THE JOURNAL OF BIOLOGICAL CHEMISTRY© 2002 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 277, No. 3, Issue of January 18, pp. 1705–1711, 2002
Printed in U.S.A.

Polyunsaturated Fatty Acids Suppress Sterol Regulatory Elementbinding Protein 1c Promoter Activity by Inhibition of Liver X
Receptor (LXR) Binding to LXR Response Elements*
Received for publication, June 20, 2001, and in revised form, November 1, 2001
Published, JBC Papers in Press, November 2, 2001, DOI 10.1074/jbc.M105711200

Tomohiro Yoshikawa, Hitoshi Shimano‡§, Naoya Yahagi, Tomohiro Ide‡,
Michiyo Amemiya-Kudo, Takashi Matsuzaka‡, Masanori Nakakuki‡, Sachiko Tomita,
Hiroaki Okazaki, Yoshiaki Tamura, Yoko Iizuka, Ken Ohashi, Akimitsu Takahashi‡,
Hirohito Sone‡, Jun-ichi Osuga, Takanari Gotoda, Shun Ishibashi, and Nobuhiro Yamada‡From the Department of Metabolic Diseases, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku,
Tokyo 113-8655, Japan and ‡Department of Internal Medicine, Institute of Clinical Medicine, University of Tsukuba,
Ibaraki 305-8575, Japan

Previous studies have demonstrated that polyunsaturated fatty acids (PUFAs) suppress sterol regulatory element-binding protein 1c (SREBP-1c) expression and,thus, lipogenesis. In the current study, the molecularmechanism for this suppressive effect was investigatedwith luciferase reporter gene assays using the
SREBP-1c promoter in HEK293 cells. Consistent withprevious data, the addition of PUFAs to the medium inthe assays robustly inhibited the SREBP-1c promoter
activity. Deletion and mutation of the two liver X receptor (LXR)-responsive elements (LXREs) in the SREBP-1c
promoter region eliminated this suppressive effect,indicating that both LXREs are important PUFAsuppressive elements. The luciferase activities of both
SREBP-1c promoter and LXRE enhancer constructs induced by co-expression of LXR␣ or -␤ were strongly
suppressed by the addition of various PUFAs (arachidonic acid > eicosapentaenoic acid > docosahexaenoic
acid > linoleic acid), whereas saturated or monounsaturated fatty acids had minimal effects. Gel shiftmobility and ligand binding domain activation assays
demonstrated that PUFA suppression of SREBP-1c expression is mediated through its competition with LXRligand in the activation of the ligand binding domain of
LXR, thereby inhibiting binding of LXR/retinoid X receptor heterodimer to the LXREs in the SREBP-1c promoter. These data suggest that PUFAs could be deeply
involved in nutritional regulation of cellular fatty acidlevels by inhibiting an LXR-SREBP-1c system crucial for
lipogenesis.

Sterol regulatory element (SRE)1-binding proteins (SREBPs)* This study was supported by the Promotion of Fundamental Studies in Health Science of the Organization for Pharmaceutical Safety andResearch (OPSR). The costs of publication of this article were defrayedin part by the payment of page charges. This article must therefore be
hereby marked “advertisement” in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Dept. of Internal
Medicine, Institute of Clinical Medicine, University of Tsukuba, 1-1-1
Tennodai, Tsukuba, Ibaraki 305-8575, Japan. Fax: 81-298-63-2170;
E-mail: shimano-tky@umin.ac.jp.
1
The abbreviations used are: SRE, sterol regulatory element;
SREBP, SRE-binding protein; PUFA, polyunsaturated fatty acid; SA,
stearic acid; OA, oleic acid; LA, linoleic acid; EPA, eicosapentaenoic
acid; DHA, docosahexaenoic acid; AA, arachidonic acid; PPAR, peroxisome proliferator-activated receptor; PPRE, PPAR response element;
LBD, ligand binding domain; LXR, liver X receptor; LXRE, LXR responThis paper is available on line at http://www.jbc.org

