Yannick Parmentier,5 Antonio Sa-Cunha,6 Bertrand Suc,7 Jean-Michel Fabre,1,8 Francis Navarro,1,9 Jeanne Ramos,10
Urs Meyer,4 Patrick Maurel,1 Marie-Jose Vilarem,1 and Jean-Marc Pascussi1
The pregnane X receptor (PXR) initially isolated as a nuclear receptor regulating xenobiotic anddrug metabolism and elimination, seems to play an endobiotic role by affecting lipid homeosta-sis. In mice, PXR affects lipid homeostasis and increases hepatic deposit of triglycerides. In thisstudy, we show that, in human hepatocyte, PXR activation induces an increase of de novolipogenesis through the up-regulation of S14. S14 was first identified as a thyroid-responsivegene and is known to transduce hormone-related and nutrient-related signals to genes involvedin lipogenesis through a molecular mechanism not yet elucidated. We demonstrate that S14 is anovel transcriptional target of PXR. In addition, we report an increase of fatty acid synthase(FASN) and adenosine triphosphate citrate lyase genes expression after PXR activation in humanhepatocyte, leading to an increase of fatty acids accumulation and de novo lipogenesis. RNAinterference of the expression of S14 proportionally decreases the FASN induction, whereas S14overexpression in human hepatic cells provokes an increase of fatty acids accumulation andlipogenesis. These results demonstrate for the first time that xenobiotic or drug-activated PXRpromote aberrant hepatic de novo lipogenesis via activation of the nonclassical S14 pathway. Inaddition, these data suggest that the up-regulation of S14 by PXR may promote aberrant hepaticlipogenesis and hepatic steatosis in human hepatocytes. (HEPATOLOGY 2009;49:2068-2079.)
Lipid homeostasis is achieved by complex physio-logical mechanisms. Disruptions of lipid forma-tion and catabolism have been implicated in
various metabolic diseases such as obesity and diabetes.Hepatic lipid homeostasis is tightly maintained by bal-anced lipid synthesis, catabolism (�-oxidation), and up-take/secretion. The liver is a major organ for lipogenesisand expresses high levels of lipogenic enzymes, such asfatty acid synthase (FASN), adenosine triphosphate ci-trate lyase, and stearoyl-CoA desaturase-1. Several recep-tors have been implicated in lipid homeostasis, such as theliver X receptor alpha and beta isoforms (LXR� andLXR�1) or thyroid hormone receptor (TR2). The effect ofLXR on lipogenesis involves both direct and indirectmechanisms. LXR/retinoid X receptor (RXR) het-erodimers bind lipogenic gene promoters, such as FASN,or regulate lipogenic gene expression by controlling levelsof sterol regulatory element binding protein (SREBP)-1c,a transcriptional factor known to regulate the expressionof a battery of lipogenic enzymes.3-5 In addition, thyroidhormone (T3) is known to regulate hepatic lipogenesisafter binding to TR, which binds to the target DNA se-quence thyroid response element (TRE), leading to anincreased transcription of several genes involved in lipo-genesis.6,7
The pregnane X receptor (PXR), initially isolated as anuclear receptor regulating xenobiotic and drug metabo-lism and elimination,8 plays an endobiotic role by affect-
Abbreviations: cDNA, complementary DNA; ChREBP, carbohydrate response ele-ment binding protein; DMSO, dimethylsulfoxide; FASN, fatty acid synthase; GFP,green fluorescent protein; HHPC, lipogenic pathway in human hepatocytes; HNF,hepatocyte nuclear factor; LXR, liver X receptor; mRNA, messenger RNA; PCN, preg-nenolone 16 alpha-carbonitrile; PCR, polymerase chain reaction; PPAR�, peroxisomeproliferator-activated receptor gamma; PXR, pregnane X receptor; RT-PCR, reversetranscription polymerase chain reaction; RXR, retinoid X receptor; SFN, 4-methylsulfi-nylbutyl isothiocyanate; siNC, irrelevant control gene; siPXR, duplexes of small interfer-ing RNA targeting human pregnane X receptor; siRNA, small interfering RNA; SREBP,sterol-regulatory element binding protein; TAG, triacylglycerol; T3, thyroid hormone;TR, thyroid hormone receptor; TRE, thyroid response element.
From 1Institut National de la Sante et de la Recherche Medicale (Inserm), U632,Montpellier, F-34293 France; Universite Montpellier 1, UMR-S632, Montpellier,F-34293 France; 2INSERM ERI22/EA 4173, Faculte de Medecine Rockefeller—F69373 Lyon, France; 3Sanofi-Aventis, Montpellier, France; 4Biozentrum, Basel,Switzerland; 5Technologie Servier, Orleans, France; 6Service de Chirurgie Diges-tive, Hopital Haut-Leveque, Pessac, France; 7Service de Chirurgie Digestive, CHURangueil, Toulouse, France; 8Service de Chirurgie Digestive II, CHU Saint Eloi,Montpellier, France; 9Service Medico-Chirurgical des Maladies de l’Appareil Di-gestif et de Transplantation Hepatique, CHU Saint Eloi, Montpellier, France; and10Service d’Anatomie Pathologique, CHU Gui de Chauliac, Montpellier, France.
Received December 15, 2008; accepted February 4, 2009.Supported by ANR JCJC-05-47810 (J.M. P & C. T), the Steroltalk European
project (J.M. P), and Technologie Servier Orleans (A.M.).Address reprint requests to: Jean-Marc Pascussi, Inserm, U632, 1919 Route de
Mende, F-34293 Montpellier, France. E-mail: [email protected]; fax:(33)-4-67-52-36-81.
article.Potential conflict of interest: Nothing to report.
