University of Groningen

The enterohepatic circulation of bile salts in health and diseaseHulzebos, Christian Victor

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Chapter 5

Enterohepatic circulation of bile salts in farnesoid X receptor-defi cient mice:

effi cient intestinal bile salt absorption in the absence of ileal bile acid-binding protein

Tineke Kok*1, Christian V. Hulzebos*1, Henk Wolters1,Rick Havinga1, Luis B. Agellon2, Frans Stellaard1, Bei Shan3,

Margrit Schwarz4, Folkert Kuipers1

*equally contributed to this study

1 Department of Pediatrics, Center for Liver, Digestive and Metabolic Diseases, University Hospital Groningen, Groningen, The Netherlands.

2 Department of Biochemistry, University of Alberta, Edmonton, Canada.3 Tularik Inc., South San Francisco, California, USA.

4 F.Hoffmann-La Roche Ltd., Pharmaceuticals Division Vascular and Metabolic Diseases, Basel, Switzerland.

The Journal of Biological Chemistry 2003;278:41930-41937© 2003 by the American Society for Biochemistry and Molecular Biology,

Inc. (http://www.jbc.org)

84

Chapter 5

ABSTRACT

Background/Aims: The bile salt-activated farnesoid X receptor (FXR; NR1H4)

controls expression of several genes considered crucial in maintenance of bile salt

homeostasis. We evaluated the physiological consequences of FXR defi ciency on

bile formation and on the kinetics of the enterohepatic circulation of cholate, the

major bile salt species in mice.

Methods: The pool size, fractional turnover rate, synthesis rate, and intestinal

absorption of cholate were determined by stable isotope dilution and were related

to expression of relevant transporters in the liver and intestines of FXR-defi cient

(Fxr (-/-)) mice.

Results: Fxr (-/-) mice showed only mildly elevated plasma bile salt concentrations

associated with a 2.4-fold higher biliary bile salt output, whereas hepatic mRNA

levels of the bile salt export pump (Bsep) were decreased. Cholate pool size

and total bile salt pool size were increased by 67% and 39%, respectively,

in Fxr (-/-) mice compared to wild-type mice. The cholate synthesis rate was

increased by 85% in Fxr (-/-) mice, coinciding with a 2.5-fold increase in cholesterol

7α-hydroxylase (Cyp7a1) and unchanged sterol 12α-hydroxylase (Cyp8b1)

expression in the liver. Despite a complete absence of ileal bile acid-binding

protein (Ibabp) mRNA and protein, the fractional turnover rate and cycling time of

the cholate pool were not affected. The calculated amount of cholate reabsorbed

from the intestine per day was ~2-fold higher in Fxr (-/-) than in wild-type mice.

Conclusions: Absence of FXR in mice is associated with defective feedback

inhibition of hepatic cholate synthesis, which leads to enlargement of the

circulating cholate pool with an unaltered fractional turnover rate. The absence of

Ibabp does not negatively interfere with the enterohepatic circulation of cholate

in mice.

85

Enterohepatic circulation of bile salts in farnesoid X receptor-defi cient mice

INTRODUCTION

Bile salts, synthesized from cholesterol in the liver, have a number of important

physiological functions in the body. Bile salts are essential for generation of bile

fl ow and for biliary excretion of cholesterol. In the intestine, they are required

for effi cient absorption of dietary fat and fat-soluble vitamins1. Finally, there is

a growing body of evidence that bile salts are involved in the control of high

density lipoprotein (HDL)2 and very low density lipoprotein (VLDL)3-5 metabolism.

To ensure the presence of adequate concentrations of these biologically active

compounds at the sites of their actions, i.e., liver, biliary tract and intestine,

bile salts are maintained within the enterohepatic circulation by the combined

actions of transporter systems in the liver and intestine. The bile salt export pump

(Bsep; Abcb11) has been identifi ed as the major canalicular bile salt-transporting

protein6. Intestinal absorption of bile salts is mediated, to a large extent, by the

apical sodium-dependent bile salt transporter (Asbt; Slc10a2) localized in the

terminal ileum7. The Asbt splice variant t-Asbt is localized basolaterally and may

be involved in effl ux of bile salts from enterocytes toward portal blood7. Ileal

bile acid-binding protein (Ibabp) is a small soluble protein of which expression

is restricted to the terminal ileum. Ibabp is thought to be involved in facilitating

uptake of bile salts and their intracellular traffi cking in the small intestine8,9.

After their reabsorption, bile salts are taken up by hepatocytes from portal

blood via the Na+-taurocholate co-transporting polypeptide (Ntcp; Slc10a1)

and Na+-independent organic anion-transporting polypeptides, including Oatp1

(or Slc21a1)10. Only a relatively small fraction of bile salts escapes intestinal

absorption and is lost into the feces, which is compensated for by de novo bile salt

biosynthesis in the liver. Therefore, under steady state conditions, bile salt pool

size remains constant.

Recently, it has been become clear that bile salts exert regulatory actions

on expression of specifi c genes via the nuclear farnesoid X receptor (FXR;

NR1H4)8. Bile salts such as chenodeoxycholate, deoxycholate, cholate, and their

conjugates are natural ligands for FXR. Activated FXR inhibits expression of the

gene encoding cholesterol 7α-hydroxylase (Cyp7a1)11, which catalyzes the fi rst

and rate-controlling step of bile salt synthesis12. This repression is achieved

indirectly via a coordinated regulatory cascade involving FXR-mediated induction

of the small heterodimer partner (SHP; NROB2), which, in turn, inhibits the

activity of the tissue-specifi c factor liver receptor homologue-1 (LRH-1; NR5A2),

which controls expression of Cyp7a111. In this way, bile salts exert negative

feedback control on their own synthesis. Sterol 12α-hydroxylase (Cyp8b1), the

enzyme that controls the ratio in which the primary bile salt species cholate

and chenodeoxycholate are formed, seems to be under the negative control of

bile salts in an FXR-dependent manner as well13. Activated FXR also controls

expression of hepatic bile salt transporters, i.e., it induces the expression of

Bsep14,15 and down-regulates the expression of Ntcp via SHP16. A recent study17

showed that mouse (but not rat) Asbt is also subjected to negative feedback

regulation mediated by FXR via SHP-dependent repression of LRH-1 activation.

