THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 15, pp. 11681–11691, April 9, 2010
© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

Polyunsaturated Fatty Acids Selectively Suppress Sterol
Regulatory Element-binding Protein-1 through Proteolytic
Processing and Autoloop Regulatory Circuit*
Received for publication, December 17, 2009, and in revised form, February 5, 2010 Published, JBC Papers in Press, February 9, 2010, DOI 10.1074/jbc.M109.096107

Yoshinori Takeuchi‡§¶, Naoya Yahagi‡§1, Yoshihiko Izumida‡§, Makiko Nishi‡§, Midori Kubota‡§, Yuji Teraoka‡§,Takashi Yamamoto§, Takashi Matsuzaka¶, Yoshimi Nakagawa¶, Motohiro Sekiya§, Yoko Iizuka§, Ken Ohashi§,
Jun-ichi Osuga§, Takanari Gotoda§, Shun Ishibashi§, Keiji Itaka储, Kazunori Kataoka储, Ryozo Nagai§,
Nobuhiro Yamada¶, Takashi Kadowaki‡§, and Hitoshi Shimano¶2
From the ‡Laboratory of Molecular Physiology on Energy Metabolism, §Department of Internal Medicine, and 储Center for Disease
Biology and Integrative Medicine, Graduate School of Medicine, University of Tokyo, Tokyo 113-8655 and ¶Advanced Biomedical
Applications, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Ibaraki 305-8575, Japan

Polyunsaturated fatty acids (PUFA)3 have been well established as negative regulators of hepatic lipogenesis (reviewed inRef. 1). Allmann and Gibson (2) discovered that adding 2% linoleate to a high carbohydrate fat-free diet suppressed the rate ofhepatic fatty acid biosynthesis and the activities of fatty-acid
synthase and glucose-6-phosphate dehydrogenase by nearly
70% in mice. In contrast, supplementing the high carbohydrate
diet with palmitate, oleate, or cholesterol had no effect on
hepatic lipogenesis or the activity of lipogenic enzymes. Since* This work was supported by grants-in-aid from the Ministry of Science, Education, Culture and Technology of Japan (to N. Y. and H. S.) and research
grants from The Uehara Memorial Foundation, ONO Medical Research
Foundation, Takeda Science Foundation, Suzuken Memorial Foundation,
Japan Heart Foundation, Kanae Foundation for the Promotion of Medical
Science, Senri Life Science Foundation, Japan Foundation for Applied Enzymology, and Okinaka Memorial Institute for Medical Research (to N. Y.).
1
To whom correspondence may be addressed: 7-3-1 Hongo, Bunkyoku, Tokyo
113-8655, Japan. Fax: 81-3-5802-2955; E-mail: nyahagi-tky@umin.ac.jp.
2
To whom correspondence may be addressed. E-mail: shimano-tky@umin.ac.jp.
3
The abbreviations used are: PUFA, polyunsaturated fatty acid; EPA, eicosapentaenoic acid; LXR, liver X receptor; LXRE, liver X receptor-binding element; SREBP, sterol regulatory element-binding protein; SRE, SREBP-binding element; pfu, plaque-forming unit; OLA, oleic acid ethyl ester; shRNA,
short hairpin RNA; SCAP, SREBP cleavage-activating protein.

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then, a number of investigators have demonstrated that dietary
PUFA of the n-6 and n-3 families suppress hepatic lipogenesis.
This anti-lipogenic action of PUFA reflects decreases in mRNAlevels of hepatic enzymes, including acetyl-CoA carboxylase,fatty-acid synthase, and stearoyl-CoA desaturase.
The fatty acid biosynthetic pathway, composed of some 25
enzymes, has been elucidated in detail (3). For the de novo synthesis of long chain saturated fatty acids, fatty-acid synthase,the main synthetic enzyme that catalyzes the condensation ofmalonyl-CoA to produce the 16-carbon saturated fatty acid
palmitate, and acetyl-CoA carboxylase, which synthesizes malonyl-CoA from acetyl-CoA, are of particular importance. The
regulation of these lipogenic enzymes has been revealed to be
primarily controlled by a transcription factor sterol regulatory
element-binding protein (SREBP)-1c (4, 5).
SREBPs are transcription factors that belong to the basic
helix-loop-helix leucine zipper family and are considered to be
profoundly involved in the transcriptional regulation of cholesterogenic and lipogenic enzymes (6, 7). Unlike other membersof the basic helix-loop-helix leucine zipper family, SREBPs aresynthesized as precursors bound to the endoplasmic reticulumand nuclear envelope. Upon activation, SREBPs are cleaved,
and the N-terminal parts are released from the membrane into
the nucleus as mature protein by a sequential two-step proteolytic processing. To date, three SREBP isoforms, SREBP-1a, -1c,and -2, have been identified and characterized. SREBP-1a and-1c are transcribed from the same gene, each by a distinct promoter, and the predominant SREBP-1 isoform in liver is 1c
rather than 1a (8). It has been established by several lines ofevidence, especially by those from transgenic and knock-out
mouse models, that SREBP-1c controls hepatic lipogenesis,
whereas SREBP-2 plays a crucial role in regulation of cholesterol synthesis (5, 9, 10).
In 1999, we and others reported that the PUFA-specificsuppression of lipogenic enzymes is mediated by the reduction of nuclear SREBP-1c protein in the liver (11–14). Interestingly, PUFA selectively decreases SREBP-1, not affecting
SREBP-2. The mechanism by which PUFA specifically suppresses SREBP-1c nuclear abundance, however, remains
unclear, although several potential mechanisms have been
implicated, including suppression of Srebf1c gene transcription
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Sterol regulatory element-binding protein (SREBP)-1 is a key
transcription factor for the regulation of lipogenic enzyme
genes in the liver. Polyunsaturated fatty acids (PUFA) selectively
suppress hepatic SREBP-1, but molecular mechanisms remain
largely unknown. To gain insight into this regulation, we established in vivo reporter assays to assess the activities of Srebf1ctranscription and proteolytic processing. Using these in vivo
reporter assays, we showed that the primary mechanism for
PUFA suppression of SREBP-1 is at the proteolytic processing
level and that this suppression in turn decreases the mRNA transcription through lowering SREBP-1 binding to the SREBPbinding element on the promoter (“autoloop regulatory circuit”), although liver X receptor, an activator for Srebf1ctranscription, is not involved in this regulation by PUFA. The
mechanisms for PUFA suppression of SREBP-1 confirm that the
autoloop regulation for transcription is crucial for the nutritional regulation of triglyceride synthesis.

