Polyunsaturated Fatty Acids Ameliorate Hepatic
Steatosis in Obese Mice by SREBP-1 Suppression
Motohiro Sekiya,1 Naoya Yahagi,1 Takashi Matsuzaka,2 Yuho Najima,1 Masanori Nakakuki,2 Ryozo Nagai,1
Shun Ishibashi,1 Jun-ichi Osuga,1 Nobuhiro Yamada,2 and Hitoshi Shimano2
Leptin-deficient ob/ob mice show many characteristics of obesity, including excess peripheral adiposity as well as severe hepatic steatosis, at least in part, due to increased hepatic
lipogenesis. Polyunsaturated fatty acids (PUFAs) are not only ligands for peroxisome proliferator-activated receptor (PPAR) ␣ but are also negative regulators of hepatic lipogenesis,
which is thought to be mediated by the repression of sterol regulatory element-binding
protein (SREBP)-1. We have previously shown that the disruption of SREBP-1 in ob/ob micedecreased their liver triglyceride storage. To examine whether PUFAs could reduce hepatic
triglyceride deposition, we challenged ob/ob mice with dietary PUFA. It is demonstrated that
PUFA markedly decreased the mature form of SREBP-1 protein and thereby reduced theexpression of lipogenic genes such as fatty acid synthase (FAS) and stearoyl-CoA desaturase
1 (SCD1) in the livers of ob/ob mice. Consequently, the liver triglyceride content and plasma
alanine aminotransferase (ALT) levels were decreased. Furthermore, both hyperglycemia
and hyperinsulinemia in ob/ob mice were improved by PUFA administration, similar to theeffect of PPAR␣ activators. In conclusion, PUFAs ameliorate obesity-associated symptoms,
such as hepatic steatosis and insulin resistance, presumably through both down-regulationof SREBP-1 and activation of PPAR␣. (HEPATOLOGY 2003;38:1529-1539.)

S

terol regulatory element-binding proteins (SREBPs)
are members of the basic helix-loop-helix leucine
zipper family of transcription factors that regulate
fatty acid and cholesterol synthesis (reviewed in Brown
and Goldstein1). Unlike other members of the family,
SREBPs are synthesized as precursors bound to the endoplasmic reticulum and nuclear envelope and are released
from the membrane into the nucleus as mature proteinsby cleavage processes. To date, 3 isoforms of SREBP, -1a,-1c, and -2, have been identified and characterized. The
predominant SREBP-1 isoform in liver and adipose tissue
is SREBP-1c. Whereas SREBP-2 plays a crucial role inregulation of cholesterol synthesis, SREBP-1c controls
Abbreviations: SREBP, sterol regulatory element-binding protein; FAS, fatty acidsynthase; SCD, stearoyl-CoA desaturase; PUFA, polyunsaturated fatty acid(s); ACL,
ATP citrate lyase; EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid; PPAR,
peroxisome proliferator-activated receptor.
From the 1Department of Internal Medicine, Tokyo University Graduate Schoolof Medicine, Tokyo, and 2Department of Internal Medicine, Institute of Clinical
Medicine, University of Tsukuba, Ibaraki, Japan.
Received April 28, 2003; accepted September 5, 2003.
Address reprint requests to: Hitoshi Shimano, M.D., Ph.D., Department ofInternal Medicine, Institute of Clinical Medicine, University of Tsukuba, 1-1-1
Tennodai, Tsukuba, Ibaraki 305-8575, Japan. E-mail: shimano-tky@umin.ac.jp;
fax: (81) 298-63-2081.
Copyright © 2003 by the American Association for the Study of Liver Diseases.
0270-9139/03/3806-0026$30.00/0
doi:10.1016/j.hep.2003.09.028

the transcription and expression of lipogenic enzymes
such as fatty acid synthase (FAS) and stearoyl-CoA desaturase 1 (SCD1) (reviewed in Shimano2 and Horton etal.3). It is remarkable that SREBP-1c regulates not only
the synthetic rate of triglycerides but also the amount of
their storage in the liver.4,5 Thus, SREBP-1 has been revealed to be a promising target for hepatic steatosis (fatty
livers) from a therapeutic point of view.
The leptin-deficient ob/ob mouse model of obesity
exhibits severe obesity and obesity-related symptoms,
including hepatic steatosis and insulin resistance (reviewed in Bray and York6). The livers of ob/ob mice
have an increase in triglyceride content, probably because of the increased lipogenesis paralleled by elevatedmessenger RNA (mRNA) expression and enzymatic activity of several lipogenic enzymes such as FAS and
SCD1.6,7 Recently, it has been reported that both
SREBP-1c mRNA and its active nuclear protein are increased in ob/ob mouse livers.8 Furthermore, we havedemonstrated in a previous report5 that the disruption of
the SREBP-1 gene in ob/ob mice leads to marked amelioration of hepatic steatosis.
Dietary polyunsaturated fatty acids (PUFAs) of the n-6
and n-3 families are well established as negative regulatorsof hepatic lipogenesis (reviewed in Clark and Jump9).
Recently, others and we have shown that the suppressive
1529

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SEKIYA ET AL.

effects of PUFA on lipogenic enzymes are mediated bytheir reduction of mature SREBP-1 protein in theliver.10-13 The primary mechanism by which PUFAs suppress SREBP-1 is presumably by cleaving the precursor
protein as well as by the reduction of both transcription
rate and mRNA stability.14,15 Interestingly, PUFAs selectively decrease SREBP-1, not affecting SREBP-2.
Considering these findings, we hypothesized that
PUFA would be effective for the treatment of hepatic
steatosis. To test this, we examined the effects of PUFA on
hepatic steatosis in the ob/ob mouse. We show that, in
ob/ob mice, PUFAs ameliorate hepatic steatosis through areduction of mature SREBP-1 protein. In addition, theadministration of PUFA resulted in marked improvement of insulin resistance.

