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University of Groningen
The role of the farnesoid X receptor in metabolic controlStroeve, Johanna Helena Maria
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Sco pe of the dissertation
General introduction
Outline of the dissertation
Curr Opin Lipidol. 2007 Jun;18(3):289-97
1Parts of this introduction have been adapted and modifi ed from:Bile acids, farnesoid X receptor, atherosclerosis and metabolic control
Kuipers FStroeve JHMCaron S Staels B
General introduction 11
1SCOPE OF THE DISSERTATION
Body fat accumulates when energy intake, i.e., food intake, exceeds output as physical
activity. Prevalence of obesity in adults and children is reaching epidemic proportions
worldwide and is now considered one of the most serious public health problems of
the 21st century. The World Health Organization’s (WHO) latest statistics indicate that
globally in 2005 at least 20 million children under the age of 5 years were overweight.
A shift towards increased intake of energy-dense food that is high in fat and sugars and
decreased physical activity contribute to this epidemic. Obesity has adverse effects on
health, increasing the likelihood of various diseases, particularly of metabolic diseases
such as the metabolic syndrome. The metabolic syndrome is referred to as a ‘syndrome’,
because it comprises, in addition to obesity, components such as insulin resistance,
fatty liver and a disturbed lipid content in blood (dyslipidemia, characterized by high
triglycerides, low high-density lipoprotein (HDL) cholesterol and elevated low-density
lipoprotein (LDL) cholesterol). Patients with the metabolic syndrome are at increased
risk of developing type 2 diabetes and cardiovascular disease. Treatments like lifestyle
changes and pharmacological interventions are often prescribed by physicians. How-
ever, whereas lifestyle-based approaches are not always effective or suffi cient, pharma-
cological interventions often only control a single component of the metabolic syn-
drome and not the syndrome as a whole. Given its tremendous physical, emotional and
fi nancial burden, strategies to prevent and manage obesity and associated metabolic
complications are eagerly awaited.
Food is processed through a fi nely regulated network of metabolic pathways (Figure
1). Upon ingestion of a meal, food is mechanically and enzymatically digested; e.g., car-
bohydrates are broken down into simple sugars like glucose, proteins are broken down
into single amino acids and fat is broken down into fatty acids and glycerol. Absorption
of individual dietary compounds occurs in the small intestine. Blood containing the
absorbed nutrients is transported via the portal vein to the liver and subsequently to
other organs in the body. Processing by individual organs include breakdown and stor-
age of nutrients by the liver, oxidation by muscle and brown adipose tissue and storage
by white adipose tissue. Finally, it is important to realize that nutrient processing is
dependent on the health status of the subject.
To quickly and adequately respond to changes in the environment, the complex
network of metabolic pathways require a precise regulation. It becomes more and more
evident that nutrients themselves as well as certain other molecules like vitamins and
bile acids, by acting on so-called nuclear receptors, function as molecular regulators of
metabolism. Because of its broad functionality, the farnesoid X receptor (FXR, NR1H4)
is considered a promising therapeutic target for novel therapies aimed at treatment
of metabolic diseases such as obesity, diabetes, dyslipidemia and atherosclerosis. FXR
12 General introduction
is activated upon binding with bile acids. Activated FXR plays key roles in regulating
metabolism of bile acids, glucose and lipids. In recent years, our knowledge of FXR
functioning has increased substantially. At the same time, however, this new knowledge
raised many new questions. This dissertation aims to unravel some specifi c roles of FXR
activity in control of bile acid, glucose and lipid metabolism in health and disease using
innovative mouse models.
GENERAL INTRODUCTION
Nuclear receptors
Nuclear receptors are a class of proteins that are responsible for sensing the cellular
presence of nutrients, steroid hormones and certain other molecules like vitamins and
bile acids. Upon activation, these receptors work in concert with other proteins to regu-
coordination
brain
gastro-intestinal
tract
pancreas
whiteadiposetissue
muscle &brown adiposetissue
vasculartissue
bioavailability
utilization
storage & release
transport
oxidation
oxidation & storage
liver
Figure 1. The network of metabolic pathways.
Nutrients are processed through a fi nely regulated network of metabolic pathways. Main players (organs) and their
main functions in nutrient processing are indicated.
