Last Updated: October 21, 2024
Introduction to the FXR Family
The farnesoid X receptor was originally cloned as an orphan nuclear receptor. The original FXR was so-called because it was shown to be weakly activated by farnesol (an acyclic sesquiterpene alcohol extracted from several different essential oils like lemon grass and citronella) and juvenile hormone III (a acyclic sesquiterpenoid compound that is involved in insect physiology). Subsequent studies demonstrated that FXR could be activated by bile acids and so the protein has also been identified as the bile acid receptor (BAR). Given its relationship to the steroid/thyroid hormone superfamily of nuclear receptors the original FXR encoding gene is also identified as NR1H4 (for nuclear receptor subfamily 1, group H, member 4).
The NR1H4 gene is located on chromosome 12q23.1 and is composed of 14 exons that generate six mRNAs via alternative promoter usage and alternative splicing. These six NR1H4 encoded mRNAs collectively encode five distinct protein isoforms.
Two FXR protein families have been characterized in animals (identified as FXRα and FXRβ). However, in humans the FXRβ encoding gene (NR1H5P) is a pseudogene and so any functional role for FXRβ in humans remains unclear. In humans at least four functional FXR isoforms have been identified as being derived from the NR1H4 gene as a result of activation from two different promoters and the use of alternative splicing between exons 5 and 6. These isoforms are identified as FXRα1, FXRα2, FXRα3, and FXRα4. The four FXRα isoforms exhibit tissue-specific patterns of expression.
The domain structures of the FXR are similar to other members of the nuclear receptor family of transcriptional regulators. Each FXR has a DNA-binding domain (DBD) near the N-terminus and a ligand-binding domain (LBD) near the C-terminus that are separated by a hinge domain. In addition, the FXR contain two activation function (AF) domains. The AF1 is a ligand-independent domain and is located in the N-terminus proximal to the DBD. The AF2 is a ligand-dependent domain located at the C-terminus distal to the LBD. FXRα3 and FXRα4 contain an N-terminal extension, relative to FXRα1 and FXRα2, that encompasses the ligand-independent AF1 domain. FXRα1 and FXRα3 each contain an insert of four amino acids (MYTG) in the hinge domain immediately distal to the DBD.
The NR1H4 gene is expressed predominantly in the liver, intestine, kidney, and adrenal gland with low levels of expression also seen in adipose tissue and heart. The precise mechanisms that control the expression of the NR1H4 gene are not completely delineated. However, it is known that the levels of FXR mRNA increase in the liver in response to fasting. The transcriptional co-activator PGC-1α (peroxisome proliferator-activated receptor-γ co-activator-1α) is also induced in liver in response to fasting and experiments have shown that overexpression of PGC-1α in hepatocyte cell cultures results in increased expression of the FXR gene. The ability of PGC-1α to effect this change in FXR expression is likely due to PGC-1α co-activation of the transcription factor, hepatocyte nuclear factor 4α (HNF4α), which is bound to repeat elements in the two FXR promoters.
The various FXRα proteins form permissive heterodimers with the various retinoid X receptors (RXR) and as such regulate gene expression upon binding FXR ligands and RXR ligands. The FXR/RXR heterodimers bind to FXR-responsive elements (FXRE) in target DNA that consist of inverted repeats (IR) with the core sequence AGGTCA separated by one nucleotide, designated IR1 or direct repeats separated by four nucleotides (DR4), or everted repeats separated by eight nucleotides (ER8). Many FXR target genes respond to ligand activation of the transcription factor activity of FXR in an isoform-independent manner. However, several target genes such as intestinal bile acid binding protein (IBABP) and fibroblast growth factor 19 (FGF19: FGF15 in mice) have increased responsiveness to FXRα2 or FXRα4 which lack the MYTG insert.
Gene Activation by FXR
In the absence of ligand the FXR/RXR heterodimer resides in the nucleus bound to FXRE in a complex with transcriptional co-repressors such as silencing mediator of retinoic acid and thyroid hormone receptor (SMRT) and nuclear receptor co-repressor (N-CoR). Nuclear receptors that reside in the nucleus in the absence of activating ligand are classified as type II nuclear receptors, whereas, nuclear receptors that reside in the cytosol until engaging ligand are classified as type I nuclear receptors. Upon ligand binding to FXR there is a conformational change in the complex that facilitates a co-repressor for co-activator complex exchange and transcriptional activation of target genes.
