Physiol Rev 89: 147–191, 2009;
doi:10.1152/physrev.00010.2008.
Role of Bile Acids and Bile Acid Receptors
in Metabolic Regulation
PHILIPPE LEFEBVRE, BERTRAND CARIOU, FLEUR LIEN, FOLKERT KUIPERS, AND BART STAELS
I. Introduction
II. Bile Acid Metabolism
A. Bile acid synthesis in the liver
B. Bile acid biotransformations by intestinal bacteria
C. Transport of bile acids in the enterohepatic circulation
D. Kinetics of the enterohepatic circulation of bile acids
E. Bile acids and thermogenesis
III. Cloning and Characterization of Farnesoid X Receptor
A. Cloning of FXR and gene structure
B. Tissue distribution patterns of FXR
C. FXR as a ligand-regulated transcription factor
D. Natural and synthetic ligands of FXR
E. Ligand-binding properties of FXR
F. DNA-binding properties of FXR and FXR target genes
G. FXR polymorphisms
IV. Farnesoid X Receptor-Mediated Regulation of Bile Acid Synthesis and Transport
A. Transcriptional regulation of bile acid biosynthesis
B. Transcriptional regulation of bile acid export and reabsorption
C. Cholestatic liver diseases
V. Farnesoid X Receptor and Lipoprotein Metabolism
A. FXR and LDL-cholesterol metabolism
B. FXR and HDL-cholesterol metabolism
C. FXR and triglyceride metabolism
VI. Farnesoid X Receptor and Glucose Homeostasis
A. FXR expression in diabetes
B. FXR and hepatic glucose metabolism
C. FXR and insulin sensitivity
D. FXR and energy expenditure
VII. Farnesoid X Receptor and Atherosclerosis
A. A role for FXR in the vessel wall?
B. In vivo role of FXR in mouse models of atherosclerosis
VIII. Other Pathophysiological Aspects of Farnesoid X Receptor Biology
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Institut Pasteur de Lille, Institut National de la Santé et de la Recherche Médicale (INSERM) UMR 545,
and Université de Lille 2, Faculté des Sciences Pharmaceutiques et Biologiques et Faculté de Médecine, Lille;
INSERM, U 915, Université de Nantes, Faculté de Médecine, and Clinique d’Endocrinologie, Institut du
Thorax, Nantes, France; and Center for Liver, Digestive, and Metabolic Diseases, Laboratory of Pediatrics,
University Medical Center Groningen, Groningen, The Netherlands
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Lefebvre P, Cariou B, Lien F, Kuipers F, Staels B. Role of Bile Acids and Bile Acid Receptors in Metabolic
Regulation. Physiol Rev 89: 147–191, 2009; doi:10.1152/physrev.00010.2008.—The incidence of the metabolic syndrome has taken epidemic proportions in the past decades, contributing to an increased risk of cardiovascular
disease and diabetes. The metabolic syndrome can be defined as a cluster of cardiovascular disease risk factors
including visceral obesity, insulin resistance, dyslipidemia, increased blood pressure, and hypercoagulability. The
farnesoid X receptor (FXR) belongs to the superfamily of ligand-activated nuclear receptor transcription factors.
FXR is activated by bile acids, and FXR-deficient (FXR⫺/⫺) mice display elevated serum levels of triglycerides and
high-density lipoprotein cholesterol, demonstrating a critical role of FXR in lipid metabolism. In an opposite manner,
activation of FXR by bile acids (BAs) or nonsteroidal synthetic FXR agonists lowers plasma triglycerides by a
mechanism that may involve the repression of hepatic SREBP-1c expression and/or the modulation of glucosewww.prv.org
0031-9333/09 $18.00 Copyright © 2009 the American Physiological Society
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induced lipogenic genes. A cross-talk between BA and glucose metabolism was recently identified, implicating both
FXR-dependent and FXR-independent pathways. The first indication for a potential role of FXR in diabetes came
from the observation that hepatic FXR expression is reduced in animal models of diabetes. While FXR⫺/⫺ mice
display both impaired glucose tolerance and decreased insulin sensitivity, activation of FXR improves hyperglycemia
and dyslipidemia in vivo in diabetic mice. Finally, a recent report also indicates that BA may regulate energy
expenditure in a FXR-independent manner in mice, via activation of the G protein-coupled receptor TGR5. Taken
together, these findings suggest that modulation of FXR activity and BA metabolism may open new attractive
pharmacological approaches for the treatment of the metabolic syndrome and type 2 diabetes.
I. INTRODUCTION
Physiol Rev • VOL
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Bile acids (BAs) are amphipathic molecules with a
steroid backbone that are synthesized from cholesterol exclusively in parenchymal cells (hepatocytes) of the liver.
BAs and related bile alcohols with a surprising complexity in
structure and, therefore, with different physicochemical
properties have been identified in bile samples collected
from various vertebrate species (207), implying the evolution of several biochemical pathways to convert membranebound, water-insoluble cholesterol molecules into watersoluble, amphipathic molecules with detergent properties.
In fish and in several amphibians and reptiles, there is a
preponderance of bile alcohols and BAs in which the C27
steroid backbone of cholesterol is preserved. In contrast, in
most mammals, shortening of the C8 side chain of cholesterol to a C5 side chain occurs as part of the synthetic
cascade leading to BA pools in which the so-called C24 BAs
predominate. This review will focus on metabolism and on
the metabolic actions of the latter class of BAs, which are
present in humans and in rodents. The conversion of cholesterol into C24 BAs involves multiple enzymatic steps, of
which the details have been largely elucidated (see Ref. 261),
which are catalyzed by enzymes predominantly or exclusively expressed in the liver. Intriguingly, the enzymes that
catalyze the individual biosynthetic steps are localized in
different cellular compartments, i.e., endoplasmic reticulum,
cytosol, mitochondria, and peroxisomes of the hepatocytes.
How exactly BA synthesis intermediates, showing a gradual
increase in hydrophilicity, shuttle from one cellular
compartment to the other and how they pass the various intracellular membranes is still largely unknown
and represents a challenging issue in current BA research.
BAs are actively secreted by the liver into bile and
discharged into the intestinal lumen upon ingestion of a
meal. The multistep enzymatic conversion of cholesterol
into BAs confers detergent-like properties to the latter that
are crucial for their physiological functions in hepatic bile
formation and absorption of dietary lipids and fat-soluble
vitamins from the small intestine. Efficient reabsorption of
BAs in the terminal ileum results in the accumulation of a
certain mass of BAs within the body, referred to as the BA
pool, which cycles between intestine and liver in the enterohepatic circulation. The existence of this circulating pool
ensures the presence of adequate concentrations of BAs in
the intestinal lumen for digestion: each molecule is utilized
multiple times. Of special note is the magnitude of the flux
of these steroid molecules within the enterohepatic circulation: an average human BA pool of ⬃2 g that cycles ⬃12
times per day requires that liver and intestine theoretically
transport ⬃24 g of BAs each day (304). Direct assessment of
hepatic BA secretion into the duodenum in humans using
invasive indicator dilution techniques yielded values of ⬃12
g/day, i.e., in the same order of magnitude (30, 223, 328).
Highly effective transport systems in liver and intestine, of
which the individual components have largely been identified in the 1990s, have evolved to accommodate this flux.
Only ⬃5% of the pool escapes reabsorption per cycle and is
lost into the large intestine and finally into the feces. This
fecal loss of BAs, which is compensated for by de novo BA
biosynthesis in the liver to maintain pool size, represents a
major route for cholesterol turnover in humans and most
other mammals. A relatively unexplored area concerns the
functional heterogeneity of hepatic BA metabolism. It is
evident that not all hepatocytes contribute equally to the
various aspects of BA metabolism. Based on the distribution
of key synthetic enzymes across the liver acinus, it has been
concluded that under normal physiological conditions the
production of BAs predominantly occurs in the so-called
perivenous hepatocytes (322), i.e., the cells surrounding the
central hepatic vein. In contrast, BAs that return from the
intestine to the liver during the course of their enterohepatic
circulation are taken up and transported primarily by pericentral hepatocytes that surround the portal triads where
portal blood enters the liver acinus (96). The (patho)physiological relevance of this metabolic zonation, if any, remains
to be established.
During the last decades of the 20th century, BA research focused mainly on topics related to the specific physicochemical characteristics of these natural detergents, i.e.,
bile formation and cholestasis, cytotoxicity, gallstone formation, fat absorption, and on the role of BA synthesis in the
maintenance of cholesterol homeostasis. The detergent
properties of BAs, determined by the number and the orientation of hydroxyl groups present on the steroid moiety, and
influenced by the conjugation profile, are crucial for most of
their biological functions, i.e., promoting biliary excretion of
hydrophobic compounds and facilitating intestinal fat absorption. The physical characteristics of BAs, which allow
BILE ACIDS AND BILE ACID RECEPTORS IN METABOLIC REGULATION
II. BILE ACID METABOLISM
A. Bile Acid Synthesis in the Liver
The immediate products of the BA synthetic pathways are referred to as primary BAs. Cholic acid
(3␣,7␣,12␣-trihydroxy-5␤-cholanoic acid) and chenodePhysiol Rev • VOL
oxycholic acid (3␣,7␣-dihydroxy-5␤-cholanoic acid) are
the primary BAs formed in humans (see Fig. 1 for structure). In rodents, alternative hydroxylation reactions give
rise to differently structured primary BAs, particularly the
␣-, ␤- and ␥-muricholic acids (3␣,6␤,7␣-trihydroxy-5␤cholanoic acid, 3␣,6␤,7␤-trihydroxy-5␤-cholanoic acid,
and 3␣,6␣,7␤-trihydroxy-5␤-cholanoic acid, respectively).
The chemical diversity of the BA pool in the body is
further increased by the actions of the intestinal bacterial
flora, giving rise to so-called secondary BA species. In humans, deoxycholic (3␣,12␣-dihydroxy-5␤-cholanoic acid)
and lithocholic (3␣-hydroxy-5␤-cholanoic acid) acids (Fig.
1) represent the major secondary BA species that are derived from cholic and chenodeoxycholic acids, respectively.
A complete description of the BA synthesis cascades
is beyond the scope of this overview and can be found
in excellent recent review articles (47, 261). In short,
the steps leading to formation of primary BAs include the
hydroxylation of cholesterol at the C7 position of the
steroid ring structure or at the C24, C25, or C27 positions at
the side chain, subsequent further modification of the
steroid ring structure, followed by side-chain shortening.
In particular, the hydroxylation step catalyzed by the
microsomal cytochrome P-450 enzyme cholesterol 7␣hydroxylase (CYP7A1), the first step of the so-called “classical pathway” of BA biosynthesis that yields 7␣-hydroxycholesterol (see Fig. 1), is considered to be of great
regulatory importance and the activity of this enzyme is
subject to complex modes of control (see sect. V). The
physiological relevance of this step, and of BA synthesis
in general, is evident from the phenotype of CYP7A1deficient mice (132, 271): these mice display a very high
incidence of postnatal lethality as a consequence of liver
failure and fat/vitamin malabsorption. Mice that die
within the first 3 postnatal weeks produce only small
amounts of specific monohydroxy BA species that are
hepatotoxic. Animals that survive this period, by supplementation of cholic acid to the diet of the nursing dams,
start to produce “normal” BAs via pathways in which
oxysterols, rather that cholesterol, serve as substrates for
7␣-hydroxylation. However, their BA pool remains markedly compromised, i.e., at ⬃25% of normal values. A
frameshift mutation in the human CYP7A1 gene that abolishes enzyme activity has been identified in three subjects
with statin-resistant hypercholesterolemia and premature
gallstone disease (249). A 94% reduction in fecal BA output, which by definition reflects the hepatic synthesis rate,
was recorded in one of the homozygous subjects with a
clear shift towards predominant formation of chenodeoxycholic acid. The latter finding indicates a preferential formation of the remaining BAs via alternative
pathways. Unfortunately, no formal assessment of BA
synthesis and pool size has been reported so far in
CYP7A1-deficient subjects.
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them to form micelles, also impose a certain risk to cells that
are exposed to high concentrations of these natural detergents. When present at high concentrations, BAs may become cytotoxic. In particular, hepatocytes and bile duct
cells are at risk, for instance, in conditions of disturbed bile
formation or stasis of bile in the ductular system (cholestasis), and protective mechanisms appear to become active
when intracellular BA concentrations are elevated. Obviously, both the maintenance of physiological control of the
enterohepatic circulation and the initiation of cell protective
reactions require a mode of “BA sensing” in exposed cells. A
new era of BA research was initiated in 1999, when BAs
were identified as the natural ligands of the nuclear receptor
farnesoid X receptor/bile acid receptor (FXR/BAR or
NR1H4) (190, 232, 336), which uncovered a hitherto unknown function of BAs in the control of gene expression. It
became then clear that BAs themselves are directly involved
in regulation of gene expression in liver and intestine via
interaction with FXR, which provides such a sensor function.
It turned out that many of the genes encoding proteins involved in BA synthesis, metabolism and transport
in the enterohepatic circulation are strictly controlled by
BAs themselves via activation of FXR. In addition, and
more surprising, it became clear that BAs also serve
specific functions as “metabolic integrators” in the control of fat, glucose, and energy metabolism through modulation of gene expression, a signaling role which may be
partly dependent and partly independent of the FXR signaling pathway, and involve the G protein-coupled receptor TGR5/Gpbar1. This knowledge has opened up new
avenues for exploration of strategies for prevention and
treatment of metabolic diseases. This review focuses on
the current status of knowledge of FXR biology, i.e., the
cloning and characterization of FXR (sect. IV), the role of
this nuclear receptor in the control of BA synthesis and
transport (sect. V), in lipoprotein (sect. VI) and glucose
metabolism (sect. VII), the potential role of FXR in the
development of atherosclerosis (sect. VIII) and, finally, a
discussion on potential roles of FXR in other pathophysiological phenomena (sect. IX). Relevant information on
BA biology, which is essential for full appreciation of FXR
biology, will first be discussed (sect. III). From the discussion below, it will become evident that BAs fulfill a variety
of functions in the body that go far beyond the classical
role as the endogenous detergent of the body.
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It was discovered in the 1960s that side-chain hydroxylated cholesterol molecules like 24-hydroxycholesterol, 25-hydroxycholesterol, and 27-hydroxycholesterol
can serve as substrates for BA synthesis. As extensively
discussed by Russell (261), available data from mouse and
human studies indicate that formation of 24-hydroxychoPhysiol Rev • VOL
lesterol, which is particularly important for cholesterol
turnover in the brain, and 25-hydroxycholesterol contributes only very modestly to BA synthesis in quantitative
terms. In contrast, BA synthesis from 27-hydroxycholesterol occurs in relevant quantities in humans, as well as in
mice, via a route usually referred to as the “alternative” or
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FIG. 1. Bile acid (BA) metabolism in
humans. The main biochemical steps in
BA synthesis from the cholesterol backbone are shown and are detailed for biotransformations occurring in the liver.
Cholesterol is modified to cholestanoic acids (C27 BAs) then cholanoic acids (C24
BAs). The most abundant primary BAs in
human bile are chenodeoxycholic acid
and cholic acid. Primary BAs are further
modified by intestinal bacteria and converted to the secondary BAs deoxycholate
originating from cholate, and lithocholate originating from chenodeoxycholate. Cholate
and cholate derivatives amount to ⬎70 – 80%
of total BAs in humans. Most of the primary and secondary BAs are reabsorbed
through the intestine and follow the enterohepatic cycle described in the text. The
functional importance of enzymes (indicated in red) is discussed in detail in the text
(CH25H, cholesterol-25-hydrolase). The subcellular localization of enzymatic reactions
is also indicated (ER, endoplasmic reticulum). A more detailed metabolic pathway
can be found in Reference 256.
BILE ACIDS AND BILE ACID RECEPTORS IN METABOLIC REGULATION
Physiol Rev • VOL
mally and did not display any sign of liver dysfunction.
The BA pool size was slightly reduced in HSD3B7⫺/⫺
mice compared with controls, but did contain large quantities of 3␤-hydroxylated BAs (3␤,7␣,12␣-trihydroxy-5␤cholanoic acid and 3␤,6␣-dihydroxy-5␤-cholanoic acid).
Since the physicochemical characteristics of 3␤-hydroxylated and 3␣-hydroxylated BAs differ greatly, with the
3␤-hydroxylated species having much higher critical micellar concentrations than their 3␣-hydroxylated counterparts (260), it is very likely that the enrichment of the BA
pool with 3␤-hydroxylated species is responsible for the
observed strong reduction in fractional cholesterol absorption in HSD3B7⫺/⫺ mice (281). The impact of
HSD3B7 deficiency on bile formation and biliary lipid
secretion has not yet been reported but would be of
interest, in view of the severe phenotype observed in
children with autosomal recessive mutations in the
HSD3B7 gene (46, 51, 273). These children develop progressive liver disease characterized by cholestatic jaundice, likely related to the accumulation of hepatotoxic
3␤-hydroxy BA species, and malabsorption of fat and
fat-soluble vitamins. As it could be predicted, BA treatment provides an adequate and effective therapy for these
children (46).
The products of HSD3B7 activity can subsequently
take two routes: in case the 3-oxo,⌬4 intermediate interacts with microsomal sterol 12␣-hydroxylase (CYP8B1),
the end product will be cholic acid. Chenodeoxycholic
acid (or muricholic acids in rodents) will be formed when
12␣-hydroxylation does not occur (see Fig. 1). The activity of CYP8B1 thus determines the ratio in which the
primary BAs are being formed and, thereby, the physicochemical and biological properties of the BA pool. The
12␣-hydroxylated intermediates as well as those that escaped the actions of CYP8B1 are both subject to reduction of the C4 double bond by the ⌬4-3-oxosteroid 5␤reductase (AKR1D1), localized in the cytosol, implying
that at this point in the synthetic cascade intermediates
transfer from a lipophilic membraneous (endoplasmic reticulum, ER) environment to an aqueous environment.
Whether or not specific transport proteins are involved
herein is not known (261). Defective ⌬4-3-oxosteroid 5␤reductase has been identified as the cause of severe intrahepatic cholestasis in identical infant twins on the
basis of an elevated urinary excretion and predominance
of unsaturated hydroxy-oxo BAs, i.e., 7␣-hydroxy-3-oxo4-cholenoic and 7␣,12␣-dihydroxy-3-oxo-4-cholenoic acids
(⬎75% of total). Chenodeoxycholic acid and allo-BAs, i.e.,
BAs in which the A and B rings of the steroid backbone
are in the trans- rather than in the cis-configuration, could
be identified in serum and bile, albeit at low concentrations, indicative of the existence of a pathway in which
side chain oxidation (see below) can occur despite incomplete modification of the steroid nucleus (278). The
final step of ring modification in normal BA biosynthesis
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“acidic” pathway. This oxysterol, which is the most abundant oxysterol species in human plasma, is synthesized by
the mitochondrial sterol 27-hydroxylase (CYP27A1), an
ubiquitously expressed enzyme. On the basis of studies in
CYP7A1-null mice, it has been estimated that the alternative pathway contributes to ⬃25% of total BA synthesis in
the mouse (272). The relative contribution of the oxysterol-initiated pathways to total BA synthesis in humans
is difficult to assess directly. Limited data from CYP7A1deficient subjects (250) would indicate that the alternative pathway is only responsible for ⬃6% of total BA
synthesis. Yet, there are data to indicate that this relative
contribution may be larger under specific conditions, e.g.,
during fetal development (8) and in chronic liver disease
(53). For further conversion into BAs, oxysterols undergo
7␣-hydroxylation by the microsomal cytochrome P-450
enzymes CYP39A1 for 24(S)-hydroxycholesterol and
CYP7B1 for both 25- and 27-hydroxycholesterol. CYP7B1
is not only highly expressed in adult liver, but also in
kidney and brain, and is able to utilize various substrates
in addition to C27 oxysterols, e.g., dehydroepiandosterone
(261). CYP7B1-null mice display elevated plasma levels of
both 25- and 27-hydroxycholesterol and show a compensatory induction in hepatic CYP7A1 expression to maintain BA synthetic capacity, resulting in BA pool sizes and
compositions indiscernible from those in wild-type control mice (175). In contrast, a mutation in the human
CYP7B1 gene leading to an inactive truncated protein
conferred a phenotype characterized by severe cholestasis with cirrhosis and liver dysfunction in a human newborn (277). Gas chromatographic-mass spectrometric
analysis revealed accumulation of the hepatotoxic unsaturated monohydroxy 3␤-hydroxy-5-cholenoic and 3␤hydroxy-5-cholestenoic acids. The severe phenotype of
this inborn error in BA synthesis delineates the importance of the alternative pathway in early human life.