Materials and Methods
Materials. Triolein (99% grade) and fenofibrate werepurchased from Wako Pure Chemicals (Tokyo, Japan)
and Sigma (St. Louis, MO), respectively. Eicosapentaenoic acid (EPA) ethyl ester (95% grade) was provided
from Mochida pharmaceutical (Tokyo, Japan) and pioglitazone (100.0% pure) from Takeda pharmaceutical(Tokyo, Japan). Fish oils (extracted from tuna; containing
24.8% docosahexaenoic acid [DHA] and 8.1% EPA)
were provided from NOF (Tokyo, Japan). Standard laboratory chow (composed of 60% carbohydrate, 13% fat,and 27% protein on a caloric basis; Oriental MF, Oriental
Yeast, Tokyo, Japan), high-carbohydrate, fat-free diet
(70% sucrose; 10% starch; and 20% casein supplemented
with methionine, vitamins, and minerals), and high-protein, fat-free diet (100% casein and no carbohydrate supplemented with methionine, vitamins, and minerals)
were obtained from Oriental Yeast (Tokyo, Japan).
Animals. Heterozygous Lepob/⫹ C57BL/6J mice werepurchased from Jackson Laboratories (Bar Harbor, ME).
Eight-week-old wild-type and ob/ob male littermates wereused in all experiments. Genotypes at the leptin locus
were determined by a PCR-based, restricted fragment
length polymorphism analysis as described previously.5
Mice were housed in a temperature-controlled environment with a 12-hour light/dark cycle (light: 10:00 AM to
10:00 PM) and given free access to water and a standard
chow diet for the 1-week adaptation period. Each groupof 4 animals (kept all together in 1 cage) was fed a control
high-carbohydrate, fat-free chow or that supplemented
with either 15% triolein, 15% triolein plus 5% EPA ethyl
ester (approximately 5 g/kg/d), 20% fish oil, 0.125% fenofibrate, or 0.05% pioglitazone for 7 days. We determined this duration based on the preliminary data that
7-day administration of fish oil decreased the serum ala-

HEPATOLOGY, December 2003

nine aminotransferase (ALT) level to the same extent as
was observed at the 14th day, whereas 3-day feeding was
not sufficient. After collecting blood samples from theretroorbital sinus, animals were killed by cervical dislocation in an early phase of the light cycle in a nonfasted state.
SREBP-1c transgenic mice over-expressing amino acids
1– 436 of human SREBP-1c under control of the rat
PEPCK promoter (TgSREBP1) were made as previously
described.4 A line homozygous for the transgene was established and used. The diets used for TgSREBP1 miceexperiments were based on the high-protein, fat-free dietto maximize the transgene expression and mixed with
15% triolein, 15% triolein plus 5% EPA ethyl ester, or
20% fish oil. All experiments were repeated at least twice,
and the data from a representative experiment wereshown.
Blood Chemistries and Liver Lipid Analyses. Enzymatic assay kits were used for the determination of cholesterol (Determiner TC: cholesterol oxidase method;
Kyowa Medex, Tokyo, Japan), triglycerides (TG LH: lipoprotein lipase method; Wako Pure Chemicals, Tokyo,
Japan), nonesterified fatty acids (NEFA C: acyl-CoA oxidase method; Wako Pure Chemicals) and ALT (transaminase C- II: POP TOOS method; Wako Pure Chemicals).
Plasma glucose was measured by ANTSENSE II (Bayer
Medical, Tokyo, Japan) based on immobilized glucose
oxidase membrane/hydrogen peroxide electrode method.
Plasma insulin was determined by the mouse insulin
ELISA kit (Wako Pure Chemicals). The content of cholesterol and triglycerides in liver was measured as described previously.16
RNA Isolation and Northern Blotting. Total RNA
from liver and epididymal fat pad was isolated with Trizol
Reagent (Invitrogen), and 10 ␮g RNA samples wereequally pooled from 4 mice of each group and (2.5 ␮g
RNA for each mouse) were run on a 1% agarose gel containing formaldehyde and transferred to a nylon membrane. The cDNA probes used were cloned as previously
described.17 The probes were labeled with [␣-32P]dCTP
using Megaprime DNA Labelling System kit (Amersham
Biosciences). The membranes were hybridized with theradiolabeled probe in Rapid-hyb Buffer (Amersham Biosciences) at 65°C. The membranes were washed 3 times(10 minutes each) in 0.1X SSC, 0.1% SDS at 65°C. Blots
were exposed to Kodak XAR-5 film and the BAS imaging
plate for BAS2000 BIO IMAGING ANALYZER (Fuji
Photo Film, Tokyo, Japan). The quantification results
obtained with BAS2000 system were normalized to thesignal generated from 36B4 (acidic ribosomal phosphoprotein P0) mRNA.
RNase Protection Assay. RNase protection assay was
performed as described previously.18 Briefly, after linear-HEPATOLOGY, Vol. 38, No. 6, 2003

SEKIYA ET AL.

1531

separated on 8 mol/L urea/10% polyacrylamide gels. The
protected fragments for SREBP-1a and -1c contained thesame number of cytidine phosphates (32 cytidines), so theintensity of each band was proportional to the amount ofeach isoform in the samples. The gels were subjected to
Kodak XAR-5 film and the BAS2000 BIO IMAGINGANALYZER (Fuji Photo Film, Tokyo, Japan).
Immunoblotting of SREBP Proteins. Nuclear extracts and membrane fractions from mice livers were prepared as previously described.19 Aliquots of nuclear (20␮g) and membrane (30 ␮g) proteins were subjected to
SDS/PAGE. Immunoblot analysis was performed using
the ECL Western Blotting Detection System kit (Amersham Biosciences) and exposed to Kodak XAR-5 film.
The primary antibodies (rabbit polyclonal; No. 931 formouse SREBP-1a and -1c, No. 772 for SREBP-1c, which

Fig. 1. PUFAs reduce mature SREBP-1 in the nuclei from ob/ob micelivers. (A) Immunoblot analysis of SREBP-1 and -2 in nuclear extracts
and membrane fractions from livers of ob/ob mice are shown. Mice (4male ob/ob mice for each group) were fed a high-carbohydrate fat-freediet (lane 1) or a high-carbohydrate diet supplemented with 15% triolein(lane 2), 5% EPA ethyl ester plus 15% triolein (lane 3), 20% fish oil(lane 4), 0.125% fenofibrate (lane 5), or 0.05% pioglitazone (lane 6) for
7 days before being killed in a nonfasted state. Aliquots of nuclear
extracts (20 ␮g protein) and membrane proteins (30 ␮g) from pooled
livers of each group were subjected to immunoblot analysis. The primary
antibodies used were polyclonal anti-mouse SREBP-1 (1c plus 1a and1c-specific) and -2. (B) Quantification of SREBP-1a and -1c by RNase
protection assay are shown. Lanes are the same as above. The protectedfragments for SREBP-1a and -1c contain the same number of cytidine
phosphates (32 cytidines), so the intensity of each band was proportional to the amount of each isoform in the samples. Series of experiments were performed twice for the same number of animals, and thedata from a representative experiment are shown.

