From the Molecular Disease Branch, NHLBI, National
Institutes of Health (NIH), and the ¶ Laboratory of Metabolism,
NCI, NIH, Bethesda, Maryland 20892 and the
Department of
Pharmacology, Dalhousie University, Halifax, Nova Scotia B3H 4H7,
Canada
Received for publication, September 17, 2002, and in revised form, November 4, 2002
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ABSTRACT |
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To address the importance of the farnesoid
X-receptor (FXR; NR1H4) for normal cholesterol homeostasis, we
evaluated the major pathways of cholesterol metabolism in the
FXR-deficient ( The nuclear receptor superfamily represents a large group of
ligand-activated transcription factors that are involved in a variety
of physiological, developmental, and toxicological processes (1). This
group is characterized by highly conserved DNA and ligand binding
domains and includes the functionally diverse estrogen, progesterone,
peroxisome proliferator-activated, constitutive androstane, retinoic
acid, vitamin D, and thyroid hormone receptors. A growing body of
evidence indicates that an important function fulfilled by some members
of the nuclear receptor superfamily is to contribute to the homeostatic
control of lipid metabolism. In particular, LXR
(NR1H3),1 the liver
oxysterol-activated receptor (2), and FXR (NR1H4), the bile acid
(BA)-activated farnesoid X-receptor (3-5), regulate the hepatic
biosynthesis of BAs from cholesterol, a quantitatively important
route for the elimination of excess cholesterol. Cross-talk between LXR
feed-forward and FXR feed-back regulation, particularly of the
cytochrome P450 (CYP) gene encoding cholesterol 7 The role of LXR as a sterol sensor coordinating the expression of genes
involved in various aspects of sterol homeostasis has been firmly
established by the use of a synthetic LXR agonist (T0901317) and
LXR Animals and Diets--
FXR( Plasma Lipids and Lipoproteins--
Plasma total cholesterol and
triglycerides (TG) (Sigma, St. Louis, MO), as well as free cholesterol
(FC) and phospholipids (Wako, Osaka, Japan) were measured in a 12-µl
aliquot of plasma using commercial kits and the Hitachi 911 automated
chemistry analyzer (Roche Molecular Biochemicals, Indianapolis, IN).
Lipoproteins from pooled plasma samples (100 µl) were separated by
FPLC using two Superose 6HR 10/30 columns in series (Amersham
Biosciences, Piscataway, NJ). High density lipoprotein (HDL) subclasses
were resolved by two-dimensional gel electrophoresis (19). Plasma apoB
was measured by enzyme-linked immunosorbent assay (20).
Lecithin Cholesterol Acyl Transferase, LPL, and Hepatic Lipase
Activities--
Plasma LCAT activity measurements were performed as
previously described (21). Post-heparin plasma LPL and (HL) activities were assayed in triplicate using 14C-labeled triolein
substrate in 5 and 1 M NaCl as previously described (22).
Northern Blot Analysis--
Total RNA was prepared using TRIzol
reagent (Invitrogen, Carlsbad, CA), quantitated by optical densitometry
at 260 nm, and subjected to electrophoresis on a 1.0% agarose gel
containing 0.22 M formaldehyde in 20 mM MOPS, 8 mM sodium acetate, 1 mM EDTA buffer (pH 7.0).
After separation, the RNA was transferred to Immobilon-Ny+ transfer
membranes (Millipore, Bedford, MA) by downward capillary transfer in
the presence of 20× SSC buffer (3 M NaCl, 0.3 M sodium citrate, pH 7.0) and fixed by UV cross-linking.
Full-length cDNA probes were prepared by reverse transcription-PCR
from liver total RNA and 32P-labeled by the random primer
method using Prime-it RT labeling beads (Stratagene, La Jolla, CA).
Probe hybridization was carried out overnight at 65 °C in PerfectHyb
Plus hybridization buffer (Sigma). Washes (three at 30 min each) were
performed at 65 °C using 2× SSC/0.5% SDS as the buffer. Blots were
exposed to phosphorimaging screens and were visualized using a Storm
860 PhosphorImager system (Amersham Biosciences, Sunnyvale, CA).
HDL Metabolic Studies--
HDL (1.063 < d < 1.21) from control C57BL mice were labeled with
[3H]cholesterylpalmityl ether (CEt) or with
125I-apoA-I and 131I-apoA-II as previously
described (23). The radioactivity of 3H was quantitated in
a Tri-Carb 2500 TR liquid scintillation counter and that of
125I/131I was measured using a Cobra
Western Blot Analysis--
Mouse liver membrane proteins were
purified as previously described (25), resolved on Nu-PAGE 3-8% Tris
acetate gels (Novex, San Diego, CA) under reducing conditions,
transferred onto an Immobilon membrane (Millipore, Bedford, MA), and
probed with a polyclonal-IgG antibody against either low density
lipoprotein receptor (LDLr, RDI, Flanders, NJ) or scavenger receptor BI
(SRBI, Novus Biologicals, Littleton, CO). Densitometric scanning was performed using the Molecular Dynamics Personal Densitometer SI.