2068
ing lipid homeostasis.9,10 This “xenosensor” is activatedby a variety of ligands, including drugs, insecticides, pes-ticides, and nutritional compounds, and coordinates theexpression of several genes encoding the most pertinentseries of xenobiotic metabolizing and transporters systemsto inactivate or eliminate these compounds. At least fourphases occur in the xenobiotic metabolizing and trans-porters systems process: (1) xenobiotic uptake; (2) xeno-biotic oxidation, mostly performed by the cytochromeP450s (CYPs), notably CYP3A4 and CYP2B6; (3) xeno-biotic conjugation achieved by uridine 5�-diphospho-glu-curonosyltransferase, glutathione S-transferase, orsulfono-transferases; and (4) xenobiotic efflux performedby transporters such as the multi-drug resistance gene andMRP2. The detoxification proteins induced are respon-sible for the metabolism, deactivation, and transport ofnumerous environmental chemicals and several drugs,but they are also involved in bile acid, thyroid, andsteroid hormone catabolism, and this may alter theirnormal physiological function.11 In addition, althoughPXR evolved to protect the body, its activation by avariety of prescription drugs represents the molecularbasis for an important class of harmful drug– drug in-teractions.12
It has been recently observed that PXR activation per-turbs lipid homeostasis in mice by decreasing �-oxidationand concomitantly increasing free acid uptake. These al-terations result in an increase of hepatic triglycerides andhepatic steatosis in mice.9,10 PXR activation down-regu-lates the messenger RNA (mRNA) levels of carnitinepalmitoyltransferase 1A,9 which controls the entry of ac-tivated long-chain fatty acids into the mitochondria and isa main regulatory step in the �-oxidation pathway.13 Thisaction of PXR involves repression of the activity of theinsulin response forkhead factor FoxA2, of hepatocytenuclear factor (HNF) 4 alpha, and of PPAR� coactiva-tor-1 alpha (PGC1�).14-16 In addition, PXR stimulates inmice lipid uptake and synthesis by activation of cd36(cluster of differentiation 36).10 These actions are not me-diated by the classical SREPB-1c pathway; notably, Zhouand colleagues10 demonstrated the direct involvement ofPXR in the transcriptional control of cd36 gene, the pro-moter of which contains a functional PXR response ele-ment. In addition, PXR stimulates cd36 expressionindirectly through an increased expression of peroxisomeproliferator-activated receptor gamma (PPAR�).10
In the current report, we investigated the effects ofPXR activation on the expression and activity of the lipo-genic pathway in human hepatocytes (HHPC). In pre-liminary experiments, transcriptional profiling of HHPC(from three different donors) treated with rifampicin, awell-known hPXR agonist, was analyzed using Agilent
Human Genome Microarray 44K. A statistically signifi-cant increase in the expression of genes such as CYP3A4,multidrug resistance gene, CYP2B6, and ALAS1 was ob-served, as expected. In addition, expression of severalgenes whose responsiveness to PXR had not been previ-ously reported was found to be increased (data notshown). Among these, S14 (thyroid hormone–responsiveSPOT14 homolog, Gene ID: 7069) whose expressionexhibited a 3.5 � 0.9-fold increase. S14 is a small (ap-proximately 17 kDa) acidic (pI 4.65) protein with nosequence similarity to other functional motifs.17 It is pre-dominantly expressed in tissues producing lipids such asthe liver, white and brown adipose tissues, and lactatingmammary glands. Thyroid hormone regulates S14 geneexpression both in vivo and in vitro,6,18,19 and functionalTRE in the rat and human S14 gene have been mapped.18
Although the molecular mechanism of its action isunknown, it is clear that S14 protein acts to transducehormone-related and nutrient-related signals to genesinvolved in lipid metabolism,17 and accumulating evi-dence suggests that S14 could play an important rolein the induction of lipogenic enzymes, particularly bycarbohydrate feeding and thyroid hormone adminis-tration.17,20-22 Notably, antisense RNA experiments per-formed in mice hepatocytes have demonstrated that S14regulates the activation of lipogenic enzyme transcription(adenosine triphosphate citrate lyase, FASN) and triacyl-glycerol formation by stimuli such as carbohydrate feed-ing and thyroid hormone administration.20 In contrast,S14 overexpression in MCF-7 human breast cancer cellspromoted neutral lipid vacuole accumulation.21 The ob-servation that S14 protein is present in the nucleus ofhepatic cells, forms a homodimer, and physically interactswith the T3R open up the possibility that this proteinmay represent a new transcriptional cofactor involved inthe expression of lipogenic enzymes.23
Here we show that S14 is a new PXR target gene. Inaddition, we provide evidence that PXR activation stim-ulates de novo lipogenesis and lipogenic enzymes up-reg-ulation in HHPC. S14 seems to be involved in theseprocesses, because its overexpression in human hepaticcell line HepaRG provokes an increase of fatty acid accu-mulation and lipogenesis, whereas small interfering RNA(siRNA)-mediated S14 down-regulation leads to a de-crease of FASN expression and induction.
Materials and Methods
Animals and Treatment. The PXR null mice havebeen described previously.24 Mice were housed in apathogen-free animal facility under a standard 12-hourlight/dark cycle with free access to water and food. For the
HEPATOLOGY, Vol. 49, No. 6, 2009 MOREAU ET AL. 2069
pregnenolone 16 alpha-carbonitrile (PCN) treatment,mice were injected with PCN (100 mg/kg/day) in cornoil. The mice (six mice/group) were killed 12 hours later,and livers were snap-frozen in liquid nitrogen. This workwas carried out in accordance with the Guide for the Careand Use of Laboratory Animals as adopted and promul-gated by the U.S. National Institutes of Health underpermit Nr.1179 issued by the competent veterinary au-thority of the Kanton Basel-Stadt for animal experimen-tation.
Chemicals. Culture media and additives, dimethyl-sulfoxide, T3, rifampicin, RU486, paxillin, T0901317,4-methylsulfinylbutyl isothiocyanate (SFN), and rosigli-tazone, were from Sigma (Saint Louis, MO). Anotherbatch of rosiglitazone was a gift from Technologie Servier(Orleans, France). The �[32P]dATP was from GEHealthCare Sciences. SR12813 was purchased fromTebu-bio (Le Perray en Yvelines, France).
Primary Culture of Human Hepatocytes. Humanhepatocytes were prepared and cultured according to thepreviously published procedure.25 Clinical characteristicsof livers and lobectomy donors used in this work are sum-marized in Supporting Table 1.
Quantitative Polymerase Chain Reaction. TotalRNA was prepared using trizol reagent. Real-time reverse-transcription polymerase chain reaction (RT-PCR) usinga SYBR Green mix was performed as we described be-fore.26 Polymerase chain reaction (PCR) primers se-quences are listed in Supporting Table 2.
RNA Interference. Duplexes of siRNA targeting hu-man PXR (siPXR), human S14 (siS14), and an irrelevantcontrol gene (siNC) were synthesized (GeneCust Eu-rope), and sequences are listed in Supporting Table 2.Human hepatocytes were seeded in P12 well plates, and100 nM of siRNAs were transfected in Optimem-1 me-dium using Santa Cruz SiRNA transfection reagent (sc-29528). Six hours later, cells were treated as described inthe figure legends.
