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Molecular Endocrinology 17(7):1240–1254
Copyright © 2003 by The Endocrine Society
doi: 10.1210/me.2002-0190

Cross-Talk between Peroxisome ProliferatorActivated Receptor (PPAR) ␣ and Liver X Receptor(LXR) in Nutritional Regulation of Fatty Acid
Metabolism. I. PPARs Suppress Sterol Regulatory
Element Binding Protein-1c Promoter throughInhibition of LXR Signaling
TOMOHIRO YOSHIKAWA, TOMOHIRO IDE, HITOSHI SHIMANO, NAOYA YAHAGI,MICHIYO AMEMIYA-KUDO, TAKASHI MATSUZAKA, SHIGERU YATOH, TETSUYA KITAMINE,HIROAKI OKAZAKI, YOSHIAKI TAMURA, MOTOHIRO SEKIYA, AKIMITSU TAKAHASHI,
ALYSSA H. HASTY, RYUICHIRO SATO, HIROHITO SONE, JUN-ICHI OSUGA, SHUN ISHIBASHI,
AND NOBUHIRO YAMADA
Department of Metabolic Diseases (T.Y., N.Y., M.A.-K., T.K., H.O., Y.T., M.S., A.H.H., J.O., S.I.),
Faculty of Medicine, Department of Applied Biological Chemistry (R.S.), Graduate School of
Agricultural and Life Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113-8655, Japan; andDepartment of Internal Medicine (T.I., H.S., T.M., S.Y., A.T., H.S., N.Y.), Institute of Clinical Medicine,
University of Tsukuba, Ibaraki 305-8575, Japan
Liver X receptors (LXRs) and peroxisome proliferator-activated receptors (PPARs) are members of nuclear receptors that form obligate heterodimers withretinoid X receptors (RXRs). These nuclear receptors
play crucial roles in the regulation of fatty acid metabolism: LXRs activate expression of sterol regulatory element-binding protein 1c (SREBP-1c), a dominant lipogenic gene regulator, whereas PPAR␣
promotes fatty acid ␤-oxidation genes. In the current
study, effects of PPARs on the LXR-SREBP-1c pathway were investigated. Luciferase assays in humanembryonic kidney 293 cells showed that overexpression of PPAR␣ and ␥ dose-dependently inhibited
SREBP-1c promoter activity induced by LXR. Deletion and mutation studies demonstrated that the two
LXR response elements (LXREs) in the SREBP-1c
promoter region are responsible for this inhibitory
effect of PPARs. Gel shift assays indicated that

PPARs reduce binding of LXR/RXR to LXRE. PPAR␣selective agonist enhanced these inhibitory effects.
Supplementation with RXR attenuated these inhibitions by PPARs in luciferase and gel shift assays,
implicating receptor interaction among LXR, PPAR,
and RXR as a plausible mechanism. Competition of
PPAR␣ ligand with LXR ligand was observed in LXR/RXR binding to LXRE in gel shift assay, in LXR/RXR
formation in nuclear extracts by coimmunoprecipitation, and in gene expression of SREBP-1c by Northern blot analysis of rat primary hepatocytes andmouse liver RNA. These data suggest that PPAR␣
activation can suppress LXR-SREBP-1c pathway
through reduction of LXR/RXR formation, proposing
a novel transcription factor cross-talk between LXR
and PPAR␣ in hepatic lipid homeostasis. (Molecular
Endocrinology 17: 1240–1254, 2003)

S

and E-boxes, within their promoter regions (4, 5).
There are three forms of SREBP: SREBP-1a and -1c(also known as adipocyte determination and differentiation 1, ADD1) and -2 (6–8). Most organs, including
the liver and adipose tissue, express predominantly
SREBP-2 and the -1c isoform of SREBP-1 (9). Recent
in vivo studies demonstrated that SREBP-1c plays acrucial role in the dietary regulation of most hepatic
lipogenic genes, whereas SREBP-2 is actively in-

TEROL REGULATORY ELEMENT (SRE)-binding
proteins (SREBPs) are membrane-bound transcription factors that belong to the basic helix-loophelix leucine zipper family (1–3). Through sterol-regulated cleavage, SREBP enters the nucleus andactivates the transcription of genes involved in cholesterol and fatty acid synthesis by binding to a SRE or
its related sequences including SRE-like sequences
Abbreviations: ABCA1, ATP-binding cassette transporter
A1; ACO, acyl-CoA oxidase; 36B4, acidic ribosomal phosphoprotein PO; 9CRA, 9-cis-retinoic acid; CBP, cAMP response element binding protein-binding protein; CMV, cytomegalovirus; CoA, coenzyme A; DMSO, dimethylsulfoxide;
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; Pio, pioglitazone; PPAR, peroxisome
proliferator-activated receptor; PPRE, peroxisome proliferator responsive element; pSV, simian virus 40 promoter plasmid; 22RHC, 22(R)-hydroxycholesterol; RXR, retinoid X receptor; SRE, sterol regulatory element; SREBP-1c, sterol
regulatory element-binding protein 1c; TK, thymidine kinase;
Wy, Wy14,643.
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1241