ization of the template plasmid DNA with Hind III, antisense RNA probes for SREBP-1a and -1c weretranscribed with [␣-32P]CTP using bacteriophage T7
RNA polymerase (MAXIscript in vitro transcription kit;
Ambion) and purified by electrophoresis on 5% polyacrylamide gel. Then, aliquots of probes and total RNA(10 ␮g) pooled within each group (2.5 ␮g RNA for eachmouse) were mixed and subjected to the RNase protection assay using HybSpeed RPA kit (Ambion). After digestion with RNase A/T1, protected fragments were

Fig. 2. PUFAs repress lipogenic enzymes in liver. Northern blot analysis from mice livers. Wild-type (lanes 1-6) and ob/ob (lanes 7-12) mice
(4 mice for each group) were fed the indicated diet for 7 days and killed
in a nonfasted state. Diets were as follows: lanes 1 and 7, a highcarbohydrate, fat-free diet; lanes 2 and 8, a high-carbohydrate diet with
15% triolein (C18:1 n-9); lanes 3 and 9, 5% EPA ethyl ester (C20:5 n-3)
plus 15% triolein (C18:1 n-9); lanes 4 and 10, 20% tuna fish oil; lanes
5 and 11, 0.125% fenofibrate, a PPAR␣ agonist; and lanes 6 and 12,
0.05% pioglitazone, a PPAR␥ agonist. Total RNA (10 ␮g) pooled equallyfrom 4 mice was subjected to Northern blotting, followed by hybridization
with the indicated cDNA probes. SREBP1, sterol regulatory elementbinding protein-1; FAS, fatty acid synthase; S14, Spot 14; SCD1,
stearoyl-CoA desaturase 1; D5D, ⌬5-desaturase; D6D, ⌬6-desaturase;
HMGsyn, hydroxymethylglutaryl-CoA synthase. A cDNA probe for 36B4
(acidic ribosomal phosphoprotein P0) was used to confirm equal loading. Series of experiments were performed twice for the same number ofanimals, and the data from a representative experiment are shown.

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HEPATOLOGY, December 2003

Fig. 3. PUFAs induce PPAR␣-target genes. (A) Northern blot analysis from mice livers and (B) adipose tissue. Wild-type (lanes 1-6) and ob/ob(lanes 7-12) mice (4 mice for each group) were fed the indicated diet for 7 days and killed in a nonfasted state. Diets were as follows: lanes 1
and 7, a high-carbohydrate, fat-free diet; lanes 2 and 8, a high-carbohydrate diet with 15% triolein (C18:1 n-9); lanes 3 and 9, 5% EPA ethyl ester(C20:5 n-3) plus 15% triolein (C18:1 n-9); lanes 4 and 10, 20% tuna fish oil; lanes 5 and 11, 0.125% fenofibrate, a PPAR␣ agonist; and lanes
6 and 12, 0.05% pioglitazone, a PPAR␥ agonist. Total RNA (10 ␮g), pooled equally from four mice, was subjected to Northern blotting, followedby hybridization with the indicated cDNA probes. AOX, acyl-CoA oxidase; CYP4A2, cytochrome P450 4A2; PPAR, peroxisome proliferator-activatedreceptor; IRS, insulin receptor substrate; SREBP1, sterol regulatory element-binding protein-1; FAS, fatty acid synthase; S14, Spot 14; GLUT4, glucose
transporter 4. A cDNA probe for 36B4 (acidic ribosomal phosphoprotein P0) was used to confirm equal loading. The quantification results wereobtained with BAS2000 system and normalized to the signal generated from 36B4 (acidic ribosomal phosphoprotein P0) mRNA, and thefold-changes against control were displayed below each blot. Series of experiments were performed twice for the same number of animals, and thedata from a representative experiment are shown.

does not bind to SREBP-1a, and No. 528 for SREBP-2)
were as previously described.17,20
Fatty Acid Composition of Liver. 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 BioMedical Laboratories (Tokyo, Japan).21 Briefly, total
lipids in liver homogenates were extracted according to
the Folch’s procedure,22 followed by transesterification of
fatty acids with boron trifluoride-methanol at 100°C for
90 minutes. The methylated fatty acids were then extracted with hexane and analyzed using GC-17A gas chromatograph (Shimadzu Corporation, Kyoto, Japan) and
BPX70 capillary column (0.25 mm ID ⫻ 30 m, SGEInternational Ltd., Melbourne, Australia).
Statistical Analysis. Data are presented as means ⫾SE. All data were analyzed by 2-tailed Student’s t test
against control. Differences were considered statisticallysignificant at P ⬍ .05.

Results
Effects of PUFA on the Mature Form of SREBP-1
in ob/ob Mice Livers. In wild-type mice, PUFAs havebeen shown to decrease the mature form of SREBP-1 inthe liver.10-13 To examine effects of PUFA on SREBP-1 inleptin-deficient ob/ob mice livers in which SREBP-1 isdysregulated, they were fed a high-carbohydrate diet supplemented with PUFA (EPA or tuna fish oil enriched inDHA) or control diet for 7 days. As expected, immunoblot analysis of liver nuclear extracts showed that ob/ob
mice fed a diet with EPA or fish oil for 7 days had ⬃3-foldlower SREBP-1 mature protein than those fed the control
diet (high-carbohydrate without fat) (Fig. 1A). In contrast, monounsaturated fatty acids (oleate) did not reduce
mature SREBP-1.
To clarify the mechanism by which PUFAs down-regulate mature SREBP-1 protein, the amounts of SREBP-1(both precursor and mature forms in the membrane andnuclear extracts, respectively; Fig. 1A) as well as SREBP-1
mRNA levels (Fig. 1B) were compared between control
and PUFA (EPA or fish oil)-fed ob/ob mice. In contrast to
the mature protein, which was profoundly decreased by
PUFA feeding, no reduction was observed in eithermRNA or membrane-bound precursor protein levels.
This indicates that PUFAs regulate the abundance of mature SREBP-1 protein mainly at a posttranslational level,
presumably through cleavage processes.
RNase protection assay with probes that can discriminate SREBP-1a and -1c (Fig. 1B) also demonstrated thatthe isoform pattern of SREBP-1 is SREBP-1c-dominant

