From the Departments of Cardiovascular Research,
¶ Gene Regulation,
Lead Optimization Biology,
** Drug Disposition, and
Medicinal Chemistry, Eli Lilly & Co.,
Indianapolis, Indiana 46285
Received for publication, September 19, 2002, and in revised form, October 25, 2002
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ABSTRACT |
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Fenofibrate is clinically successful in treating
hypertriglyceridemia and mixed hyperlipidemia presumably through
peroxisome proliferator-activated receptor Originally developed as hypolipidemic therapeutic agents, the
fibrate class of molecules has been extensively characterized as
ligands for the nuclear receptor, peroxisome proliferator-activated receptor Conversely, under appropriate metabolic states, such as those when
starved animals are fed a high carbohydrate diet, increased lipogenesis
in liver occurs in part by means of transcriptional activation of genes
associated with de novo fatty acid biosynthesis, such as
fatty acid synthase (FAS), via the transcription factor, sterol
regulatory element-binding protein-1 (SREBP1) (3-5). Coincidentally, recent studies revealed that both oxysterols and synthetic agonists for
the nuclear receptor liver X receptor (LXR) indirectly activate the
lipogenic program by LXR-mediated induction of SREBP1, thereby leading
to the coordinate expression of major lipogenic enzymes and profound
elevation of triglyceride levels in liver (6-8). LXR responsiveness of
the FAS promoter also is attributed to a conserved LXR/RXR binding site
within the 5'-flanking region of the FAS gene (9). Moreover, LXR target
genes are pivotal to overall lipid metabolism, since potent LXR
agonists activate genes involved in catabolism of cholesterol to bile
acids, in regulation of several genes important for reverse cholesterol
transport from peripheral tissues, in high density lipoprotein
accumulation, and in cholesterol excretion into bile or intestinal
lumen. The battery of LXR target genes mediating these effects includes
Cyp7A1, ATP-binding cassette A1 (ABCA1), ABCG1, apolipoprotein E,
lipoprotein lipase, cholesterol ester transfer protein, ABCG5,
and ABCG8 (10-18). Thus, cholesterol and fatty acid homeostasis
involves integration of synthesis and degradation pathways that are
controlled by the nuclear proteins PPAR Repression of de novo fatty acid synthesis in liver by
fenofibrate has been observed, but the mechanism of repression remains unknown (19). Here we describe a mechanism by which fenofibrate ester
may decrease fatty acid synthesis both in vitro and in
vivo by decreasing expression of SREBP1 and FAS. Our studies
reveal that fenofibrate ester represses the expression of SREBP1 and FAS mRNA by directly binding to and antagonizing LXR.
Interestingly, the ester form of multiple fibrate molecules bind LXR
with greater affinity than their respective carboxylic acid derivatives
bind to PPAR Cell Culture and Transient Transfections--
The human
hepatocellular carcinoma cell line, HepG2 (HB-8065; American Type
Culture Collection, Manassas, VA), was maintained in monolayer
culture at 37 °C in 5% CO2. HepG2 cells were stably transfected at 100:1 with 3XSRE-luciferase (SRE; gtggggtgat) that is
controlled by the herpes simplex virus thymidine kinase basal promoter
from Quantitative PCR--
Total RNA was isolated from cells or
tissue by the Trizol method (Invitrogen). cDNA was synthesized
using random hexamer primers and Omniscript (Qiagen, Valencia, CA).