Finally, bile salts strongly induce the expression of intestinal Ibabp in an FXR-

dependent manner8,18.

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Chapter 5

Therefore, in general terms, FXR appears to control various crucial steps in

bile salt metabolism, which may provide possibilities for therapeutic interventions,

e.g., aimed at treatment of hypercholesterolemia or of cholestatic liver diseases.

However, in view of the wide variety of genes that are controlled by FXR, it is

essential to understand the impact of induced alterations in FXR activity, not only

on expression of individual genes, but also on metabolism at the whole body level.

Recent studies in chow-fed FXR-defi cient mice by Sinal et al.13 showed increased

hepatic Cyp7a1 mRNA levels, but, very surprisingly, reductions in total bile salt

pool size and fecal bile salt loss. Counterintuitively, these data imply that, in

the absence of functional FXR, bile salt synthesis would actually be suppressed.

Other mouse models with increased Cyp7a1 expression19,20 showed, as expected,

increased bile salt synthesis rates and expansion of bile salt pool sizes. This may

imply that FXR defi ciency has an impact on maintenance of bile salt pool size at

other levels, for instance in the intestine. To address this issue further, we have

evaluated parameters of the enterohepatic circulation of cholate, quantitatively

the major bile salt species in the mouse, in relation to bile formation and

the expression of transport proteins in a new mouse model of FXR defi ciency

generated by the ‘classical’ homologous recombination approach. For quantitation

of cholate kinetic parameters, a recently developed stable isotope dilution

technique was used21.

This study demonstrates that, in accordance with derepressed transcription

of Cyp7a1, Fxr (-/-) mice do show an increased cholate synthesis rate. Interestingly,

the calculated intestinal cholate reabsorption was markedly increased despite a

complete absence of Ibabp mRNA and protein, leading to an enlarged bile salt

pool size. This fi nding may imply that Ibabp functions as a negative regulator

rather than as a positive regulator of intestinal bile salt reabsorption in the

mouse.

EXPERIMENTAL PROCEDURES

Animals

Fxr (-/-) mice were generated by Deltagen, Inc. (Redwood City, CA) using

standard gene-targeting methods. To disrupt the Fxr locus, a 292 bp fragment

corresponding to a segment of exon 2 was replaced by a phosphoglycerate

kinase promoter-driven neomycin resistance cassette in a targeting vector. The

construct was linearized and electroporated into embryonic stem cells derived

from the 129/OlaHsd strain. Cells harboring the desired mutation were identifi ed

by positive selection and injected into recipient C57BL/6J blastocysts to produce

chimeras, which were used for the generation of F1 heterozygotes. F

2 wild-type,

heterozygous, and homozygous mice were produced from F1 intercross in the

expected mendelian ratios. Genotyping was accomplished by PCR using primer

pairs specifi c for the wild-type Fxr allele (5’-GTT GTA GTG GTA CCC AGA GGC CCT

G-3’ and 5’-TAT GCT AAC AGA ACA CGC GGC AGG C-3’) or the mutant allele (5’-

GTT GTA GTG GTA CCC AGA GGC CCT G-3’ and 5’-GGG TGG GAT TAG ATA AAT

GCC TGC TCT-3’).

Male homozygous (Fxr (-/-)), heterozygous (Fxr

(+/-)) and wild-type (Fxr (+/+)) mice

(C57BL/6Jx129/OlaHsd) of 25-30 g were bred at the animal facility of the

87

Enterohepatic circulation of bile salts in farnesoid X receptor-defi cient mice

University of Groningen and used for these studies. Mice were housed in a light-

and temperature-controlled facility. Food and water were available ad libitum,

and mice were maintained on standard laboratory chow (RMH-B; Hope Farms

BV, Woerden, The Netherlands). All experiments were approved by the Ethical

Committee on animal testing of the University of Groningen.

Materials

[2,2,4,4-2H4]-cholate ([2H

4]-cholate, isotopic purity 98%) was obtained from Isotec

(Miamisburg, OH). Cholylglycine hydrolase from Clostridium perfringens (welchii)

was purchased from Sigma Chemicals (St. Louis, MO). Pentafl uorobenzylbromide

(PFB) was purchased from Fluka Chemie (Buchs, Neu-Ulm, Switzerland). All other

chemicals and solvents used were of the highest purity commercially available.

Methods

Mice were anesthesized with a mixture of Hypnorm (1ml/kg) and Diazepam (10

mg/kg) (Janssen Pharmaceutica, Beerse, Belgium). Six Fxr (-/-), six Fxr

(+/-), and

six wild-type mice were subjected to bile duct cannulation for collection of bile22.

During the 30 min bile collection period, animals were placed in a humidifi ed

incubator to ensure maintenance of body temperature. Bile fl ow was determined

gravimetrically, assuming a density of 1 g/ml for bile. Bile was stored at -200C

until analysis. Blood was obtained by cardiac puncture and collected in EDTA-

containing tubes. Plasma was obtained by centrifugation at 9000 rpm for 10 min

and stored at -800C until analyzed. The livers were excised, weighed, cut into

small pieces, snap-frozen in liquid nitrogen, and stored at -800C until used for

isolation of RNA and for biochemical analyses. Samples for microscopic evaluation

were frozen in isopentane and stored at -800C or fi xed in paraformaldehyde

for hematoxylin/eosin and oil red O staining. The small intestine was rinsed

with phosphate-buffered saline (PBS) containing phenyl-methyl-sulfonylfl uoride

(PMSF) (Roche Applied Science) to prevent protein degradation and divided in

proximal, mid and distal parts. Tissue samples were immediately frozen in liquid

nitrogen and stored at -800C for membrane preparation and for RNA isolation.

Feces were lyophilized, weighed, and homogenized. To collect urine, four wild-type

and four Fxr (-/-) mice were placed in metabolic cages that allowed for separate

collection of feces and urine for a period of 24 h.

In a second experiment, 240 µg of [2H4]-cholate in a solution of 0.5% NaHCO

3 in

PBS (pH = 7.4) was intravenously administered to male Fxr (+/+) and Fxr

(-/-) mice.