Plasmid Constructs and Transfection Assays. Thehuman PXR complementary DNA (cDNA) and XREM-CYP3A4Luc plasmids have been described previously.27
The pcDNA3.1-hS14 and pcDNA3.1-hS14HisV5 plas-mids were obtained after amplification of hS14 cDNAsfrom human liver RNA. FG12-CMV-hS14 and FG12-CMV-hS14HisV5 were obtained after amplification ofthe CMV-hS14 or CMV-hS14HisV5 cassettes and inser-tion into Hpa-1 linearized FG12.28 The 4.3kbS14Luc(�4300/�8) was obtained from C. Mariash.18 Muta-tions in the TRE candidate region (4.3kbS14�TRELucconstruct) were prepared using the Stratagene Quick-change kit. To generate the synthetic thymidine kinase re-porter genes, two copies of the wild-type or the mutant TRE
S14 element were synthesized and inserted in thepGL3TKLuc vector. Transient transfections were per-formed on HuH7 cells ATCC (American Type CultureCollection) or human hepatocytes seeded into 24-well tissueculture plates as described previously.29 Transfected cellswere treated with drugs for 24 hours before luciferase activ-ities were assayed.
Electromobility Shift Assays. Electrophoretic mobil-ity shift assays using TNT Coupled Reticulocyte lysatesystem (Promega) in vitro synthesized receptor proteinswere performed as previously described.27 Probe se-quences are shown in Fig. 3A.
Western Blot Analysis. Whole-cell lysates were pre-pared with M-Per lysis buffer (Pierce) and anti-protease(Roche). Protein were submitted to electrophoresis, andimmunoblotting was carried out with anti-peptide S14(611813, BD Biosciences Pharmingen), anti-hS14 (Dr.Huang SM, National Defense Medical Center, Taipei,Taiwan), anti-actin (sc1616, Tebu-bio, Santa Cruz Bio-technology), anti-FASN (sc48357, Tebu-bio, Santa CruzBiotechnology), anti PPAR�1�2 (600-401-419, Rock-land) or anti-CD36 (HM2122, Hycult Biotechnologyb.v.) antibodies as previously described.26
Deuterium Enrichment in the Palmitate. The con-tribution of lipogenesis to the intracellular pool of triac-ylglycerol (TAG) was evaluated using the incorporationinto the palmitate of this TAG pool of deuterium fromdeuterated water.30 After treatment, cells were maintained16 hours in culture medium supplemented with 3% deu-terated water. Cells were collected and lipids were ex-tracted by the methods of Folch et al.31 TAGs wereseparated from other lipid fractions by thin layer chroma-tography as previously described,30 and the methylesterderivative of fatty acids of TAG was prepared by themethod of Morisson et al.32 The deuterium enrichment ofpalmitate was then measured by gas chromatographymass spectrometry as previously described and is ex-pressed as the number of excess deuterium atoms incor-porated per molecule of palmitate.30
Free Fatty Acids Quantification. The characteriza-tion of free fatty acid profiles was performed by ultraperformance liquid chromatography electro spray ioniza-tion mass spectrometry (UPLC-ESI-MS). Cells werewashed in phosphate-buffered saline and centrifuged at2000g for 10 minutes at 4°C. Cell pellets were resus-pended with 1 mL chloroform/methanol (2:1) and weredisrupted using two centrifugation steps at 5000g in aPrecellys system (Bertin Technologies). The resulting ho-mogenate was centrifuged, and the organic phase waswashed with 200 �L NaCl 0.9%. After centrifugation,the lower phase was evaporated and the dried residuesolubilized in 200 �L mobile phase (acetonitrile:isopro-
2070 MOREAU ET AL. HEPATOLOGY, June 2009
panol 1:1). The samples were analyzed by ultra-perfor-mance liquid chromatography (Waters Acquity UPLCSystem) coupled with an Applied Biosystems Q-TRAPfor free fatty acids analysis.
Cell Culture and Lentiviral Vector Transduction.HepaRG33 cells were provided by Dr. C. Guillouzoand Biopredic (Rennes France) and cultured as recom-mended. Cells were transduced with lentiviral vectors,allowing the expression of S14 or S14HisV5 and greenfluorescent protein (GFP) as a marker for transductionas previously described.26 GFP-positive cells were iso-lated using a BD FACSAria cell sorter.
Results
PXR Agonists Increase S14 Expression in HHPC.In preliminary experiments, transcriptional profiling ofHHPC treated with rifampicin suggested that S14 was a newPXR target gene. To confirm these data, human hepatocyteswere treated with several human PXR agonists, rifampicin,
RU486, SR12813, phenobarbital or T0901317 (known tobind and activate both PXR and LXR with the same poten-cy34), or vehicle (0.1% dimethylsulfoxide [DMSO]). Asexpected, CYP3A4 and CYP2B6 mRNAs were inducedby all compounds tested, whereas the �-actin mRNA levelwas unchanged (Fig. 1A). We confirmed S14 mRNAinduction by all PXR agonists. Both S14 and CYP3A4mRNAs exhibited similar time-dependent and dose-de-pendent induction in response to rifampicin or SR12813(Fig. 1B, C). Because S14 is known to be regulated byTR35 and LXR,36 we compared the inductive effect ofrifampicin, SR12813, T0901317, paxilline (LXR ago-nists37), and T3. Rifampicin, SR12813, and T3 displayedsimilar effects on S14 mRNA expression, whereas LXRagonists were more potent (Fig. 1C). CYP3A4 andCYP2B6 did not respond to paxilline or T3. Consistentwith these data, the S14-immunoreactive protein was in-duced by PXR, LXR, and TR agonists in HHPC (Fig.1D). In contrast, the expression of S14-R, a paralog S14
Fig. 1. PXR activation increases S14 expression in cultured human hepatocytes. (A, B, and C) Real-time RT-PCR analysis on the expression of�-actin, CYP3A4, CYP2B6, and S14 in human hepatocytes untreated (0.1% DMSO) or treated with 10 �M rifampicin, 500 �M phenobarbital (PB),1 �M SR12813, 10 �M RU486, 1 �M T0901317, or 100 nM T3. (A) Cells (Ft286) were treated for 36 hours. (B) Kinetics of S14 mRNA induction(FT266). (C) Dose dependence. Human hepatocytes (FT285) were cultured in the presence of increasing concentrations of rifampicin (0.3; 3 and30 �mM), or 1 �M SR12813, 10 �M paxilline, 1 �M T0901317, and 100 nM T3 for 24 hours. (D) Western blot showing the levels of S14, CYP3A4,and �-actin in human hepatocytes (FT285 and FT286) treated for 3 days with solvent or in the presence of 1 �M T3, 5 �M paxillin, 1 �M T0901317,5 �M rifampicin (RIF), or 1 �M SR12813. Two different antibody sources were used: a mice monoclonal anti-peptide antibody (B.D.) and a rabbitpolyclonal anti-full length hS14 antibody (Gift of Dr. Huang SM, National Defense Medical Center, Taipei, Taiwan).