are membrane-bound transcription factors that belong to thebasic helix-loop-helix leucine zipper family (1–3). In the absence of sterols, by means of sterol-regulated cleavage, SREBP
enters the nucleus and activates the transcription of genes
involved in cholesterol and fatty acid synthesis by binding to an
SRE or its related sequences including SRE-like sequences andE-boxes, within their promoter regions (4, 5). There are threeforms of SREBP, SREBP-1a and -1c (also known as ADD1) and-2 (6 – 8). Most organs, including the liver and adipose tissue,
predominantly express SREBP-2 and the -1c isoform of
SREBP-1 (9). Recent in vivo studies demonstrate that
SREBP-1c plays a crucial role in the dietary regulation of mosthepatic lipogenic genes, whereas SREBP-2 is actively involvedin the transcription of cholesterogenic enzymes (10). These
include studies of the effects of the absence or overexpression of
SREBP-1 on hepatic lipogenic gene expression (10 –12) as well
as physiological changes of SREBP-1c protein in normal micerefed after fasting (13–17).
Polyunsaturated fatty acid (PUFA) administration has been
well established as a negative regulator of hepatic lipogenesis
as well as an activator of peroxisome proliferator-activatedreceptor (PPAR) ␣, which is crucial for lipid degradation. Consistent with the notion that SREBP-1c is a dominant regulator
for lipogenesis, there are several reports demonstrating thatadministration of PUFA suppresses SREBP-1c protein andmRNA both in cultured cells and in animal livers (14 –16, 18).
PUFA inhibition of SREBP-1c gene expression has been reported to be at cleavage, transcriptional, and post-transcriptional levels (14 –16, 18 –21); however, the precise mechanism
for this effect remains unknown.
Recent promoter analysis reveals that the expression of the
SREBP-1c gene is regulated by two factors; they are SREBP
itself, forming an autoloop, and the liver X receptor (LXR)/
retinoid X receptor (RXR) (22, 23). LXRs belong to a subclass of
nuclear hormone receptors that form obligate heterodimers
with RXRs and are activated by oxysterols (24 –27). It has been
established that LXRs regulate intracellular cholesterol levels
by transactivating the expression of cholesterol 7␣-hydroxylase(26 –28), cholesterol ester transfer protein (29), and ATP binding cassette transporter 1, which modulates cholesterol efflux
from cells with excess cellular cholesterol and mediates reverse
cholesterol transport from peripheral tissues. LXR/RXR may

sive element; RXR, retinoid X receptor; 22RHC, 22(R)-hydroxycholesterol; 9CRA, 9-cis-retinoic acid; kb, kilobases; CMV, cytomegalovirus;
HEK, human embryonic kidney.

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PUFA Inhibition of LXR-activated SREBP-1c Promoter

FIG. 2. Inhibitory effects of various polyunsaturated fatty acids on SREBP-1c promoter activity. pBP1c2600-Luc was co-transfected into HEK293 cells with LXR␣ (0.1 ␮g), and pSV-␤gal (0.2 ␮g) asa reference plasmid. Various PUFAs (100 ␮M) dissolved in ethanol or
ethanol only (EtOH) as a control were added to the cells after transfection in medium with 10% fetal bovine serum 24 h prior to the assay.
After incubation, luciferase activity was measured and normalized to␤-galactosidase activity. The relative fold change in luciferase activity
as compared with a mock-transfected control is shown (means ⫾ SD,
three independent experiments in a duplicate assay).

FIG. 1. Polyunsaturated fatty acids suppress SREBP-1c promoter activity in HepG2 and HEK293 cells. A luciferase reporter
gene containing the mouse SREBP-1c promoter (2.6 kb); pBP1c2600Luc was co-transfected into HepG2 (A) and HEK293 (B) cells with LXR(0.1 ␮g) or an empty vector CMV-7, as a control, and pSV-␤gal, as areference plasmid. Either EPA (100 ␮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, luciferase activity
was measured and normalized to ␤-galactosidase activity. The relative
fold change in luciferase activity as compared with a mock-transfected
control is shown (means ⫾ S.D., three independent experiments in a
duplicate assay).

also be involved in cholesterol absorption in intestine (30).
Furthermore, LXR/RXR was identified as an activator of the
SREBP-1c promoter (23, 31), implicating a new link of cholesterol and fatty acid metabolism.
Extending our previous promoter analysis studies, we attempted to explore the mechanism for the transcriptional inhibition of SREBP-1c by PUFA. In the current study, we analyzed the suppressive effect of PUFAs on SREBP-1c promoter
activity. The data indicate that PUFA inhibit binding of the
LXR/RXR heterodimer to the LXR response elements (LXREs)
in the SREBP-1c promoter, a process crucial for SREBP-1c
expression.
EXPERIMENTAL PROCEDURES