In Vivo TG Production--
Mice were injected with Tyloxapol
(Sigma) (500 µg/g body weight). Plasma very low density lipoprotein
(VLDL)/chylomicron-TG clearance in mice is completely inhibited under
these conditions. Blood samples were taken from each mouse at 0, 30, 90, and 150 min after injection (26). The accumulation of newly
synthesized VLDL/chylomicron-TG was measured in plasma aliquots and
ascertained by FPLC.
Biliary Lipid Secretion--
Mice were fasted for 4 h,
weighed, and anesthetized, and their abdominal cavities opened under
sterile conditions. The cystic duct was ligated and the common bile
duct was cannulated through the sphincter of Oddi (27) using a PE-10
catheter (Becton Dickinson, Sparks, MD). The hepatic bile was collected
by gravity for 45 min. Biliary cholesterol, phospholipids, and BAs
(Sigma diagnostic kit) were measured immediately after bile collection.
Cholesterol Absorption--
Intestinal cholesterol absorption
was determined by a modification of the dual isotope plasma ratio
method (28). Anesthetized non-fasted mice were injected intravenously
with 2.5 µCi of [3H]cholesterol dissolved in 100 µl
of Intralipid. The animals were given 1 µCi of
[14C]cholesterol dissolved in 150 µl of medium chain TG
oil by gavage. After 72 h, mice were bled and plasma aliquots (100 µl) were counted for 3H and 14C. Cholesterol
absorption was calculated as: % cholesterol absorption = (% of
gavage dose Statistical Analysis--
Values are reported as mean ± S.E. Comparisons between groups of mice were made using the Student's
t test for independent samples (two-tailed).
Previously, we reported that FXR(/
) mouse model. Compared with wild-type,
FXR(
/
) mice have increased plasma high density
lipoprotein (HDL) cholesterol and a markedly reduced rate of plasma HDL
cholesterol ester clearance. Concomitantly, FXR(
/
) mice
exhibit reduced expression of hepatic genes involved in reverse
cholesterol transport, most notably, that for scavenger receptor BI.
FXR(
/
) mice also have increased: (i) plasma non-HDL cholesterol and triglyceride levels, (ii) apolipoprotein B-containing lipoprotein synthesis, and (iii) intestinal cholesterol absorption. Surprisingly, biliary cholesterol elimination was increased in FXR(
/
) mice, despite decreased expression of hepatic
genes thought to be involved in this process. These data demonstrate
that FXR is a critical regulator of normal cholesterol metabolism and
that genetic changes affecting FXR function have the potential to be pro-atherogenic.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-hydroxylase (CYP7A1), the rate-limiting enzyme of the classic BA biosynthetic pathway, permits fine-tuning of hepatic cholesterol homeostasis (6, 7).
LXR and FXR also modulate the expression of several genes involved in
lipoprotein metabolism and thus, contribute to the maintenance of
proper plasma cholesterol levels. LXR positively regulates the
expression of the genes encoding lipoprotein lipase (LPL) (8),
cholesterol ester transfer protein (9), and the ATP-binding cassette
(ABC) A1 transporter (10, 11). FXR has been shown to be a positive
regulator of the apolipoprotein (apo) C-II (12)
and the phospholipid transfer protein (PLTP) (13) genes, and
a negative regulator of apoA-I gene expression in cultured cells (14).
