Hepatic Fatty Acid Transporter Cd36 Is a Common Target of LXR, PXR, and PPARγ in Promoting Steatosis

Background & Aims: Liver X receptor (LXR) is known to promote hepatic lipogenesis by activating the lipogenic transcriptional factor sterol regulatory element-binding protein (Srebp). Pregnane X receptor (PXR), a previously known “xenobiotic receptor,” could mediate a Srebp-independent lipogenic pathway by activating the free fatty acid uptake transporter Cd36. The goal of this study is to investigate further the role of Cd36 in hepatic steatosis. Methods: Wild-type, LXR transgenic, PXR transgenic, and Cd36 null mice were used to study the regulation of Cd36 and other hepatic lipogenic genes and the implication of this regulation in hepatic steatosis. Promoter sequences of Cd36 and peroxisome proliferator-activated receptor (PPAR) γ were cloned, and their respective regulation by LXR and PXR was investigated by combinations of receptor-DNA binding and reporter gene assays. Results: We showed that genetic (transgene) or pharmacologic (ligands) activation of LXR induced Cd36. Promoter analysis established Cd36 as a novel transcription target of LXRα. Moreover, the hepatic steatosis induced by LXR agonists was largely abolished in Cd36 null mice. We also showed that PPARγ, a positive regulator of Cd36, is a transcriptional target of PXR, suggesting that PXR can regulate Cd36 directly or through its activation of PPARγ. Interestingly, both LXR-mediated Cd36 regulation and PXR-mediated PPARγ regulation are liver specific. Conclusions: We conclude that Cd36 is a shared target of LXR, PXR, and PPARγ. The network of CD36 regulation by LXR, PXR, and PPARγ establishes this free fatty acid transporter as a common target of orphan nuclear receptors in their mediation of lipid homeostasis. Background & Aims: Liver X receptor (LXR) is known to promote hepatic lipogenesis by activating the lipogenic transcriptional factor sterol regulatory element-binding protein (Srebp). Pregnane X receptor (PXR), a previously known “xenobiotic receptor,” could mediate a Srebp-independent lipogenic pathway by activating the free fatty acid uptake transporter Cd36. The goal of this study is to investigate further the role of Cd36 in hepatic steatosis. Methods: Wild-type, LXR transgenic, PXR transgenic, and Cd36 null mice were used to study the regulation of Cd36 and other hepatic lipogenic genes and the implication of this regulation in hepatic steatosis. Promoter sequences of Cd36 and peroxisome proliferator-activated receptor (PPAR) γ were cloned, and their respective regulation by LXR and PXR was investigated by combinations of receptor-DNA binding and reporter gene assays. Results: We showed that genetic (transgene) or pharmacologic (ligands) activation of LXR induced Cd36. Promoter analysis established Cd36 as a novel transcription target of LXRα. Moreover, the hepatic steatosis induced by LXR agonists was largely abolished in Cd36 null mice. We also showed that PPARγ, a positive regulator of Cd36, is a transcriptional target of PXR, suggesting that PXR can regulate Cd36 directly or through its activation of PPARγ. Interestingly, both LXR-mediated Cd36 regulation and PXR-mediated PPARγ regulation are liver specific. Conclusions: We conclude that Cd36 is a shared target of LXR, PXR, and PPARγ. The network of CD36 regulation by LXR, PXR, and PPARγ establishes this free fatty acid transporter as a common target of orphan nuclear receptors in their mediation of lipid homeostasis. Lipid homeostasis is maintained by balanced lipogenesis and fat burning. Liver plays an essential role in lipogenesis. One major source of hepatic lipids is de novo fatty acid synthesis, in which the lipogenic transcriptional factor