HEPATOLOGY, Vol. 49, No. 6, 2009 MOREAU ET AL. 2071
expressed in the liver,17 was not affected by PXR agonists(Table 1).
PXR Is Required for Drug-Induced S14 Expression.To further demonstrate the role of PXR on S14 expres-sion, HHPCs were transfected with duplexes of siRNAstargeting PXR (siPXR) or an irrelevant sequence (siNC)(Fig. 2A). Six hours later, cells were treated with rifampi-cin (5 �M). Experimental conditions did not permit eval-uation of the level of PXR protein; however, expression ofPXR mRNA was significantly inhibited by severalsiPXRs, notably siPXR-460 and siPXR-858 (S3). Wetherefore used rifampicin-mediated CYP3A4 mRNA in-duction as a surrogate marker of the repression of PXR.Drug-mediated induction of CYP3A4 mRNA was signif-icantly reduced in siPXR-460-transfected or siPXR-858-transfected cells compared with controls. Interestingly,rifampicin-mediated S14 mRNA induction was reducedas well with these siPXR duplexes. However, because ofthe poor effect observed with the siPXR approach, wedecided to pharmacologically inhibit PXR by usingSFN.38 Cells’ co-treatment with SFN efficiently inhibitedrifampicin-mediated CYP3A4 and S14 gene expression atthe protein and mRNA levels (Fig. 2B, C). To confirmthat PXR activation regulates S14 in vivo, wild-type andPXR-null (pxr�/�) mice were treated for 12 hours withthe rodent pxr activator PCN, and expression ofCyp3a11, S14, and cyclophilin (internal control) was an-alyzed in the liver by real-time RT-PCR (Fig. 2D). Asexpected, the Cyp3a11 mRNA level was significantly in-creased by PCN in wild-type mice but not in pxr�/� mice.In addition, PCN provoked a significant increase of S14mRNA expression only in wild-type mice, demonstratingthat PXR is required for this process.
S14 Is a Transcriptional Target of PXR. Examina-tion of region �5000/�8 of the human S14 promoterrevealed seven putative nuclear receptor binding sites
(Fig. 3A). Electrophoretic mobility shift assays were per-formed with in vitro synthesized proteins. PXR:RXR andTR:RXR complexes bind the previously described distalDR4 thyroid hormone response element (S14-TRE18)(Fig. 4B). None of the six other putative binding sitestested exhibited any binding with these complexes (datanot shown). According to this, we observed that full-length S14 promoter (�4300/�8) was induced in hu-man hepatocytes treated with rifampicin (3.4-foldinduction), whereas mutation of the TRE element com-pletely abolished this effect (Fig. 3C). A control CYP3A4luciferase reporter, XREM-CYP3A4Luc, containing bothproximal and distal PXREs, was also induced by rifampi-cin (8.9-fold induction) in these cells. In addition, weobserved that, in HuH7 cells cotransfected with a heter-ologous tk-luciferase reporter gene construct containingtwo copies of a native S14-TRE (TRE�2 S14) with orwithout a PXR-expressing vector, the activity of the re-porter gene was increased by PXR agonists only in pres-ence of PXR (Fig. 3D). No increase was observed whenthe TRE was mutated (�TRE�2 S14). Because S14 ap-pears to be regulated by PXR and TR through transacti-vation of the same TRE element, we evaluated the effectof a combination of these receptor agonists in HHPC. T3
alone, or in combination with rifampicin or SR12813,did not significantly affect CYP3A4 mRNA expression,whereas it clearly increased S14 mRNA. Interestingly,PXR agonists and T3 combination did not over-increaseS14 mRNA accumulation (S4).
PXR Activation Increases Lipogenic Enzymes Ex-pression and De Novo Lipogenesis in Human Hepa-tocyte. Because S14 has been proposed to control thetranscription of genes encoding lipogenic enzymes inmice,20 we investigated the effect of PXR activation onlipogenic enzyme expression and de novo lipogenesis inhuman hepatocytes. Both rifampicin and SR12813 sig-
Table 1. Rifampicin-Mediated mRNAs Induction in Cultured Human Hepatocytes (48 Hours of Treatment).
Human hepatocytes were treated with DMSO 0.1% (CTRL) or 10 �M rifampicin (RIF) for 48 hours. Mean of induction ratios (mRNA in inducer-treated cells/mRNAin untreated cells) � standard error are presented here; n, number of different hepatocyte cultures tested (prepared from different patients); n FI � 1.5 refer to thenumber of cultures in which the induction ratio was �1.5 compared with control cells; range refers to the range of variation observed in the different hepatocyte culturestested; P value: Student t test.
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nificantly induced FASN mRNA accumulation after 24to 48 hours of treatment (Fig. 4A, Table 1). Interestingly,the time-dependence of induction exhibited a delay of 12to 24 hours with respect to that of S14 or CYP3A4mRNA, suggesting an indirect mechanism of induction.Similar results were obtained with adenosine triphosphatecitrate lyase (ACLY) mRNA (Table 1 and data notshown). As expected, FASN protein level also was in-creased after 96 hours of rifampicin treatment (Fig. 4B),
leading to a significant increase of de novo lipogenesis asmeasured by deuterium enrichment of palmitate (Fig.4C). This stimulation induced by rifampicin (�33%)was less than that induced by paxillin (�60%), but com-parable to that induced by 27 mM glucose (�26%). Inaddition, we observed that rifampicin provokes an in-crease of free fatty acids accumulation in human hepato-cytes (Fig. 4D). To further evaluate the role of S14expression onto lipogenic enzymes expression, HHPC
Fig. 2. PXR is necessary for regulation of S14 in HHPC and in vivo in mouse liver. (A) RNA interference-mediated knockdown of PXR decreasesrifampicin-stimulated CYP3A4 and S14 gene expression in HHPC. Human hepatocytes (FT283) were transfected with 100-nM duplexes of smallinterfering RNA targeting PXR (siPXRs) or an irrelevant control sequence (siNC). Forty hours after transfection, cells were treated for 16 hours withrifampicin (5 �M), and the expression of glyceraldehyde 3-phosphate dehydrogenase, �-actin, S14, CYP3A4, and PXR mRNAs were then analyzedby quantitative PCR. Values were normalized with respect to glyceraldehyde 3-phosphate dehydrogenase mRNA levels and are expressed as n-foldinduction compared with untransfected cells treated with the solvent alone � standard deviation. Student t tests were calculated betweenuntransfected and siRNA transfected cells treated with rifampicin (CYP3A4 and S14 mRNAs): **P � 0.01; *P � 0.05. (B, C) SFN inhibitsrifampicin-mediated CYP3A4 and S14 gene expression. Human hepatocytes (FT296 and FT297) were cultured in the absence or presence of 5 �MSFN and treated with 10 �M rifampicin or 5 �M paxillin for 36 hours. (B) Western blot showing the levels of S14, CYP3A4, and �-actin (FT296).(C) Real-time RT-PCR analysis (FT297). Values were normalized with respect to the glyceraldehyde 3-phosphate dehydrogenase mRNA level and areexpressed as relative induction compared with untreated cells � standard deviation. (D) PXR is necessary for the up-regulation of S14 in vivo in mice.Real-time PCR analyses on liver RNA derived from the vehicle-treated or drug-treated mice from wild-type or PXR null (PXR�/�) mice treated for 12hours with solvent alone (oil) or PCN. Cyp3a11 and s14 mRNA levels were determined with cyclophilin as an internal control. The bar graphs representthe mean � standard deviation derived from each group of mice (n 6), with the average value from wild-type mice set as 1. P Student t test(drug versus solvent).