-knockout mice. T0901317 induces the intestinal expression of
ABCA1 and concomitantly decreases the absorption of cholesterol in
wild-type but not in LXR
knockout mice (15). T0901317 also
regulates the expression of two additional ABC transporters, ABCG5 and
ABCG8, responsible for the exclusion of dietary sterols others than
cholesterol from absorption by the enterocyte (16). Characterization of
FXR knockout mice has firmly established the critical role
for this receptor in bile acid homeostasis by virtue of its role as an
intracellular bile acid sensor (17). This function is achieved in large
measure through the regulation of genes involved in BA transport and
biosynthesis. These include CYP7A1, the liver canalicular, the
bile salt export pump (BSEP-ABCB11), the ileal bile acid binding
protein (I-BABP), the hepatic basolateral sodium taurocholate
co-transporter protein (NTCP), and the sterol 12
-hydroxylase
(CYP8B1), which controls the ratio of cholic acid to chenodeoxycholic
acid in the bile (18). Thus, although FXR is a key regulator of BA
homeostasis, its physiological role in the overall metabolism of
cholesterol remains to be established. The purpose of the present study
was to elucidate the role of FXR as a regulator of cholesterol
homeostasis and to gain mechanistic insight into the means by which
this is achieved.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
) mice (17) were
backcrossed to strain C57BL/6 for at least five generations. Mice were
housed in a pathogen-free animal facility under a standard 12-h
light/12-h dark cycle and were fed a standard rodent chow (AIN-93G) and
water ad libitum. For some experiments, mice were fed a
standard chow diet supplemented with 0.4% (w/w) cholic acid (Bioserv,
Frenchtown, NJ). For tissue collection, mice were euthanized by carbon
dioxide asphyxiation. The livers and ilea were quickly removed,
snap-frozen in liquid N2, and stored at
80 °C until
use. All protocols and procedures were reviewed and approved by the
National Institutes of Health animal care and use committee.
-counter (Packard, Downers Grove, IL; for all).
[3H]CEt-labeled HDL or
125I-apoA-I/131I-apoA-II-labeled HDL was
injected into the exposed saphenous veins of anesthetized mice (23).
Blood samples were collected in heparinized capillary tubes (Fisher,
Pittsburgh, PA) from the retro-orbital plexus. The plasma disappearance
curves were generated by dividing the plasma radioactivity at each time
point by the radioactivity of 1-min time point. FCR were determined
from the area under the curves using a multiexponential curve-fitting
technique on the SAAM program (24). After the 13-h blood sample was
collected, mice injected with [3H]CEt-HDL were
exsanguinated (1.2 ml of blood) and euthanized. Liver, adrenals,
testis, heart, lungs, kidneys, and spleen were harvested. CEt was
extracted in 20 volumes of chloroform-methanol, 2:1 (v/v) (34). Phases
were separated by the addition of water, and aliquots of the lower
organic phase were counted. Mean recoveries at 13 h were greater
than 90% of the injected dose (plasma volume: 3.16% of body weight).
% of intravenous dose) × 100.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
) mice maintained
on a regular chow diet exhibit profound hyperlipidemia (17). To further elucidate the role of FXR in cholesterol and lipoprotein homeostasis, plasma lipid concentrations were determined and the lipoprotein profile
of non-fasting wild-type and FXR(
/
) mice was compared using gel filtration (FPLC) analysis. Compared with wild-type mice,
FXR(
/
) mice had 2-fold higher plasma total cholesterol, cholesterol ester (CE) and FC concentrations (Table
I). Based upon this FPLC profile, it was
evident that these increases of plasma cholesterol were
primarily due to increased quantities of VLDL, low density lipoprotein
(LDL), as well as HDL cholesterol levels (Fig.
1a).
Plasma lipids and apoB levels are increased in FXR(/
) mice
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Fig. 1.
FXR( /
) mice have increased plasma HDL-,
LDL-, and TG-rich lipoproteins levels. a, cholesterol
and TG FPLC profiles of pooled plasma samples (100 µl) from (
)
wild-type and (
) FXR(
/
) mice. b,
two-dimensional gel electrophoresis analysis of 15 µl of pooled
plasma samples from wild-type (+/+) and FXR(
/
) mice. HDL subclasses
were visualized after immunoblotting against mouse apoA-I.
HDL Metabolism and Reverse Cholesterol Transport Are Impaired in
FXR(/
) Mice--
In FXR(
/
) mice, the
increase in plasma HDL cholesterol was associated with an increased HDL
particle size, as indicated by the presence of large HDL1
eluting before the main HDL2/3 peak (Fig. 1a).
Separation of plasma lipoproteins by two-dimensional native gel
electrophoresis and immunoblotting against mouse apoA-I (Fig.
1b), confirmed the increase of HDL size concomitant with a
relative diminution of the proportion of small HDL2/3
particles and an increase in the proportion of large HDL1
particles in FXR(
/
) mice. Nascent pre-
HDL levels
were also elevated in the plasma of FXR(
/
) mice (Fig.
1b). Northern blot analysis indicated that the hepatic and
intestinal mRNA expression levels of the two major protein
components of HDL, apoA-I, and apoA-II, were similar in FXR(
/
) and wild-type mice (Fig.
2). The catabolism of HDL
125I-apoA-I (fractional catabolic rate (FCR) = 2.3 ± 0.2 versus 2.4 ± 0.2 day
1)
and that of HDL 131I-apoA-II (FCR = 2.6 ± 0.5 versus 2.7 ± 0.2 d
1) were also similar
(n = 6; p > 0.5, all) in FXR(
/
)
and wild-type animals, respectively (Fig.