TAQMANTM real time PCR analysis was applied using prepared
reagents and protocols from Applied Biosystems (Foster City, CA). The
primer sequences for human SREBP1 were as follows: forward,
acagcccacaacgccatt; reverse, TGCCGAAGACAGCAGATTTATT; and probe,
cgctaccgctcctccatcaatgaca. The primer sequences for mouse SREBP1 were
as follows: forward, catcgactacatccgcttcttg; reverse,
ttgcttttgtgtgcacttcgt; and probe, cacagcaaccagaagctcaagcagga. For mouse
ABCA1, they were as follows: forward, GGTTTGGAGATGGTTATACAATAGTTGT;
reverse, TTCCCGGAAACGCAAGTC; and probe, CGAATAGCAGGCTCCAACCCTGACC. For
mouse ABCG5, they were as follows: forward, ttgcgatacacagcgatgct;
reverse, tgactgcctctaccttcttgttgt; and probe, ccctctgccgcagctccg. For
mouse ABCG8, they were as follows: forward, gctgcccgggatgatagag;
reverse, ccggaagtcattggaaatctg; and probe, ttttccaccctgatccgtcg. All
PCRs were performed in triplicate using an Applied Biosystems Prism
7900HT Sequence Detection System (Applied Biosystems). Relative levels
of mRNA are expressed as a ratio of the target gene mRNA values
to the values obtained using the 18 S rRNA probe set (Applied Biosystems).
Scintillation Proximity Assay (SPA)--
The scintillation
proximity radioligand binding assay for LXR was previously described
(21). Briefly, we utilized 800 ng of baculovirus-expressed His-tagged
LXR Animal Studies--
To determine fenofibrate ester and acid
concentrations in liver, mice (129Sv) were dosed for 7 days with 300 mg/kg/day fenofibrate, and following a final dose, perfused livers were
collected at various time points. Individual liver samples (1.0 g) were
homogenized in acetonitrile. Debris was pelleted by centrifugation.
Supernatant (50 µl) was diluted with 150 µl of 0.2% formic acid.
Fenofibrate ester and acid concentrations were measured on a Sciex API
3000 mass spectrophotometer. To measure SREBP1, FAS, ABCA1, ABCG5, and
ABCG8 gene expression, mice (129Sv) were dosed for 7 days with 50 mg/kg/day T0901317, 100 mg/kg/day fenofibrate, or a combination of
both. Liver expression of the various mRNAs was measured by quantitative PCR.
Fenofibrate Represses LXR-induced Expression of SREBP--
To
determine whether fenofibrate can repress de novo fatty acid
synthesis at the level of gene transcription, we studied the regulation
of SREBP1-mediated gene transcription by fenofibrate. A stable HepG2
cell line harboring an integrated luciferase reporter gene driven by
the minimal thymidine kinase promoter preceded by three tandem repeats
of a sterol response element was generated (HepG2 3XSRE). HepG2 3XSRE
cells were treated with the LXR agonist, T0901317, in the absence or
presence of increasing concentrations of fenofibrate (Fig.
1A). The LXR agonist
effectively caused a 5-fold increase in SREBP-induced transcription of
the reporter gene; fenofibrate alone did not significantly affect
reporter activity. When added together, fenofibrate inhibited
LXR-induced SREBP activity in a dose-dependent manner
(IC50 ~25 µM).
We explored whether fenofibrate represses LXR-mediated induction of
endogenous SREBP1 mRNA expression in HepG2 cells by quantitative PCR analysis (Fig. 1B). The LXR agonist increased SREBP1
mRNA levels 4-fold as shown previously, and fenofibrate alone had
no effect on basal levels of SREBP1 mRNA (6). The addition of fenofibrate to T0901317-treated cells resulted in a
dose-dependent reduction of induced SREBP1 mRNA levels,
which is consistent with the decrease observed in HepG2 3XSRE cells.
These data indicate that fenofibrate may repress genes required for
de novo fatty acid synthesis by disrupting LXR-mediated
SREBP1 gene activation.