The FXR were originally identified by their ability to bind farensol and its metabolites. Farnesol is a naturally occurring 15-carbon acyclic sesquiterpene alcohol. Subsequent to the identification of farnesol activating the FXR research has demonstrated that FXR are receptors for bile acids and bile acid metabolites. The activation of FXR by bile acids represents the primary mechanism by which bile acids negatively regulate genes involved in their own synthesis.
Various bile acids exert differing levels of FXR activation with chenodeoxycholic acid (CDCA) being the most potent followed in potency by deoxycholic acid (DCA), lithocholic acid (LCA), and then cholic acid (CA). In addition to binding bile acids, FXR have been shown to bind polyunsaturated fatty acids (PUFA) such as the omega-3 PUFA, docosahexaenoic acid (DHA), as well as the omega-6 PUFA arachidonic acid, and also the essential fatty acid α-linolenic acid (ALA). Most recently, FXR has been shown to bind the androgen hormone, androsterone, derived via testosterone metabolism.
FXR can activate expression of target genes as well as repress target genes via a trans-repression process which involves FRX/RXR-mediated interference with the binding of transcriptional activation complexes at the repressed gene. Interestingly, FXR has been shown to be able to activate gene expression when bound to certain target genes as a monomer. For example, FXR monomers have been shown to activate the expression of the gene (APOA1) encoding apolipoprotein A-I (apoA-I) and the gene (UGT2B4) encoding UDP-glucuronosyltransferase 2B4. UDP-glucuronosyltransferase 2B4 converts hydrophobic bile acids into more hydrophilic glucuronide derivatives.
Regulation of Bile Acid and Lipid Metabolism by FXR
FXR plays a critical role in the regulation of bile acid synthesis, lipoprotein metabolism, glucose metabolism, protection of the liver from toxic metabolites and xenobiotics (hepatoprotection), liver regeneration, and repression of overgrowth of intestinal bacteria. Numerous target genes have been identified as being either activated or repressed in response to ligand activation of FXR.
Bile acid synthesis occurs only in the liver and the synthesis and metabolism of bile acids is tightly controlled to ensure that toxic levels do not accumulate. There are two pathways for the synthesis of bile acids referred to as the classic (or neutral) pathway and the acidic pathway (see the Bile Acids: Synthesis, Metabolism and Biological Functions page for details).
One major target of FXR is the gene encoding small heterodimer partner (SHP). SHP is also a member of the nuclear receptor superfamily and is encoded by the NR0B2 (nuclear receptor subfamily 0 group B member 2) gene. Although SHP is a member of the nuclear receptor superfamily it lacks a DBD and thus, represses gene transcription by binding to and inhibiting other transcription factors. Activation of SHP expression by FXR results in inhibition of transcription of SHP target genes. Of significance to bile acid synthesis, SHP represses the expression of the cholesterol 7α-hydroxylase gene (CYP7A1). CYP7A1 is the rate-limiting enzyme in the synthesis of bile acids from cholesterol via the classic pathway.
FXR also regulates the expression of CYP7A1 via the induction of human FGF19 (see the Gut-Brain Interrelationships and the Control of Feeding page). Experiments done in mice involve FGF15 as this is the murine ortholog of human FGF19. When FGF19 is secreted it activates hepatic FGF receptor-4 (FGFR4) which then down-regulates CYP7A1 via Jun N-terminal kinase (JNK)-mediated signal transduction. FGF19 also affects the motility of the gall bladder allowing for refilling with hepatic bile from the bile canaliculi.
The bile acids are conjugated to the amino acids glycine or taurine via the sequential action of bile acid-CoA synthetase (BACS) and bile acid-CoA:amino acid N-acetyltransferase (encoded by the BAAT gene) which increases the solubility of the bile acids in the aqueous phase. The BACS enzyme is encoded by the SLC27A5 gene. The SLC27A5 encoded protein is a very long-chain acyl-CoA synthetase. Expression of both SLC27A5 and BAAT genes is regulated by FXR.