volved in the transcription of cholesterogenic enzymes(10–17). SREBP-1c seems to control hepatic lipogenic
enzymes through changing its mRNA level in a process that appears to be highly related to glucose/
insulin signaling (18).
Liver X receptors (LXRs) belong to a subclass of
nuclear hormone receptors that form obligate heterodimers with retinoid X receptors (RXR) and are
activated by oxysterols (19–22). There have been two
subtypes of LXRs identified: LXR␣ and LXR␤. LXR␣ isexpressed in liver, spleen, kidney, adipose, and small
intestine (23), whereas LXR␤ is ubiquitously expressed(24). LXRs have been established to regulate intracellular cholesterol levels by transactivating the expression of cholesterol 7␣-hydroxylase (21, 22, 25), cholesterol ester transfer protein (26), and ATP-binding
cassette transporter A1 (ABCA1), which modulatescholesterol efflux and mediates reverse cholesteroltransport from peripheral tissues. LXR/RXR also appears to be involved in cholesterol absorption in intestine (27). Furthermore, LXR/RXR was recently identified as a dominant activator of SREBP-1c promoter(28, 29), implicating a new link between cholesteroland fatty acid metabolism.
Peroxisome proliferator-activated receptors (PPARs)
belong to a ligand-activated nuclear hormone receptor
superfamily and are known to regulate the expressionof numerous genes involved in fatty acid metabolism
and adipocyte differentiation (30, 31). PPAR␣ is primarily expressed in the liver, in which it has beenshown to promote ␤-oxidation of fatty acids (30).
PPAR␥ is mainly expressed in adipose tissue (32),where it has been shown to be an essential component of the adipocyte differentiation program (33); andin macrophages, where it modulates differentiationand cytokine production (34–36). PPARs were originally identified as factors that mediate transcriptional
responses to peroxisome proliferators, a broad classof xenobiotic chemicals that include fibrate hypolipidemic drugs and other nongenotoxic rodent hepatocarcinogens (37, 38). Subsequently, PPARs wereshown to be differentially activated by a variety ofsaturated or unsaturated long chain fatty acids andlipid-like compounds (39–43), suggesting that fatty acids or fatty acid derivatives serve as physiologicalactivators.
The roles of these nutritional transcription factors inwhole body physiology and metabolism can be best
illustrated by comparing two opposite nutritional
states: fasted and refed states. In the fasted liver, fatty
acids are oxidized to acetyl-coenzyme A (CoA) andsubsequently to ketone bodies. PPAR␣, plays a majorrole in both processes, which was confirmed by observations in PPAR␣-null mice (44, 45). In contrast,
expression of SREBP-1c is reduced during fasting. In
the refed state, lipogenesis is induced through increased amount of SREBP-1, whereas PPAR␣ is decreased. This coordinated reciprocal regulation of thetwo transcription factors is key to nutritional regulationof fatty acids and triglycerides as energy storage sys-

tem and implicates the presence of a cross-talk between these factors. The involvement of LXRs and
PPARs in multiple and diverse cellular functions on the
nutritional regulation of fatty acid metabolism suggests that these receptors may be integrated withother cellular signaling pathways, in addition to thewell-characterized RXR pathway. Indeed, the reciprocal modulation of thyroid hormone and peroxisome
proliferator-responsive genes through cross-talk between thyroid hormone receptors (TRs) and PPARs
has been demonstrated (46, 47). Moreover, it is reported that LXR␣ interacts with PPAR␣ and inhibits
peroxisome proliferator signaling (48).
In the current study, we analyzed effects of PPARs
on the LXR-SREBP-1c system. The results demonstrate that activation of PPAR␣ represses LXR signaling through reduction of LXR/RXR heterodimerizationin the liver. Taken together with the accompanying
paper (49) describing LXR suppression of the PPAR␣
signaling, we propose a novel aspect of nutritional
regulation with these mutual interactions forming a
network of transcription factors regulating fatty acidmetabolism.

RESULTSPPARs Suppress SREBP-1c Promoter Activity
through LXR Response Elements (LXREs)
The SREBP-1c promoter contains two LXREs and isactivated by overexpression of LXR␣, ␤, and/or addition of an LXR agonist, 22(R)-hydroxycholesterol(22RHC) (Fig. 1) as previously reported (29). As shownin luciferase (Luc) reporter gene assays (Fig. 1), this
LXR activation of SREBP-1c promoter (2.6 kb) is efficiently suppressed by cotransfection of PPAR␣ or ␥.
Even without LXR activation, overexpression of PPAR
substantially decreased the basal activity of the
SREBP-1c promoter. PPAR inhibition was observed inboth HepG2 cells, a liver cell line, and in human embryonic kidney (HEK) 293 cells. HEK293 cells wereused for the experiments thereafter. Expression levelof transfected PPAR␣ or LXR gene in HEK293 cells
were roughly comparable to that in mouse liver as
estimated by Northern blotting, and thus was within aphysiological range (data not shown). To locate ciselement(s) responsible for this inhibitory effect in the
SREBP-1c promoter, sequential deletion constructs of
SREBP-1c promoter-Luc were estimated in light of
PPAR repression of LXR activation (Fig. 2). The inhibitory effect of PPAR on both basal and LXR␣-induced
activity was partially impaired by deletion of upstream
LXRE (LXREa) of the two LXR binding sites and wascompletely abolished in the absence of both LXREs(LXREa and b). These results suggest that PPAR overexpression cancels out LXR activation of the
SREBP-1c promoter through the two LXREs.
To further investigate the effects of PPARs on these
LXREs in the SREBP-1c promoter, an LXRE enhancer

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Yoshikawa et al. • PPARs Suppress SREBP-1c Promoter

Fig. 1. PPARs Suppress SREBP-1c Promoter Activity in HepG2 and HEK293 Cells
A Luc reporter gene containing the mouse SREBP-1c promoter (2.6 kb); pBP1c2600-Luc was cotransfected in HepG2 (A) and
HEK293 (B) cells with pCMV-LXR (0.1 ␮g), CMV-PPAR␣ or ␥ (0.5 ␮g) or an empty vector CMV-7 as a control, and pSV-␤-gal asa reference plasmid. 22RHC (10 ␮M), or ethanol (EtOH) as a control was added to the cells after transfection in medium with 10%
fetal bovine serum 24 h before the assay. After incubation, Luc activity was measured and normalized to ␤-gal activity. The relative
fold change in Luc activity as compared with a mock-transfected control is shown (means ⫾ SD, n ⫽ 3).

Fig. 2. Identification of a PPAR-Suppressive Region in the SREBP-1c Promoter by Deletion Analysis
SREBP-1c promoter Luc reporters of various lengths were constructed (left panel). The HEK293 cells were transfected witheach reporter plasmid, pCMV-LXR␣, pCMV-PPAR␣, and reference plasmid, pSV-␤-gal. After incubation, Luc activity wasmeasured and normalized to ␤-gal activity. The effect of PPAR␣ in each reporter construct without LXR␣ coexpression (basal
activity) is expressed as normalized Luc activity (means ⫾ SD, n ⫽ 3) (middle panel). The data from LXR␣ coexpression (0.1 ␮g
pCMV-LXR␣, LXR␣-induced activity) are shown as fold-change relative to mock transfected control (means ⫾ SD, n ⫽ 3) (rightpanel).

