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Molecular Endocrinology 17(7):1255–1267
Copyright © 2003 by The Endocrine Society
doi: 10.1210/me.2002-0191

Cross-Talk between Peroxisome ProliferatorActivated Receptor (PPAR) ␣ and Liver X Receptor(LXR) in Nutritional Regulation of Fatty Acid
Metabolism. II. LXRs Suppress Lipid Degradation
Gene Promoters through Inhibition of
PPAR Signaling
TOMOHIRO IDE, HITOSHI SHIMANO, TOMOHIRO YOSHIKAWA, NAOYA YAHAGI,MICHIYO AMEMIYA-KUDO, TAKASHI MATSUZAKA, MASANORI NAKAKUKI, SHIGERU YATOH,
YOKO IIZUKA, SACHIKO TOMITA, KEN OHASHI, AKIMITSU TAKAHASHI, HIROHITO SONE,
TAKANARI GOTODA, JUN-ICHI OSUGA, SHUN ISHIBASHI, AND NOBUHIRO YAMADA
Department of Internal Medicine (T.I., H.S., T.M., M.N., S.Y., A.T., H.S., N.S.), Institute of Clinical
Medicine, University of Tsukuba, Ibaraki 305-8575, Japan; and Department of Metabolic Diseases(T.Y., N.Y., M.A.-K., Y.I., S.T., K.O., T.G., J.O., S.I.), Faculty of Medicine, University of Tokyo,
Bunkyo-ku, Tokyo 113-8655, Japan
Fatty acid metabolism is transcriptionally regulated by two reciprocal systems: peroxisome
proliferator-activated receptor (PPAR) ␣ controls
fatty acid degradation, whereas sterol regulatory
element-binding protein-1c activated by liver X
receptor (LXR) regulates fatty acid synthesis. To
explore potential interactions between LXR and
PPAR, the effect of LXR activation on PPAR␣ signaling was investigated. In luciferase reporter geneassays, overexpression of LXR␣ or ␤ suppressed
PPAR␣-induced peroxisome proliferator response
element-luciferase activity in a dose-dependent
manner. LXR agonists, T0901317 and 22(R)hydroxycholesterol, dose dependently enhanced
the suppressive effects of LXRs. Gel shift assays
demonstrated that LXR reduced binding of PPAR␣/
retinoid X receptor (RXR) ␣ to peroxisome proliferator response element. Addition of increasing
amounts of RXR␣ restored these inhibitory effects
in both luciferase and gel shift assays, suggesting
the presence of RXR␣ competition. In vitro protein
binding assays demonstrated that activation of
LXR by an LXR agonist promoted formation of
LXR/RXR␣ and, more importantly, LXR/PPAR␣

heterodimers, leading to a reduction of PPAR␣/
RXR␣ formation. Supportively, in vivo administration of the LXR ligand to mice and rat primary
hepatocytes substantially decreased hepatic
mRNA levels of PPAR␣-targeted genes in bothbasal and PPAR␣ agonist-induced conditions. The
amount of nuclear PPAR␣/RXR heterodimers in
the mouse livers was induced by treatment with
PPAR␣ ligand, and was suppressed by superimposed LXR ligand. Taken together with data fromthe accompanying paper (Yoshikawa, T., T. Ide, H.
Shimano, N. Yahagi, M. Amemiya-Kudo, T. Matsuzaka, S. Yatoh, T. Kitamine, H. Okazaki, Y. Tamura,
M. Sekiya, A. Takahashi, A. H. Hasty, R. Sato, H.
Sone, J. Osuga, S. Ishibashi, and N. Yamada, Endocrinology 144:1240–1254) describing PPAR␣
suppression of the LXR-sterol regulatory elementbinding protein-1c pathway, we propose the presence of an intricate network of nutritional transcription factors with mutual interactions, resulting
in efficient reciprocal regulation of lipid degradation and lipogenesis. (Molecular Endocrinology 17:
1255–1267, 2003)

R

isome proliferator-activated receptors (PPARs) andliver X receptors (LXRs), whereas roles by sterol regulatory element-binding protein (SREBP) family havebeen established as lipid synthetic regulators. PPARs
are members of a nuclear receptor superfamily and are

ECENT PROGRESS IN studying transcriptional
regulation of lipid catabolism has unveiled thefunctions of orphan nuclear receptors such as peroxAbbreviations: ABCA1, ATP-binding cassette transporter A1;
ACO, acyl-CoA oxidase; 36B4, acidic ribosomal phosphoprotein PO; CBP, cAMP-response element binding protein-binding
protein; CMV, cytomegalovirus; CoA, coenzyme A; CPTI, Lcarnitine palmitoyltransferase; FBS, fetal bovine serum; FXR,
farnesoid X receptor; ␤-gal, ␤-galactosidase; HEK, human embryonic kidney; Luc, luciferase; LXR, liver X receptor; LXRE, liver
X receptor response element; m, mouse; mHMG-CoA syn, mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase; PGC-1,

peroxisome proliferator-activated receptor ␥ coactivator-1;
PPAR, peroxisome proliferator-activated receptor; PPRE, peroxisome proliferator responsive element; pSV, simian virus 40
promoter plasmid; 22RHC, 22(R)-hydroxycholesterol; RXR, retinoid X receptor; SREBP-1c, sterol regulatory element-binding
protein 1c; TK, thymidine kinase; TR, thyroid hormone receptor;
VDR, vitamin D3 receptor.
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Mol Endocrinol, July 2003, 17(7):1255–1267

