Bile Acid Receptors as Targets for Drug

NATURE REVIEWS | GASTROENTEROLOGY & HEPATOLOGY ADVANCE ONLINE PUBLICATION | 1

Department of Surgery, NUTRIM School of Nutrition, Toxicology and Metabolism, Maastricht University, PO Box 616, 6200 MD, Maastricht, Netherlands (F. G. Schaap). Division of Gastroenterology and Hepatology, Department of Internal Medicine III, Medical University of Vienna, Waehringer Guertel 18–20, 1090 Vienna, Austria (M. Trauner). Department of Gastroenterology and Hepatology, Academic Medical Centre, Meibergdreef 9, 1105 AZ, Amsterdam, Netherlands (P. L. M. Jansen).

Correspondence to: P. L. M. Jansen [email protected]

Bile acid receptors as targets for drug development Frank G. Schaap, Michael Trauner and Peter L. M. Jansen

Abstract | The intracellular nuclear receptor farnesoid X receptor and the transmembrane G protein-coupled receptor TGR5 respond to bile acids by activating transcriptional networks and/or signalling cascades. These cascades affect the expression of a great number of target genes relevant for bile acid, cholesterol, lipid and carbohydrate metabolism, as well as genes involved in inflammation, fibrosis and carcinogenesis. Pregnane X receptor, vitamin D receptor and constitutive androstane receptor are additional nuclear receptors that respond to bile acids, albeit to a more restricted set of species of bile acids. Recognition of dedicated bile acid receptors prompted the development of semi-synthetic bile acid analogues and nonsteroidal compounds that target these receptors. These agents hold promise to become a new class of drugs for the treatment of chronic liver disease, hepatocellular cancer and extrahepatic inflammatory and metabolic diseases. This Review discusses the relevant bile acid receptors, the new drugs that target bile acid signalling and their possible applications.

Schaap, F. G. et al. Nat. Rev. Gastroenterol. Hepatol. advance online publication 27 August 2013; doi:10.1038/nrgastro.2013.151

IntroductionTraditional Chinese medicine recognized the therapeutic value of bear bile long before the era of modern medicine. Bear bile is replete with ursodeoxycholic acid (UDCA), a bile acid encountered in appreciable amounts only in Ursidae.1 Although bear bile gained scientific interest at the turn of the previous century with the isolation of a theretofore unknown bile acid (that is, UDCA) from bile of the polar bear by Olof Hammersten of the University of Uppsala in Sweden,1 it would last nearly eight more decades before the therapeutic potential of bile acids for dissolv-ing gallstones and the treatment of bile acid biosynthesis defects and primary biliary cirrhosis (PBC) was realized.2–4 An early observation was made in 1938 by Nobel l aureate Philip Hench. He observed that rheumatic symptoms a lleviated when patients with rheumatoid arthritis become jaundiced.5 Elevation of serum bile acids has been a candi-date to explain this phenomenon ever since, and suggests an anti-inflammatory effect of bile acids. The discovery of receptors with affinity for bile acids, and subsequent X-ray crystallography studies of their ligand-binding sites, has given the development of natural, semi-synthetic and fully synthetic drugs targeting these receptors and associated pathways a strong impetus and molecular base. These new agents offer novel therapeutic possibilities for the treatment and prevention of liver disease, atherosclerosis, obesity and type 2 diabetes mellitus (T2DM). However, before this new approach can be fully appreciated, a more complete

understanding is needed of the complex n etworks that link bile acids and metabolism.

In reading this Review, one has to appreciate that much of the research in this area has been undertaken in mice, which often does not translate directly to humans. Working with human cell systems only partially remedies this situ-ation, as effects of bile acid signalling often rely on interac-tions between tissues rather than responses in single-cell systems. The final proof for the therapeutic value of bile acid analogues has to come from carefully planned, ran-domized human studies with clear end points. In this Review, we focus on bile acid signalling in humans, albeit it has to be acknowledged that much of the data and ideas are based on evidence from mouse models.

Bile acidsExcellent in-depth reviews covering the synthesis, metab-olism, transport and physicochemical functions of bile acids have been published elsewhere.6–9 We will, therefore, only mention some elemental knowledge.

Bile acids are a diverse class of water-soluble, cholesterol-derived, amphipathic molecules that are formed in the liver (primary bile acids) with microbial transformation in the gut (secondary bile acids) greatly expanding the molecu-lar repertoire. Bile acids typically occur as conjugates with either glycine or taurine, and are negatively charged over most of the physiological pH range. Although the term ‘bile salt’ would be more appropriate from a chemical perspec-tive, the expression ‘bile acid’ is mostly used throughout this Review for congruence with colloquial terminology (such as UDCA and obeticholic acid). These chemical fea-tures necessitate dedicated transport proteins for efficient cellular permeation and largely confine intracellular bile

Competing interestsM. Trauner declares associations with the following companies: Falk Pharma, Intercept, Phenex. See the article online for full details of the relationships. F. G. Schaap and P. L. M. Jansen declare no competing interests.

REVIEWS

© 2013 Macmillan Publishers Limited. All rights reserved

mailto:[email protected]

http://www.nature.com/doifinder/10.1038/nrgastro.2013.151

2 | ADVANCE ONLINE PUBLICATION www.nature.com/nrgastro

acid signalling to the small intestine and liver where such transporters are expressed.6,8,9

In contrast to intracellular nuclear receptors such as the farnesoid X receptor (FXR, also known as bile acid receptor NR1H4, discussed below), activation of the cell-surface receptor TGR5 (also known as G-protein coupled bile acid receptor 1) does not depend on trans-port systems for cellular ligand uptake. TGR5 is not only located in intestinal epithelium, Kupffer cells, sinusoidal endothelium and bile duct cells but also in tissues not participating in the enterohepatic circulation, suggesting that bile acids in the systemic circulation are relevant for activation of this receptor. This concept is entirely new as it brings into focus tissues (for example, muscle, brain, adipose tissue) and cell types (for example, macrophages and endothelial cells) that do not participate in classic bile acid cycling as possible targets of bile acid signalling.

When the gallbladder contracts after food ingestion, bile and pancreatic juice enter the intestine and lipid break-down starts. Here, bile acids in millimolar concentrations act as detergents and enable pancreatic lipase to digest emulsified fat into monoglycerides and fatty acids that together with fat-soluble vitamins can be absorbed in the proximal small intestine.9 Bile acids are also indispensable for solubilization of cholesterol in bile and absorption of cholesterol in the intestine.9 In addition, canalicular bile salt secretion is the main driving force for bile flow. Bile acids are, therefore, key agents for removal of cholest-erol from the body. These traditional roles of bile acids have been known for more than 50 years.10 In the 1990s, a number of ligand-activated transcription factors of the nuclear hormone receptor (NHR) family were discovered, later followed by recognition of bile acids as their endog-enous ligands.11,12 These findings gave rise to the idea that bile acids in micromolar concentrations can act as signal-ling molecules. Feedback regulation of bile acid synthe-sis by bile acids was already known, but the responsible receptor remained elusive until the discovery of FXR.13,14 Upon binding of bile acids, FXR induces expression of the transcriptional repressor SHP (small heterodimer partner, or NR0B2).15 SHP subsequently interferes with the tran-scription of CYP7A1, which encodes the rate-determining enzyme cholesterol 7-α-monooxygenase in the multiple-step conversion of cholesterol to the primary bile acids chenodeoxycholic acid (CDCA) and cholic acid.6 In addition, CYP7A1 and sterol-12α-hydroxylase (CYP8B1)

Key points

■ The discovery of dedicated bile acid receptors and further research defined multiple bile acid signalling routes affecting bile acid synthesis, lipid synthesis, gluconeogenesis, inflammation, liver fibrosis and cancer

■ Steroidal and nonsteroidal agonists of bile acid receptors have been developed as potential treatments for cholestatic and metabolic liver diseases, including primary biliary cirrhosis, primary sclerosing cholangitis and NASH

■ Randomized, placebo-controlled clinical trials for the treatment of primary biliary cirrhosis and NASH with the farnesoid X receptor (FXR) agonist obeticholic acid are the first clinical trials initiated

■ Future indications for FXR and TGR5 agonists include genetic cholestatic syndromes, intrahepatic cholestasis of pregnancy, atherosclerosis, IBS, bile-reflux oesophagitis, IBD, hepatocellular and colon carcinoma

are controlled via an endocrine mechanism by fibroblast growth factor 19 (FGF19, termed Fgf15 in rodents) pro-duced in the ileum upon activation of FXR.16 It turned out that bile acids not only regulate their own synthesis and secretion,13 but also have a role in many other metab-olic pathways (reviewed elsewhere17). In addition, bile acids affect such diverse processes as liver regeneration, intestinal integrity and shaping of the intestinal micro-biome.18–20 Bile acids are also known to have pro-apoptotic and proinflammatory actions.21,22

Bile acid receptorsNHRs for which bile acids are relevant ligands include FXR (NR1H4),11 pregnane X receptor (PXR, also known as NR1I2),23 vitamin D3 receptor (VDR, also known as NR1I1),24 and constitutive androstane receptor (CAR, also known as NR1I3).25 PXR and CAR are rather promis-cuous receptors responding to a large variety of structur-ally unrelated xenobiotic compounds. In addition to these intracellular receptors, TGR5 is a cell-surface receptor of the G protein-coupled receptor family that binds and is activated by bile acids.26,27

Farnesoid X receptorAmong NHRs, FXR is the receptor most dedicated to signalling by bile acids. FXR was also the first identi-fied bile acid receptor.11,12 There are two distinct FXR genes, encoding FXR-α (NR1H4) and FXR-β (NR1H5), with the latter having an unresolved function in mice and is considered a pseudogene in humans.28 Four func-tional isoforms of FXR-α have been identified that differ in transactivation potential and tissue distribution.29 Throughout this Review, the term ‘FXR’ is used to denote (an unspecified isoform of) FXR-α.

