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). Given its relationship to the steroid/thyroid hormone superfamily of nuclear receptors the FXR gene is also identified as NR1H4 (for nuclear receptor subfamily 1, group H, member 4). There are two genes encoding FXRs in humans identified as FXRα and FXRβ. In humans at least four FXR isoforms have been identified as being derived from the FXRα 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 human FXRβ gene is a pseudogene located on chromosome 1. The domain structures of the FXRs are similar to other members of the nuclear receptor family of transcriptional regulators. Each FXR has a DNA-binding domain (DBD) and a ligand-binding domain (LBD). In addition, the LBD contains an activation function domain identified as the AF2 domain. FXRα3 and FXRα4 contain an N-terminal extension, relative to FXRα1 and FXRα2, that encompasses the AF1 domain. FXRα1 and FXRα3 each contain an insert of four amino acids (MYTG) immediately adjacent to the DBD.
The FXR gene resides on chromosome 12q23.1 and contains 11 exons. The FXR 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 FXR 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 FXRs form permissive heterodimers with the RXRs and as such can regulate gene expression either upon binding FXR ligands or RXR ligands. The FXR/RXR heterodimers bind to FXR-responsive elements (FXREs) in target DNA that consist of inverted repeats (IRs) 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 FXRs 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.
In the absence of ligand the FXR/RXR heterodimer resides in the nucleus bound to FXREs 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 FXRs 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 FXRs were originally identified by their ability to bind farensol metabolites. However, subsequent research has demonstrated that FXRs are receptors for bile acids and bile acid metabolites which is the primary mechanism by which bile acids negatively regulate their own expression. Various bile acids exert differing levels of FXR activation with chenodeoxycholic acid (CDCA) being the most potent followed by lithocholic acid (LCA) and deoxycholic acid (DCA) then cholic acid (CA). In addition to binding bile acids, FXRs have been shown to bind polyunsaturated fatty acids (PUFAs) such as the omega-3 PUFAs docosahexaenoic acid (DHA), arachidonic acid, and 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 apolipoprotein A-I (apoA-I) gene and the UDP-glucuronyltransferase 2B4 gene (UGT2B4). The latter gene converts hydrophobic bile acids into more hydrophilic glucuronide derivatives.
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 page for details). One major target of FXR is the small heterodimer partner (SHP; also identified as NR0B2) gene. SHP is a member of the nuclear receptor superfamily but 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 7-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 (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 (BAT) which increases the solubility of the bile acids in the aqueous phase. Expression of both BACS and BAT 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). Both MDR3 and MRP2 are members of the ATP-binding cassette (ABC) family of transporters. MDR3 is also identified as ABCB4 and MRP2 is identified as ABCC2. The significance of the actions of these three transporters can be demonstrated from the fact that mutations in BSEP and MDR3 are associated with familial intrahepatic cholestasis type 2 and 3, respectively and mutations in MRP2 are associated with Dubin-Johnson syndrome, a form of inherited hyperbilirubinemia.
The consumption of food causes the intestine to release cholecystokinin (CKK) 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 heterodimeric 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. All of these genes have been shown to be directly regulated 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.
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 heptatic 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.
Bile acids, in excess, 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.
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. 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 FXR-activation of genes involved in thermogenesis, mitochondrial biogenesis and fatty acid oxidation.