Ide et al. • LXRs Suppress Lipid Degradation Gene Promotersstructurally related to other members such as thyroid
hormone receptor (TR) and vitamin D3 receptor (VDR)(1). PPARs are known to regulate expression of numerous genes involved in fatty acid metabolism andadipocyte differentiation (2, 3). PPAR␣ is abundantly
expressed in tissues, which have high lipid catabolic
activity, such as liver, kidney, heart, skeletal muscle,
and brown adipose tissue (4, 5). PPAR␣ is activated byfatty acids, eicosanoids, and fibrates, a known class ofhypolipidemic drugs. Like other nuclear receptors
such as: TR, VDR, and retinoic acid receptor (RAR)(6–9), PPAR␣ forms a heterodimer with RXR␣, which
enhances its binding to DNA sequence elements
termed peroxisome proliferator response elements(PPRE) (10–12). PPREs have been recently identified in
the 5⬘-flanking sequences of genes involved in lipid
degradation such as the ACO [acyl-coenzyme A (CoA)oxidase] (13), mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (mHMG-CoA Syn) (14), L-carnitine
palmitoyltransferase (CPTI) (15), and L-fatty acid binding protein genes (16). Studies using PPAR␣-deficient
mice have established that PPAR␣ plays a crucial rolein fatty acid degradation (17–19).
LXRs (LXR␣/NR1H3 and LXR␤/NR1H2) were identified as orphan nuclear receptors and are now
thought to regulate the metabolism of several important lipids, including cholesterol and bile acid (20, 21).
LXRs regulate intracellular cholesterol levels by
induction of the gene expression of cholesterol 7␣hydroxylase (22, 23), which is the rate-limiting enzymeof the classic bile acid synthesis pathway, and ATPbinding cassette transporter A1 (ABCA1) (24), which
modulates apolipoprotein mediated-efflux of cholesterol. Further evidence supporting an important role of
LXR␣ in lipid homeostasis is provided by the loss of
capacity to regulate catabolism of dietary cholesterolin LXR␣ deficient mice, an effect for which the isoform,
LXR␤, could not compensate (25). Differences in thephysiological functions between LXR␣ and LXR␤ including target and tissue specificity have been suggested (26). LXR␤ is ubiquitously expressed, whereas
LXR␣ is restricted to metabolically active tissues, suchas liver, kidney, intestines, and adrenal glands (27, 28).
Recently, we (29) and others (30) reported that both
LXR ␣ and ␤ are dominant activators for SREBP-1c/
adipocyte determination and differentiation 1 (ADD1).
Previous in vivo studies established that SREBP-1cplays a crucial role in the dietary regulation of mosthepatic genes of fatty acid synthetic enzymes (31–34).
Therefore, LXRs could be also important in fatty acidsynthesis (35).
As PPAR␣ and LXR-SREBP-1c are reciprocal regulators for fatty acid metabolism, nuclear receptors
could interact with each other as was previously observed in a cross-talk between PPAR␣ and TR (36, 37).
The current study examined the effects of LXR activation on PPAR␣ signaling. The results demonstrate that
LXR␣ ligand activation represses PPAR␣ signaling
through reduction of stimulated-PPAR␣/RXR heterodimerization in the liver. Taken together with theaccompanying paper (38) describing PPAR suppression of the LXR-SREBP-1c pathway, we propose anovel aspect of nutritional regulation with these mutualinteractions forming a network of transcription factors
regulating fatty acid metabolism.

Fig. 1. Inhibition of PPAR␣-Induced PPRE-Luc Activity by
LXR␣ and ␤
HEK293 cells were transfected with a reporter plasmid(0.25 ␮g): pPPRE-Luc (A and B) or constitutive Luc plasmid,
pTK-Luc (C), expression plasmids (0.05 ␮g): CMV-mPPAR␣with CMV-mLXR␣ (A) or CMV-mLXR␤ (B), and a reference
plasmid (0.2 ␮g): pSV-␤-gal. Cells were treated with either
ethanol as vehicle, 3 ␮M of 22(R)HC or T0901317. All Luc
activities were corrected for transfection efficiency by measuring ␤-gal activities. Results are means ⫾ SE from three
independent experiments.

Ide et al. • LXRs Suppress Lipid Degradation Gene Promoters

RESULTS
LXR Activation Represses PPAR␣-MediatedTransactivation
To estimate the effect of LXR/RXR␣ activation on
PPAR␣ signaling, transfection studies with human embryonic kidney (HEK) 293 cells were performed using
luciferase (Luc) reporter gene assays containing a
PPRE from the ACO gene promoter [pPPRE-Luc(PPRE-Luc plasmid)], a representative PPAR␣ target.
As shown in Fig. 1, the Luc activity of PPRE-Luc wasmarkedly (60-fold) induced by cotransfection of
PPAR␣ due to lack of endogenous PPAR expressionin HEK293 cells (data not shown). The PPAR␣-inducible expression of Luc activity was slightly, but dose
dependently suppressed by coexpression of LXR␣(Fig. 1A) (50% inhibition at a DNA dose ratio of 1:10).
Expression level of transfected PPAR␣ or LXR gene in
HEK293 cells were roughly comparable to that inmouse liver as estimated by Northern blotting, andthus was within a physiological range (data notshown). This observation is consistent with a previous
report describing LXR interaction with PPAR␣ signaling (39). More interestingly, addition of LXR ligands
such as 22RHC and T0901317 markedly enhanced theinhibitory effects of LXR␣. LXR␤ alone, without an LXR
ligand, substantially inhibit PPAR␣ activation of PPRELuc in a dose-dependent manner (Fig. 1B). A 50%
inhibition was observed at a DNA ratio of 1:5. Additional LXR ligands further augmented the inhibitory
effect of LXR␤. These effects by LXRs and LXR ligands
are not due to direct inhibition of Luc activity because
Luc activity from constitutive expression of Luc gene