FXR is abundantly expressed in tissues engaged in the enterohepatic circulation of bile acids, with expression in the small intestine being highest at the site of bile acid reclamation, that is, the ileum (Figure 1).30 In the mouse liver, Fxr is prominently expressed in parenchymal cells and to a lesser extent in endothelial, Kupffer and stellate cells.31,32 Studies in mice have largely focused on the func-tion of Fxr in the small intestine and liver, with little infor-mation available on functionality in the kidneys, adrenal glands, cardiovascular system, thyroid gland, lungs, skin, adipose tissue, immune cells and human cancers.33–37

Endogenous FXR agonists include (with decreasing affinity): CDCA, the secondary bile acid deoxycholic acid, cholic acid and lithocholic acid (LCA) (Table 1). Muricholic acids, present in rodents but not in humans, were identified in a study published in 2013 as Fxr antago-nistic bile acids.38 UDCA, a bile acid that is widely used in the treatment of cholestatic liver disease is not an FXR ligand. Neither is the synthetic side-chain-shortened UDCA analogue 24-nor-UDCA, despite a number of actions resembling bona fide Fxr activation (for example, maintenance of bile acid homeostasis, anti-inflammator y and anti-fibrotic effects, and improvement of serum lipids). Interestingly, 24-nor-UDCA suppresses Cyp7a1 in the context of reduced Shp expression in a genetic model of steatohepatitis in mice.39

REVIEWS



FXR exerts its functions by eliciting transcriptional alter-ations. The protein has a multidomain structure with dis-tinct regions engaged in DNA binding, ligand binding and transactivation. FXR is thought to be bound in an unligan-ded state to target promoter elements either as a monomer or as a heterodimer with the retinoic acid receptor RXR-α (RXRA, also known as NR2B1). Ligand binding results in dissociation of co-bound co-repressors and recruitment of co-activator proteins, thus, promoting target gene expres-sion. Regulatory modes distinct from direct induction, including direct repression and indirect repression via SHP, as well as effects exclusively at a post-transcriptional level, have been described for FXR.40 FXR is part of a complex network of interacting transcription factors that include hepatocyte nuclear factor 1α, peroxisome proliferator-activate d receptor α (PPARA, also known as NR1C1), PXR, oxysterols receptor LXR-α (NR1H3) and CAR.41–45

In the liver and intestine, FXR controls a number of important metabolic pathways (Figure 1, Table 1, reviewed elsewhere46,47). Its pivotal role in maintaining bile acid homeostasis and, thus, preventing bile acid toxi-city includes induction of: bile acid conjugation (upregula-tion of the conjugating enzyme bile acid-CoA:amino acid N-acetyltransferase, BAAT);48 canalicular bile salt secretion (upregulation of bile salt export pump, BSEP);49 and baso-lateral efflux (‘overflow’) systems that provide an altern-ative route of bile acid elimination (for example, OSTα/β).50 Moreover, FXR represses bile acid synthesis via upregula-tion of ileal FGF1916 and hepatic SHP.14 SHP differs from other typical NHRs in lacking a ligand-binding domain; it represses gene expression by interfering with the function of transcription factors and co-activators required for basal gene expression.14 In the case of CYP7A1, these factors include liver receptor homologue 1 (LRH1, also known as NR5A2), hepatocyte nuclear factor 4 -α (HNF4-α, also known as NR2A1) and peroxisome proliferator-activated receptor γ co-activator 1-α (PGC1-α).

FXR was reported to be expressed in hepatic stellate cells, explaining the anti-fibrotic action of FXR agonists in rat models of liver fibrosis.51 However, Fickert et al.32 have cast doubt on this concept; they found rather low levels of FXR expression in human and mouse hepatic stellate cells and portal myofibroblasts. Moreover, models of biliary type fibrosis in Fxr–/– mice showed less fibrosis than wild-type mice, and absence of Fxr had no effect in traditional models of liver fibrosis.32

Fxr–/– mice have a high incidence of hepatocellular carcinoma (HCC) and other degenerative features in the liver.52 Two studies have shown that Fxr controls the expres-sion of tumour suppressor genes. In one study, a direct effect of Fxr on Ndrg2 (N-myc downstream-regulate d gene 2) mRNA expression was shown and the other study reported that Fxr reduces gankyrin expression53,54 (this oncoprotein is a proteasomal subunit that has a role in the degradation of a number of tumour suppressor proteins). In agreement with a role for Fxr in hepatocarcinogenesis, it was shown that nonsteroidal Fxr agonists reduce tumour size in a mouse xenograft model.53

Bile acid homeostatic actions of Fxr in the small intes-tine include buffering of intracellular bile acid levels

(through upregulation of intestinal bile acid protein, Ibabp), and feedback inhibition of hepatic bile acid syn-thesis via induction of the secreted enterokine FGF19 (or Fgf15 in rodents).16 Fxr also has an important role in maintaining intestinal barrier integrity and antibacterial defence; the underlying mechanisms are poorly under-stood and seem to include induction of antibacterial p roteins such as angiogenin.19

Some of the effects of FXR are mediated by the entero-kine FGF19 (Figure 1). FGF19 is an unusual member of the FGF family in being secreted into the (portal) circula-tion and having an endocrine mode of action. Ileal bile acid uptake induces FGF19 (Fgf15 in rodents) expression via FXR-mediated transcriptional induction.16 In addi-tion, LCA might induce intestinal FGF19 expression via PXR-dependent and VDR-dependent pathways.55,56 In humans, systemic FGF19 levels peak 3–4 h after a fatty meal.57 After portal delivery, FGF19 signals in the liver via hepatocytic FGF receptor 4 (FGFR4) and possibly

CDCA

CDCA

NTCP

FGF19

CDCA

CDCA

BSEP

FGFR4

ASBTOSTα/β

Hepatocyte

Ileal epithelial cell

Bile acid synthesisLipogenesisGluconeogenesisRegeneration

FGF19

CYP7A1

FXRagonist

FXR agonist

FXR

Cholesterol Primary bile acids (e.g. CDCA)

FXR

CDCA

Bile canalicus

FXRagonist

Figure 1 | Enterohepatic actions of FXR. Bile acids (exemplified as the primary bile acid CDCA) are produced in the liver by CYP7A1-initiated conversion of cholesterol in primary bile acids. Bile salts are secreted via BSEP into the canalicular lumen. In the ileum, bile salts are reabsorbed via ASBT in terminal ileum enterocytes. Here, they bind and activate FXR and this stimulates the transcription of FGF19, which encodes a protein that is secreted into the portal circulation. In the liver, FGF19 binds to its receptor FGFR4, which activates a signalling pathway involving MAP kinases and causes repression of CYP7A1, thus downregulating bile acid synthesis. After OSTα/β-mediated secretion into the portal circulation, bile acids are taken up by the liver via NTCP, thus, completing the enterohepatic cycle. In the liver, bile acids bind to FXR, which transcriptionally upregulates a protein called SHP (not shown) that interferes with expression of CYP7A1. Oral FXR agonists will affect FXR in both liver and intestine and this strongly downregulates CYP7A1 both by FGF19-dependent and FGF19-independent effects. FGF19 additionally affects lipogenesis, gluconeogenesis and liver regeneration. Abbreviations: ASBT, apical sodium-dependent bile salt transporter; BSEP, bile salt export pump; CDCA, chenodeoxycholic acid; CYP7A1, cholesterol 7-α-monooxygenase; FGF19, fibroblast growth factor 19; FGFR4, fibroblast growth factor receptor 4; FXR, farnesoid X receptor; NTCP, Na+-taurocholate cotransporting polypeptide; OST, organic solute transporter; SHP, small heterodimer partner.

REVIEWS



FGFR1c. Physiological FGF19 signalling requires the presence of βKlotho, a membrane protein expressed in the liver and white adipose tissue, whose exact function in FGFR-mediated FGF19 signalling is being explored.58,59 FGF19-responsive receptors FGFR4 and FGFR1c are receptor tyrosine kinases that exert their effects on target gene expression via activation of signalling cascades including the mitogen-activated protein kinase pathway.59 FGF19 regulates bile acid synthesis via downregulation of CYP7A1 expression in a SHP-dependent way.60 As hepatic FXR can directly downregulate CYP7A1 expression via the induction of SHP, this mechanism seems redundant. However, tissue-specific Fxr-knockout models have shown that under normal conditions the intestinal Fxr–Fgf15 axis is more important for downregulation of hepatic Cyp7a1 than hepatic Fxr–Shp in mice.61

Apart from its role in bile acid homeostasis, post prandial actions of FGF19 include inhibition of gluconeogenesis and stimulation of glycogen synthesis.62 These metabolic actions resemble that of insulin with a notable exception that FGF19 does not stimulate hepatic lipo genesis.63 FGF19 reduces key enzymes and regulators of hepatic lipid syn-thesis.64 Studies using Fgf15–/– mice revealed that Fgf15 is involved in gallbladder relaxation. Human FGF19 can take over this function in Fgf15–/– mice.65 Whether FGF19 is also involved in relaxation of the human gallbladder is currently unknown. Evidence indicates that FGF19 has

anti-inflammatory actions66 and has a role in liver regen-eration.67 Human, but not mouse, bile contains substan-tial amounts of FGF19, produced in the biliary tree and in the gall bladder;68 its presence in bile might provide a nti-inflammatory protection to the bile ducts.

FGF19 and FGF19-inducing drugs have potential for a number of therapeutic applications. However, FGF19 transgenic mice develop HCC.69 Although this finding could be because of supraphysiological circulating levels, FGF19 might be procarcinogenic.70 This undesired activity can be eliminated by replacement of a 7-amino-acid stretch that comprises an FGFR4 interaction domain. This engi-neered variant retains the metabolic functions of FGF19, but is devoid of mitogenic activity.71

Transmembrane G protein-coupled receptorTGR5 is highly expressed in the gallbladder and bile duct epithelial cells, brown adipose tissue, muscle, intestine, kidney, placenta and brain (Figure 2). In the liver, TGR5 is expressed in sinusoidal endothelial cells, bile duct epi-thelial cells and Kupffer cells, but not in hepatocytes.72–74 LCA is the strongest natural agonist of TGR5, but TGR5 also responds to (un)conjugated deoxycholic acid, CDCA, UDCA and cholic acid26,27 (Table 1). Oleanolic acid from olive tree leaves is a nonsteroidal selective TGR5 agonist,75 whereas INT-77776 (6-ethyl-23(S)-methyl-CDCA) is a semi-synthetic TGR5 agonist (Figure 3).