Subsequently, blood samples (100 µL) were obtained at 24, 36, 48, and 60 hours

after administration of [2H4]-cholate. Plasma was obtained by centrifugation at

9000 rpm for 10 min and stored at –20ºC until analyzed. After 60 h, the mice

were anesthesized with Hypnorm and Diazepam (see above) and subjected to bile

duct cannulation22. To ensure that hepatic production was accurately measured,

bile produced during the initial 5 min after cannulation was discarded, and bile

was sampled for 30 min thereafter.

Steady-state mRNA levels determined by real-time quantitative PCR

Total RNA was isolated from frozen mouse liver and intestinal tissue using

TRIzol Reagent (Invitrogen) according to the manufacturer’s instructions. RNA

88

Chapter 5

was checked on an agarose gel for integrity, and RNA concentration was

measured using the Ribogreen RNA quantitation kit (Molecular Probes, Leiden,

The Netherlands). Reverse transcription was performed on 2.5 mg of total

RNA using random primers in a fi nal volume of 38 ml (Reverse Transcription

System, Promega, Madison, WI) for 10 min at 250C, followed by one hour at

450C. Samples were subsequently heated for 5 min at 950C to terminate the

reverse transcription reaction. Real-time quantitative PCR was performed on cDNA

samples as described by Heid et al.23 to quantify mRNA levels. Primer and probe

sequences for β-actin, Fxr (Nr1h4), Asbt (Slc10a2), truncated Asbt (t-Asbt), and

Ibabp have been described by Hulzebos et al.24. Primer and probe sequences for

Abcg5, Abcg8, Bsep (Abcb11), Cyp7a1, Cyp27, Mdr2 (Abcb4), Ntcp (Slc10a1),

and Oatp1(Slc21a1) have been described by Plösch et al.25. The following primer

sequences were used for Mrp2 (Abcc2); sense primer GGA TGG TGA CTG TGG

GCT GAT, anti-sense primer GGC TGT TCT CCC TTC TCA TGG and probe AGC TGC

ATC GTC AGG AAT TTC CTC CAC A (Accession number NM_013806). For Cyp8b1;

sense primer AAG GCT GGC TTC CTG AGC TT, anti-sense primer AAC AGC TCA

TCG GCC TCA TC and probe CGG CTA CAC CAA GGA CAA GCA GCA AG (Accession

number NM_010012). For Shp (Nr0b2); sense primer AAG GGC ACG ATC CTC TTC

AA, anti-sense primer CTG TTG CAG GTG TGC GAT GT and probe ATG TGC CAG

GCC TCC GTG CC (Accession number L76567). For Fic1 (Atp8b1); sense primer

CAC ACC AGG ATG GAG AAT CAG A, anti-sense primer GCC AGG AGC CAG TGA

TGA TTA and probe TCT CTG CGA AAT TTG CAC CTC CTG TG (Accession

number AF395823). And for Mrp3 (Abcc3); sense primer TCC CAC TTT TCG

GAG ACA GTA AC, anti-sense primer ACT GAG GAC CTT GAA GTC TTG GA

and probe CAC CAG TGT CAT TCG GGC CTA TGG C (Accession number

BF584533). Primers and detection probes for the gene of interest, labeled with

a fl uorescent reporter dye (6-carboxyfl uorescein) and a fl uorescent quenching

dye (6-carboxytetramethylrhodamine), were added. Fluorescence was measured

by an ABI Prism 7700 Sequence Detector version 1.6 software (Perkin Elmer

Life Sciences, Foster City, CA). For every PCR reaction, β-actin was used as

the internal control. The cycle number at the treshold (CT), after which the

intensity of reporter fl uorescent emission increases, was used to quantitate the

PCR product.

Preparation of intestinal membranes for protein analysis

Intestinal brush border membranes were isolated as described by Schmitz et

al.26. Total protein concentration of intestinal homogenates and brush-border

membrane fractions was determined using the method described by Lowry et

al.27.

Western blotting

Approximately 2.5 µg of protein of homogenates and of intestinal brush-border

membrane fraction of each group was separated using 4-15% Tris-HCL ready

gradient gels (Bio-Rad Laboratories, Hercules, CA) and transferred to nitrocellulose

(Amersham Biosciences, Buckinghamshire, UK) using a tankblotting system

(Bio-Rad laboratories). The Ibabp protein content of intestinal homogenates

and the Asbt protein content of brush-border membranes were determined

89

Enterohepatic circulation of bile salts in farnesoid X receptor-defi cient mice

using recombinant anti-murine Ibabp antibody28 and polyclonal anti-hamster

Asbt antibody29, respectively. The blots were incubated with the fi rst antibody

diluted in Tris-buffered saline (TBS) containing 5% dried milk powder and 0.1%

polyoxyethylene sorbitan monolaurate (Tween 20; Sigma), washed in TBS / 0.1%

Tween 20 and incubated with horseradish peroxidase-labeled donkey anti-rabbit

IgG (dilution 1:1000; Amersham Biosciences). Detection was done using the ECL

Western blot kit (Amersham Biosciences).

Analyses

Bile salt concentrations in plasma, bile, feces and urine were determined by an

enzymatic fl uorimetic assay30. Levels of biliary cholesterol and phospholipids were

measured as described by Kuipers et al.31. Aspartate transaminase (ASAT) and

alanine transaminase (ALAT) activities and total bilirubin concentrations in plasma

were determined by routine clinical chemical procedures.

Gas chromatography

Bile salt composition of bile samples was determined by capillary gas

chromatography on a Hewlett-Packard gas chromatograph (HP 5880A) equipped

with a 50 m x 0.32 mm CP-Sil-19 fused silica column (Chrompack BV, Middelburg,

The Netherlands). For this purpose, bile salts were converted to their methyl

ester/trimethylsilyl derivatives21.

Thin-layer chromatography

Conjugation patterns of biliary bile salts were analyzed by thin-layer

chromatography (TLC) on precoated silica gels (60F254, Merck, Darmstadt,

Germany) using n-butanol-acetic acid-water (10:2:1) as solvent system32.