Materials—We obtained 22(R)-hydroxycholesterol (22RHC), 9-cisretinoic acid (9CRA), Wy-14,643, stearic acid sodium salt (SA), oleic acidsodium salt (OA), linoleic acid sodium salt (LA), eicosapentaenoic acidsodium salt (EPA), docosahexaenoic acid sodium salt (DHA), andarachidonic acid sodium salt (AA) from Sigma, Redivue [␣-32P]dCTP(6,000 Ci/mmol) from Amersham Biosciences, Inc., and restriction
enzymes from New England Biolabs. T0901317 (N-methyl-N-[4(2,2,2-trifluoro-1-hydroxy-1-trifluoromethylethyl)-phenyl]-benzenesulfonamide), fenofibric acid and pioglitazone were provided by Kyorin
Pharmaceutical Co. LTD., Laboratories Fournier (Paris, France), andTakeda pharmaceutical (Osaka, Japan), respectively.
Plasmids—Luciferase gene constructs containing a 2.6-kb fragmentof the mouse SREBP-1c promoter (pBP1c2600-Luc), and other
SREBP-1 promoter luciferase constructs were prepared as previously
described (23). CMV and T7 promoter expression plasmids of human
RXR␣ (pRXR) and PPAR response element (PPRE) luciferase reporter
plasmid (pPPRE-Luc) were kind gifts from Dr. D. J. Mangelsdorf. The
expression plasmid of the Gal4 DNA binding domain fused to thehuman LXR␣-ligand binding domain (LBD) (pM-LXR␣) was provided

from Mochida Pharmaceutical co. ltd. (Tokyo, Japan). A luciferase reporter plasmid containing Gal4 binding sites (p17 m8) was a gift fromDr. S. Kato.
Transfections and Luciferase Assays—Human embryonic kidney(HEK) 293 and HepG2 cells were grown at 37 °C in an atmosphere of
5% CO2 in Dulbecco’s modified Eagle’s medium containing 25 mM glucose, 100 units/ml penicillin, and 100 ␮g/ml streptomycin sulfate supplemented with 10% fetal bovine serum. Transfection studies werecarried out with cells plated on 12-well plates as previously described(22). The indicated amount of each expression plasmid was transfected
simultaneously with a luciferase reporter plasmid (0.25 ␮g) and pSV␤gal (0.2 to 0.4 ␮g). The total amount of DNA in each transfection was
adjusted to 1.5 ␮g/well with the vector DNA, pCMV7-NotI. Each fatty
acid was dissolved in water or ethanol, 22RHC and T0901317 weredissolved in ethanol, and PPAR ligands were dissolved in dimethysulfoxide. Each agent was added to the cells immediately after transfectionin Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum
and incubated for 24 h. After incubation, the amount of luciferase
activity in transfectants was measured and normalized to the amount of
␤-galactosidase activity as measured by standard kits (Promega).
Gel Mobility Shift Assays—Gel shift assays were performed as previously described (22). Briefly, the entire open reading frames ofmLXR␣ and mPPAR␣ were amplified from the pCMV-LXR␣ andpCMV-mPPAR␣ by PCR (forward primers, 5⬘-TTGGTAATGTCCAGGG
and 5⬘-GCCATACACTTGAGTGACAAT; reverse primers, 5⬘-CTTCCAAGGCCAGGAGA and 5⬘-AGATCAGTACATGTCTCTGTAGA) andcloned into the EcoRI and NotI sites, and SalI and NotI sites of thepBluescript II SK plasmid, respectively. mLXR␣, mPPAR␣, and hRXR␣
proteins were generated from the expression vectors using a coupled invitro transcription/translation system (Promega). Double-stranded oligonucleotides used in gel shift assays were prepared by annealing bothstrands of the LXREb in the LXRE complex of the SREBP-1c promoter(23) or rat fatty acyl-CoA oxidase PPRE (32). These were then labeledwith [␣-32P]dCTP by Klenow enzyme followed by purification on G50Sephadex columns. The labeled probes (3,000 –10,000 cpm) were incubated with nuclear receptor lysates (1–1.5 ␮l) in a 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(dI-dC), 0.1%
Triton X-100, and 1 mg/ml nonfat milk for 30 min on ice. The DNAprotein complexes were resolved on a 4.6% polyacrylamide gel at 140 V
for 1 h at 4 °C. Gels were dried and exposed to BAS2000 with BAStation
software (Fuji Photo Film).
RESULTSPUFA Suppression of Mouse SREBP-1c Promoter Activity in
HepG2 and HEK293 Cells—To investigate the molecular mechanism by which dietary PUFAs decrease hepatic SREBP-1c
expression, we established mouse SREBP-1c promoter luciferase reporter gene assays in HepG2 and HEK293 cells. As an
initial study, we estimated the effect of supplementation of
EPA to the medium (100 ␮M) on mouse SREBP-1c promoter(2.6-kb 5⬘-flanking region) activity in HepG2 cells. Cells were