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were transfected with duplexes of siRNAs targeting S14(siS14) or an irrelevant sequence (siNC) (Fig. 5). Sixhours later, cells were treated with rifampicin (5 �M) for36 hours. Experimental conditions did not permit evalu-ation of the level of S14 protein; however, expression ofS14 mRNA was significantly inhibited by SiS14-370,siS14-142, and siS14-34. Interestingly, both basal andrifampicin-mediated FASN mRNA levels were reduced aswell with these siS14 duplexes. Indeed, down-regulationof S14 expression provoked to a complete inhibition ofrifampicin-mediated FASN accumulation compared withcontrol or siNC transfected hepatocytes, suggesting thatS14 is required for these effects.
S14 Overexpression Increases Free Fatty Acids Ac-cumulation and De Novo Lipogenesis in HepaRGCells. To test the effect of S14 overexpression alone ontohepatic de novo lipogenesis, we transduced HepaRG cellswith control (FG12) or hS14-expressing lentivirus vectors
(FG12-S14 and FG12-S14HisV5) that also express a hu-man UbiC-driven GFP gene to provide a marker fortracking transduced cells. HepaRG cells represent the bestsurrogate model of primary human hepatocytes.39 Cellswere sorted by GFP expression using a fluorescence-acti-vated cell sorter, amplified during 3 weeks and then cul-tured in the presence of 6 mM glucose or 27 mM glucosefor 3 days. S14-expressing HepaRG cells cultured with 6mM glucose showed an enhanced rate of de novo lipogen-esis (�37%) compared with FG12-transduced cells com-parable to that induced by 27 mM glucose (�42%),according to deuterium enrichment in the palmitate (12hours’ incubation in culture medium supplemented with3% deuterated water). In addition, we observed a signif-icant increase of free fatty acid accumulation in S14-trans-duced cells compared with control FG12 cells maintainedin 6 mM glucose (Fig. 6B), notably palmitoleic acid(threefold), palmitic acid (1.2-fold), and arachidonic acid
Fig. 3. S14 is a target gene of PXR. (A) Schematic representation of the human S14 gene promoter. The positions of putative PXR-binding elementsare indicated in boxes. The S14 TRE element is underlined, and the mutated nucleotides are indicated in bold. (B) Electrophoretic mobility shift assayswere performed using 32P-radiolabeled double-stranded oligonucleotides corresponding to �2660 TRE of S14 (S14-TRE) and in vitro RXR�, PXR, T3R,and CAR synthesized proteins. (C) Rifampicin-mediated transcriptional activation of full-length S14 promoter (�4300 to �8bp) in humanhepatocytes. Human hepatocytes (FT299) were transfected with a CYP3A4 reporter (XREM-CYP3A4Luc), the wild-type human S14-luciferase(�4.3kbS14Luc) or the TRE-mutated S14 (�4.3kbS14�TRELuc) constructs and then treated 24 hours with 5 �M rifampicin. Results shown areluciferase activities (A.U.) and represent the averages and standard deviations from quadruplicate assays. (D) PXR activates the synthetic thymidinekinase reporter construct containing two copies of the wild type (TREx2 S14) but not the mutant TRE S14 (�TRE�2 S14) element in transienttransfections and luciferase reporter gene assays in HuH7 cells. Results shown are fold induction compared with untreated cells and represent theaverages and standard deviations from quadruplicate assays.
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Fig. 4. PXR activation increases de novo lipogene-sis in HHPC. (A) PXR agonists increase FASN mRNAaccumulation. Gene expression measured by real-timePCR analysis in hepatocytes (FT266) cultured in theabsence or presence of 10 �M rifampicin or 1 �MSR12813 for 8, 24, 32, and 48 hours. Values werenormalized with respect to the glyceraldehyde 3-phos-phate dehydrogenase mRNA level and are expressedas relative induction compared with untreated cells �standard deviation. (B) Western blot showing the lev-els of FASN, CYP3A4, and �-actin after 96 hours oftreatment (FT290). (C) Rifampicin increases de novolipogenesis in human hepatocytes. Human hepato-cytes (FT285, FT286, and FT290) were treated with 5�M rifampicin, 5 �M paxillin, or 27 mM glucose for 3days, then deuterium enrichment in the palmitate ofhepatocytes triacylglycerol (mean number of excessdeuterium percent molecules of palmitate or IE MPE(isotope enrichment mole percent excess) was mea-sured after 16 hours of culture in the presence ofdeuterated water. *P � 0.05 versus the control situ-ation (6 mM glucose). (D) PXR activation increasesfree fatty acid content in human hepatocytes. Humanhepatocytes (n 6 per group) from two donors(FT280 and FT281) were treated with solvent (CTRL) or10 �M rifampicin for 96 hours. The characterization offree fatty acid profiles in human hepatocytes wasperformed by ultra-performance liquid chromatographycoupled with an Applied Biosystems Q-TRAP. ***P �0.001; **P � 0.01, *P � 0.05 versus the controlsituation. Results are expressed as nmoles/g protein.
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(1.6-fold). These accumulations were comparable tothose induced by 27 mM glucose. These results stronglysuggest that S14 overexpression by itself is sufficient topromote lipogenesis in hepatic cells even in low glucoseconcentration.
