FXR-null Mice Research Articles

Bile Acid-Induced Elevated Oxidative Stress in the Absence of Farnesoid X Receptor

The major function of farnesoid X receptor (FXR) is to maintain bile acid and lipid homeostasis. Fxr-null mice, in which the levels of hepatic bile acid and lipid have been elevated, develop spontaneous liver tumors. We evaluated differences in hepatic bile acid and triglyceride concentrations, and in generation of oxidative stress between wild-type mice and Fxr-null mice. The hepatic levels of 8-hydroxy-2'-deoxyguanosine (8OHdG), thiobarbituric acid-reactive substance (TBARS) and hydroperoxides, oxidative stress-related genes, and nuclear factor (erythroid-2 like) factor 2 (Nrf2) protein in Fxr-null mice were significantly higher than those in wild-type mice. An increase in the hepatic bile acid concentration in Fxr-null mice fed a cholic acid (CA) diet resulted in an increase in the hepatic levels of hydroperoxides, TBARS and 8OHdG, whereas a decrease in the hepatic concentration in mice fed a diet containing ME3738 (22beta-methoxyolean-12-ene-3beta,24(4beta)-diol) resulted in a decrease in these oxidative stress marker levels. A good correlation was observed between the hepatic bile acid concentrations and the hepatic oxidative stress marker levels, although there was no significant correlation between the hepatic triglyceride concentrations and oxidative stress. The results show that oxidative stress is spontaneously enhanced in Fxr-null mice, which may be attributable to a continuously high level of hepatic bile acids.

The p300 Acetylase Is Critical for Ligand-activated Farnesoid X Receptor (FXR) Induction of SHP

The primary bile acid receptor farnesoid X receptor (FXR) maintains lipid and glucose homeostasis by regulating expression of numerous bile acid-responsive genes, including an orphan nuclear receptor and metabolic regulator SHP. Using SHP as a model gene, we studied how FXR activity is regulated by p300 acetylase. FXR interaction with p300 and their recruitment to the SHP promoter and acetylated histone levels at the promoter were increased by FXR agonists in mouse liver and HepG2 cells. In contrast, p300 recruitment and acetylated histones at the promoter were not detected in FXR-null mice. p300 directly interacted with and acetylated FXR in vitro. Overexpression of p300 wild type increased, whereas a catalytically inactive p300 mutant decreased, acetylated FXR levels and FXR transactivation in cells. While similar results were observed with a related acetylase, CBP, GCN5 did not enhance FXR transactivation, and its recruitment to the promoter was not increased by FXR agonists, suggesting functional specificity of acetylases in FXR signaling. Down-regulation of p300 by siRNA decreased acetylated FXR and acetylated histone levels, and occupancy of FXR at the promoter, resulting in substantial inhibition of SHP expression. These results indicate that p300 acts as a critical coactivator of FXR induction of SHP by acetylating histones at the promoter and FXR itself. Surprisingly, p300 down-regulation altered expression of other metabolic FXR target genes involved in lipoprotein and glucose metabolism, such that beneficial lipid and glucose profiles would be expected. These unexpected findings suggest that inhibition of hepatic p300 activity may be beneficial for treating metabolic diseases.

Cholesterol Feeding Prevents Hepatic Accumulation of Bile Acids in Cholic Acid-Fed Farnesoid X Receptor (FXR)-Null Mice: FXR-Independent Suppression of Intestinal Bile Acid Absorption

Cholic acid (CA) feeding of farnesoid X receptor (Fxr)-null mice results in markedly elevated hepatic bile acid levels and liver injury. In contrast, Fxr-null mice fed cholesterol plus CA (CA+Chol) do not exhibit liver injury, and hepatic bile acid levels and bile acid pool size are reduced 51 and 40%, respectively, compared with CA-treated Fxr-null mice. These decreases were not observed in wild-type mice. Despite a reduced bile acid pool size, hepatic Cyp7a1 mRNA expression was increased in Fxr-null mice fed the CA+Chol diet, and biliary bile acid output was not changed. Analysis of other potential protective mechanisms revealed significant decreases in portal blood bile acid concentrations and a reduced ileal bile acid absorption capacity, as estimated using an in situ loop method. Fecal bile acid excretion was also increased in Fxr-null mice fed the CA+Chol versus CA diet. The decreased ileal bile acid absorption correlated with decreased ileal apical sodium-dependent bile salt transporter (ASBT) protein expression in brush-border membranes. These results suggest a critical role for ileal bile acid absorption in regulation of hepatic bile acid levels in Fxr-null mice fed CA+Chol. Furthermore, experiments with Fxr-null mice suggest that cholesterol feeding can down-regulate ASBT expression through a pathway independent of FXR.