Table 1 | Bile acid receptors and targets of action

Receptor Cytogeneticlocation

Exemplary ligands Affected pathways and/or processes

Target tissues Target disease

FXR (NR1H4)

12q23.1 Bile acids: (un)conjugated CDCA > DCA > LCA > CA159

Bile acid analogues: 6α-ethyl-CDCA (INT-747),111 6α-ethyl-3α,7α,23-trihydroxy-24-nor-5β-cholan-23-sulphate (INT-767)113

Nonsteroidal ligands: GW4064, fexaramine, GSK2324, Way362450, PX-102160–164

Bile acid synthesis14,16

Bile acid export 50

Bile formation 49

Phase I/II metabolism 48

Lipogenesis46

Gluconeogenesis46

Tumour suppression 52,53

Liver regeneration165

Liver inflammation166

Liver fibrosis32,51

Intestinal barrier function19

Liver, intestine, kidney

Cholestatic liver disease, NASH, T2DM, HCC, IBD

TGR5 2q35 Bile acids: (un)conjugated LCA > DCA > CDCA > CA,26,27102

Bile acid analogues: INT-767, 6α-ethyl-23(S)-methyl-3α,7α,12α-trihydroxy-5β-cholan-24-oic acid (INT-777)112,113

Xenobiotic ligands: oleanolic acid75

Glucose homeostasis80

Energy expenditure 82

Gallbladder relaxation84

Anti-inflammatory77

Liver, intestine, gallbladder, muscle, brain

T2DM, NASH

PXR (NR1I2)

3q13.33 Bile acids: LCA, 3-keto-LCA >> CDCA, DCA, CA167

Other steroidal ligands: pregnenolone, progesteroneXenobiotic ligands: herbal medicine107 (e.g. hyperforin, guggulsterone), drugs (e.g. rifampicin, paclitaxel, lovastin)168,169

Bile acid synthesis96

Phase I/II metabolism 94

Phase III efflux 93,94

Lipogenesis 94,98

Gluconeogenesis94

Anti-inflammatory94,95

Liver, intestine Cholestatic liver disease, pruritus, IBD

VDR (NR1I1)

12q13.11 Bile acids: (un)conjugated LCA, 3-keto-LCA24,102

Non-bile acid ligands: vitamin D3 (calcitriol) and synthetic analogues (e.g. 2MbisP, BXL-01-0772)170

Nonsteroidal ligands: LY2108491, LY2109886171

Bile salt synthesis56,101

Phase I metabolism 24

Ca2+, phosphate homeostasis100

Bone mineralization100

Antimicrobial defence105

Intestine, kidney, bone

Osteoporosis

CAR (NR1I3)

1q23.3 Bile acids: CA, 6-keto-LCA, 12-keto-LCA108,172

Xenobiotic ligands: CITCO, TCPOBOP, herbal medicine (e.g. 6,7-dimethylesculetin), drugs (e.g. phenobarbital)107

Phase I/II metabolism 45,106110

Phase III efflux 93

Lipogenesis 173

Gluconeogenesis 109,173

Liver Cholestatic liver disease, pruritus

Abbreviations: >, higher affinity than; >>, much higher affinity than; , decreased activity; , increased activity; 2MBisP, 2-methylene-19-nor-(20S)-1α-hydroxy-bishomopregnacalciferol; CA, cholic acid; CDCA, chenodeoxycholic acid; CITCO, 6-(4-Chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime; DCA, deoxycholic acid; HCC, hepatocellular carcinoma; LCA, lithocholic acid; T2DM, type 2 diabetes mellitus; TCPOBOP, 1,4-Bis-[2-(3,5-dichloropyridyloxy)]benzene, 3,3',5,5'-Tetrachloro-1,4-bis(pyridyloxy)benzene.

REVIEWS



Ligand binding to TGR5 results in stimulation of a denylate cyclase, with elevation of cAMP levels, trigger-ing subsequent activation of protein kinase A and further downstream signalling events.77 In lipopolysaccharide-stimulated mouse macrophages, activation of Tgr5 has anti-inflammatory effects by inhibiting nuclear translocation of nuclear factor κB, thereby reducing production of pro-inflammatory cytokines and mediators such as Tnf, Il-1β, Il-6, Ifn-γ and inducible nitric oxide synthase.78,79 INT-777 reduced inflammation and lipid-loading of plaque macro-phages in mice.78 Activation of Tgr5 in enteroendocrine cells leads to secretion of glucagon-like peptide 1 (GLP-1).80,81 This incretin improves pancreas function, insulin secre-tion and insulin sensitivity (Figure 2). Tgr5 in muscle and brown adipose tissue has a role in energy homeostasis via activation of cAMP-dependent iodothyronine d eiodinase 2, an enzyme that converts inactive thyroxine (T4) into active thyroid hormone (T3).82 Thus, activation of Tgr5 in these tissues leads to an increase in energy expenditure and oxygen consumption. This action probably underlies the prevention of obesity, hepatic steatosis and improvement of insulin sensitivity in mice on a high-fat diet supplemented with either bile acid or INT-777.81,82 Elevation of systemic bile acid levels in cholestatic disorders or after Roux-en-Y gastric bypass surgery in humans83 can be expected to result in metabolic effects through activation of TGR5.

Moreover, stimulation of Tgr5 in sinusoidal endothelial cells can result in nitric oxide production, which might affect the haemodynamics of sinusoidal perfusion.74 In the biliary tree, activation of Tgr5 causes gallbladder relaxation and activates the chloride channel CFTR and enhances secretion of bicarbonate.72,84 By increasing the pH of bile, a higher proportion of bile acids will be in the ionized form, which reduces their ability to diffuse into the biliary epi-thelium. This process protects bile duct epithelium against the detergent effect of bile acids.85

Pregnane X receptorIn mice, Pxr (also referred to as the steroid and xeno-biotic receptor in humans) is highly expressed through-out the gastrointestinal tract and to a lesser extent in the kidneys.30,86–89 In humans, PXR is expressed in liver, gastrointestinal tract and brain.90 In mouse liver, Pxr is expressed primarily in parenchymal cells.31 The main, if not only, bile acid ligand of PXR is LCA and its oxidized 3-keto form. LCA is a cytotoxic bile acid formed mainly by bacterial deconjugation and 7α-dehydroxylation of conjugated CDCA, and is passively absorbed in the colon where it is considered pro-oncogenic.91 LCA induces experimental cholestasis that results in extensive liver damage. PXR serves as a xenobiotic sensor, and induces phase I and II metabolism of many endogenous and exog-enous compounds, including bile acids.92 PXR-mediated detoxification of LCA includes 6α-hydroxylation by members of the CYP3A subfamily and sulphation by the bile acid sulphotransferase SULT2A1. Apart from (3-keto)LCA, a great number of prescription drugs qualify as PXR a gonists with rifampicin on top of the list (Table 1).

PXR binds as a heterodimer with RXR-α to response elements in the promoter region of target genes, which

include phase I metabolizing mono-oxygenases of the cytochrome P-450 family (for example, CYP1A, CYP2B, CYP2C, CYP3A members), phase II conjugating enzymes (for example, UDP-glucuronosyltransferases, glutathione S-transferases and sulphotransferases), and phase III efflux pumps (for example, canalicular multi-specific organic anion transporter 1 [ABCC2], also known as multidrug resistance-associated protein 2). ABCC2 is a multispecific organic anion transporter that mediates canalicular secretion of glucuronidated substrates including bilirubin and divalent bile salts; its expression is affected not only by PXR, but also by FXR and CAR.93

Besides its role in drug metabolism, PXR has additional functions in bile acid, lipid and glucose metabolism, endo-crine homeostasis, androgen metabolism, bone mineral homeostasis, vitamin D metabolism and immuno-suppression, as well as having an anti-inflammator y action.94,95 Treatment with the PXR agonist rifampicin results in downregulation of CYP7A1 expression, which seems to result from binding of PXR to HNF4-α, thereby displacing PGC1-α and interfering with HNF4-α–PGC1-α-stimulated CYP7A1 transcription.96 This SHP-independent pathway of CYP7A1 suppression causes downregulation of bile acid synthesis.

TGR5agonists

TGR5TGR5

TGR5

TGR5

GLP-1

Kupffer cellEnteroendocrinecells in intestine

Brown adiposetissue

Muscle

LPS Cytokines

T4 T3

DIO2

T4 T3

DIO2

Energy expenditureInsulin secretion and/or sensitivityIn�ammation

Insulin secretionInsulin sensitivity

cAMP cAMP

cAMPcAMP

Figure 2 | TGR5-expressing tissues and targets. TGR5 signalling in skeletal muscle and brown adipose tissue results in local activation of the deiodinase DIO2 that generates active thyroid hormone (T3), an important regulator of metabolism and energy homeostasis. Bile acids in the intestinal lumen activate TGR5 in enteroendocrine cells, resulting in release of the incretin GLP-1. In Kupffer cells and macrophages, TGR5 activation inhibits LPS-induced cytokine production. Abbreviations: DIO2, type II iodothyronine deiodinase; GLP-1, glucagon-like peptide 1; LPS, lipopolysaccharide; T3, active thyroid hormone; T4, inactive thyroxine; TGR5, transmembrane G protein-coupled receptor TGR5 (also known as GPBAR1).

REVIEWS



In general, PXR activation has a protective effect by enhancing drug metabolism and suppressing bile acid synthesis. These are favourable actions of PXR agonists. PXR agonists might therefore have value in the treat-ment of chronic inflammatory diseases of liver and bowel.95,97 However, Pxr activation leads to hepatic stea-tosis in mice,98 and enhanced drug metabolism might cause unwanted drug–drug interactions in patients who are dependent on more than one drug. This interaction could be harmful in patients with NASH and patients on anti-coagulants, chemotherapeutics or HIV inhibi-tors. Additional undesirable effects of PXR activation include osteoporosis, disturbances of the glucocorticoid and mineralocorticoid balance, disturbances of androgen metabolism and enhanced breakdown of vitamins.99

Vitamin D receptorVDR is widely expressed in human tissues such as the intestine and kidneys, as well as in pancreatic β-cells, osteo-β-cells, osteo--cells, osteo-blasts, adipocytes, vascular smooth muscle cells, mono-cytes and immune-competent cells.100 Evidence indicates that VDR is also expressed in human (but not mice) hepato cytes.31,101 VDR has a central role in mineral and bone homeostasis, and is engaged in control of cellular growth and differentiation.100

Calcitriol (1,25-dihydroxyvitamin D3), a steroid-like molecule with a disrupted steroid nucleus, and the bile acid LCA (but not CDCA and cholic acid) are endogenous VDR ligands (Table 1).102 Activation of VDR by pro-oncogenic LCA is achieved at concentrations lower than required to activate PXR.102 A special feature of VDR is its function as both an intracellular nuclear receptor and membrane recep-tor.103 Upon ligand binding, VDR moves to the nucleus where it binds to DNA response elements to modulate gene transcription. In addition, unliganded VDR resides

in caveolin domains at the cell membrane where it serves as a receptor. The genomic action is fairly slow (hours) and includes downregulation of CYP7A1 expression and induction of CYP3A4 expression, an enzyme that detoxi-fies LCA.24,101,104 By contrast, the response initiated at the membrane is rapid (minutes) and results in activation of signalling cascades that, in the liver, contribute to CYP7A1 repression. In the bile duct epithelium, VDR (together with FXR) has been shown to stimulate production of antimicro-bial proteins such as cathelicidin, which might add to the immunomodulatory function of vitamin D.105 Activation of Vdr by intraperitoneal injections of vitamin D3 in mice causes induction of Fgf15 and repression of Cyp7a1.56

Constitutive androstane receptorAs with its closest relative PXR, CAR is expressed mainly in the liver and gastrointestinal tract. Here, CAR acts in concert with PXR to detoxify and eliminate endog-enous (for example, LCA, bilirubin) and foreign com-pounds.23,92,106 In line with this function, CAR binds a broad variety of structurally diverse molecules such as certain bile acids (for example, cholic acid and 6-keto-LCA), drugs including the prototypical CAR activator phenobarbital as well as herbal medicines107 (Table 1).