Mol Endocrinol, July 2003, 17(7):1255–1267

1257

by TK promoter-Luc construct was not affected by
LXR coexpression, by addition of LXR ligand, or a
combination of both (Fig. 1C). These data demonstratethat LXRs and their ligands inhibit PPAR signaling withoverexpressing nuclear receptors system.
LXR Cannot Bind to PPRE, but Inhibits PPAR␣/
RXR␣ Binding to PPRE
The inhibitory effect of LXR was estimated in the lightof the dose dependency of LXR ligands in Fig. 2. As apositive control, a dose-response curve of LXRE (LXR
response element)-Luc activation by T0901317 and
22RHC with coexpression of LXR␣ is shown in theupper panel, which is consistent with previous data(29). With a similar efficiency, these LXR agonists inhibited PPAR␣ activation of PPRE-Luc, making bothcurves a mirror image of each other. LXR␤ coexpression
gave similar results with a slightly higher efficiency.
To further investigate the mechanism for inhibitionof PPAR␣ by LXR, gel mobility shift assays were performed. The binding specificity of LXR/RXR␣ to LXRE,
and PPAR␣/RXR␣ to PPRE was confirmed in the experiments shown in Fig. 3, A and B. RXR␣ is required
for specific binding of both LXR and PPAR. Neither
LXR␣/RXR␣ nor LXR␤/RXR␣ bound to PPRE within
the sensitivity of this assay. Coincubation of PPAR␣
and LXR␣ or ␤ essentially caused no shift using PPRE
probe. These data exclude the possibility that LXR/
RXR␣ competes with PPAR␣/RXR␣ binding to a response element. Next, PPAR␣/RXR␣ binding to PPRE
was estimated in the presence of LXR and/or its ligand(Fig. 4). PPAR␣/RXR␣ binding to PPRE was partiallyinhibited by addition of LXR protein. LXR ligand en-

Fig. 2. Dose-Dependent Reversal Effects of LXR Ligands on LXR- and PPAR␣-Mediated Transactivation
HEK293 cells were transfected with 0.25 ␮g of pLXRE-Luc (A and B) or pPPRE-Luc (C and D) as a reporter plasmid, 0.05 ␮gof pCMVmLXR␣ (A and C), pCMVmLXR␤ (B and D) as an expression plasmid and pSV-␤-gal as a reference plasmid were
cotransfected in the absence (A and B) or presence (C and D) of 0.05 ␮g of pCMV-mPPAR␣. Cells were treated with either ethanol
as vehicle or the indicated concentrations of 22(R)HC or T0901317. All Luc activities were corrected for transfection efficiency bymeasuring ␤-gal activities.

1258 Mol Endocrinol, July 2003, 17(7):1255–1267

Ide et al. • LXRs Suppress Lipid Degradation Gene PromotersFig. 3. No Binding of LXR␣ or ␤ to the PPRE
Gel shift analysis was performed using in vitro translated receptor proteins as described in Materials and Methods with an LXR
ligand (3 ␮M T0901317). In vitro translated LXR␣ or ␤ (2 ␮l), PPAR␣ (1 ␮l), and/or RXR␣ (1 ␮l) as indicated were incubated with
32
P-labeled SREBP1c-LXRE (A) or 32P-labeled ACO-PPRE (B), and resolved on a 4.5% PAGE. Excess competitor was in 50-foldmolar excess of unlabeled LXRE or PPRE.

Fig. 4. LXR␣ and ␤ Inhibition of PPAR␣/RXR␣ Binding to the
PPRE
Gel shift analysis was performed using in vitro translated
receptor proteins as indicated with LXR ligands as indicated.
In vitro translated PPAR␣ (1 ␮l) and RXR␣ (1 ␮l), with orwithout LXR␣ or ␤ (4 ␮l) in the presence or absence of LXRligands [10 ␮M 22(R)HC or T0901317] were incubated with
32
P-labeled ACO-PPRE, and resolved on a 4.5% PAGE.

hanced this inhibition. These data are consistent withresults from reporter assays, suggesting that suppression of PPAR␣ activity by LXR is mediated at least partly
through inhibition of PPAR␣/RXR␣ binding to PPRE.
RXR Restores LXR Repression of PPAR␣/RXR␣Mediated Transactivation
Because PPAR␣ and LXRs share RXR␣ as a heterodimer partner for their specific functions, it is con-ceivable that LXR inhibition of PPAR␣ activation on
PPRE-Luc is mediated through competition for RXR␣when RXR␣ is in limiting amounts in cultured cells.
Thus, the effect of supplementation with RXR␣ on the
LXR inhibition of PPRE-Luc activity was evaluated(Fig. 5). By cotransfection of an increasing amount of
RXR␣, PPAR␣-activated PPRE-Luc activity was enhanced, indicating that the amount of RXR␣ is not
saturated for PPAR␣/RXR␣ activation (Fig. 5A). Figure
5B shows effect of each amount of RXR␣ on thepercent inhibition of PPAR␣-induced PPRE-Luc values by LXR␣ activation (LXR␣ coexpression with orwithout an LXR ligand, T0901317 or 22RHC). The inhibitory efficiency of LXR activation was attenuated bycoexpressed RXR␣ in a dose-dependent manner (Fig.
5B). Similar results were obtained in the repression by
LXR␤ (Fig. 5C).
Next, we compared the inhibitory action of LXR on
PPAR signaling with other RXR heterodimer partners,
TR and farnesoid X receptor (FXR) (Fig. 6A). TR␤
strongly suppressed PPRE-Luc activity, which is consistent with a previous report (37). Inhibition by FXR
was modest. The ability of inhibitory action on PPAR␣mediated transactivation appears to be different
among LXR, TR␤, and FXR, suggesting their constitutive activity based on levels of intracellular natural
ligands may influence cross-talk in heterodimeric receptors with RXR␣.
cAMP response element binding protein-binding
protein (CBP)/p300 is known to be involved in liganddependent activation of PPARs (40–42). However, unlike RXR, overexpression of CBP/p300 did not restore
on LXR suppression of PPAR␣-induced PPRE-Luc activity (Fig. 6B).
The RXR restoration of LXR-dependent suppression
was also evaluated in gel mobility shift assays. Without
LXR, the signal of PPRE shifted by PPAR␣ was enhanced with increasing amounts of RXR␣, suggestingthat the amount of RXR␣ is a limiting factor in theIde et al. • LXRs Suppress Lipid Degradation Gene Promoters