Bacterial-dependent up-regulation of intestinal bile acid binding protein and transport is FXR-mediated following ileo-cecal resection

Bile acid (BA) reclamation following ileo-cecal resection (ICR) may prevent colonic mucosa from chronic injury. In this study, we hypothesized that in a murine model of ICR the remnant colon would upregulate the cellular machinery necessary for BA reclamation and would do so in an FXR- and bacteria-dependent manner. Conventional (WT), conventional FXR knockout (FXR null) and germ-free (GF) mice were randomized to undergo either ICR or sham operation. The ascending colon was harvested for histology and immunohistochemistry and changes in bile acid homeostatic gene expression determined by real-time polymerase chain reaction (RT-PCR) 7 days following surgery. Following ICR WT mice showed significant increases in the expression of genes regulating bile acid transport including IBABP, Asbt, Ost beta and FGF 15. Increased expression of IBABP and Asbt was confirmed by immunohistochemistry. Induction of bile acid transport genes was absent or attenuated in FXR null and GF mice. Bacterial dependent up regulation of IBABP is FXR mediated in the colon following ICR. Mice lacking microbiota (GF) or FXR are unable to increase the expression of IBABP or FGF 15.

PPARα gene expression is up-regulated by LXR and PXR activators in the small intestine

LXR, PXR, and PPARα are members of a nuclear receptor family which regulate the expression of genes involved in lipid metabolism. Here, we show the administration of T0901317 stimulates PPARα gene expression in the small intestine but not in the liver of both normal and FXR-null mice. The administration of LXR specific ligand GW3965, or PXR specific ligand PCN has the same effect, indicating that ligand-dependent activation of LXR and PXR, but not FXR, is responsible for the increased gene expression of PPARα in the mouse small intestine.

Endogenous bile acids as carcinogens

Spontaneous development of liver tumors in the absence of the bile acid receptor farnesoid X receptor. Yang F, Huang X, Yi T, Yen Y, Moore DD, Huang W. Farnesoid X receptor (FXR, NR1H4) is a member of the nuclear hormone receptor superfamily, which plays an essential role in regulating bile acid, lipid, and glucose homeostasis. Both male and female FXR(−/−) mice spontaneously developed liver tumors; however, no other tumors were developed after 15months of age. In contrast, no liver tumors were observed in wild-type mice of the same age. Histologic analyses confirm that tumors were hepatocellular adenoma and carcinoma. Although there was no obvious tumor at ages 9–12months, FXR(−/−) livers displayed prominent liver injury and inflammation. Strong labeling of apoptotic hepatocytes and liver damage-induced compensatory regeneration were observed. Deregulation of genes involved in bile acid homeostasis in FXR(−/−) mice was consistent with abnormal levels of bile acids presented in serum and liver. Genes involved in inflammation and cell cycle were up-regulated in aging FXR(−/−) mice but not in wild-type controls. Increasing the bile acid levels by feeding mice with a 0.2% cholic acid diet strongly promoted N -nitrosodiethylamine-initiated liver tumorigenesis, whereas lowering bile acid pool in FXR(−/−) mice by a 2% cholestyramine feeding significantly reduced the malignant lesions. Our results suggest an intriguing link between metabolic regulation and hepatocarcinogenesis. [Abstract reproduced by permission of Cancer Res 2007;67:863–867] Spontaneous hepatocarcinogenesis in farnesoid X receptor-null mice. Kim I, Morimura K, Shah Y, Yang Q, Ward JM, Gonzalez FJ. The farnesoid X receptor (FXR) controls the synthesis and transport of bile acids (BAs). Mice lacking expression of FXR, designated Fxr-null, have elevated levels of serum and hepatic BAs and an increase in BA pool size. Surprisingly, at 12months of age, male and female Fxr-null mice had a high incidence of degenerative hepatic lesions, altered cell foci and liver tumors including hepatocellular adenoma, carcinoma and hepatocholangiocellular carcinoma, the latter of which is rarely observed in mice. At 3months, Fxr-null mice had increased expression of the proinflammatory cytokine IL-1β mRNA and elevated β-catenin and its target gene c-myc. They also had increased cell proliferation as revealed by increased PCNA mRNA and BrdU incorporation. These studies reveal a potential role for FXR and BAs in hepatocarcinogenesis. [Abstract reproduced by permission of Carcinogenesis 2007;28:940–946]