Activation of CAR results in its nuclear trans location, a process that does not necessarily require interac-tion between ligand and the ligand-binding domain. Indeed, phenobarbital is a potent activator of CAR target gene expression yet does not bind to its ligand-binding domain.108 CAR forms a heterodimer with RXR-α and binds to retinoic-acid-response elements of target genes. Reflected in its name, CAR is unusual among NHRs in functioning as an transcriptional activator in the absence of ligand, which has been attributed to ligand-independe nt recruitment of transcriptional co-activators ,

FXR agonists

TGR5 agonists

Intercept INT-747Obeticholic acid

Phenex PX-102(PX20606)

P�zer WAY-362450(FXR450, XL335)

HO

CO2H

OH

Oleanolic acidIntercept INT-777 6-methyl-2-oxo-4-thiophen-2-yl-1,2,3,4-tetrahydro-pyrimidine-5-

carboxylic acid benzyl ester

HO

CO2H

OH

(S)

HO

CO2H

OS

O

CH2

NH

O

HN

Cl

O

HO

O

Cl Cl

O

NN

OF

F

NH

O

O

Figure 3 | Steroidal and nonsteroidal agonists of FXR and TGR5. The semi-synthetic bile acid obeticholic acid (6-ethyl-CDCA, INT-747) and non-steroidal compounds PX-102 and WAY-362450 are potent FXR agonists. Side-chain modification (for example, addition of a methyl group) turns obeticholic acid into the potent and selective TGR5 agonist INT-777. Oleanolic acid is a naturally occurring TGR5 agonist found in olive tree leaves. Abbreviations: FXR, farnesoid X receptor; TGR5, transmembrane G protein-coupled receptor TGR5 (also known as GPBAR1).

REVIEWS



with agonists (for example, TCPOBOP) promoting co-activator recruitment and CAR transactivation, and inverse agonists (for example, androstanol) inhibiting the constitutive activity of CAR by blocking co-activato r recruitment. CAR-binding bile acids seem to act as inverse agonists that repress CAR activity.108

CAR targets include genes encoding proteins involved in phase I and II drug metabolism (for example, CYP2B monooxygenases, UDP-glucuronosyltransferases and sulpho transferases) and elimination (MRP2 and MRP3). As with PXR, CAR not only targets the metabolism of xenobiotics, but also regulates lipid and glucose metabo-lism.109 Studies with Pxr/Car double knockout mice revealed that Pxr and Car cooperate in the detoxification of LCA.106,110 Fxr expression is decreased in Car–/– mice, suggesting i nteraction between Car and Fxr.45

Bile acid receptors as drug targetsThe dedicated bile acid receptors FXR and TGR5 have been prime targets for drug development. Obeticholic acid (6α-ethyl-chenodeoxycholic acid, also known as INT-747 or OCA), INT-777 (6α-ethyl-23(S)-methyl-cholic acid) and INT-767 (the 23-sulphate derivative of obeticholic acid) are agonists of FXR, TGR5 and both FXR and TGR5, respectively (Figure 3).111–113 Development of these agonists has been greatly stimulated by the elucidation of the 3D structure of the ligand-binding domains of FXR and TGR5. FXR agonists have to fit into a tight pocket in the ligand-binding domain, for binding to TGR5 both the steroid nucleus and side-chain structure are important molecu-lar features.111,112 Side-chain modification (for example, methylation or sulphation) turns bile acid derivatives into TGR5 agonists. In Mdr2–/– mice with extensive chronic cholangiopathy, the mixed Fxr–Tgr5 agonist INT-767 led to stronger improvement of liver injury parameters than obeticholic acid or INT-777 alone.114

FXR agonists have considerable therapeutic poten-tial. Obeticholic acid is currently being investigated in phase III and phase II trials in patients with PBC and NASH, respectively.111,115 Synthetic FXR agonists include fexaramine, PX-102, GSK2324 and WAY-362450 (Figure 3). Synthetic FXR agonists are in phase I and phase II of drug development.37

In view of the pleiotropic spectrum of FXR actions, unexpected effects and off-target effects might occur, which could complicate drug development. For instance, FXR agonists cause elevation of circulating FGF19 levels. FGF19 transgenic mice develop HCC.69 The question therefore arises as to what the effect would be of chronic treatment with FXR agonists and persistent elevation of FGF19. Currently, no answer is available, and the role of FGF19 in human tumorigenesis is unresolved.

For application of FXR agonists in atherosclerosis, the picture is not entirely clear either. The potent FXR agonists obeticholic acid and WAY-362450 both reduced aortic plaque formation in Apoe–/– mice on a high-fat diet.116,117 By contrast, the FXR antagonist guggulsterone decreased hepatic cholesterol in wild-type mice and Fxr deficiency reduced atherosclerosis in Ldlr–/– and Apoe–/– mice. In both studies, mice were fed a high-fat diet.118–120 Furthermore,

another study showed that FXR activation induces hepatic expression of a flavin-containing monooxygenase (that is, FMO3) that converts the choline metabolite trimethyl-amine into atherogenic trimethylamine N-oxide.121 How these seemingly disparate findings in mice could lead to useful drug therapy in humans requires further study.

Synthetic FXR and TGR5 agonists have been devel-oped to serve as next-generation drugs for the treat-ment of metabolic diseases and cholestatic liver disease (Figure 3).37,76,122,123 Of note, these drugs undergo entero-hepatic cycling and will be most effective in liver and intestine. Apical sodium-dependent bile salt transporter (ASBT, also known as ileal sodium/bile acid cotrans-porter or SLC10A2) inhibitors and bile salt sequestrants (discussed later) are developed as possible drugs for treat-ment of hypercholesterolaemia, cholestatic liver disease and T2DM.124 In addition, colestyramine, originally devel-oped for treatment of hypercholesterolaemia, improves insulin sensitivity and causes weight loss; as such, colesty-ramine, colesevalam and other sequestrants are being re-investigate d for treatment of T2DM.125

UDCA and bile salt sequestrantsAlthough UDCA is not an FXR agonist and only a weak PXR agonist, it has a number of actions that contribute to UDCA being a drug for the treatment of PBC and intra-hepatic cholestasis of pregnancy.126 UDCA stimulates bile flow by virtue of its own biliary secretion, by stimulating biliary bicarbonate secretion, and by promoting insertion of transporter proteins into the canalicular membrane.127–129 In a study published in 2013, Gohlke et al.130 showed that α5β1 integrin functions as an intracellular sensor for tauroUDCA and might be involved in the latter process. Whether this activity is maintained in the cholestatic liver, and to what extent previously reported actions of UDCA as Ca2+ agonist and activator of protein kinase C also has a role in stimulating hepatocyte secretion in the cholestatic liver, needs to be studied.127,131

Reabsorption of (de)conjugated bile acids in the terminal ileum occurs mainly via ASBT (or SLC10A2) (Figure 1).132 Via this transporter ~90% of luminal bile acids are reclaimed. Inhibition of bile acid transport in the ileum will lead to spillover of bile acids into the colon where at high concentrations (millimolar range) they stimulate chloride and water secretion and cause bile acid diarrhoea.133 Bile acids, brought into the colon by nonabsorbable anion exchange resins, activate Tgr5 in enteroendocrine L cells leading to release of the incretin Glp-1.134 Against this background, inhibition of bile salt reabsorption by anion exchange resins or ASBT inhibitors might be considered as a possible therapy for T2DM.134,135 Moreover, the ASBT inhibitor SC-435 decreased LDL-cholesterol and reduced atherosclerosis in Apoe–/– mice, indicating that these inhibi-tors have potential in the treatment of hypercholesterol-aemia.136 Inhibition of bile salt reabsorption decreases the circulating bile salt pool, reduces FGF19 levels, increases the conversion of cholesterol into bile acids and reduces serum cholesterol levels.137 Similar effects on serum glucose and serum LDL-cholesterol levels can be achieved with the bile acid sequestrant colesevalam, an anion exchange

REVIEWS



resin that is used for treatment of bile acid diarrhoea, h ypercholesterolaemia and T2DM.138,139

Candidate diseasesMost studies on the effect of FXR and TGR5 agonists have been done in mouse models of human disease. Randomized trials in humans are still in their infancy. The first human trials focus on the efficacy of obeticholic acid in PBC (phase III, NCT01473524) and NASH (phase II, FLINT trial, NCT01265498).140,141 End points of these studies are decreased alkaline phosphatase levels after 6 and 12 months in PBC,140 and improvement of histological score after 72 weeks treatment in NASH.141 A phase II trial on the effect of the addition of obeticholic acid to UDCA that was completed in 2012, revealed a ~22% decrease of alkaline phosphatase levels after 12 weeks in the obeticholic acid group (NCT00550862).142

The rationale of the PBC studies is that bile acid toxic-ity contributes to the symptomatology of this cholestatic disorder. In addition, bile acid toxicity causes necrosis and apoptosis of resident hepatocytes and leads to inflam-mation, fibrosis, cirrhosis and HCC. However, severe c holestasis leads to an adaptive response by endogenous FXR activation.143 Thus, questions arise on what would be the best disease phase for optimal use of FXR agonists; they will probably work best in moderate c holestasis when endogenous FXR activation is suboptimal. FXR agonists

are strong repressors of de novo bile acid synthesis. This aspect is particularly important when bile flow is obstructed. In this setting, input of extra bile acid into the biliary space would cause bile acid overload and bile-acid-mediated necrosis and apoptosis.144

Pruritus has been noted as an adverse effect of obeti-cholic acid in the treatment of PBC;145 a potential draw-back for the use of the drug in PBC as pruritus is already a cumbersome symptom in baseline PBC. Cholestatic p ruritus has long been attributed to bile acids, but studies indicate that lysophosphatidic acid produced by the enzyme autotaxin might be the responsible prurito-gen.146,147 However, this issue has not been settled yet as bile acids might still have a role. In a study published in 2013, bile acids and TGR5 have been implicated in the development of cholestatic pruritus.148 Highly selective FXR agonists, without any activity against TGR5, might therefore be better c andidates for the treatment of PBC.