PUFA Inhibition of LXR-activated SREBP-1c Promoter

1707

FIG. 3. Identification of the polyunsaturated fatty acid-suppressive region in the SREBP1c-promoter by deletional analysis.
SREBP-1c promoter luciferase reporters of various lengths (as indicated) were constructed (left panel). The HEK293 cells were transfected witheach reporter plasmid, pCMV-LXR␣, and reference plasmid, pSV-␤gal. Either EPA (300 ␮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, luciferase activity was measured and normalizedto ␤-galactosidase activity. The effect of EPA in each construct without LXR␣ co-expression (Basal activity) is expressed as normalized luciferase
activity (means ⫾ S.D., three independent experiments in a duplicate assay) (middle panel). The data from LXR␣ co-expression (0.1 ␮g ofpCMV-LXR␣, LXR␣-induced activity) are shown as fold change relative to mock-transfected control (means ⫾ S.D., three independent experiments
in a duplicate assay) (right panel). bp, base pairs.

FIG. 4. Inhibitory effect of polyunsaturated fatty acid 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 (23). B, the LXRE complex in the SREBP-1c promoter was fused to a luciferase reporter plasmid, which contained an SV40
promoter (pGL2 promoter vector). This enhancer construct (pLXRE-Luc) or the indicated mutant construct was co-transfected into HEK293 cellswith pCMV-LXR␣, -␤, or an empty vector, CMV-7 as a control, and pSV-␤gal as a reference plasmid. C, pBP1c90b-Luc, which contained an SRE
complex but no LXRE complex, was co-transfected into HEK293 cells with pCMV-SREBP-1c or an empty vector (CMV-7) as a control and pSV-␤gal
as a reference plasmid. Either EPA (100 ␮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, luciferase activity was measured and normalized to ␤-galactosidase activity. The fold change
by LXRs or their ligands in the luciferase activity (means ⫾ S.D., three independent experiments in a duplicate assay) as compared with therespective control is shown. bp, base pairs.

co-transfected with LXR␣ or -␤. These conditions have beenshown to activate SREBP-1c promoter through LXREs (23).
Consistent with previous in vivo observations that PUFA suppresses SREBP-1c expression (14), current data indicate that
EPA considerably decreases the SREBP-1c promoter activity
when LXR/RXR is activated (Fig. 1A). This suppressive effect of
EPA was similarly observed in HEK293 cells (Fig. 1B). Afterthese studies, effects of various PUFAs were tested in HEK293
cells co-transfected with LXR␣. As shown in Fig. 2, SREBP-1c
promoter activity was reduced by the addition of each PUFA(AA ⬎ EPA ⬎ DHA ⬎ LA). In contrast, saturated fatty acid(SA) had no effect, and the result of the addition of OA wasminimal. These data indicate that SREBP-1c promoter assays
can reflect PUFA suppression of SREBP-1c expression reported

by us and others (14 –16, 18) and that the cis-element(s) responsible for this PUFA effect should be located within this
2.6-kb 5⬘-flanking sequence of the mouse SREBP-1c gene.
LXREs as PUFA-suppressive Elements in the SREBP-1c Promoter—To locate the PUFA-suppressive element in the
SREBP-1c promoter, we estimated the inhibitory effects of EPA
on reporter genes containing the SREBP-1c promoter of various sizes. As shown in Fig. 3, the lack of LXREa, the upstream
LXRE site of the two LXREs in the SREBP-1c promoter, caused
a partial decrease in the inhibitory effect of EPA, and deletionof both LXREa and -b abolished the effect completely. These
data suggest that the region containing the two LXREs are the
PUFA-responsive elements of the SREBP-1c promoter. To explore this more precisely, we constructed an enhancer lucifer-