3). The catabolism of HDL
[3H]CEt, a non-degradable analogue of CE, was
significantly delayed in FXR(
/
) compared with wild-type
mice (FCR = 3.2 ± 0.2 versus 4.1 ± 0.3 d
1, respectively, n = 8, p < 0.02) (Fig. 3) due to impaired hepatic uptake of
HDL [3H]CEt (64 ± 2% versus 78 ± 1% of injected dose, respectively, n = 8, p < 0.01). The uptake of HDL [3H]CEt by
other organs (i.e. adrenals, testis, heart, lungs, kidneys, and spleen) was not significantly different between both groups (data
not shown). All together, these data demonstrate that the selective
uptake of HDL-CE by the liver is impaired in FXR(
/
) mice.
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|
To determine the molecular mechanisms responsible for delayed HDL-CE
hepatic uptake in FXR(/
) livers, mRNA levels of the major hepatic genes involved in HDL metabolism were measured in this
animal model. The expression of LCAT, HL, and of SRBI, the HDL
receptor, which promotes the selective uptake (29) of HDL-CE, were
decreased, whereas that of ABCA1 and PLTP were not changed in the
livers of FXR(
/
) mice compared with wild-type (Figs. 2
and 4a). However, the plasma
LCAT activity (20 ± 2 versus 18 ± 1 nmol of
CE/ml/h, n = 8, p > 0.5), as well as
the post-heparin HL activity (67 ± 23 versus 58 ± 10 nmol of free fatty acid (FFA)/ml/h, respectively,
n = 8, p > 0.5) was similar in both
mouse models. In agreement with the mRNA data, Western blotting
revealed that the hepatic expression of SRBI was significantly reduced
(
63%) in FXR(
/
) (Fig. 4, b and
c) versus wild-type mice, consistent with the HDL
kinetic analysis data (Fig. 3). When the mice were placed on a diet
containing 0.4% of the FXR agonist cholic acid, the hepatic SRBI
mRNA and protein expression increased in the wild-type but not in
the FXR(
/
) (Fig. 4, b and c).
Taken together, these data demonstrate that FXR deletion in
mice results in increased HDL cholesterol due to impaired hepatic
selective removal of HDL-CE mediated by SRBI. Thus, FXR is a positive
regulator of SRBI expression and thereby of HDL mediated reverse
cholesterol transport in vivo.
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Synthesis of Apolipoprotein B-containing Lipoprotein (LpB) Is
Increased in FXR(/
) Mice--
In agreement with our previous
studies (17), plasma apoB and non-HDL cholesterol levels were increased
1.6- and 2.6-fold in FXR(
/
) compared with wild-type
mice, respectively (Table I). By FPLC, both VLDL and LDL cholesterol
levels were increased in FXR(
/
) mice (Fig.
1a). FXR(
/
) mice also had 1.7-fold increased plasma phospholipids (PL) and TG levels, compared with controls. Whereas the distribution of PL among the various classes of
lipoproteins was similar to that of cholesterol (not shown), more than
90% of the TG in FXR(
/
) plasma was associated with
VLDL-sized particles (Fig. 1a). To examine the mechanism
underlying the increased plasma LpB levels in FXR(
/
)
mice, we measured the expression of the LDL receptor (LDLr), the major
receptor for LpB uptake by Northern and Western (Fig. 4, b
and c) blots. The expression of the LDLr was similar in
FXR(
/
) and wild-type livers, ruling out delayed hepatic
catabolism to significantly account for elevated LpB in FXR(
/
) mice. Particularly vexing were the parallel
increases in plasma TG levels and post-heparin LPL activity (306 ± 49 versus 179 ± 18 nmol of FFA/ml/h,
n = 8, p < 0.05) observed in
FXR(
/
) compared with wild-type mice.
We reasoned that if the catabolism of LpB cholesterol and TG was not
significantly impaired in FXR(/
) mice, the production of
LpB would be increased in this animal model. By Northern blot analysis, the hepatic and ileal expression of apoB, apoE,
apoC-II, and apoC-III, the major proteins of LpB, were similar in
FXR(
/
) and wild-type mice (Fig. 2). The expression of
the microsomal triglyceride transfer protein (MTP), which plays a major
role in LpB assembly (30), and of the
3-hydroxy-3-methylglutaryl-coenzymeA (HMG-CoA) reductase, the
rate-limiting enzyme in the cholesterol biosynthetic pathway (31), were
reduced (
67% and
42%, respectively) in the liver but not the
ileum of FXR(
/
) mice (Fig. 2). However, despite reduced
hepatic MTP and HMG-CoA reductase expression, the accumulation of
plasma TG was dramatically increased in FXR(
/
) mice
compared with wild-type (Fig.