Spontaneous hepatocarcinogenesis in farnesoid X receptor-null mice

The farnesoid X receptor (FXR) controls the synthesis and transport of bile acids (BAs). Mice lacking expression of FXR, designated Fxr-null, have elevated levels of serum and hepatic BAs and an increase in BA pool size. Surprisingly, at 12 months of age, male and female Fxr-null mice had a high incidence of degenerative hepatic lesions, altered cell foci and liver tumors including hepatocellular adenoma, carcinoma and hepatocholangiocellular carcinoma, the latter of which is rarely observed in mice. At 3 months, Fxr-null mice had increased expression of the proinflammatory cytokine IL-1beta mRNA and elevated beta-catenin and its target gene c-myc. They also had increased cell proliferation as revealed by increased PCNA mRNA and BrdU incorporation. These studies reveal a potential role for FXR and BAs in hepatocarcinogenesis.

Farnesoid X Receptor Agonist Reduces Serum Asymmetric Dimethylarginine Levels through Hepatic Dimethylarginine Dimethylaminohydrolase-1 Gene Regulation

The farnesoid X receptor (FXR, NR1H4) is a bile acid-responsive nuclear receptor that plays critical roles in the transcriptional regulation genes involved in cholesterol, bile acid, triglyceride, and carbohydrate metabolism. By microarray analysis of hepatic genes from female Zucker diabetic fatty (ZDF) rats treated with the FXR agonist GW4064, we have identified dimethylarginine dimethylaminohydrolase-1 (DDAH1) as an FXR target gene. DDAH1 is a key catabolic enzyme of asymmetric dimethylarginine (ADMA), a major endogenous nitric-oxide synthase inhibitor. Sequence analysis of the DDAH1 gene reveals the presence of an FXR response element (FXRE) located 90 kb downstream of the transcription initiation site and within the first intron. Functional analysis of the putative FXRE demonstrated GW4064 dose-dependent transcriptional activation from the element, and we have demonstrated that the FXRE sequence binds the FXR-RXR heterodimer. In vivo administration of GW4064 to female ZDF rats promoted a dose-dependent and >6-fold increase in hepatic DDAH1 gene expression. The level of serum ADMA was reduced concomitantly. These findings provide a mechanism by which FXR may increase endothelium-derived nitric oxide levels through modulation of serum ADMA levels via direct regulation of hepatic DDAH1 gene expression. Thus, beneficial clinical outcomes of FXR agonist therapy may include prevention of atherosclerosis and improvement of the metabolic syndrome.

Effects of FXR in foam-cell formation and atherosclerosis development

Farnesoid X receptor (FXR), a bile-acid-activated member of the nuclear receptor superfamily, is essential in regulating bile-acid, cholesterol, and triglyceride homeostasis. Disruption of the FXR gene in mice results in a proatherosclerotic lipid profile with increased serum cholesterols and triglycerides. However, the role of FXR in foam-cell formation and atherosclerosis development remains unclear. The current study showed that the peritoneal macrophages isolated from FXR-null mice took up less oxidized LDL-cholesterol (oxLDL-C), which was accompanied by a marked reduction in CD36 expression in these cells. This result appears to be FXR-independent, as FXR was not detected in the peritoneal macrophages. To assess to what extent FXR modulates atherosclerosis development, FXR/ApoE double-null mice were generated. Female mice were used for atherosclerosis analysis. Compared to ApoE-null mice, the FXR/ApoE double-null mice were found to have less atherosclerotic lesion area in the aorta, despite a further increase in the serum cholesterols and triglycerides. Our results indicate that disruption of the FXR gene could attenuate atherosclerosis development, most likely resulting from reduced oxLDL-C uptake by macrophages. Our study cautions the use of serum lipid levels as a surrogate marker to determine the efficiency of FXR modulators in treating hyperlipidemia.