PUFA Selectively Suppresses SREBP-1

and proteolytic processing as well as enhancement of proteasomal degradation and mRNA decay (11, 15–18). As for the suppression of Srebf1c gene transcription by PUFA, we have previously identified liver X receptor-binding element (LXRE) and
SREBP-binding element (SRE) on the Srebf1c promoter region
by a series of promoter analyses (19, 20), and we have also suggested that PUFA can antagonize LXR in an in vitro setting (16).
These situations prompted us to clarify the molecular mechanism underlying the suppressive effect of PUFA on nuclear
SREBP-1 abundance, especially in the in vivo setting. Because
the inhibitory effect of fatty acids on SREBP-1 was specific andclear for PUFA in in vivo experiments, whereas many previous
reports using in vitro system have failed to show this specificity(21, 22), we adopted an approach of in vivo reporter assays
utilizing the in vivo imaging system (IVISTM; Xenogen, Alameda, CA). First, to examine the transcriptional mechanism, in

11682 JOURNAL OF BIOLOGICAL CHEMISTRY

vivo promoter analyses were performed, and the responsible
cis-element on the Srebf1c promoter was located at SRE, not at
LXRE. Next, the mechanism by which PUFA decreases thenuclear form of SREBP-1 was explored by another reporter system detecting proteolytic activity for the precursor form of
SREBP-1, demonstrating that PUFA suppresses the maturationof SREBP-1 through proteolytic processes. From these experiments, we concluded that the primary mechanism for PUFA
suppression of SREBP-1 expression is at the proteolytic processing level and that this suppression in turn decreases the
Srebf1c mRNA transcription through lowering SREBP-1 binding to SRE on the promoter (“autoloop regulation” (19)).

EXPERIMENTAL PROCEDURES
Materials—Eicosapentaenoic acid (EPA) ethyl ester (95%
grade) was provided from Mochida Pharmaceutical (Tokyo,
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FIGURE 1. PUFA selectively decreases SREBP-1, not affecting SREBP-2. A, Northern blot analysis of SREBP-1 and SREBP-2 from livers. Total RNA (7.5 ␮g) fromlivers pooled equally from two mice for each group was subjected to Northern blotting to determine SREBP-1, SREBP-2, and 36B4 (used as a loading control)mRNA levels. ICR male mice were fed a high carbohydrate fat-free diet and treated orally with EPA at indicated doses once a day for 4 consecutive days. Control
mice were treated orally with 7.5 g/kg water. Mice were sacrificed in a nonfasted state. B, immunoblot (IB) analysis of mature and precursor SREBP-1 and
SREBP-2 proteins from livers. Aliquots of nuclear extracts (10 ␮g) and total proteins (50 ␮g) from livers pooled equally from four male mice for each group were
subjected to immunoblot analysis. The primary antibodies used were polyclonal anti-mouse SREBP-1 and polyclonal anti-mouse SREBP-2. C, plasma triglycerides in EPA-treated mice. D, Northern blot analysis of SREBP-1 from livers. ICR male mice were fed a high carbohydrate fat-free diet and treated orally with 7.5
g/kg OLA or EPA once a day for 4 days. Total RNA (7.5 ␮g) from livers pooled equally from two mice for each group was subjected to Northern blotting todetermine SREBP-1 and 36B4 (used as a loading control (CTL)) mRNA levels. E, plasma triglycerides in OLA- and EPA-treated mice. These data are representativeof at least two independent experiments (n ⫽ 4 mice/group). Results are means ⫾ S.E. **, p ⬍ 0.01; N.S., not significant.