construct (LXRE-Luc) was used. This construct was
shown to be activated by LXR␣, or ␤, or an LXR ligand
in a very similar fashion to the native SREBP-1c promoter Luc. Reflecting the observation from the 2.6-kb
SREBP-1c promoter, LXRE-Luc showed a very similar
repression pattern by either PPAR␣ or ␥ coexpression(Fig. 3A). When either LXREa or b was disrupted bypositionall mutation in LXRE-Luc, the inhibitory effectof PPAR was partially impaired. Mutation of both
LXREs abolished the PPAR inhibition of LXR activation. Figure 3B shows that the LXR (0.1 ␮g DNA)-

induced LXRE-Luc activity is inhibited by PPAR␣ and␥ overexpression in a dose-dependent manner. Fifty
percent inhibition was observed at 0.025 ␮g DNA of
PPAR. We have previously shown that the SREBP-1c
promoter contains an SRE, an activation site for
SREBPs, mediating an auto-loop activation of the
SREBP-1c-lipogenic gene expression system (50).
The 90-bp SREBP-1c promoter construct containing
the SRE, but not the upstream LXREs was activated bycoexpression of nuclear SREBP-1a or -1c, but not by
LXR. The activity of this reporter construct was notYoshikawa et al. • PPARs Suppress SREBP-1c Promoter

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Fig. 3. Inhibitory Effect of PPARs on SREBP-1c Promoter Activity Is Mediated by the LXRE Complex in the SREBP-1c Promoter
A, The LXRE complex containing two LXREs (LXREa and b) was located at ⫺249 to ⫺148 bp in the SREBP-1c promoter as
described previously (29). The LXRE complex in the SREBP-1c promoter was fused to a luciferase reporter plasmid, whichcontained a simian virus 40 promoter (pGL2 promoter vector). This enhancer construct [pBP1c(LXRE)-Luc] or the indicatedmutant construct was cotransfected into HEK293 cells with pCMV-LXR␣, ␤ (0.1 ␮g, each), pCMV-PPAR␣, ␥ (0.5 ␮g, each), or an
empty vector, CMV-7 as a control and pSV-␤-gal as a reference plasmid. B, Dose-dependent suppression of the LXRE complex
enhancer in the SREBP-1c promoter by PPARs. pBP1c(LXRE)-Luc and pSV-␤-gal were cotransfected into HEK293 cells with
pCMV-LXR␣, pCMV-PPAR␣, pCMV-PPAR␥ or an empty vector, CMV-7 as a control. After the 24-h incubation, Luc activity wasmeasured and normalized to ␤-gal activity. The fold change by PPARs in the Luc activity (means ⫾ SD, n ⫽ 3) as compared with
the LXR␣ (0.1 ␮g)-induced control is shown. C, pBP1c(90b)-Luc, which contained SRE-complex but not LXRE-complex, wascotransfected into HEK293 cells with pCMV-SREBP-1a, pCMV-SREBP-1c, pCMV-LXR␣, or an empty vector, CMV-7 in thepresence or absence of pCMV-PPAR␣, or pCMV-PPAR␥ as a control and pSV-␤-gal as a reference plasmid. 22RHC (10 ␮M), or
ethanol (EtOH) as a control was added to the cells after transfection in medium with 10% fetal bovine serum 24 h before the assay.
After incubation, Luc activity was measured and normalized by ␤-gal activity. The fold change by LXRs or their ligands in the Luc
activity (means ⫾ SD, n ⫽ 3) as compared with the respective control is shown.

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Yoshikawa et al. • PPARs Suppress SREBP-1c Promoter

changed by PPAR␣ or ␥ coexpression (Fig. 3C), indicating that PPAR does not affect SREBP activation of
the SREBP-1c promoter through SRE.
PPAR Agonists Enhance PPARs Repression of
SREBP-1c Promoter Activity

Fig. 4. Suppression of the SREBP-1c Promoter (LXRE Complex Luc) Activity by PPAR Ligand-Activation of PPARs
Activation of BP1c(LXRE)-Luc by LXR was competed withligand activation of PPAR␣ (A) or PPAR␥ (C). Ligand-specificactivation of PPAR␣ (B) or PPAR␥ (D) was confirmed by
PPRE-Luc. Effects of ligands in the absence of receptor
cotransfection on BP1c(LXRE)-Luc (E) and PPRE-Luc (F)were also estimated. A, pBP1c(LXRE)-Luc (0.25 ␮g) was
cotransfected into HEK293 cells with pCMV-LXR␣ (0.1 ␮g),
pCMV-PPAR␣ (0.05 ␮g), and pSV-␤-gal (0.25 ␮g) as a reference plasmid. A PPAR␣ pharmacological ligand; Wy (1, 10,100 ␮M), a PPAR␥ pharmacological ligand; Pio (1, 10, 100␮M), or DMSO was added to the cells after transfection. B,
pPPRE-Luc (0.25 ␮g) was cotransfected into HEK293 cellswith pCMV-PPAR␣ (0.01 ␮g), and pSV-␤-gal (0.25 ␮g). Wy(10 ␮M), Pio (10 ␮M), or DMSO was added to the cells without
22RHC after transfection. C, pBP1c(LXRE)-Luc (0.25 ␮g) was
cotransfected into HEK293 cells with pCMV-LXR␣ (0.1 ␮g),
pCMV-PPAR␥ (0.05 ␮g), and pSV-␤-gal (0.25 ␮g). Wy (1, 10,100 ␮M), Pio (1, 10, 100 ␮M), or DMSO was added to the cells
without 22RHC after transfection. D, pPPRE-Luc (0.25 ␮g)

Activities of PPARs are usually thought to depend
upon the presence of PPAR agonists. We estimatedthe effects of pharmacological PPAR agonists on
PPAR inhibition of LXRE-Luc. We used Wy14,643(Wy), a specific agonist for PPAR␣, and pioglitazone(Pio) for PPAR␥. Their specific actions on PPRE-Luc
were shown in Fig. 4, B and D. The suppression of
LXRE-Luc activity was enhanced by addition of each
PPAR specific agonist in a dose-dependent manner(Fig. 4, A and C). Without overexpression of PPARs,
PPAR␣ and ␥ agonists had no effect on LXRE-Luc
activity induced by LXR␣ activation (Fig. 4E). As was
expected, PPAR␣ and ␥ agonists did not influence
PPRE-Luc activity without overexpression of PPAR␣and ␥, respectively (Fig. 4F). These results are presumably due to lack of endogenous PPAR expression in
HEK293 cells (data not shown).
To examine this suppressive action with endogenous nuclear receptors, we performed reporter assay
using rat primary hepatocytes. An LXR ligand,T0901317 highly induced LXRE luciferase activity inthese cells. Wy dose dependently suppressed thisinduced activity (Fig. 5A). Thus, PPAR␣ interference of
the LXR␣-SREBP-1c pathway could be observed under physiological expression levels of these receptors.
Meanwhile, Wy alone slightly increased basal LXRE
Luc activity, presumably due to a slight induction ofendogenous LXR␣ expression, to lesser extent, but
consistent with previous reports in macrophages (51–53). We also examined the reciprocal effects of the
PPAR ligand with a dual reporter system using
SREBP-1c(LXRE)-renilla and PPRE-firefly luciferases
in the same setting of HEK 293 cells. After transfectionof both PPAR␣ and LXR␣, addition of Wy increased
PPRE-firefly Luc activity and simultaneously decreased BP1c(LXRE)-renilla Luc activity (Fig. 5, B and
C). Conversely, addition of T0901713 increased
SREBP1c(LXRE) renilla and decreased PPRE-firefly.