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Fig. 4. PUFAs ameliorate hepatic
steatosis of ob/ob mice. (A) liver
triglyceride (TG) and total cholesterol (TC) content per body weight inwild-type (lanes 1-6) and ob/ob
mice (lanes 7-12). Diets are as follows: lanes 1 and 7, a high-carbohydrate, fat-free diet; lanes 2 and 8,
15% triolein (C18:1 n-9); lanes 3
and 9, 5% EPA ethyl ester (C20:5n-3) plus 15% triolein (C18:1 n-9);
lanes 4 and 10, 20% tuna fish oil;lanes 5 and 11, 0.125% fenofibrate, a PPAR␣ agonist; and lanes
6 and 12, 0.05% pioglitazone, a
PPAR␥ agonist. Each group consisted of 4 male mice except forlanes 7 and 9 (n ⫽ 8). (B) Plasma
ALT levels. Lanes are the same asabove. (C) Liver lipid content of wildtype and SREBP-1 transgenic mice.
Lanes are as follows: lane 1, wildtype mice fed with a high-carbohydrate, fat-free diet; lane 2, SREBP-1
transgenic (TgSREBP1) mice fed
with a high-protein, fat-free diet;lane 3, TgSREBP1 mice fed with 5%
EPA ethyl ester plus 15% triolein;lane 4, TgSREBP1 mice fed with
20% fish oil. Bars ⫽ SE. *Denotes
significance vs. control (lane 1 or 7)
at P ⬍ .05 (Student’s t test). Seriesof experiments were performed 3
times for the same number of animals, and the data from a representative experiment are shown.

in ob/ob mice livers, which is the same as in wildtype mice.18 This was further supported by Western
blot analysis with an SREBP-1c-specific antibody(Fig. 1A).
Fenofibrate and pioglitazone, peroxisome proliferatoractivated receptor (PPAR) ␣ and ␥ agonists, respectively,
were also tested, but neither altered mature SREBP-1 levels. The amount of mature SREBP-2 protein did notchange with any of these diets.
Suppression of Lipogenic Gene Expressions by
PUFA. We compared the mRNA levels of genes encoding lipogenic enzymes in the livers of mice fed diets withor without PUFA (EPA or fish oil) for 7 days as measured
by Northern blot analysis. Consistent with previous reports, dietary PUFA suppressed hepatic expression of
SREBP-1-target lipogenic genes such as FAS and SCD1
in wild-type mice (Fig. 2). Similar suppression of thesegenes was also observed in livers from ob/ob mice fed thePUFA diets. Meanwhile, suppression of these lipogenic
genes was not observed in white adipose tissue from eithergroup (Fig. 3).
The ⌬5- and ⌬6-desaturases, key players in the production of EPA and DHA, have been reported to be suppressed by PUFA.23,24 We previously reported that they
are dually regulated by SREBP-1 and PPAR␣ and suppressed by PUFA in wild-type mouse liver.25 However,
because PUFAs suppress SREBP-1 and at the same time
activate PPAR␣, it is unclear whether PUFAs decrease or
increase these desatureases as an overall effect in various
nutritional situations. In the current study, it was revealed
that, in both wild-type and ob/ob mice, PUFA diets reduced the expression of these enzymes, whereas PPAR␣
activation by fenofibrate consistently increased their expression.

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HEPATOLOGY, December 2003

Table 1. Representative Profile of Wild-Type and ob/ob Mice Used for the ExperimentWild-type
Body weight (g)Liver weight (g)WAT weight (g)Plasma data
ob/ob
Body weight (g)Liver weight (g)WAT weight (g)Plasma dataPre
At sacrifice

TC (mg/dL)
TG (mg/dL)
Pre
At sacrifice

TC (mg/dL)
TG (mg/dL)

1

2

3

4

5

6

26.4 ⫾ 1.9
26.3 ⫾ 2.1
1.35 ⫾ 0.17
0.28 ⫾ 0.08
72.5 ⫾ 16.0
64.2 ⫾ 10.2

26.4 ⫾ 1.9
26.8 ⫾ 1.8
1.43 ⫾ 0.13
0.33 ⫾ 0.05
121.9 ⫾ 5.5*
88.5 ⫾ 11.1

26.4 ⫾ 1.6
27.0 ⫾ 1.5
1.25 ⫾ 0.09
0.53 ⫾ 0.16
61.2 ⫾ 9.3
55.1 ⫾ 2.8

26.5 ⫾ 1.5
25.1 ⫾ 0.7
1.40 ⫾ 0.10
0.28 ⫾ 0.05
57.2 ⫾ 8.3
46.9 ⫾ 2.8

26.4 ⫾ 1.3
25.5 ⫾ 1.1
2.33 ⫾ 0.10*
0.20 ⫾ 0.00
109.1 ⫾ 7.4
66.7 ⫾ 3.8

26.4 ⫾ 1.2
26.8 ⫾ 1.0
1.53 ⫾ 0.05
0.40 ⫾ 0.07
106.0 ⫾ 5.9
46.4 ⫾ 2.2

50.5 ⫾ 2.5
52.3 ⫾ 2.1
6.12 ⫾ 0.31
3.20 ⫾ 0.31
265.1 ⫾ 28.0
115.9 ⫾ 11.8

50.3 ⫾ 2.4
53.6 ⫾ 2.5
5.50 ⫾ 0.20
3.30 ⫾ 0.11
284.5 ⫾ 10.3
145.6 ⫾ 26.8

50.6 ⫾ 2.1
51.8 ⫾ 2.4
4.58 ⫾ 0.40*
3.25 ⫾ 0.41
229.6 ⫾ 8.0
79.6 ⫾ 0.6*

50.4 ⫾ 2.0
51.8 ⫾ 2.0
4.60 ⫾ 0.31*
3.62 ⫾ 0.13
330.6 ⫾ 24.4
147.0 ⫾ 25.7

50.1 ⫾ 1.9
51.3 ⫾ 1.7
5.35 ⫾ 0.15
3.48 ⫾ 0.15
267.1 ⫾ 13.4
79.6 ⫾ 4.0*

50.4 ⫾ 1.6
54.1 ⫾ 1.8
5.54 ⫾ 0.32
3.63 ⫾ 0.16
191.5 ⫾ 18.5
69.2 ⫾ 10.0*

NOTE. Diets are as follows: 1, a high-carbohydrate fat-free diet; 2, a high-carbohydrate diet with 15% triolein (C18:1 n-9); 3, 5% EPA ethyl ester (C20:5 n-3) plus
15% triolein (C18:1 n-9); 4, 20% tuna fish oil; 5, 0.125% fenofibrate, a PPAR␣ agonist; 6, 0.05% pioglitazone, a PPAR␥ agonist. Each value is mean ⫾ SE.
Abbreviations: TC, total cholesterol; TG, triglycerides.
*Significance vs. control at P ⬍ .05 by Student’s t test.