Gas-liquid chromatography/electron capture negative chemical ionization

mass spectrometry

Plasma samples were prepared for isotopic analysis of bile salts by gas

chromatography mass spectrometry (GC-MS) as described21. All analyses

were performed on a Finnigan SSQ7000 Quadrupole GC-MS instrument. Gas

chromatographic separation was performed on a 15m x 0.25 mm column,

0.25-mm fi lm thickness (AT-5MS, Alltech Associates Inc., Deerfi eld, IL).

Isotope dilution technique calculations

The isotope dilution technique has been described in detail by Hulzebos et al.21.

Enrichment was defi ned as the increase of M4-cholate/ M0-cholate relative to

baseline measurements after administration of [2H4]-cholate and is expressed as

the natural logarithm of atom percent excess (ln APE) value. The decay of ln

APE in time was calculated by linear regression analysis for the individual mice.

From the linear decay curve thus obtained, the fractional turnover rate (FTR)

and pool size of cholate were calculated. The FTR (per day) equals the slope

of the regression line. The pool size (mmol/100g) was determined according to

the formula: (D . b . 100) / ea ) - D, where ‘D’ is the administered amount of

label, ‘b’ is the isotopic purity, and ‘a’ is the intercept on the y-axis of the ln APE

versus time curve. The cholate synthesis rate (mmol/100g/day) was determined

90

Chapter 5

by multiplying pool size and FTR.

Enterohepatic cycling time and intestinal reabsorbtion of cholate

The cholate cycling time, i.e., the time it takes the cholate pool to circulate a

single time in the enterohepatic circulation, was calculated by dividing the cholate

pool size (mmol/100g) by the biliary secretion rate of cholate (mmol/100g/h).

The cholate biliary secretion rate was calculated by multiplying the bile fl ow

(mL/100g/h) by the cholate concentration (mM) in a single 30 min fraction,

obtained from 5 to 35 min after cannulation of the gallbladder. The amount of

cholate reabsorbed per day was calculated by multiplying cholate pool size by

cycling frequency and subsequent subtraction of the daily cholate synthesis rate.

Statistical analyses

All results are presented as means ± SD. Differences between the two or three

groups were determined by t test or one-way analysis of variance (ANOVA),

with post hoc comparison by Newman-Keuls t test, respectively. The level of

signifi cance for all statistical analyses was set at p < 0.05. Analyses were

performed using SPSS for Windows software (SPSS, Chicago, IL).

Figure 1. Targeted disruption of the murine Fxr gene. (A) Genomic organization of the

wild-type allele and of the disrupted allele arising after homologous recombination. Neor,

neomycin resistance cassette; pA, polyadenylation signal. (B) PCR genotyping of wild-type

(Fxr (+/+)), Fxr

(+/-) and Fxr (-/-) mice. Genomic DNA was isolated from animals of the indicated

genotype and amplifi ed via PCR using allele-specifi c primers described under Experimental

Procedures. Products were separated by agarose gel electrophoresis. (C) Northern blot

analysis of hepatic RNA from wild-type(+/+) and Fxr (-/-) mice. Poly (A+) RNA was isolated

from livers and pooled within genotype groups (n=5). Aliquots (3 µg) were separated by

gel electrophoresis, transferred to nylon membranes, and hybridized to a radiolabeled FXR

probe representing a 837-bp BamHI fragment containing the ligand-binding domain (upper

panel). The fi lters were stripped and reprobed with a cDNA encoding rat cyclophilin (lower

panel).

91

Enterohepatic circulation of bile salts in farnesoid X receptor-defi cient mice

RESULTS

Deletion of the Fxr gene

Mutation of the Fxr gene was accomplished by replacement of 292 bp from exon

2 with a neomycin resistance cassette conferring antibiotic resistance (Figure 1A).

Homologous recombination in embryonic stem cells, injection into blastocysts,

and transmission of the mutation through the mouse germ line were carried

out by standard methods. The loss of ~97 amino acids encoded by exon 2 was

anticipated to remove a large part of the DNA-binding domain of the FXR protein.

PCR analysis (Figure 1B) of genomic DNA confi rmed the predicted recombinations.

RNA blotting revealed a Fxr transcript of ~2.0 kb in the livers of wild-type mice,

whereas no transcript was detectable in Fxr (-/-) mice (Figure 1C).

Animal characteristics

The body and liver weights of Fxr (-/-) and Fxr

(+/-) mice at three months of age

were slightly higher than those of wild-type mice, but liver weight/body weight

ratios were not affected (Table 1). There were no differences in plasma alanine

transaminase (ASAT), aspartate transaminase (ALAT) and bilirubin concentrations

between Fxr (-/-), Fxr

(+/-) and Fxr (+/+) mice. Plasma bile salt concentrations were

only slightly increased in Fxr (-/-) mice, in contrast to previously reported data in

another strain of Fxr (-/-) mice described by Sinal et al.13. Accordingly, urinary bile

salt loss was not signifi cantly different between wild-type and Fxr (-/-) mice, i.e.,

67.4 ± 14.6 nmol/day vs. 43.4 ± 11.0 nmol/day, respectively. Examination of

hematoxylin- and eosin-stained liver sections of wild-type, Fxr (+/-) and Fxr

(-/-) mice

did not reveal any overt abnormalities in FXR-defi cient mice (data not shown).

Table 1. Body and liver weights and plasma liver function parameters in wild-type, Fxr

(+/-) and Fxr (-/-) mice

Strain wild-type Fxr(+/-) Fxr(-/-)

Body weight (g) 23.0 ± 1.1 26.5 ± 1.7* 29.2 ± 3.1* Liver weight (g) 1.1 ± 0.1 1.4 ± 0.1* 1.5 ± 0.2* Ratio LW/BW 0.049 ± 0.004 0.051 ± 0.006 0.053 ± 0.006 ASAT (U/L) 98 ± 18 83 ± 14 120 ± 64 ALAT (U/L) 40 ± 8 36 ± 12 63 ± 32Bilirubin (mmol/L) 13.6 ± 2.6 13.0 ± 1.9 12.5 ± 1.0 Bile salts (mmol/L) 18.1 ± 3.6 21.6 ± 3.9 27.2 ± 7.4*

Values are expressed as means ± SD (n = 6 per group). *Signifi cant difference between wild-type and Fxr

(+/-) or Fxr (-/-) mice. BW, body weight; LW, liver weight; ASAT, aspartate

transaminase; ALAT, alanine transaminase.