The 7␣-hydroxy intermediates that are formed by
CYP7A1, CYP39A1, or CYP7B1 are subsequently converted into 3-oxo,⌬4 intermediates by the actions of microsomal 3␤-hydroxy-⌬5-C27-steroid oxidoreductase
(HSD3B7). HSD3B7 catalyzes isomerization of the double
bond from the C5 to the C4 position and oxidation of
the 3␤-hydroxyl to a 3-oxo group. This step is critical for
the epimerization of the 3␤-hydroxyl group of the original
cholesterol molecule to the 3␣-orientation that is characteristic for virtually all BAs. This step is crucial for BA
function, as recently established by Shea et al. (281) upon
generation of HSD3B7⫺/⫺ mice. Similar to Cyp7A1⫺/⫺
mice, the HSD3B7⫺/⫺ mice showed no abnormalities at
birth, but the majority failed to grow and died within the
first 3 wk of life. Postnatal death could be prevented by
feeding the nursing dams with cholic acid- and vitaminsupplemented diets. Adult HSD3B7⫺/⫺ mice maintained
on the supplemented diets until weaning and then
switched to standard laboratory chow developed nor-
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disease states. The percentage of taurine conjugates is, in
general, higher than normal in cholestatic liver disease
and lower in situations characterized by increased conjugation requirements, such as during treatment with BA
sequestrants or external biliary drainage. Conjugation appears to be a prerequisite for efficient secretion of the
common C24 BAs into bile. Furthermore, conjugation with
glycine or taurine lowers the pKa value by ⬃1.3 and 5
units, respectively, compared with the unconjugated parent compound, and therefore renders conjugated BAs
fully ionized at physiological pH. This will prevent backdiffusion of conjugated BAs during their passage down
the biliary tree, as well as their passive reabsorption from
the small intestine (see below). The conjugation reaction
is highly efficient, because essentially all biliary BAs in all
mammalian species studied are conjugated (207). BA infusion studies in rodents showed that conjugation is virtually complete after a single pass through the liver (100).
BAAT resides in peroxisomes with variable amounts
present in the cytosol of both human and rat liver (109,
294). This has led to the speculation that peroxisomal
BAAT is responsible for conjugation of newly synthesized
BAs present in these organelles, whereas cytosolic BAAT
is involved in reconjugation of free BAs returning to the
liver after deconjugation in the intestine (see sect. IIIC).
However, a recent study by Pellicoro et al. (240) firmly
established the presence of both human and rat BAAT
exclusively in peroxisomes: this finding implies that unconjugated BAs must be taken up by peroxisomes and
that conjugated BAs need to be released from these organelles before they can be secreted into the bile. Putative
transporter proteins involved in the various obligatory
steps in intracellular BA (intermediate) transport have
remained elusive so far.
Very recently, fatty acid transport protein 5 (FATP5)
has been identified as another player in BA conjugation
based on the phenotype of FATP5-deficient mice (68,
122). FATP5 is a liver-specific member of the FATP/Slc27
family that exhibits both fatty acid transport and BA-CoA
ligase activity in vitro. It was reported that, although total
BA concentrations were unchanged in bile, urine, and
feces, the majority of gallbladder BAs was actually in
unconjugated form in FATP5-null mice. Only primary BAs
were found to be conjugated with taurine, indicative of a
specific requirement for FATP5 in reconjugation during
enterohepatic cycling. Potential interactions and redundancies between the various pathways involved in BA
conjugation now need to be defined.
Although the term conjugation is generally used to
denote the N-acyl amidation of BAs with glycine or taurine, it is important to realize that BAs can be subjected to
other conjugation reactions, i.e., phase II metabolic reactions similar to those involved in the inactivation of endoand xenobiotics. Sulfation was first recognized as a pathway in BA metabolism by Palmer (229), who identified the
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involves reduction of the 3-oxo group to a 3␣-hydroxyl
group by 3␣-steroid dehydrogenase (AKR1C4), a cytosolic
enzyme that belongs to the aldo-keto reductase family. As
stated previously, the presence of this hydroxyl group in
the 3␣ orientation is a main determinant of the physicochemical characteristics and, therefore, of the biological
actions of the end product.
The next steps in the biosynthetic cascade leading to
primary BA production involve the progressive oxidation
and shortening of the side chain. The first three steps of
this process are performed by mitochondrial CYP27A1,
previously shown to be involved in formation of 27hydroxycholesterol in the alternative route of BA synthesis. The actions of CYP27A1 introduce a hydroxyl group at
C27, then oxidize this group to an aldehyde then to a
carboxylic acid (243). The ensuing oxidized intermediates
are then transferred from mitochondria to peroxisomes
for shortening of the side chain by yet unresolved mechanisms. This process, in which the three terminal C atoms
are removed in a series of reactions that are analogous to
those in fatty acid ␤-oxidation, involves the sequential
actions of BA coenzyme A ligase to activate the BA intermediate, of 2-methylacyl-coenzyme A racemase to convert the coenzyme A conjugate to the proper 25(S)-isomer, and of branched chain acyl-coenzyme A oxidases
(ACOX1 or ACOX2) to yield 24,25-trans-unsaturated derivatives. The next step involves hydration and oxidation
at the ⌬ 24 bond by the D-bifunctional enzyme. Consequently, mice as well as humans deficient in D-bifunctional protein not only accumulate very-long-chain fatty
acids (e.g., pristanic acid) but also unsaturated C27 BA
intermediates. The last step of side chain shortening is
catalyzed by peroxisomal thiolase 2, which cleaves the
C24-C25 bond to yield a C24 BA-coenzyme A and propionylcoenzyme A. As a consequence of the prominent role of
peroxisomes in BA biosynthesis, various inborn errors of
metabolism caused by peroxisomal dysfunction are characterized by the presence of abnormal BA species with
diagnostic value (25, 80, 302).
Prior to their secretion into the bile canalicular lumen, primary BAs are conjugated at their side chains with
either taurine or glycine, further enhancing their hydrophilicity. This reaction, i.e., the formation of an amide
linkage between the amino acid and the C24 BA-coenzyme
A thioester, is catalyzed by bile acid coenzyme A:amino
acid N-acyltransferase (BAAT) (78). Most mammals are
able to form both taurine and glycine conjugates: the ratio
in which they are formed is determined by the availability
of taurine since the enzyme has a greater affinity for this
amino acid. BAAT is solely responsible for the conjugation reaction, as delineated by the fact that both taurine
and glycine conjugates are absent in serum of patients
with familial hypercholanaemia homozygous for a mutation in the BAAT gene (39). The normal 3:1 ratio of glycine
to taurine conjugates in human bile is altered in various
BILE ACIDS AND BILE ACID RECEPTORS IN METABOLIC REGULATION
B. Bile Acid Biotransformations
by Intestinal Bacteria
uptake is highly effective (⬃95%), frequent cycling of the
BA pool, i.e., up to 12 cycles/day in humans, results in
deposition of 400 – 800 mg BAs into the colon for bacterial
biotransformation each day. Major structural modifications that occur in the large intestine include deconjugation, oxidation of hydroxy groups at C3, C7 and C12, and
7␣/7␤-dehydroxylation. Deconjugation, i.e., enzymatic hydrolysis of the N-acylamide bond between BA and glycine
or taurine at the C24 position, is essentially complete in
the human large intestine. BA hydrolases belonging to the
family of choloyl hydrolases (EC 3.5.1.24) have been isolated and/or characterized from several species of bacteria (257). Oxidation and epimerization of C3, C7, and C12
hydroxy groups is performed by hydroxysteroid dehydrogenases expressed by various strains of bacteria. Epimerization, i.e., interchange between ␣- and ␤-configuration,
involves the generation of stable oxo-BAs of which several can be found in human feces (276). Since deoxycholic
and lithocholic acids represent the predominant BA species in human feces (276), 7␣-dehydroxylation of cholic
and chenodeoxycholic acids represents the most quantitatively important biotransformation in the human colon.
Surprisingly, current estimates indicate that this pathway
is found in only 0.0001% of total colonic flora, by species
belonging to the Clostridium genus (142). Importantly,
the dehydroxylation reaction appears to be restricted to
free BAs, implicating a functional interplay between deconjugation and dehydroxylation activities in the large
intestine (257). This combined action increases hydrophobicity and pKa of the BAs, thereby permitting their absorption by passive diffusion across the colonic epithelium and entry into the BA pool. Because the human liver
is incapable to rehydroxylate deoxycholic acid, unlike
rodent liver, this secondary BA constitutes a significant
part of the circulating BA pool in humans (⬃35%). In
contrast, lithocholic acid does not build up in the pool:
this has been ascribed to its efficient sulfation prior to
secretion into bile which limits intestinal reabsorption,
possibly due to interactions with calcium in the small
intestinal lumen (161), hence preventing its accumulation
in the enterohepatic circulation.
C. Transport of Bile Acids in
the Enterohepatic Circulation
The microbial flora of the large intestine strongly
impacts on BA metabolism and, vice versa, bile and BAs
contribute to suppression of significant bacterial colonization of the small intestine. The intensive interactions
between intestinal flora and BAs and their implications
have been the subject of excellent recent reviews (16,
257). Within the context of the current review, it is of
importance to appreciate that colonic bacteria contribute
to the salvage of BAs that escape from uptake in the distal
ileum and thus spill over in the colon. Although ileal BA
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Conservation of the pool of hydrophilic, largely ionized BA molecules within the enterohepatic circulation
requires the coordinate action of several transporter proteins expressed at the apical and basolateral membranes
of liver and intestinal epithelial cells. In view of the size of
the BA pool, physiological fluctuations in BA flux through
the enterohepatic system during the day (because the
dynamics hereof are controlled by ingestion of meals) and
the relatively small escape of BAs from the circulation, it
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3␣-sulfates of glyco- and taurolithocholic acid in human
bile. Since then, ester glucuronidation at the C24 position,
ether glucuronidation at the C3 and C6 nuclear sites, and
N-acetylglucosaminidation at the C7 position of BAs with
a ␤-hydroxy group such as ursodeoxycholic acid have
been described (see Ref. 318 for review). Sulfation of BAs,
i.e., the transfer of a sulfonyl group from 3-phosphoadenosine-5-phosphosulfate to the 3␣-hydroxy group of
hydrophobic BA species like lithocholic acid, is catalyzed
by the sulfotransferase SULT2A1 (SULT2A9 in mice) (42).
Glucuronide conjugation of BAs can be catalyzed by several of the ubiquitous UDP glucuronosyltransferases. In
humans, UGT2B4 mediates glucuronide conjugation at
the 6␣-hydroxy position while UGT2B7 transfers glucuronosyl moieties to 3␣- and 6␣-hydroxy groups (87, 244,
320). UGT1A3 uniquely modifies the C24-carboxyl group at
the side chain of BAs, giving rise to BA ester glucuronides
(319). Whereas sulfated glycolithocholic and taurolithocholic acids are low abundant but normal components of
the human BA pool (229), BA glucuronide formation appears to be a quantitatively minor pathway in human BA
metabolism (113, 306). BA conjugation has been proposed
as an adaptive response in cholestatic liver diseases to
allow for effective urinary clearance of these more hydrophilic metabolites. Indeed, increased concentrations of
sulfated and glucuronidated BAs have been found in urine
of patients with cholestatic liver diseases. However, in
the absence of cholestasis, sulfated and glucuronidated
BAs are primarily excreted into bile, in humans (306) as
well as in rodents (159, 162). In contrast to expectations,
it appeared that neither sulfation nor glucuronidation reduced hepatotoxicity of monohydroxylated BAs in rodents. Sulfated glycolithocholic acid (160, 355), but not its
taurine-conjugated counterpart, as well as lithocholic
acid-3-O-glucuronide (226) showed a cholestatic potency
equal to or higher than that of the parent compound
lithocholic acid upon intravenous administration to rats.
The functional consequences of sulfation or glucuronidation on newly identified BA signaling pathways (see below) have not been systematically addressed.
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portal blood via the Na⫹-taurocholic acid cotransporting
polypeptide (NTCP or Slc10a1), which has a substrate
specificity essentially limited to conjugated BAs (102), or
by Na⫹-independent systems represented by members of
the superfamily of organic anion transporting polypeptides (OATP/SLCO) (103). Of these, OATP1A2, OATP1B1,
and OATP1B3 exhibit overlapping substrate specificities
for conjugated and unconjugated BAs as well as neutral
steroids, steroid sulfates, and selected organic cations
(103). After their uptake and, if necessary, reconjugation,
BAs must be transported to the canalicular pole of the
hepatocytes to be secreted again into the bile, which
completes their enterohepatic circulation. Processes involved in the transcellular movement of BAs across the
hepatocytes are yet poorly defined. Several intracellular
binding proteins (e.g., glutathione-S-transferases, fatty
acid binding proteins, 3-hydroxysteroid dehydrogenase)
have been implicated, but their roles have not been formally proven. Additionally, free BAs may reach the canalicular membrane by rapid diffusion or, when high BA
loads need to be translocated, partitioning of hydrophobic
BAs into organelles such as ER and Golgi apparatus may
occur. Whether or not physiological changes in transcellular BA flux, e.g., during the postprandial phase or in
conjunction with (diet-induced) changes in BA pool size
or composition (23), affect intracellular localization of
BAs is not known.
D. Kinetics of the Enterohepatic Circulation
of Bile Acids
During the day, there is a massive transport of BAs
through different compartments of the body. The magnitude of this flux is not constant but highest during the
immediate postprandial phase, since gallbladder contraction initiates BA cycling. Although hepatic extraction of BAs by the liver from portal blood is highly
efficient, a postprandial rise of plasma BA levels occurs
in healthy subjects, which may be of relevance for the
signaling functions in peripheral tissues and organs
(see below). On their way through the enterohepatic
circulation, BAs can interact with various potential
“modulators” of their metabolism, such as food components with BA-binding properties in the intestinal lumen and biotransforming enzyme systems present in
the intestinal flora and in the liver. Furthermore, the
dynamics of enterohepatic cycling will be influenced by
mechanical factors such as gallbladder contraction and
intestinal motility. To be able to understand the physiology of BA metabolism as well as pathophysiological
consequences of disturbances in the various processes
described in the preceeding paragraphs, it is necessary
to have an accurate insight in the concentrations of
individual BA species in the various compartments as
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is evident that the capacity of the transporter systems
involved must be important. The molecular characterization, function, and modes of regulation of the various BA
transporters have been reviewed in a recent issue of
Physiological Reviews (317) and several other excellent
overview articles (27, 305). In addition, defects in transporter systems related to human cholestatic liver diseases
have been subject of several, well-referenced reviews (76,
163, 235). Hence, only a condensed description of the
systems involved will be provided here.
Newly synthesized BAs and those returning from the
intestine during the course of their enterohepatic cycle
are actively secreted by the hepatocytes into the bile
canalicular lumen by the action of the Bile Salt Export
Pump (BSEP), a member of the ATP binding cassette
superfamily of transporter molecules (symbol ABCB11).
Active secretion of BAs provides a major driving force for
bile formation (317) and is therefore of crucial importance for hepatobiliary removal of a variety of endo- and
xenobiotics from the body via the hepatobiliary pathway.
In addition, BA secretion drives the secretion of phospholipids and cholesterol into bile. Secretion of these lipids
also depends critically on the activities of ABC transporters, i.e., MDR3 (ABCB4) and the ABCG5/ABCG8 heterodimer for phospholipids and cholesterol, respectively
(76). Cosecretion of BAs and these lipids, allowing for
mixed micelle formation in bile, is crucial for protection
of the biliary system from the detergent actions of high
biliary BA concentrations (291).
Primary bile, containing BAs and associated lipids in
the millimolar concentration range, is stored in the gallbladder, where bile is further concentrated by absorption
of water, and discharged into the intestinal lumen upon
ingestion of a fatty meal through cholecystokinin (CCK)induced gallbladder contraction. In the small intestinal
lumen, BAs act as detergents to solubilize and facilitate
the absorption of dietary fats and lipid-soluble vitamins in
the small intestine. Due to their relatively low pKa value,
passive absorption of conjugated BAs is minimal and their
intraluminal concentrations remain high along the length
of the small intestine. BAs are actively and very effectively reabsorbed by the actions of specific transporter
systems only in the terminal ileum, i.e., the apical sodiumdependent BA transporter (ASBT/Slc10a2) and the basolaterally localized heterodimeric organic solute transporter Ost␣/Ost␤. The role in vectorial transport of BAs of
the cytosolic intestinal BA-binding protein (IBABP) that is
attached, in the cytoplasmic compartment, to ASBT (93)
through the ileal enterocytes remains enigmatic (158).
BAs escape reabsorption and enter the colon where they
may be converted into secondary BAs that can be either
passively absorbed to enter the BA pool or are lost from
the body via the feces. The mixture of primary and secondary BAs that is absorbed returns to the liver via the
portal system. BAs are then taken up by hepatocytes from
BILE ACIDS AND BILE ACID RECEPTORS IN METABOLIC REGULATION
Physiol Rev • VOL
reflects the fractional turnover (FTR in pools/day) of the
pool, i.e., the fraction that is renewed per time interval.
Multiplying pool size and FTR establishes a value for the
absolute turnover, which equals the hepatic synthesis rate
under steady-state conditions.
Pioneering mass spectrometric studies by Klein et al.
(156) in the 1970s allowed for the replacement of radioactive
tracers by stable isotopically labeled (2H, 13C) BAs for kinetic measurements, thereby facilitating their use in patient
groups also including pregnant women and children (e.g.,
Ref. 344). Although avoiding radiation hazard, this technique
remained invasive because bile sampling was part of the
procedure. Owing to major advances in gas chromatography-mass spectrometric techniques and novel derivatization
procedures, Stellaard et al. (304) completed the technical
development enabling accurate isotope ratio measurements
for BAs in serum samples in the normal concentration
range. Direct comparison of the serum sampling technique
with the bile sampling technique proved isotopic equilibrium
between serum and biliary BAs in the postprandial state.
The technique, originally designed for simultaneous assessment of kinetic parameters of both primary BAs, i.e., cholic
and chenodeoxycholic acids, was subsequently extended to
also include deoxycholic acid pool size, fractional turnover,
and the input rate of this secondary BA from the colon (303).
This technique has been successfully applied to establish
kinetic parameters of BA metabolism in various patient
populations (20, 234). Furthermore, its application allowed
the demonstration of strong effects of diet composition on
human BA metabolism (23, 135), implying that diet may
affect BA signaling functions (see below). Finally, refinement of analytical techniques has allowed for a significant
down-scaling of the amount of plasma required for accurate
measurements which makes the technique applicable for
use in rodents (125). In this case, because biliary BA output
rates can also be determined in the same experiment, an
estimate of intestinal absorption efficiency and of the cycling time of the pool can be obtained (158). For instance, it
was found in rats that the frequently used immunosuppressive drug cyclosporin A reduced hepatic cholic acid synthesis by 71% without inducing significant changes in cholic
acid pool size, due to an accompanying 65% reduction in the
fractional turnover rate of the pool. More efficient cholic
acid reabsorption (1.4 ⫾ 0.5% loss per cycle in treated rats
versus 4.3 ⫾ 0.5% loss per cycle in controls) associated with
upregulation of ileal ASBT protein expression appeared to
account for maintenance of BA pool size under these conditions (126). This finding illustrates the large metabolic
consequences of a relatively small increase in absorption
efficiency from 95.7% in controls to 98.6% in treated animals,
underscoring the important role of the intestine in the maintenance of BA homeostasis. Similar reductions in BA synthesis were observed in children treated with cyclosporin A
after liver transplantation (118).