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Fig. 5. Effect of Supplementation with RXR␣ on LXR Inhibition of PPAR␣/RXR␣-Induced PPRE-Luc Activity
HEK293 cells were transfected with pPPRE-Luc as reporter plasmid (0.25 ␮g), pCMVmPPAR␣ (0.05 ␮g), the indicated amountof pCMV-RXR␣ as expression plasmids and SV-␤-gal (0.2 ␮g) as a reference plasmid were cotransfected (A). Inhibition of
PPAR␣-mediated PPRE-Luc activation by LXR was evoked by cotransfection with CMV-mLXR␣ (B) or CMV-mLXR␤ (C) (0.05 ␮g).
RXR-restoration was shown by further cotransfection with the indicated amount of pCMV-RXR␣ (B and C). Cells were treated witheither ethanol as vehicle, 22(R)HC or T0901317 (3 ␮M). All Luc activities were corrected for transfection efficiency by measuring
␤-gal activities. Results are means ⫾ SE from three independent experiments.

Fig. 6. Effects of RXR Heterodimers (A) and CBP/p300 (B) on PPRE-Luc Activity
A, pPPRE-Luc and pSV-renilla Luc were cotransfected into HEK293 cells with indicated expression plasmids: pCMV-mPPAR␣(0.25 ␮g), pCMV-LXR␤ (0.25 ␮g), pCMV-TR␤ (0.25 ␮g), and pCMV-FXR (0.25 ␮g). After incubation for 24 h, Luc activity wasmeasured and normalized to renilla Luc activity. B, pPPRE-Luc and pSV-renilla Luc were cotransfected into HEK293 cells withindicated expression plasmids: pCMV-PPAR␣ (0.25 ␮g), pCMV-LXR␣ (0.25 ␮g), pCMV-CBP (0.5 ␮g), pCMV-p300 (0.5 ␮g), andpCMV-RXR␣ (0.5 ␮g). Cells were treated with or without 3 ␮M Wy14,643 and/or 3 ␮M T0901317. After incubation for 24 h, Luc
activity was measured and normalized to renilla Luc activity. All results are means ⫾ SE from three independent experiments.

range of PPAR␣/RXR␣ ratio used here (Fig. 7A). With
the lowest amount of RXR␣, addition of LXRs andT0901317 caused a significant decrease in PPAR␣/
RXR␣ binding to PPRE (Fig. 7, A and B). The inhibitory
effect of LXR␣ activation was abolished with increasing concentrations of RXR␣. In the case of LXR␤, RXR
restoration of the binding was partial even at the highest amount, reflecting stronger LXR␤ inhibitory effect.
These data are consistent with the results from cell

reporter assays (Fig. 5), providing additional evidence
for the hypothesis that LXR inhibits PPAR␣ activationby competing with it for heterodimerization with RXR␣.
LXR Can Bind to PPAR␣ and Inhibits Formationof PPAR␣/RXR␣ Heterodimer
To explore the heterodimerization of the three nuclear
receptors, mutual protein-protein interactions were in-1260 Mol Endocrinol, July 2003, 17(7):1255–1267

Ide et al. • LXRs Suppress Lipid Degradation Gene PromotersFig. 7. Effect of Supplementation with RXR␣ on LXR Inhibition of PPAR␣/RXR␣ Binding to PPRE
Gel shift analysis was performed using in vitro translated receptor proteins as indicated. In vitro translated PPAR␣ (1 ␮l), RXR␣(1, 2, and 4 ␮l), and LXR␣ (2 ␮l, A) or LXR ␤ (2 ␮l, B) were incubated with 32P-labeled PPRE in the absence or presence ofT0901317 (3 ␮M) and resolved on a 4.5% PAGE.

Fig. 8. LXR Inhibits PPAR␣/RXR␣ Heterodimerization by the Formation of LXR/RXR␣ and LXR/PPAR␣In vitro translated receptor proteins as indicated were used for protein-protein interaction assay. Indicated proteins were usedin the absence or presence of T0901317. Coimmunoprecipitation of the unlabeled RXR␣ and 35S-labeled LXR␣ (A) or ␤ (B) withan anti-RXR antibody; unlabeled PPAR␣, and 35S-labeled LXR␣ (C) or ␤ (D); 35S-labeled PPAR␣, unlabeled RXR␣, and unlabeled
LXR␣ (E) or ␤ (F). After precipitation with protein G-sepharose, samples were run on SDS-PAGE.

vestigated (Fig. 8). First, LXR␣ protein was labeledwith 35S-methionine, incubated with unlabeled RXR␣protein, and precipitated with anti-RXR␣ antibody.
Resolution of immunocomplex on SDS-PAGE de-tected labeled LXR␣, indicating formation of an LXR␣/RXR␣ heterodimer. The complex formation was markedly enhanced by addition of T0901317, suggestingthat LXR ligand increases affinity for LXR␣/RXR␣ het-