PUFA Selectively Suppresses SREBP-1
Japan) and GW532 (SCAP ligand) from GlaxoSmithKline (LesUlis Cedex, France). The synthetic LXR agonist T0901317 was
Fatty acid composition in the liver was analyzed by gas chromatography (n ⫽ 4purchased from Cayman Chemical (Ann Arbor, MI). Standard
mice/group). Results are means ⫾ S.E.
laboratory diet (CRF-1, composed of 60% carbohydrate, 13%
EPA (g/kg/day)
fat, and 27% protein on a caloric basis) and high carbohydrate
0
2.5
5.0
7.5
fat-free diet (70% sucrose and 20% casein supplemented with
mg/g liver weight
methionine, vitamins, and minerals) were obtained from OriC12:0
0.00 ⫾ 0.00 0.00 ⫾ 0.00
0.00 ⫾ 0.00
0.00 ⫾ 0.00
C14:0
0.22 ⫾ 0.05 0.09 ⫾ 0.03
0.05 ⫾ 0.01a 0.07 ⫾ 0.02a
ental Yeast (Tokyo, Japan). Other materials were purchased
C14:1n-5
0.00 ⫾ 0.00 0.00 ⫾ 0.00
0.00 ⫾ 0.00
0.00 ⫾ 0.00
from Sigma unless indicated otherwise.
C16:0
10.11 ⫾ 1.07 8.06 ⫾ 0.60
6.48 ⫾ 0.40a 7.97 ⫾ 0.58
Animals—Seven- to 9-week-old ICR male mice were pur0.75 ⫾ 0.13b
C16:1n-7
2.06 ⫾ 0.26 1.10 ⫾ 0.18a 0.70 ⫾ 0.04b
C18:0
3.12 ⫾ 0.09 3.72 ⫾ 0.17
3.13 ⫾ 0.18
4.15 ⫾ 0.18b
chasedfrom CLEA (Tokyo, Japan). All animals were mainC18:1n-9 14.77 ⫾ 2.96 7.76 ⫾ 1.82a 3.70 ⫾ 0.54a 4.88 ⫾ 1.43a
tained in a temperature-controlled environment with a 12-h
C18:2n-6
1.76 ⫾ 0.32 1.74 ⫾ 0.23
1.92 ⫾ 0.27
1.65 ⫾ 0.30
C18:3n-6
0.00 ⫾ 0.00 0.00 ⫾ 0.00
0.03 ⫾ 0.00
0.03 ⫾ 0.00
light/dark cycle and were given free access to standard laboraC18:3n-3
0.00 ⫾ 0.00 0.00 ⫾ 0.00
0.03 ⫾ 0.00
0.00 ⫾ 0.00
tory diet and water. Four days before the start of indicated fatty
C20:0
0.04 ⫾ 0.01 0.07 ⫾0.02
0.08 ⫾ 0.02
0.08 ⫾ 0.02
C20:1n-90.64 ⫾ 0.17 0.22 ⫾ 0.09
0.10 ⫾ 0.02a 0.12 ⫾ 0.04a
acid administration, the basal diet was switched to a high carC20:2n-6
0.07 ⫾ 0.01 0.05 ⫾ 0.00
0.05 ⫾ 0.01
0.04 ⫾ 0.00abohydrate fat-free diet. EPA or oleic acid ethyl ester (OLA) was
C20:3n-90.93 ⫾ 0.06 0.18 ⫾ 0.10b
0.07 ⫾ 0.01b
0.08 ⫾ 0.02b
C20:3n-6
0.44 ⫾ 0.09 0.19 ⫾ 0.02a 0.17 ⫾ 0.03a 0.15 ⫾ 0.01a
administered orally once a day for 4 days. GW532 (0 –15
C20:4n-6
2.37 ⫾ 0.33 1.79 ⫾ 0.05
1.64 ⫾ 0.15
1.54 ⫾ 0.08a
mg/kg/day) or vehicle (0.9% carboxymethylcellulose, 9.95%
C20:5n-30.12 ⫾ 0.02 3.15 ⫾ 0.31b
3.06 ⫾ 0.17b4.42 ⫾ 0.35b
C22:0
0.09 ⫾ 0.02 0.14 ⫾ 0.03
0.16 ⫾ 0.03
0.17 ⫾ 0.04
polyethylene glycol 400, and 0.05% Tween 80) was adminisC22:1n-9
0.04 ⫾ 0.01 0.00 ⫾ 0.00
0.03 ⫾ 0.01
0.03 ⫾ 0.01
tered orally once a day for 3 days. Mice were sacrificed in the
C22:4n-6
0.11 ⫾ 0.02 0.07 ⫾ 0.01
0.06 ⫾ 0.01a 0.06 ⫾ 0.01a
C22:5n-3
0.05 ⫾ 0.01 1.21 ⫾ 0.33a 1.50 ⫾ 0.23b
2.01 ⫾ 0.44b
light phase in a nonfasted state. All experiments were repeated
C22:6n-3
2.88 ⫾ 0.23 3.60 ⫾ 0.32
3.33 ⫾ 0.34
3.65 ⫾ 0.09a
at least twice. All animals studied were anesthetized and euthaC24:0
0.07 ⫾ 0.01 0.10 ⫾ 0.02
0.12 ⫾ 0.01a 0.13 ⫾ 0.02a
C24:1n-90.18 ⫾ 0.01 0.18 ⫾ 0.01
0.12 ⫾ 0.02a 0.15 ⫾ 0.01
nized according to protocol approved by the Tokyo Universityap ⬍ 0.05.
Animal Care and Use Committee.
bp ⬍ 0.01.
RNA Isolation and Northern Blotting—Total RNA from mouse liver
was extracted using TRIzol reagent(Invitrogen), and a 7.5-␮g RNA
sample equally pooled among eachgroup was run on a 1% agarose gel
containing formaldehyde and transferred to a nylon membrane. The
cDNA probes for mouse SREBP-1,
SREBP-2, and 36B4 were cloned as
described previously (11). The probes
were labeled with [␣-32P]dCTP using Megaprime DNA labeling system (Amersham Biosciences). The
membranes were hybridized withthe radiolabeled probe in RapidHyb Buffer (Amersham Biosciences)and washed in 0.1⫻ SSC, 0.1% SDS
at 65 °C. Blots were exposed to BAS
imaging plate for the BAS2000 BIO
imaging analyzer (Fuji Photo Film).
The quantification results obtainedwith the BAS2000 system were normalized to the signal generated
from 36B4 mRNA.
Nuclear Protein Extraction from
Liver—Nuclear extract protein from
FIGURE 2. PUFA suppresses Srebf1c promoter activity. 2.2-kbp Srebf1c-Luc adenovirus (Ad-2.2k- Srebf1c-1c-Luc) mouse or rat liver was prepared as(6.0 ⫻ 106 pfu/body) was intravenously injected into ICR male mice. After 4 days, mice (n ⫽ 4 for each group) were described previously (23). Briefly,
treated orally with EPA, OLA, or water (CTL) at indicated doses once a day for 4 days. A, on day 0 (before) and day 4 excised livers (0.5 g) were homogefrom the first treatment of EPA, luciferin was injected intraperitoneally in nonfasted mice, and the luminescencefrom liver was captured with IVIS. The color overlay on the image represents the photons/s emitted from the animal nized in a Polytron in 5 ml of buffer
with a range of 1.5 ⫻ 105–1.5 ⫻ 106 photons/s (day 0), 5.0 ⫻ 105–5.0 ⫻ 106 photons/s (day 4), as indicated by the A, which consisted of 10 mM HEPES,
color scale next to the images. B and C, quantification of luciferase activity with LivingImage software. Fold changesof luciferase activity on day 4 versus day 0 are shown. These data are representative of at least two independent pH 7.9, 25 mM KCl, 1 mM EDTA, 2 M
experiments (n ⫽ 4 mice/group). Results are means ⫾ S.E. *, p ⬍ 0.05; **, p ⬍ 0.01, respectively. N.S., not significant. sucrose, 10% glycerol, 0.15 mM
TABLE 1
Fatty acid composition in the liver