General introduction 13
1late the expression of specifi c genes, thereby controlling metabolism. Whereas C. el-
egans possesses 270 nuclear receptors[1], humans, mice and rats ‘only’ have 48, 49 and
47 nuclear receptors[2], respectively. Examples of nuclear receptors, their endogenous
ligands and their main functions are the peroxisome proliferator-activated receptors
(PPARα, NR1C1; PPARβ/δ, NR1C2; PPARγ, NR1C3) that upon activation by free fatty acids
function in cellular differentiation and lipid metabolism, liver X receptors (LXRα, NR1H3;
LXRβ, NR1H2) that upon activation by oxysterols function in cholesterol metabolism
and the farnesoid X receptors (FXRα, NR1H4; FXRβ, NR1H5) that upon activation by bile
acids function in bile acid, glucose and lipid metabolism.
Organization and classifi cation
The classical view of the structural organization divides a nuclear receptor into 5 do-
mains: the N-terminal regulatory domain (AF1), the DNA-binding domain (DBD; pro-
viding the ability to directly bind to DNA), a fl exible hinge region, a ligand-binding
domain (LBD) and a C-terminal domain (AF2)[3,4] (Figure 2A). Nuclear receptors can
bind DNA as homodimers, heterodimers or monomers[5]. In addition, some nuclear
receptors have been identifi ed that do not bind DNA directly but instead function by
interacting with other transcription factors and altering their activity[6,7]. Nonetheless,
most nuclear receptors, including FXR[5], bind DNA as heterodimers with the common
partner, retinoid X receptor (RXRα, NR2B1; RXRβ, NR2B2).
Functioning: co-activators and co-repressors
In the cell nucleus the nuclear receptor binds to a specifi c sequence of DNA (see Figure
2B). Upon DNA binding, nuclear receptors recruit a signifi cant number of proteins (so-
called transcriptional co-regulators) including transcription factors, co-activators and
co-repressors and the RNA polymerase (RNAPII) machinery itself[8]. The unliganded
receptor has an open conformation that allows interaction with co-repressors through
the co-repressor motif, the so-called co-repressor nuclear receptor (CoRNR) box mo-
tif[9,10]. Upon agonist binding, a conformational change in the receptor causes loss of
binding of co-repressors and replacement by co-activators. The co-activators interacts
with the receptor through a highly conserved ‘signature sequence’ called the nuclear
receptor box[11].
Complexity of gene regulation is further increased by the realization that transcrip-
tion factors must regulate gene expression from the genome that is compacted in the
form of chromatin[12,13]. Through acetylation, methylation, phosphorylation, ubiqui-
tylation, sumoylation and ADP-ribosylation co-activators and co-repressors alter the
chromatin structure (for reviews, see [14,15,16]). Whereas co-activators weaken the as-
sociation of histones with DNA to induce gene transcription, co-repressors strengthen
the association of histones with DNA to repress gene transcription.
14 General introduction
The farnesoid X receptor
Gene structure FXRαThe farnesoid X receptor (FXRα, NR1H4, from now on referred to as FXR) is a member of
the nuclear receptor superfamily. Independently, FXR was identifi ed in 1995 by Forman
et al.[17] and by Seol et al.[18]. The Fxr gene is composed of 11 exons and 10 introns
(Figure 3A). From this single gene, four FXR isoforms are transcribed[19,20]; FXRα1,
FXRα2, FXRα3 and FXRα4. Figure 3B and C show that FXRα1/2 and FXRα3/4 differ at
their amino terminus due to the existence of two alternative promoters. FXRα1 and
FXRα3 have a four-amino (MYTG) residue insertion in the hinge region immediately
adjacent to the DNA binding domain, resulting from an alternative splice donor site. As
of today, little is known about the specifi c functions of each FXR isoform.
Tissue distribution of FXR
FXR is expressed at high levels in liver and small intestine[19]. FXR is also expressed in
white adipose tissue, kidney, adrenal glands, stomach, heart and pancreas[21,22]. In
A/B EC D F
N-terminaldomain
DNA bindingdomain (DBD)
hingeregion
ligand bindingdomain (LBD)
C-terminaldomain
A.
DBD DBD
LBD
LBD
RE RE
Ac Ac Ac
TATA
RNAPII
L L
B.
co-repressor complex
co-activator complex
Figure 2. Nuclear receptors.