Bile acids, bile acid metabolites, and various related hydroxysteroid compounds are cytotoxic if present at high concentration. Therefore, the liver must modify these various compounds into less toxic, more water soluble metabolites. As indicated above, FXR activates the UGT2B4 gene involved in glucuronidation of bile acids making them more water soluble. Another modifying enzyme induced in the liver by FXR is dehydroepiandrosterone sulfotransferase (SULT2A1) which conjugates sulfur to hydroxysteroids.
Bile acid and related compound efflux from hepatocytes into the bile canaliculi, that lead to the gall bladder, is enhanced by activation of FXR. The genes encoding three transporters involved in this efflux process are induced by FXR including bile salt export protein (BSEP), multi-drug resistance protein 3 (MDR3), and multi-drug resistance-associated protein 2 (MRP2). All three transporters are members of the ATP-binding cassette (ABC) family of transporters. BSEP is encoded by the ABCB11 gene, MDR3 is encoded by the ABCB4 gene, and MRP2 is encoded by the ABCC2 gene. The significance of the actions of these three transporters can be demonstrated from the fact that mutations in ABCB11 (BSEP) and ABCB4 (MDR3) are associated with familial intrahepatic cholestasis type 2 and 3, respectively. Mutations in ABCC2 (MRP2) gene are associated with Dubin-Johnson syndrome, a form of inherited hyperbilirubinemia.
The consumption of food causes the intestine to release cholecystokinin (CCK) which stimulates the gall bladder to contract and expel bile acids into the duodenum. The bile emulsifies lipids in the food allowing for more effective uptake by the gut. Approximately 95% of the bile acids and bile acid metabolites in the intestine are reabsorbed and returned to the liver via the enterohepatic circulation. Transporters involved in this reabsorption are apical sodium-dependent bile acid transporter (ASBT) and the heterodimeric transporter composed of organic solute transporters-α and -β (OSTα and OSTβ). Within the intestine the protein, intestinal bile acid-binding protein (IBABP), binds bile acids and may protect the liver from excess uptake of bile acids by OSTα and OSTβ.
ASBT, OSTα, and OSTβ are members of the solute carrier (SLC) family of transporters. ASBT is encoded by the SLC10A2 gene, OSTα is encoded by the SLC51A gene, and OSTβ is encoded by the SLC51B gene. All three of the genes encoding these proteins have been shown to be directly regulated by FXR.
The significance of FXR, in the regulation of bile acid homeostasis, is demonstrated by the clinical consequences of mutations in the NR1H4 gene. Loss of FXR activity is associated with a form of severe progressive familial intrahepatic cholestasis (PFIC) identified as PFIC5. This form of PFIC is associated with low to normal gamma-glutamyl transferase (GGT), increased alpha-fetoprotein, and a vitamin K-independent coagulopathy. PFIC5 is an autosomal recessive disorder that is a rapidly progressing lethal form of liver failure.
Regulation of Lipoprotein Metabolism by FXR
That FXR is involved in triglyceride and lipoprotein metabolism was demonstrated in individuals who were given CDCA or CA for the treatment of gall stones. In these individuals the levels of plasma triglycerides and HDLs were reduced whereas the levels of LDLs were increased. Given that CDCA and CA are known activators of FXR it became clear that bile acids also regulated lipid homeostasis via FXR activation. The mechanism of FXR involvement in lipid homeostasis is in part via FXR-mediated repression of the master transcriptional regulator SREBP-1c. SREBP-1c regulates the expression of numerous genes involved in fatty acid and triglyceride synthesis. Thus it is not surprising that FXR activation, which reduces SREBP-1c levels, results in reduced hepatic triglyceride synthesis and secretion.
FXR activation in the liver also results in increased expression of receptors that are involved in lipoprotein clearance (VLDL receptor and syndecan-1). FXR also increases expression of apolipoprotein C-II (apoC-II) which is necessary for the activation of lipoprotein lipase (LPL) and decreases expression of apoC-III which inhibits LPL activity. Fatty acid oxidation is enhanced by FXR via its ability to activate transcription of PPARα which is a transcription factor that promotes the β-oxidation of fatty acids. Activation of FXR also results in increased expression of the scavenger receptor type I BI (SR-BI) which is important in the process of reverse cholesterol transport via hepatic cholesterol uptake from HDLs.