was cotransfected into HEK293 cells with pCMV-PPAR␥(0.01 ␮g), and pSV-␤-gal (0.25 ␮g). Wy (10 ␮M), Pio (1 ␮M), or
DMSO was added to the cells without 22RHC after transfection. E, pBP1c(LXRE)-Luc (0.25 ␮g) and pSV-␤-gal (0.25 ␮g)were cotransfected into HEK293 cells without pCMV-LXR␣.
Wy (10 ␮M), Pio (1 ␮M), or DMSO (and/or ethanol) was added
to the cells with or without 22RHC (10 ␮M) after transfection.
F, pPPRE-Luc (0.25 ␮g) and pSV-␤-gal (0.25 ␮g) were cotransfected into HEK293 cells without pCMV-LXR␣ andpCMV-PPAR␣ or ␥. Wy (10 ␮M), Pio (1 ␮M), or DMSO (and/or
ethanol) was added to the cells with or without 22RHC (10␮M) after transfection. After incubation for 24 h, Luc activity(means ⫾ SD, n ⫽ 3) was measured and normalized to ␤-gal
activity.

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Mol Endocrinol, July 2003, 17(7):1240–1254

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Fig. 5. Effect of PPAR␣ Ligand on the SREBP-1c Promoter (LXRE Complex Luc) Activity in Rat Primary Hepatocytes (A) and on
Dual Reporter Assays for LXRE (B) and PPRE (C) in HEK293 Cells
A, Reporter plasmid, pBP1c(LXRE)-firefly Luc, and reference plasmid, pSV-renilla Luc, were transfected into rat primary
hepatocytes. T0901317 and/or Wy were added to the cells after transfection of pBP1c(LXRE)-Luc and pSV-renilla Luc 24 h prior
to the assay. After incubation, firefly Luc activity was measured and normalized to renilla Luc activity. B and C, Dual reporters
assay was performed with pBP1c(LXRE)-renilla Luc, pACO-PPRE firefly Luc, and pSV-␤-gal that were cotransfected into HEK293
cells with pCMV-LXR␣ (0.25 ␮g) and/or pCMV-PPAR␣ (0.25 ␮g). The cells were cultured with T0901317 or Wy for 24 h after
transfection. After incubation, firefly and renilla Luc activities were measured and normalized to ␤-gal activity. The fold changeby their ligands in the Luc activity (means ⫾ SD, n ⫽ 3) as compared with the control is shown.

These results from dual reporter system confirmed
consistency of the data from individual reporter system, providing further evidence for the possibility ofcross-talk between PPAR and LXR signaling in thecultured cells [see the accompanying paper (49)].
Because PPARs and LXR share RXR as an obligate
heterodimer partner, it is conceivable that PPAR repression of LXR activation on the LXRE-containing
promoter is mediated through reduction of LXR/RXR
heterodimers when RXR levels are limiting. To explore
this possibility, the effect of RXR overexpression
and/or addition to an RXR ligand [9-cis-retinoic acid
(9CRA)] was estimated (Fig. 6). Under basal conditionsof LXRE-Luc without LXR overexpression, coexpression of PPAR␣ or ␥ caused approximately a 50%
reduction. This PPAR inhibition was essentially abolished by coexpression of the same amount of RXR.
Although LXR␣ (0.1 ␮g) coexpression induced LXRELuc activity by 6-fold, the percent inhibition by PPARs
was more prominent (65%) than that without LXR
overexpression. RXR overexpression also increased
LXRE-Luc activity, and dose dependently suppressed
PPAR inhibition, although the percent restoration wasslightly less than in the absence of LXR overexpression. When an RXR ligand, 9CRA was added, LXRELuc was activated robustly because of LXR/RXR activation. Even in this condition, PPAR␣ and ␥
overexpression efficiently suppressed its activity.
Overexpression of RXR substantially, although notcompletely, restored the activity. These data sug-gested that PPAR inhibition of LXR activated
SREBP1c-LXRE-luc is at least partly due to RXR
competition.
RXR competition as possible mechanism for PPAR
inhibition of LXR signaling prompted us to study theeffects of TR and farnesoid X receptor (FXR), other
RXR heterodimer partners. Whereas each nuclear receptor was active for luciferase reporter containing its
own target cis-element (Fig. 7A), PPAR␣ was the mosteffective at inhibiting LXR induced-SREBP1c promoter activity (Fig. 7B), highlighting the importance ofthe cross-talk between PPAR ␣ and LXR. TR␤ hassome inhibitory effect. Inhibition by FXR was barely
detectable. Although recruitment of coactivators is important for activation of receptors (54) including
PPARs, overexpression of neither cAMP response element binding protein-binding protein (CBP) nor p300
(data not shown) restored PPAR␣ suppression of
LXR␣-induced LXRE-Luc activity (Fig. 7C).
PPARs Inhibit LXR␣/RXR Binding to LXRE of
SREBP-1c Promoter
The PPAR inhibition of LXR activation observed inreporter assays was also evaluated in gel mobility shift
assays (Fig. 8). The LXRE DNA probe from the
SREBP-1c promoter was shifted by coincubation withrecombinant LXR and RXR. This LXR/RXR binding to
LXRE was specific and required RXR because none

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Yoshikawa et al. • PPARs Suppress SREBP-1c Promoter

Fig. 6. Inhibitory Effects of PPARs on the SREBP-1c Promoter (LXRE Complex Luc) Activity Are Restored by Overexpressionof RXR
pBP1c(LXRE)-Luc was cotransfected into HEK293 cells with pCMV-LXR␣ (0.1 ␮g), PPAR␣ (0.01 ␮g), PPAR␥ (0.01 ␮g),
pCMV-RXR (0.02, 0.1, and 0.5 ␮g), and pSV-␤-gal as a reference plasmid. 9CRA (10 ␮M) was added to the cells after transfection.
After incubation for 24 h, Luc activity was measured and normalized by ␤-gal activity. The percent inhibition of Luc activity by
PPAR␣ or ␥ (0.5 ␮g) as compared with the mock, LXR␣ (0.1 ␮g)-, or 9CRA (10 ␮M)-induced controls is shown (means ⫾ SD, n ⫽3). The number in parentheses indicates the fold induction by LXR␣ (0.1 ␮g) or 9CRA (10 ␮M) in Luc activity as compared withbasal BP1c(LXRE)-Luc activity.