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PUFA Selectively Suppresses SREBP-1
glucose, 100 units/ml penicillin, and 100 ␮g/ml streptomycin
sulfate supplemented with 10% fetal bovine serum. For luciferase assay, HEK293 cells were seeded in a 48-well plate andincubated until 80% confluent. The indicated amounts of expression plasmids, firefly luciferase reporter plasmid, and pSV40Renilla luciferase plasmid were co-transfected into HEK293
cells using SuperFect transfection reagent (Qiagen) according
to the manufacturer’s protocol. Total amounts of transfected
DNA were adjusted with empty vector. The luciferase activity
in transfectants was measured on a luminometer. Renilla luciferase activities were used to normalize transfection efficiencies.
In Vivo Imaging of Luciferase Activity—In vivo imaging was
performed as described previously (27). Mice were anesthetizedwith isoflurane/oxygen, and 3.0 mg of luciferin dissolved in 0.4
ml of phosphate-buffered saline (7.5 mg/ml) was injected into
the intraperitoneal cavity. Mice were imaged from the ventral
side using an In Vivo Imaging System (IVISTM, Xenogen) 15
min following the injection of luciferin. Relative photon emission over the liver region was quantified using LivingImageTM
software (Xenogen).
Plasmid Construction—To construct expression plasmids
for GAL4-DNA binding domain (GAL4-DBD) and VP16transactivation domain (VP16-AD) fused to human SREBPs,
VP16-AD from pACT vector (Promega) was inserted intopM vector (Clontech) with various lengths of DNA fragmentof human SREBP-1c (amino acids 1–1123 (“FL” for fulllength), 1– 436 (“Nuc” for N-terminal nuclear part), or 431–
1123 (“Reg” for C-terminal regulatory domain) or human
SREBP-2 (amino acids 14 –1141 (FL) or 450 –1141 (Reg))retrieved by PCR from pTK-HSV-hSREBP-1c, pTK-HSVhSREBP-2, and pcDNA3.1(⫹)-SREBP-1c (28, 29). Gal4-RELuc plasmid was described previously (30). For the construction of an expression plasmid for mouse Insig-1, cDNA
fragment was amplified by PCR with primers 5⬘-GGATCCATGCCCAGGCTGCACGACCACG-3⬘ and 5⬘-CTCGAGTCAGTCACTGTGAGGCTTTTCCG-3⬘ and cloned into pcDNA3
vector with hemagglutin tag at the N terminus using BamHI
and XhoI. The expression plasmid for SCAP is a kind gift fromDr. Nakakuki.
Preparation of Recombinant Adenoviruses—To constructvarious lengths of mouse Srebf1c promoter luciferase reporter

FIGURE 3. PUFA suppresses Srebf1c promoter activity through SRE site. A, adenoviruses encoding various lengths of Srebf1c promoter, as well as two LXRE
sites on Srebf1c promoter (⫺239 to ⫺165) with SV40 promoter and SRE mutant version of 90-bp Srebf1c promoter, attached with luciferase (Luc) (6.0 ⫻ 106
pfu/body) were injected intravenously into ICR male mice. After 4 days, mice (n ⫽ 4 –7 for each group) were treated orally with 7.5 g/kg EPA or water (CTL) oncea day for 4 days. On day 0 and 4 after the first treatment of EPA, luciferin was injected intraperitoneally, and the luminescence from liver was captured with IVIS.
B, Ad-LXRE-Luc injected mice were administered orally with 50 mg/kg T0901317 or vehicle (0.9% carboxymethylcellulose, 9.95% polyethylene glycol 400, and0.05% Tween 80) after fasting overnight. At 0 and 16 h following T0901317 treatment in fasted states, luciferin was injected intraperitoneally, and theluminescence from liver was captured with IVIS. Fold change of luciferase activity at 16 versus 0 h was presented. C, E, F, and I, knockdown of hepatic LXR␣/␤ byadenoviral expression of shRNA. 250-bp Srebf1c-Luc adenovirus (Ad-250-bp- Srebf1c-Luc; 6.0 ⫻ 106 pfu/body) plus adenovirus expressing LXR␣/␤-specific or
LacZ-specific shRNA (Ad-LXRi or Ad-LacZi, respectively; 2.5 ⫻ 108 pfu/body) were intravenously injected into ICR male mice. After 4 days, the mice (n ⫽ 4 foreach group) were treated orally with 7.5 g/kg EPA or water (CTL) once a day for 4 days. On day 0 and 4 after the first treatment of EPA, luciferin was injected
intraperitoneally, and the luminescence from liver was captured with IVIS. D, G, H, and J, inhibitory effect of PUFA on Srebf1c gene transcription is not affectedby LXR agonist. 250-bp Srebf1c-Luc adenovirus (Ad-250bp- Srebf1c-Luc) (6.0 ⫻ 106 pfu/body) was intravenously injected into ICR male mice. After 6 days, themice (n ⫽ 4 for each group) were treated orally with 7.5 g/kg EPA or water (CTL) once a day for 4 days. At day 3 of EPA treatment, the mice were administeredorally with 10 mg/kg T0901317 or vehicle. On days 0 and 4 after the first treatment of EPA, luciferin was injected intraperitoneally, and the luminescence fromliver was captured with IVIS. C and D, quantification of luciferase activity with LivingImage software. Fold changes of luciferase activity on day 4 versus day 0 are
shown. E and G, Northern blot analysis of SREBP-1 from livers. Total RNA (7.5 ␮g) from livers pooled equally from mice of each group was subjected to Northernblotting to determine SREBP-1 and 36B4 (used as a loading control) mRNA levels. F and H, quantification of the data shown in E and G. The fold change is therelative ratio of each signal versus the control mice. I and J, immunoblot (IB) analysis of mature and precursor SREBP-1 proteins and LXR␣/␤ in livers. Aliquotsof nuclear extracts (10 ␮g) and total proteins (50 ␮g) from livers pooled equally from four male mice of each group were subjected to immunoblot analysis. The
primary antibodies used were polyclonal anti-mouse SREBP-1 and polyclonal anti-mouse LXR␣/␤. These data are representative of at least two independent
experiments (n ⫽ 4 –7 mice/group). Results are means ⫾ S.E. *, p ⬍ 0.05, and **, p ⬍ 0.01 versus controls.