sterol regulatory element-binding protein (SREBP) has an important role. Activation of SREBP induces the expression of a battery of lipogenic enzymes including fatty acid synthase (Fas), stearoyl CoA desaturase-1 (Scd-1), and acetyl CoA carboxylase 1 (Acc-1).1Repa J.J. Liang G. Ou J. et al.Regulation of mouse sterol regulatory element-binding protein-1c gene (SREBP-1c) by oxysterol receptors, LXRα and LXRβ.Genes Dev. 2000; 14: 2819-2830Crossref PubMed Scopus (1399) Google Scholar, 2Schultz J.R. Tu H. Luk A. et al.Role of LXRs in control of lipogenesis.Genes Dev. 2000; 14: 2831-2838Crossref PubMed Scopus (1380) Google Scholar, 3Peet D.J. Turley S.D. Ma W. et al.Cholesterol and bile acid metabolism are impaired in mice lacking the nuclear oxysterol receptor LXR α.Cell. 1998; 93: 693-704Abstract Full Text Full Text PDF PubMed Scopus (1230) Google Scholar, 4Joseph S.B. Laffitte B.A. Patel P.H. et al.Direct and indirect mechanisms for regulation of fatty acid synthase gene expression by liver X receptors.J Biol Chem. 2002; 277: 11019-11025Crossref PubMed Scopus (615) Google Scholar, 5Chawla A. Repa J.J. Evans R.M. et al.Nuclear receptors and lipid physiology: opening the X-files.Science. 2001; 294: 1866-1870Crossref PubMed Scopus (1671) Google Scholar, 6Repa J.J. Mangelsdorf D.J. The liver X receptor gene team: potential new players in atherosclerosis.Nat Med. 2002; 8: 1243-1248Crossref PubMed Scopus (337) Google Scholar, 7Francis G.A. Annicotte J.S. Auwerx J. Liver X receptors: Xcreting Xol to combat atherosclerosis.Trends Mol Med. 2002; 8: 455-458Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar, 8Zelcer N. Tontonoz P. Liver X receptors as integrators of metabolic and inflammatory signaling.J Clin Invest. 2006; 116: 607-614Crossref PubMed Scopus (756) Google Scholar Another major source of hepatic lipids is circulating free fatty acids (FFAs).9Bradbury M.W. Lipid metabolism and liver inflammation. I. Hepatic fatty acid uptake: possible role in steatosis.Am J Physiol Gastrointest Liver Physiol. 2006; 290: G194-G198Crossref PubMed Scopus (257) Google Scholar Upon uptake by hepatocytes, FFAs can be converted to triglycerides, especially when intrahepatic FFAs are in excess.9Bradbury M.W. Lipid metabolism and liver inflammation. I. Hepatic fatty acid uptake: possible role in steatosis.Am J Physiol Gastrointest Liver Physiol. 2006; 290: G194-G198Crossref PubMed Scopus (257) Google ScholarThe activation of orphan nuclear receptors, including the liver X receptors (LXRs), peroxisome proliferator-activated receptor γ (PPARγ), and pregnane X receptor (PXR), has been associated with hepatic steatosis. Both LXR α and β have been shown to promote lipogenesis by activating SREBP-1c and its target genes.1Repa J.J. Liang G. Ou J. et al.Regulation of mouse sterol regulatory element-binding protein-1c gene (SREBP-1c) by oxysterol receptors, LXRα and LXRβ.Genes Dev. 2000; 14: 2819-2830Crossref PubMed Scopus (1399) Google Scholar, 2Schultz J.R. Tu H. Luk A. et al.Role of LXRs in control of lipogenesis.Genes Dev. 2000; 14: 2831-2838Crossref PubMed Scopus (1380) Google Scholar, 3Peet D.J. Turley S.D. Ma W. et al.Cholesterol and bile acid metabolism are impaired in mice lacking the nuclear oxysterol receptor LXR α.Cell. 1998; 93: 693-704Abstract Full Text Full Text PDF PubMed Scopus (1230) Google Scholar, 4Joseph S.B. Laffitte B.A. Patel P.H. et al.Direct and indirect mechanisms for regulation of fatty acid synthase gene expression by liver X receptors.J Biol Chem. 2002; 277: 11019-11025Crossref PubMed Scopus (615) Google Scholar, 5Chawla A. Repa J.J. Evans R.M. et al.Nuclear receptors and lipid physiology: opening the X-files.Science. 