FXR activation protects against NAFLD via bile-acid-dependent reductions in lipid absorption.

FXR agonists are used to treat non-alcoholic fatty liver disease (NAFLD), in part because they reduce hepatic lipids. Here, we show that FXR activation with the FXR agonist GSK2324 controls hepatic lipids via reduced absorption and selective decreases in fatty acid synthesis. Using comprehensive lipidomic analyses, we show that FXR activation in mice or humans specifically reduces hepatic levels of mono- and polyunsaturated fatty acids (MUFA and PUFA). Decreases in MUFA are due to FXR-dependent repression of Scd1, Dgat2, and Lpin1 expression, which is independent of SHP and SREBP1c. FXR-dependent decreases in PUFAs are mediated by decreases in lipid absorption. Replenishing bile acids in the diet prevented decreased lipid absorption in GSK2324-treated mice, suggesting that FXR reduces absorption via decreased bile acids. We used tissue-specific FXR KO mice to show that hepatic FXR controls lipogenic genes, whereas intestinal FXR controls lipid absorption. Together, our studies establish two distinct pathways by which FXR regulates hepatic lipids.

Transcription Factor MAFF (MAF Basic Leucine Zipper Transcription Factor F) Regulates an Atherosclerosis Relevant Network Connecting Inflammation and Cholesterol Metabolism.

BACKGROUND: Coronary artery disease (CAD) is a multifactorial condition with both genetic and exogenous causes. The contribution of tissue-specific functional networks to the development of atherosclerosis remains largely unclear. The aim of this study was to identify and characterize central regulators and networks leading to atherosclerosis. METHODS: Based on several hundred genes known to affect atherosclerosis risk in mouse (as demonstrated in knockout models) and human (as shown by genome-wide association studies), liver gene regulatory networks were modeled. The hierarchical order and regulatory directions of genes within the network were based on Bayesian prediction models, as well as experimental studies including chromatin immunoprecipitation DNA-sequencing, chromatin immunoprecipitation mass spectrometry, overexpression, small interfering RNA knockdown in mouse and human liver cells, and knockout mouse experiments. Bioinformatics and correlation analyses were used to clarify associations between central genes and CAD phenotypes in both human and mouse. RESULTS: The transcription factor MAFF (MAF basic leucine zipper transcription factor F) interacted as a key driver of a liver network with 3 human genes at CAD genome-wide association studies loci and 11 atherosclerotic murine genes. Most importantly, expression levels of the low-density lipoprotein receptor (LDLR) gene correlated with MAFF in 600 CAD patients undergoing bypass surgery (STARNET [Stockholm-Tartu Atherosclerosis Reverse Network Engineering Task]) and a hybrid mouse diversity panel involving 105 different inbred mouse strains. Molecular mechanisms of MAFF were tested in noninflammatory conditions and showed positive correlation between MAFF and LDLR in vitro and in vivo. Interestingly, after lipopolysaccharide stimulation (inflammatory conditions), an inverse correlation between MAFF and LDLR in vitro and in vivo was observed. Chromatin immunoprecipitation mass spectrometry revealed that the human CAD genome-wide association studies candidate BACH1 (BTB domain and CNC homolog 1) assists MAFF in the presence of lipopolysaccharide stimulation with respective heterodimers binding at the MAF recognition element of the LDLR promoter to transcriptionally downregulate LDLR expression. CONCLUSIONS: The transcription factor MAFF was identified as a novel central regulator of an atherosclerosis/CAD-relevant liver network. MAFF triggered context-specific expression of LDLR and other genes known to affect CAD risk. Our results suggest that MAFF is a missing link between inflammation, lipid and lipoprotein metabolism, and a possible treatment target.

A MAFG-lncRNA axis links systemic nutrient abundance to hepatic glucose metabolism.