Fenofibrate Binds to the LXR Ligand Binding Domain--
Whether
fenofibrate does indeed alter LXR-mediated transactivation was tested
by assessing the ability of fenofibrate to inhibit an LXRE-luciferase
reporter gene in cells expressing exogenous human LXR
To test the possibility that fenofibrate competes directly for agonist
binding to the ligand binding domain (LBD) of LXR, we used chimeric
receptors encoding the LXR-LBD fused to the Gal4 DNA binding domain
that were co-transfected with a Gal4-responsive luciferase reporter
into HEK293 cells. The LXR agonist elicited a robust transcriptional
response from Gal4-LXR Fibrate Esters, but Not Fibric Acids, Bind to LXR and Inhibit LXR
Transactivation--
Several fibrate derivatives have been used
clinically in the treatment of hyperlipidemia, namely bezafibrate,
gemfibrozil (Lopid®), and fenofibrate (Tricor®). In contrast to
fenofibrate, which is formulated in its ester form, the other fibrates
are utilized as carboxylic acids. We examined both the ester and acid derivatives of each fibrate molecule and determined whether other fibrate esters or fibric acid derivatives could modulate
LXR-dependent transcription. HEK293 cells transiently
transfected either with LXR Fenofibrate Ester Accumulates in Liver and Represses LXR-mediated
Induction of SREBP1 and FAS--
The fenofibrate ester is rapidly
converted into the carboxylic acid form by esterases in the liver and
plasma (25). It has been assumed that the pharmacologically relevant
form of fenofibrate is the acid form due to its specificity for PPAR Fenofibrate Ester Does Not Inhibit LXR Transactivation of ABC
Transporters in Liver and Macrophages--
Increasing evidence
supports the notion that LXRs serve as sensors that respond to elevated
intracellular sterol concentrations by increasing the expression of
genes that reduce the sterol burden. ApoA1-mediated cholesterol efflux
from macrophages to high density lipoprotein requires the expression of
the ABCA1 transporter, which is controlled, in part, by LXR/RXR
heterodimers (12, 26-33). Likewise, LXR regulates the expression of
ABCG5 and ABCG8 genes that promote secretion of sterols from the liver
and decrease intestinal sterol retention, thereby facilitating
coordinate regulation of sterol efflux and elimination. We explored the
possibility that fenofibrate ester could adversely repress LXR-induced
ABC gene expression both in liver and in THP-1 macrophages. The same liver samples from mice treated with T0901317 and fenofibrate in Fig. 4
were evaluated for ABCA1, ABCG5, and ABCG8 gene expression levels. Both
ABCA1 and ABCG5 mRNA levels were increased ~5-fold, and ABCG8
increased 3-fold upon treatment with T0901317 as compared with the
vehicle-treated mice (Fig.
5A). Administration of
fenofibrate ester alone caused slight yet significant increases in
ABCA1, ABCG5, and ABCG8 mRNA levels (p < 0.05). In
contrast to the ability of fenofibrate to antagonize LXR-induced
expression of SREBP1 and FAS mRNA, fenofibrate did not reduce
LXR-induced expression of the ABC transporters in liver. Likewise, the
addition of fenofibrate either to 22RHC- or to T0901317-stimulated
THP-1 macrophage cells did not diminish LXR-induced expression of
endogenous ABCA1 mRNA (Fig. 5B). The observed increase
in ABC transporter expression could be due to PPAR As a transcriptional regulator of genes, such as the ABC
transport proteins, apolipoprotein E, lipoprotein lipase, and
cholesterol 7 By directly binding to the LXR ligand binding domain, various fibrate
ester compounds displace a naturally occurring LXR ligand in
vitro, and they selectively repress LXR agonist-induced liver target gene transcription in vivo. We propose that
fenofibrate could induce distinct structural changes in LXR that
influence its ability to interact with other proteins, such as
transcription factors residing at selective target gene promoters or
with coactivators or corepressors that are critical for target gene
regulation. For other nuclear receptors such as the estrogen and
progesterone receptors, the relative expression of various cofactors in
target tissues and the promoter context have been demonstrated to be essential for the determination of the agonist/antagonist character of
a particular ligand (38-41). Similarly, the action of fibrate esters
on LXR may be determined by the exact sequence of the LXRE, the
contribution of additional transcription factors, and the influence of
local chromatin structure of selected LXR target genes. Whether these
specific promoters differentially utilize coactivators and
corepressors, as is the case for estrogen-, raloxifene-, and
tamoxifen-stimulated gene expression, also remains to be investigated (39).
Although obscured by mixed PPAR Fenofibrate ester is subject to esterases and is rapidly converted to a
carboxylic acid in the plasma and liver. Indeed, we readily detected
fenofibric acid in mouse plasma samples but were unable to detect
fenofibrate ester in plasma, yet fenofibrate ester reached steady-state
levels in liver that persisted for at least 24 h (Fig.