of LXRs alone, PPAR␣/RXR, nor PPAR␣/LXR bound
LXRE (Fig. 8A). Figure 8B shows that addition of
PPAR␣ or ␥ to the incubation diminished the shiftedsignal in a dose-dependent manner. A 4-fold increasein amount of PPAR compared with LXR nearly abolished the shifted signal. Addition of PPAR agonists(fenofibrate, Wy, or Pio) essentially did not change
LXR/RXR binding to LXRE without PPAR␣ or ␥. However, these ligands slightly enhanced the inhibitory
effect of PPAR on the binding of LXR to LXRE. As
shown in Fig. 8C, supplementation with RXR, did notchange the basal binding of LXR/RXR to the LXRE,
suggesting that the amount of RXR added in the basal
condition of this assay was sufficient to form an LXR/RXR heterodimer. The decreased LXR/RXR binding to
the LXRE by PPAR was completely restored by further
addition of RXR. The data suggest that PPAR suppression of LXR-activated SREBP-1c promoter, as observed in the reporter assays, was mediated throughinhibiting LXR/RXR binding to the LXRE, which waspresumably caused by reduction of LXR/RXR heterodimer formation.
PPAR␣ Activation Represses LXR AgonistInduced SREBP-1c Expression
To further evaluate the in vivo physiological significance of PPAR␣ interference with LXR signaling, westudied hepatic gene expression in mice treated with
the PPAR␣ ligand and/or the LXR ligand. The mice
were fasted to induce hepatic endogenous PPAR␣.

The activation of LXR by T0901317 was confirmed byobservational increases in SREBP-1 and ATP-binding
cassette transporter A1 (ABCA1) mRNA levels, both ofwhich are well-known LXR target genes. Wy alone
minimally increased basal ABCA1 mRNA, which isconsistent with reports of PPAR-LXR-ABCA1 pathway(51, 52). The hepatic mRNA levels of ABCA1 and
SREBP1 induced by the LXR ligand was suppressedby Wy (Fig. 9A). Coimmunoprecipitation experiments
were performed to estimate LXR␣/RXR heterodimers
in hepatic nuclear extracts. As a control experiment,
LXR␣ antibody was shown to coimmunoprecipitate
successfully the in vitro translated LXR␣/RXR proteinsas detected by RXR antibody (Fig. 9B). These assays
demonstrated that the appreciable increase in amountof LXR␣/RXR heterodimers in hepatic nuclear extracts
that was observed in the mice treated with T0901317
was completely suppressed by concomitant Wy treatment (Fig. 9C). These results suggest that PPAR␣
agonist could cancel hepatic LXR ligand-induced
SREBP-1c gene induction via reduction of nuclear
LXR/RXR formation. We confirmed that PPAR␣/RXR
complex formation was enhanced by PPAR␣ agonist
using coimmunoprecipitation of in vitro transcription/translation protein (Fig. 9D), as was previously reported (41). Effects of ligand activation of PPAR␣ on
LXR/RXR binding to LXRE was also assessed by gel
mobility shift assays using hepatic nuclear extracts
from fasted mice as physiological receptors. As shownin Fig. 9E, the signal was enhanced by T0901317, andthis induction was canceled by further addition of Wy.

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Fig. 7. Effects of RXR Heterodimers and CBP/p300 on SREBP-1c Promoter Activity
HEK293 were grown at 37 C in an atmosphere of 5% CO2 in DMEM supplemented with 10% FBS. Transfection studies werecarried out with cells plated on 12-well plates. A, pPPRE-Luc [(ACO-PPRE)3-TK-Luc], pLXRE-Luc (BP1c-LXREb-Luc), pTRE-Luc[(DR4)2-Ld40-Luc], or FXRE-Luc (human ileal bile acid-binding protein promoter-Luc (⫺862/⫹ 30-Luc, relative translation startsite) and pSV-renilla Luc were cotransfected into HEK293 cells with indicated expression plasmids (0.25 ␮g): pCMV-LXR␣,pCMV-LXR␤, pCMV-PPAR␣, pCMV7-TR␤, pCMV-FXR, and/or pCMV-RXR␣ (0.25 ␮g). After incubation for 24 h, Luc activity wasmeasured and normalized to renilla Luc activity. B, pBP1c(LXRE)-Luc and pSV-renilla Luc were cotransfected into HEK293 cellswith indicated expression CMV-promoter plasmid (0.25 ␮g). C, pBP1c(LXRE)-Luc and pSV-renilla Luc were cotransfected into
HEK293 cells with indicated expression plasmids; pCMV-LXR␣ (0.25 ␮g), pCMV-PPAR␣ (0.25 ␮g), pCMV-RXR␣ (0.5 ␮g),
pCMV-CBP (0.5 ␮g), and pCMV-p300 (0.5 ␮g). Cells were treated with or without 3 ␮M Wy. After incubation for 24 h, Luc activity
was measured and normalized to renilla Luc activity. The fold change in the Luc activity as compared with the control is shown(means ⫾ SD, n ⫽ 3).

Studies with receptor antibodies and T0901317 indicated that shifted band of LXRE probe incubated with
nuclear extracts was mainly caused by LXR (Fig. 9E),
although the complex contain some other proteins.
The data also suggest that the PPAR␣ agonist inhibits
LXR ligand-induced LXR/RXR binding to LXRE in acompetitive manner.
The observational inhibition of SREBP-1c gene expression by PPAR␣ activation in transfection studies
and mouse livers was also estimated in rat primary
hepatocytes. As evaluated by Northern blot analysis,
the hepatocytes showed consistent PPAR␣ activation
by Wy (Fig. 10A). Addition of Wy to the medium
caused dose-dependent increases in PPAR␣ target
gene, mHMG-CoA synthase, and acyl-CoA oxidase(ACO). LXR␣ expression was induced as previously
described (51–53) but only modestly. PPAR␣ gene
was also significantly induced by Wy owing to autoregulation as was recently described (55). As shown in
Fig. 10B, when the cells were incubated with T0901317,
SREBP-1c expression was robustly induced through
LXR activation. This induction was suppressed in adose-dependent manner by coincubation with Wy (Fig.
10B). The inhibitory effect of Wy was also observed withcycloheximide treatment, suggesting that this action
might be independent of de novo protein synthesis (Fig.
10C). These data suggest that suppression of the LXRSREBP1c pathway by the PPAR␣ ligand is mediatedthrough its PPAR␣ activation and is more prominent
under conditions of LXR activation.