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well as in kinetic parameters that define their behavior
in the enterohepatic circulation, i.e., the size of the
circulating pool, its (fractional) turnover rate as a measure of absorption efficiency, and the rate at which BAs
are being synthesized.
Over the years, there has been considerable
progress in analytical techniques that allow for accurate assessment of concentrations of individual BA species and their conjugates in plasma, bile, urine and
feces, which were largely developed for diagnostic purposes. Application of these techniques has been of
great benefit for the identification of inborn errors in
BA metabolism and, at the same time, of major relevance for phenotyping of new mouse models. However,
concentration measurements only do not provide full
insight in the dynamics of (disturbances in) BA metabolism. Hepatic BA synthesis, an important determinant
of whole body cholesterol turnover, can be derived
from the rate of fecal BA output since, under steadystate conditions, fecal loss equals hepatic synthesis
rate. As already discussed by Setchell et al. (276), however, accurate measurement of fecal BA loss in humans
is not trivial. Other approaches have been developed
for this purpose, such as the rate of release of 14CO2
from orally administered [26-14C]cholesterol (70) or of
3
H2O from [24,25-3H]cholesterol (58) as a measure of
cholesterol side chain oxidation. Both methods were
found to correlate reasonably well with fecal BA output, yet the use of radiolabeled tracers evidently limits
the use of these approaches in human studies. As alternatives, quantitative measurement of intermediates in
the BA synthetic cascade in blood plasma, such as
7␣-hydroxycholesterol (61) and 7␣-hydroxy-4-cholesten-3-one
(89), have been proposed to reflect hepatic CYP7A1 activity
and thus BA synthesis. These assays are definitively very
useful for comparison of (patient) groups (19) and for monitoring of physiological variations in CYP7A1 activity that
occur during the day-night cycle (88, 185); however, they do
not provide insight in actual quantitative changes in BA
synthesis.
For the actual determination of BA synthesis rate and
other relevant parameters such as pool size and fractional
turnover rate, tracer methodologies are required. Already
in 1957, Lindstedt (179) introduced an isotope dilution
approach for assessment of cholic acid kinetic parameters in human volunteers, involving oral administration of
[24-14C]cholic acid and repeated sampling of duodenal
bile. Later, Duane et al. (71) have validated the use of
[22,23-3H]cholic acid for this purpose. On the basis of the
assumption of a one-compartment model, the decay of
specific activity of cholic acid in subsequent bile samples
is described by an exponential curve. Conversion to the
logarithm and extrapolation of the curve to time point
zero, being the time of administration, allowed for assessment of pool size. The slope of the logarithmic curve
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E. Bile Acids and Thermogenesis
III. CLONING AND CHARACTERIZATION OF
FARNESOID X RECEPTOR
A. Cloning of FXR and Gene Structure
Using a yeast-based interaction trap with the human
nuclear receptor RXR␣ ligand binding domain (LBD) as a
bait, Seol et al. (274) isolated mouse liver cDNAs encoding for the RXR-interacting protein 14, which exhibited a
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A novel concept indicating a signaling role of BAs in
the control of energy metabolism independent of FXR
was conceived in 2006 (342), based on the observation
that addition of cholic acid to the diet increases energy
expenditure in brown fat in mice and prevents the development of high fat-induced obesity and insulin resistance.
This metabolic effect of BAs was found to be critically
dependent on induction of the cAMP-dependent thyroid
hormone activation enzyme D2 (type 2 iodothyronine
deiodinase, DIO2) since it was lost in D2-deficient mice.
DIO2 converts inactive thyroxine into active 3,5,3⬘-triiodothyronine in cells and plays a crucial role in regulating thermogenesis in BAT by controlling the expression of
the uncoupling protein (UCP)-1. These effects appeared
to be independent of FXR and were suggested to be
mediated by cAMP production induced by BAs binding to
the G protein-coupled receptor TGR5 (also known as
Gpbar1). This intriguing concept requires further evaluation, e.g., identification of the high-fat-associated factor
that is essential for the effect to occur, and assessment of
its relevance in species like humans that lack brown fat,
etc. In addition, data obtained with recently generated
Gpar1-deficient mice are not entirely consistent with the
proposed sequence of events. Vassileva et al. (331) demonstrated that Gpbar1 is predominantly expressed in gallbladders rather than in organs/tissues involved in energy
metabolism and showed that Gpbar1-deficient mice
are resistant to cholesterol gallstone formation upon feeding with a lithogenic diet. The authors showed that
Gpbar1⫺/⫺ mice display disturbed feedback inhibition of
CYP7A1 gene expression upon BA feeding. Yet, in vivo
studies in Gpbar1⫺/⫺ mice yielded apparently conflicting
results, since weight gain upon high-fat, lithogenic [0.5%
(wt/wt) cholic acid] diet feeding was found to be similar
(331) or significantly increased in Gpbar1⫺/⫺ compared
with wild-type mice. In the latter case, a gender-dependent manner effect was noted (female, but not male,
Gpbar1⫺/⫺ mice showed increased body weight) (195).
Clearly, the exact role of the Gpbar1 in BA-mediated
effects on energy metabolism still needs to be defined,
although these results highlight the crucial role of BAs in
regulating body weight.
primary sequence with notable homologies with other
members of the nuclear receptor superfamily (RIP14).
RIP14 expression was detected in liver and kidney. These
cDNAs corresponded to distinct isoforms of the same
protein with either a 38-amino acid NH2-terminal extension (RIP14-2), or a 4-amino acid (MYTG) central insertion (RIP14-1). RIP14 bound as a heterodimer with RXR␣
to various previously identified hormone response elements, such as the ecdysone response element (EcRE)
from the Drosophila hsp27 promoter, direct repeats with
a 5-bp spacer (DR5), DR2 and DR4, and on inverted
repeats of the IR0 and IR1 types. On the contrary, RXRRIP14 heterodimers were devoid of any affinity for DR0,
DR1, DR3, and IR2 to IR5 response elements. A general
decrease of the DNA binding activity was noted for the
RIP14-2 isoform, harboring the NH2-terminal extension. A
few months later, Forman et al. (83) reported the cloning
of the rat homolog of RIP14, a 469-amino acid protein able
to dimerize with RXR␣, by screening a regenerating rat
liver cDNA library with a truncated mouse RIP14 cDNA
(83). Intermediates of the mevalonate pathway such as
juvenile hormone and farnesol activated RXR-rRIP14 heterodimers at 50 ␮M, and a synthetic RXR ligand (LG1069)
could synergize with farnesol in transcriptional assays.
The subsequently called “farnesoid X receptor” was
shown, as RIP14, to be expressed mostly in liver and
kidney, and also in the adrenal cortex and in intestinal
villi from E19.5 rat embryo and adults. Of note however,
recent reports described the RXR ligand 9-cis-retinoic
acid as an inhibitor of FXR transcriptional activity (48,
144), suggesting that distinct RXR ligands impact differently on FXR-controlled transcription. Farnesol was also
described as a PPAR␣ ligand (104, 225), and interestingly,
farnesyl pyrophosphate, an intermediate on the cholesterol synthesis pathway, is unable to activate FXR, but is
a promiscuous ligand activating the thyroid hormone receptor ␣ and other steroid receptors (57).
In 1997, a report showed that RIP14/mouse FXR
could not be activated by farnesol. However, a high concentration (5 ␮M) of the synthetic retinoid TTNPB {[E]4-[2-(5,6,7,8-tetrahydro-5, 5,8,8-tetramethyl-2-naphthalenyl)propen-1-yl]benzoic acid} potently activated mouse FXR in
transient transfection assays in trophoblast tumor JEG3
cells (360). TTNPB was later identified as a FXR ligand
based on coactivator recruitment assays (232). FXR was
deorphanized 2 yr later, with the recognition that it could
be activated by major BAs (190, 232, 336) and thus renamed BAR, for bile acid receptor. Chenodeoxycholic
acid (CDCA), cholic acid (CA), deoxycholic acid (DCA),
and lithocholic acid (LCA) were shown to activate FXR at
physiological concentrations (⬃100 ␮M). Interestingly,
the 7␤ epimer of CDCA, ursodeoxycholic acid (UDCA),
was inactive in such assays, hinting at specific structureactivity relationships which were explored later and
which are detailed below. Due to the lack of a labeled
BILE ACIDS AND BILE ACID RECEPTORS IN METABOLIC REGULATION
lectively by FXR ligands (367). Huber et al. (123) showed
that the structures of the FXR gene and of the encoded
isoforms are highly homologous across several species
(Syrian hamster, mouse, rat, and human) and that the
human gene also generates four isoforms (123). As of
today, little is known about the specific biological functions of each isoform, although functional differences
have been highlighted (see below).
B. Tissue Distribution Patterns of FXR
High levels of FXR isoforms were detected in the
adult C57BL/6J female mouse liver (367). Mouse liver and
small intestine express equal amounts of each FXR isoform, whereas kidney and stomach exclusively express
FXR␣3/4. In contrast, heart and adrenal glands express
only FXR␣1/2. Other mouse organs, such as lung and fat,
exhibit low amounts of FXR␣1/2, whereas FXR is undetectable in brain, spleen, or skeletal muscle. In adult
human tissues, an exclusive FXR␣1/2 expression is found
in adrenals and liver, whereas colon and kidney exclusively express FXR␣3/4. Equal amounts of these isoforms
are found in small intestine and duodenum, and FXR is
not detectable in brain, heart, lung, and skeletal muscle
(123). However, histochemical techniques suggest the
presence of FXR in biopsies of human cardiac muscle,
small intestine, and adrenal glands (22). A role in vascular
biology can be inferred from FXR expression in human
vascular smooth muscle cells from coronary arteries and
aortas, as well as in atherosclerotic plaques (22) and in
FIG. 2. Structure of the mouse farnesoid X receptor (FXR) gene and transcripts. A: structure of the FXR gene with exons numbered from 1 to
11. B: pre-mRNAs with the alternative splice site at exon 5, introducing a 12-bp sequence encoding for the “MYTG” insert. C: mRNAs encoding for
each isoform. D: FXR proteins structures. The A/B, C (DNA binding domain), D (hinge region), and E (ligand binding) domains are indicated.
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high-affinity reference ligand, evidence for a direct interaction of FXR with BAs came initially from coactivator
recruitment assays, which probe for ligand-induced structural transitions occurring in the LBD. Furthermore, the
identification of FXR as a BA-activated transcriptional
regulator of the mouse intestinal BA binding protein (IBABP) and indirectly of CYP7A1 in human hepatoma
cells (HepG2) clearly pointed to a critical role of FXR in
BA metabolism.
Both the unique mouse and human gene (NR1H4)
encode four isoforms, initially termed ␣1 (RIP14-2), ␣2,
␤1, and ␤2 (RIP14-1) (367). The identification of FXR␤
(NR1H5), a pseudogene in primates (227) but a receptor
for lanosterol, an intermediate in the cholesterol synthesis pathway, in other mammals, led to a new nomenclature designing the four NR1H4 isoforms as FXR␣1 (previously mFXR␣1), FXR␣2 (previously mFXR␣2), FXR␣3
(previously mFXR␤1), and FXR␣4 (previously FXR ␤2)
(Fig. 2).
The murine FXR␣ gene (termed hereafter FXR) is
composed of 11 exons and 10 introns. Two distinct promoters, located upstream of exon 1 and exon 3, drive the
expression of either FXR␣1 and ␣2 or FXR␣3 and ␣4,
respectively. As a consequence, FXR␣1 and -2 isoform
transcripts emanate from exon 1, whereas FXR␣3 and -4
transcripts start from exon 3. An alternative splice donor
site is located downstream of exon 5 and produces either
␣1 and -3 isoforms, which contain a MYTG insertion, or
isoforms which lack this additional sequence (␣2 and -4)
(see Fig. 2). Exons 4 –11 are shared between all isoforms,
suggesting that all FXR isoforms will be activated nonse-
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Therefore, FXR is a nuclear receptor predominantly
expressed in the gastrointestinal tract, whose regulation
hints at roles in glucose and lipid metabolism. However,
other roles have yet to be explored when considering the
distribution of FXR expression. Notably, the regulation of
the FXR promoter by HNF1␣/TCF1 (182), which is mutated in the rare monogenic form of maturity-onset diabetes of the young (MODY)-3 and implicated in the
growth and function of islet ␤ cells, suggests additional
role(s) which still have to be deciphered.
C. FXR as a Ligand-Regulated Transcription Factor
As a member of the nuclear receptor (NR) family,
FXR acts as a ligand-modulated transcription factor
whose role is to increase or decrease the transcriptional
activity of regulated promoters in a coordinate fashion.
Two general mechanisms contribute to the transcriptional regulation of eukaryotic promoters: step 1) the
nuclear receptor releases corepressor (CoR) complexes
and interacts with multiprotein coactivator complexes
upon agonist binding, which modify the chromatin structure, yielding access to general transcription factors
(GTFs) and RNA polymerase II (pol2) to promoter DNA;
step 2) the activator stimulates pol2 and GTFs binding to
DNA to promote the formation of the preinitiation complex (PIC). About 300 coactivators for NRs that favor step
1, including SWI/SNF complexes, CBP/p300, or p160related factors (SRC-1, -2 and -3), have been described
(see Refs. 169, 181 for reviews). Step 2 seems to rely
mostly, if not exclusively, on the mediator complex,
which is a molecular bridge between activators and pol2,
although a repressive role has also been described both in
yeast (9) and in mammalian cells (82). Being necessary
for the activation process, mediator also plays a role in
transcriptional initiation (335).
Several coactivators play a role in FXR-mediated
transcription. DRIP205, a subunit of the mediator complex interacting with a number of NRs, is recruited by
FXR in a ligand-dependent manner and is required for
FXR-induced transcription as demonstrated by siRNA
studies (246). FXR ligand-dependent loading of CARM-1, a
secondary coactivator for nuclear receptors with protein
methylase activity, is detected on the BSEP promoter in
HepG2 cells. This interaction is correlated to methylation
of histone H3 (R17) at this locus, a posttranslational
modification associated with gene activation (14), thus
suggesting a direct correlation between FXR activation,
CARM1 recruitment, chromatin structure alteration, and
transcriptional activation (4). Quite similarly, another
protein methylase, PRMT-1, has also been described as a
coactivator for FXR (259). CARM1 being known to interact indirectly with nuclear receptors through members of
the p160 family (SRC1, SRC2/TIF2/GRIP1, SRC3/AIB1/
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cultured rat endothelial cells (110). FXR is also detected
in white adipose tissue from mice (37, 258) and is one of
the numerous nuclear receptors expressed in human immune cells. FXR mRNA is indeed detected in human
peripheral blood mononuclear cells and subsets of lymphocytes and monocytes (270), but not in mouse peritoneal macrophages (98). The placenta transfers a large
variety of substances from the mother to the fetus, and is
also the main route for the elimination of toxic compounds produced by the fetal liver, including BAs. The
trophoblast is a layer of chorionic villi and is a key element in the placental barrier function. FXR mRNA is
expressed in cytokeratin-7-positive human trophoblast
cells isolated from human placenta at term, and also in
three human choriocarcinoma cell lines. Therefore, FXR
may play a role in the control of the hepatobiliary-like
excretory function of the placenta (275).
FXR expression is affected in several conditions.
FXR is not detectable during rat kidney embryonic development, but is highly expressed in proximal tubules of
adult animals (310). Similarly, the expression of FXR in
the rat ileum increases from birth to adulthood (128).
Metabolic and hormonal cues also modulate FXR expression. For example, FXR expression is repressed during
the acute phase response and by interleukin-1 and tumor
necrosis factor-␣ (153). High glucose concentrations (25
mM) upregulate and insulin represses FXR expression in
rat primary hepatocytes (74). Glutamine, an amino acid
whose facilitated uptake through the transporter ASCT2
is essential for the rapid growth of cancer cells, induces
FXR expression in HepG2 hepatoma cells (31, 198). Finally, fasting, which is known to promote overexpression
of the transcriptional regulator PGC1␣ (PPAR coactivator
1␣), upregulates FXR␣3/4 expression in the liver. A mechanistic study showed that this process is dependent on a
DR1 response element located upstream of the second,
internal FXR promoter. This response element binds the
nuclear receptors PPAR-␥ and HNF4, whose transcriptional activity increases upon interaction with PGC1␣,
establishing a molecular link between fasting and FXR
expression (366).
Several pathological conditions have been correlated
to an altered FXR expression. FXR expression is decreased in the liver of models of genetically or chemically
induced diabetes in rats (74). In contrast, diabetic db/db
mice display a higher expression of FXR isoforms in liver
(364). Two mutations (⫺1g⬎t and M1V) in the FXR sequence have been isolated from patients with intrahepatic
cholestasis of pregnancy (ICP), resulting in a reduced
FXR expression (329). FXR isoform expression is directly
correlated to ATP8B4/FIC1 (a P-type adenosine triphosphatase) activity or expression (2, 44), whose deregulation through mutation(s) leads to progressive familial
intrahepatic cholestasis type 1 (PFIC1).
BILE ACIDS AND BILE ACID RECEPTORS IN METABOLIC REGULATION
Physiol Rev • VOL
apprehended, without forgetting corepressors, the role of
which remains to be fully evaluated.
D. Natural and Synthetic Ligands of FXR
Seminal papers published in 1999 already mentioned
above identified the major mammalian BAs as activators
of FXR (190, 232, 336). The use of bile extracts, as well as
other natural extracts from plants, used to treat lipid
metabolism disorders, prompted the search for potential
FXR ligands in these extracts. The observation that the
synthetic retinoid TTNPB could activate FXR gave the
impetus for a search for nonsteroidal compounds, activating or repressing this nuclear receptor. These three
axis of research (BA derivatives, natural extracts, synthetic FXR ligands) yielded an impressive amount of data
identifying FXR modulators. Reports are sometimes contradictory or to be interpreted with caution, due to the
multiplicity of experimental models and/or the poor selectivity of the identified ligands. Most BAs indeed display
significant cell toxicity at concentrations above 100 ␮M,
limiting their use as chemical tools to study FXR functions. In this respect, it is important to take into account
experimental conditions that led to the classification of
FXR ligands (see Table 1), and to keep in mind that BAs
are promiscuous ligands; BAs also activate VDR [LCA,
LCA-acetate (1, 189)] and PXR [LCA (299, 347)] or repress
CAR (206). These properties, underlining the concerted
action of VDR, FXR, PXR, and CAR in protecting against
BA toxicity in the liver and intestinal tract (97, 262), may
be confounding when analyzing structure-activity relationships. Finally, FXR-independent activities of BAs,
such as activation of the TGR5 membrane receptor or of
kinase-controlled pathways (see below), are potential
confounders when studying FXR biological properties.
Despite these difficulties, a few rules in structure-activity
relationships have emerged, and they will be described
after reviewing the main structural properties of FXR
ligands and its LBD.
1. Bile acids and bile acids derivatives
The primary BA CDCA was initially described as the
most potent FXR ligand in transient transfection assays,
with an EC50 of ⬃50 ␮M (190). DCA and LCA also displayed a significant ability to activate FXR, albeit with a
lower maximal efficiency. UDCA was inactive in these
classical transcriptional assays, but turned out to be a
partial agonist, able to inhibit CDCA activity (33, 116).