2001; 294: 1866-1870Crossref PubMed Scopus (1671) Google Scholar, 6Repa J.J. Mangelsdorf D.J. The liver X receptor gene team: potential new players in atherosclerosis.Nat Med. 2002; 8: 1243-1248Crossref PubMed Scopus (337) Google Scholar, 7Francis G.A. Annicotte J.S. Auwerx J. Liver X receptors: Xcreting Xol to combat atherosclerosis.Trends Mol Med. 2002; 8: 455-458Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar, 8Zelcer N. Tontonoz P. Liver X receptors as integrators of metabolic and inflammatory signaling.J Clin Invest. 2006; 116: 607-614Crossref PubMed Scopus (756) Google Scholar In addition to the LXR-SREBP pathway, accumulating evidence has suggested that LXRs have additional transcriptional targets, such as PPARγ,10Seo J.B. Moon H.M. Kim W.S. et al.Activated liver X receptors stimulate adipocyte differentiation through induction of peroxisome proliferator-activated receptor γ expression.Mol Cell Biol. 2004; 24: 3430-3444Crossref PubMed Scopus (206) Google Scholar carbohydrate response element binding protein (ChREBP),11Cha J.Y. Repa J.J. The liver X receptor (LXR) and hepatic lipogenesis The carbohydrate-response element-binding protein is a target gene of LXR.J Biol Chem. 2007; 282: 743-751Crossref PubMed Scopus (362) Google ScholarSCD-1,12Chu K. Miyazaki M. Man W.C. et al.Stearoyl-coenzyme A desaturase 1 deficiency protects against hypertriglyceridemia and increases plasma high-density lipoprotein cholesterol induced by liver X receptor activation.Mol Cell Biol. 2006; 26: 6786-6798Crossref PubMed Scopus (179) Google Scholar and lipoprotein lipase (LPL),13Zhang Y. Repa J.J. Gauthier K. et al.Regulation of lipoprotein lipase by the oxysterol receptors, LXRα and LXRβ.J Biol Chem. 2001; 276: 43018-43024Crossref PubMed Scopus (242) Google Scholar which may have also contributed to the lipogenic effect.Other than its role in adipocyte differentiation, PPARγ is also known to promote hepatic steatosis. Increased expression of PPARγ has been reported in high-fat diet-induced liver steatosis.14Inoue M. Ohtake T. Motomura W. et al.Increased expression of PPARγ in high fat diet-induced liver steatosis in mice.Biochem Biophys Res Commun. 2005; 336: 215-222Crossref PubMed Scopus (296) Google Scholar Over expression of PPARγ1 in liver of PPARα null mice induced the expression of lipogenesis-related genes, leading to hepatic steatosis.15Yu S. Matsusue K. Kashireddy P. et al.Adipocyte-specific gene expression and adipogenic steatosis in the mouse liver due to peroxisome proliferator-activated receptor γ1 (PPARγ1) overexpression.J Biol Chem. 2003; 278: 498-505Crossref PubMed Scopus (503) Google ScholarPPARγ2 has also been reported to induce lipid accumulation in hepatocytes.16Schadinger S.E. Bucher N.L.R. Schreiber B.M. et al.PPARγ2 regulates lipogenesis and lipid accumulation in steatotic hepatocytes.Am J Physiol Endocrinol Metab. 2005; 288: E1195-E1205Crossref PubMed Scopus (305) Google Scholar One of the lipogenic genes induced by PPARγ was Cd36,13Zhang Y. Repa J.J. Gauthier K. et al.Regulation of lipoprotein lipase by the oxysterol receptors, LXRα and LXRβ.J Biol Chem. 2001; 276: 43018-43024Crossref PubMed Scopus (242) Google Scholar a membrane receptor capable of uptaking modified forms of low-density lipoproteins (LDL) and fatty acids from circulation.17Abumrad N.A. El-Maghrabi M.R. Amri E.Z. et al.Cloning of a rat adipocyte membrane protein implicated in binding or transport of long-chain fatty acids that is induced during preadipocyte differentiation Homology with human CD36.J Biol Chem. 1993; 268: 17665-17668Abstract Full Text PDF PubMed Google Scholar, 18Endemann G. Stanton L.W. Madden K.S. et al.CD36 is a receptor for oxidized low-density lipoprotein.J Biol Chem. 1993; 268: 11811-11816Abstract Full Text PDF PubMed Google Scholar, 19Cohen P.A. Frazier W.A. Hoff H.F. et al.Macrophage scavenger receptor CD36 is the major receptor for LDL modified by monocyte-generated reactive nitrogen species.J Clin Invest. 