5a) after tyloxapol injection, which blocks the hydrolysis (26) of TG by LPL, demonstrating that the
production of LpB is increased upon FXR deletion.
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Furthermore, the expression of apoA-IV, an important protein component
of newly synthesized LpB, particularly chylomicrons (32), was increased
5.1-fold in the liver and 4.5-fold in the ileum of
FXR(/
) compared with wild-type animals (Fig. 2). Next, we measured the absorption of dietary cholesterol in
FXR(
/
) and wild-type mice by the dual isotope plasma
ratio method (28). Control mice absorbed only 62 ± 4% of the
ingested cholesterol dose, whereas 97 ± 2% was absorbed by the
intestine of FXR(
/
) mice (n = 6, p < 0.003). Taken together, these results demonstrate that FXR deletion in mouse results in increased plasma
non-HDL cholesterol and TG levels caused by an increase in LpB
synthesis associated with an elevation in intestinal cholesterol
absorption. Thus, FXR is a negative regulator of dietary cholesterol
absorption and of chylomicrons and/or VLDL secretion.
FXR Regulates the Enterohepatic Circulation of
Cholesterol--
To elucidate the molecular mechanism responsible
for increased dietary cholesterol absorption in FXR(/
)
mice, we measured the ileal mRNA levels of ABC transporters
proposed to be involved in sterol absorption. Surprisingly, the
expression of ABCA1, a transporter that is thought to reduce
cholesterol absorption in the enterocyte (6), was increased 3-fold in
the ileum of FXR(
/
) compared with wild-type (Fig. 2). In
contrast, the expression of one of the two ABCG transporters
cooperatively involved in the efflux of sterols from the enterocyte
into the intestinal lumen (16) was sharply decreased in the ileum of
FXR(
/
) mice compared with wild-types (Fig. 2). Hence,
ABCG8 expression was reduced (
82%) in FXR(
/
) mice,
but that of ABCG5 was not significantly altered.
Because FXR plays a critical role in BA homeostasis, a function
achieved through the regulation of genes involved in BA transport and
biosynthesis (17), we also measured the mRNA expression of a series
of genes proposed to play a role in biliary cholesterol secretion in
FXR(/
) and wild-type mice. In the liver, ABCG5/8 have
been proposed to play a key role in the biliary excretion of sterols
(7, 33). Both ABCG5 (
76%) and ABCG8 (
66%) were down-regulated in
the liver of FXR(
/
) compared with wild-type mice. The
expression of the cholesterol ester hydrolase (CEH), an enzyme
responsible for the conversion (34) of intra-cellular CE into FC and of
the sterol carrier protein (SCP), a protein thought to be involved in
the hepatocellular trafficking of FC to the canalicular membrane (35,
36) were also reduced in the liver of FXR(
/
) mice (Fig.
4a). Together, these data suggest that CEH-mediated
mobilization, SCP-mediated intracellular transport, and
ABCG5/8-mediated biliary secretion of FC may be decreased in
FXR(
/
) mice.
However, very surprisingly, the flow of biliary cholesterol, but also
the flow of biliary PL and bile salts, measured after cannulation of
the common bile duct (5), were increased in FXR(/
) mice
compared with wild-types (Fig. 5b). In separate experiments
the mice were challenged for 3 or 5 days with a diet containing 0.4%
cholic acid to saturate their hepatic biliary secretion ability, and
their flow of biliary PL, FC, and BAs was measured. As anticipated, the
biliary flow of BAs increased in wild-type mice upon cholic acid
feeding (Fig. 5b). Conversely, there was a non-significant
trend toward a decrease in the biliary flow of BAs in
FXR(
/
) mice maintained 3 and 5 days on a 0.4% cholic
acid diet, indicating that the hepatic BAs secretion ability of
FXR(
/
) mice was saturated under these experimental
conditions (Fig. 5b). The biliary flow of PL and FC were
sharply decreased in FXR(
/
) compared with wild-type mice
maintained for 5 days on 0.4% cholic acid. Together, these data
indicate that FXR deletion in mouse results in an apparent
increase in bile flow, despite the down-regulation of a series of genes
involved in hepatic mobilization, transport, and canalicular secretion
of biliary acids and lipids. However, upon cholic acid feeding, which
saturates canalicular BA secretion in FXR(
/
), the
biliary cholesterol and PL flow was reduced dramatically, whereas it
increased in wild-type mice.