Regulation of Glucose Homeostasis by FXR
FXR activation has also been shown to regulate glucose homeostasis. Activation of FXR in mouse models of diabetes results in reduced plasma glucose and increased insulin sensitivity. These effects are due to hepatic regulation of gluconeogenesis, glycogen synthesis, and insulin responsiveness in the liver. FXR activation results in reduced expression of hepatic phosphenoylpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase both of which are important in hepatic gluconeogenesis. The reduction in expression of these two enzymes explains the reduced hepatic glucose output upon FXR activation. A reduction in glucose-6-phosphatase activity would result in higher levels of substrate for hepatic glycogen synthesis. Indeed, FXR activation in diabetic mice results in increased hepatic glycogen levels. The increase in glycogen synthesis is associated with increased phosphorylation of glycogen synthase kinase 3β (GSK3β). GSK3β normally phosphorylates and inhibits glycogen synthase. When insulin binds its receptor GSK3β becomes phosphorylated and inhibited. Thus, FXR activation exerts an effect on glycogen synthesis that is associated with increased insulin responsiveness.
Clinical and Pharmacological Relevance of FXR
Bile acids, in excess, particularly in the liver, have been associated with pathophysiology for quite some time. Given that activation of hepatic FXR synthesis has been shown to suppress bile acid synthesis, thereby reducing the overall bile acid pool, it has been suggested that FXR agonists would be useful in the treatment of disorders related to bile acid metabolism and hepatic health. Given the critical role of the liver in glucose and lipid homeostasis, it is understandable why pharmacologic regulation of FXR is expected to have benefits in the treatment of obesity and diabetes.
Indeed, in 2016 the US FDA approved the use of obeticholic acid (6α-ethyl-chenodeoxycholic acid) as a second-line treatment for primary biliary cholangitis (PBC). PBC is an auto-immune disease that is associated with a slow, progressive destruction of the small bile ducts of the liver. Obeticholic acid is sold under the trade name of Ocaliva. Numerous other FXR agonists are currently in various stages of clinical trials for use in the treatment of PBC and non-alcoholic steatohepatitis (NASH). NASH is now referred to as metabolic dysfunction-associated steatohepatitis (MASH).
When FXR is activated in the intestines the major effect is the activation of expression of the growth factor, FGF19. FGF19 activity contributes to control of overall bile acid homeostasis. In addition, FXR activation in the liver contributes to glucose, lipid, and cholesterol homeostasis as well as being able to promote liver regeneration, further contributing to the potential pharmacologic benefits of FXR agonists. The potential for pharmacologic targeting of FXR was initially demonstrated in 2003 when a novel compound, called fexamarine, was shown to be a potent FXR agonist.
When bile acids are released to the duodenum from the gallbladder following a meal there is selective activation of intestinal FXR. When given orally, fexaramine is not absorbed resulting in gut-restricted activation of FXR. This effect leads to alterations in bile acid composition without concurrent activation of FXR target genes in the liver. This is due to the activation of intestinal FGF19 expression with the hormone entering the enteric circulation and exerted specific effects, particularly in the liver. Initial experiments have demonstrated that oral fexaramine results in reduced weight gain, decreased inflammation, browning of WAT, and increased insulin sensitization in the diet-induced obesity mouse model. These effects of fexaramine were exerted due to systemic effects of FGF19 on the expression of genes involved in thermogenesis, mitochondrial biogenesis and fatty acid oxidation.
Oral fexaramine induction of intestinal FGF19 expression results in the reduced expression of cholesterol 7α-hydroxylase (CYP7A1) and oxysterol 7α-hydroxylase (CYP7B1) in the liver while simultaneously leading to increased expression of the genes encoding G-protein coupled bile acid receptor 1 (GPBAR1), SHP (NR0B2), OSTα (SLC51A), and OSTβ (SLC51B). In the case of the CYP7A1 and CYP7B1 genes their reduced level of expression results in reduced production of bile acids. Oxysterol 7α-hydroxylase is a minor enzyme in hepatic bile acid synthesis. Increased expression of the NR0B2 gene results in reduced expression of the CYP7A1 gene and thus reduced bile acid synthesis since the encoded protein, SHP, is a transcriptional inhibitor of the CYP7A1 gene. In the case of the SLC51A, and SLC51B genes their increased expression results in increased efflux of bile acids from the liver into the bile canaliculi and eventually into the gallbladder for excretion.