Obesity and type 2 diabetes mellitus are global emergencies and long noncoding RNAs (lncRNAs) are regulatory transcripts with elusive functions in metabolism. Here we show that a high fraction of lncRNAs, but not protein-coding mRNAs, are repressed during diet-induced obesity (DIO) and refeeding, whilst nutrient deprivation induced lncRNAs in mouse liver. Similarly, lncRNAs are lost in diabetic humans. LncRNA promoter analyses, global cistrome and gain-of-function analyses confirm that increased MAFG signaling during DIO curbs lncRNA expression. Silencing Mafg in mouse hepatocytes and obese mice elicits a fasting-like gene expression profile, improves glucose metabolism, de-represses lncRNAs and impairs mammalian target of rapamycin (mTOR) activation. We find that obesity-repressed LincIRS2 is controlled by MAFG and observe that genetic and RNAi-mediated LincIRS2 loss causes elevated blood glucose, insulin resistance and aberrant glucose output in lean mice. Taken together, we identify a MAFG-lncRNA axis controlling hepatic glucose metabolism in health and metabolic disease.

Intracellular Localization of Endogenous Mouse ABCG1 Is Mimicked by Both ABCG1-L550 and ABCG1-P550-Brief Report.

OBJECTIVE: In a recent article in Arteriosclerosis, Thrombosis, and Vascular Biology, it was reported that ATP-binding cassette transporter G1 (ABCG1) containing leucine at position 550 (ABCG1-L550) was localized to the plasma membrane, whereas ABCG1-P550 (proline at position 550) was intracellular. Because the published data on the subcellular localization of ABCG1 are controversial, we performed additional experiments to determine the importance of leucine or proline at amino acid 550. APPROACH AND RESULTS: We transfected multiple cell lines (CHO-K1, Cos-7, and HEK293 [human embryonic kidney]) with untagged or FLAG-tagged ABCG1 containing either leucine or proline at position 550. Immunofluorescence studies demonstrated that in all cases, ABCG1 localized to intracellular endosomal vesicles. We also show that both ABCG1-L550 and ABCG1-P550 are equally active in both promoting the efflux of cellular cholesterol to exogenous high-density lipoprotein and in inducing the activity of sterol regulatory element-binding protein-2, presumably as a result of redistributing intracellular sterols away from the endoplasmic reticulum. Importantly, we treated nontransfected primary peritoneal macrophages with a liver X receptor agonist and demonstrate, using immunofluorescence, that although endogenous ABCG1 localizes to intracellular endosomes, none was detectable at the cell surface/plasma membrane. CONCLUSIONS: ABCG1, irrespective of either a leucine or proline at position 550, is an intracellular protein that localizes to vesicles of the endosomal pathway where it functions to mobilize sterols away from the endoplasmic reticulum and out of the cell.

Focal middle cerebral artery ischemia in rats via a transfemoral approach using a custom designed microwire.

OBJECTIVES: The aim of this study was to develop a reliable and repeatable method of inducing focal middle cerebral artery occlusion (MCAo) in rats without ligation of the external carotid artery (ECA), while reducing the risk of subarachnoid hemorrhage. METHODS: We prototyped microwires with different diameters (0.0120 inch, 0.0115 inch, 0.0110 inch), materials, and construction methods (coil-on-core, extruded polymer jacket-on-core). Under fluoroscopic guidance and using femoral artery access, the microwires were navigated into the internal carotid artery of male Wistar rats (n=50, weight 376±64 g) to induce MCAo for 1 or 2 h. We performed neurological assessments at baseline, and at 3, 24, 72, and 168 h after MCAo. MRI measurements were performed on a 9.4 T scanner at 1 and 7 days post-injury. RESULTS: The 0.0115 inch microwire with polymer jacket-on-core provided the most successful outcome. At 1 and 7 days post-injury, we observed similar infarction volumes for 1 and 2 h MCAo in the MRI study. Infarcted lesion volumes in both MCAo groups were significantly reduced at 7 days compared with 1 day post-injury. The trend in longitudinal changes for the scores of different neurological assessments was confirmed to be significant after the injury, but both groups showed a similar trend of neurological deficits over the course of the study. CONCLUSIONS: We have developed a reliable and repeatable MCAo method in rats, allowing for precise occlusion of the MCA under direct fluoroscopic visualization without alteration of the cerebral hemodynamics associated with ECA ligation. The custom designed microwire can also be sized for targeted focal ischemia in larger animals.