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DISCUSSION |
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In the present report, we address the physiological role of the
nuclear hormone receptor FXR on cholesterol homeostasis in vivo. We have shown that FXR(/
) mice have increased
plasma HDL cholesterol due to defective HDL-CE removal associated with
decreased hepatic SRBI expression. FXR(
/
) mice also had
increased LpB cholesterol and TG plasma levels caused by an increase in
LpB synthesis and intestinal cholesterol absorption. Very surprisingly, the biliary flow was increased in FXR(
/
) mice, despite
decreased expression of genes involved in the mobilization,
intracellular transport, and canalicular secretion of cholesterol and
bile acids, a phenomenon reversed upon cholic acid feeding. A schematic
representation of the integrated role of FXR in bile acid and
cholesterol metabolism is presented in Fig.
6. This model is based upon our current
and previous (17) studies, as well as contributions from a number of
independent laboratories (15, 16, 18).
|
Our study establishes that the increase in plasma HDL levels in
FXR(/
) mice does not result from altered mRNA
expression of apoA-I and apoA-II, or hepatic ABCA1, a major contributor
to plasma HDL levels (20, 37), indicating that the production of HDL is
not altered in FXR(
/
) mice. Consistent with our data, Fuchs et al. (38) demonstrated that gallstone-susceptible
C57L/J or gallstone-resistant AKR mice had similar apoA-I mRNA and
protein levels when placed either on chow or on 0.5% cholic acid diet. In contrast, Claudel et al. (14) reported that
administration of the FXR agonist taurocholate repressed apoA-I
expression in human hepatoma HepG2 cells as well as in human
apoA-I transgenic mice. The differences between murine and
human apoA-I promoters may account for the apparent
discrepancy between these studies. FXR(
/
) mice exhibited
reduced mRNA expression of LCAT and HL, but their plasma LCAT and
HL enzymatic activities were similar to those of wild-types.
FXR(
/
) mice exhibited reduced mRNA expression of
LCAT and HL. Hepatocyte nuclear factor
(HNF)-1
-deficient mice also present with
decreased hepatic LCAT and HL mRNA levels, accompanied by a
concomitant reduction in FXR expression (39), which suggests that FXR
may be a modulator of LCAT and HL expression in vivo. However, FXR(
/
) and wild-type mice had similar plasma
LCAT and HL enzymatic activities. Thus, the definitive role of FXR on
the hepatic production, secretion, and/or plasma activation of these two enzymes remains to be established.
After ruling out differential HDL production or plasma enzymatic
remodeling to account for elevated plasma HDL levels in
FXR(/
) mice, we showed that there was delayed selective
uptake of HDL-CE by FXR(
/
) livers, a mechanism mediated
primarily by the HDL receptor (29) SRBI. A similar increase in the
proportion of large HDL1 particles is also observed with
partial (40) or total (41) SRBI deficiency. Consistent with our
finding, SRBI expression is induced in the liver of C57L/J mice upon
cholic acid feeding (38). In the present report, we also showed that
cholic acid positively regulates SRBI mRNA and protein expression
in the liver of wild-type but not in that of FXR(
/
)
mice. These combined findings establish for the first time that FXR is
a physiological modulator of SRBI expression; thereby enhancing HDL
mediated reverse cholesterol transport.
Next, we assessed the major aspects of LpB metabolism in
FXR(/
) and wild-type mice. Plasma LpB levels were
sharply increased in FXR(
/
) mice. The degradation of LpB
cholesterol and TG did not appear to be significantly impaired in
FXR(
/
) mice, because neither the hepatic LDLr protein
expression nor the plasma LPL activity were reduced in this animal
model. Despite no change in the expression of the major LpB
apolipoproteins, we observed a dramatic increase of LpB production in
FXR(
/
) mice. Likewise, CYP7A1 overexpression
(42) results in increased secretion of LpB. FXR(
/
) mice
also present with increased hepatic CYP7A1, due to the lack
of repression (17), but unlike CYP7A1 transgenic mice,
FXR(
/
) animals do not have increased hepatic lipogenic enzymes (data not shown) and furthermore have decreased hepatic HMG-CoA
reductase and MTP expression, suggesting that, unlike the CYP7A1
transgenic mouse model, the liver is not responsible for increased LpB
production in FXR(
/
) mice. These results are intriguing,
because, despite a large reduction in hepatic MTP expression, LpB
production increases. The intestine may account for an significant
proportion of increased LpB production in FXR(
/
) mice,
particularly in the post-prandial state, as discussed below. We cannot
rule out that FXR(
/
) livers secrete dysfunctional VLDL
(e.g. rich in apoA-IV and/or apoC-III or poor in apoC-II), resistant to vascular and circulating lipases catabolism and/or uptake.