DiscussionIn this work, we demonstrate that S14 is a PXR target
gene, and we provide evidence that PXR activation leadsto enhanced de novo lipogenesis in human hepatocytesthrough S14 up-regulation. These conclusions are basedon the following arguments: (1) PXR agonists induce S14mRNA and protein accumulation; (2) siRNA-mediateddown-regulation of PXR is accompanied by a paralleldown-regulation of both CYP3A4 and S14 mRNAs; (3)the PXR antagonist SFN inhibits PXR-mediated S14 in-duction, (4) induction of S14 by PCN is abrogated inPXR knock-out mice; (5) electrophoretic mobility shiftassay and transfection assays show that PXR binds to andtransactivates the TRE of S14 promoter; (6) rifampicinincreases lipogenic enzymes expression, de novo synthesisof palmitate, and fatty acids content in human hepato-cyte, whereas S14 overexpression in HepaRG promotesan enhancement of lipogenesis.
Besides TR and LXR, several transcription factors andnuclear receptors control the expression of lipogenic en-zymes and S14. Notably, SREBP-1c40 and carbohydrateresponse element binding protein (ChREBP)41 areknown to control S14 expression. We found that SREBP-1c, HNF1�, CD36, PPAR�1/2, ChREBP, and its targetgene liver-pyruvate kinase (L-PK)41 expressions were notaffected on rifampicin-mediated PXR activation (mRNAand protein levels; Table 1, Supporting TABLES 4, 5).Similar results were obtained with SR12813 (data notshown). Control experiments revealed that glucose-medi-ated ChREBP activation induced S14 and L-PK mRNAs,whereas LXR agonists induced SREBP-1c and PPAR�1/2mRNA, and rosiglitazone induced CD36 mRNA, as ex-pected. These results rule out a significant role of theseclassical lipogenic pathways in the PXR-mediated up-reg-ulation of S14, because the expression of these transcrip-tion factors, or their prototypical target genes, is notaffected by PXR activators in human hepatocytes.
If most of the data described above corroborate andexpand some observations concerning the association be-tween PXR activation and hepatic lipid accumulation inmice, several other observations are in contradiction, sug-gesting that functional interactions between xenosensorsand signaling pathways controlling lipid homeostasis ex-hibit species-dependent specificities. For example,whereas Zhou et al.10 and Yu et al.42 reported that LXRand PXR mediates the induction of PPAR� and CD36 in
Fig. 5. RNA interference-mediated knockdown of S14 decreasesFASN gene expression in HHPC. Human hepatocytes (FT299) weretransfected with 100-nM duplexes of siRNA targeting PXR (siPXRs) or anirrelevant control sequence (siNC). Forty hours after transfection, cellswere treated for 36 hours with rifampicin (5 �M), and the expression ofglyceraldehyde 3-phosphate dehydrogenase, �-actin, S14, and FASNmRNAs were then analyzed by quantitative PCR. Values were normalizedwith respect to glyceraldehyde 3-phosphate dehydrogenase mRNA levelsand are expressed as n-fold induction compared with untransfected cellstreated with the solvent alone � standard deviation. (A) Student t testswere calculated between siNC and siS14s transfected untreated cells(CTRL) (B) Student t tests were calculated between siNC and siS14stransfected cells treated with rifampicin (S14 and FASN mRNAs). (A, B)P � 0.01.
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mice liver, no induction of these genes were observed inhuman hepatocytes. The possibility that the regulationpathways are affected or deficient in these cultures wasruled out by the finding that rosiglitazone, a PPAR� ag-onist, induced CD36 gene expression whereas paxillineinduced PPAR�1/2 gene expression. Thus, although fattyacid uptake by CD36 gene activation seems to play amajor part in the control of liver triglyceride concentra-tion in LXR-mediated or PXR-mediated liver steatosis inmice, the situation appears different in human hepato-cytes. In these cells, CD36 is not activated, and triglycer-ide accumulation results rather from increased fatty acidsynthesis and decreased fatty acid �-oxidation. This haspotential therapeutic implications because CD36 wouldtherefore not be an appropriated target for the treatmentof steatosis in humans, whereas S14 may represent a newcandidate.
Interestingly, S14 mRNA expression was significantlyup-regulated in PXR�/� mice compared with PXR�/�
mice. This is reminiscent of the up-regulation of Scd1 andFae observed in PXR�/� mice, as well as the accumulationof triglycerides and large lipid droplets in the liver ob-served in PXR�/� mice.9,10 Although it remains to befurther investigated, there is the possibility that the en-dogenous PXR (PXR not activated by drugs) is capable ofinhibiting genes expression through repressor recruit-ment. In addition, even if the induction of unsaturatedfatty acids was dramatically greater than that for saturatedones, we observed that the expression of fatty acid desatu-rase stearoyl-CoA desaturase-1 and stearoyl-CoA desatu-rase-5 mRNA were not affected by rifampicin orSR12813 (data not shown). This may reflect the fact that,although palmitate biosynthesis is a limiting factor, de-saturase expressions or enzymatic activities are not limit-ing in these cells.
Although the precise physiologic role of S14 remainsunknown and requires additional investigations, it isclearly implicated as a regulator of the lipogenic process.Indeed, we observed that down-expression of S14 in hu-man hepatocytes provoked a decrease of FASN expres-sion. In addition, we observed that overexpression of S14in HepaRG cells increased fatty acid accumulation andlipogenesis even in low carbohydrate culture condition,whereas others reported a strong accumulation of neutrallipid droplets in MCF-7 stably transfected with S14.21
S14 has been reported to be regulated at the transcrip-tional level by TR, LXR, ChREBP, and SREBP-1c. Ourdata demonstrate that PXR and TR co-regulate S14mRNA expression, through the S14 TRE motif, whereasPXR cooperates with LXR and ChREBP for S14 generegulation. In addition, additional data obtained in ourgroup strongly suggest that S14 is also a constitutive an-drostane receptor (CAR) target gene (Moreau, manu-script in preparation). This makes S14 the convergencepoint not only of different lipogenic signaling pathways,but also of the xenobiotic detoxication pathways such asCAR and PXR. Indeed, it would be very important toinvestigate how the perturbation of one of these pathwayscan lead to significant changes in net activity of S14 inpatients, given that nature tends to maintain homeostasiswhenever possible. However, even if the exact relation-ship between PXR activation, S14 expression, andenhanced lipogenesis certainly warrants further investiga-tion, our results are in agreement with many clinical ob-servations showing that drugs now identified as PXRactivators affect lipid metabolism and induce steatosis inpatients. Notably, rifampicin was shown to provoke liversteatosis in tuberculosis patients.43 In addition, nifedipineand carbamazepine, two recently characterized PXR acti-vators,44,45 are known to induce sporadic events of steato-
Fig. 6. Ectopic S14 expression in HepaRG cell increased free fatty acids. HepaRG cells were transduced with control (FG12) and S14-expressinglentivirus vectors (FG12-S14 and FG12-S14HisV5). Cells were sorted by GFP expression using a fluorescence-activated cell sorter and amplifiedduring 2 weeks in 6 mM glucose. (A) Western blot showing the levels of S14, FASN, and �-actin in sorted cells. (B) Characterization of free fattyacid profiles in S14-expression HepaRG cells. Free fatty acid compositions in transduced-HepaRG cells maintained 3 days in 6 mM or 27 mM glucose(n 5 in each group). ***P � 0.001; **P � 0.01, *P � 0.05 versus the control situation (FG12 in 6 mM glucose). Results are expressed asnmoles/g protein.