MAFG is a transcriptional repressor of bile acid synthesis and metabolism.

Specific bile acids are potent signaling molecules that modulate metabolic pathways affecting lipid, glucose and bile acid homeostasis, and the microbiota. Bile acids are synthesized from cholesterol in the liver, and the key enzymes involved in bile acid synthesis (Cyp7a1, Cyp8b1) are regulated transcriptionally by the nuclear receptor FXR. We have identified an FXR-regulated pathway upstream of a transcriptional repressor that controls multiple bile acid metabolism genes. We identify MafG as an FXR target gene and show that hepatic MAFG overexpression represses genes of the bile acid synthetic pathway and modifies the biliary bile acid composition. In contrast, loss-of-function studies using MafG(+/-) mice causes de-repression of the same genes with concordant changes in biliary bile acid levels. Finally, we identify functional MafG response elements in bile acid metabolism genes using ChIP-seq analysis. Our studies identify a molecular mechanism for the complex feedback regulation of bile acid synthesis controlled by FXR.

Upregulation of endogenous farnesyl diphosphate synthase overcomes the inhibitory effect of bisphosphonate on protein prenylation in Hela cells.

Nitrogen-containing bisphosphonates (N-BPs) such as zoledronic acid (ZOL) are the gold standard treatment for diseases of excessive bone resorption. N-BPs inactivate osteoclasts via inhibition of farnesyl diphosphate synthase (FPPS), thereby preventing the prenylation of essential small GTPases. Not all patients respond to N-BP therapy to the same extent, and some patients, for example with tumour-associated bone disease or Paget's disease, appear to develop resistance to N-BPs. The extent to which upregulation of FPPS might contribute to these phenomena is not clear. Using quantitative PCR and western blot analysis we show that levels of FPPS mRNA and protein can be upregulated in HeLa cells by culturing in lipoprotein deficient serum (LDS) or by over-expression of SREBP-1a. Upregulated, endogenous FPPS was predominantly localised to the cytosol and did not co-localise with peroxisomal or mitochondrial markers. Upregulation of endogenous FPPS conferred resistance to the inhibitory effect of low concentrations of ZOL on the prenylation of the small GTPase Rap1a. These observations suggest that an increase in the expression of endogenous FPPS could confer at least partial resistance to the pharmacological effect of N-BP drugs such as ZOL in vivo.

LXRs regulate ER stress and inflammation through dynamic modulation of membrane phospholipid composition.

The fatty acyl composition of phospholipids determines the biophysical character of membranes and impacts the function of membrane proteins. Here, we define a nuclear receptor pathway for the dynamic modulation of membrane composition in response to changes in cellular lipid metabolism. Ligand activation of liver X receptors (LXRs) preferentially drives the incorporation of polyunsaturated fatty acids into phospholipids through induction of the remodeling enzyme Lpcat3. Promotion of Lpcat3 activity ameliorates endoplasmic reticulum (ER) stress induced by saturated free fatty acids in vitro or by hepatic lipid accumulation in vivo. Conversely, Lpcat3 knockdown in liver exacerbates ER stress and inflammation. Mechanistically, Lpcat3 modulates inflammation both by regulating inflammatory kinase activation through changes in membrane composition and by affecting substrate availability for inflammatory mediator production. These results outline an endogenous mechanism for the preservation of membrane homeostasis during lipid stress and identify Lpcat3 as an important mediator of LXR effects on metabolism.