Other primary and secondary BAs such as CA and muricholic acid (MCA), as well as their conjugated derivatives were inactive. However, FXR activation by CA and
conjugated BA activity was restored when the BA transporter IBAT was expressed in target cells, indicating
that BA derivatives exhibit a tissue-specific activity that
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pCIP) (43, 297), p160 proteins are thus likely to play a role
in FXR-mediated transcriptional activation. SRC-2 interacts in a ligand-dependent manner with DNA-bound RXRFXR heterodimers, and SRC-1, widely used in cell-free
systems as a probe for ligand-induced FXR transconformation, coactivates FXR (4, 69, 86, 172, 246). However,
SRC-1 and SRC-2/TIF-2 do not seem to have a physiological role in FXR-mediated transcription, since SRC-1
and/or SRC-2 gene invalidation does not have any detectable influence on hepatic physiology (242). PGC-1␣, a
well-characterized coactivator for PPAR with broad metabolic functions, also interacts physically and functionally
with FXR (266, 366), although the territory of expression
of PGC-1␣ in normal conditions (brown fat, skeletal muscle) as well as the lack of effect of PGC-1␣ gene knockout
on hepatic physiology (295) raise questions about the
physiological relevance of this interaction. However,
PGC-1␣ expression is known to vary in several instances,
including fasting, upon stimulation of the cAMP signaling
pathway and in type 2 diabetes animal models (354). In
these situations, PGC-1␣ might become a prominent FXR
coactivator, as its FXR coactivating properties are more
potent than those of SRC-1 (141). How PGC-1␣ interacts
with FXR remains a matter of debate, since FXR has been
shown to either interact with the NH2 terminus of PGC1␣
(1– 400) through its DBD (366), or to bind to the FXR LBD
in an AF2- and charge clamp-dependent manner, or even
to exhibit a RXR activation-dependent binding mode (140,
141, 266).
Thus the ligand-specific recruitment of cofactors to
FXR is a common theme in the literature which, however,
remains poorly defined, despite its major implications for
understanding the biology of FXR and exploiting its full
potential as a therapeutic target. Moreover, most investigations have focused on coactivators already identified as
partners for other nuclear receptors, leaving open the
possibility that other proteins might modulate significantly FXR transcriptional activity in vivo. Of note, coactivators such as PGC1␤, Tip60, and pCAF do not exhibit
significant coactivating properties on FXR-mediated transcription (141). The G protein pathway suppressor 2
(GSP2) component of the NCoR corepressor complex
acts unexpectedly as a potentiator of FXR action on the
human CYP8B1 FXR response element (263). The transformation/transcription domain associated protein TRRAP,
a constituent of the so-called “TBP-free TAF II-containingtype HAT” coactivator complexes has also been shown to
possess FXR coactivating properties (324). Finally, much
like other intracellular, small lipophilic transport proteins
such as CRABP-2 or FABP (64, 314) which act as coactivators for retinoic acid receptors or PPARs, IBABP binds
to and coactivates FXR in intestinal cells (211). The
multiplicity and diversity of FXR coactivators probably
reflect the existence of very subtle regulatory pathways of
FXR transcriptional activity, which remain to be fully
159
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R
R
H
H
BAT
BSEP/ABCB11
BSEP/ABCB11
BSEP/ABCB11
IR-1
IR-1
IR-1
IR-2
IR-1
R
R
AT2R
BACS
negFXRE
(DR-1)
IR-1
M, H
ApoCIII
IR-1
IR-1
IR-8
negFXRE
IR-1
IR-1
Response
Element
ASCT2
H
ApoCII
H
ApoCII
H
ApoAI
H
H
␣2-Crystallin
ApoAV
H
ALAS-1
Species
⫺63
⫺64
⫹2615
⫺531
⫺238
⫺592
⫺739
HepG2
⫹214*
Up
100 ␮M CDCA, 100 ␮M CA
HepG2, primary
human
hepatocytes
HepG2
HepG2
Up
Up
Up
100 ␮M CDCA, 1 ␮M GW4064
100 ␮M CDCA
Up
Up
Up
100 ␮M DCA, 100 ␮M CDCA,
10 ␮M LCA
RAMSC
50 ␮M CDCA, 10 ␮M GW4064
CV1, in vivo (rat,
1 ␮M GW4064, 100 ␮M CDCA
GW4064100 mg/kg,
7 days)
CV1, in vivo (rat,
1 ␮M GW4064, 100 ␮M CDCA
GW4064 100 mg/
kg, 7 days)
Up
Down
Up
Up
Down
75 ␮M CDCA, 5 ␮M GW4064
1 ␮M GW4064, 100 ␮M CDCA
Up
Up
Regulation
250 ␮M CDCA, 10 ␮M
GW4064
50 ␮M CDCA, 50 ␮M CA,
50 ␮M UDCA, 25 ␮M LCA,
1 ␮M GW4064
Ligands
HepG2, in vivo (1%
100 ␮M CDCA, DCA, LCA,
cholate for 5 days)
CA; 1 ␮M GW4064, 100 ␮M
Androsterone
HepG2, mouse and
GW4064
human primary
hepatocytes,
in vivo
Hep3B, CV1
Cholangiocytes,
hepatocytes,
HepG2
HepG2, human
primary
hepatocytes
Primary human
hepatocytes, liver
slices
Models
⫺103
⫹1037
⫺13606
Position
Main features of direct target genes for FXR
Gene Name
1.
Comments
Claudel et al.,
2003
Kast et al., 2003
Kardassis et al.,
2003
Prieur et al., 2003
Claudel et al.,
2002
Lee et al., 2005
Peyer et al., 2007
Reference
Induction potentialized by
1 ␮M 9-cis-retinoic acid
Ananthanarayanan
et al., 2001
Bungard et al.,
2005
Zhang et al., 2007
The rexinoid LG1305 inhibits Pircher et al., 2003
CDCA-mediated activation
of the promoter
The BAT FXRE is not
Pircher et al., 2003
inducible in CV1 cells. The
rexinoid LG1305
potentiates CDCAmediated activation of the
promoter
Not inducible in NIH 3T3,
Gerloff et al., 2002
MDCK, and weakly
responsive in Caco2 cells
LCA decreases BSEP
Yu et al., 2002
expression
The DR-1 binds also RXRHNF4 heterodimers
FXR binds to this negative
FXRE as a monomer. The
negative FXRE has a
sequence
(GATCCTTGAACTCT)
which is close to the
negative FXRE found in
the UGT2B7 promoter
Not inducible in HepG2 cells
nor in primary
hepatocytes from
cynomolgus liver
The IR-1 overlaps a T3
response element.
*Numbering refers to the
HCR-1 locus in the ApoE/
C gene cluster
FXR is permissive to RXRmediated activation
The IR-1 FXRE is not
conserved in rodents, and
GW4064 represses ALAS-1
expression in mice
FXR␣2 and -␣4 activate this
gene more efficiently
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TABLE
160
LEFEBVRE ET AL.
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M
H
H
M
H
FGF15
FGF19
Fibrinogen ␣, ␤, and ␥
IBABP
IBABP
H
FAS
H
R
Fetuin-B
H
DHEA
sulfotransferase/SULT2A4
eNOS
H
Cyp8B1
R
H
CYP3A4
DDAH1
H
M
Species
BSEP/ABCB11
Complement C3
Gene Name
1.—Continued
⫺148
Position
?
ND
IR-1
IR-1
IR-1
IR-1
IR-1
IR-2
IR-0
IR-1
IR-1
Down
1 ␮M GW4064
CaCO2, in vivo (0.5% 100 ␮M CDCA
CDCA and TCA)
Caco2
250 ␮M DCA or LCA or
UDCA
⫺160
ND
Primary human
100 ␮M CDCA, 1 ␮M GW4064
hepatocytes, HuH7
In vivo (1% CA diet
1 ␮M GW4064
for 5 days)
50 ␮M CDCA
⫹3322
⫺1592, ⫺1559
⫺632
HepG2
Endothelial cells
5 ␮M GW4064, 50 ␮M CDCA
(RPMVEC, BAEC),
CV1
HEK
1 ␮M GW4064
100 ␮M CDCA
⫺641
⫺185
Up
Up
Up
Up
Up
Up
Up
Up
Up
10 ␮M GW4064, 100 ␮M
CDCA
1 ␮M GW4064, 100 ␮M CDCA
Up
Up
Regulation
100 ␮M CDCA
1 ␮M GW4064, 100 ␮M CDCA
Ligands
HEK, in vivo
(GW4064 for
9 days)
Caco2
HepG2
In vivo (single dose
100 mg/kg
GW4064), Caco2
In vivo (GW4064 at
40 mg/kg, single
injection) HepG2,
HuH7
HepG2
Models
⫹90,000
⫺2730
IR-1, DR-3, ⫺7740, ⫺7733,
ER-8
⫺7675
IR-1
IR-1
Response
Element
Comments
Murakami et al.,
2007
Li et al., 2005
Holt et al., 2003
Matsukuna et al.,
2006
Echchgadda et al.,
2004
Li et al., 2007
Hu et al., 2006
Sanyal et al., 2007
Gnerre et al., 2004
Plass et al., 2004
Li et al., 2005
Reference
Nakahara et al.,
2005
The promoter region
Anisfield et. al.,
conferring FXR
2005
responsiveness maps from
⫺2281 to ⫺1700, with no
bona fide FXRE. FXR␣2
and -␣4 (without the
MYTG insertion) isoforms
are upregulating fibrinogen
expression
CDCA is inactive in vivo
Grober et al.,1999
Ortholog of human FGF19
Conserved in mouse, rat,
and human promoters. A
LRH-1 binding site is
embedded into the IR-1
sequence. The IR-1
sequence is located ⬃20
bp downstream a
functional LXRE
IR-0 is also a target for VDR
and PXR
IR-1 not found in mouse or
rat gene. Cyp8B1 is also
negatively regulated by
FXR through a SHPmediated inhibition of
HNF4
The IR-1 motif overlaps the
DR-3 motif
IR-1 FXRE conserved in
mouse and human
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TABLE
BILE ACIDS AND BILE ACID RECEPTORS IN METABOLIC REGULATION
161
Physiol Rev • VOL
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C
H
M
H
H
M
H
H
M
OST␣ and -␤
OST␣ and -␤
OST␣ and -␤
PLTP
PLTP
PLTP
PPAR-␣
PXR
H
OAT2
OATP8/SLC21A8
M
H
MDR3
NaS-1/Slc13a1
H
Kininogen
R
M
INSIG-1
MRP2/ABCC2
M
Species
ICAM-1
Gene Name
1.—Continued
IR-1
Imperfect
DR5
IR-1
IR-1
IR-1
IR-1
IR-1
IR-1
IR-1
DR-1
IR-1
ER-8
IR-1
IR-1
IR-1
IR-1
Response
Element
In vivo (30 mg/kg
GW4064 for
4 days, ex vivo
(adrenals), H295R
LMH
100 ␮M CDCA, DCA, CA,
UDCA; 50 ␮M LCA
1 ␮M GW4064
Up
1 ␮M GW4064
⫺40,000
⫺599
⫺339
⫺407, ⫺393
⫺1375, ⫺1295
(OST␣);
⫺464
(OST␤)
⫺249
Up
Up
100 ␮M CDCA
100 ␮M CDCA, 1% CA
(30 days)
5 ␮M GW4064, 50 ␮M CDCA
Up
Up
Up
Up
GW4064, CDCA
50 ␮M CDCA, 0.2 ␮M
GW4064
HepG2, primary
human
hepatocytes
In vivo (CA-rich diet, 1 ␮M GW4064
450 mg/kg or 50
mg/kg GW4064),
HEK
CV1, in vivo
Murine primary
macrophages,
HuH7, HepG2
HepG2
HepG2, HuH7, ileal
human biopsies
Up
Up
Up
Down
Up
Up
100 ␮M CDCA
⫺1221 (OST␣); In vivo (0.2% CA diet 100 ␮M CDCA
⫺5 (OST␤)
or 100 mg/kg
GW4064 for
5 days), CT26
⫺1375, ⫺1295
(OSTalpha);
⫺134 (OST
beta)
ca. ⫺10,000
⫺401
CDCA, GW4064
Primary human
hepatocytes
HepG2, primary
mouse hepatocyte
In vivo (GW4064,
30 mg/kg, 4 days),
HepG2
⫺1970
Up
CDCA, GW4064
Primary human
hepatocytes,
HepG2
⫺66
HepG2, in vivo
(CA-rich diet)
Up
⫺309
1 ␮M GW4064, 100 ␮M CDCA
Regulation
⫹6733, ⫹11543 In vivo (GW4064
10 days), AML12
Ligands
Up
Models
1 ␮M GW4064
Position
Comments
Reference
Landrier et al.,
2006
Frankenberg et al.,
2006
Lee et al., 2006
Popowski et al.,
2005
Jung et al., 2002
Three functional IR-1 were
identified in intron 2
The IR-1 is not conserved in
human
FXR is not permissive to
RXR-mediated activation
The mouse PPAR-␣ gene is
not regulated by FXR
Jung et al., 2006
Pinneda et al.,
2003
Tu and Albers,
2001
Urizar et al., 2000
The FXRE is also responsive Mak et al., 2002
to LXR␣ and -␤
IR-1 FXRE conserved in
mouse and human. The
FXRE are also regulated
by RXR-LXR heterodimers
(Okuwaki et al., 2007)
Opposite effect of FXR
through SHP-mediated
inhibition of LRH-1 and
through FXRE-mediated
activation
Acts by competing with
HNF4␣
Lee et al., 2007
Kast et al., 2002
The IR-1 is embedded into
Qin et al., 2005
an imperfect DR5 retinoic
acid response element
Regulated by two intronic
Hubbert et al.,
FXREs; FXR␣2 is a more
2007
potent activator of this
promoter than FXR␣1
RXR acts synergistically with Zhao et al., 2003
FXR, and the RXR-FXR is
permissive to rexinoidmediated transcription
Huang et al., 2003
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TABLE
162
LEFEBVRE ET AL.
89 • JANUARY 2009 •
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H
UGT2B7
IR-1
Negative
FXRE
B4-BARE
Caco2, HepG2
⫺1193
⫺148
Down
30 ␮M LCA, 75 ␮M
CDCA
Up
Up
Up
Up
Regulation
5 ␮M GW4064, 50 ␮M
CDCA
10 ␮M GW4064, 250 ␮M
CDCA
25 ␮M CDCA
1 ␮M GW4064, 100 ␮M
CDCA
Ligands
Gallbladder epithelial 20 ␮M GW4064
cells
Primary human
hepatocytes,
HepG2
⫺921
⫺919, ⫹33,
⫹156
HepG2, HuH7
⫺193
Models
In vivo (rat, 30 mg/
kg GW4064 for 7
days), primary
hepatocytes
HepG2, Caco2
⫺291
Position
Reference
FXR is permissive to RXRmediated activation
The SCD promoter is
specifically responsive to
FXR␣2 and -␣3 isoforms
RXR activation decreases
FXR-mediated
transcriptional activation
and decreases FXR
binding to the B4-BARE
The negative FXRE has a
sequence
(GATCCTTGATATTA) that
is close to the negative
FXRE found in the
hApoAI promoter
9-cis-retinoic inhibits
GW4064-mediated
activation of VPAC1 gene.
The functionality of
FXREs has not been
established
Chignard et al.,
2005
Lu et al., 2005
Barbier et al.,
2003
Anisfeld et al.,
2003
Song et al., 2001
The IR-1 is conserved in
Goodwin et al.,
human, mouse (⫺320) and
2000
rat (⫺309)
Comments
Species: H, human; M, mouse; R, rat. The “models” column indicates cellular or animal models in which the activity of the FXRE has been characterized; the “ligands” column indicates
which ligand(s) and concentration have been used. Comments highlight some of the salient features of the FXR-regulated promoter.
h
H
UGT2B4
DR-1
IR-0
IR-1
Response
Element
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Physiol Rev • VOL
VPAC1
H
SYNDECAN
H
Species
R
Gene Name
1.—Continued
STD
SHP
TABLE
BILE ACIDS AND BILE ACID RECEPTORS IN METABOLIC REGULATION
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164
LEFEBVRE ET AL.
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2. Natural extracts
The extract from the gum resin of Commiphora
mukul, guggulipid, is used in traditional medicine for its
hypolipidemic effects. This has prompted investigators to
isolate the active principle from this extract (327, 346). A
mix of Z- and E-[4,17(20)-pregnadiene-3,16-dione] or guggulsterone (GS) was initially shown to antagonize FXR
target genes activation by CDCA in vitro [IBABP, SHP,
BSEP (228, 327, 346)] and to decrease hepatic cholesterol
in vivo, a surprising observation when considering the
phenotype of FXR⫺/⫺ mice. On the contrary, GS was
reported to increase BSEP expression in another study
(56), a complex effect that may be attributable to GSmediated control of AP-1 activity (66). However, GS displayed, like BAs, agonist activity on CYP7A1 expression
in HepG2 cells, an effect probably relayed through activation of the pregnane X receptor (PXR). Indeed, further
investigations demonstrated that GS is a promiscuous
ligand able to inhibit (327) or activate PXR in HepG2 cells
(29, 228). A 10 ␮M GS concentration also significantly
induces progesterone receptor (PR) activity in transient
transfection assays (29, 32). Indeed, GS binds to PR and to
other steroid receptors with an affinity in the high nanomolar range and is an antagonist for the androgen, glucocorticoid, and mineralocorticoid receptors (32). Thus,
despite its relative lack of selectivity, GS is a gene-selective modulator of FXR activity, since displaying a molecular behavior typical of antagonists in cell-free coactivator binding assays (327, 346) but exhibiting gene-specific
agonist or antagonist activities. Molecular modeling suggested that this peculiar behavior may be attributed to the
occurrence of a specific GS docking site in the FXR LBD,
although this has not been backed up by crystallographic
or mutational studies (199).
Other natural extracts have also been shown to contain FXR activators. Stigmasterol, a soy lipid-derived phytosterol, suppresses ligand-induced expression of FXR target
genes involved in adaptation to cholestasis in HepG2 cells
(40). The diterpene cafestol, isolated from unfiltered coffee brew, is a hypercholesterolemic agent and an agonist
for FXR and PXR displaying an efficiency in cultured
hepatoma cells (HepG2) similar to that of reference compounds (CDCA and PCN, respectively) (255). More surprisingly, cafestol did not induce FXR target genes in the
liver in vivo, but activated the FGF15 gene in the small
intestine, hence triggering an inhibitory signaling enterohepatic loop (129). This probably reflects the low bioavailability of cafestol, or its rapid metabolism in the liver
(255) and further illustrates the role of the ileum as a
critical FXR target organ (115). The prenylated chalcone
xanthohumol from beer hop activates the BSEP promoter
in transcriptional assays in hepatoma cells (EC50⫽ 5 ␮M),
and lowers hepatic and plasma triglycerides in diabetic
KK-Ay mice, suggesting that it may be a FXR activator
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may be conditioned by the expression of membrane
transporter(s). This barrier may be circumvented by
converting the carboxylic extremity of BA into an alcoholic function (85). Classification of BAs as FXR ligands is based on their behavior in cell-free coactivator
recruitment assays. While CDCA was consistently reported as a molecule promoting SRC-1 recruitment, and
thus classified as an agonist (EC50 ⫽ 8 ␮M) (190, 232,
237, 359), DCA, LCA, and CA were inactive in this assay
but antagonized CDCA-induced SRC-1 recruitment
(172, 190, 232, 359). Intriguingly, LCA could be classified as a weak agonist on the basis of its capacity to
induce BSEP expression in HepG2 cells (359), much
like UDCA. There is therefore no strict correlation
between BA affinity for FXR, its capacity to promote
SRC-1 interaction with FXR, and its biological activity,
suggesting that either coactivator recruitment assays
are not predictive of the transcriptional outcome, or
that SRC-1 is not the appropriate model system, or that
the transcriptional outcome is a function of multiple
parameters including the dimerization partner and the
DNA binding sequence, as suggested for FXR (172) and
demonstrated for other nuclear receptors (204).