In contrast, these lipids did not affect genes involved incholesterol synthesis, such as hydroxymethylglutaryl(HMG)-CoA synthase, that are mainly regulated by
SREBP-2 (Fig. 3). These results are consistent with thedata shown in Fig. 1 in which PUFA did not affect theamount of mature SREBP-2.
Effects on PPAR␣-Regulated Genes. It is wellknown that PUFAs are ligands for PPAR␣,26,27 and somebiologic effects of PUFA such as stimulation of peroxisomal and microsomal fatty acid oxidation are mediated by
PPAR␣.28 Therefore, we assessed PUFA effects on thehepatic PPAR␣ pathway. As expected, mRNA levels of
PPAR␣-target genes such as PPAR␣ itself, acyl-CoA oxidase (AOX), and cytochrome P-450 4A2 (CYP4A2)
were induced by PUFA (Fig. 3). In particular, fish oil(tuna, rich in DHA) had more potent effects than EPA on
PPAR␣-mediated pathways in both wild-type and ob/ob
mice.
Effects of PUFA on Liver Lipid Content. To determine whether the effects of PUFA reduced hepatic steatosis in ob/ob mice, liver triglyceride content was measured(Fig. 4). In both wild-type and ob/ob mice, PUFA (EPA
and fish oil) significantly (P ⬍ .05) decreased hepatic
triglyceride content per body weight. During the feeding
period, mean body weights were not different among eachgroup (Table 1), suggesting that the decrease in hepatic
triglycerides was not due to reduced food intake.
Reduction of ALT by PUFA. ALT is a well-known
marker for evaluation of liver injury and is elevated inanimals with hepatic steatosis, including ob/ob mice. We
determined plasma ALT levels by enzymatic assay. Although there were no differences among each dietarygroup in wild-type mice, PUFA (EPA or fish oil) lowered
the elevated ALT levels in ob/ob mice (Fig. 4). These data

are consistent with our finding that PUFA ameliorated
hepatic steatosis.
Effects of PUFA on Triglyceride Content in TgSREBP1 Mice Livers. To investigate whether the reduction of triglyceride in liver can be ascribed to the decreasein mature SREBP-1, we examined a transgenic mousemodel, which forcibly expresses a mature form of
SREBP-1 protein in the liver (TgSREBP1). Consistent
with a previous report,10 dietary PUFA did not decrease
the mature SREBP-1 protein or the mRNA levels of lipogenic genes in the liver of TgSREBP1 mice (data notshown). Accordingly, the triglyceride content of EPA-fed
mice was almost equal to mice fed the control diet (Fig.
4). Intriguingly, the fish oil administration still caused aremarkable reduction in the triglyceride content elevatedby the over-expressed SREBP-1. These data suggest thatfish oil has some additional effects beyond those mediatedby SREBP-1.
Effects on Fatty Acid Composition in Liver. We
measured the composition of fatty acids in liver total lipids by gas chromatography (Table 2, Fig. 5). The fatty
acids that can be synthesized de novo by lipogenic enzymes
such as palmitate (C16:0), palmitoleate (C16:1 n-7), andoleate (C18:1 n-9) were reduced by PUFA. Because thesefatty acids dominate liver lipids, this resulted in an overall
reduction in the total content of fatty acids in PUFA-fed
mice. In the animals fed fish oil, EPA (C20:5 n-3) andDHA (C22:6 n-3) were abundantly distributed in theliver in both wild-type and ob/ob mice. However, mice
given EPA showed similarly low levels of DHA comparedwith control animals, despite the availability of EPA for
metabolism to DHA by desaturation and elongation.
PUFA Effects on Insulin Resistance. To evaluate
insulin sensitivity, we also examined some markers of glu-

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Table 2. Fatty Acid Composition in the Liver
Wild-type (␮g/g body weight)
C12:0
C14:0
C14:1 n-5
C16:0
C16:1 n-7
C18:0
C18:1 n-9
C18:2 n-6
C18:3 n-6
C18:3 n-3
C20:0
C20:1 n-9
C20:2 n-6
C20:3 n-9
C20:3 n-6
C20:4 n-6
C20:5 n-3
C22:0
C22:1 n-9
C22:4 n-6
C22:5 n-3
C22:6 n-3
C24:0
C24:1 n-9
ob/ob (␮g/g body weight)
C12:0
C14:0
C14:1 n-5
C16:0
C16:1 n-7
C18:0
C18:1 n-9
C18:2 n-6
C18:3 n-6
C18:3 n-3
C20:0
C20:1 n-9
C20:2 n-6
C20:3 n-9
C20:3 n-6
C20:4 n-6
C20:5 n-3
C22:0
C22:1 n-9
C22:4 n-6
C22:5 n-3
C22:6 n-3
C24:0
C24:1 n-9

1

2

3

4

5

6

0.22 ⫾ 0.22
6.88 ⫾ 0.64
0.00 ⫾ 0.00
327.2 ⫾ 24.0
77.2 ⫾ 12.8
106.6 ⫾ 13.4
475.5 ⫾ 25.0
114.5 ⫾ 32.2
2.14 ⫾ 1.12
0.52 ⫾ 0.52
2.38 ⫾ 0.25
19.23 ⫾ 1.33
3.58 ⫾ 1.06
24.18 ⫾ 7.49
19.38 ⫾ 2.72
85.6 ⫾ 11.5
4.38 ⫾ 0.7
4.19 ⫾ 0.95
1.03 ⫾ 0.37
3.86 ⫾ 1.44
4.13 ⫾ 0.94
117.9 ⫾ 19.4
2.79 ⫾ 0.76
5.47 ⫾ 1.06