Impaired cholesterol efflux in senescent macrophages promotes age-related macular degeneration.

Pathologic angiogenesis mediated by abnormally polarized macrophages plays a central role in common age-associated diseases such as atherosclerosis, cancer, and macular degeneration. Here we demonstrate that abnormal polarization in older macrophages is caused by programmatic changes that lead to reduced expression of ATP binding cassette transporter ABCA1. Downregulation of ABCA1 by microRNA-33 impairs the ability of macrophages to effectively efflux intracellular cholesterol, which in turn leads to higher levels of free cholesterol within senescent macrophages. Elevated intracellular lipid polarizes older macrophages to an abnormal, alternatively activated phenotype that promotes pathologic vascular proliferation. Mice deficient for Abca1, but not Abcg1, demonstrate an accelerated aging phenotype, whereas restoration of cholesterol efflux using LXR agonists or miR-33 inhibitors reverses it. Monocytes from older humans with age-related macular degeneration showed similar changes. These findings provide an avenue for therapeutic modulation of macrophage function in common age-related diseases.

Pleiotropic roles of bile acids in metabolism.

Enzymatic oxidation of cholesterol generates numerous distinct bile acids that function both as detergents that facilitate digestion and absorption of dietary lipids, and as hormones that activate four distinct receptors. Activation of these receptors alters gene expression in multiple tissues, leading to changes not only in bile acid metabolism but also in glucose homeostasis, lipid and lipoprotein metabolism, energy expenditure, intestinal motility and bacterial growth, inflammation, liver regeneration, and hepatocarcinogenesis. This review covers the roles of specific bile acids, synthetic agonists, and their cognate receptors in controlling these diverse functions, as well as their current use in treating human diseases.

MicroRNA-144 regulates hepatic ATP binding cassette transporter A1 and plasma high-density lipoprotein after activation of the nuclear receptor farnesoid X receptor.

RATIONALE: The bile acid receptor farnesoid X receptor (FXR) regulates many aspects of lipid metabolism by variouscomplex and incompletely understood molecular mechanisms. We set out to investigate the molecular mechanisms for FXR-dependent regulation of lipid and lipoprotein metabolism. OBJECTIVE: To identify FXR-regulated microRNAs that were subsequently involved in regulating lipid metabolism. METHODS AND RESULTS: ATP binding cassette transporter A1 (ABCA1) is a major determinant of plasma high-density lipoprotein (HDL)-cholesterol levels. Here, we show that activation of the nuclear receptor FXR in vivo increases hepatic levels of miR-144, which in turn lowers hepatic ABCA1 and plasma HDL levels. We identified 2 complementary sequences to miR-144 in the 3' untranslated region of ABCA1 mRNA that are necessary for miR-144-dependent regulation. Overexpression of miR-144 in vitro decreased both cellular ABCA1 protein and cholesterol efflux to lipid-poor apolipoprotein A-I protein, whereas overexpression in vivo reduced hepatic ABCA1 protein and plasma HDL-cholesterol. Conversely, silencing miR-144 in mice increased hepatic ABCA1 protein and HDL-cholesterol. In addition, we used tissue-specific FXR-deficient mice to show that induction of miR-144 and FXR-dependent hypolipidemia requires hepatic, but not intestinal, FXR. Finally, we identified functional FXR response elements upstream of the miR-144 locus, consistent with direct FXR regulation. CONCLUSIONS: We have identified a novel pathway involving FXR, miR-144, and ABCA1 that together regulate plasma HDL-cholesterol.

Role of ABC transporters in lipid transport and human disease.