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Downloaded from www.jbc.org at University of Tokyo Library, on April 3, 2010spermine, and 2 mM spermidine, supplemented with proteaseinhibitors (6 ␮g/ml N-acetyl-leucyl-leucyl-norleucinal (ALLN,
Calbiochem), 2.5 ␮g/ml pepstatin A, 2 ␮g/ml leupeptin, 0.1 mM
phenylmethylsulfonyl fluoride and 2.5 ␮g/ml aprotinin).
Pooled homogenate was then subjected to one stroke of a Teflon pestle in a Potter-Elvehjem homogenizer, followed by filtration through two layers of cheesecloth, and layered over 10 mlof buffer A. After centrifugation at 24,000 rpm on a Beckman
SW28 rotor for 1 h at 4 °C, the resulting nuclear pellet was
resuspended in a buffer containing 10 mM HEPES, pH 7.9, 100
mM KCl, 2 mM MgCl2, 1 mM EDTA, 1 mM dithiothreitol, and
10% glycerol supplemented with protease inhibitors, after
which 0.1 volume of 5 M NaCl was added. Each mixture wasagitated gently for 30 min at 4 °C and then centrifuged at 89,000rpm on a Himac S120AT2 rotor (Hitachi, Tokyo, Japan) for 30
min at 4 °C. The supernatant was used as nuclear extract.
Immunoblotting of SREBP Proteins—Aliquots of nuclear
extract (10 ␮g) and total lysate (50 ␮g) proteins were subjected
to SDS-PAGE. Immunoblot analysis was performed using the
ECL Western blotting detection system (Amersham Biosciences) and exposed to XAR-5 film (Eastman Kodak Co.). The
primary antibodies for SREBPs (rabbit polyclonal; number 931
for mouse SREBP-1 and number 528 for SREBP-2) were used as
described previously (24). The primary antibody for LXR␣/␤
(H-144; sc-13068) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
Fatty Acid Composition of Liver—Fatty acid composition wasmeasured as described previously (24). An aliquot (0.1 g) of liver
samples snap-frozen by liquid nitrogen was homogenized in 1
ml of normal saline. The fatty acid composition was measured
by gas chromatography at Bio-Medical Laboratories (Tokyo,
Japan) (25) Briefly, total lipids in liver homogenates wereextracted according to the Folch’s procedure (26), followed bytransesterification of fatty acids with boron trifluoride/methanol at 100 °C for 90 min. The methylated fatty acids were thenextracted with hexane and analyzed using a GC-17A gas chromatograph (Shimadzu Corp., Kyoto, Japan) and BPX70 capillary column (0.25 mm inner diameter ⫻ 30 m, SGE International Ltd., Melbourne, Australia).
Transfection and Luciferase Assays—HEK293 cells were cultured in Dulbecco’s modified Eagle’s medium containing 25 mM

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PUFA Selectively Suppresses SREBP-1

RESULTSPUFA Selectively Decreases SREBP-1, Not Affecting SREBP-2—
In our first series of experiments, we verified the effects of
PUFA on SREBP-1 and -2 expression in the liver. As expected,
administration of EPA, one of the major n-3 PUFA in mammals, down-regulated SREBP-1 mRNA and protein expression(Fig. 1, A, B, D, and E), leading to a decrease in plasma triglycerides concentration (Fig. 1, C and F) as compared with control
or oleate. The delivery of orally administered EPA to the liver
was confirmed by gas chromatography analysis (Table 1). EPA
administration did not affect the body weights of mice (data notshown). The dose-response relationship data suggested that thenuclear form is more sensitive to PUFA than the membrane
form of protein or mRNA. In contrast, SREBP-2 mRNA andprotein levels were not altered by PUFA.
PUFA Suppresses Srebf1c Promoter Activity through SRE Site—
Next, we attempted to estimate the Srebf1c promoter activity inthe liver with an in vivo luciferase reporter analysis. The luciferase reporter gene driven by the 2200-bp Srebf1c promoter
was adenovirally transduced into mouse liver, and the transcriptional activity was assessed by measuring luciferase activ-