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Fig. 9. PPAR␣ Activator Inhibits LXR Ligand-Induced LXR/RXR␣ Binding to PPRE in Hepatic Nuclear Extracts
A, Protein and mRNA levels of PPAR␣, LXR␣, and RXR␣ in hepatic nuclear extracts from fasted or refed mice. For fasting andrefeeding treatment, mice were fasted for 24 h and fed a high sucrose/fat-free diet for 12 h. Equal protein amounts (20 ␮g) innuclear extracts were subjected to SDS-PAGE, and immunoblotted with an anti-PPAR␣, anti-LXR␣, or anti-RXR␣ antibody. Total
RNA was isolated from the livers of mice from each group, pooled, and subjected to Northern blot analysis with the indicatedcDNA probes. B, Confirmation of presence of PPAR␣ in Wy14,643 induced-complexes of nuclear proteins/ACO-PPRE in nuclear
extracts from fasted mice. The 32P-labeled ACO-PPRE probe and hepatic nuclear extracts (1 mg) from fasted mice wereincubated with Wy14,643 in the presence or absence of anti-PPAR␣ (H-98, sc-9000, Santa Cruz Biotechnology Inc.) or anti-RXRa(D-20, sc-553, Santa Cruz Biotechnology Inc.) antibody. The DNA-protein complexes were resolved in a 4.5% PAGE. C,
Wy14,643 induced-PPAR␣/RXR␣ binding to PPRE was inhibited by addition of T0901317. 32P-labeled ACO-PPRE probe andhepatic nuclear extracts from fasted mice were incubated with Wy14,643 and/or T0901317. The DNA-protein complexes wereresolved in a 4.5% PAGE.

erodimerization (Fig. 8A). We also tested the possibilityof formation of PPAR␣/LXR␣ heterodimers, as waspreviously described (39). Labeled LXR␣ was incubated with PPAR␣ and immunoprecipitated with antiPPAR␣ antibody. PPAR␣/LXR␣ complex was detected with an intensity comparable to LXR␣/RXR␣,suggesting that LXR␣ can heterodimerize with PPAR␣with similar efficiency to RXR␣ (Fig. 8C). T0901317
enhanced this interaction. LXR␤ gave very similar results (Fig. 8, B and D). The gel shift assay (Fig. 3B)
showed no retardation of the PPRE probe in the presence of both PPAR␣ and LXR␣, indicating that
PPAR␣/LXR␣ heterodimers do not bind to PPRE.
Therefore, LXR␣ could show inhibition of PPAR␣ signaling by forming not only LXR␣/RXR␣, but also
PPAR␣/LXR␣ in a competition with functional PPAR␣/RXR formation. To show this, effects of LXRs on
PPAR␣/RXR␣ interaction were estimated in this protein interaction assay. PPAR␣ protein was labeled,incubated with RXR␣ in the presence or absence of

LXR␣, precipitated with anti-RXR␣ antibody, and run
on gels. PPAR␣/RXR␣ heterodimer formation was inhibited by addition of LXR␣ or LXR␤ with T0901317(Fig. 8, E and F), suggesting that PPAR␣/LXR as wellas LXR/RXR␣ could prevent formation of PPAR␣/
RXR␣ and participate in repressing PPAR␣ signaling.
LXR Activation Inhibits PPAR␣/RXR␣ Binding to
PPRE in Hepatic Nuclear Extracts
To examine suppressive action of LXR on PPAR/RXR
binding to PPRE in a more physiological condition, gelmobility shift assays were performed using hepatic
nuclear extracts from fasted mice. In nuclear extracts
from fasted mice, LXR␣ and RXR␣ were abundant on
immunoblot analysis, although PPAR␣ was decreased
after refeeding (Fig. 9A) as well as at mRNA level. Gel
shift mobility assays demonstrated that the PPRE
probe was shifted by coincubation with hepatic nuclear extracts. The signal of the complexes was stron-

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Ide et al. • LXRs Suppress Lipid Degradation Gene PromotersFig. 10. LXR Ligand Suppresses PPAR␣-Target Gene Expression in Rat Primary Hepatocytes
A, T0901317 inhibited gene expression of mHMG-CoA Syn in rat primary hepatoctyes. The cells were incubated with theindicated concentration of T0901317 for 24 h. B, T0901317 suppressed induction of mHMG-CoA Syn gene expression by
Wy14,643 treatment. The hepatocytes were incubated with the indicated concentration of T0901317 in the absence or presenceof Wy14643 for 24 h. Total RNA (10 ␮g) was extracted and Northern blot analysis was performed with the indicated cDNA probes.
Fold changes of expression relative to corresponding with controls are shown.

ger from fasted mice than in a refed state, was enhanced by addition of Wy14,643 and declined after
addition of anti-PPAR␣ or anti-RXR␣ antibody, confirming the presence of PPAR␣/RXR␣ in the hepatic
nuclear extracts from fasted mice and its specificbinding to PPRE (Fig. 9B). The PPRE complex also
contained a small amount of unidentified RXR heterodimer(s). T0901713 suppressed the signal of the
PPAR␣/RXR␣-PPRE complex induced by Wy14,643(Fig. 9C). Thus, these data indicate that LXR ligand
suppressed PPAR␣ ligand-induced PPAR␣/RXR␣PPRE complexes in a condition with physiological levels and spectrum of various nuclear receptors.
LXR Activation Represses PPAR␣ Target Gene inPrimary Hepatocytes and Mouse Livers
To extend these findings to a system with more physiological relevance, LXR interference with PPAR␣ signaling was estimated using their ligands in rat primary
hepatocytes. Hepatocytes were incubated with increasing amounts of T0901317 and expression of related target genes was estimated by Northern blot
analysis (Fig. 10). LXR activation was confirmed by adose-dependent increases in SREBP-1c and ABCA1mRNA levels (Fig. 10A). LXR ligand dose dependently
suppressed a representative PPAR␣ gene, mHMG-