4A). Therefore, since fenofibrate ester did reach
significant steady-state levels in the liver, it is reasonable to
propose that fibrate esters (i.e. Tricor® and Lopid®)
may decrease hepatic triglyceride levels, at least in part, by
decreasing the expression of SREBP1 and FAS through modulation of
LXR.
The observation that other acidic PPAR In summary, we have demonstrated that not only does the fibrate class
of hypolipidemic compounds function through activation of PPAR (PPAR
)-dependent induction of genes that control fatty
acid
-oxidation. Lipid homeostasis and cholesterol metabolism also
are regulated by the nuclear oxysterol receptors, liver X receptors
and
(LXR
and LXR
). Here we show that fenofibrate ester, but
not fenofibric acid, functions as an LXR antagonist by directly binding
to LXRs. Likewise, ester forms, but not carboxylic acid forms, of other
members of the fibrate class of molecules antagonize the LXRs. The
fibrate esters display greater affinity for LXRs than the corresponding
fibric acids have for PPAR
. Thus, these two nuclear receptors
display a degree of conservation in their recognition of ligands; yet, the acid/ester moiety acts as a chemical switch that determines PPAR
versus LXR specificity. Consistent with its LXR
antagonistic activity, fenofibrate potently represses LXR
agonist-induced transcription of hepatic lipogenic genes. Surprisingly,
fenofibrate does not repress LXR-induced transcription of various
ATP-binding cassette transporters either in liver or in macrophages,
suggesting that fenofibrate manifests variable biocharacter in the
context of differing gene promoters. These findings provide not only an
unexpected mechanism by which fenofibrate inhibits lipogenesis but also
the basis for examination of the pharmacology of an LXR ligand in humans.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(PPAR
)1 (1).
In humans, the major pharmacological effects of PPAR
activation by
fibrates are the reduction of plasma cholesterol and triglyceride
levels. Mechanistically, decreases in triglyceride-rich plasma
lipoprotein levels could occur via accelerated catabolism, decreased
synthesis, or both. Indeed, activation of PPAR
by fibrates promotes
-oxidation of fatty acids in the peroxisome as well as in the
mitochondria, thus reducing the fatty acid available to the liver for
triglyceride synthesis (2).
, SREBP, and LXR.
. Importantly, the effects of fenofibrate on LXR target gene repression appear promoter-specific; fenofibrate does not repress
LXR agonist-induced ABC transporter gene expression either in liver
or in macrophages. These findings provide an unexpected mechanism
by which fenofibrate ester inhibits SREBP1 transcription and
lipogenesis without negating the beneficial role LXR that plays in
mediating secretion of sterols from the liver and in increasing
reverse cholesterol transport from macrophages.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
105 to +51 (20) and pHygEGFP (Clontech, Palo Alto, CA). Stable cells were selected in medium containing 800 µg/ml hygromycin. For transient transfection of HEK293 cells, 4 × 104 cells were plated into 24-well dishes. Each
transfection contained 100 ng of luciferase reporter, 100 ng of
CMV-driven expression plasmid where indicated (pM LXR
LBD; amino
acids 162-447, pCMV6 LXR
; NR1H3, accession number NM_005693), and
50 ng of CMV
-galactosidase. The luciferase reporter plasmids that
were used include pGL3B-E1b-3XLXRE and pG5luc (Promega,
Madison, WI). Firefly luciferase activity was measured using standard
luciferase substrate reagents (BD PharMingen, San Diego, CA) and was
corrected using
-galactosidase activity as transfection control.
LBD protein (amino acids 162-447) or 600 ng of LXR
LBD protein
(amino acids 202-461), 25 nM
[3H]25-Hydroxycholesterol (Amersham Biosciences), 0.05 mg
of yttrium silicate polylysine-coated SPA beads (Amersham Biosciences),
and varying concentrations of competitor per well of a 96-well
OptiPlate (Packard Instrument Co.). Protein, radioligand, and
competitor were added to the plate. SPA beads were then added to the
assay plate followed by 10 min of gentle shaking at room temperature protected from light. The plates were incubated in the dark at room
temperature for 2 h prior to reading in a TopCount plate reader
(Packard Instrument Co.). PPAR
binding assays were performed using
the ABCD assay (22) as described previously (23).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Fenofibrate represses LXR-induced SREBP
expression. A, SREBP1 transactivation in HepG2 cells.