ity with the IVIS imaging system. The physiological activity ofthis promoter had previously been confirmed by transgenic
mice (31). As shown in Fig. 2, EPA decreased the Srebf1c promoter activity in a dose-dependent manner.
To determine the responsible region for PUFA suppression,a promoter deletion study was performed; six adenovirus constructs containing different lengths of the Srebf1c promoter(ranging from 2200 to 58 bp) were transduced into mouse livers, and the suppressive effect of EPA on promoter activity wasassessed with IVIS (Fig. 3A). From this experiment, the responsible element was located at the SRE site within ⫺90 to ⫺60 bp
upstream of the transcription start site. This result was confirmed by another experiment using a mutant SRE construct.
LXR Is Not Involved in the PUFA Suppression of Srebf1c Gene
Expression in Vivo—Furthermore, it was clarified from theseries of deletion studies that EPA did not suppress the promoter activity of a construct containing only LXRE (Fig. 3A),
demonstrating that EPA does not antagonize LXR binding to
LXRE on the Srebf1c promoter at least in the in vivo setting.
Moreover, as shown in Fig. 3, C, E, F, and I, it was demonstrated
that the simultaneous knockdown of both LXR␣ and -␤ did notaffect the suppressive effect of PUFA on Srebf1c promoter
activity. Furthermore, the stimulation of LXR by an LXR agonist T0901317 was also shown to be independent of the inhibitory effect of PUFA, as shown in Fig. 3, D, G, H, and J. Based onthese findings, we concluded that LXR is not the direct target of
PUFA regulation in the in vivo setting, although LXR is a determinant of the expression level of SREBP-1 mRNA.
Inhibitory Effect of PUFA on Srebf1c Gene Transcription Is
Mediated through SREBP-1 Itself—To determine whether
the trans-acting factor for SRE is SREBP-1 or -2 or both,
SREBP-1/2 was knocked down by RNA interference. As
shown in Fig. 4, A and B, the effects of PUFA administrationand knocking down SREBP-1 were essentially equal, and when
SREBP-1 was knocked down, the promoter activity of Srebf1c
gene was reduced by half, and PUFA showed no additive effects.
In contrast, knocking down SREBP-2 did not affect the promoter activity of Srebf1c as assessed by luciferase reporter
expression (Fig. 4, E and F), demonstrating that SREBP-2 is not
involved in the transcriptional regulation of Srebf1c gene
expression. From these findings, it was concluded that thetrans-acting factor for SRE on the Srebf1c promoter is SREBP-1
and not SREBP-2. This indicates that SREBP-1 constitutes anautoloop regulatory circuit.
PUFA Suppresses the Proteolytic Activation of SREBP-1, Not
Affecting SREBP-2—SREBP-1 is synthesized as a precursor
bound to the endoplasmic reticulum and nuclear envelope

FIGURE 4. Inhibitory effect of PUFA on Srebf1c gene transcription is mediated through SREBP-1 itself. A–H, knockdown of hepatic SREBP-1 (A–D) or
SREBP-2 (E–H) by adenoviral expression of shRNA. 250- or 90-bp Srebf1c-Luc adenovirus (Ad-250bp- Srebf1c-Luc or Ad-90bp- Srebf1c-Luc; 6.0 ⫻ 106
pfu/body) plus adenovirus expressing SREBP-1-specific, SREBP-2-specific, or LacZ-specific shRNA (Ad-SREBP1i, Ad-SREBP2i, or Ad-LacZi; 2.5 ⫻ 108
pfu/body) were intravenously injected into ICR male mice. After 4 days, the mice (n ⫽ 3– 6 for each group) were treated orally with 7.5 g/kg EPA or water(CTL) once a day for 4 days. On days 0 and 4 after the first treatment of EPA, luciferin was injected intraperitoneally, and the luminescence from liver was
captured with IVIS. A, B, E, and F, quantification of luciferase activity with LivingImage software. Fold changes of luciferase activity on day 4 versus day 0 are
shown. C and G, Northern blot analysis of SREBP-1 and -2 in the liver. Total RNA (7.5 ␮g) from livers pooled equally from mice for each group was subjected to
Northern blotting to determine SREBP-1, -2, and 36B4 (used as a loading control) mRNA levels. D and H, immunoblot (IB) analysis of mature and precursor
SREBP-1 and -2 proteins in the liver. Aliquots of nuclear extracts (10 ␮g) and total proteins (50 ␮g) from livers pooled equally from four male mice of each groupwere subjected to immunoblot analysis. The primary antibodies used were polyclonal anti-mouse SREBP-1 and polyclonal anti-mouse SREBP-2. These data arerepresentative of at least two independent experiments (n ⫽ 3– 6 mice/group). Results are means ⫾ S.E. *, p ⬍ 0.05, and **, p ⬍ 0.01, respectively.