CoA syn mRNA level. In the presence of Wy14,643 at
a concentration sufficient to induce mHMG-CoA syn, aclear dose-dependent suppression by T0901317 wasstill observed (Fig. 10B). These data demonstrate that
LXR activation by its ligand suppresses PPAR␣-target
genes, in a competitive manner with PPAR␣ activation, supporting LXR interference with PPAR␣ signaling in primary hepatocytes. To further evaluate the invivo physiological significance of LXR interference to
PPAR␣-mediated transactivation, similar conditions inmouse livers were recreated. Mice were fasted to induce hepatic endogenous PPAR␣ and its downstream
genes (17, 31) and were treated with T0901317 toactivate LXR. Various PPAR␣-target genes in the livers
such as mHMG CoA Syn, ACO, CPTI, and cytochrome
P450A2 were estimated by Northern blot analysis.
Wy14,643 was also administered to fasted mice as apositive control for activation of PPAR␣ target genes.
As shown in Fig. 11A, hepatic mRNA levels of these
PPAR␣-regulated genes from T0901317 treated animals were considerably decreased, whereas Wy14643
treatment increased expression of these genes. The
activation of LXR by T0901317 was confirmed by observational increases in SREBP-1 and ABCA1 mRNAlevels, both of which are well-known LXR target genes(Fig. 11B). These data demonstrate that LXR activation

Ide et al. • LXRs Suppress Lipid Degradation Gene Promoters

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1263

Fig. 11. Effect of LXR Ligand on PPAR␣ Target Gene Expression in the Livers of Fasted Mouse
A–C, Mice (n ⫽ 5) were fasted and treated with T0901317 (50 mg/kg) or Wy14,643 (50 mg/kg) for 18 h. D, Mice (n ⫽ 3) were
fasted and treated with T0901317 (50 mg/kg), Wy14,643 (50 mg/kg), or both these ligands for 18 h. Total RNA was isolated fromthe livers of mice from each group, pooled, and subjected to northern blot analysis with the indicated cDNA probes. Foldincreases of expression relative to corresponding with vehicle-treated controls are shown. E, T0901317 inhibited Wy14,643induced LXR/RXR␣ heterodimer formation in the liver from C57BL6 mice. Hepatic nuclear extracts were prepared from eachgroup. Equal protein amounts of nuclear extracts were subjected to immunoprecipitation by using anti-PPAR␣ antibody, which
had been coupled to protein G-sepharose beads. Immunoprecipitates were subjected to SDS-PAGE, and immunoblotted withan anti-RXR␣ antibody. Arrowheads represent RXR␣ or IgG signal.

by LXR ligand can affect PPAR␣ signaling in mouse
livers. In contrast, Wy14,643 alone slightly increased
ABCA1 mRNA (Fig. 11B), which is consistent withrecently reports of PPAR-LXR-ABCA1 pathway inmacrophages (43–45). Moreover, we studied LXR
interference in mice treated with both PPAR␣ and
LXR ligands. As estimated by Northern blot analysis
of mHMG-CoA syn and ACO mRNA levels, these
PPAR␣-target genes were suppressed by T0901317
in both Wy14,643-induced and untreated conditions(Fig. 11D). Immunoprecipitation assays of liver nuclear extracts from these mice demonstrated that
PPAR␣/RXR␣ heterodimers were increased by
Wy14,643 treatment and this induction was suppressed by T0901317. These results suggested that
LXR␣ ligand could repress hepatic expression of the
PPAR␣ target genes via reduction of PPAR␣/RXR␣
formation in liver nucleus (Fig. 11E), extending the
hypothesis from transfection studies to in vivo
conditions.

DISCUSSION
The current studies demonstrate that LXR activationby either overexpression of LXR or its ligand activation
causes suppression of PPAR␣ signaling. The Luc, gelshift, and protein-protein binding assays show that
LXR activation inhibits formation of PPAR␣/RXR␣, andthus, their binding to PPRE. RXR␣ supplementation
restored this LXR inhibition of PPAR action, indicatingthat the mechanism could be at least partially explained by RXR␣ competition between PPAR and LXR.
Protein-protein interaction experiments establishedthe formation of PPAR␣/LXR␣ and PPAR␣/LXR␤ as
well as LXR/RXR heterodimers. Direct binding between PPAR␣ and LXR␣ has been found in a twohybrid system, and involvement of LXR␣ in PPAR signaling has been implicated (39). We further found that
PPAR␣/LXR formation is enhanced by LXR ligand.
PPAR␣/LXR complex cannot bind to either PPRE or
LXRE. Therefore, this heterodimer can interfere with

1264 Mol Endocrinol, July 2003, 17(7):1255–1267

Fig. 12. Mutual Interactions between PPAR␣ and LXRSREBP-1c in Reciprocal Regulation of Fatty Acid Metabolism
Current cross-talk of PPAR␣, LXR, and SREBP-1c in nutritional regulation is schematized, based upon the current
study and the accompanying paper (38).