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PUFA Inhibition of LXR-activated SREBP-1c Promoter

FIG. 5. Dose-dependent suppression of the LXRE enhancer
complex in the SREBP-1c promoter by various polyunsaturated
fatty acids. Indicated PUFA or ethanol (control) was added to the
HEK293 cells after transfection of pLXRE-Luc, pCMV-LXR␣, and pSV␤gal. After a 24-h incubation, luciferase activity was measured andnormalized to ␤-galactosidase activity. The percent inhibition by PUFAs in the luciferase activity (means ⫾ S.D., three independent experiments in a duplicate assay) as compared with the LXR␣ (0.1 ␮g)induced control is shown.

ase construct containing the two LXREs (pLXRE-Luc). As
shown in Fig. 4, pLXRE-Luc was activated by overexpression ofeither LXR␣ or -␤. EPA suppression was observed in both
LXR␣- and LXR␤-activated LXRE-Luc activities. Introductionof a mutation in either LXREa or LXREb caused a partial
impairment in this EPA inhibitory effect. Disruption of bothelements abolished the EPA suppression completely. Another
SREBP-1c promoter luciferase reporter (pBP1c90-Luc) containing an SRE, but no LXREs, was activated by co-expressionof nuclear SREBP-1c. No EPA inhibition was observed in thisconstruct, even when SREBP-1c was overexpressed. These
data confirm that both LXREs are responsible for PUFA suppression of the SREBP-1c promoter.
Using the LXRE complex construct, we demonstrated a dosedependent inhibition of SREBP-1c promoter activity by a variety of fatty acids. As shown in Fig. 5, PUFA, AA, EPA, DHA,
and LA all suppressed pLXRE-Luc in a dose-dependent manner. OA showed a weak suppression, whereas SA did not haveany effect. These data are consistent with results from theoriginal 2.6-kb SREBP-1c construct and indicate that two
LXREs are responsible for PUFA suppression.
PUFA are known to be PPAR ligands (33, 34). However,previous in vivo observations on PUFA inhibition of SREBP-1csuggest that the effect was PPAR-independent (35). The present study indicates that the element responsible for PUFA
suppression is the LXREs. To rule out the possibility that
PUFA inhibition of pLXRE-Luc is mediated through PPAR␣,
we used synthetic PPAR ligands such fenofibric acid and Wy14,643 for PPAR␣ and pioglitazone for PPAR␥ (Fig. 6). In
contrast to EPA, these pharmacological PPAR agonists did not
suppress pLXRE-luc, whereas these ligands did activate theirrespective target pPPRE-luc constructs. These data indicatethat PPAR activation is not involved in PUFA inhibition of
LXRE and SREBP-1c.
PUFA Inhibition of Ligand-activated LXR-RXR Binding to the
LXRE—To further investigate the molecular mechanism bywhich PUFAs suppress LXREs in the SREBP-1c promoter, gelshift mobility assays were performed. In vitro translated LXR␣and RXR recombinant proteins were used to confirm binding of
LXR␣/RXR heterodimer to the LXREb probe as estimated by theshifted band. Fig. 7A shows that the shifted signal was enhancedby the direct addition of T0901713, an artificial LXR ligand, in adose-dependent manner, demonstrating ligand activation of LXR
binding to LXRE. The addition of PUFA inhibited the shifted