The use of BA derivatives in ligand-binding and
transcriptional assays has provided interesting structure-activity relationships that are in line with the reported crystallographic structure of the FXR LBD (see
below). Taken together, these studies (52, 85, 221, 222,
236, 237) have shown that 1) introduction of a 6␣ alkyl
substituent is compatible with in vitro and in vivo
activity, but polar and hydrogen-bonding substituents
are deleterious for activity; 2) 6␣-ethyl CDCA (6␣ECDCA)
is a potent FXR agonist with an activity in transcriptional
assays three orders of magnitude higher than the parent
compound CDCA; 3) introduction of a bulky substituent
in either the 3␤ or the 7␤ position led to a decreased
activity of the molecule; 4) derivatization of the C24 group
by carbamate moieties generates molecules exhibiting
agonist or antagonistic properties; 5) an intact cholesterol
side chain (C27) is not compatible with FXR agonism or
antagonism, but a C25 or C26-hydroxylated bile alcohol is
as effective as CDCA; 6) epimerization at C12 of the
hydroxyl group abolished FXR activation; and 7) the 5␣
epimerization yields bile alcohols that are agonists, on the
basis of a coactivator recruitment assay, whereas 5␤ bile
alcohols are antagonists. Removal of the 3-hydroxyl group
has no effect on coactivator recruitment. Other steroidal
molecules such as androsterone also activate FXR (340).
Interestingly, BA “extranuclear” activities, dependent on
the binding of BA to the G protein-coupled membrane
receptor TGR5, can be triggered selectively with enantiomeric LCA and CDCA (147) or other substituted BAs
(239).
BILE ACIDS AND BILE ACID RECEPTORS IN METABOLIC REGULATION
(224). Sesterterpenes extracted from a marine sponge
displayed FXR antagonistic properties, although some of
them exhibited strong cell toxicity (213, 214).
3. Synthetic compounds
Physiol Rev • VOL
produce BAs at nonnegligible rates [58 ␮g/96 h, equivalent
to 16 ␮M for HepG2 cells (171)], and that, as mentioned
above, BAs may elicit FXR-independent effects. Finally,
1,1-biphosphonate esters are potent FXR agonists displaying hypocholesterolemic and antiproliferative effects
(218), and the sulfonamide T0901317 activates FXR in the
micromolar range, concentrations well above its reported
affinity for LXR and PXR (20 – 40 nM) (203).
E. Ligand-Binding Properties of FXR
An important step in exploiting FXR as a molecular
target in metabolic diseases is to establish structureactivity relationships, with the aim of identifying ligands
with optimized biological properties. Early mutational
studies identified several amino acids of the FXR LBD that
are critical for ligand binding or ligand discrimination.
Considering sequence homologies with LXR, Urban et al.
(325) suggested that F268 in the human FXR LBD is
critical for ligand recognition. Based on the differential
response of hFXR vs mFXR to CDCA, N354 and I372 were
identified as responsible for the high sensitivity of hFXR
to CDCA, and mutation of the corresponding amino acids
in mFXR could “humanize” this receptor (55). However,
these amino acids are not involved in a direct interaction
with CDCA (see Fig. 3C).
Crystallographic data of fexaramine and of 3-deoxyCDCA or 6ECDCA complexed to the human or rat FXR
LBD, respectively (69, 200), brought significant insights in
the mechanism of activation of FXR by its cognate ligands
(Fig. 3). Of note, amino acids involved in ligand-receptor
interaction are conserved between rat and human FXRs.
These studies have revealed not only features of the FXR
LBD similar to those of other nuclear receptor LBDs, such
as its general organization in a 12 ␣-helix bundle, but also
unanticipated characteristics. As for other NRs, the
COOH-terminal LBD acts as a molecular switch, undergoing major structural transitions enabling the selective recruitment of coactivators by forming a charge clamp and
a hydrophobic groove with which LXXLL motifs from
coactivator molecules will interact (69, 200). More unanticipated was the opposite orientation of these ligands in
the LBD compared with other steroid receptors, as well as
the occurrence of a second docking site for the LXXLL
motif. However, a recent report described the binding
mode of synthetic MFA-1 FXR agonist as similar to that of
steroids to steroid receptors, underlining a structural versatility which expands drug design strategies in this field
(293).
When considering BAs, the ligand-binding pocket
(LBP) has to accommodate a steroid nucleus with specific
conformational and physicochemical properties, with a
concave hydrophilic face (␣ face) and a convex hydrophobic face (␤ face), which is the privileged site for
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As mentioned previously, naturally produced FXR
ligands have a moderate selectivity, thus prompting the
development of synthetic molecules displaying a strong
selectivity and affinity for FXR. The initial observation
that TTNPB activates FXR, albeit with a low affinity (360),
provided a lead compound for the synthesis of stilbene
derivatives (192). GW9047 emerged as a compound fulfilling the “selectivity” criterion, but is a weak activator
in transcriptional assays. Further pharmacomodulation
around this structure led to the identification of GW4064
[3-(2,6-dichlorophenyl)-4-(3⬘-carboxy-2-chloro-stilben-4yl)-oxymethyl-5-isopropyl-isoxazole], a potent and selective FXR agonist (EC50⫽ 90 nM), active both in vivo and
in vitro (94, 192, 288). Although displaying a limited bioavailability, GW4064 has gained a widespread use as a
powerful and selective FXR ligand and has reached the
status of “reference compound” in this field. Using again
TTNPB as a lead compound, Dussault et al. (75) adopted
a strategy to reinforce its selectivity towards FXR. They
modified the TTNPB backbone to prevent RAR binding,
and such a pharmacophore modification yielded AGN29
and AGN31. These molecules turned out to be efficient
agonists of FXR with an EC50 value around 1 ␮M, and as
well as of RXR, as expected from the property of the
parent compound (24). Further pharmacomodulation
around retinoid structures led to the identification of
AGN34, a dual RXR-FXR antagonist displaying genespecific properties. Of note, the synthetic RXR agonist
LG268 was shown to activate FXR/RXR heterodimers
through the RXR moiety (75), but was later shown to
inhibit GW4064, 6␣EDCA, and CDCA-induced FXR transcriptional activity (144, 259). Using a benzopyran structure to identify novel FXR ligands, Downes et al. (69)
optimized the structure of lead compounds to identify
biaryl cinnamates, amongst which fexaramine and fexarine
emerged as potent and highly selective FXR agonists
(EC50⫽ 38 and 36 nM, respectively). A comparative gene
profiling study underlined distinctive properties of fexaramine, GW4064, and CDCA to activate target genes in
primary human hepatocytes, demonstrating that each of
these ligands, in addition to the AGN compounds, may be
considered as selective BA receptor modulators or
SBARMs. Possible explanations for these differential activities could be a differential positioning and filling of the
ligand-binding pocket by these ligands, or a RXR-induced
allosteric regulation of FXR ligand-binding property. Indeed, GW4064 requires RXR to exhibit maximal efficacy
in transcriptional assays. However, a difficulty in comparing ligand effects is that target cells, such as hepatocytes,
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LEFEBVRE ET AL.
interaction with other hydrophobic molecules (Fig. 3).
The ␣ face may harbor several hydroxyl groups in the 3, 7,
and/or 12␣ positions, and these hydroxyl groups directly
affect BA affinity for FXR. Indeed, the 7␣-hydroxy group
interacts with rY366, conferring a high affinity to CDCA,
whereas the lack of this hydroxyl group in LCA decreases
the affinity for FXR. Introducing bulky substituent on the
␤ face has a general deleterious impact on the affinity for
FXR (85). Importantly, optimal activation of FXR is dependent on the proper positioning of helix H3 versus helix
H12, generating the LXXLL docking groove. CDCA, like
fexaramine, engages interactions with helix 3 and helix 7
Physiol Rev • VOL
through the steroid 7␣ hydroxy group, resulting in a
highly stable H3-H12 structure and full agonist activity. In
contrast, partial agonists such as DCA and LCA lack the
7␣-hydroxyl group, loosening this structure and compromising stable coactivator recruitment. Furthermore, the
FXR LBP cannot accommodate a 12␣-hydroxyl group.
Therefore, CA and DCA display a weaker affinity for FXR.
Generating additional contacts within a hydrophobic cavity strongly increases the affinity for FXR, as seen for
6-ECDCA. Quite strikingly, the carboxylic extremity of
BAs is oriented towards the entry of the LBP, thus explaining why derivatization of this function, as seen in
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FIG. 3. Ligand-receptor interactions. A: the planar structure of deoxycholic acid is shown, together with its typical tridimensional arrangement
defining the ␣-face harboring the hydroxyl groups (in red). Both deoxycholic acid and chenodeoxycholic acid are hydroxylated in 3, whereas the
second hydroxyl group is present in 12 in the DCA molecule and in 7 for CDCA. The fexaramine molecule exhibits a totally different structure,
allowing an optimal filling of the ligand-binding pocket of FXR, without protruding from this cavity. B: three-dimensional structure of the FXR LBD
bound to fexaramine. C: molecular fingerprints of ligand-FXR interactions. A schematic structure of the human FXR LBD secondary structure
indicates the 12 ␣-helices present in this domain. Ligand-LBD contact points are indicated for fexaramine, 6␣-ethylCDCA, and MFA-1 (68, 196, 293).
Numbering corresponds to the amino acid positon in the human FXR␣2 sequence. Contact residues common for all ligands are indicated by an
empty box, whereas contacts specific for each ligand are indicated by a black box. Residues that contact two out of the three ligands are indicated
by a green box. Images were generated from the pdb (http://www.rcsb.org/pdb) or the PDBsum (http://www.ebi.ac.uk/pdbsum/) websites.
BILE ACIDS AND BILE ACID RECEPTORS IN METABOLIC REGULATION
IV. FARNESOID X RECEPTOR-MEDIATED
REGULATION OF BILE ACID SYNTHESIS
AND TRANSPORT
F. DNA-Binding Properties of FXR and FXR
Target Genes
FXR can bind to DNA to so-called FXREs either as a
monomer or a heterodimer with RXR. The latter configuration is the most common and generally associated with
gene activation, whereas the monomeric form may also
be associated with gene repression. Early studies (165,
274) established that the preferred consensus FXRE is an
inverted repeat (IR) of the core hexanucleotidic AGGTCA
sequence separated by one base pair (IR-1). Sequences
flanking this IR-1 were not found to influence RXR-FXR
affinity for DNA. In addition, FXR could bind in vitro to
IR-0, but not to IR-2, IR-3, IR-4, and IR-5 sites. A direct
repeat (DR) geometry with a similar core sequence
AGGTCA is compatible with RXR-FXR binding, and DR-2,
DR-4, and DR-5, but not DR-0, DR-1, or DR-3 sequences,
can form complexes with RXR-FXR heterodimers. The
identification of FXR target genes, which regulate diverse
cellular processes (Fig. 4), has shown that FXR can bind
FXREs with a remarkably diverse geometry, ranging from
IR-1 to everted repeat (ER)-8 sites (summarized in Table
1). While the structure of hormone response elements has
been shown to dictate the activity of several other nuclear
receptors such as the thyroid hormone receptor (117,
332), the glucocorticoid receptor (292, 298) or the retinoic
acid receptor (208), this phenomenon has not yet been
appreciated for FXR.
G. FXR Polymorphisms
The etiology of some BA metabolism disorders is
poorly characterized and thus, in view of the central role
Physiol Rev • VOL
of FXR in the maintenance of BA homeostasis, genetic
variations in its sequence have been sought in several
pathologies. As mentioned above, heterozygous variants
of FXR␣1&2 have been isolated from ICP patients (329).
The ⫺1g⬎t, ⫹1a⬎g (M1V), and 518t⬎c (M173T) affect the
stability or the activity of FXR, whereas the 238c⬎t
(W80R) did not display any functional defect in vitro. The
⫺1g⬎t mutation affects protein translation efficiency, although this destabilization was not observed when assessing the translation efficiency of this cDNA in in vitro
heterologous systems (196). Other single nucleotide polymorphisms were detected in several ethnic groups, albeit
with a much lower frequency compared with the ⫺1g⬎t
mutation (2.5–12.1% vs. 0 – 0.5%). Two SNPs altered the
coding sequence [⫹643c⬎t (H215Y), ⫹646g⬎t (A216S)],
but the functional consequences of these mutations were
not studied (196). A study involving three cases of idiopathic BA malabsorption did not reveal any mutation in
the FXR gene (205).
As described in section II, hepatic BA biosynthesis
and intestinal reabsorption must be rigorously coordinated to maintain a constant BA pool size. The biosynthetic and transport processes involved in enterohepatic
cycling are essentially regulated through complex transcriptional networks involving not only FXR and other
nuclear receptors, but also other signaling pathways.
A. Transcriptional Regulation of Bile
Acid Biosynthesis
The importance of CYP7A1 and of CYP8B1 in generating an appropriate primary BA pool has been described
in section II. Studies with FXR-null (FXR⫺/⫺) mice have
unequivocally demonstrated the physiological role of this
BA-activated nuclear receptor in the control of BA metabolism (288). Increased hepatic CYP7A1 mRNA levels but,
very surprisingly, reductions of total bile salt pool size
and fecal bile salt loss are observed in FXR⫺/⫺ mice.
Counterintuitively, these data would imply that in the
absence of functional FXR, BA synthesis would actually
be suppressed. Other mouse models with increased
CYP7A1 expression showed, as expected, increased BA
synthesis rates and expansion of BA pool sizes. This may
imply that FXR deficiency has an impact on the maintenance of BA pool size at other levels, for instance, in the
intestine. To address this issue, Kok et al. (158) evaluated
parameters of the enterohepatic circulation of CA, quantitatively the major BA species in the mouse, in relation to
bile formation and the expression of transport proteins in
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conjugated BAs, does not preclude high-affinity binding to
FXR and allows FXR activation by these compounds.
However, structural variations introduced on the carboxyl end of BAs may yield substantial variations in FXR
activity (238).
Isolated LXXLL motifs bind to the FXR LBD with a
low affinity (⬃160 –500 ␮M), whereas receptor interaction
domains (RIDs) from coactivators encompassing two or
three LXXLL motifs display an increased affinity (⬃1 ␮M)
for the FXR LBD. Structural and physical studies of the
FXR-coactivator complex showed that the FXR LBD
binds 2 LXXLL motifs in an anti-parallel fashion, with one
being located in the “classical” groove and secured by a
charge clamp, whereas the second binding site lacks this
charge clamp and is located on helix 3. Data were compatible with the association of a single coactivator
polypeptide to the FXR LBD, suggesting that the second
LXXLL interaction surface increases FXR affinity for a
single coactivator molecule (200). However, this hypothesis has not yet been tested in mutational studies.
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FIG. 4. The FXR-controlled gene regulation network. The main biological functions regulated by FXR are indicated on the right. Genes
upregulated by FXR are indicated by green arrows, and those that are downregulated by red arrows. Indirect regulation modes are indicated on the
right, underlining the important role of SHP in this pathway. When known, the intermediate signaling molecule or the component(s) of the signaling
cascade are indicated. Species-specific regulation is mentioned when known (h, human; Rb, rabbit). When known, the type of FXRE is indicated (IR,
inverted repeat; DR, direct repeat, ER, everted repeat).
another mouse model of FXR deficiency. For quantitation
of cholic acid kinetic parameters, a stable isotope dilution
technique was used. This study demonstrated that, in
accordance with derepressed transcription of CYP7A1,
FXR⫺/⫺ mice showed an increased cholic acid synthesis
rate without any effect on its fractional turnover rate.
Interestingly, the calculated intestinal cholic acid reabsorption was markedly increased, leading to a significantly enlarged BA pool size. The different procedures
used for generation of FXR null mice might be responsible
for the apparently discrepant findings of Sinal et al. (288)
and Kok et al. (158): a direct comparison of both strains
using the same methodologies to analyze bile acid metabolism has not been reported so far.
Both CYP7A1 and CYP8B1 are transcriptionally regulated by several nuclear receptors. A complex regulatory
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sequence containing overlapping binding sites for the orphan NRs HNF4␣ and LRH-1, and the retinoic acid receptors, conveys a positive regulation of the Cyp7A1 gene
upon HNF4␣ or LRH-1 binding. Interestingly, HNF4␣ is
coregulated by the transcriptional coactivator PGC1␣,
whose expression and activity are modulated by insulin
and fasting, two signals known to modulate BA synthesis
(59, 321). The FXR-mediated repression of CYP7A1 occurs through a direct regulation of the small heterodimer
partner (SHP) gene by FXR. SHP, a peculiar NR devoid of
a DNA-binding domain, exerts a repressive activity upon
dimerization with several transcription factors, including
LRH-1 and HNF4␣ (94, 184). This occurs through diverse
mechanisms, which may involve the recruitment of a
Sin3A/Swi-Snf complex to the CYP7A1 promoter (15,
149). A similar SHP-mediated feedback regulation of the
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α
BILE ACIDS AND BILE ACID RECEPTORS IN METABOLIC REGULATION
B. Transcriptional Regulation of Bile Acid Export
and Reabsorption
Hepatic BA transporter expression is, to a considerable extent, regulated by FXR, as detailed by Trauner and
Boyer (317). On the one hand, secretion of BAs by hepatocytes into the canalicular lumen is essentially dependent on the expression of the ATP-dependent bile salt
export pump BSEP/ABCB11, whose expression is directly
regulated by FXR (3). The functional importance of BSEP
is underlined by pathological consequences of BSEP mutations, leading to PFIC type 2 (308). The ABC transporters MDR3/ABCB4 and MRP2/ABCC2, responsible for
hepatobiliary transport of phospholipids and of hydrophilic organic anions like divalent BA conjugates, respectively, are also upregulated by FXR (120, 145). On the
other hand, FXR downregulates the expression of the
sinusoidal/basolateral sodium-taurocholate cotransporting polypeptide NTCP/SLC10A1, through which 80% of
BA uptake occurs. This regulation is species specific and
may involve SHP (67). More surprisingly, the expression
of another basolateral BA import pump, OATP1B3/
SLC01B3, is upregulated by FXR. This unexpected response has been interpreted as a means for the hepatoPhysiol Rev • VOL
cyte to maintain its xenobiotics disposal potential, even
during cholestasis (139).
BA uptake by enterocytes is a highly critical step in
BA metabolism (see sect. II). The expression of the murine apical sodium-dependent BA transporter (ABST)/intestinal BA transporter (IBAT) is positively controlled by
LRH-1, which, as described for CYP7A1 in hepatocytes, is
repressed by a FXR-induced SHP-mediated repression
(45). Repression of hABST expression is likely the result
of a distinct mechanism, involving a negative interference
of SHP with the positive regulation by the glucocorticoid
and/or retinoic acid receptors (137, 215). The expression
of the fatty acid binding protein 6 (FABP6/I-BABP),
whose role is likely to sequester excess intracellular BA
and to serve as a coactivator for FXR, results from a
direct transcriptional regulation by FXR (95, 128).
C. Cholestatic Liver Diseases
Cholestasis, functionally defined as an impairment or
absence of bile flow, may result from functional defects in
the primary bile formation process at the level of the
hepatocytes or the cholangiocytes (intrahepatic cholestasis) or from a (usually mechanical) obstruction of bile
flow at the level of bile ducts (extrahepatic cholestasis).
Impaired function of hepatobiliary transport systems
(sect. IIC) due to genetic defects, e.g., BSEP/ABCB11 in
PFIC2 or MDR3/ABCB4 in PFIC3 (235) or secondary to
exposure to certain drugs, hepatotoxic endobiotics, or the
presence of sepsis, are key in the pathogenesis of most
forms of intrahepatic cholestasis (317). Treatment options are very limited and, apart from liver transplantation
for some of the inherited forms of cholestasis, mainly
aimed at reduction of symptoms (jaundice, pruritus). Independent of the underlying origin, cholestasis results in
intrahepatic and systemic accumulation of substances
normally excreted into bile (cholephils). Particularly, accumulation of detergent BAs that are normally secreted
into bile in millimolar amounts may lead to liver cell
damage, inflammation, and eventually organ failure. A
series of adaptive reactions occurs during cholestasis that
contributes to a diminished BA toxicity: inhibition of
endogenous synthesis, induction of phase I and phase II
detoxification reactions to render BAs more hydrophilic,
and induction of alternative transport routes to eliminate
BAs. FXR, but also other nuclear receptors like PXR,
VDR, and CAR, are involved in the orchestration of these
compensatory reactions (370). Modulation of FXR activity
has been proposed as an attractive target for treatment of
cholestatic liver diseases. Given the complexity of the bile
formation process and the various etiologies that may
underlie cholestatic liver disease, it is evident that FXR
agonism will not provide a panacea. Therapeutic strategies should be focused not (only) on restoration of biliary
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human CYP8B1 gene has been reported (62, 353, 362).