both PPAR and LXR action and can contribute to
PPAR inhibition of the LXR-SREBP-1c system as proposed in the accompanying paper (38) as well as LXR
inhibition of PPAR signaling as described in the current study. Consistently, the combined treatment withboth PPAR␣ and LXR agonists resulted in simultaneous reduction of both PPAR␣/RXR␣ and LXR/RXR␣
[Fig. 11E in this paper and Fig. 9B in the accompanying paper (38)], implicating an important contributionof PPAR␣/LXR formation in reciprocal inhibition of LXR
and PPAR␣ signaling. This can also explain the restoration by supplementation with RXR, not only by providing sufficient RXR for functional RXR heterodimers,
but also by preventing nonfunctional PPAR␣/LXR
formation.
LXR inhibition of PPAR␣ signaling observed fromtransfection studies with cultured cells is likely to beextendable to the in vivo regulation of the hepaticenergy metabolism. It is noteworthy that administration of LXR agonist reduced hepatic nuclear PPAR␣/RXR and impaired expression of hepatic fatty aciddegradation enzyme genes both in rat primary hepatocytes and livers of fasted mice (Figs. 10 and 11). Ina fasted state, hepatic PPAR␣ expression is highly
induced and fatty acids are recruited as ligands for
PPAR␣ activation (17, 18). Nutritional condition does
not markedly change the expression of hepatic LXR␣(29). Therefore, LXR ligand can activate hepatic LXR␣and cause cross-talk with PPAR␣/RXR leading to suppression of expression of PPAR␣ target genes involved in lipid degradation. These data demonstratethat LXR interference with PPAR signaling can occur invivo. In the accompanying paper (38), we observed amirror image observation, that PPAR␣ activation inhibits ligand-induced LXR signaling in hepatocytes.

Ide et al. • LXRs Suppress Lipid Degradation Gene Promoters

The triangle relationship among LXR, PPAR, andRXR could be crucial for mutual regulation of both LXR
and PPAR activities, and thus nutritional regulation of
their downstream genes. PPAR␣ is involved in fatty
acid degradation as an adaptic control of energy depletion. Meanwhile, SREBP-1c, whose expression isdominated by LXR is involved in fatty acid synthesis
for storage of excess energy. These opposite nutritional regulators are reciprocally up- and down-regulated depending upon energy states. As summarized
in Fig. 12, our findings suggest that the mutual suppression efficiently facilitates these reciprocal actionsof PPAR␣ and LXR-SREBP-1c systems. These studies
should open up a new paradigm of a novel cross-talkof nutritional transcription factors in energy metabolism where the nuclear concentrations of each receptor and ligand are crucial for nutritional regulation forfatty acid metabolism. It is also important to investigate whether the cross-talk of these receptors involves a potential competition for their coactivatorssuch as CBP/p300 (42, 46, 47), steroid receptor
coactivator-1 (48–50), and PPAR␥ coactivator-1 (51,52). Further studies should focus on the precise mechanisms for this network.

MATERIALS AND METHODS
Materials
Anti-PPAR␣ sc-9000, RXR␣ D20 sc-553, and LXR␣ C-19
sc-1201 antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA), Redivue [␣-32P]deoxy-CTP(6,000 Ci/mmol) and Protein G Sepharose from Amersham
Biosciences Inc. (Uppsala, Sweden), in vitro transcription andtranslation kit (TNT Coupled Reticulocyte Lysate System)from Promega (Madison, WI), and other chemicals from
Sigma (St. Louis, MO). T0901317 (N-methyl-N-[4-(2, 2, 2trifluoro-1-hydroxy-1-trifluoromethylethyl)-phenyl]benzenesulfonamide) was provided by Kyorin Pharmaceutical Co. Ltd. (Tochigi, Japan).
Plasmids
Cytomegalovirus (CMV) promoter expression plasmid of human RXR␣ and the Luc reporter gene construct, PPRE from
ACO gene promoter fused upstream from thymidine kinase(TK) promoter (pPPRE-Luc), and pCMV-RXR were kind gifts
from Dr. D. J. Mangelsdorf (University of Texas Southwestern
Medical Center, Dallas, TX). pCMV-FXR, pCMX-TR␤, andpCMV-CBP were from Dr. H. Fujii (Graduate School of Agricultural and Life Science, The University of Tokyo, Tokyo,
Japan), Dr. R. M. Evans (The Salk Institute for BiologicalStudies, La Jolla, CA), and Dr. T. Nakajima (St. Marianna
University School of Medicine, Kawasaki, Japan), respectively. The LXRE of SREBP-1c promoter-Luc construct(pLXRE-Luc), pCMV-mLXR␣ and pCMV-mLXR␤ were prepared as previously described (29). CMV or T7 promoter
expression plasmid of mouse (m) PPAR␣ (1–468, amino acid)(16) was prepared by PCR, and the cDNA was introduced into
pCMV7 or pBluescript II SK vector, respectively. T7 promoter
expression plasmids of mLXR␣ and mLXR␤ were preparedusing pBluescript II SK vector.

Ide et al. • LXRs Suppress Lipid Degradation Gene Promoters

Mol Endocrinol, July 2003, 17(7):1255–1267

1265

Transfections and Luc Assays

Animals

HEK293 were grown at 37 C in an atmosphere of 5% CO2 in
DMEM containing 25 mM glucose, 100 U/ml penicillin, and100 ␮g/ml streptomycin sulfate supplemented with 10% fetal
bovine serum (FBS). Transfection studies were carried out
with cells plated on 12-well plates as previously described(29). The indicated amount of each expression plasmid wastransfected simultaneously with a Luc reporter plasmid (0.25
␮g) and simian virus 40-␤-galactosidase (␤-gal) plasmid (0.2
␮g) or (0.05 ␮g). The total amount of DNA in each transfection
was adjusted to 1.5 ␮g/well with the vector DNA, pCMV7.
22RHC and T0901713 were dissolved in ethanol. Each agent
was treated to the cells immediately after transfection in
DMEM with 10% FBS, and incubated for 24 h. After incubation, the amount of Luc activity in transfectants was measured and normalized to the amount of ␤-gal activity.