Role for enhanced faecal excretion of bile acid in hydroxysteroid sulfotransferase-mediated protection against lithocholic acid-induced liver toxicity

The efficient clearance of toxic bile acids such as lithocholic acid (LCA) requires drug-metabolizing enzymes. We therefore assessed the influence of pregnenolone 16α-carbonitrile (PCN) treatment on LCA-induced hepatotoxicity and disposition of LCA metabolites using female farnesoid X receptor (FXR)-null and wild-type mice. Marked decreases in serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities, and hepatic tauroLCA (TLCA) concentrations were found in LCA-fed wild-type mice co-treated with PCN. Whereas induction of Cyp3a and hydroxysteroid sulfotransferase (Sult2a) proteins was observed in FXR-null and wild-type mice, clear increases in biliary 3α-sulfated TLCA but not total 6α-hydroxy LCA (taurohyodeoxycholic acid and hyodeoxycholic acid) were only observed in PCN-treated wild-type mice. Biliary 3α-sulfated TLCA output rate was increased 7.2-fold, but accounts for only 4.2% of total bile acid output rate in LCA and PCN-co-treated wild-type mice. Total 3α-sulfated LCA (LCA and TLCA) was, however, the most abundant bile acid component in faeces suggesting that efficient faecal excretion of biliary 3α-sulfated TLCA through escape from enterohepatic circulation. FXR-null mice, which have constitutively high levels of the Sult2a protein, were fed a diet supplemented with 1% LCA and 0.4% dehydroepiandrosterone (DHEA), a typical Sult2a substrate/inhibitor. The faecal total 3α-sulfated bile acid excretion was reduced to 62% of FXR-null mice fed only the LCA diet. Hepatic TLCA concentration and serum AST activity were significantly higher in FXR-null mice fed DHEA and LCA diet than in FXR-null mice fed the LCA diet or DHEA diet. These results suggest that hepatic formation of 3α-sulfated TLCA is a crucial factor for protection against LCA-induced hepatotoxicity.

Farnesoid X receptor is essential for normal glucose homeostasis

The bile acid receptor farnesoid X receptor (FXR; NR1H4) is a central regulator of bile acid and lipid metabolism. We show here that FXR plays a key regulatory role in glucose homeostasis. FXR-null mice developed severe fatty liver and elevated circulating FFAs, which was associated with elevated serum glucose and impaired glucose and insulin tolerance. Their insulin resistance was confirmed by the hyperinsulinemic euglycemic clamp, which showed attenuated inhibition of hepatic glucose production by insulin and reduced peripheral glucose disposal. In FXR-/- skeletal muscle and liver, multiple steps in the insulin signaling pathway were markedly blunted. In skeletal muscle, which does not express FXR, triglyceride and FFA levels were increased, and we propose that their inhibitory effects account for insulin resistance in that tissue. In contrast to the results in FXR-/- mice, bile acid activation of FXR in WT mice repressed expression of gluconeogenic genes and decreased serum glucose. The absence of this repression in both FXR-/- and small heterodimer partner-null (SHP-/-) mice demonstrated that the previously described FXR-SHP nuclear receptor cascade also targets glucose metabolism. Taken together, our results identify a link between lipid and glucose metabolism mediated by the FXR-SHP cascade.

Upregulation of a basolateral FXR-dependent bile acid efflux transporter OSTα-OSTβ in cholestasis in humans and rodents

Organic solute transporter (OSTalpha-OSTbeta) is a novel heteromeric bile acid and sterol transporter expressed at the basolateral membranes of epithelium in the ileum, kidney, and liver. To determine whether OSTalpha-OSTbeta undergoes farnesoid X receptor (FXR)-dependent adaptive regulation following cholestatic liver injury, mRNA and protein expression levels were analyzed in patients with primary biliary cirrhosis (PBC) and following common bile duct ligation (CBDL) in rats and Fxr null and wild-type mice. Hepatic OSTalpha and OSTbeta mRNA increased 3- and 32-fold, respectively, in patients with PBC compared with controls, whereas expression of Ostalpha and Ostbeta also increased in the liver of rats and mice following CBDL. In contrast, expression of Ostalpha and Ostbeta mRNA was generally lower in Fxr null mice, and CBDL failed to enhance expression of Ostalpha and Ostbeta compared with wild-type mice. HepG2 cells treated for 24 h with chenodeoxycholic acid, a selective FXR ligand, had higher levels of OSTalpha and OSTbeta mRNA and protein. Increases in OST protein were visualized by confocal microscopy at the plasma membrane. These results indicate that expression of Ostalpha and Ostbeta are highly regulated in response to cholestasis and that this response is dependent on the FXR bile acid receptor.