FIG. 6. No suppression of the SREBP-1c promoter by pharmacological PPAR activators. pLXRE-Luc was co-transfected into HEK293
cells with pCMV-LXR␣ or an empty vector, CMV-7 as a control andpSV-␤gal as a reference plasmid (A). pPPRE-Luc was co-transfected into
HEK293 cells with pCMV-PPAR␣, pCMV-PPAR␥, or an empty vector,
CMV-7 as a control, and pSV-␤gal as a reference plasmid (B). LXR ligand
22RHC (10 ␮M), EPA (100 ␮M), PPAR␣ pharmacological ligands fenofibric
acid (Feno, 10 ␮M) and Wy-14,643 (WY, 10 ␮M), PPAR␥ pharmacologicalligand pioglitazone (Pio, 1 ␮M), and ethanol (or dimethysulfoxide) as acontrol were added to the cells after transfection of pLXRE-Luc andpSV-␤gal 24 h before the assay. After incubation, luciferase activity wasmeasured and normalized to ␤-galactosidase activity. The percent inhibition of luciferase activity by EPA or PPAR ligands in the luciferase
activity (means ⫾ S.D., n ⫽ 3) as compared with the LXR␣ (0.1 ␮g)- or
22RHC (10 ␮M)-induced controls (A) and fold change by EPA or PPAR
ligands in the luciferase activity (means ⫾ S.D., three independent experiments in a duplicate assay) as compared with the PPAR␣ (0.01 ␮g)- or
PPAR␥ (0.01 ␮g)-induced controls, are shown.

bind, whereas SA and OA had a minimal effect. The rank orderof the potency of the inhibitory effect of each fatty acid was
similar to that observed in the luciferase assays. The inhibitory
effect of AA was the strongest (Fig. 7B). Fig. 7C shows thecompetition between AA and LXR ligand. The signal from the
LXR/RXR䡠LXRE complex was inhibited by the addition of AA in
a dose-dependent manner, and further addition of T0901713
dose-dependently blocked the inhibitory effect of AA. These results strongly suggest that inhibition of LXR/RXR binding to
LXRE by PUFA was mediated through antagonizing the ligand
effect on the LBD of LXR. For comparison, effects of PUFA on
shifts of a PPRE probe by PPAR␣ and RXR proteins were also
tested (Fig. 7D). PPAR␣/RXR binding to PPRE was not affectedby SA but was enhanced by OA, PUFA, and PPAR ligands,
demonstrating that blocking the effects of PUFA on LXR/RXRLXRE binding are not due to nonspecific inhibition of the assays
by the fatty acid moiety. The data suggest that PUFA directly
inhibits LXR/RXR binding to LXRE or LXR/RXR heterodimers.
PUFA Competition with an LXR Ligand in the Activation of
the LBD of LXR—To clarify the molecular mechanism by which
PUFA inhibits LXR/RXR binding to LXRE, LBD activation
assays of LXR␣ were performed. In these assays, an expression
plasmid of the LBD of LXR␣ fused to the Gal4 DNA binding
domain was co-transfected with a luciferase reporter containing Gal4 binding sites to estimate the specific ligand binding of
the samples to LBD of LXR␣. The addition of 22RHC, a well

PUFA Inhibition of LXR-activated SREBP-1c Promoter

1709

FIG. 7. Polyunsaturated fatty acids
inhibit LXR-RXR binding to LXREs
in the SREBP1c-promoter as measured by gel-mobility shift assays. The
indicated fatty acid, PPAR ligands, or ethanol (or dimethysulfoxide) as controls
were incubated with in vitro synthesized
LXR␣, PPAR␣, and RXR (1–1.5 ␮l of programmed reticulocyte lysate, TNT Quick
Coupled Transcription/Translation Systems, Promega) for 30 min on ice. Afterincubation, labeled LXR-response element (LXREb) in the SREBP-1c promoter(A–C) or labeled PPRE in the acyl-CoAoxidase promoter (D) was added and incubated for 30 min on ice. 30 ␮M PUFA, 10
␮M Wy-14,643 (WY), and 1 ␮M pioglitazon(Pio) were used in panels B and D. The
DNA-protein complexes were resolved ina 4.8% polyacrylamide gel. DM, dimethyl
sulfoxide.