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plasmids, DNA fragments retrieved from pGL2 vectors constructed previously (19, 20, 31) were inserted into pGL3 basic
vector plasmids (Promega). The fragments including promoter
region linked to luciferase reporter gene were inserted into the
Gateway entry vector pENTR4 (Invitrogen) and generated byhomologous recombination between the entry vector and thepAd promoterless vector (Invitrogen). The fragments of human
SREBPs GAL4-DBD and VP16-AD fusion protein from various
GAL4-SREBP vectors were inserted into pENTR4 and generated by homologous recombination with the pAd/CMV/V5DEST vector (Invitrogen). Adenoviruses encoding SREBP-1specific and LacZ-specific shRNA for RNA interference(SREBP1i and LacZi, respectively) were described previously(32). Adenovirus construct encoding SREBP-2-specific shRNA(SREBP2i) targeting 5⬘-GGAGCAGTCTCAACGTCAACG-3⬘
sequence on SREBP-2 was subcloned into U6 entry vector(Invitrogen) and generated by homologous recombination withthe pAd promoterless vector. Adenovirus construct encoding
both LXR␣ and LXR␤ shRNA (LXRi) targeting 5⬘-ACAGCTCCCTGGCTTCCTA-3⬘ sequence on LXR␣ (33) and 5⬘-CTACAACCACGAGACAGAA-3⬘ sequence on LXR␤, respectively,
was subcloned into U6 entry vector (Invitrogen) and generated
by homologous recombination with the pAd promoterless vector. Recombinant adenoviruses were propagated in HEK293
cells and purified by CsCl gradient centrifugation as described
previously (34).

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PUFA Selectively Suppresses SREBP-1

DISCUSSION
This study has clearly demonstrated that the primarymechanism of the inhibitory effect of PUFA is the suppres-

FIGURE 6. Schematic representation of molecular mechanisms for inhibitory effects of PUFA on SREBP-1. The primary mechanism for PUFA suppression of SREBP-1 is at the proteolytic processing level, and this suppression in turn decreases the mRNA transcription through lowering SREBP-1
binding to SRE on the promoter (autoloop regulatory circuit), although LXR is
not involved in this regulation.

sion of the proteolytic activation of SREBP-1 and that thetranscriptional regulation is secondary to this post-translational suppression of mature SREBP-1 that itself binds to the
SRE site on the Srebf1c promoter (autoloop regulation (19)),
whereas LXR is not involved in the transcriptional regulation
by PUFA (Fig. 6).
This is the first report that has clearly demonstrated theinhibitory effect of PUFA on the SREBP-1 proteolytic processing in vivo. This result is consistent with our previous reportshowing that PUFA decreases nuclear SREBP-1, although it
does not affect the nuclear abundance of the truncated form of
SREBP-1 expressed from a transgene, suggesting that PUFAFIGURE 5. PUFA suppresses the proteolytic activation of SREBP-1, not affecting SREBP-2. A, various regions of human SREBP-1c (amino acids (a.a.)
1–1123 (FL), 431–1123 (Reg), 1– 436 (Nuc)) and human SREBP-2 (amino acids 14 –1141 (FL), 450 –1141 (Reg)) were fused to Gal4-DNA binding domain
and VP16-transactivation domain. FL, full length; Nuc, nuclear; Reg, regulatory; bHLH-Zip, basic helix-loop-helix leucine zipper. GAL4-VP16-SREBP fusion
protein is attached to the endoplasmic reticulum (ER) similarly as endogenous SREBP protein. Upon activation, its N-terminal region is released from themembrane into the nucleus by a cleavage process. The GAL4-VP16 promotes the luciferase (Luc) reporter gene expression by binding to GAL4-RE
containing eight copies of upstream activation sequence, Gal4-binding site. B, regulation of cleavage of GAL4-VP16 fusion SREBP protein by SCAP and
INSIG-1. HEK293 cells in a 48-well plate were co-transfected with GAL4-RE-Luc plasmid (100 ng/well), expression plasmids of GAL4-VP16 fusion SREBP
protein (100 ng/well), SCAP (250 ng/well), INSIG-1 (100 ng/well), and pSV40-Renilla plasmid (50 ng/well). Cells were harvested 24 h after transfection.
The firefly luciferase activity was measured and normalized by Renilla luciferase activity. All experiments were performed in triplicate. C–E, regulation ofcleavage of GAL4-VP16 fusion SREBP protein by EPA in vivo. GAL4-RE-Luc adenovirus (Ad-GAL4-RE-Luc, 2.0 ⫻ 108 pfu/body) plus adenovirus expressing
GAL4-VP16 fusion SREBP protein (Ad-Gal4-FL-SREBP-1c, Ad-Gal4-FL-SREBP-2 or Ad-Gal4-Nuc-SREBP-1c, 1.0 ⫻ 108 pfu/body) were intravenously
injected into ICR male mice. After 2 days, the mice (n ⫽ 7–9 for each group) were treated orally with 7.5 g/kg EPA or water (CTL) once a day for 4 days.
C, 0 and 4 days after the fast treatment of EPA, luciferin was injected intraperitoneally, and the luminescence from liver was captured with IVIS. The color
overlay on the image represents the photons/s emitted from the animal with a range of 4.0 ⫻ 105–2.5 ⫻ 106 photons/s (Ad-Gal4-FL-SREBP-1c) and 1.0 ⫻106– 8.0 ⫻ 106 photons/s (Ad-Gal4-FL-SREBP-2), as indicated by the color scale next to the images. D, quantification of luciferase activity with LivingImage
software. Fold changes of luciferase activity on day 4 versus day 0 are shown. E, immunoblot analysis of mature and precursor SREBP-1 proteins fromlivers. Aliquots of nuclear extracts (10 ␮g) and total proteins (50 ␮g) from livers pooled equally from male mice for each group were subjected toimmunoblot analysis. The primary antibodies used were polyclonal anti-mouse SREBP-1. N.S., not significant. F–H, SCAP ligand GW532 accelerates
SREBP-1 cleavage. F, regulation of cleavage of GAL4-VP16 fusion SREBP protein by GW532. HEK293 cells in a 48-well plate were co-transfected with
GAL4-RE-Luc plasmid (100 ng/well), expression plasmids for GAL4-VP16 fusion SREBP protein (100 ng/well), and pSV40-Renilla plasmid (50 ng/well). 3 hafter transfection, GW532 (1 ␮M) or EtOH was added to media, and cells were harvested 24 h after transfection. The firefly luciferase activity wasmeasured and normalized by Renilla luciferase activity. All experiments were performed in triplicate. G and H, GAL4-RE-Luc adenovirus (Ad-GAL4-RELuc, 2.0 ⫻ 108 pfu/body) plus adenovirus expressing GAL4-VP16 fusion SREBPs protein (Ad-Gal4-FL-SREBP-1c or Ad-Gal4-FL-SREBP-2, 1.0 ⫻ 108 pfu/body) were intravenously injected into ICR male mice. After 2 days, the mice (n ⫽ 8 for each group) were administered orally with GW532 or vehicle atindicated doses. 6 h after the last treatment, luciferin was injected intraperitoneally, and the luminescence from liver was captured with IVIS. G, quantification of luciferase activity with LivingImage software. Fold changes on day 3 versus day 0 are shown. H, immunoblot analysis of mature and precursor
SREBP-1 and SREBP-2 proteins from livers. Aliquots of nuclear extracts (10 ␮g) and total proteins (50 ␮g) from livers pooled equally from four male micefor each group were subjected to immunoblot (IB) analysis. The primary antibodies used were polyclonal anti-mouse SREBP-1 and anti-mouse SREBP-2.
These data are representative of at least two independent experiments (n ⫽ 3– 8 mice/group). Results are means ⫾ S.E. *, p ⬍ 0.05.