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sis/steatohepatitis.46,47 Whether these effects areresponsible for the nonalcoholic steatohepatitis syn-drome, more and more frequently observed in patientswith acquired immune deficiency syndrome under pro-tease inhibitor (another series of PXR agonists) treatment,remains to be established. In addition, it remains to bedetermined whether this new PXR-mediated and S14-mediated lipogenesis pathway plays a role in lipid-associ-ated metabolite diseases, such as obesity and diabetes.
Acknowledgment: The authors acknowledge the giftof anti-hS14 from Dr. Huang SM, National DefenseMedical Center, Taipei, Taiwan. We thank LydianePichard-Garcia for human hepatocyte preparation, andthe MRI-IGMM cytometry facility.
References1. Repa JJ, Mangelsdorf DJ. The liver X receptor gene team: potential new
players in atherosclerosis. Nat Med 2002;8:1243-1248.2. Sugden MC, Watts DI, Marshall CE. Regulation of hepatic lipogenesis in
starved and diabetic animals by thyroid hormone. Biosci Rep 1981;1:757-764.
3. Joseph SB, Laffitte BA, Patel PH, Watson MA, Matsukuma KE, WalczakR, et al. Direct and indirect mechanisms for regulation of fatty acid syn-thase gene expression by liver X receptors. J Biol Chem 2002;277:11019-11025.
4. Laffitte BA, Chao LC, Li J, Walczak R, Hummasti S, Joseph SB, et al.Activation of liver X receptor improves glucose tolerance through coordi-nate regulation of glucose metabolism in liver and adipose tissue. Proc NatlAcad Sci U S A 2003;100:5419-5424.
5. Repa JJ, Liang G, Ou J, Bashmakov Y, Lobaccaro JM, Shimomura I, et al.Regulation of mouse sterol regulatory element-binding protein-1c gene(SREBP-1c) by oxysterol receptors, LXRalpha and LXRbeta. Genes Dev2000;14:2819-2830.
6. Feng X, Jiang Y, Meltzer P, Yen PM. Thyroid hormone regulation ofhepatic genes in vivo detected by complementary DNA microarray. MolEndocrinol 2000;14:947-955.
7. Jiang W, Miyamoto T, Kakizawa T, Sakuma T, Nishio S, Takeda T, et al.Expression of thyroid hormone receptor alpha in 3T3–L1 adipocytes; tri-iodothyronine increases the expression of lipogenic enzyme and triglycer-ide accumulation. J Endocrinol 2004;182:295-302.
8. Willson TM, Kliewer SA. PXR, CAR and drug metabolism. Nat Rev DrugDiscov 2002;1:259-266.
9. Nakamura K, Moore R, Negishi M, Sueyoshi T. Nuclear pregnane Xreceptor cross-talk with FoxA2 to mediate drug-induced regulation of lipidmetabolism in fasting mouse liver. J Biol Chem 2007;282:9768-9776.
10. Zhou J, Zhai Y, Mu Y, Gong H, Uppal H, Toma D, et al. A novel pregnaneX receptor-mediated and sterol regulatory element-binding protein-inde-pendent lipogenic pathway. J Biol Chem 2006;281:15013-15020.
11. Pascussi JM, Gerbal-Chaloin S, Duret C, Daujat-Chavanieu M, VilaremMJ, Maurel P. The tangle of nuclear receptors that controls xenobioticmetabolism and transport: crosstalk and consequences. Annu Rev Pharma-col Toxicol 2007.
12. Kliewer SA, Goodwin B, Willson TM. The nuclear pregnane X receptor: akey regulator of xenobiotic metabolism. Endocr Rev 2002;23:687-702.
13. McGarry JD, Brown NF. The mitochondrial carnitine palmitoyltrans-ferase system: from concept to molecular analysis. Eur J Biochem 1997;244:1-14.
14. Louet JF, Hayhurst G, Gonzalez FJ, Girard J, Decaux JF. The coactivatorPGC-1 is involved in the regulation of the liver carnitine palmitoyltrans-ferase I gene expression by cAMP in combination with HNF4 alpha andcAMP-response element-binding protein (CREB). J Biol Chem 2002;277:37991-38000.
15. Bhalla S, Ozalp C, Fang S, Xiang L, Kemper JK. Ligand-activated preg-nane X receptor interferes with HNF-4 signaling by targeting a commoncoactivator PGC-1alpha: functional implications in hepatic cholesteroland glucose metabolism. J Biol Chem 2004;279:45139-45147.
16. Li T, Chiang JY. Mechanism of rifampicin and pregnane X receptor inhi-bition of human cholesterol 7 alpha-hydroxylase gene transcription. Am JPhysiol Gastrointest Liver Physiol 2005;288:G74-G84.
18. Campbell MC, Anderson GW, Mariash CN. Human spot 14 glucose andthyroid hormone response: characterization and thyroid hormone re-sponse element identification. Endocrinology 2003;144:5242-5248.
19. Oppenheimer JH, Kinlaw WB, Wong NC, Schwartz HL, Mariash CN.Regulation of gene S-14 by triiodothyronine in liver. Horm Metab ResSuppl 1987;17:1-5.
20. Kinlaw WB, Church JL, Harmon J, Mariash CN. Direct evidence for a roleof the “spot 14” protein in the regulation of lipid synthesis. J Biol Chem1995;270:16615-16618.
21. Sanchez-Rodriguez J, Kaninda-Tshilumbu JP, Santos A, Perez-Castillo A.The spot 14 protein inhibits growth and induces differentiation and celldeath of human MCF-7 breast cancer cells. Biochem J 2005;390:57-65.