Indeed the expression of apoA-IV was increased in the liver as well as
in the ileum of FXR(
/
) mice. Because, of the various
apolipoproteins, only apoA-IV synthesis and secretion is stimulated by
fat absorption (32), we measured the absorption of dietary cholesterol
in FXR(
/
) and wild-type animals. The intestinal
absorption of cholesterol was dramatically increased upon FXR
deficiency, consistent with increased plasma apoB48/apoB100 ratio (17),
indicating increased chylomicron production in FXR(
/
) mice. Of note, the intestinal expression of HMG-CoA reductase and MTP
was not decreased in FXR(
/
) ileum. Taken together, these results demonstrate that FXR deletion in mouse results in
increased plasma non-HDL cholesterol and triglyceride levels, increased LpB (mostly chylomicrons) synthesis, and an elevation in intestinal cholesterol absorption. We conclude that FXR is a negative regulator of
dietary cholesterol absorption and of the subsequent intestinal LpB secretion.
The expression of ABCG8 but not of ABCG5, two transporters
cooperatively involved in the efflux of sterols from the enterocyte into the intestinal lumen (16), was sharply decreased in the ileum of
FXR(/
) mice. These data indicate that the
down-regulation of at least one of these two cooperative ABCG
transporters may account for a diminution in dietary sterol exclusion,
and thus for an increase in intestinal cholesterol absorption by
FXR(
/
) mice. However, feeding the FXR agonist
chenodeoxycholic acid does not induce ABCG5 and ABCG8 expression in the
jejunum of mice (33). The differential regulation pattern of ABCG5/8
expression along the cephalo caudal axis of the intestine may explain
this discrepant finding. More likely, because the ileum is the portion
of the intestine where BAs are preferentially absorbed (43) and where FXR expression levels are the greatest (44), FXR, and consequently its
target genes, are not efficiently activated in the upper intestine. Surprisingly, the expression of ABCA1, also proposed to reduce cholesterol absorption in the enterocyte (33, 45), was increased in the
ileum of FXR(
/
) mice. The physiological significance of
this result is difficult to establish, because ABCA1 is primarily expressed in the duodenum and jejunum (6) and because the exact role
played by ABCA1 in intestinal cholesterol absorption remains controversial (33, 46).
Finally, we have shown that FXR(/
) mice have decreased
hepatic mRNA expression of several genes encoding enzymes (CEH,
SCP2) and transporters (ABCG5, ABCG8) involved in the biliary secretion of cholesterol (Fig. 6). This is consistent with recent data indicating that BA feeding of wild-type mice results in increased hepatic ABCG5/8
mRNA levels and that both synthetic and natural FXR ligands increase the expression of ABCG5/8 in hepatoma cells (33).
Surprisingly, the biliary flow of cholesterol, PL, and BAs was
increased in FXR(
/
) mice, despite decreased expression
of the BSEP-ABCB11. With respect to the biliary output of cholesterol
and PL, this finding is consistent with the phenotype of BSEP-ABCB11
knockout mice (47). Recent studies have established that, upon abnormal physiological conditions where BSEP-ABCB11 is impaired, elevated intracellular concentrations of toxic BAs may activate another nuclear
hormone receptor, the pregnane X-receptor (PXR; NR1I2). A number of
target genes encoding proteins with relevance to BA metabolism are
regulated by PXR. For example, the gene encoding CYP3A, an enzyme
capable of the metabolism and detoxification of certain BAs (48,
49), is regulated in a positive manner by PXR. The gene encoding
multiple drug resistance-associated protein-2, which mediates the
efflux of conjugated compounds into the bile, (50) is also activated by
PXR. We propose that, in the absence of FXR, alternate PXR-regulated
pathways act to limit the excessive accumulation of bile acids and
lipids and to maintain bile flow (Fig. 6). It is also possible that
FXR(
/
) mice exhibit increased reabsorption of bile salts
and/or lipids by epithelial cells of the gallbladder (43). When
FXR(
/
) mice were fed cholic acid to saturate this
alternate pathway, their ability to excrete biliary cholesterol and
phospholipids was impaired. Studies are currently underway in
PXR/FXR double-knockout mice to definitively establish the physiological importance of the PXR-regulated pathway for
backup biliary secretion. Nevertheless, since on chow diet (i)
the net output of fecal BA is decreased in FXR(
/
) mice
(17) and (ii) 97% of intestinal cholesterol is absorbed by
FXR(
/
) enterocytes, compared with only 62% in
wild-types, our data indicate that FXR plays a major role in the
enterohepatic circulation of sterols and thereby is a positive
regulator of excess cholesterol disposal out of the body (Fig. 6).
Considering the established role of FXR as the primary regulator of
bile acid homeostasis and the intimate linkage between bile acid
biosynthesis and cholesterol elimination, targeted disruption of the
FXR gene might be expected to promote hepatic cholesterol elimination due to an absence of feedback repression of CYP7A1 and
CYP8B1 expression. Indeed, a recent study (51) demonstrated that
administration of guggulsterone, a traditional medicine in India
used to treat hyperlipidemia, blocked hepatic cholesterol accumulation in cholesterol-fed wild-type but not FXR(/
)
mice. The authors also demonstrated that guggulsterone is an effective antagonist of FXR in transient transfection assays, a finding that has
been independently confirmed (52). Thus, the observation that
FXR gene deletion results in hepatic cholesterol
accumulation (17) and hypercholesterolemia is intriguing. A number of
possibilities may account for the apparent discrepancy of these
results. For example, the developmental and lifelong absence of FXR may
result in abnormal metabolic effects that are quite different from
those caused by acute, transient antagonism of this receptor. An
alternative explanation is that one or more of the sites of
pharmacological action of guggulsterone do not include all of the
tissues in which FXR is functional. That is, although FXR expression is
uniformly absent in all tissues of the FXR(
/
) mouse
model, guggulsterone may antagonize FXR only within a subset of these
sites. This is further complicated by expression of multiple splice
variants of FXR in mouse (53) and human and hamster (54).
Developmental and tissue-specific patterns of expression of these
isoforms may provide for cell-specific FXR-dependent
functions and response to ligands. The current absence of in
vivo data regarding the modulation of FXR target gene expression
by guggulsterone (51) makes comparisons between our data and the
actions of this antagonist very difficult. Added to this is the recent
demonstration that the FXR agonist taurocholate reduced serum HDL and
apoA-I expression in human apoA-I transgenic mice (14). Nevertheless,
it is clear that modulation of FXR function, either genetically or
pharmacologically, has a profound impact on cholesterol homeostasis.
In conclusion, our study demonstrates that FXR (i) is a positive
regulator of SRBI expression and thereby of HDL-mediated reverse
cholesterol transport, (ii) is a negative regulator of the intestinal
absorption of cholesterol and synthesis of LpB, and (iii) plays a
pivotal and complex role in the enterohepatic circulation of
cholesterol (Fig. 6). It is clear that important differences in lipid
metabolism exist between mice and humans. However, a number of
fundamental aspects of reverse cholesterol transport are common to both
organisms. Thus, there is a real possibility that mutations of the
human FXR gene will prove to have a pro-atherogenic affect. Our data
also indicate that FXR is a promising target to establish original
therapeutic strategies for the treatment of dyslipoproteinemias as well
as cholestatic diseases.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Chloé Champion for excellent technical assistance as well as Alan Remaley for reviewing the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by the Région des Pays de Loire (to G. L.), Nestlé France (to G. L.), and the Canadian Institutes for Health Research (to C. J. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Current address, INSERM U539, Centre Hospitalier Universitaire Hotel Dieu, Nantes 44000, France.
** To whom correspondence should be addressed: Dalhousie University, Sir Charles Tupper Medical Bldg., 5859 University Ave., Halifax, Nova Scotia B3H 4H7, Canada. Tel.: 902-494-2347; Fax: 902-494-1388; E-mail: csinal@dal.ca.
Published, JBC Papers in Press, November 5, 2002, DOI 10.1074/jbc.M209525200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: LXR, liver oxysterol receptor; ABC, ATP-binding cassette; apo, apolipoprotein; BA, bile acid; BSEP, bile salt export protein; CE, cholesterol ester; CEH, cholesterol ester hydrolase; CEt, cholesterylpalmityl ether; CYP, cytochrome P450; FC, free cholesterol; FFA, free fatty acid; FXR, farnesoid X-receptor; HDL, high density lipoprotein; HL, hepatic lipase; HMG-CoA, 3-hydroxy-3-methylglutaryl-coenzymeA; LCAT, lecithin cholesterol acyl transferase; LDL, low density lipoprotein; LDLr, low density lipoprotein receptor; LpB, apolipoprotein B-containing lipoprotein; LPL, lipoprotein lipase; MTP, microsomal triglyceride transfer protein; PXR, pregnane X-receptor; SCP, sterol carrier protein; SRBI, scavenger receptor BI; TG, triglycerides; VLDL, very low density lipoprotein; FCR, fractional catabolic rate; MOPS, 4-morpholinepropanesulfonic acid.
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