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and is released from the membrane into the nucleus as amature protein by a cleavage process (7). To explore themolecular mechanism by which PUFA decreases the nuclear
form of SREBP-1, another reporter system to detect proteolytic activity for the precursor form of SREBPs was constructed (Fig. 5A). In this system, Gal4-DNA binding domain
and VP16 activation domain were attached to the N terminus of SREBPs, and the nuclear entry of this N-terminal fragment was measured by the Gal4-UAS system to assess theproteolytic release of the SREBP N termini. Using this cleavage-detecting reporter system, the proteolytic activities forprecursor forms of SREBP-1 or -2 were tested in vitro with
SCAP and insulin-induced gene (INSIG)-1 overexpressions as
an activator and an inhibitor for cleavage of SREBP, respectively. As expected, the SCAP overexpression drasticallyenhanced the cleavage of N-terminal fragments of both
SREBP-1 and -2, and the co-expression of INSIG1 reversed theeffect of SCAP overexpression (Fig. 5B), indicating that thisreporter system reflects the physiological regulation of SREBP
activation processes by proteolysis. Next, we transduced thereporter genes into mouse livers using adenoviruses and examined the effect of PUFA in the in vivo setting. As shown in Fig. 5,
C and D, EPA suppressed only the SREBP-1 cleavage-detecting
reporter but did not affect the SREBP-2 reporter. Additionally,
EPA did not decrease the reporter activity from the constructthat contains only the N terminus (designated as Nuc-SREBP1c) and enters the nucleus without cleavage, demonstratingthat EPA did not accelerate the degradation of reporter fragment. In contrast, GW532, a SCAP activator, enhanced thecleavage of both SREBP-1 and -2 in the in vitro (Fig. 5F) and invivo (Fig. 5H) situations.

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does not accelerate the degradation of nuclear SREBP-1 protein(11).
We have shown that two mechanisms are involved in the
PUFA regulation on SREBP-1; one is at the proteolytic processing level, and the other is at the transcription level. What is thephysiological role of this two-step regulation? As we haveshown in Fig. 1, the proteolytic mechanism is high sensitive,
although the transcriptional mechanism is low sensitive, consistent with the previous report by Ezaki and co-workers (35).
The combination of these two steps of regulation with different
sensitivity connected in series probably helps to achieve thebroader responsive range of the amount of PUFA.
One of our conclusions is that LXR is not involved in thetranscriptional regulation by PUFA, but controversy exists overthis point; several previous reports suggested the involvementof LXR in this regulation (15, 16), whereas others did not (36).
These previous studies have been all performed in in vitro settings, and therefore we evaluated the contribution of the LXR
pathway in the in vivo setting for the first time, and we haveconcluded that the involvement of LXR is not detectable,
although LXR is an important determinant of the SREBP-1
expression level.
Because an in vitro model is always only a small part of thewhole in vivo system, how large or small the contribution of aregulatory pathway elucidated in vitro is in the whole in vivo
system cannot be estimated until it is assessed in the in vivo
setting. To address these issues, our approach of the extensionof in vitro reporter assays to in vivo settings will be very useful invarious situations.
We have demonstrated that PUFA selectively suppresses
the proteolytic processing of SREBP-1, but the molecularmechanism underlying this SREBP-1-specific regulation iscurrently unknown. SCAP escorts both SREBP-1 and -2 to
the Golgi, and SREBP-1-specific adaptor protein has notbeen reported yet. Recently, an endoplasmic reticulum
membrane protein TRC8 (translocation in renal cancer fromchromosome 8) has been documented to hamper endoplasmic reticulum-to-Golgi transport of SREBP-2/SCAP and
reduce SREBP-2 cleavage specifically (37). Perhaps there
might be some adaptor molecule that specifically interacts
with SREBP-1 and mediates the suppressive effect of PUFA,
although we have no evidence. If the molecular mechanism
underlying this SREBP-1-specific effect of PUFA is clarified
in the future, it will be a potential molecular target for new
lipid-lowering drugs. In conclusion, the primary mechanism
for PUFA suppression of SREBP-1 expression is at the proteolytic processing level and that this suppression in turndecreases the transcription of Srebf1c through lowering
SREBP-1 binding to SRE on the promoter.

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Fukamizu, A., and Yamada, N. (2004) Nat. Cell Biol. 6, 351–357
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