As a result of their anti-inflammatory and anti-fibrotic properties, FXR agonists are candidate drugs for treatment of primary sclerosing cholangitis, a disease p rimarily affecting the large intrahepatic and extra-hepatic bile ducts and giving rise to biliary fibrosis of the liver. FXR agonists have anti-inflammatory effects in the inflamed colon of mouse models of IBD.149 80% of patients with primary sclerosing cholangitis also have ulcerative colitis or Crohn’s disease and there are good

Diet

InsulinHyperglycaemia

Liver

Muscle Adipocytes

FFA FFA FFAAMPK

ROSATP

Intestine

LPS

Kupffer cell

OxLDLFibrosis

Stellate cell

Apoptosis

TGF-β

Oxidativestress

TriglyceridesSREBP-1

TNF, IL-1,chemokines

FXR agonistTGR5 agonist

In�ammation

Pancreas

Microbiome

FGF19GLP-1

VLDL

Fatty liver

FGFR4

Figure 4 | Potential targets of FXR and TGR5 agonists in NASH. NASH is characterized by hepatic fat accumulation with concurrent inflammation and fibrosis. Steatosis is the result of ongoing lipogenesis, in the face of elevated influx of fatty acids derived from adipocyte lipolysis and impaired VLDL-lipid export. Inflammation can arise from compromised gut barrier function, lipotoxic effects of adipocyte-derived fatty acids and modified lipoproteins such as oxLDL. Inflammatory stimuli result in activation of resident Kupffer cells and release of inflammatory factors that recruit immune cells and initiate fibrogenesis through activation of stellate cells. NASH can progress through fibrotic and cirrhotic stages to hepatocellular carcinoma. Possible sites of action of FXR (blue) and TGR5 (green) agonists are indicated by arrows, and are discussed in more detail in the main text. Abbreviations: FFA, free fatty acid; FGF19, fibroblast growth factor19; FXR, farnesoid X receptor; GLP-1, glucagon-like peptide 1; LPS, lipopolysaccharide; oxLDL, oxidized LDL; ROS, reactive oxygen species; SREBP-1, sterol regulatory element-binding protein 1; TGR5, transmembrane G protein-coupled receptor TGR5 (also known as GPBAR1); VLDL, very low density lipoprotein.

REVIEWS



arguments for an aetiological role of an impaired intes-tinal integrity in the pathogenesis of primary sclerosing cholangitis.150 Drugs that target both diseases (primary sclerosing cholangitis and IBD) are therefore particu-larly attractive. However, it should be realized that FXR agonists are stimulators of bile flow and caution is needed in using these drugs in patients with severely damaged extrahepatic bile ducts as seen in primary sclerosing chol-angitis. Moreover, Fickert et al.32 reported that loss of Fxr in mouse models of biliary fibrosis decreased fibrosis. How this translates to humans needs to be studied.

Other indications for FXR-agonist-mediated repres-sion of bile acid synthesis include inborn errors of bile acid synthesis, progressive familial intrahepatic cholestasis (in particular type 1 and 3) and cholestasis of pregnancy. Increased bile acid synthesis, with spillover of bile acids into the colon, might have a role in bile-acid-induced diar-rhoea and perhaps also in IBS; conversely, impaired bile acid excretion has been linked to constipation.151

In view of the actions of FXR and TGR5 on lipid and glucose metabolism, FXR and/or TGR5 agonists are candidate drugs for the treatment of NASH, hyper-cholesterolaemia, hypertriglyceridaemia and T2DM. The action of FXR and TGR5 agonists in NASH is shown in Figure 4. A phase II trial of obeticholic acid, administered in two different doses (25 and 50 mg per day) during 6 weeks to patients with NAFLD and T2DM, showed an increase in insulin sensitivity, a decrease in levels of γ-glutamyltransferase and alanine aminotransferase, and dose-dependent weight loss.152

An interesting application of FXR agonists might be in the treatment of diabetic renal disease. Fxr deficiency leads to acceleration of diabetic nephropathy in a mouse model for type 1 diabetes, and obeticholic acid improved proteinuria and podocyte function and decreased renal tubulointerstitial fibrosis and inflammation.153 Testing FXR agonists in diabetic nephropathy would therefore be of interest. In addition, FXR agonists have potential for the prevention and treatment of HCC, atherosclerosis, (bile-) reflux oesophagitis, IBD and colon carcinoma.

Bariatric surgery, in particular Roux-en-Y gastric bypass surgery provides an interesting example of the actions of FXR, TGR5 and VDR in the intestine (Figure 5). After this procedure, digestive juices and food mix late in the small intestine. Thus, limited spillover of bile acids into the colon occurs, where bile acids activate TGR5 and VDR resulting in secretion of GLP-1 and, possibly, FGF19. GLP-1 will improve insulin sensitivity and secretion and FGF19 might reduce hepatic steatosis by inhibiting lipo-genesis. Weight reduction is obtained mainly by incom-plete absorption of nutrients in the small intestine. In line with this model, GLP-1 and FGF19 levels are increased after Roux-en-Y gastric bypass surgery,83,154 which makes this procedure a plausible treatment for NASH in patients with major overweight.

After an episode of bile duct obstruction, owing to sepsis, liver surgery or drug exposure, it might take days to weeks before the jaundice improves. The term ‘severe persistent hepatocellular secretory failure’ has been introduced for this situation. A short treatment with the

PXR agonist rifampicin greatly shortened the time to recovery.155 Moreover, PXR and CAR agonists have been widely used in the treatment of unconjugated hyperbili-rubinaemia as occurring in Gilbert syndrome and type 2 Crigler–Najjar syndrome.156–158

ConclusionsThe endocrine-like signalling function of bile acids and insight into the 3D structure of bile acid receptors have given momentum to a new field of pharmacology with the promise of new drugs for difficult-to-treat metabolic and liver disorders. Some of these new drugs are already the subject of randomized controlled trials. New mol-ecules are in the pipeline and these factors might undergo clinical testing in the near future. Many of the concepts discussed in this Review are based on in vitro studies and work with knockout mice. The bridge from basic studies to clinical medicine still needs to be crossed.

Liver

Bilio-pancreaticloop

Alimentaryloop

FGF19

TGR5

FGF19?

GLP-1

Bile acidNutrients

Insulin sensitivity

LipogenesisGluconeogenesis

FGFR4

FXR

VDR

Figure 5 | Bile acid receptor activation after Roux-en-Y gastric bypass surgery. After Roux-en-Y gastric bypass surgery, partially digested food (red filled circles) and bile acids (green filled circles) mix at a distal site in the small intestine. Because of incomplete admixture of chyme and bile–pancreatic juice, bile acids spill into the large intestine where they activate TGR5 and VDR. As a result, secreted factors are produced that signal to the liver (FGF19) and pancreas (GLP-1) to reduce hepatic lipogenesis and improve insulin sensitivity and insulin secretion. Abbreviations: FGF19, fibroblast growth factor 19; FXR, farnesoid X receptor; GLP-1, glucagon-like peptide 1; TGR5, transmembrane G protein-coupled receptor TGR5 (also known as GPBAR1); VDR, vitamin D receptor.

Review criteria

This Review is based on a PubMed search of relevant topics without time constraints. Key references are cited for original data. This Review is obviously a selection of available literature and we apologize for not citing other relevant work. For more general statements we cite reviews published in high-impact, peer-reviewed journals. This Review is not meant to be a ‘systematic review’.

REVIEWS



1. Hagey, L. R. et al. Ursodeoxycholic acid in the Ursidae: biliary bile acids of bears, pandas, and related carnivores. J. Lipid Res. 34, 1911–1917 (1993).

2. Maton, P. N., Murphy, G. M. & Dowling, R. H. Ursodeoxycholic acid treatment of gallstones. Dose-response study and possible mechanism of action. Lancet 2, 1297–1301 (1977).

3. Poupon, R. et al. Is ursodeoxycholic acid an effective treatment for primary biliary cirrhosis? Lancet 1, 834–836 (1987).

4. Salen, G., Meriwether, T. W. & Nicolau, G. Chenodeoxycholic acid inhibits increased cholesterol and cholestanol synthesis in patients with cerebrotendinous xanthomatosis. Biochem. Med. 14, 57–74 (1975).

5. Hench, P. S. Effect of jaundice on rheumatoid arthritis. Br. Med. J. 2, 394–398 (1938).

6. Russell, D. W. The enzymes, regulation, and genetics of bile acid synthesis. Annu. Rev. Biochem. 72, 137–174 (2003).

7. Ridlon, J. M., Kang, D. J. & Hylemon, P. B. Bile salt biotransformations by human intestinal bacteria. J. Lipid Res. 47, 241–259 (2006).

8. Dawson, P. A., Lan, T. & Rao, A. Bile acid transporters. J. Lipid Res. 50, 2340–2357 (2009).

9. Hofmann, A. F. Biliary secretion and excretion in health and disease: current concepts. Ann. Hepatol. 6, 15–27 (2007).

10. Borgstrom, B., Dahlqvist, A., Lundh, G. & Sjovall, J. Studies of intestinal digestion and absorption in the human. J. Clin. Invest. 36, 1521–1536 (1957).

11. Forman, B. M. et al. Identification of a nuclear receptor that is activated by farnesol metabolites. Cell 81, 687–693 (1995).

12. Seol, W., Choi, H. S. & Moore, D. D. Isolation of proteins that interact specifically with the retinoid X receptor: two novel orphan receptors. Mol. Endocrinol. 9, 72–85 (1995).

13. Shefer, S., Hauser, S., Bekersky, I. & Mosbach, E. H. Feedback regulation of bile acid biosynthesis in the rat. J. Lipid Res. 10, 646–655 (1969).

14. Goodwin, B. et al. A regulatory cascade of the nuclear receptors FXR, SHP-1, and LRH-1 represses bile acid biosynthesis. Mol. Cell 6, 517–526 (2000).

15. Seol, W., Choi, H. S. & Moore, D. D. An orphan nuclear hormone receptor that lacks a DNA binding domain and heterodimerizes with other receptors. Science 272, 1336–1339 (1996).

16. Inagaki, T. et al. Fibroblast growth factor 15 functions as an enterohepatic signal to regulate bile acid homeostasis. Cell. Metab. 2, 217–225 (2005).

17. Houten, S. M., Watanabe, M. & Auwerx, J. Endocrine functions of bile acids. EMBO J. 25, 1419–1425 (2006).

18. Huang, W. et al. Nuclear receptor-dependent bile acid signaling is required for normal liver regeneration. Science 312, 233–236 (2006).

19. Inagaki, T. et al. Regulation of antibacterial defense in the small intestine by the nuclear bile acid receptor. Proc. Natl Acad. Sci. USA 103, 3920–3925 (2006).

20. Alemi, F. et al. The receptor TGR5 mediates the prokinetic actions of intestinal bile acids and is required for normal defecation in mice. Gastroenterology 144, 145–154 (2013).

21. Higuchi, H., Grambihler, A., Canbay, A., Bronk, S. F. & Gores, G. J. Bile acids up-regulate death receptor 5/TRAIL-receptor 2 expression via a c-Jun N.-terminal kinase-dependent pathway involving Sp1. J. Biol. Chem. 279, 51–60 (2004).