However, SHP-independent regulatory pathways have
also been described, which may account for the fact that
the loss of SHP in mice increased the levels of hepatic
CYP7A1 and CYP8B1 by only twofold, which is much less
than observed in mice with a depleted BA pool size (150,
338). Further investigations pointed to the role of FXRmediated upregulation of FGF19 expression in human
hepatocytes (114), and of FGF15 in murine intestine
(129), initiating an autocrine/paracrine loop activating the
FGF-receptor 4 (FGFR4). Of note, FGF’s action requires
the presence of Klotho and ␤Klotho, which are single-pass
transmembrane proteins, to activate the FGFR4 signaling
pathway. The synthesis and excretion of BAs are strongly
increased in ␤Klotho-deficient mice, as well as the expression of CYP7A1 and CYP8B1. BA-dependent induction of
SHP is significantly attenuated in the liver, but not in the
intestine of ␤Klotho-null mice (133). Mice deficient for
FGFR4 exhibit an increased BA pool size and increased
CYP7A1 expression (357). FGFR4-overexpressing mice
display an opposite phenotype (358). The role of FGF19
signaling in the maintenance of diurnal variation in hepatic BA synthesis in humans was elegantly demonstrated
by Lundasen et al. (185). Interestingly, these molecular
investigations shed light on the nature of the putative
intestine-derived molecule regulating BA synthesis that
explains the paradox that intravenous TCA administration fails to regulate CYP7A1 in rats while duodenal TCA
infusion does (230).
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LEFEBVRE ET AL.
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beneficial effects of FXR stimulation, either as a consequence of cholestasis or induced by a ligand, may be
secondary to activation of PXR gene transcription by FXR
(138). PXR is known to induce a variety of BA-detoxifying
pathways, including cytochrome P-450s and several of the
transporter genes, and to repress CYP7A1 expression
(299). Recent data have also implicated the xenobiotic
receptor CAR in protection from BA-induced hepatotoxicity (97, 361). Thus understanding the complex network
of adaptative responses that collectively protect liver
cells, as well as other cells in the body, from the toxic
actions of BAs, and potentially also other harmful cholephiles, provides opportunities for development of new
therapeutics. This will however require a tailored design
for specific disease entities and will strongly depend on
whether or not bile flow is completely absent. Particularly
important in this respect is the role of the intestine in
conservation of BAs. Lanzini et al. (168) reported a
strongly reduced fractional turnover of the BA pool in
patients with primary biliary cirrhosis (PBC). Thus, in this
case, there seems to be an undesired, more effective
conservation of BAs while enhanced removal would be
preferable. As argued by Hofmann (112), this appears to
be an adaptive response to low intraluminal BA concentrations with the undesired consequence of exposing the
liver to even more BAs. In addition, low intraluminal BAs
will be associated with low intestinal FGF19 expression
and, hence, a less effective suppression of hepatic BA
synthesis. Whether induction of intestinal FGF15 contributes to the suppression of CYP7A1 expression in cholestatic rodents treated with an FXR agonist remains to be
established.
V. FARNESOID X RECEPTOR AND
LIPOPROTEIN METABOLISM
The ability of BAs to affect lipid metabolism is known
since the 1970s. Since its discovery in 1995, a role for FXR
in the control of both triglyceride and cholesterol metabolism has been established, identifying FXR as a molecular link between BAs and plasma lipids.
A. FXR and LDL-Cholesterol Metabolism
FXR impacts on cholesterol metabolism through the
repression of CYP7A1, the rate-controlling enzyme for
cholesterol catabolism into BAs. CYP7A1 induction stimulates the conversion of cholesterol to BAs, resulting in
a relative deprivation of hepatic microsomal cholesterol content, followed by upregulation of LDL-receptor
(LDL-R) expression and activity which consequently reduces plasma LDL-cholesterol (LDL-C) levels. This mechanism underlies the hypocholesterolemic effect of BA
sequestrants, like cholestyramine and colesevelam (131).
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excretion function but predominantly on the reduction of
hepatocellular uptake via NTCP/OATPs, stimulation of
detoxification systems (hydroxylation, conjugation), and
alternative pathways for elimination of BAs via the kidneys. Studies in bile duct-ligated (BDL) mice, a model for
obstructive extrahepatic cholestasis, would indicate that,
particularly for this form of cholestasis, a FXR antagonist
might be beneficial. FXR-null mice were found to be less
sensitive to BDL-induced liver damage (301, 334) likely
due to the fact that these mice, in contrast to wild-type
mice, did not maintain BSEP/ABCB11 expression. Maintenance of BA transporting capacity into the occluded
biliary system resulted in increased biliary pressure and
induction of bile infarcts, as well as to bile ductular
proliferation in wild-type mice (334). In addition, FXRnull mice were reported to adapt to biliary obstruction by
enhanced BA hydroxylation, likely catalyzed by induction
of CYP3A11, leading to enhanced urinary excretion (194).
Studies in rat models of chemically induced intrahepatic
cholestasis and also, counterintuitively, of BDL-induced
extrahepatic cholestasis showed that activation of FXR
with the synthetic agonist GW4064 resulted in significant
reductions in serum alanine aminotransferase, aspartate
aminotransferase, as well as other markers of liver damage (180). GW4064 also decreased the incidence and extent of necrosis as well as decreased inflammatory cell
infiltration and bile duct proliferation. On the basis of
analysis of gene expression profiles, the beneficial effects
of FXR activation have been ascribed to reduction of BA
synthesis genes, such as CYP7A1, and induction of genes
involved in biliary transport, such as the phospholipid
transporter Mdr2/Abcb4 (180). Similarly, 6-ECDCA has
been shown to exert protective effects in estrogen-induced cholestasis in rats by suppression of BA synthesis
genes, reduction of NTCP expression and induction of
BSEP/ABCB11, Mdr2/ABCB4 as well as Mrp2/ABCC2,
the latter being involved in hepatobiliary transport of
divalent BA conjugates (81). Feeding of the hepatotoxic
monohydroxy BA lithocholic acid resulted in a more severe cholestatic phenotype in wild-type mice than in FXRnull mice, ascribed to higher LCA sulfotransferase activity
in the latter (155). Lithocholic acid has been proposed to
act as a FXR antagonist and, thereby, to decrease BSEP/
ABCB11 expression (359). FXR activation has also been
shown to induce overall gene expression of alternative
basolateral BA transporters like Mrp3/ABCC3 and Mrp4/
ABCC4, specific for divalent BA conjugates, and of the
bidirectional BA transporter Ost␣/Ost␤ (371). FXR induces UGT2B4 expression and activity in human hepatocytes, indicating a feed-forward reduction of BA toxicity
in humans by glucuronidation (12). In addition, upregulation of Mrp2/ABCC2, Mrp4/ABCC4 and Ost␣/Ost␤ on the
basolateral surface of renal tubular cells in the kidney will
increase the overall elimination capacity for such hydrophilic BA metabolites from the body (10). Part of the
BILE ACIDS AND BILE ACID RECEPTORS IN METABOLIC REGULATION
Physiol Rev • VOL
Clearly, additional in vivo studies with specific FXR
agonists are needed to resolve the question of the role of
FXR in controlling LDL-C levels.
B. FXR and HDL-Cholesterol Metabolism
HDL carries cholesterol from the peripheral organs
to the liver, where it can be excreted into the bile either as
free cholesterol or after conversion into BAs, in a process
called reverse cholesterol transport. FXR has a profound
effect on HDL metabolism and remodeling, which stems
seemingly from liver-specific processes. On the one hand,
FXR⫺/⫺ mice display elevated plasma HDL-C and apolipoprotein (apo) A-I concentrations (158, 288). FXR represses human and murine apoA-I gene expression both
in vitro and in vivo, via a monomeric FXR binding site in
the promoter (50). This negative FXRE is also a functional
LRH-1 binding site, suggesting that FXR-mediated repression may also involve a FXR-induced SHP-mediated inhibition of LRH-1 transcriptional activity (63). Consistent
with this, feeding apoA-I transgenic mice with CA lowers
plasma HDL cholesterol and apo-AI levels. On the other
hand, hepatic and ileal apo-AI expression is unaffected in
FXR⫺/⫺ mice (166). In vivo kinetic studies indicated that
elevated plasma HDL-C levels observed in FXR⫺/⫺ mice
are due to a reduced, selective uptake of HDL-cholesteryl
esters by the liver, potentially linked to a decreased expression of the scavenger receptor B1 (SR-B1), rather
than to enhanced production. Accordingly, a CA-enriched
diet decreases hepatic SR-B1 expression, both at the
mRNA and protein level (166). Similar to FXR-mediated
repression of CYP7A1, the reported BA-induced downregulation of SR-BI may implicate the activation of the
FXR ⬎ SHP ⬎ LRH-1 cascade (191). FXR enhances the
expression of the phospholipid transfer protein (PLTP;
Ref. 326) and represses the expression of hepatic lipase
(290), demonstrating a role for FXR in HDL remodeling.
In vivo treatment with the FXR agonist GW4064 significantly improves hypercholesterolemia in mouse models of insulin resistance (37, 364), an effect unfortunately
associated with a decrease in HDL-C (343). These latter
results are in accordance with human studies in which BA
sequestrants increase HDL-C concentrations (5, 282),
whereas CDCA administration results in decreased
HDL-C levels (13, 170). Additional studies are needed to
establish whether FXR activation may modify HDL composition, hence its function. Intriguingly, treatment with
CDCA of patients with cerebrotendinous xanthoma increased cholesteryl ester transfer protein (CETP) activity
(154). Kinetic studies in humans are clearly required to
investigate further the relationships between FXR and
HDL formation, catabolism, and functions. In addition,
the contribution of intestinal factors to the regulation of
HDL metabolism should be evaluated.
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Moreover, human CYP7A1 deficiency results in a statinresistant hypercholesterolemia (250). The fact that BA
sequestrants lower LDL-C suggests that a synthetic FXR
antagonist might reduce LDL-C levels. In traditional Indian medicine, oleogum resin (known as guggul) from the
guggul tree, Commiphora mukul, has been used to treat
various diseases, including hypercholesterolemia. A number of uncontrolled clinical studies carried out in India
demonstrated the hypolipidemic activity of guggulsterone
with, on average, 10 –30 and 10 –20% decreases in total
cholesterol and triglyceride, respectively. However, individual variations in response to guggulsterone treatment
have been noted with ⬃70 – 80% responders and 20 –30%
nonresponders (65, 323). In contrast to these data, a
United States randomized controlled trial demonstrated
that guggulsterone treatment did not improve the plasma
lipid profile of hypercholesterolemic patients, and even
leads to a modest increase in LDL-C (313). This demonstrates that guggulsterone does not reduce LDL-C in humans at the doses commonly used. However, it cannot be
definitively excluded that a more potent or specific FXR
antagonist might have the potential to decrease LDL-C
levels.
On the other hand, in vitro studies performed in
human hepatocyte cell lines demonstrate that CDCA increases LDL-R gene expression and activity, a potential
beneficial effect in the prevention of atherosclerosis (38,
210, 315). This CDCA-mediated induction of LDL-R may
involve a mitogen-activated protein (MAP) kinase-mediated stabilization of LDL-R mRNA (210). More recently,
CDCA, as well as GW4064, have been shown to potentiate
LDL-R activity in response to statin treatment in human
hepatocytes. It has been proposed that FXR activation
leads to the repression of the Proprotein Convertase Subtilisin/Kexin type 9 (PCSK9), a natural inhibitor of
LDL-R, thereby enhancing LDL-R activity (167). These in
vitro studies suggest that FXR activation by CDCA or
synthetic agonists may be useful for lowering LDL-C levels in humans. It should be noted, however, that a 3-wk
treatment with CDCA reduced LDL-R mRNA levels in the
liver of subjects who underwent cholecystectomy for gallstone disease, while PCSK9 mRNA levels were not altered
(219). In humans, short-term (20 days) treatment with
CDCA did not alter LDL-C levels (341), whereas long-term
administration (2 yr) of CDCA in patients with gallstone
disease resulted in a 10% increase of LDL-C (269). The
reason for this discrepancy between in vitro and in vivo
studies remains unclear, but in vivo treatment with CDCA
may impact additional signaling pathways in the liver and
in the intestine. FXR also controls intestinal cholesterol
absorption since FXR⫺/⫺ mice have been reported to
exhibit increased dietary cholesterol absorption (166).
However, this may simply represent a secondary consequence of the enlarged BA pool size in these animals.
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C. FXR and Triglyceride Metabolism
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Already in the 1970s, several clinical studies highlighted a reciprocal relationship between BA and triglyceride metabolism. The first evidence came from the observation that treatment of dyslipidemic patients with
BA-sequestering resins, such as cholestyramine, resulted
in an increase in plasma triglyceride and very-low-density
lipoprotein (VLDL) levels, which are potentially undesirable effects (5, 17, 54). Furthermore, elevated plasma
triglyceride concentrations were found in patients exhibiting decreased BA synthesis linked to CYP7A1 deficiency
(250), and some patients with monogenic familial hypertriglyceridemia were found to have a defect in ileal BA
absorption (5). Conversely, CDCA used for the treatment
of patients with cholesterol gallstone disease decreased
plasma triglyceride levels and was therefore suggested as
a potential drug for the treatment of hypertriglyceridemia
(5, 13, 18). The ability of endogenous and synthetic FXR
agonists to act as hypotriglyceridemic agents has also
been evaluated in rodent models. CA lowers hepatic triglyceride accumulation, VLDL secretion, and elevated serum triglycerides in KK-A(y) mice, a mouse model of
hypertriglyceridemia (343). GW4064 treatment lowered
plasma triglyceride levels in rats (192), as well as in
chow-fed mice in an FXR-dependent manner (49, 186,
364). Furthermore, GW4064 treatment also improves hypertriglyceridemia in mouse models of insulin resistance
such as ob/ob and db/db mice (36, 364). Interestingly,
CDCA significantly reduces plasma triglycerides and
VLDL-cholesterol in fructose-fed hamsters, due to a reduction in the rate of VLDL production (21). Altogether,
these results suggest that FXR activation may improve
combined hyperlipidemia, a common feature of the metabolic syndrome in humans. Interestingly, FXR agonists
also reduced the elevated free fatty acid (FFA) levels in
insulin-resistant rodents, although the underlying mechanisms remain elusive (21, 364).
Both the production of triglyceride-rich VLDL particles and their clearance from the circulation have been
proposed as key processes regulated by several BAs.
Indeed, BAs inhibit production of VLDL by cultured human (178), rat (177), and mouse (77) hepatocytes in a
dose-dependent and BA species-dependent fashion. Some
of these in vitro effects of BAs may be FXR independent,
since taurocholic acid is equally effective in suppressing
VLDL production in hepatocytes isolated from wild-type
and FXR⫺/⫺ mice (77). Since treatment with UDCA,
which is unable to activate FXR, does not alter plasma
triglyceride levels in humans, FXR may act as a molecular
link between BA and triglyceride metabolism (41, 170). In
accordance with this hypothesis, FXR⫺/⫺ mice exhibit a
lipid profile characterized by increased serum triglyceride
levels, linked to an increased hepatic synthesis of apoBcontaining lipoproteins (mainly VLDL) (166, 288).
FXR activation impacts on triglyceride metabolism at
different levels (Fig. 5). FXR is involved in the control of
hepatic de novo lipogenesis, one source of fatty acids
used for the assembly of VLDL. FXR activation by BAs or
synthetic agonists represses the expression of the transcription factor SREBP-1c and its lipogenic target genes
in mouse primary hepatocytes and in liver (343, 366).
Based on studies in SHP-deficient (SHP⫺/⫺) mice, it has
been proposed that the induction of SHP mediates the
downregulation of SREBP-1c expression in response to
BA treatment (338, 343). Counterintuitively, decreased,
and not increased, SREBP-1c mRNA was observed in
ob/ob/SHP⫺/⫺ double mutants (119). More disturbing
was the observation that transgenic mice constitutively
expressing SHP in the liver exhibited an enhanced expression of SREBP-1c mRNA levels and a concomitant accumulation of hepatic triglycerides (28). Thus acute induction of SHP expression following FXR activation and
transgenic SHP overexpression have opposite effects
on triglyceride accumulation in the liver. Additional studies tend to call into question the central role of SREBP-1c
as a mediator of the hypotriglyceridemic effect of FXR.
First, in combined hyperlipidemic hamsters, CDCA treatment did not significantly decrease SREBP1-c mRNA levels, whereas triglyceride production was efficiently reduced (21). Second, hepatic SREBP-1c mRNA levels are
not increased, but reduced, in FXR⫺/⫺ mice compared
with controls (73, 366). However, it has not yet been
determined whether FXR alters SREBP-1c cleavage,
hence modulating the level of biologically active nuclear
SREBP-1c protein. Third, reduced SREBP-1c levels are
observed in livers of diabetic mice fed with the BA-binding resin colestimide (157). To complicate the issue further, a recent study demonstrated that FXR enhances the
transcription of fatty acid synthase (FAS), a key lipogenic
enzyme, through direct binding to an IR-1 site in the FAS
promoter (197). Such a FXR-mediated induction of FAS
could be a mechanism by which BAs may bypass SHP
inhibition on lipogenesis, thus maintaining an adequate
fatty acid pool for cholesterol esterification in specific
situations, for instance when cholesterol and BAs are
chronically elevated. Taken together, these data suggest
that FXR activation may control de novo lipogenesis
through both SREBP-1c-dependent and -independent
mechanisms. As detailed in section VIIB, FXR modulates
the kinetics of the response to dietary carbohydrate intake, since the maximal induction of glycolytic and lipogenic genes occurs earlier during the refeeding phase in
FXR⫺/⫺ than in wild-type mice. Lack of FXR therefore
leads to an enhanced glycolytic flux which provides substrates for lipogenesis (Fig. 6) (74). Triglycerides derived
from de novo lipogenesis efficiently mobilize apoB and
induce VLDL assembly (72, 92). Consistent with this observation, hepatic VLDL production is significantly increased upon refeeding FXR⫺/⫺ mice with a carbohy-
BILE ACIDS AND BILE ACID RECEPTORS IN METABOLIC REGULATION
173
α
drate-rich diet (73). It is of importance to note, however,
that this FXR-lipogenesis-VLDL hypothesis is mainly
based on gene expression data and that no actual measurements of lipogenesis rate have been reported so far.
Moreover, hepatic lipogenesis should not be considered
as the sole driving force for VLDL production by the liver.
Indeed, various animal models have been described in
which increased hepatic lipogenesis is associated with
liver steatosis without any effect on VLDL production (11,
345).
Several other mechanisms may contribute to the triglyceride-lowering effect of FXR activation (Fig. 5). In
human primary hepatocytes, FXR ligands induce the expression of PPAR␣ and of its target gene pyruvate dehydrogenase kinase-4 (PDK-4), which may promote fatty
acid oxidation (245, 265). CDCA treatment represses the
expression of microsomal triglyceride transfer protein
(MTP), which plays a critical role in the assembly and
secretion of VLDL (111). Notably, FXR also controls genes
governing triglyceride clearance. Indeed, FXR activation
increases apoC-II expression (146), an activator of lipoprotein lipase (LPL) activity and decreases both apoCPhysiol Rev • VOL
III (49) and ANGPTL3 (342), which are LPL inhibitors. In
addition, FXR induces the expression of the VLDL receptor in human and mouse liver, possibly enhancing the
clearance of triglyceride-rich lipoproteins (289). Finally,
the expression of syndecan-1, a transmembrane heparin
sulfate proteoglycan involved in the clearance of remnant
particles, is induced by FXR ligands in cultured human
liver cells (7). Altogether, these observations suggest that
FXR activation also promotes the clearance of circulating
triglycerides. Additional studies, including lipoprotein kinetic studies in humans, are necessary to delineate the
precise role of FXR activation on triglyceride metabolism.