Stably transfected HepG2 cells harboring a 3XSRE-thymidine
kinase-luciferase reporter construct were treated either with T0901317
(10 µM), with a dose-response of fenofibrate (as
indicated), or with T0901317 (10 µM) in combination with
a dose-response of fenofibrate. Following 24 h of treatment, cells
were lysed, and firefly luciferase activity was measured.
RLU, relative light units. Values are mean ± S.D.
(n > 6). B, SREBP1 mRNA expression in
HepG2 cells. HepG2 cells were treated with T0901317 (10 µM) in the absence or presence of a dose response of
fenofibrate (as indicated) for 24 h. Total RNA was isolated, and
cDNA was generated by random-primed reverse transcription. SREBP1
mRNA and 18 S rRNA were measured in triplicate by quantitative PCR
(see "Experimental Procedures"). The results are expressed as a
ratio of SREBP1 target gene mRNA transcripts to 18 S rRNA
transcripts. These results are representative of at least two separate
studies.
. Treatment of
transfected HEK293 cells with T0901317 caused powerful induction of the
reporter gene. Fenofibrate inhibited T0901317-induced reporter activity
in a dose-dependent manner with an IC50 of
~25 µM (Fig.
2A). Similarly, the natural
LXR agonist (22R)-hydroxycholesterol (22RHC) induced LXR
activity that was potently inhibited by fenofibrate (data not shown).
Interestingly, basal LXR
activity also was inhibited, suggesting
either the presence of an endogenous ligand or low constitutive
activity of the receptor that can be inhibited by an antagonist (24). Similar results were obtained with LXR
(data not shown). Hence, fenofibrate can inhibit LXR-dependent transcription,
irrespective of the nature of the LXR ligand.
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Fig. 2.
Fenofibrate represses
ligand-dependent activation of LXR. A,
transactivation of LXR is repressed by fenofibrate. HEK293 cells
were co-transfected with 100 ng of pGL3B E1b 3XLXRE luciferase plasmid,
100 ng of pCMV6 LXR
, and 50 ng of CMV
-galactosidase. Cells were
treated either with T0901317 (10 µM) (
), with a
dose-response of fenofibrate (
) (as indicated on the
x-axis) or with T0901317 (10 µM) in
combination with a dose response of fenofibrate (
). Following
24 h of treatment, cells were lysed, and firefly luciferase
activity was measured. RLU, relative light units. Values are
mean ± S.D. (n > 6). B, fenofibrate
represses LXR-LBD transactivation. HEK293 cells were co-transfected
with the Gal4-responsive luciferase plasmid (pG5luc), an
expression plasmid encoding LXR
-LBD fused to the GAL4 DNA-binding
domain (pM LXR
-LBD), and CMV
-galactosidase as control. Cells
were treated either with T0901317 (10 µM), with a dose
response of fenofibrate (as indicated), or with T0901317 (10 µM) in combination with a dose response of fenofibrate.
Following 24 h of treatment, cells were lysed, and firefly
luciferase activity was measured. R.L.U., relative light
units. Values are mean ± S.D. (n > 6).
C, fenofibrate binds LXR-LBD directly. The scintillation
proximity radioligand binding assay for LXR was previously described
(21). Receptor protein (His6-hLXR
LBD, amino acids
162-447, 800 ng) immobilized on SPA beads was incubated with 25 nM 3H-labeled 25-hydroxycholesterol in the
presence of increasing amounts of fenofibrate ester or 22RHC. These
results are representative of at least two separate studies.