1248 Mol Endocrinol, July 2003, 17(7):1240–1254

Yoshikawa et al. • PPARs Suppress SREBP-1c Promoter

Fig. 8. PPARs Inhibit LXR/RXR Binding to LXREs in the SREBP1c-Promoter in Gel Mobility Shift Assay
A, PPAR/RXR or PPAR/LXR had no ability to bind to LXRE. 32P-labeled LXREb in the SREBP-1c promoter was incubated within vitro synthesized LXR␣, PPAR␣, and/or RXR␣ (1.5 ␮l of programmed reticulocyte lysate) as indicated for 30 min on ice. The
DNA-protein complexes were resolved on a 4.6% PAGE. B, PPARs and their activators inhibited LXR/RXR binding to LXRE.
Complexes of LXR/RXR-LXREb were incubated with 2- or 4-fold higher amounts of PPAR␣ or PPAR␥ in the absence or presenceof fenofibric acid (Feno, 100 ␮M), Wy (100 ␮M), or Pio (10 ␮M). C, PPAR␣ or PPAR␥ inhibition of LXR/RXR binding to LXREb as
observed in panel B was restored by addition of a 4-fold higher amount of RXR.

DISCUSSION
The current studies demonstrate that PPAR␣ activation suppresses LXR-mediated SREBP-1c gene expression. Evidence for this was first shown in luciferase assays in cultured cells and was confirmed in rat
primary hepatocyte cultures and mouse livers. Overexpression of PPARs represses LXR/RXR activation of
LXRE containing promoters such as the SREBP-1c
promoter. This inhibitory effect was enhanced by addition of PPAR ligands. In the liver nuclei, addition of
PPAR␣ agonist enhanced binding of PPAR␣ to RXR,
decreased the amount of LXR/RXR heterodimers,
leading to suppression of LXR ligand-activated
SREBP-1c expression. RXR supplementation experiments suggest that the mechanism for PPAR inhibitionof LXR/RXR activity could be at least partly RXR competition between PPAR and LXR. Involvement of RXR
competition in the regulation of activities of nuclear
receptors has been well established for PPAR and TR(46, 47, 56). It has been also reported that LXR␣ interacts with PPAR␣ and inhibits peroxisome proliferator signaling (48). As described in the accompanying
paper (49), PPAR␣ heterodimerizes with both LXR␣and ␤ as efficiently as with RXR. Because PPAR␣/
LXR␣ (␤) cannot bind to LXRE, this complex formation
could interfere with the formation of LXR␣/RXR and
activation of LXRE containing promoters by LXR␣/RXR. Therefore, as schematized in Fig. 11, it is possible that abundant PPAR␣ can absorb LXR as well as
RXR, resulting in inhibition of LXR/RXR formation and
SREBP-1c promoter activation. Reduction of hepatic
nuclear LXR/RXR from fasted mice doubly treated with
LXR and PPAR␣ ligands supports this hypothesis.

Whether the dominant mechanism for PPAR inhibitionof SREBP-1c expression is RXR competition between
PPAR and LXR, or LXR competition between PPAR␣
and RXR is currently unknown. The amounts of three
nuclear receptor proteins in the nucleus, concentrationof each ligand, and affinity for one another, possible
involvement of other nuclear receptors and cofactors
will all be involved in determining the overall effect.
Further studies will be needed to determine the precise mutual interactions in other combinations of nuclear receptors.
LXR␣ expression was recently shown to be regulated by PPAR (51–53). PPAR enhances cholesterol
efflux in macrophages via PPAR-LXR␣-ABCA1 pathway, and a combination treatment with both PPAR
and LXR agonists result in a further induction in
ABCA1 mRNA in macrophages (51, 52). We also observed a relatively small PPAR␣ ligand-gene inductionof LXR. We propose that the PPAR-LXR␣-ABCA1
pathway has been established in macrophages,
PPAR-LXR-SREBP1c pathway may not be very potent
in liver. Lack of hepatic SREBP-1c induction after adenoviral overexpression of PPAR␥ supports this (57).
Each receptor, ligand, and cofactor concentration
could be important for this tissue specificity of crosstalk of transcription factors.
The physiological relevance of the cross-talk between PPAR␣ and LXR could be extended to nutritional regulation of energy metabolism by a mutualinteraction between PPAR␣ and SREBP-1c, whose
expression are dominated by LXR. Hepatic fatty aciddegradation is activated in an energy-depleted statesuch as fasting to produce alternate energy substrates