0.00 ⫾ 0.00
4.93 ⫾ 0.19*
0.00 ⫾ 0.00
342.6 ⫾ 23.4
51.7 ⫾ 3.4
125.1 ⫾ 11.3
499.7 ⫾ 19.2
113.0 ⫾ 17.0
1.62 ⫾ 0.56
0.44 ⫾ 0.44
3.00 ⫾ 0.03*
19.35 ⫾ 0.59
3.14 ⫾ 0.60
21.95 ⫾ 2.35
25.77 ⫾ 3.04
104.0 ⫾ 11.4
5.07 ⫾ 0.4
4.68 ⫾ 0.21
1.50 ⫾ 0.08
2.41 ⫾ 0.30
3.68 ⫾ 0.37
100.9 ⫾ 7.6
2.86 ⫾ 0.34
6.59 ⫾ 0.70

0.00 ⫾ 0.00
2.94 ⫾ 4.58*
0.00 ⫾ 0.00
251.9 ⫾ 15.5*
20.1 ⫾ 2.8*
118.5 ⫾ 5.8
286.6 ⫾ 43.9*
93.7 ⫾ 16.1
0.36 ⫾ 0.36
2.42 ⫾ 0.45*
2.75 ⫾ 0.26
8.01 ⫾ 1.36*
1.13 ⫾ 0.40
0.95 ⫾ 0.55*
3.94 ⫾ 0.80*
23.9 ⫾ 1.6*
201.9 ⫾ 16.6*
4.41 ⫾ 0.44
1.32 ⫾ 0.10
0.00 ⫾ 0.00*
58.34 ⫾ 5.65*
103.0 ⫾ 15.4
3.48 ⫾ 0.36
5.00 ⫾ 0.71

0.34 ⫾ 0.34
3.54 ⫾ 0.73*
0.00 ⫾ 0.00
394.9 ⫾ 13.3*
20.9 ⫾ 3.3*
167.1 ⫾ 10.2*
175.4 ⫾ 52.6*
156.8 ⫾ 53.0
1.33 ⫾ 0.89
3.54 ⫾ 1.16
3.61 ⫾ 0.13*
7.10 ⫾ 3.02*
2.13 ⫾ 0.92
1.26 ⫾ 0.88*
5.67 ⫾ 1.93*
107.4 ⫾ 8.9
54.9 ⫾ 8.1*
7.06 ⫾ 1.51
0.43 ⫾ 0.43
1.90 ⫾ 1.00
29.95 ⫾ 9.42*
460.4 ⫾ 64.8*
3.33 ⫾ 0.64
5.15 ⫾ 0.84

0.00 ⫾ 0.00
9.40 ⫾ 1.02
0.00 ⫾ 0.00
1084.0 ⫾ 96.0*
248.0 ⫾ 30.7*
165.6 ⫾ 19.5*
975.8 ⫾ 106.9*
254.0 ⫾ 19.7*
5.69 ⫾ 0.73*
0.59 ⫾ 0.59
3.09 ⫾ 0.31
20.14 ⫾ 2.57
5.24 ⫾ 0.40
27.58 ⫾ 5.42
90.98 ⫾ 8.43*
221.4 ⫾ 25.3*
15.5 ⫾ 0.8*
5.44 ⫾ 0.58
0.00 ⫾ 0.00*
9.53 ⫾ 1.13*
13.77 ⫾ 0.51*
169.5 ⫾ 13.0
3.31 ⫾ 0.34
8.73 ⫾ 0.96

0.00 ⫾ 0.00
4.84 ⫾ 0.22*
0.00 ⫾ 0.00
431.8 ⫾ 30.8*
97.5 ⫾ 8.3
118.8 ⫾ 6.7
481.9 ⫾ 43.3
64.4 ⫾ 4.4
0.83 ⫾ 0.48
0.00 ⫾ 0.00
2.00 ⫾ 0.09
14.78 ⫾ 1.70
1.46 ⫾ 0.06
67.52 ⫾ 7.16*
19.58 ⫾ 1.08
102.2 ⫾ 5.9
6.1 ⫾ 0.4
4.32 ⫾ 0.21
0.38 ⫾ 0.38
2.29 ⫾ 0.19
3.53 ⫾ 0.20
89.6 ⫾ 5.1
2.95 ⫾ 0.35
5.85 ⫾ 0.30

4.52 ⫾ 0.35
169.3 ⫾ 7.2
7.72 ⫾ 0.706397 ⫾ 198
1673 ⫾ 153
587.6 ⫾ 28.2
12964 ⫾ 214
1260.3 ⫾ 242.7
12.83 ⫾ 2.18
45.6 ⫾ 9.7
7.48 ⫾ 0.56
386.1 ⫾ 22.1
31.4 ⫾ 6.2
37.61 ⫾ 4.07
85.2 ⫾ 10.8
134.6 ⫾ 41.8
54.6 ⫾ 6.0
4.61 ⫾ 0.48
9.7 ⫾ 0.5
11.3 ⫾ 1.3
64.3 ⫾ 9.4
350.2 ⫾ 34.3
2.4 ⫾ 0.8
12.0 ⫾ 0.8

3.99 ⫾ 0.47*
159.3 ⫾ 22.7
5.75 ⫾ 1.27
5797 ⫾ 553
1458 ⫾ 231
490.5 ⫾ 13.3*
13207 ⫾ 1229
1308.2 ⫾ 22.3
11.67 ⫾ 0.92
51.4 ⫾ 2.8
6.34 ⫾ 0.52
412.3 ⫾ 60.0
37.6 ⫾ 0.6
36.39 ⫾ 2.85
91.2 ⫾ 2.1
164.4 ⫾ 2.6
57.9 ⫾ 1.3
2.29 ⫾ 0.80*
7.5 ⫾ 0.5*
12.3 ⫾ 0.7
74.2 ⫾ 3.4
378.9 ⫾ 8.6
1.4 ⫾ 0.8
11.3 ⫾ 0.5