(A) The structural organization of a nuclear receptor (from left to right): A/B, N-terminal regulatory domain (AF1); C, the
DNA-binding domain (DBD); D, a fl exible hinge region; E, a ligand-binding domain (LBD) and F, a C-terminal domain
(AF2). (B) The nuclear receptor functioning. NRs bind to specifi c sequences of DNA (response elements, REs). Upon
ligand binding (L), the co-repressor complex is replaced by the co-activator complex. After recruiting the co-regulators
and the RNA polymerase (RNAPII) machinery, gene transcription is induced ().
General introduction 15
1
addition, FXR was found in different parts of the circulatory system; e.g., in endothe-
lial cells[23], cardiac muscle, vascular smooth muscle cells and also in atherosclerotic
plaques[24] and immune cells[25]. Conversely, FXR is undetectable in spleen and skel-
etal muscle[20].
Natural and synthetic ligands of FXR
In initial studies, supraphysiological levels of farnesol were shown to activate FXR[17].
Farnesol is a natural alcohol and is used in perfumery to emphasize the odors of sweet
fl oral perfumes (Note: farnesol was named after the fl ower Acacia farnesiana, in turn,
named after Cardinal Odoardo Farnese (1573-1626) maintainer of the Farnese botanical
gardens in Rome). Farnesol in vertebrates is also an intermediate in cholesterol bio-
synthesis. In 1999, several groups independently identifi ed bile acids as endogenous
ligands of FXR[26,27,28]. Recently, 6-ethyl-chenodeoxycholic acid (6E-CDCA, INT-
747[29]) and some nonsteroid molecules, such as GW4064[30], fexaramine[31] and a
azepino[4,5-b]indole (XL335, WAY-362450)[32] have been developed as FXR agonists.
C. MmFXRa1/2
MmFXRa3/4
-- - M N L I G H S - - - H L Q A T D E F S L S - - - - - - - - - E S L F G
M V M Q F Q G L E N P I Q I S L H H S H R L S G F V P E G M S V K P A K G
B. MmFXRa1/2
MmFXRa3/4 A/B EC D FN C
MYTG
MYTG
A/B EC D FN C
A. MmFXR 1 4 5 6 97 83 101**
12bpFXRa1/2 FXRa3/4
2
Figure 3. The farnesoid x receptor.
(A) The murine farnesoid x receptor (FXR, NR1H4) gene structure, consisting of 11 exons and 10 introns, two alternative
promoters giving rise to FXR isoforms FXRα1/2 and FXRα3/4, respectively, and a 12 base pair alternative splice donor
site, which determines the difference between isoforms FXRα1/3 and FXRα2/4. , transcription start site; *, translation
start site; T, translation stop site. Exon parts in white represent untranslated regions and black and gray correspond to
the structural organization represented in (B). (B) The structural organization of the murine FXR isoforms (from left to
right): distinct N-terminal regulatory domain; A/B, N-terminal regulatory domain (AF1); C, the DNA-binding domain
(DBD); the four-amino (MYTG) residue insertion in FXRα1/3; D, a fl exible hinge region; E, a ligand-binding domain
(LBD), and F, a C-terminal domain (AF2). (C) Amino acid sequences of the two distinct N-terminal regulatory domains
of FXRα1/2 and FXRα3/4, respectively. These distinct amino termini originate from two alternative promoters in the
FXR gene. Mm, Mus Musculus.
16 General introduction
DNA binding properties of FXR
FXR can bind to so-called FXREs in DNA. The FXR-RXR heterodimer binds with highest
affi nity to direct inverted repeats of the hexanucleotidic AGGTCA sequence separated
by 1 bp (IR-1)[17].
FXRβRecently, FXRβ (NR1H5) was identifi ed as a novel family member[33]. FXRβ shares about
50% amino acid identity with FXRα. In primates, including humans, FXRβ has been clas-
sifi ed as a pseudogene, since hFXRβ contains two stop codons and three frame shifts. In
contrast, it has functional relevance in other mammals, like mice and rats. Interestingly,
FXRβ does not share common ligands with FXRα; i.e., bile acids. Instead, lanosterol, an
intermediate of the cholesterol biosynthetic pathway, has been proposed as endog-
enous ligand for FXRβ[33].