Almost half of the 48 human ATP-binding cassette (ABC) transporter proteins are thought to facilitate the ATP-dependent translocation of lipids or lipid-related compounds. Such substrates include cholesterol, plant sterols, bile acids, phospholipids, and sphingolipids. Mutations in a substantial number of the 48 human ABC transporters have been linked to human disease. Indeed the finding that 12 diseases have been associated with abnormal lipid transport and/or homeostasis demonstrates the importance of this family of transporters in cell physiology. This review highlights the role of ABC transporters in lipid transport and movement, in addition to discussing their roles in cellular homeostasis and inherited disorders.

Trimethylamine-N-oxide, a metabolite associated with atherosclerosis, exhibits complex genetic and dietary regulation.

Circulating trimethylamine-N-oxide (TMAO) levels are strongly associated with atherosclerosis. We now examine genetic, dietary, and hormonal factors regulating TMAO levels. We demonstrate that two flavin mono-oxygenase family members, FMO1 and FMO3, oxidize trimethylamine (TMA), derived from gut flora metabolism of choline, to TMAO. Further, we show that FMO3 exhibits 10-fold higher specific activity than FMO1. FMO3 overexpression in mice significantly increases plasma TMAO levels while silencing FMO3 decreases TMAO levels. In both humans and mice, hepatic FMO3 expression is reduced in males compared to females. In mice, this reduction in FMO3 expression is due primarily to downregulation by androgens. FMO3 expression is induced by dietary bile acids by a mechanism that involves the farnesoid X receptor (FXR), a bile acid-activated nuclear receptor. Analysis of natural genetic variation among inbred strains of mice indicates that FMO3 and TMAO are significantly correlated, and TMAO levels explain 11% of the variation in atherosclerosis.

Loss of FXR protects against diet-induced obesity and accelerates liver carcinogenesis in ob/ob mice.

Farnesoid X receptor (FXR) is known to play important regulatory roles in bile acid, lipid, and carbohydrate metabolism. Aged (>12 months old) Fxr(-/-) mice also develop spontaneous liver carcinomas. In this report, we used three mouse models to investigate the role of FXR deficiency in obesity. As compared with low-density lipoprotein receptor (Ldlr) knockout (Ldlr(-/-)) mice, the Ldlr(-/-)Fxr(-/-) double-knockout mice were highly resistant to diet-induced obesity, which was associated with increased expression of genes involved in energy metabolism in the skeletal muscle and brown adipose tissue. Such a striking effect of FXR deficiency on obesity on an Ldlr(-/-) background led us to investigate whether FXR deficiency alone is sufficient to affect obesity. As compared with wild-type mice, Fxr(-/-) mice showed resistance to diet-induced weight gain. Interestingly, only female Fxr(-/-) mice showed significant resistance to diet-induced obesity, which was accompanied by increased energy expenditure in these mice. Finally, we determined the effect of FXR deficiency on obesity in a genetically obese and diabetic mouse model. We generated ob(-/-)Fxr(-/-) mice that were deficient in both Leptin and Fxr. On a chow diet, ob(-/-)Fxr(-/-) mice gained less body weight and had reduced body fat mass as compared with ob/ob mice. In addition, we observed liver carcinomas in 43% of young (<11 months old) Ob(-/-)Fxr(-/-) mice. Together these data indicate that loss of FXR prevents diet-induced or genetic obesity and accelerates liver carcinogenesis under diabetic conditions.

Dancing with the sterols: critical roles for ABCG1, ABCA1, miRNAs, and nuclear and cell surface receptors in controlling cellular sterol homeostasis.

ATP binding cassette (ABC) transporters represent a large and diverse family of proteins that transport specific substrates across a membrane. The importance of these transporters is illustrated by the finding that inactivating mutations within 17 different family members are known to lead to specific human diseases. Clinical data from humans and/or studies with mice lacking functional transporters indicate that ABCA1, ABCG1, ABCG4, ABCG5 and ABCG8 are involved in cholesterol and/or phospholipid transport. This review discusses the multiple mechanisms that control cellular sterol homeostasis, including the roles of microRNAs, nuclear and cell surface receptors and ABC transporters, with particular emphasis on recent findings that have provided insights into the role(s) of ABCG1. This article is part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945-2010).