-LBD. Whereas fenofibrate by itself did not
induce transcription (Fig. 2B), it caused potent inhibition
of LXR agonist-induced LBD-mediated transcriptional activation. Thus,
it became apparent that fenofibrate action might be due to direct
modulation of LXR. If true, fenofibrate should bind to recombinant
LXR-LBD proteins in vitro; therefore, an SPA was developed
using radiolabeled 25-hydroxycholesterol as ligand (Fig. 2C)
(21). Fenofibrate bound to LXR
, potently displacing the tritiated
25-hydroxycholesterol. These findings suggest that in addition to
increasing
-oxidation of fatty acids via activation of PPAR
,
fenofibrate also may modulate LXR-mediated transcription of genes
involved in the lipogenic signaling pathway by direct antagonism of
LXR.
and LXRE-luciferase plasmids or with
Gal4-LXR
and 5XUAS-luciferase plasmids were treated with T0901317 in
the absence or presence of increasing concentrations of the fibrate
esters and acids. The LXR agonist induced transcriptional
activity, which was sustained in the presence of all carboxylic acid
PPAR
ligands (Fig. 3). However, each
of the corresponding fibrate esters repressed agonist-induced transcription with varying potencies. Consistent with this observation, the fibrate esters bound only to the LXRs, whereas the fibric acids
were specific for PPAR
(Table I).
These data expose a degree of promiscuity and similarity in recognition
of ligands by these two nuclear receptors. Clearly, the ester/acid
moiety acts as a switch that determines LXR versus PPAR
affinity, yet the remaining portion of the ligand seems to have
relatively little impact on this selectivity. That this relationship is
not restricted to a single structural class of ligands (fibrates
versus Wy14643) indicates that ligand sharing by PPAR
and
the LXRs may be common. Indeed, the primary amino acid sequences of
human PPAR
and human LXR
ligand binding domains are 30%
identical and share 50% similarity. Since PPAR
and the LXRs are
essential regulators of lipid metabolism, related natural ligands
that regulate both receptors may exist.
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Fig. 3.
Fibrate esters, but not fibric acids,
antagonize LXR. A-E, fenofibrate ester represses
LXR-LBD transactivation. HEK293 cells were co-transfected either with
100 ng of pGL3B E1b 3XLXRE luciferase plasmid, 100 ng of pCMV6 LXR ,
and 50 ng of CMV
-galactosidase (gray bars) or
with the Gal4-responsive luciferase plasmid (pG5luc), an
expression plasmid encoding LXR
-LBD fused to the GAL4 DNA-binding
domain (pM LXR
-LBD), and CMV
-galactosidase, as control
(black bars). After transfection, cells were
treated either with T0901317 (10 µM), with a dose
response of fibrate esters (50, 100, and 150 µM; except
fenofibrate (25, 50, and 100 µM)) or carboxylic acids
(50, 150, and 300 µM) or with T0901317 (10 µM) in combination with a dose-response of fibrate esters
or carboxylic acids. Following 24 h of treatment, cells were
lysed, and firefly luciferase activity was measured. R.L.U.,
relative light units (mean ± S.D.; n > 6).
Fibrate esters bind the LXR-LBD
and the lack of detectable ester in the plasma. However, since the
primary target tissue for the hypolipidemic effects of the fenofibric acid (PPAR
) and ester (LXR) is the liver, we compared the relative levels of these two compounds in the liver in order to determine the
relevance of the LXR antagonist component of fenofibrate action. Mice
were treated with fenofibrate for duration of 7 days followed by
collection of plasma and liver at multiple time points after the final
dose administration. Consistent with previous reports, we observed that
fenofibrate ester was undetectable in mouse serum, whereas the
fenofibric acid metabolite was easily detected (data not shown). In
contrast, fenofibrate ester was readily detectable in the liver (Fig.
4A). The highest concentration
of fenofibrate ester, 3.7 µg/g, was present at 30 min. After 1 h, fenofibrate ester reached steady-state levels in the liver that
persisted for 24 h. Although the ester levels were lower than the
acid levels in the liver, the relative amount is consistent with the
potential for affecting LXR activity, based on the greater affinity of
the ester for the LXRs relative to acid affinity for PPAR
.