known LXR ligand, increased the Gal4 activity. As shown in
Fig. 8, the addition of each PUFA showed a dose-dependent
inhibition of the LBD activation in a very similar pattern to theeffects observed in the LXRE luciferase assay under 22RHCactivated conditions, suggesting that PUFA inhibits LXR ligand binding to LXR/RXR.
Subsequently, the PUFA inhibitory effect on SREBP-1c promoter activity was re-estimated by competition between EPA
and 22RHC in the presence of an abundant amount of LXR␣ byco-transfection (0.1 ␮g DNA). The addition of 22RHC without
EPA resulted in a dose-dependent increase in the luciferase
activity (Fig. 9, left). As shown in Fig. 9, right, in the presenceof 10 and 30 ␮M of 22RHC, the percent inhibition curve of
LXRE-LBD binding activity by EPA was shifted to the right,
suggesting a competition between 22RHC and EPA in theactivation of LXR.
No Involvement of RXR on PUFA Suppression of the
SREBP-1c Promoter—We also investigated the possibility that
PUFA inhibition of LXR/RXR binding to LXRE might be mediated through an interaction of PUFA to RXR. Fig. 10 showsthat overexpression of RXR by co-transfection minimallychanged LXRE-enhancer luciferase activity, suggesting that
RXR is not a limiting factor for LXR/RXR binding to LXRE in
this system. If PUFA could interact with RXR to modify LXR/RXR binding to LXRE, overexpression of RXR should absorb
and repress this PUFA effect on LXR/RXR. However, inhibitory
effects of PUFA on LXRE-enhancer activity (Fig. 10) and the
2.6-kb SREBP-1c promoter activity (data not shown) were notaffected by RXR overexpression in the RXR-co-transfected
cells. The addition of 9CRA, an RXR ligand, increased the effectof LXR/RXR but did not markedly affect the inhibitory efficiency of PUFA. These results indicate that the inhibitory
effect of PUFA may be independent of the RXR portion of the
LXR/RXR heterodimer.

FIG. 8. Polyunsaturated fatty acids inhibit the LBD activation
assay of LXR. The Gal4-driven luciferase reporter construct, p17m8
was co-transfected into HEK293 cells with an expression vector containing the Gal4 DNA binding domain fused to the ligand binding
domain of LXR␣, pM-LXR␣, and a control plasmid, pSV-␤gal. After thetransfection, the cells were incubated with various PUFAs in the presence of their ligand, 22RHC for 24 h. After incubation, luciferase activity was measured and normalized to ␤-galactosidase activity. The percent inhibition in luciferase activity by polyunsaturated fatty acids(means ⫾ S.D., three independent experiments in a duplicate assay) in
the presence of 22RHC (10 ␮M) is shown.
DISCUSSION

In the current study, we located PUFA suppressive elementsin the mouse SREBP-1c promoter. The responsible elements
correspond to two LXREs that were previously identified as
LXR/RXR activation sites (23). Further luciferase studies, gelshift assays, and LBD activation assays demonstrated that
PUFAs suppress SREBP-1c expression through interactingwith the LBD of LXR and inhibiting LXR/RXR binding to
the LXREs crucial for SREBP-1c expression as schematized inFig. 11.

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PUFA Inhibition of LXR-activated SREBP-1c Promoter

FIG. 9. Competition between 22(R)-hydroxycholesterol and
EPA in LXR-induced SREBP-1c promoter activity. pLXRE-Luc(0.25 ␮g) was co-transfected into HEK293 cells with pCMV-LXR␣ (0.1
␮g) or an empty vector, CMV-7, as a control, and pSV-␤gal, as areference plasmid. An indicated concentration of 22RHC 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 (left panel). In theright panel, an indicated amount of EPA was also added. After incubation, luciferase activity was measured and normalized to ␤-galactosidase activity. The fold induction by LXR␣ and 22RHC in luciferase
activity as compared with control (mock-transfected cells without
22RHC addition) is shown in the left panel. The percent inhibition by
EPA in the luciferase activity is shown (right panel). M, mock.

FIG. 10. Inhibitory effect of eicosapentaenoic acid on
SREBP-1c promoter activity is not affected by the overexpression of RXR or 9CRA addition. pLXRE-Luc was co-transfected into
HEK293 cells with pCMV-LXR␣ (0.1 ␮g), pCMV-RXR (0.5 ␮g), or an
empty vector, pCMV-7 as a control, and pSV-␤gal as a reference plasmid. EPA (100 ␮M), 9-cis retinoic acid (9CRA, 10 ␮M), or ethanol (EtOH)
as a control were added to the cells after transfection in medium with
10% fetal bovine serum 24 h before the assay. After incubation, luciferase activity was measured and normalized to ␤-galactosidase activity.
The fold induction by RXR and 9CRA in luciferase activity (means ⫾S.D., three independent experiments in a duplicate assay) as comparedwith control (MOCK and ethanol) is shown.