ATP binding cassette transporter G1 (ABCG1) is an intracellular sterol transporter.

Four members of the mammalian ATP binding cassette (ABC) transporter G subfamily are thought to be involved in transmembrane (TM) transport of sterols. The residues responsible for this transport are unknown. The mechanism of action of ABCG1 is controversial and it has been proposed to act at the plasma membrane to facilitate the efflux of cellular sterols to exogenous high-density lipoprotein (HDL). Here we show that ABCG1 function is dependent on localization to intracellular endosomes. Importantly, localization to the endosome pathway distinguishes ABCG1 and/or ABCG4 from all other mammalian members of this superfamily, including other sterol transporters. We have identified critical residues within the TM domains of ABCG1 that are both essential for sterol transport and conserved in some other members of the ABCG subfamily and/or the insulin-induced gene 2 (INSIG-2). Our conclusions are based on studies in which (i) biotinylation of peritoneal macrophages showed that endogenous ABCG1 is intracellular and undetectable at the cell surface, (ii) a chimeric protein containing the TM of ABCG1 and the cytoplasmic domains of the nonsterol transporter ABCG2 is both targeted to endosomes and functional, and (iii) ABCG1 colocalizes with multiple proteins that mark late endosomes and recycling endosomes. Mutagenesis studies identify critical residues in the TM domains that are important for ABCG1 to alter sterol efflux, induce sterol regulatory element binding protein-2 (SREBP-2) processing, and selectively attenuate the oxysterol-mediated repression of SREBP-2 processing. Our data demonstrate that ABCG1 is an intracellular sterol transporter that localizes to endocytic vesicles to facilitate the redistribution of specific intracellular sterols away from the endoplasmic reticulum (ER).

Hepatic hepatocyte nuclear factor 4α is essential for maintaining triglyceride and cholesterol homeostasis.

OBJECTIVE: Loss-of-function mutations in human hepatocyte nuclear factor 4α (HNF4α) are associated with maturity-onset diabetes of the young and lipid disorders. However, the mechanisms underlying the lipid disorders are poorly understood. In this study, we determined the effect of acute loss or augmentation of hepatic HNF4α function on lipid homeostasis. METHODS AND RESULTS: We generated an adenovirus expressing LacZ (Ad-shLacZ) or short hairpin RNA of Hnf4α (Ad-shHnf4α). Tail vain injection of C57BL/6J mice with Ad-shHnf4α reduced hepatic Hnf4α expression and resulted in striking phenotypes, including the development of fatty liver and a >80% decrease in plasma levels of triglycerides, total cholesterol, and high-density lipoprotein cholesterol. These latter changes were associated with reduced hepatic lipogenesis and impaired very-low-density lipoprotein secretion. Deficiency in hepatic Hnf4α did not affect intestinal cholesterol absorption despite decreased expression of genes involved in bile acid synthesis. Consistent with the loss-of-function data, overexpression of Hnf4α induced numerous genes involved in lipid metabolism in isolated primary hepatocytes. Interestingly, many of these HNF4α-regulated genes were not induced in wild-type mice that overexpressed hepatic Hnf4α. Because of selective gene regulation, mice overexpressing hepatic Hnf4α had unchanged plasma triglyceride levels and decreased plasma cholesterol levels. CONCLUSIONS: Loss of hepatic HNF4α results in severe lipid disorder as a result of dysregulation of multiple genes involved in lipid metabolism. In contrast, augmentation of hepatic HNF4α activity lowers plasma cholesterol levels but has no effect on plasma triglyceride levels because of selective gene regulation. Our data indicate that hepatic HNF4α is essential for controlling the basal expression of numerous genes involved in lipid metabolism and is indispensable for maintaining normal lipid homeostasis.

Regulation of ABCG1 expression in human keratinocytes and murine epidermis.