22. Allen, K., Jaeschke, H. & Copple, B. L. Bile acids induce inflammatory genes in hepatocytes:

a novel mechanism of inflammation during obstructive cholestasis. Am. J. Pathol. 178, 175–186 (2011).

23. Baes, M. et al. A new orphan member of the nuclear hormone receptor superfamily that interacts with a subset of retinoic acid response elements. Mol. Cell Biol. 14, 1544–1552 (1994).

24. Makishima, M. et al. Vitamin D receptor as an intestinal bile acid sensor. Science 296, 1313–1316 (2002).

25. Choi, H. S. et al. Differential transactivation by two isoforms of the orphan nuclear hormone receptor CAR. J. Biol. Chem. 272, 23565–23571 (1997).

26. Maruyama, T. et al. Identification of membrane-type receptor for bile acids (M-BAR). Biochem. Biophys. Res. Commun. 298, 714–719 (2002).

27. Kawamata, Y. et al. A G protein-coupled receptor responsive to bile acids. J. Biol. Chem. 278, 9435–9440 (2003).

28. Otte, K. et al. Identification of farnesoid X receptor beta as a novel mammalian nuclear receptor sensing lanosterol. Mol. Cell Biol. 23, 864–872 (2003).

29. Zhang, Y., Kast-Woelbern, H. R. & Edwards, P. A. Natural structural variants of the nuclear receptor farnesoid X receptor affect transcriptional activation. J. Biol. Chem. 278, 104–110 (2003).

30. Bookout, A. L. et al. Anatomical profiling of nuclear receptor expression reveals a hierarchical transcriptional network. Cell 126, 789–799 (2006).

31. Li, Z., Kruijt, J. K., van der Sluis, R. J., Van Berkel, T. J. & Hoekstra, M. Nuclear receptor atlas of female mouse liver parenchymal, endothelial, and Kupffer cells. Physiol. Genomics 45, 268–275 (2013).

32. Fickert, P. et al. Farnesoid X receptor critically determines the fibrotic response in mice but is expressed to a low extent in human hepatic stellate cells and periductal myofibroblasts. Am. J. Pathol. 175, 2392–2405 (2009).

33. Bishop-Bailey, D., Walsh, D. T. & Warner, T. D. Expression and activation of the farnesoid X receptor in the vasculature. Proc. Natl Acad. Sci. USA 101, 3668–3673 (2004).

34. Lee, H. et al. FXR regulates organic solute transporters α and β in the adrenal gland, kidney, and intestine. J. Lipid Res. 47, 201–214 (2006).

35. Cariou, B. et al. The farnesoid X receptor modulates adiposity and peripheral insulin sensitivity in mice. J. Biol. Chem. 281, 11039–11049 (2006).

36. Jiang, T. et al. Farnesoid X receptor modulates renal lipid metabolism, fibrosis, and diabetic nephropathy. Diabetes 56, 2485–2493 (2007).

37. Abel, U. et al. Synthesis and pharmacological validation of a novel series of non-steroidal FXR agonists. Bioorg. Med. Chem. Lett. 20, 4911–4917 (2010).

38. Sayin, S. I. et al. Gut microbiota regulates bile acid metabolism by reducing the levels of tauro-β-muricholic acid, a naturally occurring FXR antagonist. Cell. Metab. 17, 225–235 (2013).

39. Beraza, N. et al. Nor-ursodeoxycholic acid reverses hepatocyte-specific nemo-dependent steatohepatitis. Gut 60, 387–396 (2011).

40. Gardmo, C., Tamburro, A., Modica, S. & Moschetta, A. Proteomics for the discovery of nuclear bile acid receptor FXR targets. Biochim. Biophys. Acta 1812, 836–841 (2011).

41. Jung, D. & Kullak-Ublick, G. A. Hepatocyte nuclear factor 1 α: a key mediator of the effect of bile acids on gene expression. Hepatology 37, 622–631 (2003).

42. Jung, D., Mangelsdorf, D. J. & Meyer, U. A. Pregnane X receptor is a target of farnesoid X

receptor. J. Biol. Chem. 281, 19081–19091 (2006).

43. Inoue, J. et al. PPARα gene expression is up-regulated by LXR and PXR activators in the small intestine. Biochem. Biophys. Res. Commun. 371, 675–678 (2008).

44. Renga, B. et al. Farnesoid X receptor suppresses constitutive androstane receptor activity at the multidrug resistance protein-4 promoter. Biochim. Biophys. Acta 1809, 157–165 (2011).

45. Beilke, L. D. et al. Constitutive androstane receptor-mediated changes in bile acid composition contributes to hepatoprotection from lithocholic acid-induced liver injury in mice. Drug Metab. Dispos. 37, 1035–1045 (2009).

46. Claudel, T., Staels, B. & Kuipers, F. The Farnesoid X receptor: a molecular link between bile acid and lipid and glucose metabolism. Arterioscler. Thromb. Vasc. Biol. 25, 2020–2030 (2005).

47. Wang, Y. D., Chen, W. D., Moore, D. D. & Huang, W. FXR: a metabolic regulator and cell protector. Cell Res. 18, 1087–1095 (2008).

48. Pircher, P. C. et al. Farnesoid X receptor regulates bile acid-amino acid conjugation. J. Biol. Chem. 278, 27703–27711 (2003).

49. Ananthanarayanan, M., Balasubramanian, N., Makishima, M., Mangelsdorf, D. J. & Suchy, F. J. Human bile salt export pump promoter is transactivated by the farnesoid X receptor/bile acid receptor. J. Biol. Chem. 276, 28857–28865 (2001).

50. Boyer, J. L. et al. Upregulation of a basolateral FXR-dependent bile acid efflux transporter OSTα-OSTβ in cholestasis in humans and rodents. Am. J. Physiol. Gastrointest. Liver Physiol. 290, G1124–G1130 (2006).

51. Fiorucci, S. et al. The nuclear receptor SHP mediates inhibition of hepatic stellate cells by FXR and protects against liver fibrosis. Gastroenterology 127, 1497–1512 (2004).

52. Kim, I. et al. Spontaneous hepatocarcinogenesis in farnesoid X receptor-null mice. Carcinogenesis 28, 940–946 (2007).

53. Deuschle, U. et al. FXR controls the tumor suppressor NDRG2 and FXR agonists reduce liver tumor growth and metastasis in an orthotopic mouse xenograft model. PLoS ONE 7, e43044 (2012).

54. Jiang, Y. et al. Farnesoid X receptor inhibits gankyrin in mouse livers and prevents development of liver cancer. Hepatology 57, 1098–1106 (2013).

55. Wistuba, W., Gnewuch, C., Liebisch, G., Schmitz, G. & Langmann, T. Lithocholic acid induction of the FGF19 promoter in intestinal cells is mediated by PXR. World J. Gastroenterol. 13, 4230–4235 (2007).

56. Schmidt, D. R. et al. Regulation of bile acid synthesis by fat-soluble vitamins A and D. J. Biol. Chem. 285, 14486–14494 (2010).

57. Schreuder, T. C. et al. The hepatic response to FGF19 is impaired in patients with nonalcoholic fatty liver disease and insulin resistance. Am. J. Physiol. Gastrointest Liver Physiol. 298, G440–G445 (2010).

58. Triantis, V., Saeland, E., Bijl, N., Oude-Elferink, R. P. & Jansen, P. L. Glycosylation of fibroblast growth factor receptor 4 is a key regulator of fibroblast growth factor 19-mediated down-regulation of cytochrome P450 7A1. Hepatology 52, 656–666 (2010).

59. Wu, X. et al. Co-receptor requirements for fibroblast growth factor-19 signaling. J. Biol. Chem. 282, 29069–29072 (2007).

60. Kir, S., Zhang, Y., Gerard, R. D., Kliewer, S. A. & Mangelsdorf, D. J. Nuclear receptors HNF4α and LRH-1 cooperate in regulating Cyp7a1 in vivo. J. Biol. Chem. 287, 41334–41341 (2012).

REVIEWS



61. Kim, I. et al. Differential regulation of bile acid homeostasis by the farnesoid X receptor in liver and intestine. J. Lipid Res. 48, 2664–2672 (2007).

62. Potthoff, M. J. et al. FGF15/19 regulates hepatic glucose metabolism by inhibiting the CREB-PGC-1α pathway. Cell. Metab. 13, 729–738 (2011).

63. Kir, S. et al. FGF19 as a postprandial, insulin-independent activator of hepatic protein and glycogen synthesis. Science 331, 1621–1624 (2011).

64. Miyata, M., Sakaida, Y., Matsuzawa, H., Yoshinari, K. & Yamazoe, Y. Fibroblast growth factor 19 treatment ameliorates disruption of hepatic lipid metabolism in farnesoid X receptor (Fxr)-null mice. Biol. Pharm. Bull. 34, 1885–1889 (2011).

65. Choi, M. et al. Identification of a hormonal basis for gallbladder filling. Nat. Med. 12, 1253–1255 (2006).

66. Drafahl, K. A., McAndrew, C. W., Meyer, A. N., Haas, M. & Donoghue, D. J. The receptor tyrosine kinase FGFR4 negatively regulates NF-κB signaling. PLoS ONE 5, e14412 (2010).

67. Uriarte, I. et al. Identification of fibroblast growth factor 15 as a novel mediator of liver regeneration and its application in the prevention of post-resection liver failure in mice. Gut 62, 899–910 (2013).

68. Zweers, S. J. et al. The human gallbladder secretes fibroblast growth factor 19 into bile: towards defining the role of fibroblast growth factor 19 in the enterobiliary tract. Hepatology 55, 575–583 (2012).

69. Nicholes, K. et al. A mouse model of hepatocellular carcinoma: ectopic expression of fibroblast growth factor 19 in skeletal muscle of transgenic mice. Am. J. Pathol. 160, 2295–2307 (2002).

70. Lin, B. C. & Desnoyers, L. R. FGF19 and cancer. Adv. Exp. Med. Biol. 728, 183–194 (2012).

71. Wu, X. et al. Separating mitogenic and metabolic activities of fibroblast growth factor 19 (FGF19). Proc. Natl Acad. Sci. USA 107, 14158–14163 (2010).

72. Keitel, V. et al. The membrane-bound bile acid receptor TGR5 is localized in the epithelium of human gallbladders. Hepatology 50, 861–870 (2009).

73. Keitel, V. et al. The bile acid receptor TGR5 (Gpbar-1) acts as a neurosteroid receptor in brain. Glia 58, 1794–1805 (2010).

74. Keitel, V. et al. The G.-protein coupled bile salt receptor TGR5 is expressed in liver sinusoidal endothelial cells. Hepatology 45, 695–704 (2007).

75. Sato, H. et al. Anti-hyperglycemic activity of a TGR5 agonist isolated from Olea europaea. Biochem. Biophys. Res. Commun. 362, 793–798 (2007).