FXR-mediated regulation of bile acid metabolism
Bile acid metabolism
Bile acids are synthesized from cholesterol exclusively by the liver in a cascade of reac-
tions that converts hydrophobic, water-insoluble cholesterol into more water-soluble
bile acids that confer detergent-like properties (Figure 4A). Immediate products of the
cascade are referred to as primary bile acids. Chemical diversity of the bile acid pool is
increased by the actions of intestinal bacteria, giving rise to so-called secondary and
tertiary bile acid species[34,35]. Primary bile acids are conjugated with either taurine
or glycine. Upon ingestion of a meal, bile acids are secreted into the intestinal lumen
where they facilitate the digestion and absorption of dietary lipids and lipid-soluble vi-
tamins. In the terminal ileum, bile acids are effectively reabsorbed mainly by the actions
of specifi c transporter systems. Only ~5% of bile acids escape reabsorption and enter
the colon. Here they can be converted into secondary bile acids that can be passively
absorbed or are lost from the body through the feces. The mixture of primary and sec-
ondary bile acids that is absorbed returns back to the liver via the portal system, thereby
completing the so-called enterohepatic circulation of bile acids (see Figure 4B). The
fraction of bile acids that is lost from the body is compensated for by de novo synthesis
from cholesterol. Total bile acid synthesis in humans amounts to ~500 mg/day, account-
ing for about 90% of the cholesterol that is actively metabolized.
FXR-mediated regulation of bile acid metabolism
The physical characteristics of bile acids, which allow them to form micelles, impose a
threat to cells that are exposed to high concentrations of these natural detergents. Ob-
viously, both maintenance of physiological control of the enterohepatic circulation and
General introduction 17
1
FXR
BA
intestine
lumen
duodenumileum
gallbladder
liver
blood
BA
BA
BA
FXRFGF15
BA BA
FGFR4 JNKP
acetate
cholesterol
CYP7A1
BA
A.
B.
C
O
R2
R1
R3
OH
glycine/taurine
CA
CDCA
DCA
LCA
UDCA
αMCA
βMCA
R1
αOH
αOH
H
H
βOH
αOH
βOH
R2
αOH
H
αOH
H
H
H
H
R3
H
H
H
H
H
βOH
βOH
FXR
++
++
++
+
-
-
-
FGF1
5
FGF15FGFR3
BA
bile
duc
t
Figure 4. Bile acids and their enterohepatic circulation.
(A) Structure of major naturally occuring bile acids. CA, cholic acid; CDCA, chenodeoxycholic acid; DCA, deoxycholic
acid; LCA, lithocholic acid; UDCA, ursodeoxycholic acid; αMCA, α-muricholic acid; βMCA, β-muricholic acid. (B) The
enterohepatic circulation of bile acids (BAs). Bile acids are synthesized from cholesterol in hepatocytes and are actively
secreted into bile canaliculi that drain the common bile duct. Bile acids are stored in the gallbladder, which contracts
and expels its contents into the intestinal lumen upon ingestion of a meal. In the intestine, bile acids facilitate the
absorption of dietary lipids. Bile acids themselves are actively taken up in the ileum: only a relatively small fraction
escapes reabsorption and is eventually lost via the feces. In the ileal enterocytes, bile acids activate FXR which, among
other effects, induces the expression of FGF15, which is released into the circulation. FGF15 evokes a number of
physiological responses. First, it is involved in relaxation of the musculature of the gallbladder, allowing its refi lling in
preparation for the next meal. Secondly, it is involved in suppression of hepatic bile acid synthesis in the liver, which is
a key event in the maintenance of bile acid pool size. Bile acids returning to the liver after their reabsorption contribute
to this negative feedback loop by suppression of Cyp7A1 expression via activation of hepatic FXR. In this manner, the
hepatic synthesis of bile acids is regulated to compensate accurately for fecal loss. Black arrows represent the fl ux of bile
acids in the enterohepatic circulation. Gray arrows correspond to bile acid-induced actions, with arrowheads indicating
stimulatory actions and blocked arrows indicating inhibitory actions. The dashed rings around the gallbladder indicate
FGF15-mediated gallbladder relaxation. BA, bile acid; FXR, farnesoid X receptor; FGF15, fi broblast growth factor 15;
FGFR3-4, FGF receptor isotype 3-4; JNK, c-Jun N-terminal kinase; CYP7A1, cholesterol 7a-hydroxylase.