22. Zhu Q, Anderson GW, Mucha GT, Parks EJ, Metkowski JK, MariashCN. The Spot 14 protein is required for de novo lipid synthesis in thelactating mammary gland. Endocrinology 2005;146:3343-3350.
23. Chou WY, Cheng YS, Ho CL, Liu ST, Liu PY, Kuo CC, et al. Human spot14 protein interacts physically and functionally with the thyroid receptor.Biochem Biophys Res Commun 2007;357:133-138.
24. Roth A, Looser R, Kaufmann M, Blattler SM, Rencurel F, Huang W, et al.Regulatory cross-talk between drug metabolism and lipid homeostasis:constitutive androstane receptor and pregnane X receptor increase Insig-1expression. Mol Pharmacol 2008;73:1282-1289.
25. Pichard L, Fabre I, Fabre G, Domergue J, Saint Aubert B, Mourad G, et al.Cyclosporin A drug interactions: screening for inducers and inhibitors ofcytochrome P-450 (cyclosporin A oxidase) in primary cultures of humanhepatocytes and in liver microsomes. Drug Metab Dispos 1990;18:595-606.
26. Pascussi JM, Robert A, Moreau A, Ramos J, Bioulac-Sage P, Navarro F, etal. Differential regulation of constitutive androstane receptor expression byhepatocyte nuclear factor4alpha isoforms. HEPATOLOGY 2007;45:1146-1153.
27. Drocourt L, Ourlin JC, Pascussi JM, Maurel P, Vilarem MJ. Expression ofCYP3A4, CYP2B6, and CYP2C9 is regulated by the vitamin D receptorpathway in primary human hepatocytes. J Biol Chem 2002;277:25125-25132.
28. Qin XF, An DS, Chen IS, Baltimore D. Inhibiting HIV-1 infection inhuman T cells by lentiviral-mediated delivery of small interfering RNAagainst CCR5. Proc Natl Acad Sci U S A 2003;100:183-188.
29. Pascussi JM, Busson-Le Coniat M, Maurel P, Vilarem MJ. Transcriptionalanalysis of the orphan nuclear receptor constitutive androstane receptor(NR1I3) gene promoter: identification of a distal glucocorticoid responseelement. Mol Endocrinol 2003;17:42-55.
30. Diraison F, Pachiaudi C, Beylot M. Measuring lipogenesis and cholesterolsynthesis in humans with deuterated water: use of simple gas chromato-graphic/mass spectrometric techniques. J Mass Spectrom 1997;32:81-86.
31. Folch J, Lees M, Sloane Stanley GH. A simple method for the isolation andpurification of total lipides from animal tissues. J Biol Chem 1957;226:497-509.
32. Morrison WR, Smith LM. Preparation of fatty acid methyl esters anddimethylacetals from lipids with boron fluoride–methanol. J Lipid Res1964;5:600-608.
33. Guillouzo A, Corlu A, Aninat C, Glaise D, Morel F, Guguen-Guillouzo C.The human hepatoma HepaRG cells: a highly differentiated model forstudies of liver metabolism and toxicity of xenobiotics. Chem Biol Interact2007;168:66-73.
34. Mitro N, Vargas L, Romeo R, Koder A, Saez E. T0901317 is a potent PXRligand: implications for the biology ascribed to LXR. FEBS Lett 2007;581:1721-1726.
2078 MOREAU ET AL. HEPATOLOGY, June 2009
35. Jump DB, Narayan P, Towle H, Oppenheimer JH. Rapid effects of tri-iodothyronine on hepatic gene expression: hybridization analysis of tissue-specific triiodothyronine regulation of mRNAS14. J Biol Chem 1984;259:2789-2797.
36. Pawar A, Botolin D, Mangelsdorf DJ, Jump DB. The role of liver Xreceptor-alpha in the fatty acid regulation of hepatic gene expression. J BiolChem 2003;278:40736-40743.
37. Bramlett KS, Houck KA, Borchert KM, Dowless MS, Kulanthaivel P,Zhang Y, et al. A natural product ligand of the oxysterol receptor, liver Xreceptor. J Pharmacol Exp Ther 2003;307:291-296.
38. Zhou C, Poulton EJ, Grun F, Bammler TK, Blumberg B, Thummel KE,et al. The dietary isothiocyanate sulforaphane is an antagonist of the hu-man steroid and xenobiotic nuclear receptor. Mol Pharmacol 2007;71:220-229.
39. Aninat C, Piton A, Glaise D, Le Charpentier T, Langouet S, Morel F, et al.Expression of cytochromes P450, conjugating enzymes and nuclear recep-tors in human hepatoma HepaRG cells. Drug Metab Dispos 2006;34:75-83.
40. Dentin R, Pegorier JP, Benhamed F, Foufelle F, Ferre P, Fauveau V, et al.Hepatic glucokinase is required for the synergistic action of ChREBP andSREBP-1c on glycolytic and lipogenic gene expression. J Biol Chem 2004;279:20314-20326.
41. Ishii S, Iizuka K, Miller BC, Uyeda K. Carbohydrate response elementbinding protein directly promotes lipogenic enzyme gene transcription.Proc Natl Acad Sci U S A 2004;101:15597-15602.
42. Zhou J, Febbraio M, Wada T, Zhai Y, Kuruba R, He J, et al. Hepatic fattyacid transporter Cd36 is a common target of LXR, PXR, and PPARgammain promoting steatosis. Gastroenterology 2008;134:556-567.
43. Morere P, Nouvet G, Stain JP, Paillot B, Metayer J, Hemet J. Informationobtained by liver biopsy in 100 tuberculous patients [in French]. Sem Hop1975;51:2095-2102.
44. Drocourt L, Pascussi JM, Assenat E, Fabre JM, Maurel P, Vilarem MJ.Calcium channel modulators of the dihydropyridine family are humanpregnane X receptor activators and inducers of CYP3A, CYP2B, andCYP2C in human hepatocytes. Drug Metab Dispos 2001;29:1325-1331.
45. Luo G, Cunningham M, Kim S, Burn T, Lin J, Sinz M, et al. CYP3A4induction by drugs: correlation between a pregnane X receptor reportergene assay and CYP3A4 expression in human hepatocytes. Drug MetabDispos 2002;30:795-804.
46. Babany G, Uzzan F, Larrey D, Degott C, Bourgeois P, Rene E, et al.Alcoholic-like liver lesions induced by nifedipine. J Hepatol 1989;9:252-255.
47. Grieco A, Forgione A, Miele L, Vero V, Greco AV, Gasbarrini A, et al.Fatty liver and drugs. Eur Rev Med Pharmacol Sci 2005;9:261-263.
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