76. Sato, H. et al. Novel potent and selective bile acid derivatives as TGR5 agonists: biological screening, structure-activity relationships, and molecular modeling studies. J. Med. Chem. 51, 1831–1841 (2008).

77. Pols, T. W., Noriega, L. G., Nomura, M., Auwerx, J. & Schoonjans, K. The bile acid membrane receptor TGR5 as an emerging target in metabolism and inflammation. J. Hepatol. 54, 1263–1272 (2011).

78. Pols, T. W. et al. TGR5 activation inhibits atherosclerosis by reducing macrophage inflammation and lipid loading. Cell. Metab. 14, 747–757 (2011).

79. Wang, Y. D., Chen, W. D., Yu, D., Forman, B. M. & Huang, W. The G-protein-coupled bile acid

receptor, Gpbar1 (TGR5), negatively regulates hepatic inflammatory response through antagonizing nuclear factor κ light-chain enhancer of activated B cells (NF-κB) in mice. Hepatology 54, 1421–1432 (2011).

80. Katsuma, S., Hirasawa, A. & Tsujimoto, G. Bile acids promote glucagon-like peptide-1 secretion through TGR5 in a murine enteroendocrine cell line STC-1. Biochem. Biophys. Res. Commun. 329, 386–390 (2005).

81. Thomas, C. et al. TGR5-mediated bile acid sensing controls glucose homeostasis. Cell. Metab. 10, 167–177 (2009).

82. Watanabe, M. et al. Bile acids induce energy expenditure by promoting intracellular thyroid hormone activation. Nature 439, 484–489 (2006).

83. Jansen, P. L. et al. Alterations of hormonally active fibroblast growth factors after Roux-en-Y gastric bypass surgery. Dig. Dis. 29, 48–51 (2011).

84. Li, T. et al. The G protein-coupled bile acid receptor, TGR5, stimulates gallbladder filling. Mol. Endocrinol. 25, 1066–1071 (2011).

85. Beuers, U. et al. The biliary HCO3– umbrella:

a unifying hypothesis on pathogenetic and therapeutic aspects of fibrosing cholangiopathies. Hepatology 52, 1489–1496 (2010).

86. Zhang, H. et al. Rat pregnane X receptor: molecular cloning, tissue distribution, and xenobiotic regulation. Arch. Biochem. Biophys. 368, 14–22 (1999).

87. Kliewer, S. A. et al. An orphan nuclear receptor activated by pregnanes defines a novel steroid signaling pathway. Cell 92, 73–82 (1998).

88. Lehmann, J. M. et al. The human orphan nuclear receptor PXR is activated by compounds that regulate CYP3A4 gene expression and cause drug interactions. J. Clin. Invest. 102, 1016–1023 (1998).

89. Bertilsson, G. et al. Identification of a human nuclear receptor defines a new signaling pathway for CYP3A induction. Proc. Natl Acad. Sci. USA 95, 12208–12213 (1998).

90. Lamba, V. et al. PXR (NR1I2): splice variants in human tissues, including brain, and identification of neurosteroids and nicotine as PXR activators. Toxicol. Appl. Pharmacol. 199, 251–265 (2004).

91. Vogel, S. M. et al. Lithocholic acid is an endogenous inhibitor of MDM4 and MDM2. Proc. Natl Acad. Sci. USA 109, 16906–16910 (2012).

92. Wang, Y. M., Ong, S. S., Chai, S. C. & Chen, T. Role of CAR and PXR in xenobiotic sensing and metabolism. Expert Opin. Drug Metab. Toxicol. 8, 803–817 (2012).

93. Kast, H. R. et al. Regulation of multidrug resistance-associated protein 2 (ABCC2) by the nuclear receptors pregnane X receptor, farnesoid X-activated receptor, and constitutive androstane receptor. J. Biol. Chem. 277, 2908–2915 (2002).

94. Ihunnah, C. A., Jiang, M. & Xie, W. Nuclear receptor PXR, transcriptional circuits and metabolic relevance. Biochim. Biophys. Acta 1812, 956–963 (2011).

95. Wallace, K. et al. The PXR is a drug target for chronic inflammatory liver disease. J. Steroid Biochem. Mol. Biol. 120, 137–148 (2010).

96. Li, T. & Chiang, J. Y. Mechanism of rifampicin and pregnane X receptor inhibition of human cholesterol 7 α-hydroxylase gene transcription. Am. J. Physiol. Gastrointest. Liver Physiol. 288, G74–G84 (2005).

97. Cheng, J., Shah, Y. M. & Gonzalez, F. J. Pregnane X receptor as a target for treatment

of inflammatory bowel disorders. Trends Pharmacol. Sci. 33, 323–330 (2012).

98. Zhou, J. et al. Hepatic fatty acid transporter Cd36 is a common target of LXR, PXR, and PPARγ in promoting steatosis. Gastroenterology 134, 556–567 (2008).

99. Jonker, J. W., Liddle, C. & Downes, M. FXR and PXR: potential therapeutic targets in cholestasis. J. Steroid Biochem. Mol. Biol. 130, 147–158 (2012).

100. Norman, A. W. Minireview: vitamin D receptor: new assignments for an already busy receptor. Endocrinology 147, 5542–5548 (2006).

101. Han, S. & Chiang, J. Y. Mechanism of vitamin D receptor inhibition of cholesterol 7α-hydroxylase gene transcription in human hepatocytes. Drug Metab. Dispos. 37, 469–478 (2009).

102. Adachi, R. et al. Structural determinants for vitamin D receptor response to endocrine and xenobiotic signals. Mol. Endocrinol. 18, 43–52 (2004).

103. Huhtakangas, J. A., Olivera, C. J., Bishop, J. E., Zanello, L. P. & Norman, A. W. The vitamin D receptor is present in caveolae-enriched plasma membranes and binds 1α,25(OH)2-vitamin D3 in vivo and in vitro. Mol. Endocrinol. 18, 2660–2671 (2004).

104. Han, S., Li, T., Ellis, E., Strom, S. & Chiang, J. Y. A novel bile acid-activated vitamin D receptor signaling in human hepatocytes. Mol. Endocrinol. 24, 1151–1164 (2010).

105. D’Aldebert, E. et al. Bile salts control the antimicrobial peptide cathelicidin through nuclear receptors in the human biliary epithelium. Gastroenterology 136, 1435–1443 (2009).

106. Saini, S. P. et al. A novel constitutive androstane receptor-mediated and CYP3A-independent pathway of bile acid detoxification. Mol. Pharmacol. 65, 292–300 (2004).

107. Chang, T. K. Activation of pregnane X receptor (PXR) and constitutive androstane receptor (CAR) by herbal medicines. AAPS J. 11, 590–601 (2009).

108. Moore, L. B. et al. Pregnane X receptor (PXR), constitutive androstane receptor (CAR), and benzoate X receptor (BXR) define three pharmacologically distinct classes of nuclear receptors. Mol. Endocrinol. 16, 977–986 (2002).

109. Miao, J., Fang, S., Bae, Y. & Kemper, J. K. Functional inhibitory cross-talk between constitutive androstane receptor and hepatic nuclear factor-4 in hepatic lipid/glucose metabolism is mediated by competition for binding to the DR1 motif and to the common coactivators, GRIP-1 and PGC-1α. J. Biol. Chem. 281, 14537–14546 (2006).

110. Uppal, H. et al. Combined loss of orphan receptors PXR and CAR heightens sensitivity to toxic bile acids in mice. Hepatology 41, 168–176 (2005).

111. Pellicciari, R. et al. 6α-ethyl-chenodeoxycholic acid (6-ECDCA), a potent and selective FXR agonist endowed with anticholestatic activity. J. Med. Chem. 45, 3569–3572 (2002).

112. Pellicciari, R. et al. Discovery of 6α-ethyl-23(S)-methylcholic acid (S-EMCA, INT-777) as a potent and selective agonist for the TGR5 receptor, a novel target for diabesity. J. Med. Chem. 52, 7958–7961 (2009).

113. Rizzo, G. et al. Functional characterization of the semisynthetic bile acid derivative INT-767, a dual farnesoid X receptor and TGR5 agonist. Mol. Pharmacol. 78, 617–630 (2010).

114. Baghdasaryan, A. et al. Dual farnesoid X receptor/TGR5 agonist INT-767 reduces liver injury in the Mdr2–/– (Abcb4–/–) mouse cholangiopathy model by promoting biliary HCO3

– output. Hepatology 54, 1303–1312 (2011).

REVIEWS



115. Adorini, L., Pruzanski, M. & Shapiro, D. Farnesoid X receptor targeting to treat nonalcoholic steatohepatitis. Drug Discov. Today 17, 988–997 (2012).

116. Hartman, H. B. et al. Activation of farnesoid X receptor prevents atherosclerotic lesion formation in LDLR–/– and apoE–/– mice. J. Lipid Res. 50, 1090–1100 (2009).

117. Mencarelli, A., Renga, B., Distrutti, E. & Fiorucci, S. Antiatherosclerotic effect of farnesoid X receptor. Am. J. Physiol. Heart Circ. Physiol. 296, H272–H281 (2009).

118. Urizar, N. L. et al. A natural product that lowers cholesterol as an antagonist ligand for FXR. Science 296, 1703–1706 (2002).

119. Zhang, Y. et al. FXR deficiency causes reduced atherosclerosis in Ldlr – /– mice. Arterioscler Thromb. Vasc Biol. 26, 2316–2321 (2006).

120. Guo, G. L. et al. Effects of FXR in foam-cell formation and atherosclerosis development. Biochim. Biophys. Acta 1761, 1401–1409 (2006).

121. Bennett, B. J. et al. Trimethylamine-N-oxide, a metabolite associated with atherosclerosis, exhibits complex genetic and dietary regulation. Cell. Metab. 17, 49–60 (2013).

122. Marinozzi, M. et al. Pyrazole[3,4-e][1 4]thiazepin-7-one derivatives as a novel class of farnesoid X receptor (FXR) agonists. Bioorg. Med. Chem. 20, 3429–3445 (2012).

123. Herbert, M. R. et al. Synthesis and SAR of 2-aryl-3-aminomethylquinolines as agonists of the bile acid receptor TGR5. Bioorg. Med. Chem. Lett. 20, 5718–5721 (2010).

124. Sakamoto, S. et al. Glucuronidation converting methyl 1-(3,4-dimethoxyphenyl)-3-(3-ethylvaleryl)-4-hydroxy-6, 7,8-trimethoxy-2-naphthoat e (S-8921) to a potent apical sodium-dependent bile acid transporter inhibitor, resulting in a hypocholesterolemic action. J. Pharmacol. Exp. Ther. 322, 610–618 (2007).

125. Kobayashi, M. et al. Prevention and treatment of obesity, insulin resistance, and diabetes by bile acid-binding resin. Diabetes 56, 239–247 (2007).