VI. FARNESOID X RECEPTOR
AND GLUCOSE HOMEOSTASIS
A. FXR Expression in Diabetes
Several groups have demonstrated that the BA profile is
perturbed in diabetes (296). The BA pool is significantly
increased in several rat models of type 1 diabetes such as
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FIG. 5. Overview of FXR action on triglyceride metabolism. Endogenous (i.e., chenodeoxycholic acid) or synthetic (i.e., GW4064) ligands directly bind
and activate hepatic FXR, leading to a variety of responses modulating triglyceride metabolism. FXR inhibits hepatic lipogenesis both by interfering with
the promoters (ChOREs) of glucose-regulated genes and by decreasing the expression of SREBP-1c in a SHP-dependent manner. Conversely, FXR may
promote FFA catabolism by increasing PPAR expression. FXR also controls the assembly of VLDL by repressing the expression of MTP. FXR may increase
the clearance of triglycerides by stimulating LPL activity, as well as the hepatic uptake of remnants and LDL by inducing syndecan-1 and VLDL-R. Finally,
FXR may promote adipose TG storage by stimulating adipocyte differentiation (see the text for more details and references). *The proven FXR dependency
of gene regulation established on the basis of either one of the following criteria: 1) identification of a functional FXRE in the promoter or 2) loss of gene
regulation upon FXR deficiency using KO models or siRNA knockdown. CA, cholic acid; CDCA, chenodeoxycholic acid; SHP, small heterodimer partner;
SREBP-1c, sterol regulatory element binding protein-1c; ChORE, carbohydrate response element; GK, glucokinase; G6-P, glucose-6-phosphate; LPK,
L-pyruvate kinase; FAS, fatty acid synthase; ACC, acetyl CoA carboxylase-1; MTP, microsomal triglyceride transfer protein; LPL, lipoprotein lipase; VLDL,
very-low-density lipoprotein; IDL, intermediate-density lipoprotein; LDL, low-density lipoprotein; VLDL-R, VLDL-receptor; LDL-R, LDL-receptor; PCSK9,
pro-protein convertase subtilisin/kexin 9; FFA, free fatty acid.
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LEFEBVRE ET AL.
spontaneously diabetic Wistar rats (108) and streptozotocinor alloxan-treated rats (216). Changes in the BA pool size
and hepatobiliary BA secretion were directly dependent
on the insulin-deficient state and were reversed with insulin therapy (217, 330). Moreover, liver-specific insulin
receptor knockout mice (LIRKO) exhibited enlarged gallbladders with increased bile volume, suggestive for, but
not proving, the existence of an enlarged BA pool (201).
Altogether, these results suggest that the negative-feedback mechanism regulating BA synthesis is defective in
type 1 diabetes. More recent studies indicated that FXR
might be one of the molecular links between altered BA
metabolism and diabetic states. Indeed, hepatic FXR gene
expression is decreased in both streptozotocin-treated
rats and Zucker diabetic fatty rats, a model of type 2
diabetes (74). Accordingly, liver CYP7A1 mRNA levels are
increased in such diabetic animals, likely contributing to
the enlarged BA pool. Insulin therapy restores FXR as
well as CYP7A1 gene expression in streptozotocin-treated
rats (74). Moreover, physiological concentrations of insulin directly reduce BA synthesis in vitro in rat hepatocytes
through suppression of CYP7A1 expression (321). However, hepatic FXR mRNA levels are increased in diabetic
db/db mice (364). The reason for this discrepancy between diabetic rat and mouse models remains unclear.
However, these latter results in db/db mice are consistent
with in vitro data, since FXR gene expression is stimulated by glucose and repressed by insulin in vitro in
primary rat hepatocytes (74). FXR appears to be regulated
by glucose via the pentose phosphate pathway because
the addition of xylitol, a precursor of xylulose-5-phosphate, increases FXR expression to a comparable level to
that induced by D-glucose (74). In addition, glucose increases nuclear FXR DNA-binding activity and subsePhysiol Rev • VOL
quently FXR transcriptional activity on target genes such
as SHP and apo-CIII (74).
In a more physiological setting, FXR mRNA levels
vary with the nutritional status. While fasting specifically
increases hepatic FXR␣ 3/4 expression, a high-carbohydrate refeeding subsequently reduces FXR mRNA levels
(73, 366). This fasting-induced FXR expression appears to
be sustained by cAMP and PGC-1␣, both of which enhance FXR gene transcription (366). Furthermore, hepatic expression of FXR␤, but not of FXR␣, displays a
clear diurnal rhythm, like the majority of other nuclear
receptors involved in metabolic homeostasis. White adipose FXR␣ and FXR␤ expression is also subjected to
circadian variation (352). Therefore, FXR expression appears to be tightly regulated by the metabolic status,
indirectly supporting a role for this nuclear receptor in
metabolic diseases.
B. FXR and Hepatic Glucose Metabolism
In the fasted state, the flux of newly synthesized
glucose is sustained by the activation of gluconeogenesis.
To date, the precise role of FXR in the regulation of
hepatic glucose metabolism remains a controversial issue. A first observation in this field was the observation
that FXR activation induces phosphoenolpyruvate carboxykinase (PEPCK) expression (35, 300). For a long
time, PEPCK has been considered to be the rate-controlling enzyme of gluconeogenesis. However, in vivo studies
clearly demonstrate that mice with a 95% reduction in
PEPCK mRNA have a nearly normal blood glucose concentration after a 24-h fast (279, 280). Other rate-controlling steps in gluconeogenesis are catalyzed by glucose-6phosphatase (G6Pase) and fructose 1,6-bisphosphatase
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FIG. 6. FXR controls the adaptive response of the liver during the fasting-refeeding transition. After a meal, BAs, stored into
the gallbladder during the interprandial period, are discharged into the intestine to stimulate absorption of lipids that will form chylomicrons in the lymph. Carbohydrates, like
glucose, are absorbed and reach the liver
through the portal vein. BAs enter the enterohepatic circulation and return to hepatocytes
where they activate FXR. Hepatic FXR inhibits BA synthesis by a feedback mechanism
involving SHP-mediated CYP7A1 repression.
Additionally, ileal FXR induces FGF19 secretion, which in turn represses CYP7A1
through FGFR4-mediated signaling. Activation of FXR interferes with glucose metabolism by inhibiting glycolysis, via inhibition of
L-PK expression, therefore increasing glycogen storage. FXR activation also represses de
novo lipogenesis via inhibition of lipogenic
enzymes as ACC-1, FAS, and S14 leading to a
reduced hepatic VLDL output.
BILE ACIDS AND BILE ACID RECEPTORS IN METABOLIC REGULATION
(FBP1). Gluconeogenic enzymes are regulated at the transcriptional level by hormones controlling glucose homeostasis. Glucagon and glucocorticoids, which have
strong gluconeogenic actions, induce PEPCK expression,
whereas insulin, which suppresses hepatic gluconeogenesis, represses PEPCK expression (106). Since type 2
diabetes is characterized by an increased hepatic glucose
output, which contributes to fasting hyperglycemia, pharmacological modulation of gluconeogenic gene expression appears an attractive possibility for type 2 diabetes
treatment (188).
1. In vitro studies
Physiol Rev • VOL
FXR activation participates in the induction of gluconeogenesis. In agreement with this hypothesis, FXR activation
by CDCA, GW4064, or fexaramine leads to a dose-dependent increase of PEPCK mRNA levels in rat hepatoma cell
lines, as well as in primary rat hepatocytes (300). Of
potential functional relevance, FXR activation also increases glucose output by primary rat hepatocytes in vitro
(300). Results are more discordant in primary mouse
hepatocytes: GW4064 either induces (364) or has no
effect on PEPCK gene expression (36). However, basal
(i.e., nonpharmacologically stimulated) PEPCK mRNA
levels are decreased by 60% in hepatocytes isolated
from FXR⫺/⫺ mice compared with controls, suggesting
that FXR expression is required for proper PEPCK
regulation (36).
2. In vivo studies
In accordance with a putative repressive effect of
FXR on gluconeogenic genes expression, feeding
C57BL/6J mice with 1% CA-supplemented diet during 7– 8
days decreases hepatic PEPCK, G6Pase, and FBP1 mRNA
levels (60, 349). Furthermore, feeding a CA-enriched diet
has been shown to decrease PEPCK and G6Pase mRNA
levels in wild-type but not FXR⫺/⫺ or SHP⫺/⫺ mice. This
decrease was associated with decreased fasting blood
glucose only in wild-type mice, reinforcing the hypothesis
that the FXR-SHP negative regulatory cascade represses
gluconeogenesis (186). However, these results remain difficult to reconcile with those obtained in SHP⫺/⫺ mice.
Both PEPCK and G6Pase basal expression are indeed
reduced in the liver of SHP⫺/⫺ mice. This expression
profile is well correlated with a reduced in vivo glucose
output from livers of SHP⫺/⫺ mice (337). The reason for
this apparent discrepancy remains unclear. In contrast to
the results with CA feeding, in vivo treatment with the
FXR agonist GW4064 has been shown to induce PEPCK
mRNA levels in a FXR-dependent manner. However, this
regulation was also associated with a decrease in plasma
glucose levels (300, 364). Taken together, these results
suggest that BAs may impact additional FXR-independent
signaling pathways involved in glucose metabolism that
make the integrated response in vivo more complex. To
complicate the issue further, the regulation of gluconeogenic enzymes expression by FXR seems to be modulated
by the diabetic state. In contrast to what was observed in
C57Bl/6J mice, Zhang et al. (364) found that oral GW4064
treatment (30 mg⫺1 䡠kg⫺1 䡠day⫺1) reduced PEPCK and
G6Pase expression, and improved hyperglycemia in db/db
diabetic mice. Similar results were obtained following
adenoviral-mediated hepatic overexpression of constitutively active FXR (364). In contrast, another study performed in female ZDF rats showed that oral GW4064
administration increased PEPCK mRNA levels in a dosedependent manner, whereas high doses of GW4064 (30
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Numerous in vitro studies have focused on the transcriptional regulation of gluconeogenic enzymes by BAs
and led to conflicting results. On the one hand, CDCA
treatment of human hepatoma cell lines has been shown
to decrease PEPCK, as well as G6Pase and FBP1 expression (60, 349). It is still unclear whether these pathways
are regulated in a FXR-dependent manner. As on the
CYP7A1 promoter, CDCA decreases the activity of hepatocyte nuclear factor 4-␣ (HNF-4␣), a positive regulator of
PEPCK gene expression (60). Chromatin immunoprecipitation experiments demonstrated that CDCA impairs the
recruitment of PGC-1 and CBP [cAMP response elementbinding protein (CREB) binding protein] by HNF-4 to the
PEPCK promoter (60). Importantly, treatment with the
nonsteroidal FXR agonist GW4064 did not modify HNF-4
activity, suggesting an FXR-independent mechanism (60).
Other data suggest that FXR may decrease gluconeogenic
enzyme expression via induction of SHP. Notably, SHP
has been shown to prevent CBP recruitment by HNF-4␣
on the PEPCK and FBP1 promoters, and by Foxo1 on the
G6Pase promoter (349). However, the FXR dependency
of this pathway remains to be established. In keeping with
a potential role for SHP in gluconeogenesis, transfected
SHP inhibits PEPCK promoter transactivation by the glucocorticoid receptor (26), HNF-4␣ (26), HNF-3 (152), and
C/EBP (231). More recently, it has also been suggested
that CDCA represses PGC-1 promoter activity in a SHPdependent manner (348). Unexpectedly, basal glucose
production was found to be significantly lower in SHP⫺/⫺
primary hepatocytes (337), raising doubts about the
proposed role and mechanisms of SHP in gluconeogenesis.
On the other hand, a role for FXR in the induction of
gluconeogenic gene expression can be inferred from several observations. Fasting markedly induces hepatic expression of PGC-1, which subsequently stimulates the
entire program of genes involved in hepatic gluconeogenesis by acting as a coactivator for GR and HNF-4␣ (248).
PGC-1 coactivates FXR-target gene promoter activity in
vitro, either in a ligand-independent (141) or in a liganddependent manner (266, 366). Thus it is plausible that
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sion, which may contribute to a redirection of the glucose
flux to glycolysis in the interprandial state (Fig. 6).
An additional argument for a link between BA and
glucose metabolism arises from the observation that BA
sequestrants reduce blood glucose levels in animal and
human studies (reviewed in Ref. 296). Recently, colestimide treatment has been shown to prevent weight gain,
insulin resistance, and diabetes in a rodent model of
diet-induced obesity (157). In humans, a slight, but significant, improvement of glycemic control was initially reported upon cholestyramine treatment (90). Similar results were subsequently reported for colesevelam (369)
and colestimide (350). While the precise mechanisms sustaining this hypoglycemic effect remain unclear, it has
been suggested that BA sequestrants may interfere with
the enteroinsular axis. In a small open-labeled study of
patients with type 2 diabetes, a 1-wk treatment with colestimide increased the 2-h postprandial plasma glucagonlike peptide(GLP)-1 level. In parallel with this stimulation
of GLP-1 secretion, both 1-h and 2-h postprandial plasma
glucose levels were significantly decreased in the same
study (311). In addition, in vitro studies demonstrated that
BAs promote GLP-1 secretion through the activation of
TGR5 in the murine enteroendocrine STC-1 cell line (143).
C. FXR and Insulin Sensitivity
Three independent studies identified a role for FXR
in regulating insulin sensitivity (37, 186, 364). FXR deficiency leads to impaired glucose tolerance and insulin
resistance. Hyperinsulinemic-euglycemic clamp studies
confirmed that FXR⫺/⫺ mice display a peripheral insulin
resistance reflected by a reduced peripheral glucose disposal (37, 186). Consistent with these observations, insulin signaling is impaired in peripheral insulin-sensitive
tissues such as skeletal muscle and white adipose tissue
(37, 186). Discordant data concerning the level of hepatic
insulin sensitivity in FXR⫺/⫺ mice are reported in the
literature. Some studies found a reduced inhibition of
hepatic glucose output during low-dose insulin clamp
(186) and an impaired hepatic insulin signaling in FXR⫺/⫺
mice (186, 364). In contrast, FXR deficiency was also
shown to be associated with normal hepatic insulin sensitivity and signaling (37, 73). The reason for this discrepancy is unclear, but may be linked to different genetic
backgrounds [C57Bl6/J (186, 364) vs. C75Bl6/N (37, 73)] of
the mice and/or in the insulin dose used during the clamp.
Based on these results, a prediction would be that FXR
activation promotes insulin sensitivity. In accordance
with this hypothesis, treatment with GW4064 improved
insulin sensitivity in both db/db, KK-A(y) (364), and ob/ob
(37) mice. Similar results were obtained following adenoviral-mediated overexpression of constitutively active
FXR in the liver of db/db mice (364).
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mg⫺1 䡠kg⫺1 䡠day⫺1) decreased G6Pase mRNA levels. Unfortunately, blood glucose levels were not reported in this
work (118).
A complementary approach to assess the role of FXR
in hepatic glucose metabolism is based on the phenotypic
analysis of the FXR⫺/⫺ mice. Metabolic adaptation to
prolonged fasting was investigated in this mouse model.
While the long-term fasting adaptation is not altered by
FXR deficiency, the kinetics of metabolic changes during
short-term fasting are altered, leading to an accelerated
and transient drop in glycemia (36). In line with this
observation, the short-term fasting-associated induction
of PEPCK (36, 186) and of PGC-1 (186) mRNA levels is
blunted in FXR⫺/⫺ mice. This fasting-hypoglycemia may
also be linked to an impaired glycogenolysis since hepatic
glycogen content is significantly reduced in FXR⫺/⫺ mice
(36). Conversely, the FXR agonist GW4064 increases hepatic glycogen synthesis and storage in vitro in primary
hepatocytes, as well as in vivo in db/db mice (364). In
accordance with a role for FXR in the control of the
kinetics of glucose metabolism, FXR⫺/⫺ mice subjected to
an overnight fasting followed by refeeding with a highcarbohydrate diet exhibit an accelerated induction of glycolytic and lipogenic genes compared with wild-type mice
(73). The accelerated response of FXR⫺/⫺ mice to highcarbohydrate refeeding seems to be due to a potentiation
of carbohydrate-induced signaling. FXR activation by
GW4064 attenuated glucose-induced mRNA expression as
well as promoter activity of several glucose-regulated
genes, such as L-pyruvate kinase and acetylCoA carboxylase 1, in rodent primary hepatocytes (73). Additional
studies are needed to determine whether FXR can interfere with the transcriptional activity of the carbohydrate
response element-binding protein (ChREBP), a major mediator of glucose action (247).
Taken together, these data provide evidence for a
critical role of BAs in the dynamic regulation of glucose
metabolism in the liver. As underlined above, FXR acts
through a complex network to drive the expression of
gluconeogenic genes. The exact underlying molecular
mechanisms, as well as the relative contribution of FXRdependent and FXR-independent pathways, remain to be
determined. One might suggest, however, that FXR acts
mainly as a metabolic sensor to direct metabolic fluxes
during the fasting-refeeding transition. The enhanced expression of Cyp7A1 during fasting would increase the BA
pool, allowing improved digestion and absorption of fats
after a meal. Concomitantly, FXR expression is induced,
but its transcriptional activity is likely decreased due to
the absence of endogenous ligands during fasting caused
by impaired enterohepatic cycling. Upon meal ingestion,
the increased enterohepatic circulation of BAs activates
FXR, leading to inhibition of de novo lipogenesis and
glycolysis, to promote glycogen storage. Then, the rise of
plasma insulin levels upon refeeding reduces FXR expres-
BILE ACIDS AND BILE ACID RECEPTORS IN METABOLIC REGULATION
Physiol Rev • VOL
contribute to the decreased hepatic triglyceride content and improved insulin sensitivity in response to
increased FGF19 levels (84, 316). More recently, FGF19
has been shown to increase glucose uptake in 3T3-L1
adipocytes in vitro (164). Activation of FGF19 signaling
in response to injected FG19 is less prominent in white
adipose tissue than in liver, suggesting that this potential extrahepatic activity of FGF19 is dependent on
supraphysiological circulating levels of FGF19, as observed in transgenic mice (164). On the basis of these
results, it can be suggested that part of the FXR-mediated insulin-sensitizing effect may involve FGF19 signaling pathways. Thus study of tissue-specific FXR
knockout mice is essential to unravel the respective
contribution of hepatic, intestinal, and adipose FXR in
the control of insulin sensitivity.
D. FXR and Energy Expenditure
Adaptive thermogenesis is defined as heat production in response to variations in environmental temperature or diet. Hence, it protects the organism from cold
exposure and regulates energy balance after dietary
changes (183). In rodents, brown adipose tissue (BAT) is
the major site of thermogenesis.
FXR mRNA expression is not detected in mouse BAT
(34, 342), excluding a direct action of FXR on adaptive
thermogenesis. In contrast, SHP, a direct FXR target gene,
appears to be a negative regulator of thermogenesis in
BAT by inhibiting PGC-1 expression. Accordingly, SHP⫺/⫺
mice show increased energy expenditure, and resistance
to diet-induced obesity (339). However, two additional
independent studies failed to detect SHP mRNA in
mouse BAT (34, 342), raising doubts about the physiological role of SHP in this tissue. Interestingly, SHP
mutations have been associated with low birth weight,
insulin resistance, and obesity in a Japanese cohort of
young children (220). However, these findings were not
confirmed in two subsequent studies on Caucasian subjects performed in the United Kingdom (127, 202). FXR
deficiency does not alter the metabolic rate measured
under basal unstressed conditions, despite elevated
plasma BA concentrations (34). In contrast, FXR appears to be involved in the regulation of adaptive thermogenesis since FXR⫺/⫺ mice display an accelerated
entry into torpor upon fasting and cold intolerance.
