDF PubMed Scopus (5454) Google Scholar, 39.Hadari Y.R. Gotoh N. Kouhara H. Lax I. Schlessinger J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8578-8583Crossref PubMed Scopus (242) Google Scholar, 40.Nakae J. Park B.C. Accili D. J. Biol. Chem. 1999; 274: 15982-15985Abstract Full Text Full Text PDF PubMed Scopus (401) Google Scholar, 41.Ong S.H. Hadari Y.R. Gotoh N. Guy G.R. Schlessinger J. Lax I. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6074-6079Crossref PubMed Scopus (263) Google Scholar, 42.Rena G. Woods Y.L. Prescott A.R. Peggie M. Unterman T.G. Williams M.R. Cohen P. EMBO J. 2002; 21: 2263-2271Crossref PubMed Scopus (201) Google Scholar). This redistribution and inhibition of FoxO1 occurs in response to insulin and other nutrient and growth signals that activate the PI 3-kinase pathway in many tissues and cell types including liver (42.Rena G. Woods Y.L. Prescott A.R. Peggie M. Unterman T.G. Williams M.R. Cohen P. EMBO J. 2002; 21: 2263-2271Crossref PubMed Scopus (201) Google Scholar, 43.Barthel A. Schmoll D. Unterman T.G. Trends Endocrinol. Metab. 2005; 16: 183-189Abstract Full Text Full Text PDF PubMed Scopus (488) Google Scholar, 44.Brunet A. Park J. Tran H. Hu L.S. Hemmings B.A. Greenberg M.E. Mol. Cell. Biol. 2001; 21: 952-965Crossref PubMed Scopus (715) Google Scholar, 45.Woods Y.L. Rena G. Morrice N. Barthel A. Becker W. Guo S. Unterman T.G. Cohen P. Biochem. J. 2001; 355: 597-607Crossref PubMed Scopus (220) Google Scholar). Overall, nuclear localization and activation of FoxO1 is associated with stressful conditions, which in the liver occurs during fasting (43.Barthel A. Schmoll D. Unterman T.G. Trends Endocrinol. Metab. 2005; 16: 183-189Abstract Full Text Full Text PDF PubMed Scopus (488) Google Scholar). Key hepatic FoxO1 target genes include phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (46.Altomonte J. Richter A. Harbaran S. Suriawinata J. Nakae J. Thung S.N. Meseck M. Accili D. Dong H. Am. J. Physiol. 2003; 285: E718-E728Crossref PubMed Scopus (178) Google Scholar, 47.Hall 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 (244) Google Scholar, 48.Nakae J. Kitamura T. Silver D.L. Accili D. J. Clin. Investig. 2001; 108: 1359-1367Crossref PubMed Scopus (510) Google Scholar) that encode key gluconeogenic enzymes, which are turned on to produce glucose when serum levels are low and insulin signaling is compromised. Signaling through both insulin and FGF target the PI 3-kinase pathway (38.Brunet A. Bonni A. Zigmond M.J. Lin M.Z. Juo P. Hu L.S. Anderson M.J. Arden K.C. Blenis J. Greenberg M.E. Cell. 1999; 96: 857-868Abstract Full Text Full Text PDF PubMed Scopus (5454) Google Scholar, 40.Nakae J. Park B.C. Accili D. J. Biol. Chem. 1999; 274: 15982-15985Abstract Full Text Full Text PDF PubMed Scopus (401) Google Scholar, 42.Rena G. Woods Y.L. Prescott A.R. Peggie M. Unterman T.G. Williams M.R. Cohen P. EMBO J. 2002; 21: 2263-2271Crossref PubMed Scopus (201) Google Scholar, 49.Lamothe B. Yamada M. Schaeper U. Birchmeier W. Lax I. Schlessinger J. Mol. Cell. Biol. 2004; 24: 5657-5666Crossref PubMed Scopus (68) Google Scholar), and both inhibit CYP7A1. Because FoxO1 is negatively regulated by PI 3-kinase signaling and both FoxO1 and CYP7A1 are induced by fasting, we hypothesized that FoxO1 might be involved in the negative regulation of CYP7A1. Here, we show that FoxO1 directly regulates CYP7A1 gene expression and that, similar to insulin, FGF15/19 signaling leads to a PI 3-kinase-dependent phosphorylation and inhibition of FoxO1 in mice and in cultures of primary hepatocytes. Thus, FoxO1 is at a critical junction in hepatic physiology where it links bile acids with glucose metabolism. Plasmids—The rat pGL3R7α-342 has been described previously (a gift from Dr. G. Gil, Virginia Commonwealth