126. European Association for the Study of the Liver. EASL Clinical Practice Guidelines: management of cholestatic liver diseases. J. Hepatol. 51, 237–267 (2009).

127. Beuers, U. et al. Tauroursodeoxycholic acid inserts the apical conjugate export pump, Mrp2, into canalicular membranes and stimulates organic anion secretion by protein kinase C-dependent mechanisms in cholestatic rat liver. Hepatology 33, 1206–1216 (2001).

128. Garcia-Marin, J. J., Dumont, M., Corbic, M., de Couet, G. & Erlinger, S. Effect of acid-base balance and acetazolamide on ursodeoxycholate-induced biliary bicarbonate secretion. Am. J. Physiol. 248, G20–G27 (1985).

129. Kurz, A. K., Graf, D., Schmitt, M., Vom Dahl, S. & Haussinger, D. Tauroursodesoxycholate-induced choleresis involves p38(MAPK) activation and translocation of the bile salt export pump in rats. Gastroenterology 121, 407–419 (2001).

130. Gohlke, H., Schmitz, B., Sommerfeld, A., Reinehr, R. & Haussinger, D. α5 β1-integrins are sensors for tauroursodeoxycholic acid in hepatocytes. Hepatology 57, 1117–1129 (2013).

131. Bouscarel, B., Fromm, H. & Nussbaum, R. Ursodeoxycholate mobilizes intracellular Ca2+ and activates phosphorylase a in isolated hepatocytes. Am. J. Physiol. 264, G243–G251 (1993).

132. Wong, M. H., Oelkers, P., Craddock, A. L. & Dawson, P. A. Expression cloning and characterization of the hamster ileal sodium-dependent bile acid transporter. J. Biol. Chem. 269, 1340–1347 (1994).

133. Mekjian, H. S., Phillips, S. F. & Hofmann, A. F. Colonic secretion of water and electrolytes induced by bile acids: perfusion studies in man. J. Clin. Invest. 50, 1569–1577 (1971).

134. Harach, T. et al. TGR5 potentiates GLP-1 secretion in response to anionic exchange resins. Sci. Rep. 2, 430 (2012).

135. Chen, L. et al. Inhibition of apical sodium-dependent bile acid transporter as a novel treatment for diabetes. Am. J. Physiol. Endocrinol. Metab. 302, E68–E76 (2012).

136. Bhat, B. G. et al. Inhibition of ileal bile acid transport and reduced atherosclerosis in apoE–/– mice by SC-435. J. Lipid Res. 44, 1614–1621 (2003).

137. Li, H. et al. Inhibition of ileal bile acid transport lowers plasma cholesterol levels by inactivating hepatic farnesoid X receptor and stimulating cholesterol 7 α-hydroxylase. Metabolism 53, 927–932 (2004).

138. Insull, W. Jr et al. Effectiveness of colesevelam hydrochloride in decreasing LDL cholesterol in patients with primary hypercholesterolemia: a 24-week randomized controlled trial. Mayo Clin. Proc. 76, 971–982 (2001).

139. Hansen, M. et al. Effect of bile acid sequestrants on glycaemic control: protocol for a systematic review with meta-analysis of randomised controlled trials. BMJ Open 2, e001803 (2012).

140. US National Library of Medicine. ClinicalTrials.gov [online], http://www.clinicaltrials.gov/ct2/show/NCT01473524?term=NCT01473524 &rank=1 (2012).



143. Schaap, F. G., van der Gaag, N. A., Gouma, D. J. & Jansen, P. L. High expression of the bile salt-homeostatic hormone fibroblast growth factor 19 in the liver of patients with extrahepatic cholestasis. Hepatology 49, 1228–1235 (2009).

144. Fickert, P. et al. Regurgitation of bile acids from leaky bile ducts causes sclerosing cholangitis in Mdr2 (Abcb4) knockout mice. Gastroenterology 127, 261–274 (2004).

145. Fiorucci, S. et al. Farnesoid X receptor agonist for the treatment of liver and metabolic disorders: focus on 6-ethyl-CDCA. Mini Rev. Med. Chem. 11, 753–762 (2011).

146. Kremer, A. E. et al. Lysophosphatidic acid is a potential mediator of cholestatic pruritus. Gastroenterology 139, 1008–1018 (2010).

147. Kremer, A. E. et al. Serum autotaxin is increased in pruritus of cholestasis, but not of other origin, and responds to therapeutic interventions. Hepatology 56, 1391–1400 (2012).

148. Alemi, F. et al. The TGR5 receptor mediates bile acid-induced itch and analgesia. J. Clin. Invest. 123, 1513–1530 (2013).

149. Gadaleta, R. M. et al. Farnesoid X receptor activation inhibits inflammation and preserves the intestinal barrier in inflammatory bowel disease. Gut 60, 463–472 (2011).

150. Terjung, B. et al. p-ANCAs in autoimmune liver disorders recognise human β-tubulin isotype 5

and cross-react with microbial protein FtsZ. Gut 59, 808–816 (2010).

151. Wong, B. S. et al. Increased bile acid biosynthesis is associated with irritable bowel syndrome with diarrhea. Clin. Gastroenterol. Hepatol. 10, 1009–1015 e3 (2012).

152. Mudaliar, S. et al. Efficacy and safety of the farnesoid X receptor agonist obeticholic acid in patients with type 2 diabetes and nonalcoholic fatty liver disease. Gastroenterology http://dx.doi.org/10.1053/ j.gastro.2013.05.042.

153. Wang, X. X. et al. Diabetic nephropathy is accelerated by farnesoid X receptor deficiency and inhibited by farnesoid X receptor activation in a type 1 diabetes model. Diabetes 59, 2916–2927 (2010).

154. Peterli, R. et al. Improvement in glucose metabolism after bariatric surgery: comparison of laparoscopic Roux-en-Y gastric bypass and laparoscopic sleeve gastrectomy: a prospective randomized trial. Ann. Surg. 250, 234–241 (2009).

155. van Dijk, R., Kremer, A. F., Enemuo, V., Jansen, P. L., Beuers, U. Clinical and molecular aspects of rifampicin-mediated attenuation of severe persistent hepatocellular secretory failure. Hepatology 56, 549A (2012).

156. Ohkubo, H., Okuda, K. & Iida, S. Effects of corticosteroids on bilirubin metabolism in patients with Gilbert’s syndrome. Hepatology 1, 168–172 (1981).

157. Arias, I. M., Gartner, L. M., Cohen, M., Ezzer, J. B. & Levi, A. J. Chronic nonhemolytic unconjugated hyperbilirubinemia with glucuronyl transferase deficiency. Clinical, biochemical, pharmacologic and genetic evidence for heterogeneity. Am. J. Med. 47, 395–409 (1969).

158. Ellis, E. et al. Successful treatment of severe unconjugated hyperbilirubinemia via induction of UGT1A1 by rifampicin. J. Hepatol. 44, 243–245 (2006).

159. Makishima, M. et al. Identification of a nuclear receptor for bile acids. Science 284, 1362–1365 (1999).

160. Downes, M. et al. A chemical, genetic, and structural analysis of the nuclear bile acid receptor FXR. Mol. Cell 11, 1079–1092 (2003).

161. Hambruch, E. et al. Synthetic farnesoid X receptor agonists induce high-density lipoprotein-mediated transhepatic cholesterol efflux in mice and monkeys and prevent atherosclerosis in cholesteryl ester transfer protein transgenic low-density lipoprotein receptor(–/–) mice. J. Pharmacol. Exp. Ther. 343, 556–567 (2012).

162. Flatt, B. et al. Discovery of XL335 (WAY-362450), a highly potent, selective, and orally active agonist of the farnesoid X receptor (FXR). J. Med. Chem. 52, 904–907 (2009).

163. Soisson, S. M. et al. Identification of a potent synthetic FXR agonist with an unexpected mode of binding and activation. Proc. Natl Acad. Sci. USA 105, 5337–5342 (2008).

164. Bass, J. Y. et al. Conformationally constrained farnesoid X receptor (FXR) agonists: heteroaryl replacements of the naphthalene. Bioorg. Med. Chem. Lett. 21, 1206–1213 (2011).

165. Chen, W. D. et al. Farnesoid X receptor alleviates age-related proliferation defects in regenerating mouse livers by activating forkhead box m1b transcription. Hepatology 51, 953–962 (2010).

166. Hollman, D. A., Milona, A., van Erpecum, K. J. & van Mil, S. W. Anti-inflammatory and metabolic actions of FXR: insights into molecular mechanisms. Biochim. Biophys. Acta 1821, 1443–1452 (2012).

REVIEWS


http://www.clinicaltrials.gov/ct2/show/NCT01473524?term=NCT01473524&rank=1









http://dx.doi.org/10.1053/j.gastro.2013.05.042

http://dx.doi.org/10.1053/j.gastro.2013.05.042


167. Staudinger, J. L. et al. The nuclear receptor PXR is a lithocholic acid sensor that protects against liver toxicity. Proc. Natl Acad. Sci. USA 98, 3369–3374 (2001).

168. Jones, S. A. et al. The pregnane X receptor: a promiscuous xenobiotic receptor that has diverged during evolution. Mol. Endocrinol. 14, 27–39 (2000).

169. Moore, L. B. et al. St. John’s wort induces hepatic drug metabolism through activation of the pregnane X receptor. Proc. Natl Acad. Sci. USA 97, 7500–7502 (2000).

170. Ogura, M. et al. Vitamin D3 modulates the expression of bile acid regulatory genes and represses inflammation in bile duct-ligated

mice. J. Pharmacol. Exp. Ther. 328, 564–570 (2009).

171. Ma, Y. et al. Identification and characterization of noncalcemic, tissue-selective, nonsecosteroidal vitamin D receptor modulators. J. Clin. Invest. 116, 892–904 (2006).

172. Maglich, J. M. et al. Nuclear pregnane x receptor and constitutive androstane receptor regulate overlapping but distinct sets of genes involved in xenobiotic detoxification. Mol. Pharmacol. 62, 638–646 (2002).

173. Gao, J. & Xie, W. Targeting xenobiotic receptors PXR and CAR for metabolic diseases. Trends Pharmacol. Sci. 33, 552–558 (2012).

AcknowledgementsThe authors of this Review are supported by grants P19118-B05, F3008 and F3517-B20 from the Austrian Science Foundation and European Community’s Seventh Framework Program (FP7/2007-2013) under grant agreement HEALTH-F2-2009-241762 for the project FLIP (M. Trauner) and the Dutch Digestive Diseases Foundation (MLDS WO 08-69; to P. L. M. Jansen and F. G. Schaap).

Author contributionsP. L. M. Jansen and F. G. Schaap wrote the article. M. Trauner reviewed and edited the manuscript before submission.

REVIEWS


Documents

Bile Acid Receptors as Targets for Drug