18 General introduction
initiation of cell protective reactions require a system for sensing bile acids in specifi c
cell types. It is now clear that bile acids interact with FXR, which mediates the control
of bile acid synthesis and transport. For instance, FXR activity regulates the rate-con-
trolling enzyme in hepatic bile acid synthesis, CYP7A1. This regulation is implemented
through direct inhibitory actions of activated hepatic FXR[36,37] and indirect FXR-FGF15
mediated signaling from the small intestine[38,39] (Figure 4B). Down-regulation of Cy-
p7A1 by hepatic FXR occurs through an indirect cascade involving the induction of
the expression of small heterodimer partner (SHP, NR0B2). SHP, in turn, inhibits a posi-
tive regulator of the Cyp7A1 promoter, the liver receptor homologue 1 (LRH1, NR5A2)
[36,37,40]. Possibly contributing to this process are the recently identifi ed microRNAs,
miR-122a and miR-422a, that were found to destabilize Cyp7A1 mRNA upon CDCA and
GW4064 treatment of primary human hepatocytes[41].
Loss of Fxr in mice resulted in an increased cholic acid synthesis rate, biliary bile acid
output and bile acid pool size due to an increased expression of Cyp7A1[42]. A reduc-
tion in plasma bile acid levels can be brought about by bile acid sequestrant treatment.
Bile acid sequestrants bind bile acids in the intestinal lumen, making bile acids escape
small intestinal reabsorption and excrete from the body. Like Fxr-defi ciency, the loss of
bile acids, i.e., loss of FXR ligands, results in increased hepatic de novo bile acid synthe-
sis by increasing Cyp7A1 expression. Unlike Fxr-defi ciency, bile acid sequestration has
no effect on bile acid pool size, however did cause a shift from bile acid reabsorption
to de novo synthesis[43].
FXR in cholestatic liver disease
Since FXR activity promotes bile acid clearance from the body and represses de novo
bile acid synthesis, FXR activation might be a useful strategy in the treatment of choles-
tatic liver disease. Indeed, treatment of rat models of intra- and extrahepatic cholestasis
with the synthetic FXR agonist GW4064 protects against cholestatic liver damage as
evidenced by signifi cant reductions in liver damage evidenced by signifi cant reductions
in, among others, the incidence and extent of necrosis and infl ammatory cell infi ltra-
tion[44]. Supportive for a causative role of lack of FXR activity in cholestatic liver disease
is that two mutations in the Fxr sequence, reducing Fxr expression, have been isolated
from patients with intrahepatic cholestasis of pregnancy (ICP)[45].
Bile acid metabolism in metabolic derangements
Interestingly, the knowledge about possible disturbances in FXR activity and bile acid
metabolism in obesity and type 2 diabetes is very limited. Diabetic rats are reported to
have decreased Fxr expression in the liver[46]. Recently, Kemper et al.[47] reported that
acetylation of FXR is constitutively increased in metabolic diseases due to alterations in
the dynamic interaction of FXR with deacetylases like SIRT1 and p300. As a result, FXR
General introduction 19
1heterodimerization with RXR and DNA binding are inhibited[47]. With respect to bile
acid metabolism, diabetic patients were found to exhibit an increased cholic acid syn-
thesis rate and deoxycholic acid pool size[48]. Also, in several animal models of type 1
and 2 diabetes the bile acid pool was shown to be increased[43,49,50].