Furthermore, the concentration of fenofibrate that is required to
inhibit LXR transactivation might be achievable in human liver, given
commonly prescribed doses of fenofibrate ester (160 mg/day). In order
to determine whether indeed there is an LXR-mediated effect of
fenofibrate treatment in vivo, we examined the ability of
this drug to antagonize the expression of lipogenic LXR target genes in
T0901317 treated mice. After 7 days of treatment either with vehicle,
fenofibrate, T0901317, or a combination of T0901317 and fenofibrate,
the level of SREBP1 and FAS mRNA expression in liver was measured
by quantitative PCR. Mice treated with T0901317 expressed 6-fold higher
levels of SREBP1 mRNA and 12-fold higher levels of FAS mRNA, as
compared with the vehicle-treated mice (Fig. 4B).
Administration of fenofibrate ester alone promoted a decrease in the
basal level of SREBP1 and FAS expression. This may be due to antagonism
of endogenous LXR agonists in the liver. In agreement with the ability
of fenofibrate to antagonize LXR-induced expression of SREBP1 mRNA
in vitro, we observed an ~3-fold reduction in
T0901317-induced SREBP1 and FAS mRNA levels in vivo.
Thus, the novel LXR antagonist action of the fenofibrate ester may be
relevant in vivo.
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Fig. 4.
LXR activation of SREBP1 and FAS gene
transcription is antagonized by fenofibrate in
vivo. A, time course of fenofibrate ester
and fenofibric acid accumulation in liver tissue. Mice (129Sv) were
dosed for 7 days with 300 mg/kg/day fenofibrate, and following a final
dose, perfused livers were collected at the various time points
(n = 3 for each time point). Individual liver samples
(1.0 g) were homogenized in acetonitrile, and debris was pelleted by
centrifugation. Supernatant (50 µl) was diluted with 150 µl of
0.2% formic acid. Fenofibrate ester and acid concentrations were
measured on a Sciex API 3000 mass spectrophotometer. B,
LXR-induced SREBP1 and FAS mRNA expression is repressed in liver
upon fenofibrate administration. Mice (129Sv) were dosed for 7 days
either with vehicle, 50 mg/kg/day T0901317, 100 mg/kg/day fenofibrate,
or a combination (n = 3 in each group). Liver
expression of the mRNAs was measured in triplicate by quantitative
PCR (see "Experimental Procedures"). The results are expressed as a
ratio of target gene mRNA transcripts to 18 S rRNA transcripts
(mean ± S.D.). These results are representative of two separate
studies.
activation of the
endogenous LXR
promoter causing increased in vivo levels
of LXR
(34, 35), or alternatively, the fibrate ester may be
displaying promotor/tissue selective partial agonism/antagonism that
has been reported for ligands of other nuclear receptors such as the
selective estrogen receptor modulators. Unlike the endogenous LXR
antagonist geranylgeranyl pyrophosphate, fenofibrate does not negate
the beneficial role LXR plays in reverse cholesterol transport in
macrophages (36, 37). Rather, fenofibrate exhibits LXR antagonist
activity on SREBP1 and FAS gene transcription and either neutral or
partial agonist activity on ABC transporter expression in liver and
macrophage cells.
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Fig. 5.
Fenofibrate ester does not antagonize LXR
transactivation of ABCA transporter gene expression in liver and in
macrophages. A, mice (129Sv) were dosed for 7 days
either with vehicle, 50 mg/kg/day T0901317, 100 mg/kg/day fenofibrate,
or a combination (n = 3 in each group, same mice as in
Fig. 4). Liver expression of ABCA1, ABCG5, and ABCG8 mRNAs was
measured in triplicate by quantitative PCR (see "Experimental
Procedures"). The results are expressed as a ratio of target gene
mRNA transcripts to 18 S rRNA transcripts (mean ± S.D.).