Effects of FXR defi ciency on bile formation and bile composition

FXR defi ciency in mice was associated with an increase in bile fl ow (Table 2). The

concentrations of phospholipids and cholesterol in bile did not differ among the

three groups, but the biliary bile salt concentration was signifi cantly increased in

Fxr (-/-) mice. As a consequence, biliary output rates of bile salts were signifi cantly

increased in these animals. Output rates of phospholipid and cholesterol tended to

92

Chapter 5

be increased in Fxr (-/-) mice, but differences did not reach statistical signifi cance.

Because biliary secretion of phospholipids and cholesterol is tightly coupled to that

of bile salts32, the output rates of these biliary lipids were also expressed relative

to those of bile salts (Table 2). It is evident that, both for phospholipids and

cholesterol, these ratios were lower in Fxr (-/-) mice than in wild-type mice. When

Figure 2. Relationship between bile fl ow and biliary bile salt secretion in wild-type,

Fxr (+/-) and Fxr

(-/-) mice. Bile was collected for 30 min, and production rates were

determined gravimetrically. Biliary bile salt concentrations were determined as described

under Experimental Procedures. The combined data from wild-type (white circles), Fxr (+/-)

(grey circles) and Fxr (-/-) (black circles) mice reveal the characteristic linear relationship

between biliary bile salt output and bile fl ow that has been reported in several species.

Bile fl ow at the hypothetical zero value of bile salt output represents the magnitude of the

bile salt-independent fraction of bile fl ow (3 µl/min/100g BW), whereas the slope of the

relationship (8 µl/µmol) represents the choleretic activity of biliary bile salts.

Table 2. Concentrations of organic solutes in bile and biliary output rates in wild-type, Fxr (+/-) and Fxr (-/-) mice

Strain wild-type Fxr(+/-) Fxr(-/-)

Bile (mmol/l) Bile salts (BS) 51.3 ± 17.6 52.6 ± 9.5 82.6 ± 13.5* Phospholipids (PL) 6.65 ± 1.68 5.72 ± 1.36 6.74 ± 1.26 Cholesterol (CH) 0.40 ± 0.07 0.42 ± 0.06 0.43 ± 0.10

Bile fl ow (ml/min/100g body wt) 4.92 ± 1.70 6.40 ± 1.99 7.73 ± 1.5 Biliary output (nmol/min/100g body wt) Bile salts 272 ± 183 347 ± 158 640 ± 157* Phospholipids 32.8 ± 14.4 36.0 ± 13.3 51.3 ± 11.0 Cholesterol 1.99 ± 0.76 2.69 ± 0.98 3.32 ± 0.95PL/BS output ratio 0.14 ± 0.04 0.11 ± 0.04 0.08 ± 0.02* CH/BS output ratio 0.009 ± 0.003 0.008 ± 0.001 0.005 ± 0.002 Values are expressed as means ± SD ( n = 6 per group). *Signifi cant difference between wild-type and Fxr (+/-) or Fxr (-/-) mice.

93

Enterohepatic circulation of bile salts in farnesoid X receptor-defi cient mice

Table 3. Biliary bile salt composition (% total) in wild-type, Fxr(+/-) and Fxr(-/-) Mice

Strain wild-type Fxr(+/-) Fxr(-/-)

Deoxycholate 2.7 ± 1.1 3.2 ± 1.3 3.3 ± 1.8 α-Muricholate 3.8 ± 0.4 3.0 ± 0.6* 1.7 ± 0.3* β-Muricholate 16.0 ± 2.5 16.3 ± 2.5 11.8 ± 4.5 ω-Muricholate 9.8 ± 2.2 9.1 ± 3.2 6.3 ± 3.4 HDC 3.4 ± 3.0 1.9 ± 1.9 0.6 ± 0.2 Chenodeoxycholate 2.7 ± 1.0 2.0 ± 0.3 1.4 ± 0.4* Cholate 61.6 ± 4.7 64.6 ± 4.1 74.5 ± 7.3*

Values are expressed as means ± SD (n = 6 mice per group). *Signifi cant difference

between wild-type and Fxr (+/-) or Fxr

(-/-) mice.

biliary bile salt output rates were plotted against bile fl ow for the individual mice of

the three groups, the classical linear relationship between these parameters was

observed (Figure 2). This strongly indicates that the bile formation process itself

is not affected by FXR defi ciency and that the higher bile fl ow rate in Fxr (-/-) mice

is caused by the higher bile salt output.

Analysis of biliary bile salt composition (Table 3) revealed that, in all three groups,

cholate constituted the major fraction of biliary bile salts and that this fraction

was higher in Fxr (-/-) mice than in the other two groups. Accordingly, the relative

contents of α-muricholate and chenodeoxycholate were signifi cantly decreased

in Fxr (-/-) mice. Thin-layer chromatography revealed that essentially all biliary

cholate was conjugated to taurine in wild-type as well as in Fxr (-/-) mice (data not

shown). Despite the fact that bile salt-conjugated enzymes have recently been

identifi ed as FXR target genes34, unconjugated bile salts were undetectable by this

procedure in bile of wild-type and Fxr (-/-) mice.

Steady-state mRNA levels of genes involved in bile salt synthesis and bile

formation

Real-time quantitative PCR was used to evaluate hepatic expression of specifi c

genes as infl uenced by FXR defi ciency (Figure 3). As expected, expression of

Shp tended to be lower in Fxr (-/-) mice. Cyp7a1 was clearly increased in Fxr

(-/-)

mice, whereas the expression levels of Cyp27 and Cyp8b1 were not signifi cantly

affected (Figure 3A). The mRNA levels of the gene encoding the canalicular

bile salt transporter Bsep, a well-known FXR target gene14,15, were signifi cantly

decreased in Fxr (-/-) mice (Figure 3B). Expression of other transporters genes

relevant to bile formation, such as the phospholipid translocator Mdr2, Ntcp,

and the putative cholesterol transporters Abcg5/g8, was not changed by FXR

defi ciency. Likewise, no effects on expression of Oatp1, Mrp2, and Mrp3 were

observed.