Chenodeoxycholic Acid-mediated Activation of the Farnesoid X Receptor Negatively Regulates Hydroxysteroid Sulfotransferase

Hydroxysteroid sulfotransferase catalyzing bile acid sulfation plays an essential role in protection against lithocholic acid (LCA)-induced liver toxicity. Hepatic levels of Sult2a is up to 8-fold higher in farnesoid X receptor-null mice than in the wild-type mice. Thus, the influence of FXR ligand (chenodeoxycholic acid (CDCA) and LCA) feeding on hepatic Sult2a expression was examined in FXR-null and wild-type mice. Hepatic Sult2a protein content was elevated in FXR-null and wild-type mice fed a LCA (1% and 0.5%) diet. Treatment with 0.5% CDCA diet decreased hepatic Sult2a to 20% of the control in wild-type mice, but increased the content in FXR-null mice. Liver Sult2a1 (St2a4) mRNA levels were reduced to 26% in wild-type mice after feeding of a CDCA diet, while no decrease was observed on Sult2a1 mRNA levels in FXR-null mice after CDCA feeding. A significant inverse relationship (r(2)=0.523) was found between hepatic Sult2a protein content and small heterodimer partner (SHP) mRNA level. PCN-mediated increase in Sult2a protein levels were attenuated by CDCA feeding in wild-type mice, but not in FXR-null mice. Human SULT2A1 protein and mRNA levels were decreased in HepG2 cells treated with the FXR agonists, CDCA or GW4064 in dose-dependent manners, although SHP mRNA levels were increased. These results suggest that SULT2A is negatively regulated through CDCA-mediated FXR activation in mice and humans.

Regulation of the mouse organic solute transporter α-β, Ostα-Ostβ, by bile acids

The mechanisms responsible for bile acid regulation of mouse intestinal organic solute transporter alpha-beta (Ostalpha-Ostbeta) expression were investigated. Expression of Ostalpha-Ostbeta mRNA was increased in cecum and proximal colon of cholic acid-fed mice and in chenodeoxycholate-treated mouse CT26 colon adenocarcinoma cells. Sequence analysis revealed potential cis-acting elements for farnesoid X receptor (FXR) and liver receptor homolog-1 (LRH-1) in the mouse Ostalpha and Ostbeta promoters and reporter constructs containing Ostalpha and Ostbeta 5'-flanking sequences were positively regulated by bile acids. Expression of a dominant-negative FXR, reduction of FXR with interfering small RNA (siRNA), or mutation of the potential FXR elements decreased Ostalpha and Ostbeta promoter activity and abolished the induction by chenodeoxycolic acid. Negative regulation of the Ostalpha and Ostbeta promoters by bile acids was mediated through LRH-1 elements. Ostalpha and Ostbeta promoter activities were increased by coexpression of LRH-1 and decreased by coexpression of SHP. Mutation of the potential LRH-1 elements and siRNA-mediated reduction of LRH-1 expression decreased basal promoter activity. As predicted from the promoter analyses, ileal Ostalpha and Ostbeta mRNA expressions were increased in wild-type mice administered the FXR agonist GW4064 and decreased in FXR-null mice. Immunoblotting analysis revealed that Ostalpha and Ostbeta intestinal protein expressions correlated with mRNA expression. The mouse Ostalpha and Ostbeta promoters are unusual in that they contain functional FXR and LRH elements, which mediate, respectively, positive and negative feedback regulation by bile acids. Although the positive regulatory pathway appears to be dominant, this arrangement provides a mechanism to finely titrate Ostalpha-Ostbeta expression to the bile acid flux.