FXR-mediated regulation of glucose metabolism and insulin resistance
FXR in gluconeogenesis
To date, the precise role of FXR activity in the regulation of glucose metabolism remains
controversial. The fi rst evidence of a contribution of FXR activity to glucose homeosta-
sis is from 2005, when it was demonstrated that FXR activation induced whereas Fxr-
defi ciency reduced the expression of the gluconeogenic enzyme phosphoenolpyruvate
carboxykinase (PEPCK, PCK1) in hepatocytes[51,52]. In vivo, however, FXR-dependent
regulation of Pepck expression was dependent on the FXR agonist used; i.e., cholic ac-
id-supplementation decreased[53,54,55] whereas GW4064 treatment increased[52,56]
Pepck mRNA levels in mice. Next to the question if FXR activity regulates Pepck, there
is debate about the general contribution of PEPCK to gluconeogenesis. For a long time,
PEPCK was considered to be the rate-controlling enzyme in gluconeogenesis. However,
unexpectedly, mice with a 95% reduction in Pepck mRNA expression preserve eugly-
cemia during starvation[57]. Also, in Fxr-defi cient mice the adaptive response to long-
term fasting was preserved[51,58]. Nevertheless, the kinetics of short-term fasting were
changed upon Fxr-defi ciency in the sense that the induction of Pepck was attenuat-
ed[55], which led to an accelerated transient drop in glycemia[51]. To increase the com-
plexity even further, the regulation of the expression of Pepck by FXR activity appears to
be modulated in the diabetic state. However, again results are confl icting; i.e., GW4064
treatment of db/db diabetic mice reduced Pepck[56] whereas GW4064 treatment of ZDF
rats increased Pepck expression[59].
FXR in glycolysis
Besides regulation of the gluconeogenic gene Pepck by FXR activity, a role for FXR
activity is proposed in controlling glucose breakdown, i.e., glycolysis. Supportive is the
fi nding that livers of Fxr-defi cient mice contained less glycogen[51]. Conversely, FXR ac-
tivation by GW4064 treatment increased hepatic glycogen stores[56]. It was later dem-
onstrated in primary rat hepatocytes that FXR activation reduces glucose-induced gene
expression and activity of L-pyruvate kinase (L-PK, PKLR), which catalyzes the fi nal step
in the glycolytic pathway[58]. Accordingly, Fxr-defi cient mice exposed to an overnight
fast followed by refeeding a high-carbohydrate diet displayed an accelerated induction
of L-PK compared with wild-type mice[58].
20 General introduction
Bile acids in glucose metabolism
Besides a direct role of FXR activity in the regulation of glucose metabolism, FXR activity
regulates glucose metabolism indirectly, via bile acids. Consistent is the fi nding that bile
acid sequestrant treatment both in mice[60], rats[61,62] and humans[63,64,65,66,67]
reduced blood glucose levels. Possibly underlying is the recently identifi ed bile acid-
mediated activation of the G protein-coupled receptor TGR5 (also referred to as G
protein-coupled bile acid receptor 1, GPBAR1) in enteroendocrine cells. Activation of
this receptor in vitro[68] and in vivo[69] induced glucagon-like peptide 1 (GLP1, GCG)
release, leading to improved liver and pancreatic function and enhanced glucose toler-
ance in obese mice.
FXR in insulin resistance
Regarding insulin resistance, Fxr-defi ciency in lean mice has been shown to cause im-
paired glucose tolerance and insulin resistance[21,55,56]. Accordingly, GW4064 treat-
ment in diabetic and obese animal models (db/db, KK-A(y)[56] and ob/ob[21]) improved
insulin sensitivity. Hyperinsulinemic-euglycemic clamp studies showed that Fxr-defi -
ciency leads to insulin resistance in the periphery[21,55]. This fi nding was confi rmed by
reduced insulin signaling in skeletal muscle and white adipose tissue. Data on hepatic
insulin resistance, however, are less clear. While some studies describe reduced insulin
sensitivity[55,56], others describe normal hepatic insulin sensitivity[21,58]. Underlying
this discrepancy might be the different genetic backgrounds of the mice. To explain the
contribution of FXR activity to insulin resistance, several hypotheses have been made.
The fi rst hypothesis proposes that Fxr-defi ciency promotes ectopic lipid deposition as
refl ected by increased circulating free fatty acids (FFA)[21,55] and increased storage
of triglycerides in muscle and liver of Fxr-defi cient mice[55,58]. Possibly contributing
is the recently proposed role for FXR activity in adipocyte differentiation, i.e., in vi-
tro Fxr-defi ciency reduced[21,70] while FXR activation stimulated[22,71] adipogenesis.
FXR activation also led to improved insulin signaling and insulin-induced glucose up-
take in 3T3-L1 adipocytes[21,22]. A second hypothesis is based on indirect FXR-FGF15
mediated signaling from the small intestine[38,39]. Mouse fi broblast growth factor 15
(FGF15) and its human orthologue FGF19 are endocrine factors involved in a variety of
biological processes including the control of bile acid synthesis by the liver. The hypoth-
esis suggests that Fxr-defi ciency leads to impaired hepatic fatty acid oxidation[72] and
adipose tissue glucose uptake[73] due to a reduced level of FGF15.