2000; 105: 1095-1108Crossref PubMed Scopus (8) Google Scholar, 20Febbraio M. Guy E. Coburn C. et al.The impact of overexpression and deficiency of fatty acid translocase (FAT)/CD36.Mol Cell Biochem. 2002; 239: 193-197Crossref PubMed Scopus (70) Google Scholar Mice deficient of Cd36 exhibited a wide array of phenotypes, including defective uptake and utilization of fatty acids in skeletal muscle and adipose tissue, and inhibition of macrophage foam cell formation and atherogenesis.21Coburn C.T. Knapp Jr, F.F. Febbraio M. et al.Defective uptake and utilization of long-chain fatty acids in muscle and adipose tissues of CD36 knockout mice.J Biol Chem. 2000; 275: 32523-32529Crossref PubMed Scopus (517) Google Scholar, 22Febbraio M. Abumrad N.A. Hajjar D.P. et al.A null mutation in murine CD36 reveals an important role in fatty acid and lipoprotein metabolism.J Biol Chem. 1999; 274: 19055-19062Crossref PubMed Scopus (651) Google Scholar, 23Febbraio M. Podrez E.A. Smith J.D. et al.Targeted disruption of the class B scavenger receptor CD36 protects against atherosclerotic lesion development in mice.J Clin Invest. 2000; 105: 1049-1056Crossref PubMed Scopus (814) Google Scholar, 24Rahaman S.O. Lennon D.J. Febbraio M. et al.A CD36-dependent signaling cascade is necessary for macrophage foam cell formation.Cell Metab. 2006; 4: 211-221Abstract Full Text Full Text PDF PubMed Scopus (374) Google Scholar Activation of Cd36 by PPARγ in the liver was consistent with an earlier report that Cd36 is a PPARγ target gene in macrophages.25Tontonoz P. Nagy L. Alvarez J.G. et al.PPARγ promotes monocyte/macrophage differentiation and uptake of oxidized LDL.Cell. 1998; 93: 241-252Abstract Full Text Full Text PDF PubMed Scopus (1602) Google Scholar Hepatic fatty acid uptake, such as that mediated by Cd36, plays an important role in steatosis.9Bradbury M.W. Lipid metabolism and liver inflammation. I. Hepatic fatty acid uptake: possible role in steatosis.Am J Physiol Gastrointest Liver Physiol. 2006; 290: G194-G198Crossref PubMed Scopus (257) Google Scholar LXR has been reported to activate PPARγ in the liver.10Seo J.B. Moon H.M. Kim W.S. et al.Activated liver X receptors stimulate adipocyte differentiation through induction of peroxisome proliferator-activated receptor γ expression.Mol Cell Biol. 2004; 24: 3430-3444Crossref PubMed Scopus (206) Google Scholar It is unknown whether LXR can regulate Cd36 directly or through its activation of PPARγ, and, if so, the regulation of Cd36 may represent a novel mechanism by which LXR promotes hepatic steatosis.PXR also has a role in hepatic steatosis. We showed recently that activation of PXR in mice was sufficient to induce hepatic triglyceride accumulation in a Srebp-1c-independent manner.26Zhou J. Zhai Y. Mu Y. et al.A novel pregnane X receptor-mediated and sterol regulatory element-binding protein-independent lipogenic pathway.J Biol Chem. 2006; 281: 15013-15020Crossref PubMed Scopus (290) Google Scholar The PXR-induced lipid accumulation was associated with increased expression of Cd36 and several accessory lipogenic enzymes, including Scd-1 and long-chain free fatty acid elongase (Fae). Further studies established the mouse Cd36 as a direct transcriptional target of PXR.26Zhou J. Zhai Y. Mu Y. et al.A novel pregnane X receptor-mediated and sterol regulatory element-binding protein-independent lipogenic pathway.J Biol Chem. 2006; 281: 15013-15020Crossref PubMed Scopus (290) Google Scholar In the same study, PPARγ was shown to be activated by PXR, but it is unknown whether PPARγ is a direct transcriptional target of PXR.In this study, we show that LXR regulates Cd36 expression in the liver and that the steatotic effect of LXR agonists is largely abolished in the Cd36 null mice. We also show PPARγ is a transcriptional target of PXR, suggesting that PXR can regulate Cd36 directly or through its activation of PPARγ. We propose that Cd36 is a common target of LXR, PXR, and PPARγ in promoting hepatic steatosis.Materials and MethodsAnimals and DrugsFABP-VP-hPXR transgenic,27Gong H. Singh S.V. Singh S.P. et al.Orphan nuclear receptor pregnane X receptor sensitizes oxidative stress responses in transgenic mice and cancerous cells.Mol Endocrinol. 2006; 20: 279-290Crossref PubMed Scopus (84) Google Scholar FABP-VP-LXR transgenic,28Uppal H. Saini S.P.S. Moschetta A. et al.Activation of LXRs prevents bile acid toxicity and cholestasis in female mice.Hepatology. 2007; 45: 422-432Crossref PubMed Scopus (104) Google Scholar PXR null mice, and Cd36 null mice22Febbraio M. Abumrad N.A. Hajjar D.P. et al.A null mutation in murine CD36 reveals an important role in fatty acid and lipoprotein metabolism.J Biol Chem. 1999; 274: 19055-19062Crossref PubMed Scopus (651) Google Scholar have been previously described. VP-LXR/VP-PXR double transgenic mice were created by crossbreeding. Transgenic mice are in mixed background of C57BL/6J and SvJ129, and Cd36 null mice are in sixth generation of backcross to C57BL/6J mice. Background and age (8–10 weeks old)-matched littermate controls were used for all experiments. Wild-type CD-1 female mice purchased from Charles River (Wilmington, MA) were used for hydrodynamic liver transfection. The use of mice has complied with federal guidelines and institutional policies. 22(R)-hydroxycholesterol and T0901317 (TO1317) were purchased from Cayman (Ann Arbor, MI). GW3965 was synthesized in-house following a published scheme.29Collins J.L. Fivush A.M. Watson M.A. et al.Identification of a nonsteroidal liver X receptor agonist through parallel array synthesis of tertiary amines.J Med Chem. 2002; 45: 1963-1966Crossref PubMed Scopus (366) Google ScholarNorthern Blot Analysis and Real-Time Reverse-Transcription Polymerase Chain ReactionTotal RNA was prepared using TRIZOL reagent. Northern blot analysis was performed as previously described.30Saini S.P. Mu Y. Gong H. et al.Dual role of orphan nuclear receptor pregnane X receptor in bilirubin detoxification in mice.Hepatology. 2005; 41: 497-505Crossref PubMed Scopus (76) Google Scholar, 31Uppal H. Toma D. Saini S.P. et al.Combined loss of orphan receptors PXR and CAR heightens sensitivity to toxic bile acids in mice.Hepatology. 2005; 41: 168-176Crossref PubMed Scopus (96) Google Scholar Membranes were stripped and reprobed with Gapdh complementary DNA (cDNA) for loading control. Real-time reverse-transcription polymerase chain reaction (RT-PCR) using the CYBR-green reagents was performed as we described before.26Zhou J. Zhai Y. Mu Y. et al.A novel pregnane X receptor-mediated and sterol regulatory element-binding protein-independent lipogenic pathway.J Biol Chem. 2006; 281: 15013-15020Crossref PubMed Scopus (290) Google Scholar PCR primer sequences are listed in the Supplemental Table (see Supplemental Table online at www.gastrojournal.org).Supplemental TablePrimer SequencesPrimers for real-time PCR: Cd36 5′-GGAACTGTGGGCTCATTGC-3′ 5′-CATGAGAATGCCTCCAAACAC-3′ CD36 5′-AAATAAACCTCCTTGGCCTGA-3′ 5′-GCAACAAACATCACCACACC-3′ Fae 5′-TTCCGAGTCTCCCGGAAGT-3′ 5′-ACAGCCCATCAGCATCTGAGT-3′ FAE 5′-TTCCGAGTCTCCCGGAAGT-3′ 5′-ACAGCCCATCAGCATCTGAGT-3′ Fas 5′-GCTGCGGAAACTTCAGGAAAT-3′ 5′-AGAGACGTGTCACTCCTGGACTT-3′ Lpl 5′-GCCCAGCAACATTATCCAGT-3′ 5′-AGCCCTTTCTCAAAGGCTTC-3′ Lxrα 5′-GCCTCAATGCCTGATGTTTC-3′ 5′-CTGCATCTTGAGGTTCTGTCTTC-3′ Pparγ 5′-CACAATGCCATCAGGTTTGG-3′ 5′-GCTGGTCGATATCACTGGAGATC-3′ PPARγ 5′-CGAGAAGGAGAAGCTGTTGG-3′ 5′-TCAGCGGGAAGGACTTTATG-3′ Pxr 5′-CAAGGCCAATGGCTACCA-3′ 5′-CGGGTGATCTCGCAGGTT-3′ Scd-1 5′-TGCCCCTGCGGATCTT-3′ 5′-GCCCATTCGTACACGTCATT-3′ SCD-1 5′-TGTTCGTTGCCACTTTCTTG-3′ 5′-GCTAATGTTCTTGTCATAAGGAC-3′ Srebp-1c 5′-GGCACTAAGTGCCCTCAACCT-3′ 5′-GGCACTAAGTGCCCTCAACCT-3′ VP-PXR 5′-CTACGGCGCTCTGGATATGG-3′ 5′-CTCTGTGTCCTCACAGTGTAC-3′Primers to clone the mouse PPARγ2 gene promoter: Forward 5′-CGGGGTACCGAAAGGAAGGGAAGGAGGAA-3′ Reverse 5′-CGGGATCCGCTCTGGGTCAACAGGAGAA-3′Primers for ChIP assays: Cd36/DR-7 5′-CCCCTTCTATACTTTGTTTTCCATT-3′ 5′-CTGAAAGTCTTCAGGTTCATGCTA-3′ Srebp-1c 5′-CTCTTTTCGGGGATGGTTG-3′ 5′-GGTTTCTCCCGGTGCTCT-3′ PPARγ2/DR3-1 5′-CACTGAATTATATTAGGTC-3′ 5′-TACCCTGCTAGGTTGGCAAG-3′ PPARγ2/DR3-2 5′-ACCAGGTGAAATTGCTGACC-3′ 5′-GAAAGTGGGGGCTGGTCTA-3′ Cyp3a11/DR-3 5′-ATGGGTAGACCGTGACAAC-3′ 5′-GATCAAGCCAGTCGATGGATC-3′ Gapdh 5′-TACTAGCGGTTTTACGGGCG-3′ 5′-TCGAACAGGAGGAGCAGAGAGCGA-3′ Open table in a new tab Plasmid Constructs and TransfectionsThe mouse Cd36 promoter reporter gene was described previously.26Zhou J. Zhai Y. Mu Y. et al.A novel pregnane X receptor-mediated and sterol regulatory element-binding protein-independent lipogenic pathway.J Biol Chem. 2006; 281: 15013-15020Crossref PubMed Scopus (290) Google Scholar The 5′ regulatory sequence (−2326 base pair [bp] to +104 bp) of the mouse PPARγ2 gene promoter (NCBI Genbank NM_011146) was cloned by PCR see (Supplemental Table online at www.gastrojournal.org for sequences). The PCR-amplified sequences were cloned into the pGL3 vector, and site-directed mutagenesis was performed by the PCR overextension method.32Xie W. Barwick J.L. Simon C.M. et al.Reciprocal activation of xenobiotic response genes by nuclear receptors SXR/PXR and CAR.Genes Dev. 2000; 14: 3014-3023Crossref PubMed Scopus (457) Google Scholar To generate synthetic tk reporter genes, 3 copies of the wild-type or mutant Cd36/DR-7 or PPARγ2/DR-3 elements were synthesized and inserted into the tk-Luc vector. Transient transfections were performed on HepG2 cells seeded onto 48-well tissue culture plates.26Zhou J. Zhai Y. Mu Y. et al.A novel pregnane X receptor-mediated and sterol regulatory element-binding protein-independent lipogenic pathway.J Biol Chem. 2006; 281: 15013-15020Crossref PubMed Scopus (290) Google Scholarpparγ small interfering (sirna) (catalogue No. sc-29455) and the control scrambled siRNA (catalogue No. 1027284) were purchased from Santa Cruz Biotechnology, Inc (Santa Cruz, CA) and QIAGEN (Valencia, CA), respectively. Transfected cells were then treated with drugs for 24 hours before luciferase assayed. Luciferase activity was normalized against the cotransfected β-galactosidase activity.Electrophoretic Mobility Shift AssayElectrophoretic mobility shift assays (EMSAs) using TNT in vitro synthesized receptor proteins were performed as previously described.33Saini S.P. Sonoda J. Xu L. et al.A novel constitutive androstane receptor-mediated and CYP3A-independent pathway of bile acid detoxification.Mol Pharmacol. 2004; 65: 292-300Crossref PubMed Scopus (210) Google Scholar Probe sequences are shown in Figures. Fifty-fold excess of unlabeled DNA was used for competitions.Chromatin ImmunoprecipitationEight-week-old, wild-type, female CD-1 mice were treated with intraperitoneal (IP) injection of dimethyl sulfoxide (DMSO) or pregnenolone-16α-carbonitrile (PCN) (40 mg/kg) or TO1317 (50 mg/kg) 30 minutes before being transfected with the pCMX-HA-mPXR/pCMX-HA-mLXRα or pCMX-HA control plasmid by a hydrodynamic gene delivery method.26Zhou J. Zhai Y. Mu Y. et al.A novel pregnane X receptor-mediated and sterol regulatory element-binding protein-independent lipogenic