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Fig. 9. Inhibition by PPAR␣ Ligand of Gene Induction of LXR Target Genes in Mouse Livers (A), LXR Ligand-Induced LXR/RXR
Heterodimer Formation (B and C), and Binding to LXRE (D)A, Mice (n ⫽ 3) were fasted and treated with T0901317 (T, 50 mg/kg), Wy (50 mg/kg), or both agonists for 18 h. Total RNA was
isolated from the livers of mice and subjected to Northern blot analysis with the indicated cDNA probes. Fold increases of
expression relative to corresponding with vehicle-treated controls are shown. Data are means ⫾ SEM. The statistical significanceof differences between T0901317-treated and both T0901317- and Wy-treated mice was assessed with the Sheffé test. B,
Confirmation of immunoprecipitation of LXR␣/RXR␣ heterodimer by anti-LXR␣ antibody. In vitro translated RXR␣ and LXR␣proteins were incubated with or without T0901317, and the reaction mixtures were incubated with anti-LXR␣ antibody (H-144
sc-13068, Santa Cruz Biotechnology, Inc.), followed by binding to protein G-sepharose. The precipitations were washed andsubjected to immunoblotting with an anti-RXR␣ antibody (D-20, sc-553, Santa Cruz Biotechnology, Inc.). C, Wy inhibited
T0901317-induced LXR/RXR␣ heterodimer formation in the liver from C57BL6 mice. Mice were fasted and treated with T0901317(50 mg/kg), Wy (50 mg/kg), or both agonists for 18 h. Hepatic nuclear extracts were prepared from each group. Nuclear extracts(equalized by protein concentrations) were subjected to immunoprecipitation using anti-LXR␣ antibody (H-144 sc-13068, Santa
Cruz Biotechnology, Inc.) coupled to protein G-sepharose beads. Immunoprecipitates were subjected to SDS-PAGE, andimmunoblotted with an anti-RXR␣ antibody (D-20 sc-553, Santa Cruz Biotechnology, Inc.). Arrowheads represent the RXR␣ or
IgG signal. D, Wy potentiates PPAR␣ binding to RXR␣. Coimmunoprecipitation of unlabeled RXR␣ and 35S-labeled PPAR␣ withan anti-RXR␣ antibody in the absence or presence of Wy. After immunoprecipitation with protein G-sepharose beads, samples
were run on SDS-PAGE. T0901317 induced LXR/RXR␣ binding to LXRE was inhibited by addition of Wy. 32P-labeled SREBP1cLXRE probe and hepatic nuclear extracts from fasted mice were incubated with T0901317 and/or Wy. The DNA-protein
complexes were resolved on a 4.6% PAGE. E, T0901317 induced-proteins/LXRE complexes were reduced by an anti-LXR␣
antibody (H-144 sc-13068, Santa Cruz Biotechnology, Inc.) or anti-RXR␣ antibody (D-20 sc-553, Santa Cruz Biotechnology, Inc.).
32
P-labeled SREBP1c-LXRE probe and hepatic nuclear extracts (1 ␮g) from fasted mice were incubated with 20 ␮l buffer (bufferA) 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 1 h on ice. The DNA-protein complexes were resolved in a 4.6% PAGE.

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Yoshikawa et al. • PPARs Suppress SREBP-1c Promoter

Fig. 10. Ligand-Activation of PPAR␣ Suppresses LXR Ligand Induced-SREBP1 Gene Expression in Primary Rat Hepatocytes
A, Wy induced gene expression of mHMG-CoA Syn, ACO, SREBP-1, PPAR␣, and LXR␣. Rat primary hepatoctyes wereprepared as described in Materials and Methods. The cells were incubated with the indicated concentration of Wy for 24 h. B,
Wy suppressed induction of SREBP-1 gene expression by T0901317 (T) treatment. The hepatocytes were incubated with theindicated concentration of T0901317 in the absence or presence of Wy for 24 h. C, Cycloheximide had no effect in the inhibitionof Wy on T0901317 induced-SREBP-1 gene expression. Total RNA was extracted and Northern blot analysis was performed with
the indicated cDNA probes. Fold changes of expression relative to corresponding with vehicle-treated controls are shown.

such as ketone bodies. PPAR␣ activation plays a keyrole in this adaptic gene induction (44, 45). As has
been shown for other nuclear receptors, PPAR␣ activation requires binding of its ligands, one of which
could be polyunsaturated fatty acids, presumably lipolyzed from adipose tissues. More importantly, we(12) and other group (44) have shown that hepatic
PPAR␣ expression is nutritionally regulated. Hepatic
PPAR␣ level is induced by fasting and suppressed byrefeeding. This nutritional regulation of PPAR␣ expression suggests that changes in the amount of PPAR
protein could also control its downstream genes oflipid oxidation as well as changes in the ligand concentration. In a fasted state, lipogenesis should be
declined in a coordinated fashion with activation of
fatty acid degradation. Marked induction of PPAR␣ in
the livers of fasted mice might play a role in an efficient
suppression of lipogenic genes by inhibition of
SREBP-1c gene expression through a mechanism thatis proposed in the current study. Consistently, it has
recently been reported that PPAR␣-null mice show dysregulation of hepatic lipogenic genes (58). Polyunsaturated fatty acid could contribute to this reciprocal energy
regulation by PPAR␣ and SREBP-1c through activationof PPAR␣ ligands and direct inhibition of SREBP-1c asrecently reported (59).
In the current study, we also observed similar inhibitory effect by overexpression of PPAR␥ on SREBP-1c
expression. In a regular nutritional state, PPAR␥ expression is extremely low in the liver, and contributionof PPAR␥ expression to hepatic regulation of SREBP-1c
is unlikely. In contrast, PPAR␥ is highly induced andinvolved in adipogenesis. Previous reports suggest thatthe role of adipocyte determination and differentiation 1(ADD1)/SREBP-1c in the adipose tissue is positioned
upstream of PPAR␥ activation through two different
mechanisms: ligand production (60) and direct induction(61). PPAR␥ has recently been reported to activate LXR␣gene expression (52), which could in turn activate
SREBP-1c gene expression, leading to a speculationthat these three factors could form an auto-loop activation in adipogenesis. PPAR␥ inhibition of SREBP-1c inthe current study could antagonize this potential autoloop and be involved in the complex transcriptionalcross-talk among PPAR␥, SREBP-1c, and LXR in the
adipocytes.
Taken together with the accompanying paper (49)showing LXR inhibition of PPAR␣ signaling, we show

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1251

SREBP-1c promoter luciferase constructs, and expression
plasmids, pCMV (cytomegalovirus)-mLXR␣ and pCMVmLXR␤ were prepared as previously described (29). Expression plasmids, pCMV-PPAR␣ and pCMV-PPAR␥ were prepared by subcloning PCR products from mouse liver cDNA
into CMV-7. CMV-T7 promoter expression plasmid of human
RXR␣ (pCMV-RXR) was a kind gift from Dr. D. J. Mangelsdorf(University of Texas Southwestern Medical Center, Dallas,
TX). pCMV-FXR, pCMX-TR␤, and pCMV-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 Biological Studies, La Jolla, CA), and Dr. T. Nakajima (St. Marianna University School of Medicine, Kawasaki, Japan), respectively.
Transfections and Luciferase Assays

Fig. 11. Mechanism by which PPAR Suppresses the
SREBP-1c Promoter Activity through Interfering with LXRRXR.
1, LXR/RXR has been shown to bind and activate LXREs in
the SREBP-1c promoter. 2, Reciprocally, PPAR␣/RXR activates gene expression crucial for lipid degradation throughPPREs. 3 and 4, The current study proposes interference of
LXR/RXR signaling by PPAR/RXR and PPAR/LXR. 5, PUFA
(polyunsaturated fatty acid), representative PPAR␣ ligands,
can suppress LXR activation independently of PPAR␣ (59,64). The accompanying paper (49) describes LXR suppress
PPAR␣-targeted gene expression crucial for lipid catabolism
by inhibiting PPAR-RXR binding to the PPRE [see Fig. 12 of
the accompanying paper (49)].

the mutual interaction between PPAR␣ and LXRs in
the reciprocal regulation of PPAR␣ and SREBP-1ctarget genes. The data suggest that cross-talk of these
nuclear factors could play crucial roles in nutritional
regulation of fatty acid metabolism (see Fig. 11). However, as recent studies show that there could be a
global and complex network of nutritional transcriptional factors, further studies are needed to evaluate
the physiological relevance of the cross-talk between
these and other nuclear transcription factors.