The order of inhibitory magnitude of each long chain fatty
acid on SREBP-1c expression is as follows: AA ⬎ EPA ⬎DHA ⬎ LA ⬎⬎ OA ⬎ SA ⫽ 0. This order was essentiallyconsistent among the luciferase assays with the 2.6-kb
SREBP-1c promoter and LXRE-enhancer as well as in gel shift
and LBD activation assays. Furthermore, the same order oflong chain fatty acid effects on SREBP-1c suppression has beenshown in diet studies with mice.2 Furthermore, these datasuggest that the inhibitory effect of PUFA is primarily attributed to their blocking effect on the LBD of LXR. The degree of
unsaturation of the fatty acids might be a factor for this inhibitory effect, but whether they are n-3 or n-6 appears to beirrelevant.
The LXR/RXR complex has been established as a nuclear
receptor for oxysterols, controlling regulation of excess cellular
2

N. Yahagi, unpublished data.

FIG. 11. Mechanism by which polyunsaturated fatty acids suppresses the SREBP-1c promoter activity through affecting LXRRXR activation pathway. PUFAs suppress SREBP-1c gene expression crucial for lipogenesis by inhibiting LXR-RXR binding to the
LXREs. Reciprocally, PUFAs promote PPAR-activated genes expression crucial for lipid degradation through activation of PPAR-RXR
binding to the PPREs.

cholesterol (24 –27, 30). It is interesting to speculate that oxidative modification of PUFA during incubation might makethem eligible to interact with LXR. However, the addition ofseveral kinds of antioxidants such as probucol and vitamin E
did not change the ability of PUFA to inhibit SREBP-1c activation (data not shown). Finally, even after direct addition of
PUFA to the incubation mixture for gel shift assays, PUFA still
showed inhibitory effects on LXR/RXR binding to LXRE,
strongly suggesting a direct action of PUFA. The current study
demonstrates that PUFA can be an antagonist for LXR/RXR. It
seems that PUFA binds to the LBD of LXRs in a fashion that iscompetitive with an endogenous LXR ligand, thereby repressing LXR/RXR transactivity. During the process of preparing
this manuscript, an antagonizing effect of PUFA in competition
with an LXR pharmacological ligand, as measured by
SREBP-1c expression in RNA protection and LXR coactivator
recruitment assays in rat hepatoma cells, was reported (21).
The conclusion was compatible with our present data. Further
studies on LXR ligands in relation to oxysterols and PUFA are
needed. Structure analysis on ligand binding and DNA binding
domains of LXR in the presence of LXR ligands and LXRE
should assist in understanding the complex nature of thissystem.
Our new finding on PUFA inhibition of LXR-SREBP-1cbrings up an intriguing speculation for a mechanism of energy
regulation as depicted in Fig. 11. Previous work by our laboratory (23) and others (31, 36) suggests that LXR/RXR is a dominant activator for expression of SREBP-1c, a transcription
factor that is a crucial factor for hepatic lipogenesis that isnecessary for storage of excess energy as observed in a refedstate. Meanwhile, PUFAs can function as ligands for PPAR␣(33, 34), another transcription factor that plays a crucial rolefor fatty acid oxidation in an energy-depleted state such as
fasting (37, 38). In a fasted state, PUFAs can be released from
adipose tissue by lipolysis. Taken up by the liver, PUFAs canbind to and activate PPAR␣ to induce ␤-oxidation of othersaturated or monounsaturated fatty acids. At the same time,
PUFA antagonize LXR/RXR, leading to suppression of
SREBP-1c and minimizing lipogenesis. Therefore, PUFA might
have efficient regulatory roles for adaptic control of two extreme nutritional states by having reciprocal effects on LXRSREBP-1c and PPAR␣ (Fig. 11). In addition, post-transcriptional regulation of the SREBP-1c gene by PUFA has been also
proposed (15, 20, 35). Further studies are needed to clarify thePUFA Inhibition of LXR-activated SREBP-1c Promoter
relative roles of transcriptional, post-transcriptional, andcleavage regulation of SREBP-1c by PUFA in vivo.
The current data suggest that PUFAs could be intricately
involved in nutritional regulation by affecting the LXRSREBP-1c system that is crucial for lipogenesis as well as
having a well established role as ligands for PPAR␣. Thismight open up a new aspect of nutritional regulation involving
essential fatty acids as well as energy fuels.
Acknowledgment—We thank A. H. Hasty for critical reading of themanuscript.
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