THE JOURNAL OF BIOLOGICAL CHEMISTRY© 1999 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 274, No. 50, Issue of December 10, pp. 35840 –35844, 1999
Printed in U.S.A.

A Crucial Role of Sterol Regulatory Element-binding
Protein-1 in the Regulation of Lipogenic Gene Expression by
Polyunsaturated Fatty Acids*(Received for publication, June 25, 1999, and in revised form, September 10, 1999)

Naoya Yahagi, Hitoshi Shimano‡, Alyssa H. Hasty§, Michiyo Amemiya-Kudo, Hiroaki Okazaki,
Yoshiaki Tamura, Yoko Iizuka, Futoshi Shionoiri, Ken Ohashi, Jun-ichi Osuga, Kenji Harada,
Takanari Gotoda, Ryozo Nagai, Shun Ishibashi, and Nobuhiro Yamada¶From the Department of Metabolic Diseases, Faculty of Medicine, University of Tokyo, Tokyo 113-8655, Japan

Dietary polyunsaturated fatty acids (PUFA) are negative regulators of hepatic lipogenesis that exert theireffects primarily at the level of transcription. Sterol regulatory element-binding proteins (SREBPs) are transcription factors responsible for the regulation of cholesterol, fatty acid, and triglyceride synthesis. In
particular, SREBP-1 is known to play a crucial role in
the regulation of lipogenic gene expression in the liver.
To explore the possible involvement of SREBP-1 in the
suppression of hepatic lipogenesis by PUFA, we challenged wild-type mice and transgenic mice overexpressing a mature form of SREBP-1 in the liver with dietary
PUFA. In the liver of wild-type mice, dietary PUFA drastically decreased the mature, cleaved form of SREBP-1
protein in the nucleus, whereas the precursor, uncleaved form in the membranes was not suppressed. The
decreases in mature SREBP-1 paralleled those in
mRNAs for lipogenic enzymes such as fatty acid synthase and acetyl-CoA carboxylase. In the transgenic
mice, dietary PUFA did not reduce the amount of transgenic SREBP-1 protein, excluding the possibility that
PUFA accelerated the degradation of mature SREBP-1.
The resulting sustained expression of mature SREBP-1
almost completely canceled the suppression of lipogenic
gene expression by PUFA in the SREBP-1 transgenic
mice. These results demonstrate that the suppressive
effect of PUFA on lipogenic enzyme genes in the liver iscaused by a decrease in the mature form of SREBP-1
protein, which is presumably due to the reduced cleavage of SREBP-1 precursor protein.

The liver, the principal lipogenic organ, is responsible for theconversion of excess dietary carbohydrates to triglycerides. A
high carbohydrate diet induces the synthesis of several lipogenic and glycolytic enzymes including acetyl-CoA carboxylase* This work was supported in part by Promotion of Fundamental
Studies in Health Science of the Organization for Pharmaceutical
Safety and Research and Health Sciences Research Grants (Researchon Human Genome and Gene Therapy) from the Ministry of Health andWelfare. The costs of publication of this article were defrayed in 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.
§ Recipient of a fellowship under the Postdoctoral Fellowship Program for Foreign Researchers from the Japan Society for the Promotionof Science.
‡ To whom correspondence should be addressed. 7-3-1 Hongo,
Bunkyo-ku, Tokyo, 113-8655, Japan. Tel.: 81-3-3815-5411 (ext. 33113);
Fax: 81-3-5802-2955; E-mail: shimano-tky@umin.ac.jp.
¶ Present address: Division of Endocrinology and Metabolism, Dept.
of Internal Medicine, University of Tsukuba, Ibaraki 305-8575, Japan.

(ACC),1 fatty acid synthase (FAS), stearoyl-CoA desaturase,
ATP citrate lyase, malic enzyme, glucose-6-phosphate dehydrogenase, and pyruvate kinase (PK) (1–3). This coordinate induction of enzymes is due to increased mRNA levels, resulting
primarily from the accelerated transcription.
Dietary polyunsaturated fatty acids (PUFA) have been wellestablished as negative regulators of hepatic lipogenesis. Allmann and Gibson (4) discovered that adding 2% linoleate to ahigh carbohydrate fat-free diet suppressed the rate of hepatic
fatty acid biosynthesis and the activities of FAS and glucose6-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 theactivity of lipogenic enzymes. Since then, several 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 mRNA levels of hepatic enzymes
including ACC, FAS, stearoyl-CoA desaturase, ATP citratelyase, malic enzyme, glucose-6-phosphate dehydrogenase, and
PK. The regulation by PUFA has been shown to be primarily at
the transcriptional level; however, the precise mechanism forthis action remains unknown (5–7).
Sterol regulatory element-binding proteins (SREBPs) aretranscription factors that belong to the basic helix-loop-helixleucine zipper family and regulate enzymes responsible for thesynthesis of cholesterol, fatty acids, and triglycerides (8). Unlike other members of the basic helix-loop-helix-leucine zipper
family, SREBPs are synthesized as precursors bound to theendoplasmic reticulum and nuclear envelope. Upon activation,
SREBPs are released from the membrane into the nucleus as amature protein by a sequential two-step cleavage process. To
date, three SREBP isoforms, SREBP-1a, -1c and -2, have been
identified and characterized. The predominant SREBP-1 isoform in the liver is SREBP-1c. Whereas SREBP-2 is relatively
selective in transcriptionally activating cholesterol biosynthetic genes, SREBP-1c has a greater role in regulating fatty
acid synthesis than cholesterol synthesis in the liver (9 –11, 30).
The role of SREBP-1 for the regulation of hepatic lipogenesis
has been recently established. Changes in hepatic mature
SREBP-1c protein levels were shown to parallel those ofmRNAs for lipogenic genes in the liver using a dietary manipulation and a transgenic technology (12). Moreover, SREBP-1
has been demonstrated to be crucial for the carbohydrate stimulation of lipogenic genes in mice with a targeted disruption of
SREBP-1 (30).
1
The abbreviations used are: ACC, acetyl-CoA carboxylase; FAS,
fatty acid synthase; PK, pyruvate kinase; PUFA, polyunsaturated fatty
acids; SREBP, sterol regulatory element-binding protein; EPA, eicosapentaenoic acid; PPAR, peroxisome proliferator-activated receptor;
PEPCK, phosphoenolpyruvate carboxykinase.