B, differentiated THP-1 macrophage cells were treated with
either 22RHC (10 µM) or T0901317 (10 µM),
with a dose response of fenofibrate (as indicated) or with T0901317 (10 µM) or 22RHC (10 µM) in combination with a
dose response of fenofibrate, as indicated. 20 µg of total RNA was
subjected to Northern analysis using an ABCA1 cDNA fragment as
probe; 36B4 served as a loading control. Gene expression was
quantitated by scanning densitometry and is shown in the
histogram below.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-hydroxylase, LXR has emerged as an attractive target
for the development of drugs for cardiovascular disease therapy (11, 12, 14-16, 18). However, in addition to the desirable pharmacologic effects of increasing reverse cholesterol transport from the periphery and hepatic cholesterol catabolism upon activating LXR target gene
transcription, the undesirable side effect of increasing hepatic
lipogenesis occurs in the presence of LXR agonists. Here, we have
described a novel mechanism by which the clinically utilized hypolipidemic compound, fenofibrate, antagonizes LXR and represses lipogenic gene expression in liver, but not members of the ABC transporter gene family (ABCA1, ABCG5, and ABCG8).
/LXR activity, fenofibrate ester is
the first clinical compound known to target LXR, and it may be a useful
tool to explore LXR pharmacology. Notably, fibrate esters bound to LXR
with higher affinity than was anticipated, based on fibric acid
potencies in PPAR
-based assays (Table I) (42). Combined with further
identification of molecules that interact with LXR, these studies may
contribute to the design of new LXR modulators that can meet the
clinical need of decreasing atherosclerotic lesions without promoting hypertriglyceridemia.
agonists can be transformed
into LXR antagonists by simple modification to an ester indicates
similarity in ligand recognition. This suggests that there may be
structurally similar natural ligands for these two receptors that
underlie the evolutionary need for this degree of conservation of
ligand recognition between PPAR
and the LXRs. In fact, certain fatty
acids already have been shown to function both as PPAR
agonists and
as LXR antagonists. Arachidonate (C20:4) binds to and activates human
PPAR
at micromolar concentrations (IC50 = 1.2 µM), and at approximately the same concentration, arachidonate antagonizes synthetic ligand binding to LXR and
transcription of the LXR target gene, SREBP1 (43). Interestingly,
antagonism of LXR by polyunsaturated fatty acids such as
C20:4,n6 or C22:6,n3 occurs in an isoform-selective manner;
LXR
is antagonized, whereas LXR
is not (44). Such observations of
isoform-selective modulation further imply the molecular possibility of
regulating certain LXR target genes while having no effect on others
through the use of distinct, isoform-selective LXR ligands.
,
leading to induction of genes that control fatty acid
-oxidation;
they also decrease SREBP1 and FAS gene expression by antagonizing
LXR-mediated transcription. Like the activities of some selective
estrogen receptor modulators, fibrate esters display LXR partial
agonist/antagonist activity that is dependent on the target gene
context. These data ascribe a novel regulatory function to the
clinically utilized fibrate drugs and have therapeutic implications for
identification of compounds that increase cellular cholesterol efflux
through LXR yet counteract the accumulation of triglycerides by
utilizing a promoter-selective mode of action.
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ACKNOWLEDGEMENTS |
---|
We thank Drs. Marian Mosior, Minmin Wang, and Nathan Mantlo for valuable discussions and Samuel Oldham and Rick Zink for technical support.
![]() |
FOOTNOTES |
---|
* 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.
§ These authors contributed equally to this work.
§§ To whom correspondence should be addressed: Eli Lilly and Co., Cardiovascular Research, DC 0520, Indianapolis, IN 46285. Tel.: 317-433-9468; Fax: 317-433-2815; E-mail: laura_michael@lilly.com.
Published, JBC Papers in Press, November 18, 2002, DOI 10.1074/jbc.M209629200
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ABBREVIATIONS |
---|
The abbreviations used are:
PPAR, peroxisome
proliferator-activated receptor
;
FAS, fatty acid synthase;
SRE, sterol regulatory element;
SREBP1, sterol regulatory element-binding
protein-1;
LXR, liver X receptor;
CMV, cytomegalovirus;
SPA, scintillation proximity assay;
22RHC, (22R)-hydroxycholesterol;
LBD, ligand binding domain;
ABC, ATP-binding cassette.
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