Effects of FXR defi ciency on kinetic parameters of cholate metabolism

To evaluate the physiological consequences of the observed changes in expression

of bile salt synthesis and transporter genes, kinetic parameters of the enterohepatic

circulation of cholate were determined by stable isotope dilution21. Because of the

small differences between the heterozygotes and wild-type mice with respect to

94

Chapter 5

Figure 4. Decay of intravenously administered [2H4]-cholate in wild-type and Fxr

(-/-) mice.

A dose of 240 µg of [2H4]-cholate was intravenously injected into wild-type (open symbols)

and Fxr (-/-) (closed symbols) mice, and blood samples were collected at 24, 36, 48 and 60

h after injection for determination of plasma cholate enrichments by GC-MS as described

under Experimental Procedures. Values are expressed in a logarithmic fashion, and the pool

size (y-intercept), fractional turnover rate (slope of the curve), and synthesis rate (pool size

x fractional turnover rate) were calculated for individual mice. Data are means ± standard

deviation of n = 5 mice per group.

Figure 3. Steady-state mRNA levels of genes involved in bile salt synthesis and transport in

livers of wild-type, Fxr (+/-) and Fxr

(-/-) mice. Total mRNA was isolated form the livers of wild-

type (white bars), Fxr (+/-) (grey bars) and Fxr

(-/-) (black bars) mice, transcribed into cDNA,

and subjected to real-time PCR as described in Experimental Procedures. (A) Hepatic mRNA

levels of Fxr, Shp, Cyp7a1, Cyp27 and Cyp8b1. (B) Hepatic mRNA levels of Bsep, Mdr2,

Abcg5, Abcg8, Ntcp, Oatp1, Mrp2 and Mrp3. n = 5 for all groups. *Signifi cant difference

between wild-type and Fxr (+/-) or Fxr

(-/-) mice.

95

Enterohepatic circulation of bile salts in farnesoid X receptor-defi cient mice

bile formation and gene expression patterns, kinetic studies were conducted in

wild-type and Fxr (-/-) mice only. Analysis of plasma cholate enrichments over time

(Figure 4) demonstrated that the cholate pool size, calculated from the y-intercept

of the linear regression line shown in Figure 4, was larger in Fxr (-/-) mice than

in wild-type mice (Figure 5A: 42 ± 8 µmol/100g vs. 23 ± 3 µmol/100g, Fxr (-/-)

mice vs. wild-type; p < 0.0001). The percentage of cholate in hepatic bile, as

determined by gas chromatographic analysis in individual mice (Table 3), was

used to calculate total bile salt pool sizes. Under the assumption that all bile salt

species displayed a similar cycling frequency, the calculated total pool sizes of

non-cholate bile salts were similar (22 ± 4 mmol/100g vs. 23 ± 9 mmol/100g,

Fxr (-/-) vs. wild-type mice, respectively, NS), leading to calculated total bile salt

pool sizes of 64 ± 12 and 46 ± 13 mmol/100g in Fxr (-/-) and wild-type mice,

respectively (p < 0.05). Deuterated cholate disappeared from plasma at the same

rate in Fxr (-/-) and wild-type mice (Figure 4). The fractional turnover rate of

cholate, calculated from the slope of the linear regression curve, was similar in

Figure 5. Effects of FXR defi ciency on pool size (A), fractional turnover rate (B), synthesis

rate (C), cycling time (D), and daily intestinal reabsorption (E) of cholate as derived from

[2H4]-cholate isotope enrichment measurements in plasma of wild-type and Fxr (-/-) mice.

The pool size, fractional turnover rate (FTR), synthesis rate, cycling time and daily intesti-

nal reabsorption were calculated in wild-type (white bars) and Fxr (-/-) (black bars) mice as

described under Experimental Procedures. Data are means ± standard deviation of n = 5

mice per group. *Signifi cant difference between wild-type and Fxr (-/-) mice.

96

Chapter 5

both groups of mice. (Figure 5B: 0.5 ± 0.1 per day vs. 0.5 ± 0.2 per day, Fxr (-/-)

vs. wild-type, respectively, NS).

In the Fxr (-/-) mice, the calculated cholate synthesis rate (Figure 5C) was two

times increased compared to the wild-type mice (22 ± 2 mmol/100g/day vs.

11 ± 3 mmol/100g/day, Fxr (-/-) vs. wild-type mice; p < 0.001). In accordance with

the increased cholate synthesis rate determined by stable isotope dilution, fecal

loss of bile salts was increased by ~ 70 % (4.1 ± 1.1 mmol/day vs. 2.3 ± 0.7

mmol/day, Fxr (-/-) vs. wild-type mice; p < 0.05). The calculated cholate cycling

time (Figure 5D) was not affected by FXR defi ciency (4.4 ± 1.3 h vs. 4.3 ± 0.7 h,

Fxr (-/-) mice vs. wild-type mice; NS). The calculated absolute amount of cholate

reabsorbed in the intestines of Fxr (-/-) mice (Figure 5E) was ~ 2 fold larger than

that in the intestines of wild-type mice (227 ± 81 mmol/100g/day vs. 121 ± 11

mmol/100g/day, Fxr (-/-) mice vs. wild-type mice; p < 0.05).

Figure 6. mRNA and protein levels of intestinal bile salt transporters in wild-type and Fxr (-/-)

mice. (A) Steady-state mRNA levels of Asbt, Ibabp, t-Asbt, and Fic-1 in the ilea of wild-

type (white bars) and Fxr (-/-) (black bars) mice. Total mRNA was isolated from the ileum,

transcribed into cDNA, and subjected to real-time PCR as described under Experimental Pro-

cedures. *Signifi cant difference between wild-type and Fxr (-/-) mice. (B) Western blot analy-

sis of Asbt and Ibabp on brush-border membranes and liver homogenates, respectively,

from the ilea of wild-type and Fxr (-/-) mice. Apparent molecular masses are indicated to the

right.

