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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