University) (50.del Castillo-Olivares A. Gil G. Nucleic Acids Res. 2000; 28: 3587-3593Crossref PubMed Google Scholar). Point mutations were introduced into putative FoxO1 binding sites designated FoxO1/1 or FoxO1/2 by QuikChange site-directed mutagenesis (Stratagene) to generate pGL3R7α-342/M1, pGL3R7α-342/M2, pGL3R7α-342/M1 and -2, and pGL3R7α-228/M1. The following primers were used: M1, 5′-CTAGTAGGAGGACAAATAGTGGGTGCTTTGGTCACTCAAGTTCA-3′; M2, 5′-TGACAGATGTGCTCATCTGGGTACTTCTTTTTCTACACACAG-3′. pGL3R7α-228 was constructed by PCR-based amplification using the following primers: forward primer, 5′-ATGTTATGTCAGCACATGAGG-3′; reverse primer, 5′-AAAAGCAGGAAAATTTCCAAAGG-3′. The PCR product was digested with SstI and HindIII, which were added to the forward and reverse primers, respectively, for cloning purpose and inserted into the SstI and HindIII sites of pGL3-basic. The human CYP7A1 promoter construct ph-371/+24-Luc (36.Wang 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) was obtained from Dr. J. Chiang (Northeastern Ohio University). Cytomegalovirus-SHP was a gift from Dr. D. Mangelsdorf (University of Texas Southwestern Medical Center). FLAG-FoxO1/WT, FLAG-FoxO1/TSS, and TK-IRS3 were gifts from Dr. T. Unterman (University of Illinois College of Medicine). pCMV6-Akt-WT and pCMV6-Akt-K179M were provided by T. Franke (Columbia University) through Dr. D. Fruman (University of California, Irvine, CA). Cell Culture and Transient Transfection Assay—HepG2 cells were maintained in minimum essential medium supplemented with 10% fetal bovine serum at 37 °C and 5% CO2. Transient transfection was performed by the calcium phosphate coprecipitation method as described (33.Shin D.-J. Campos J.A. Gil G. Osborne T.F. J. Biol. Chem. 2003; 278: 50047-50052Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Values represent the mean of duplicates ± S.D. Each experiment was repeated at least three times. Animal Studies—All animal experiments were approved by the Institutional Animal Care and Use Committee at UC Irvine (protocol 97–1545). FoxO1flox/flox mice expressing Cre recombinase under the α1-antitrypsin promoter (51.Matsumoto M. Pocai A. Rossetti L. Depinho R.A. Accili D. Cell Metab. 2007; 6: 208-216Abstract Full Text Full Text PDF PubMed Scopus (480) Google Scholar) were obtained from Dr. D. Accili (Columbia University). These mice were crossed, generating liver-specific FoxO1 knock-out mice (confirmed by PCR of genomic DNA as described (51.Matsumoto M. Pocai A. Rossetti L. Depinho R.A. Accili D. Cell Metab. 2007; 6: 208-216Abstract Full Text Full Text PDF PubMed Scopus (480) Google Scholar)) and their littermates, FoxO1flox/flox, which were used as controls. These mice are in the FVB/N strain. Fasting experiments were performed with 8–9-week-old male mice, as described below. In other studies 4-week-old B6129 male mice were obtained from Taconic and maintained on a 12-h light/dark cycle with free access to food and water. The mice were allowed to adapt to new environments for at least 1 week before experiments. At 8 weeks of age mice were fasted for 24 h, and all mice were sacrificed at 8:00 a.m. (end of the dark cycle). Livers and ileums were removed and frozen in liquid nitrogen and stored at −80 °C until RNA was isolated. For the induction of diabetes, 4-week-old 129SVE male mice (Taconic) were treated with streptozotocin by intraperitoneal injections (100 μg/g of body weight) daily for 3 days to induce type I diabetes, as described previously (33.Shin D.