Male mice (C57BL/6J) were obtained from Charles River
Japan (Yokohama, Japan). All mice were given a standard
diet and tap water ad libitum. All institutional guidelines foranimal care and use were applied in this study. Vehicle (0.5%carboxymethyl-cellulose) T0901317 (50 mg/kg), Wy14,643(50 mg/kg), or both their agonists was orally administered tothe mice before 18 h fasting. For fasting and refeeding treatment, mice were fasted for 24 h and fed a high sucrose/fat
free diet for 12 h as described (55). Hepatic nuclear protein
were prepared from the livers as previously described (56),were subjected to immunoblotting with the anti-PPAR␣, antiLXR␣, or anti RXR␣ antibodies.

Gel Mobility Shift Assays
Gel mobility shift assays were performed as previously described (53). Briefly, mLXR␣, mLXR␤, mPPAR␣, and human(h) RXR␣ proteins were generated from the expression vectors using a coupled in vitro transcription/translation system(Promega). Double-stranded oligonucleotides used in gel
mobility shift assays were the LXREb of the SREBP-1c promoter (29) and PPRE of the ACO promoter (16). These werethen labeled with [␣-32P]deoxy-CTP by klenow enzyme, followed by purification on sephadex G50 columns. The labeledprobes (30,000–100,000 cpm) were incubated with lysates (1to 4 ␮l), or hepatic nuclear extract (1 ␮g) in a mixture (20 ␮l)containing 10 mM HEPES (pH 7.6), 50 mM KCl, 0.05 mM
EDTA, 2.5 mM MgCl2, 8.5% glycerol, 1 mM dithiothreitol, 0.4
␮g/ml poly(deoxyinosine-deoxycytidine), 0.1% Triton X-100,
and 1 mg/ml nonfat milk for 60 min on ice. The DNA-protein
complexes were resolved on a 4.5% PAGE at 100 V for 2 h at
4 C. Gels were dried and exposed to the filter of BAS2000
with BAStation software (Fuji Photo Film, Kanagawa, Japan).

Northern Blot Analysis
Total RNA was extracted from livers and rat primary hepatocytes using TRIZOL Reagent (Invitrogen Corp., Carlsbad,
CA). Equal aliquots of total RNA from mice in each groupwere pooled (total 10 ␮g), subjected to formalin-denatured
agarose electrophoresis, and transferred to nylon membrane(Hybond N, Amersham Pharmacia Biotech, Uppsala, Sweden). Blot hybridization was performed with the cDNA probes
labeled with [␣-32P]CTP (6000 Ci/mmol) using the Megaprime
DNA Labeling System (Amersham Biosciences Inc.). The
cDNA probes for SREBP-1, fatty acid synthase, ACO, cytochrome P450A2, PPAR␣, LXR␣ and ␤, ABCA1 and acidic
ribosomal phosphoprotein PO (36B4) were prepared as previously described (29, 31). The cDNA probes for L-FABP,
CPTI, and mHMG-CoA Syn were provided by Kyorin Pharmaceutical Co. LTD. Each signal was analyzed with BAS2000
and BAStation software (Fuji Photo Film).

Acknowledgments
We are grateful to A. H. Hasty for critical reading of themanuscript. We also thank T. Kitamine, H. Okazaki, Y.
Tamura, and M. Sekiya for helpful discussion.

Coimmunoprecipitation of Receptors
In vitro translated [35S]methionine-labeled receptors with unlabeled receptors or hepatic nuclear extracts from ligandtreated mice were brought to a final volume of 20 or 200 ␮lwith buffer containing 10 mM HEPES (pH 7.6), 50 mM KCl,
0.05 mM EDTA, 2.5 mM MgCl2, 8.5% glycerol, 1 mM dithiothreitol, 0.1% Triton X-100, and 1 mg/ml nonfat milk for 2 h at
4 C and incubated with 10 ␮l of rabbit or goat polyclonal
antibodies binding to protein G-sepharose for overnight at 4
C. The precipitations were washed with PBS containing 0.2%
Tween-20 and 3% BSA. After microcentrifugation, the pellet
was washed four times with 1 ml of ice-cold PBS containing
0.2% Tween-20. Twenty microliters of SDS-PAGE sample
buffer were added to the final pellet and boiled for 5 min at 95
C. The supernatant was subjected to electrophoresis on 10%
SDS-PAGE.
Hepatocyte Isolation and Culture
Primary hepatocytes were isolated from male SpragueDawley rats (160–180 g, Japan Clea, Tokyo, Japan) using thecollagenase perfusion method as described previously (54).
The viability of isolated cells was over 90% as determined by
the trypan blue. Cells were resuspended in DMEM containing
100 U/ml penicillin and 100 ␮g/ml streptomycin sulfate supplemented with 5% FBS, seeded on collagen-coated dishes
100 mm at a final density of 4 ⫻ 104 cells/cm2. After anattachment for 4 h, cells were cultured with medium containing the indicated agonists for 24 h.

Received May 23, 2002. Accepted April 14, 2003.
Address all correspondence and requests for reprints to:
Hitoshi Shimano, M.D., Ph.D., Department of Internal Medicine, Institute of Clinical Medicine, University of Tsukuba,
1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan. E-mail:shimano-tky@umin.ac.jp.
This study was supported by the Promotion of Fundamental Studies in Health Science of the Organization for Pharmaceutical Safety and Research.
T.I. and H.S. equally contributed to this work.

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