General introduction 21
1FXR and metabolism of triglycerides
Bile acids/FXR in triglyceride metabolism
The existence of a relationship between bile acid and triglyceride metabolism has been
recognized for many years. This is primarily based on the clinical observation that treat-
ment of patients with bile acid sequestrants results in increased plasma triglyceride
levels[74,75], while treatment with CDCA has the opposite effect. In line with the former,
in humans an inherited genetic defect in bile acid synthesis due to Cyp7A1-defi ciency is
also associated with increased plasma triglyceride levels[76]. As in humans, in mice bile
acid metabolism is related to triglyceride metabolism as indicated by the hypertriglyc-
eridemia in Fxr-defi cient mice[40,77].
FXR in VLDL metabolism
Both the production of triglyceride-rich VLDL (very low density lipoprotein) particles
and their clearance from the circulation have been proposed as main processes infl u-
enced by FXR activity. Supportive is the fi nding that Fxr-defi cient mice have increased
hepatic VLDL production[77]. Several potential mechanisms have been proposed by
which FXR activity contributes to control VLDL metabolism. A fi rst mechanism is formed
by FXR-mediated regulation of hepatic lipogenesis. However, two points should be
noted. First, lipogenesis per se should not be considered as a driving force for VLDL
production by the liver. Indeed, several animal models have been described in which
(massively) increased hepatic lipogenesis had no effect on VLDL production (e.g., [78]
and [79]). Second, the data on the FXR-mediated regulation of lipogenic genes are
inconclusive. For example, one study described FXR-mediated repression of Srebp1c
(Srebf1) expression (sterol regulatory element-binding protein-1c, a transcription factor
positively regulating lipogenic genes)[80], while others in which FXR activation was re-
duced by bile acid sequestrant treatment showed unchanged[81] or even repressed[60],
but not the expected increased, Srebp1c expression levels. Another potential mecha-
nism for the increased VLDL production in Fxr-defi cient mice, is the FXR-mediated re-
pression of microsomal triglyceride transfer protein (MTTP), essential for lipidation of
apolipoprotein (apo) B during VLDL assembly[82]. A fi nal, third, mechanism is based on
the fact that activation of FXR impacts on the expression of genes that control clearance
of plasma triglycerides; FXR activation induces the expression of Apoc2[83], an activator
of lipoprotein lipase (LPL) activity and suppresses expression of Apoc3[84] and Ang-
ptl3[80], which are both LPL inhibitors. Additionally, FXR activity induces the expression
of the VLDL receptor[85] and of syndecan-1[86], which may contribute to accelerated
clearance of VLDL as well (for review, see [87]).
22 General introduction
FXR in hypertriglyceridemia
Since FXR activity reduces the assembly and induces the clearance of triglyceride-rich
VLDL particles, FXR activation might be a useful strategy in treating hypertriglyceride-
mia that occurs, for example, in obesity and the metabolic syndrome. This strategy has
been tested in several animal models and the results indicated that GW4064 modestly
lowered plasma triglyceride levels in chow-fed mice and effectively reduced elevated
plasma triglycerides in ob/ob and db/db mice[55,56,84]. Likewise, CDCA treatment pre-
vented hypertriglyceridemia and decreased the VLDL production rate in fructose-fed
hamsters[88]. Interestingly, FXR activation also lowered the elevated FFA levels in mod-
els of insulin resistance. So far, however, the underlying mechanism(s) of this benefi cial
effect has remained elusive.
FXR-mediated regulation of cholesterol metabolism and atherosclerosis
Recent work indicates a potential role for FXR activity in the pathophysiology of athero-
sclerosis, independent of the profound effects of FXR activity on both LDL- and HDL-
cholesterol metabolism and remodeling. Although FXR-mediated regulation of cho-
lesterol metabolism and atherosclerosis is beyond the scope of this dissertation, it has
been the subject of several recent reviews, e.g., [89], [90] and [91].
OUTLINE OF THE DISSERTATION
In this dissertation, we aimed to unravel some specifi c roles of FXR activity in
control of bile acid, glucose and lipid metabolism in health and disease using in-
novative mouse models.