Role of FXR in Regulating Bile Acid Homeostasis and Relevance for Human Diseases

Recent studies reveal that bile acids are signalling molecules that activate several nuclear receptors and regulate many physiological pathways and processes to maintain bile acid and cholesterol homeostasis. Analysis of orphan receptor expression patterns in enterohepatic tissues identified bile acids as ligands for farnesoid X receptor (FXR). The primary bile acid chenodeoxycholic acid (CDCA) was shown to be the most potent FXR ligand in vitro at an EC50 of 10-50 microM. FXR can also be activated by the secondary bile acids lithocholic acid (LCA) and deoxycholic acid (DCA). Upon activation FXR heterodimerises with 9-cis retinoic X receptor (RXR) and regulates a cohort of genes involved in cholesterol catabolism and bile acids biosynthesis. Thus bile acid-activated FXR directly induces expression of Small Heterodimer Partner (SHP), a nuclear receptor that suppresses bile acid biosynthesis down-regulates the Na+ taurocholate cotransport peptide (NTCP), a pump depicted to transport bile acids from the lumen into hepatocyte, and induces expression of bile salt export pump (BSEP), the principal bile acid efflux transporter in the liver. As demonstrated by the Fxr null mice, FXR defends the liver against cholestasis. The 6-ethyl derivative of CDCA (6-ECDCA) is approximately 100 fold more potent than CDCA in activating FXR in vitro. In vivo administration of 6-ECDCA protects against cholestasis induced by estrogen and LCA in rats providing evidence that development of potent FXR agonists might represent a new approach for the treatment of cholestastic disorders.

Bile acid transporter (Ntcp, Bsep) protein levels and bile acid synthetic enzyme activities in wild type and FXR-null mice fed cholic acid and chow diets: Differential regulation of mRNA and protein expression: Jaya Bollineni, UMDNJ School of Medicine, Newark, NJ; I'Kyori Swaby, The Mount Sinai Medical Center, New York, NY; Christopher Sinal, Frank Gonzalez, National Institutes of Health, Bethesda, MD;

Bile acid transporter (Ntcp, Bsep) protein levels and bile acid synthetic enzyme activities in wild type and FXR-null mice fed cholic acid and chow diets: Differential regulation of mRNA and protein expression: Jaya Bollineni, UMDNJ School of Medicine, Newark, NJ; I'Kyori Swaby, The Mount Sinai Medical Center, New York, NY; Christopher Sinal, Frank Gonzalez, National Institutes of Health, Bethesda, MD;

Role of farnesoid X receptor in the enhancement of canalicular bile acid output and excretion of unconjugated bile acids: a mechanism for protection against cholic acid-induced liver toxicity.

Mice lacking the farnesoid X receptor (FXR) involved in the maintenance of hepatic bile acid levels are highly sensitive to cholic acid-induced liver toxicity. Serum aspartate aminotransferase (AST) activity was elevated 15.7-fold after feeding a 0.25% cholic acid diet, whereas only slight increases in serum AST (1.7- and 2.5-fold) were observed in wild-type mice fed 0.25 and 1% cholic acid diet, respectively. Bile salt export pump mRNA and protein levels were increased in wild-type mice fed 1% cholic acid diet (2.1- and 3.0-fold) but were decreased in FXR-null mice fed 0.25% cholic acid diet. The bile acid output rate was 2.0- and 3.7-fold higher after feeding of 0.25 and 1.0% cholic acid diet in wild-type mice, respectively. On the other hand, no significant increase in bile acid output rate was observed in FXR-null mice fed 0.25% cholic acid diet in contrast to a significant decrease observed in mice fed a 1.0% cholic acid diet in spite of the markedly higher levels of hepatic tauro-conjugated bile acids. Unconjugated cholic acid was not detected in the bile of wild-type mice fed a control diet, but it was readily detected in wild-type mice fed 1% cholic acid diet. The ratio of biliary unconjugated cholic acid to total cholic acid (unconjugated cholic acid and tauro-conjugated cholic acid) reached 30% under conditions of hepatic taurine depletion. These results suggest that the cholic acid-induced enhancement of canalicular bile acid output rates and excretion of unconjugated bile acids are involved in adaptive responses for prevention of cholic acid-induced toxicity.

Reduced hepatic expression of farnesoid X receptor in hereditary cholestasis associated to mutation in ATP8B1.