pathway.J Biol Chem. 2006; 281: 15013-15020Crossref PubMed Scopus (290) Google Scholar Mice were killed 8 hours after transfection, and liver tissues were harvested for chromatin immunoprecipitation (ChIP) assay using a hemagglutinin (HA) antibody (Santa Cruz Biotechnology; catalogue No. sc-805) as we described previously.26Zhou J. Zhai Y. Mu Y. et al.A novel pregnane X receptor-mediated and sterol regulatory element-binding protein-independent lipogenic pathway.J Biol Chem. 2006; 281: 15013-15020Crossref PubMed Scopus (290) Google Scholar An HA-antibody from Cell Signaling (catalogue No. 2367) (Danvers, MA) was used for Western blot analysis of the HA-tagged protein expression. PCR primer sequences are listed in the Supplemental Table (see Supplemental Table online at www.gastrojournal.org). The PCR conditions are 95°C, 30 seconds; 55°C, 30 seconds; 72°C, 40 seconds for 28 cycles.Measurements of Circulating and Tissue LipidsPlasma levels of triglycerides and cholesterol were measured in overnight-fasted mice using assay kits from Stanbio Laboratory (Boerne, TX). To measure liver lipid content, tissues were homogenized, and lipids were extracted before measurements as we previously described.26Zhou J. Zhai Y. Mu Y. et al.A novel pregnane X receptor-mediated and sterol regulatory element-binding protein-independent lipogenic pathway.J Biol Chem. 2006; 281: 15013-15020Crossref PubMed Scopus (290) Google Scholar The liver triglycerides were measured in the fed state, and the cholesterol measurements refer to both free and esterified cholesterol.Primary Macrophage IsolationSix-week-old C57BL/6J male mice received an IP injection of 0.5 mL concanavolin A (80 μg/mL) (Sigma Chemical Co, St. Louis, MO; catalogue No. 11028-71-0) 3 days prior to death. Peritoneal macrophages were obtained by IP injecting and withdrawing of 5 mL phosphate-buffered saline. Cell pellets were washed and then plated in minimum essential medium.ResultsActivation of LXR Induced the Expression of Cd36 in a Liver-Specific MannerWe have recently created transgenic mice that express the activated LXRα (VP-LXR) or PXR (VP-PXR) in the liver and intestine under the control of the rat liver fatty acid-binding protein promoter.26Zhou J. Zhai Y. Mu Y. et al.A novel pregnane X receptor-mediated and sterol regulatory element-binding protein-independent lipogenic pathway.J Biol Chem. 2006; 281: 15013-15020Crossref PubMed Scopus (290) Google Scholar, 27Gong H. Singh S.V. Singh S.P. et al.Orphan nuclear receptor pregnane X receptor sensitizes oxidative stress responses in transgenic mice and cancerous cells.Mol Endocrinol. 2006; 20: 279-290Crossref PubMed Scopus (84) Google Scholar, 28Uppal H. Saini S.P.S. Moschetta A. et al.Activation of LXRs prevents bile acid toxicity and cholestasis in female mice.Hepatology. 2007; 45: 422-432Crossref PubMed Scopus (104) Google Scholar VP-LXR and VP-PXR were created by fusing the VP16 activation domain of the herpes simplex virus to the amino terminal of mouse LXRα and human PXR, respectively. Our recent study showed that PXR promotes an Srebp-independent lipogenic pathway by activating Cd36, as well as PPARγ, Fae, and Scd-1.26Zhou J. Zhai Y. Mu Y. et al.A novel pregnane X receptor-mediated and sterol regulatory element-binding protein-independent lipogenic pathway.J Biol Chem. 2006; 281: 15013-15020Crossref PubMed Scopus (290) Google Scholar To determine whether LXR can also mediate Srebp-independent steatosis, the hepatic expression of these genes in VP-LXR mice was evaluated and compared with that in VP-PXR mice.Real-time RT-PCR analysis showed that the expression of Cd36, PPARγ, Fae, and Scd-1 was induced in VP-PXR transgenic mice as expected (Figure 1A).26Zhou J. Zhai Y. Mu Y. et al.A