Both these altered responses may be linked to the
lipoatrophic phenotype of FXR⫺/⫺ mice, with reduced
glycogen and triglyceride stores in liver and adipose
tissue, respectively (34). Under these dynamic conditions, FXR contributes to the control of metabolic fuel
stores that are essential to sustain heat production by
BAT. These results identify a role for FXR as a modulator of energy homeostasis.
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The molecular mechanisms behind the insulin-sensitizing effect of FXR remain ill-defined. Since FXR is not
expressed in skeletal muscle, it is conceivable that FXR
deficiency alters indirectly insulin signaling in this tissue.
A hypothesis is that FXR deficiency promotes ectopic
lipid deposition in insulin target tissues, a phenomenon
usually referred to as “lipotoxicity” (264). Indeed, FXR⫺/⫺
mice have elevated circulating FFA levels (37, 186) and
increased intramuscular triglyceride and FFA contents
(186). Furthermore, the level of insulin receptor substrate-1 (IRS-1) serine-307 phosphorylation, which is
linked to fatty acid-induced insulin resistance (356), is
increased in skeletal muscle of FXR⫺/⫺ mice (186). A
similar mechanism could also operate in liver, since hepatic triglyceride content is increased in FXR⫺/⫺ mice (73,
186). Conversely, GW4064 treatment reduces neutral lipid
accumulation in the liver of db/db mice (364). It should be
underlined that the contribution of the FXR-SHP cascade
in the insulin-sensitizing effect of FXR ligands seems unlikely, since SHP⫺/⫺ mice show increased whole body
insulin sensitivity in wild-type and ob/ob mice (119, 337).
Recent data suggest that FXR plays a role in adipocyte
differentiation and function. FXR expression increases
progressively during adipocyte differentiation in vitro,
both in 3T3-L1 cells and mouse embryonic fibroblasts
(MEFs) cells (37, 258). With the use of MEFs as a model
system, it has been shown that FXR deficiency leads to an
impaired adipogenic program with a defective triglyceride
accumulation (37). Conversely, the synthetic FXR ligand
6␣-ECDCA/INT-747 promotes adipocyte differentiation
and lipid storage in 3T3-L1 adipocytes (258). Consistent
with these in vitro data, FXR⫺/⫺ mice exhibit a moderate
lipoatrophic phenotype that may contribute to their impaired insulin sensitivity. Moreover, GW4064 treatment
improves insulin signaling and insulin-induced glucose
uptake in 3T3-L1 differentiated adipocytes (37, 258). However, several questions remain to be answered to better
understand the role of FXR in white adipose tissue. What
is the molecular nature of the endogenous FXR ligands in
the adipocyte? What are the FXR target genes in this
tissue?
Recently, fibroblast growth factor 19 (FGF19, or
the mouse ortholog FGF15), a member of the FGF
family of proteins, has emerged as a new regulator of
metabolic homeostasis (307). FGF19 expression is induced in a FXR-dependent manner in intestinal epithelial cells, in response to BA released into the intestinal
lumen upon feeding. Then, FGF19 circulates through
the portal vein to act on hepatocytes to reduce BA
synthesis (114, 129). Interestingly, resistance to both
diet-induced obesity and insulin desensitization were
observed in transgenic mice overexpressing human
FGF19 (316). A direct inhibition of hepatic acetyl CoA
carboxylase 2 and stearoyl-coenzyme A desaturase-1,
and a subsequent increase in fatty acid oxidation, may
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LEFEBVRE ET AL.
inflammation, neointimal proliferation, and blood pressure levels. However, additional studies, especially in
FXR⫺/⫺ mice, are needed to further characterize the effect of FXR activation in an intact organism and to assess
whether these signaling pathways are FXR dependent.
VII. FARNESOID X RECEPTOR
AND ATHEROSCLEROSIS
A. A Role for FXR in the Vessel Wall?
Recently, several studies have focused on a potential
new role for FXR in the vasculature. In vitro studies
demonstrated that FXR is expressed in both vascular
smooth muscle cells (VSMCs; Refs. 22, 363) and endothelial cells (110, 253). In contrast, FXR is not expressed in
macrophages, such as differentiated THP-1 macrophagelike cells (241) and peritoneal macrophages (98).
As detailed in section IIIB, FXR is expressed in the
vessel wall, in VSMCs from the coronary artery and aorta,
as well as within atherosclerotic lesions and human primary saphenous vein smooth muscle cells (22, 174, 363),
although expression is lower than in rat liver or HepG2
human hepatoma cells. Activation of FXR with the synthetic FXR ligands 6ECDCA (237) and GW4064 (192) induces expression of the FXR target genes SHP and PLTP
in rat smooth muscle cells (RASMCs) (174, 363). This
effect was also observed in response to CDCA treatment
in one study (363) but not in another (22). This discrepancy casts doubt on the identity of endogenous FXR
ligands outside the enterohepatic circulation. From a
functional point of view, FXR activation has been shown
to induce RASMC apoptosis. Interestingly, FXR activators
synergize with the RXR ligand 9-cis-retinoic acid to induce cell death (22). In addition, 6ECDCA and GW4064
inhibit VSMCs inflammatory responses and migration.
Synthetic FXR ligands inhibit interleukin-1␤-induced expression of inducible nitric oxide synthase (iNOS) and of
cyclooxygenase-2 in RASMCs, by reducing NF-␬B activation in a SHP-dependent manner (174). FXR ligands also
reduce the migration of both RASMCs and human aortic
vascular smooth muscle cells induced by platelet-derived
growth factor (PDGF)-␤ (174). Moreover, FXR directly
enhances the transcription of the angiotensin type 2
receptor (AT2R) in RASMCs by binding to an IR2 FXRE in
the AT2R promoter. The upregulation of AT2R results in
an inhibition of angiotensin II-mediated ERK signaling
in RASMCs (363). Of clinical relevance, overexpression of
AT2R and inactivation of the ERK signaling pathway has
been suggested to prevent neointimal proliferation (212),
to partly mediate the hypotensive effects of AT1R blockers (267), and to mediate the cardioprotective effect of
several PPAR␥ ligands (204). While FXR activation does
not alter expression of the AT1R in RASMCs, it should be
kept in mind that CDCA represses, in hepatocytes, angiotensinogen expression in a SHP-dependent manner (286).
Altogether, these in vitro results suggest that FXR activation in VSMCs may have a beneficial effect on vascular
Physiol Rev • VOL
A low expression level (⬃1,000-fold less than in the
liver) of FXR was detected in several models of endothelial
cells such as rat pulmonary artery endothelial cells (RPAEC)
(110), human (HAEC) (253), and bovine aortic endothelial
cells (BAEC) (173), as well as primary human umbilical vein
endothelial cells (HUVEC) (253). Like in VSMCs, activation
of FXR by CDCA leads to SHP induction in RPAEC (110).
Interestingly, FXR is positively autoregulated in RPAEC, since
CDCA treatment strongly increases FXR expression (110). The
endothelium plays a crucial role in regulating the vascular tone.
Imbalance between the vasodilating agent NO and the vasoconstrictor endothelin-1 (ET-1) contributes to endothelial dysfunction, especially in insulin resistance and diabetes (254).
The FXR ligands CDCA and GW4064 repress the basal and
lipopolysaccharide-stimulated ET-1 expression and secretion.
FXR was reported to modulate ET-1 expression via inhibition
of the activator protein AP-1 (110). Therefore, it can be hypothesized that FXR may have anti-inflammatory actions through a
transrepression mechanism, similar to that observed with peroxisome proliferator-activated receptors (PPARs) (256). FXR
also directly enhances transcriptional activation of the endothelial nitric oxide synthase (eNOS) gene promoter, leading to
increased NO production in vascular endothelial cells (173).
Thus FXR may positively influence the vascular tone. In accordance with these data, arterial hypotension and reduced pressor effects of vasoconstrictors have been described in patients
with severe cirrhosis, in which circulating BAs levels are significantly increased (187). In this context, it should also be
noted that the G protein-coupled BA receptor TGR5 is expressed in liver sinusoidal endothelial cells, and that BAs induce the expression and the activation of eNOS in liver (148).
Therefore, additional studies are needed to unravel the respective contribution of the BA receptors FXR and TGR5 in the
BA-mediated induction of eNOS. Interestingly, FXR also modulates NO levels by directly enhancing the transcription of
dimethylarginine dimethylaminohydrolase-1 (DDAH1) in the
liver (118). DDAH1 is a key catabolic enzyme of asymmetric
dimethylarginine (ADMA), a major endogenous NOS inhibitor.
FXR agonist GW4064 treatment in ZDF rats dose-dependently
decreases serum ADMA levels. Collectively, these results suggest that FXR may be a regulator of NO metabolism.
Bile duct ligation in rats, leading to abnormally high
plasma BA levels, promotes severe endothelial injury
(233). In accordance with this deleterious effect of elevated circulating BAs levels, PFIC patients display endothelial dysfunction with increased wall stiffness and
intimal-medial thickness (209). CDCA, DCA, LCA, but
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1. VSMCs
2. Endothelial cells
BILE ACIDS AND BILE ACID RECEPTORS IN METABOLIC REGULATION
3. Liver-mediated vascular effects
FXR can also impact on vascular functions via its
actions in the liver. Consequences of FXR activation on
lipoprotein metabolism are reviewed above (see sect. VI).
Atherosclerosis is a chronic inflammatory disease.
Excessive inflammation within the arterial wall is a risk
factor for cardiovascular disease and can promote atherogenesis (107). High concentrations of hydrophobic BAs
induce the expression of a number of inflammatory genes
in the liver, which may contribute to atherogenesis in
mice fed an atherogenic diet supplemented with 0.5%
cholate (176, 252, 333). In addition to a direct cytotoxic
effect, this inflammatory response is also mediated
through activation of several protein kinases including
PKC (252, 309), ERK (251), and c-Jun NH2-terminal kinase
(JNK) (99). Interestingly, hepatic FXR appears to be
downregulated during the acute phase response in rodents, similar to other nuclear receptors such as PPAR␣
and LXR (79, 153). This suggests that FXR may modulate
the expression of genes participating in the inflammatory
response. Notably, treatment of mice with CA induces the
expression of ICAM-1, VCAM-1, serum amyloid A2, and
TNF-␣. Moreover, in vitro experiments in human hepatocytes demonstrate that FXR increases the transcriptional
activity of the human ICAM-1 promoter (252). Based on
these results, FXR activation in liver could be associated
with a deleterious proinflammatory profile.
Recent data indicate that FXR activation may alter
anticoagulation processes. The first indication came with
the demonstration of a FXR-mediated upregulation of
kininogen expression in human hepatocytes (368).
Human kininogen is the precursor for the two-chain kininfree kininogen, which exerts antiadhesion, anti-inflammation, and antiplatelet aggregation activities, and for bradykinin which is a potent vasodilatator and a mediator of
inflammation (193). Thus the increased production of human kininogen may lead to antiadhesion and antithrombosis. In contrast, another study found that FXR induces
human fibrinogen expression in an isoform- and speciesPhysiol Rev • VOL
specific manner (6). Fibrinogen is a key component of the
coagulation pathway, and it has been suggested to be a
cardiovascular risk factor in a prospective cohort study
(268). Therefore, additional in vivo studies are needed to
assess the physiological consequences of FXR activation
on coagulation processes.
Finally, concordant data have demonstrated that
FXR is involved in the BA-mediated repression of paraoxonase-1 (PON1) (101, 285). PON1 is an enzyme that
circulates in plasma associated with HDL. By preventing
LDL oxidation, PON1 exerts anti-inflammatory effects and
appears to be atheroprotective in both humans and mice
(284). A CA-containing atherogenic diet reduces the expression of PON1 in C57Bl/6J mice (283). Studies with
FXR⫺/⫺ mice and FXR agonists demonstrate that BAmediated repression of PON1 expression is mediated by
FXR. As previously reported for CYP7A1 (129), FXRmediated repression of PON1 involves the induction of
FGF-19, its subsequent binding to FGFR4, and activation
of the JNK pathway (101, 285). Thus FXR activation in the
intestine may promote atherosclerosis risk by repressing
both hepatic CYP7A1 and PON1 through the ileal induction of FGF19.
B. In Vivo Role of FXR in Mouse Models
of Atherosclerosis
Since in vitro studies have led to conflicting conclusions concerning the potential role of FXR in the pathogenesis of atherosclerosis (Fig. 7), evaluation of the overall
consequence of FXR deficiency in a whole body organism
is mandatory to fully appreciate its role in cardiovascular
diseases. Unfortunately, the reported data are controversial, and discrepancies might stem from the distinct atherosclerosis mouse models, gender, or the diet used in
each study (98, 105, 365). On the basis of the observation
that FXR⫺/⫺ mice exhibit a proatherogenic lipid profile
(288), accelerated atherosclerosis would be anticipated in
these mice. However, FXR⫺/⫺ mice did not display enhanced atherosclerosis susceptibility even when fed a
high-fat/high-cholesterol diet (105, 365). Therefore, backcrosses with genetic mouse models of accelerated atherosclerosis were used to assess the consequence of FXR
deficiency on atherosclerosis progression in vivo. Hanniman et al. (105) found that male FXR⫺/⫺ mice bred on an
ApoE-deficient genetic background (FXR⫺/⫺/ApoE⫺/⫺)
fed an atherogenic diet exhibited more severe atherosclerosis and a significantly reduced survival rate. In striking
contrast, Guo et al. (98) reported fewer atherosclerotic
lesions in female FXR⫺/⫺/ApoE⫺/⫺ mice, and male
FXR⫺/⫺ mice crossed with LDL-R-deficient mice (FXR⫺/⫺/
LDL-R⫺/⫺) also displayed less pronounced atherosclerotic lesions than LDL-R⫺/⫺ mice (365). The atheroprotective effect of FXR deficiency in both male FXR⫺/⫺/
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not CA, induce the expression of intracellular adhesion
molecule-1 (ICAM-1), vascular cellular adhesion molecule-1 (VCAM-1), and E-selectin in a dose-dependent
manner in HUVEC. In contrast, GW4064 only increases
adhesion molecule expression at high concentrations
(5 ␮M) and to a lesser extent than CDCA, suggesting
that BAs operate in a FXR-independent manner. This
effect seems to be mediated through the elevation of
reactive oxygen species and the subsequent stimulation
of the NF-␬B and p38 MAP kinase signaling pathways.
Of functional relevance, this induction of adhesion molecules promotes adhesion of monocytes to endothelial
cells, a crucial strep in the initiation of the atherosclerosis process (253).
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LEFEBVRE ET AL.
LDL-R⫺/⫺ and female FXR⫺/⫺/ApoE⫺/⫺ mice may be
linked to a change in lipoprotein composition with a
decrease of LDL-C, paralleled by an increase of VLDL-C
concentrations (98, 365). Such a LDL-C decrease was not
observed in female FXR⫺/⫺/LDL-R⫺/⫺ mice, which, accordingly, did not display reduced atherosclerotic lesions
(365). More disturbing is the observation that LDL-C concentrations are increased in male FXR⫺/⫺/ApoE⫺/⫺ (105).
The reason(s) for this discrepancy is currently unclear.
Interestingly, FXR deficiency was found to be associated
with reduced foam-cell formation in macrophages isolated from both female FXR/⫺ and male FXR⫺/⫺/LDLR⫺/⫺ mice (98). While the cholesterol efflux rate is similar
between both genotypes, oxidized LDL-C uptake is reduced in peritoneal macrophages isolated from FXR⫺/⫺
mice (98, 365). Accordingly, the expression of CD36, a
fatty acid transporter that participates in oxidized LDL-C
uptake in macrophages, is significantly decreased in macrophages from FXR⫺/⫺ mice (98, 365). Moreover, ADRP
adipose differentiation-related protein (ADRP) mRNA
levels, which are crucial for the formation of lipid
droplets in macrophages, are reduced in FXR⫺/⫺ mice
Physiol Rev • VOL
(365). Because FXR is not expressed in macrophages,
the observed changes in CD36 and ADRP mRNA expression must stem from an indirect mechanism. FXR
deficiency does not alter the expression of the nuclear
receptors PPAR␥ and LXR␣ in macrophages, although
it cannot be excluded that FXR-mediated changes in
lipid metabolism and inflammation may modulate their
transcriptional activity by altering the levels of their
endogenous ligands.
In conclusion, although it appears that FXR modulates both systemic and vascular parameters of atherosclerosis, its exact effect on the atherogenic process is far
from clear. Hence, it is currently impossible to predict
vascular consequences of in vivo FXR modulation with
specific synthetic agonists or antagonists. It would be of
great interest to assess this question in an atherosclerosisprone animal model, such as the rabbit or the guinea pig.
Moreover, the use of tissue-specific FXR knockout mice
would be of considerable interest to determine the relative contribution of systemic lipidic and/or inflammatory
indirect effects versus a direct impact of FXR in the
vasculature.
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FIG. 7. Effects of FXR potentially affecting the pathogenesis of atherosclerosis. FXR is expressed in both endothelial and vascular smooth
muscle cells. FXR activation may impact on atherogenesis directly in the vessel wall or in an indirect manner through liver-mediated effects. At the
present time, it is still unclear whether FXR activation is beneficial or deleterious in atherosclerosis (see the text for more details and references).
AII, angiotensin II; AT2R, angiotensin II type 2 receptor; NO, nitric oxide; iNOS, inducible nitric oxide synthase; eNOS, endothelial nitric oxide
synthase; COX-2, cyclooxygenase-2; ICAM-1, intracellular adhesion molecule-1; VCAM-1, vascular cellular adhesion molecule-1; ET-1, endothelin-1;
DDAH1, dimethylarginine dimethylaminohydrolase-1; ADMA, asymmetric dimethylarginine; VSMC, vascular smooth muscle cell; PON-1, paraoxonase-1; SAA, serum amyloid-A; SHP, small heterodimer partner.
BILE ACIDS AND BILE ACID RECEPTORS IN METABOLIC REGULATION
VIII. OTHER PATHOPHYSIOLOGICAL ASPECTS
OF FARNESOID X RECEPTOR BIOLOGY
Physiol Rev • VOL
ACKNOWLEDGMENTS
Address for reprint requests and other correspondence: B.
Staels, Département d’Athéroslérose, UMR545 INSERM, Université de Lille 2, Institut Pasteur de Lille, 1 rue Calmette BP245,
59019 Lille cedex, France (e-mail: [email protected]).
GRANTS
This work was supported by grants from Institut National
de la Santé et de la Recherche Médicale, the Région Nord-Pas de
Calais/FEDER, “Agence Nationale pour la Recherche” (A05056GS,
PPV06217NSA, and ANR-06-PHYSIO-027-01, Project R06510NS),
ALFEDIAM and the EU Grant Hepadip (018734).
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As it could be predicted from its expression pattern, FXR regulates not only metabolism, but also processes critical for the functioning of several organs and
protection of the organism. Elevated BAs stimulate
liver growth after partial hepatectomy, a phenomenon
which is FXR dependent (121). FXR activation contributes to cell cycle entry of hepatocytes by inducing the
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although they appear somewhat contradictory. The proliferation of breast cancer MCF7 cells is induced by
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ER-positive (MCF7) and ER-negative (MDA-MB-468)
cell lines exhibited an increased apoptosis in response
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ER-negative MDA-MB-231 cells (282). Consistent with a
protective role for FXR, FXR activation decreased aromatase expression, probably through SHP-mediated inhibition of LRH-1 (312).
Obstruction of bile flow is known to promote intestinal bacterial overgrowth and systemic infection. FXR⫺/⫺
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epithelial barrier function (130), underlining the complexity of the BA enterohepatic cycle which also relies on the
conversion of primary BA to secondary BA by the gut
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diet on renal disease. FXR activation counteracted lipidinduced SREBP1c and proinflammatory cytokine expression in renal mesangial cells and in high-fat diet-fed mice.
Moreover, FXR activation prevented induced synthesis of
oxidative stress enzymes, glomerulosclerosis, and proteinuria in vivo, suggesting that FXR modulation could be
a new therapeutic avenue for treating diabetic kidney
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