Farnesoid X receptor (FXR) is a transcription factor that controls bile acid homeostasis. The phenotype of Fxr null mice is characterized by hypercholanaemia, impaired secretion of bile acids and failure to thrive. Human disorders with these characteristics include FIC1 disease (caused by mutations in ATP8B1, which encodes a putative aminophospholipid translocase, FIC1, whose function in bile handling is unknown) and bile salt export pump (BSEP) disease (caused by mutation in ABCB11, which encodes BSEP, the primary canalicular bile salt export pump). We investigated the possibility of hepatic down-regulation of FXR in FIC1 disease and BSEP disease. Three siblings with this phenotype, born to consanguine parents, were initially studied. The children were demonstrated to be compound heterozygotes for missense and nonsense mutations in ATP8B1. Expression of specific genes in liver was analysed, comparing one of these siblings with a child homozygous for missense mutation in ABCB11, as well as with a child having idiopathic cholestatic liver disease, a child with extrahepatic biliary atresia and a normal organ donor. The expression of two main FXR isoforms was specifically decreased in the liver of the FIC1 disease patient. A consistent and concomitant reduction in messenger RNA levels of FXR targets, such as BSEP and small heterodimer partner, was also found. Gene-profiling experiments identified 163 transcripts whose expression changed significantly in FIC1-disease liver. Of note was that several genes involved in synthesis, conjugation and transport of bile acids were down-regulated. A cluster of genes involved in lipid metabolism was also differentially expressed. Our findings suggest that hepatic down-regulation of FXR contributes to the severe cholestasis of FIC1 disease.

Regulation of Drug Transporters by the Farnesoid X Receptor in Mice

The farnesoid X receptor (FXR, NR1H4) regulates bile acid and lipid homeostasis by acting as an intracellular bile acid-sensing transcription factor, resulting in altered expression of enzymes and transporters involved in bile acid synthesis and transport. Here, we quantitatively analyzed the alterations in expression levels of drug transporters, mainly organic anion-transporting polypeptides (oatp), in wild-type and FXR-null mice to evaluate the role of FXR in their expression and regulation by cholic acid. Changes in the mRNA amounts in liver, kidney, small intestine, and testis in FXR-null mice fed with or without a supplement of 0.5% cholic acid in the diet were analyzed by semiquantitative RT-PCR. In FXR-null mice, the mRNA levels of oatp1, oatp2, oatp3, and octn1 were lower than those of wild-type mice in kidney and testis, while there was no difference in liver or small intestine. Cholic acid feeding led to significantly decreased levels of expression of oatp1 and oct1 and an increased level of expression of oatp2 in wild-type mouse liver. In FXR-null mice, oatp1 and other transporters were downregulated in liver, kidney, and testis, whereas small intestine ASBT, octn2, and pept1 were upregulated. Our results suggested that FXR is involved in the transcriptional regulation of oatp and other transporters in a tissue-specific manner. Furthermore, the effect of cholic acid treatment indicates the involvement of regulatory mechanism(s) other than FXR.

Peroxisome proliferator-activated receptor-gamma coactivator 1alpha (PGC-1alpha) regulates triglyceride metabolism by activation of the nuclear receptor FXR.

Peroxisome proliferator-activated receptor-gamma coactivator 1alpha (PGC-1alpha) has been shown to regulate adaptive thermogenesis and glucose metabolism. Here we show that PGC-1alpha regulates triglyceride metabolism through both farnesoid X receptor (FXR)-dependent and -independent pathways. PGC-1alpha increases FXR activity through two pathways: (1) it increases FXR mRNA levels by coactivation of PPARgamma and HNF4alpha to enhance FXR gene transcription; and (2) it interacts with the DNA-binding domain of FXR to enhance the transcription of FXR target genes. Ectopic expression of PGC-1alpha in murine primary hepatocytes reduces triglyceride secretion by a process that is dependent on the presence of FXR. Consistent with these in vitro studies, we demonstrate that fasting induces hepatic expression of PGC-1alpha and FXR and results in decreased plasma triglyceride levels in wild-type but not in FXR-null mice. Our data suggest that PGC-1alpha plays an important physiological role in maintaining energy homeostasis during fasting by decreasing triglyceride production/secretion while it increases fatty acid beta-oxidation to meet energy needs.