97

Enterohepatic circulation of bile salts in farnesoid X receptor-defi cient mice

Intestinal expression of genes involved in bile salt transport

To provide an explanation for the high bile salt absorption rate in Fxr (-/-) mice,

the mRNA levels of several genes considered to be involved in intestinal bile salt

absorption were determined in the terminal ileum. These studies showed that

expression of Ibabp, a well known FXR target gene8,18 thought to be involved

in intracellular bile salt traffi cking and active bile salt reabsorption8,9, was very

strongly decreased at the mRNA level in Fxr (-/-) mice (Figure 6A). FXR defi ciency

did not affect expression of transporter protein-encoding genes Asbt, responsible

for the major part of active ileal bile salt reabsorption, and of t-Asbt, putatively

involved in basolateral bile salt effl ux. Expression of Fic1 (Atp8b1), a P-type

ATPase proposed to function as an aminophospholipid translocator and essential

for normal bile salt metabolism35, also did not differ at the mRNA level between

wild-type and Fxr (-/-) mice. Western blot experiments on brush-border membrane

fractions and homogenates of the terminal part of the ileum (Figure 6B) showed

that Asbt protein levels were similar in wild-type and Fxr (-/-) mice, whereas the

protein levels of Ibabp were essentially non-detectable in the Fxr (-/-) mice.

DISCUSSION

This study has established the physiological consequences of FXR defi ciency on

bile formation and on the kinetics of enterohepatic bile salt circulation employing

an FXR-null mouse model generated by homologous recombination. A microscale

stable isotope dilution technique21 was used to quantify important parameters

of bile salt metabolism. Data show that the bile formation process per se was

not affected by FXR defi ciency and that effects on bile fl ow seen in Fxr (-/-) mice

were secondary to alterations in bile salt metabolism. In accordance with current

concepts of the role of FXR in control of bile salt synthesis36,37, hepatic Cyp7a1

mRNA levels were signifi cantly increased in Fxr (-/-) mice and were associated with

an increased cholate synthesis rate. Enhanced bile salt synthesis was confi rmed

by increased fecal bile salt loss in Fxr (-/-) mice, although the absolute difference

was somewhat less pronounced among the strains using this methodology. We

attribute the discrepancy between outcome of fecal excretion and the isotope

dilution method to the fact that no stool marker has been applied, which is

required to correct for fecal balance measurements. Furthermore, the intrinsic

diffi culties of quantitative fecal bile salt analysis have been extensively reviewed

by Setchell et al.38. As a consequence of defective feedback inhibition of hepatic

bile salt synthesis, Fxr (-/-) mice developed an increased bile salt pool size, which

implies that potential adaptive responses of intestinal bile salt reabsorption were

not effective in maintenance of the bile salt pool size. No change in intestinal Asbt

mRNA and protein levels was found in FXR-defi cient mice. By contrast, the well

known FXR target gene Ibabp was not expressed at all in the terminal ileum of

Fxr (-/-) mice. Despite the absence of Ibabp, our kinetic study revealed that the

absolute amount of bile salts reabsorbed from the intestine was not reduced, but

was actually enhanced by 2-fold in Fxr (-/-) mice. These fi ndings suggest that Ibabp

may not function as a ‘facilitator’9, but rather as a negative regulator of intestinal

bile salt absorption under physiological conditions in the mouse.

FXR has been shown to be involved in control of various steps of bile

98

Chapter 5

salt metabolism, i.e., synthesis and transport13,36,37,39, as well as in regulation

of plasma lipoprotein metabolism2,3,13,40. FXR-defi cient mice have been very

informative in elucidation of the various functions of this nuclear bile salt-activated

receptor. Studies by Sinal et al.13 and Lambert et al.40 were performed with

FXR-defi cient mice (C57BL/6J-SV129 background) that were generated by Cre-

mediated deletion of a fragment containing the last exon of the Fxr gene, encoding

the ligand-binding/dimerization domain, and the 3’-untranslated region of the

Fxr mRNA. In theory, a truncated protein containing the DNA-binding domain

could be formed that might affect expression of FXR target genes. In this study,

we used an FXR knockout model (C57BL/6J-129/OlaHsd background) generated

by homologous recombination, in which 292 bp of exon 2, encoding a part of

the DNA-binding domain, were deleted. These mice showed plasma HDL and

triglyceride levels (Elzinga et al., unpublished) that were elevated to a similar

extent as reported earlier13. In our study, liver function parameters were found

to be unaffected in Fxr (-/-) mice. Plasma bile salt concentrations were only slightly

increased in Fxr (-/-) mice compared with wild-type mice, in marked contrast to the

8-fold increase in plasma bile salt concentration in Fxr (-/-) mice reported by Sinal et

al.13. These strongly elevated plasma bile salt concentrations have been attributed

to defective hepatobiliary bile salt transport due to down-regulation of the major

canalicular bile salt export pump (Bsep)13. However, we have shown that a similar

or even more pronounced down-regulation of Bsep expression in mice was not

associated with impaired biliary bile salt secretion41. Furthermore, the more than

4-fold increase in biliary bile salt secretion during bile salt feeding in mice42 is

accommodated by a very modest increase in hepatic Bsep expression. These

data have been interpreted to indicate that Bsep at normal expression levels has

a marked overcapacity in mice. In fact, in this study, biliary bile salt secretion

was more than 2-fold increased despite a 40% reduction of Bsep expression.

Therefore, it appears that the livers of Fxr (-/-) mice are well able to handle the

(increased) bile salt load. The discrepancy between both strains with respect to

control of plasma bile salt concentrations remains unexplained at the moment.

FXR defi ciency was associated with an enhanced bile fl ow, as determined

during a 30 min period of bile collection. Biliary bile salt concentrations and

secretion rates were clearly enhanced in Fxr (-/-) mice, reinforcing the issue that

decreased Bsep expression levels do not necessarily correlate with defective bile

salt transport. The increase in bile fl ow in Fxr (-/-) mice appeared to be exclusively

due to the higher bile salt output, as is evident from the linear relationship

between bile fl ow and biliary bile salt output that was obtained when data from

wild-type, Fxr (+/-) mice and Fxr

(-/-) mice were combined (Figure 2). The fact that

values of individual mice from the three groups fi tted well to this relationship

indicates that the actual bile formation process was not affected by FXR defi ciency.