1.67 ⫾ 0.60
84.0 ⫾ 15.3*
1.52 ⫾ 0.98*
3328 ⫾ 544*
753 ⫾ 141*374.7 ⫾ 36.4*
7661 ⫾ 1563*
921.5 ⫾ 87.7
4.21 ⫾ 0.81*
53.0 ⫾ 4.8
5.88 ⫾ 0.91
177.9 ⫾ 44.9*
20.0 ⫾ 3.0
13.71 ⫾ 1.89*
41.5 ⫾ 5.2*
76.2 ⫾ 9.2
387.8 ⫾ 13.8*
3.23 ⫾ 0.23*
1.4 ⫾ 0.8*
3.8 ⫾ 0.6*
266.4 ⫾ 28.4*
317.4 ⫾ 33.3
2.4 ⫾ 0.8
9.6 ⫾ 0.6

2.71 ⫾ 0.95
90.9 ⫾ 16.1*
1.97 ⫾ 0.69*
3488 ⫾ 554*
912 ⫾ 136*
363.2 ⫾ 62.0*6551 ⫾ 1137*
1033.6 ⫾ 146.4
6.53 ⫾ 1.01*
79.9 ⫾ 17.3
8.51 ⫾ 3.02
148.1 ⫾ 26.1*
19.9 ⫾ 2.4
10.19 ⫾ 0.79*
38.5 ⫾ 3.7*
152.3 ⫾ 18.1
382.1 ⫾ 70.6*
3.09 ⫾ 1.15
0.6 ⫾ 0.6*
11.0 ⫾ 1.2
221.3 ⫾ 33.6*
1130.7 ⫾ 168.8*
2.2 ⫾ 0.8
10.2 ⫾ 1.1

4.02 ⫾ 0.36
128.4 ⫾ 4.2*
3.47 ⫾ 0.12*5932 ⫾ 289
1563 ⫾ 60
416.6 ⫾ 18.1*
11014 ⫾ 656*
1113.5 ⫾ 93.1
10.14 ⫾ 1.02
35.1 ⫾ 3.6
6.47 ⫾ 0.73
286.9 ⫾ 23.4*
21.8 ⫾ 2.9
26.86 ⫾ 3.19
82.4 ⫾ 5.7
140.1 ⫾ 8.6
65.4 ⫾ 2.1
1.50 ⫾ 0.87*
5.6 ⫾ 0.7*
5.4 ⫾ 1.1*
38.5 ⫾ 3.0*
244.7 ⫾ 11.8*
2.0 ⫾ 0.7
9.9 ⫾ 0.4

5.08 ⫾ 0.23
191.9 ⫾ 9.0
9.47 ⫾ 0.846553 ⫾ 477
2375 ⫾ 155*464.2 ⫾ 29.1*
15045 ⫾ 639*
986.2 ⫾ 106.9
9.77 ⫾ 1.86
24.5 ⫾ 2.2
13.21 ⫾ 1.24
535.6 ⫾ 13.1*
24.4 ⫾ 3.5
59.54 ⫾ 8.41
70.8 ⫾ 6.1
146.7 ⫾ 14.2
45.7 ⫾ 4.1
2.44 ⫾ 0.88
13.0 ⫾ 0.5*
10.3 ⫾ 1.7
38.1 ⫾ 3.7*
185.9 ⫾ 20.9*
0.6 ⫾ 0.6
11.3 ⫾ 0.7

NOTE. Diets are as follows: 1, a high-carbohydrate fat-free diet; 2, a high-carbohydrate diet with 15% triolein (C18:1 n-9); 3, 5% EPA ethyl ester (C20:5 n-3) plus
15% triolein (C18:1 n-9); 4, 20% tuna fish oil; 5, 0.125% fenofibrate, a PPAR␣ agonist; 6, 0.05% pioglitazone, a PPAR␥ agonist. Each value is mean ⫾ SE.
*Significance vs. control at P ⬍ .05 by Student’s t test.

cose-lipid metabolism (Fig. 6). It has been suggested that
PPAR␣ agonists reduce insulin resistance,29,30 and, in thisstudy, fenofibrate did ameliorate hyperinsulinemia andhyperglycemia in the ob/ob mice, although to a lesser degree than the established insulin sensitizer pioglitazone.
Intriguingly, PUFA also reduced serum glucose and insu-

lin levels nearly to the same level as fenofibrate. By Northern blot analysis, the expression of insulin receptor
substrate (IRS)-2, a key molecule in insulin signaling, inboth liver and adipose tissue of ob/ob mice were shown tobe lowered, but its expression was slightly restored by the
PUFA administration (Fig. 3).

1536

SEKIYA ET AL.

HEPATOLOGY, December 2003

Fig. 5. Effects of PUFA on fatty
acid composition in the liver of wildtype and ob/ob mice. The composition of fatty acids in liver wasmeasured by gas chromatography.
Based on the data shown in Table 2,n-3, n-6, or n-7/n-9/saturated fatty
acids (SFA) content is calculated
and illustrated. Diets are as follows:
lanes 1 and 7, a high-carbohydrate,
fat-free diet; lanes 2 and 8, a highcarbohydrate diet with 15% triolein(C18:1 n-9); lanes 3 and 9, 5% EPA
ethyl ester (C20:5 n-3) plus 15%
triolein (C18:1 n-9); lanes 4 and
10, 20% tuna fish oil; lanes 5 and
11, 0.125% fenofibrate, a PPAR␣
agonist; and lanes 6 and 12, 0.05%
pioglitazone, a PPAR␥ agonist.
Lanes 1 to 6 are data from wild-type
mice, and lanes 7 to 12 are fromob/ob mice (n ⫽ 4 for each group).
Bars ⫽ SE. *Denotes significancevs. control (lane 1 or 7) at P ⬍ .05(Student’s t test). Series of experiments were performed twice for thesame number of animals, and thedata from a representative experiment are shown.