MATERIALS AND METHODS
Materials
Anti-RXR␣ (D-20, sc-553) and LXR␣ (H-144, sc-13068) antibody were purchased from Santa Cruz Biotechnology Inc.
(Santa Cruz, CA), 22RHC, 9CRA, and Wy were purchasedfrom Sigma (St. Louis, MO), Redivue [␣-32P]deoxy-CTP (6000
Ci/mmol) from Amersham Biosciences Inc. (Amersham,
Buckinghamshire, UK), and restriction enzymes from New
England Biolabs (Boston, MA). Fenofibric acid and Pio were
provided by Laboratories Fournier (Paris, France), andTakeda Pharmaceutical (Osaka, Japan), respectively.
Plasmid
Luc gene constructs containing a 2.6-kb fragment of themouse SREBP-1c promoter (pBP1c2600-Luc), other

HEK293 and HepG2 cells were grown at 37 C in an atmosphere of 5% CO2 in DMEM containing 25 mM glucose, 100
U/ml penicillin, and 100 ␮g/ml streptomycin sulfate supplemented with 10% fetal bovine serum (FBS). Transfection
studies were carried out with cells plated on 12-well plates aspreviously described (50). The indicated amount of each expression plasmid was transfected simultaneously with a Luc
reporter plasmid (0.25 ␮g) and pSV (simian virus 40)-␤galactosidase (␤-gal) (0.2–0.4 ␮g) or pSV-renilla Luc (0.05
␮g). The total amount of DNA in each transfection was adjusted to 1.5 ␮g/well with pCMV7. 22RHC was dissolved inethanol and PPAR ligands in dimethylsulfoxide (DMSO). Eachagent was added 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 or renilla Luc
activity as measured by standard kits (Promega, Madison,
WI). pBP1c(LXRE)-firefly Luc (1.5 ␮g) and pSV-renilla Luc (0.5
␮g) were transfected into rat primary hepatocytes by using
lipofectin reagent (Invitrogen). After incubation for 24 h, theamount of firefly Luc activity in transfectants was measured
and normalized to the amount of renilla Luc activity. Dual
reporter assay was performed with pBP1c(LXRE)-firefly Luc(0.25 ␮g), PPRE-renilla Luc (0.25 ␮g), and pSV-␤-gal (0.2 ␮g).
Gel Mobility Shift Assays
Gel mobility shift assays were performed as previously described (29). Briefly, the entire open reading frames of mouse(m) LXR␣ and mPPAR␣ were amplified from the pCMV-LXR␣
and pCMV-mPPAR␣ by PCR (forward primers, 5⬘-TTGGTAATGTCCAGGG and 5⬘-GCCATACACTTGAGTGACAAT; reverse primers, 5⬘-CTTCCAAGGCCAGGAGA and 5⬘-AGATCAGTACATGTCTCTGTAGA) and cloned into the EcoRI and
NotI sites, and SalI and NotI sites of the pBluescript II SK
plasmid, respectively. mLXR␣, mPPAR␣, and human (h) RXR
proteins were generated from the expression vectors using acoupled in vitro transcription/translation system (Promega).
Double-stranded oligonucleotides used in gel shift assays
were prepared by annealing both strands of the LXREb in the
LXRE complex of the SREBP-1c promoter (29). 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 nuclear
receptor lysates (1–1.5 ␮l) or hepatic nuclear extract (1 ␮g) ina mixture (20 ␮l) containing 10 mM Tris-HCl, pH 7.6; 50 mM
KCl; 0.05 mM EDTA; 2.5 mM MgCl2; 8.5% glycerol; 1 mM
dithiothreitol; 0.5 ␮g/ml poly (deoxyinosine-deoxycytidine),
0.1% Triton X-100; and 1 mg/ml nonfat milk for 30 min on ice.
The DNA-protein complexes were resolved on a 4.6% PAGE
at 140 V for 1 h at 4 C. Gel were dried and exposed toBAS2000 filters with BAStation software (Fuji Photo Film,
Kanagawa, Japan).

1252 Mol Endocrinol, July 2003, 17(7):1240–1254

Animals
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), Wy (50 mg/kg), or both their agonists was orally administered to the 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 (13). Hepatic nuclear extracts was prepared from the livers as previously described (62).
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 ␮l withbuffer 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 andincubated with 10 ␮l of anti-PPAR␣ (H-98 sc-9000, Santa
Cruz Biotechnology, Inc.) or anti-LXR␣ (H-144 sc-13068,
Santa Cruz Biotechnology, Inc.) polyclonal antibody 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 washedfour times with 1 ml of ice-cold PBS containing 0.2% Tween20. Twenty microliters of SDS-PAGE sample buffer wereadded to the final pellet and boiled for 5 min at 95 C. The
supernatant was subjected to electrophoresis on 8 or 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 (63).
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 with or without 5 ␮M cycloheximide
for 24 h.
Northern Blot Analysis
Total RNA was extracted using TRIZOL Reagent (Invitrogen
Corp., Carlsbad, CA). Equal aliquots of total RNA from micein each group were 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 thecDNA probes labeled with [␣-32P]CTP (6000 Ci/mmol) using
the Megaprime DNA Labeling System (Amersham Biosciences Inc.). The cDNA probes for SREBP-1, ACO, PPAR␣,
LXR␣ and ␤, ABCA1, and acidic ribosomal phosphoprotein
PO (36B4) were prepared as previously described (12, 29).
The cDNA probes for mHMG-CoA Syn were provided by
Kyorin Pharmaceutical Co. Ltd. (Tochigi, Japan). Each signal
was analyzed with BAS2000 and BAStation software (Fuji
Photo Film).

Acknowledgments
We thank M. Nakakuki, Y. Iizuka, S. Tomita, and K. Ohashi
for helpful discussion.
Received May 23, 2002. Accepted April 14, 2003.

Yoshikawa et al. • PPARs Suppress SREBP-1c Promoter

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, 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.Y. and T.I. equally contributed to this work.

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