Our fi rst study focused on the relative contribution of the intestinal, FXR-controlled
FGF15 pathway in the regulation of hepatic bile acid synthesis in mice (chapter 2). A
recent study by Kim et al.[92] and earlier work[90,93] led to the general assumption
that the intestinal FXR-FGF15 pathway provides a major contribution to control hepatic
bile acid synthesis. However, the conclusions of Kim et al.[92] were based on rather
moderate differences at the gene expression level. In our study, we focused not only on
changes in gene expression, but, more importantly, also on determining the effect of
intestine-specifi c deletion of Fxr on physiological parameters. We showed that effects
of disrupting intestinal Fxr on hepatic Cyp7A1 expression are dependent on the time
of the day. Nevertheless, the disruption of intestinal Fxr resulted in an increased cholic
acid pool size leading to an increased biliary bile acid secretion and therefore to an
increased bile fl ow. Modulation of the bile acid pool, however, minimized the role of
intestinal FXR-FGF15 signaling present under normal dietary conditions. Chapter 3 and
General introduction 23
14 examine the effects of Fxr-defi ciency on glucose and bile acid metabolism and liver
disease in obesity by studying, among others, Fxr-defi cient ob/ob mice. Chapter 3 as-
sessed glucose metabolism and body weight control in these mice. We show that obesity
was attenuated upon Fxr-defi ciency in both genetic (ob/ob) and high-fat diet-induced
obesity models. Glucose homeostasis was improved predominantly due to enhanced
peripheral glucose clearance by white adipose tissue. Conversely, high-fat diet-fed liver-
specifi c Fxr-defi cient mice showed no signs of protection against obesity or glucose
and insulin intolerance, indicating a role for non-hepatic FXR activity, most probably in
white adipose tissue, in control of body weight and glucose homeostasis in obesity. The
improved glucose homeostasis in obese Fxr-defi cient mice was accompanied by high
plasma bile acid concentrations and exaggerated hepatic steatosis. In chapter 4 we,
therefore, addressed the role of FXR activity in hepatic bile acid metabolism and devel-
opment of liver disease in the ob/ob mouse model of obesity. We demonstrated that
the high plasma bile acid concentrations in these mice are the consequence of a marked
reduction in hepatic ABCB11-mediated hepatobiliary bile acid transport resulting in
a marked reduced bile acid secretory rate maximum (SRm). These data imply a novel
interplay between leptin and FXR. Moreover, livers of Fxr-defi cient ob/ob mice showed
characteristics of non-alcoholic steatohepatitis (NASH), including, next to steatosis, liver
injury (e.g., ballooning), monocyte infi ltration, mild fi brosis and expansion of the hepat-
ic stem cell niche. These hepatic alterations were partially normalized by withdrawal of
bile acids from the enterohepatic circulation using a highly effective sequestrant. These
fi ndings indicate that, in obesity caused by leptin-defi ciency, FXR directly or indirectly
via bile acids plays a role in the progression of (non-alcoholic) hepatic steatosis to ste-
atohepatitis. Since chapter 3 and other previous studies[21,22,70,71] emphasized a role
for FXR activity in adipose tissue biology, we developed an adipocyte-specifi c FXR over-
expressing mouse model (chapter 5). We showed that overexpression of FXR in adipose
tissue caused increased lipid uptake by adipocytes resulting in adipocyte hypertrophy.
Moreover, high FXR levels limited the expandability of white adipose tissue during high-
fat diet- and age-induced obesity, among others, by extensive extracellular matrix (ECM)
production and remodeling. In brown adipose tissue (BAT), high FXR levels reduced adi-
pocyte differentiation. Consequently, brown adipose tissue functioning was attenuated
upon cold exposure as evidenced by reduced expression of BAT genes like Ucp1, Dio2
and Elovl3 and reduced oxygen consumption by these mice. Nevertheless, the mice
were able to maintain their body temperature due to the induction of additional stress
responses such as lowering physical activity. These studies established that FXR activity
is a determinant in fat tissue development and function and that FXR activity in adipose
tissue may be implicated in obesity and obesity-associated pathologies such as ectopic
lipid deposition. Taken together, our studies provided novel insights in the role of FXR
activity in murine integrative physiology in health and disease.
24 General introduction
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