novel pregnane X receptor-mediated and sterol regulatory element-binding protein-independent lipogenic pathway.J Biol Chem. 2006; 281: 15013-15020Crossref PubMed Scopus (290) Google Scholar To our surprise, the VP-LXR transgene also induced the expression of Cd36, in addition to its expected activation of PPARγ, Fae, and Scd-1.10Seo J.B. Moon H.M. Kim W.S. et al.Activated liver X receptors stimulate adipocyte differentiation through induction of peroxisome proliferator-activated receptor γ expression.Mol Cell Biol. 2004; 24: 3430-3444Crossref PubMed Scopus (206) Google Scholar, 12Chu K. Miyazaki M. Man W.C. et al.Stearoyl-coenzyme A desaturase 1 deficiency protects against hypertriglyceridemia and increases plasma high-density lipoprotein cholesterol induced by liver X receptor activation.Mol Cell Biol. 2006; 26: 6786-6798Crossref PubMed Scopus (179) Google Scholar, 34Matsuzaka T. Shimano H. Yahagi N. et al.Cloning and characterization of a mammalian fatty acyl-CoA elongase as a lipogenic enzyme regulated by SREBPs.J Lipid Res. 2002; 43: 911-920Abstract Full Text Full Text PDF PubMed Google Scholar Activation of Cd36 was also seen in wild-type mice treated with the LXR agonist 22(R)-hydroxycholesterol (22(R)) (Figure 1B). A similar 22(R) effect on CD36 expression was observed in primary human hepatocytes (Figure 1C), suggesting that this regulation is conserved in humans.The VP-LXR transgene was targeted to both liver and intestine.28Uppal H. Saini S.P.S. Moschetta A. et al.Activation of LXRs prevents bile acid toxicity and cholestasis in female mice.Hepatology. 2007; 45: 422-432Crossref PubMed Scopus (104) Google Scholar Northern blot analysis showed that the activation of Cd36 was liver specific because the transgene had little effect on intestinal Cd36 expression (Figure 1D). The liver-specific Cd36 activation was also confirmed in LXR agonists-treated wild-type mice (data not shown). 22(R) also failed to induce CD36 in THP-1 human macrophages or primary mouse macrophages (Figure 1E). In the same macrophage cultures, CD36 was effectively activated by the PPARγ agonist troglitazone (Figure 1E). We found that the activation of PPARγ in VP-LXR transgenic mice was also liver specific (Figure 1F).Cd36 Is a Transcriptional Target of LXRTo determine whether Cd36 is a transcriptional target of LXR, we analyzed 5′ regulatory sequences of the mouse Cd36 gene and found a DR-7 type nuclear receptor response element located from nucleotide (nt) −998 to −1117 (Figure 2A). EMSA showed that the VP-LXRα/RXR heterodimers bound to the radiolabeled Cd36/DR-7 sequence (Figure 2B, upper panel). The binding was specific, as evidenced by the efficient competition by excess unlabeled wild-type Cd36/DR-7 or Srebp/DR-4. The wild-type LXRα/RXR heterodimers also bound to Cd36/DR-7 (data not shown). When radiolabeled Srebp/DR-4 probe was used, the cold DR-7 can compete for the binding, but the competition was not as efficient as DR-4 (Figure 2B, bottom panel), suggesting that the binding affinity of DR-7 is not as high as DR-4. ChIP assay was used to demonstrate the recruitment of LXRα onto the Cd36 promoter in vivo. In this experiment, an HA-tagged LXRα or an HA vector control plasmid was transfected into wild-type mouse liver.26Zhou J. Zhai Y. Mu Y. et al.A novel pregnane X receptor-mediated and sterol regulatory element-binding protein-independent lipogenic pathway.J Biol Chem. 2006; 281: 15013-15020Crossref PubMed Scopus (290) Google Scholar Transfected mice were then treated with DMSO solvent or TO1317 (40 mg/kg) for 8 hours before death, and the ChIP assay was performed utilizing an anti-HA antibody.26Zhou J. Zhai Y. Mu Y. et al.A novel pregnane X receptor-mediated and sterol regulatory element-bind