-J. Campos J.A. Gil G. Osborne T.F. J. Biol. Chem. 2003; 278: 50047-50052Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Mice were sacrificed, and livers were processed as described above. Overexpression of FGF15 or FoxO1 Δ256 in mice was achieved through adenoviral delivery. For this, recombinant adenoviruses expressing either GFP, FGF15 (a gift from Dr. S. Kliewer, University of Texas Southwestern Medical Center), or FoxO1 Δ256 (a gift from D. Accili, Columbia University) were first propagated in 293 cells and purified by CsCl gradient centrifugation. A total of 1 × 109 plaque-forming units of each adenovirus was administered into 8-week-old 129SVE male mice (Taconic) by intravenous injection (4 animals in each group). 7 days after adenovirus inoculation mice were sacrificed for RNA and protein analysis. Where indicated, mice were fasted before sacrifice as described above. Primary Mouse Hepatocyte Preparation—Primary mouse hepatocytes were isolated from 7–9-week-old C57BL/6 male mice (The Jackson Laboratory) by liver collagenase perfusion as described previously (52.LeCluyse E.L. Bullock P.L. Parkinson A. Hochman J.H. Pharm. Biotechnol. 1996; 8: 121-159Crossref PubMed Scopus (116) Google Scholar) with minor modifications. Briefly, mice were anesthetized using a ketamine/xylazine mixture. The liver was perfused with Earle's balanced salt solution (EBSS) (Invitrogen) supplemented with 0.5 mm EGTA through the cannulated portal vein at a flow rate of 4 ml/min for 15 min followed by perfusion with EBSS supplemented with 0.3 mg/ml collagenase (Wako Chemicals) and 4.8 mm CaCl2 for 20 min. The liver was dissected from the mouse, and dissociated cells were dispersed gently in Williams' E medium supplemented 10% fetal bovine serum. Cells were filtered through a 100-μm nylon cell strainer (BD Falcon), and hepatocytes were separated by a density gradient centrifugation using 45% Percoll (Sigma) solution. Hepatocyte viability was monitored by trypan blue exclusion, and more than 90% of cells were consistently viable. The isolated cells were plated in collagen-coated 6-well dishes at a density of 6 × 105/well. After a 4-h attachment, cells were overlaid with Matrigel (Collaborative Biomedical Products, Bedford, MA) and maintained in serum-free Williams' E medium supplemented with 10 nm dexamethasone, 2 mm glutamine, 100 units/ml penicillin, 100 units/ml streptomycin. 48 h after isolation, cells were treated with 100 nm human insulin (Sigma) for 24 h or 80 ng/ml FGF19 (R&D Systems) for 6 h and harvested for RNA analysis. Insulin or FGF19 additions were staggered so that cells were harvested at the same time. For the dose-response experiment, cells were treated with 0, 40, or 80 ng/ml FGF19 for 6 h, and RNA was isolated. Cells were treated with 100 nm wortmannin (Calbiochem) for 30 min before the treatment with FGF19. For Western analysis, cells were treated with 30 nm insulin or 80 ng/ml FGF19 for 30 min, and total cell lysates were prepared. Cells were treated with 100 nm wortmannin (Calbiochem) or 20 μm SP600125 (Calbiochem) 2.5 μm PIK 90 (Axon Medchem BV, The Netherlands) for 30 min before the treatment with FGF19 or insulin where indicated. RNA Isolation and Analysis—Total RNA was isolated using TRIzol (Invitrogen) according to the manufacturer's instructions. For quantitative real time RT-PCR (q-PCR), RNA was reverse-transcribed using the iScript cDNA synthesis kit (BioRad) at 42 °C for 30 min. cDNA was amplified and quantified using the iQ SYBR Green Supermix (Bio-Rad) according to the manufacturer's instructions in the iQ5 real-time PCR detection system (Bio-Rad) under the following conditions: initial denaturation at 95 °C for 5 min and 40 cycles of 95 °C for 30 s, 58 °C for 30 s, and 72 °C for 30 s. q-PCR was performed in triplicate for each sample and repeated at least three times. Ct values were used to calculate the relative expression level normalized to the expression of the housekeeping ribosomal protein L32. The results were expressed as the mean ± S.D. A melting curve analysis was performed