Discussion
The current study clearly demonstrates that PUFAs
reduce mature SREBP-1 and ameliorate hepatic steatosis
in ob/ob mice. Based on our previous findings that PUFAs
suppress SREBP-110 and that SREBP-1-null ob/ob mice
have less triglycerides in liver,5 we hypothesized that
PUFA would decrease hepatic triglyceride storage
through the reduction of SREBP-1. Our results provide astrong clinical implication that PUFAs are potentially
useful for the treatment of hepatic steatosis. In addition,
we provided further evidence that SREBP-1 regulates thestorage of triglycerides in liver. In this regard, we speculatethat the reduction of hepatic lipogenesis by PUFA resulted in a decrease in triglyceride storage. Two lines ofevidence indicate decreased lipogenesis in PUFA-fed
mouse livers: a decrease in lipogenic enzymes mRNA levels as shown by Northern blot analysis (Fig. 2) and thehepatic fatty acid composition (Table 2, Fig. 5), demon-

strating that PUFA administration decreased fatty acids
such as oleate (C18:1 n-9), palmitate (C16:0), and palmitoleate (C16:1 n-7) that can be endogenously synthesized
more profoundly than fatty acids such as linoleate (C18:2
n-6) that cannot be biosynthesized de novo.
In the present study, we show that EPA administration
decreased hepatic triglyceride content in ob/ob mice,
whereas it did not in a transgenic mouse model, which
forcibly expressed a mature form of SREBP-1 protein in
the liver (TgSREBP1). These findings indicate that theeffect of EPA on the improvement of hepatic steatosis ismainly mediated by SREBP-1 reduction. In contrast,
tuna oil, rich in DHA, was also effective in the TgSREBP1 mice. These data indicate that tuna oil may haveadditional positive effects beyond the suppression of
SREBP-1. The difference between them might be derived
from differences in composition and action of EPA versus
DHA. In fact, although EPA can be converted to DHA

HEPATOLOGY, Vol. 38, No. 6, 2003

SEKIYA ET AL.

1537

Fig. 6. PUFAs improve hyperglycemia and hyperinsulinemia of ob/ob
mice. Plasma glucose, insulin, andfree fatty acids (FFA) of wild-type(WT) and ob/ob (OB) mice (n ⫽ 4
for each group) are shown. Diets are
as follows: lane 1, a high-carbohydrate, fat-free diet; lane 2, 15% triolein (C18:1 n-9); lane 3, 5% EPA
ethyl ester (C20:5 n-3) plus 15%
triolein (C18:1 n-9); lane 4, 20%
tuna fish oil; lane 5, 0.125% fenofibrate, a PPAR␣ agonist; lane 6,
0.05% pioglitazone, a PPAR␥ agonist. Bars ⫽ SE. *Denotes significance vs. control (lane 1) at P ⬍.05 (Student’s t test). Series of experiments were performed twice forthe same number of animals, andthe data from a representative experiment are shown.

endogenously with elongase and ⌬6-desaturase via the
Sprecher pathway,31 the hepatic content of DHA in miceadministered EPA was not elevated, as shown in Fig. 5,
presumably because of the suppression of ⌬6-desaturase
expression by PUFA.24,25 Although EPA and DHA are
considered equally effective as suppressors of SREBP-1,10

Fig. 7. Schematic representation of PUFA effects on obesity. PUFAs
suppress SREBP-1 and activate PPAR␣, thereby preventing excess triglyceride accumulation and ameliorating obesity-associated symptoms,
including hepatic steatosis and insulin resistance.

the mechanisms by which PUFAs suppress triglyceride
synthesis are known to be divergent,32 and they might
have different potency for the SREBP-1-independent
pathways. It is also possible that some unknown substancein tuna oil could be responsible for the SREBP-1-independent action.
We showed that PUFA not only ameliorated hepatic
steatosis but also attenuated insulin resistance in ob/ob
mice. Although the precise mechanisms are currently unknown, we are speculating that this effect of PUFA wasmediated at least partially by the PPAR␣ receptor because
PUFAs are well known PPAR␣ ligands,27 and a PPAR␣
agonist, fenofibrate, was also demonstrated to improve
insulin sensitivity in ob/ob mice as shown in Fig. 6. We
presume that this effect of PUFA is not mediated by
SREBP-1 because it was previously shown that the disruption of SREBP-1 in ob/ob mice did not influence theirinsulin resistance.5 However, it should be noted that EPA
was equally competent in improving the hyperinsulinemia of ob/ob mice, whereas EPA was much weaker in
PPAR␣ activation as estimated by Northern blot analysis

1538

SEKIYA ET AL.

for PPAR␣ target genes. It is tempting to speculate that
there could also be other mechanisms for insulin sensitization by PUFA than the PPAR␣ pathway; for instance, it
is possible that altered phospholipid composition andthereby membrane fluidity might be involved.33,34
We also showed that PUFAs significantly (P ⬍ .05)
reduce hepatic cholesterol content in wild-type mice (Fig.
4A). This is in accordance with previous reports35,36 thatfish oil feeding decreased hepatic cholesterol content inrodents. The mechanism by which this occurs is currently
unknown, but it was documented that EPA has an inhibitory effect on hepatic cholesterol biosynthesis and an enhancing effect on hepatic biliary secretion.37 Although themRNA expression levels of cholesterol biosynthetic genes
were not shown to be lowered by PUFA in Northernblotting (Fig. 2), these analyses do not account for possible posttranscriptional effects.
As we summarized in Fig. 7, PUFAs down-regulate
SREBP-1 and, thereby, triglyceride synthesis and at thesame time activate the PPAR␣ receptor, leading to theincreased ␤-oxidation of fatty acids. Hence, PUFA canshift the energy balance from storage to consumption.
The antilipogenic action of PUFA through SREBP-1 suppression has been proven to be independent of the
PPAR␣ pathway by previous studies using PPAR␣knockout mice.28
From a therapeutic point of view, it was notable that
PUFA also decreased plasma ALT levels. This means that
PUFAs attenuate the liver injury resulting from excess
triglyceride deposition. Although the precise pathophysiology as to how hepatic steatosis leads to steatohepatitis
and even to cirrhosis remains unclear, liver disease might
be a major contributor to overall obesity-related morbidity and mortality.38 Our data suggest that PUFA might beof clinical use to prevent the liver dysfunction associatedwith over nutrition.
In conclusion, we demonstrate that PUFAs ameliorate
hepatic steatosis through the suppression of SREBP-1 in
ob/ob mice. Furthermore, PUFAs were suggested to bepresumably of clinical value as a therapeutic drug for insulin resistance.
Acknowledgment: The authors thank Takashi Yano,
Hiroyuki Kawano, Masaaki Soma, and Toyonori Kato forcontinuous support of the project; Alyssa H. Hasty forcritical reading of the manuscript; and Kumiko Kitaoka
for assistance with the experiments.

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