The value found for the choleretic activity (8 µl/µmol) of biliary bile salts is similar

to that reported earlier in rodents43. The bile salt-independent fraction of bile

fl ow (3 µl/min/100g body weight) was unaffected by FXR defi ciency, which is

in accordance with unchanged expression of Mrp2. Mrp2 is crucially involved in

hepatobiliary transport of glutathione44, which represents the major driving force

for the generation of bile salt-independent fl ow in rodents43,45. Although Mrp2 has

been identifi ed as an FXR-target gene46, Fxr (-/-) mice did not show reduced Mrp2

99

Enterohepatic circulation of bile salts in farnesoid X receptor-defi cient mice

mRNA levels in this study or in a study by Schuetz et al.47. Biliary secretion rates

of both cholesterol and phospholipids were slightly enhanced in Fxr (-/-) mice as

compared to wild-type mice. Secretion of cholesterol and phospholipids into bile

is coupled to that of bile salts33. Cholesterol secretion appears to involve the

activity of Abcg5/Abcg8 dimers48,49, although the exact role of this twin transporter

remains to be defi ned50, whereas phospholipid secretion critically depends on the

activity of the Mdr2 P-glycoprotein51,52. Because expression of Abcg5/Abcg8 as

well as of Mdr2 was unaffected in Fxr (-/-) mice (Figure 3B), it is plausible to ascribe

the slight stimulation of biliary lipid secretion entirely to the enhanced bile salt

secretion. It should be noted that Lambert et al.40 did report reduced hepatic

Abcg5/Abcg8 expression in their strain of Fxr (-/-) mice, but this was found to

be associated with increased biliary cholesterol output rates. The reason for the

discrepancy in hepatic Abcg5/Abcg8 expression between both strains of mice is

not clear.

We have focused on the effects of FXR defi ciency on the kinetics of cholate

metabolism. For this purpose, we used a novel microscale isotope dilution

technique, applicable in unanesthetized animals21. FXR defi ciency was associated

with an increased cholate synthesis rate, in accordance with increased hepatic

Cyp7a1 mRNA levels in Fxr (-/-) mice. Although FXR has been advocated as the

major regulator of hepatic bile salt synthesis36,39, the effects of FXR defi ciency

on the basal expression of Cyp7a1 (~ +150%) and cholate synthesis (~ +67%)

were relatively modest. This fi nding underscores the importance of the recently

described FXR/SHP-independent mechanisms of regulation. Recent studies in bile

salt-fed SHP knockout mice53,54 have clearly demonstrated the existence of FXR/

SHP-independent repression of Cyp7a1 expression. Cyp27 and Cyp8b1 expression

levels were not signifi cantly affected in Fxr (-/-) mice, although the latter showed

tendency to increase, in accordance with an increase in the fractional contribution

of cholate in the bile salt pool of Fxr (-/-) mice. The fecal loss of bile salts was

increased by ~70 % in Fxr (-/-) mice. Because the mass of bile salts excreted

into feces is, by defi nition, directly proportional to the amount synthesized in

the liver36, the data on fecal loss confi rm a generalized derepression of bile salt

synthesis in FXR-defi cient mice. This again is at variance with the study of Sinal et

al.13, who reported increased expression of Cyp7a1, but decreased fecal bile salt

loss in Fxr (-/-) mice.

The fractional turnover rate of cholate was similar in wild-type and Fxr (-/-)

mice, whereas the cholate pool size was increased 2-fold in Fxr (-/-) mice, implying

enhanced intestinal cholate reabsorption in Fxr (-/-) mice. The calculated total bile

salt pool size was increased by ~40% in Fxr (-/-) mice. This is in contrast to the

situation reported by Sinal et al.13, i.e., a reduction of the total bile salt pool

size by ~50% in Fxr (-/-) mice fed a chow diet. In this case, bile salt pool size

was measured in homogenates of gallbladder, the liver immediately surrounding

the gall bladder, and the entire small intestine harvested after termination of

the animals. The bile salt contents of the homogenates, which were extracted

into ethanol, were determined colorimetrically. Whether methodological or strain-

differences underlie the deviating results between both studies in not clear: the

stable isotope dilution method is a well established procedure to quantify bile salt

kinetics in humans55 and in laboratory animals21.

100

Chapter 5

Maintenance of bile salt pool size can theoretically be regulated at the level

of the intestine by controlled reabsorption in the terminal ileum. Asbt had been

identifi ed as the major transporter involved in this process. However, expression

of Asbt was not different between wild-type and Fxr (-/-) mice. In contrast, Chen et

al.17 reported an increase in Asbt protein levels in Fxr (-/-) mice, in accordance with

the presence of LRH-1 sites in the promoter of the murine Asbt gene identifi ed by

these authors. t-Abst and Fic1 expression was also not changed at the mRNA level

between wild-type and Fxr (-/-) mice and therefore does not seem to play a role

in enhanced bile salt reabsorption effi ciency. Ibabp, a well known FXR gene, was

drastically down-regulated at mRNA and protein level in the ilea of Fxr (-/-) mice, in

accordance with earlier studies13,17. Ibabp is thought to be involved in intracellular

bile salt traffi cking and to facilitate reuptake of bile salts in the small intestine8,9.

Yet despite the complete absence of Ibabp protein in the ilea of the Fxr (-/-) mice,

daily intestinal cholate reabsorption was much higher than in wild-type mice. This

suggests that, under physiological conditions, Ibabp functions as a negative rather

than as a positive regulator of intestinal bile salt reabsorption in the mouse.

In conclusion, this work shows that the absence of FXR in vivo in mice is

associated with defective feedback inhibition of hepatic cholate synthesis, which

leads to an enlarged circulating cholate pool with an unaltered fractional turnover

rate. The absence of Ibabp does not negatively interfere with the enterohepatic

circulation of cholate in mice.

ACKNOWLEDGMENTS

We thank Sara Berdy, Theo Boer, Renze Boverhof, Anke ter Harmsel, Bert Hellinga,

Laura Hoffmann, Karen Siegler and Fjodor van der Sluijs for excellent technical

assistance. This work was supported by grant 902-23-191 from The Netherlands

Organization for Scientifi c Research (NWO).

101

Enterohepatic circulation of bile salts in farnesoid X receptor-defi cient mice

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