for each sample after PCR amplification to verify that the amplicon is homogeneous in the absence of primer dimmers and DNA contamination. The sequences of primers used in q-PCR are available upon request. RNA from individual animals (4–6 animals in each group) was analyzed separately, and a Student t test was used for comparative statistical analysis as indicated in the individual figure legends. Glucose Production Assay—Primary mouse hepatocytes were isolated from 8-week-old C57BL/6 male mice after overnight fasting, as described above. After a 4-h attachment in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), primary cells were cultured overnight in DMEM with 5% FBS. The medium was then replaced with serum- and glucose-free Dulbecco's modified Eagle's medium (pH 7.4) supplemented with 2 mm sodium lactate without phenol red. Where indicated, 20, 40, or 80 ng/ml FGF19 was added to the medium. After a 3-h incubation, the medium was collected, and glucose concentrations were measured using the Amplex red glucose assay kit (Invitrogen). Electrophoretic Mobility Shift Assays—Nuclear extracts were prepared from 293T cells transfected with a vector expressing FLAG-FoxO1, and 5 μg of nuclear protein was used in electrophoretic mobility shift assays as described previously (33.Shin D.-J. Campos J.A. Gil G. Osborne T.F. J. Biol. Chem. 2003; 278: 50047-50052Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). For supershift experiments anti-FLAG M2 mouse monoclonal (Sigma) was incubated with the nuclear extract for 20 min on ice before incubation with the labeled probes. Competition experiments were performed in binding reactions where a 100-fold molar excess of unlabeled probes was incubated with nuclear extracts for 20 min before incubation with the labeled probes. The sequences of one strand of the complementary oligonucleotide probes were as follows: wild type FoxO1/1, 5′-AGGACAAATAGTGTTTGCTTTGGTCACTCA-3′; mutant FoxO1/1, 5′-AGGACAAATAGTGggTGCTTTGGTCACTCA-3′; wild type FoxO1/2, 5′-TGTGCTCATCTGTTTACTTCTTTTTC-3′; mutant FoxO1/2, 5′-TGTGCTCATCTGggTACTTCTTTTTC-3′; insulin-like growth factor binding protein-1/insulin response element, 5′-CACTAGCAAAACAAACTTATTTTGAACAC-3′. Protein Isolation and Blotting—Total cell lysates from primary mouse hepatocytes or mouse livers infected with either adenovirus expressing GFP or FGF15 were fractionated on 8% SDS-PAGE, transferred to nitrocellulose membranes, and analyzed first by Ponceau S staining to confirm that equal amounts of total protein were both loaded and transferred in each lane. Then the blot was incubated with an indicated antibody followed by a secondary antibody conjugated to horseradish peroxidase. Reactivity was then detected with the ECL kit (Pierce). The primary antibodies were obtained as follows: anti-hemagglutinin (HA) (clone 12CA5) from Roche Applied Science, total FoxO1, phospho-FoxO1 (Thr-24) and phospho-Akt (Ser473) from Cell Signaling Technology, total Akt from Santa Cruz Biotechnology, anti-FLAG M2 mouse monoclonal from Sigma, mouse FGFR4 from R&D systems (AF2265), and mouse β-actin from Sigma (A1978). Antibody against hepatic nuclear factor 4 was from Dr. F. Sladek (University of California, Riverside, CA). Adenovirus Infection of Primary Mouse Hepatocytes—Prima" @default.
• W2011132677 created "2016-06-24" @default.
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• W2011132677 date "2009-04-01" @default.
• W2011132677 modified "2023-10-18" @default.
• W2011132677 title "FGF15/FGFR4 Integrates Growth Factor Signaling with Hepatic Bile Acid Metabolism and Insulin Action" @default.
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• W2011132677 doi "https://doi.org/10.1074/jbc.m808747200" @default.
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