ABCG1, a member of the ATP binding cassette superfamily, facilitates the efflux of cholesterol from cells to HDL. In this study, we demonstrate that ABCG1 is expressed in cultured human keratinocytes and murine epidermis, and induced during keratinocyte differentiation, with increased levels in the outer epidermis. ABCG1 is regulated by liver X receptor (LXR) and peroxisome proliferator-activated receptor-δ (PPAR-δ) activators, cellular sterol levels, and acute barrier disruption. Both LXR and PPAR-δ activators markedly stimulate ABCG1 expression in a dose- and time-dependent fashion. PPAR-γ activators also increase ABCG1 expression, but to a lesser degree. In contrast, activators of PPAR-α, retinoic acid receptor, retinoid X receptor, and vitamin D receptor do not alter ABCG1 expression. In response to increased intracellular sterol levels, ABCG1 expression increases, whereas inhibition of cholesterol biosynthesis decreases ABCG1 expression. In vivo, ABCG1 is stimulated 3-6 h after acute barrier disruption by either tape stripping or acetone treatment, an increase that can be inhibited by occlusion, suggesting a potential role of ABCG1 in permeability barrier homeostasis. Although Abcg1-null mice display normal epidermal permeability barrier function and gross morphology, abnormal lamellar body (LB) contents and secretion leading to impaired lamellar bilayer formation could be demonstrated by electron microscopy, indicating a potential role of ABCG1 in normal LB formation and secretion.

Activation of the farnesoid X receptor provides protection against acetaminophen-induced hepatic toxicity.

The nuclear receptor, farnesoid X receptor (FXR, NR1H4), is known to regulate cholesterol, bile acid, lipoprotein, and glucose metabolism. In the current study, we provide evidence to support a role for FXR in hepatoprotection from acetaminophen (APAP)-induced toxicity. Pharmacological activation of FXR induces the expression of several genes involved in phase II and phase III xenobiotic metabolism in wild-type, but not Fxr(-/-) mice. We used chromatin immunoprecipitation-based genome-wide response element analyses coupled with luciferase reporter assays to identify functional FXR response elements within promoters, introns, or intragenic regions of these genes. Consistent with the observed transcriptional changes, FXR gene dosage is positively correlated with the degree of protection from APAP-induced hepatotoxicity in vivo. Further, we demonstrate that pretreatment of wild-type mice with an FXR-specific agonist provides significant protection from APAP-induced hepatotoxicity. Based on these findings, we propose that FXR plays a role in hepatic xenobiotic metabolism and, when activated, provides hepatoprotection against toxins such as APAP.

Genome-wide interrogation of hepatic FXR reveals an asymmetric IR-1 motif and synergy with LRH-1.

We used mouse hepatic chromatin enriched with an FXR antibody and chromatin immunoprecipitation-sequencing (ChIP-seq) to evaluate FXR binding on a genome-wide scale. This identified 1656 FXR-binding sites and 10% were located within 2 kb of a transcription start site which is much higher than predicted by random occurrence. A motif search uncovered a canonical nuclear receptor IR-1 site, consistent with in vitro DNA-binding studies reported previously. A separate nuclear receptor half-site for monomeric receptors such as LRH-1 was co-enriched and FXR activation of four newly identified promoters was significantly augmented by an LRH-1 expression vector in a co-transfection assay. There were 1038 genes located within 20 kb of a peak and a gene set enrichment analysis showed that genes identified by our ChIP-seq analysis are highly correlated with genes activated by an FXR-VP16 adenovirus in primary mouse hepatocytes providing functional relevance to the genome-wide binding study. Gene Ontology analysis showed FXR-binding sites close to many genes in lipid, fatty acid and steroid metabolism. Other broad gene clusters related to metabolism, transport, signaling and glycolysis were also significantly enriched. Thus, FXR may have a much wider role in cellular metabolism than previously appreciated.