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This paper is available on line at http://www.jbc.org

PUFA Regulation of SREBP-1

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These findings led us to hypothesize that the suppressive
effect of PUFA on lipogenic gene transcription in liver is mediated through a decrease in mature SREBP-1 levels. To verify
this hypothesis, the current studies were designed using wildtype mice and transgenic mice overexpressing mature
SREBP-1c in the liver (TgSREBP-1c). First, we show that theamount of mature SREBP-1 in the liver is decreased by dietary
PUFA. Next, we demonstrate that the expression of lipogenic
genes in the liver of TgSREBP-1c mice is maintained at a
relatively high level and that this expression follows the pattern of SREBP-1 levels that remain elevated even in the presence of dietary PUFA. These results indicate that the suppression of hepatic gene expression of lipogenic enzymes by PUFA
is most likely due to the decrease in the mature SREBP-1
protein.
EXPERIMENTAL PROCEDURES

Materials—Triolein (95% grade) and ethyl linoleate were purchasedfrom Sigma, and tristearin and casein were purchased from Wako Pure
Chemical Industries (Osaka, Japan). Eicosapentaenoic acid (EPA) ethyl
ester (95% grade) was provided by Mochida Pharmaceutical (Tokyo,
Japan), fenofibrate by Laboratoires Fournier (Paris, France), and troglitazone by Sankyo pharmaceutical (Tokyo, Japan). Fish oils (sardine
and tuna) were provided by NOF (Tokyo, Japan). Standard laboratory
chow, high carbohydrate fat-free diet (70% sucrose and 20% casein
supplemented with methionine, vitamins and minerals), and high protein fat-free diet (90% casein and no carbohydrate with methionine,
vitamins, and minerals) were obtained from Oriental Yeast (Tokyo,
Japan).
Animals and Diets—7-week-old male C57BL/6J mice (21–23 g) werepurchased from CLEA (Tokyo, Japan) and adapted to the environmentfor 1 week prior to study. SREBP-1c transgenic mice overexpressing
amino acids 1– 436 of human SREBP-1c under control of the rat PEPCK
promoter (TgSREBP-1c) were made as described previously (9). A line
homozygous for the transgene was established. All mice were housed ina controlled environment with a 12-h light/dark cycle and free access towater and diet. Prior to sacrifice, each group of three animals was fed adiet containing the indicated fatty acids, prepared fresh daily, for 7
days. The diet for wild-type mice consisted of a high carbohydrate
fat-free chow supplemented with 20% (w/w) tristearin, 20% triolein,
20% fish oil (sardine or tuna), 5% linoleic acid, or 5% EPA ethyl ester.
The diet for TgSREBP-1c mice was a high protein diet mixed with 20%
triolein, 20% triolein plus 5% EPA ethyl ester, or 20% fish oil (sardine).
For the comparison of high protein and high carbohydrate diets, wildtype mice were examined on diets composed of high protein or highcarbohydrate chow mixed with 20% triolein or 20% fish oil (sardine). All
mice were sacrificed during the early phase of the light cycle in a
nonfasted state.
Immunoblotting of SREBP Proteins—Nuclear extracts and membrane fractions from mice livers were prepared as described previously(13). Aliquots of nuclear (20 mg) and membrane (30 mg) proteins were
subjected to SDS-polyacrylamide gel electrophoresis. Immunoblot analysis was performed using the ECL Western Blotting Detection Systemkit (Amersham Pharmacia Biotech) and exposed to Eastman Kodak Co.
XAR-5 film. The primary antibodies (polyclonal) were as described
previously (14, 15).
Northern Blotting—Total hepatic RNA was isolated with Trizol reagent (Life Technologies), and 10-mg RNA samples (equally pooled fromthree mice) were run on a 1% agarose gel containing formaldehyde andtransferred to a nylon membrane. The cDNA probes used were cloned
as described previously (15, 30). The probes were labeled with[a-32P]dCTP using the Megaprime DNA Labeling System kit (Amersham Pharmacia Biotech). The membranes were hybridized with theradiolabeled probe in Rapid-hyb Buffer (Amersham Pharmacia Biotech)at 65 °C and washed in 0.13 SSC, 0.1% SDS at 65 °C. Blots were
exposed to Kodak XAR-5 film.
RESULTSThe Effect of PUFA on the Mature Form of SREBP-1 Protein
in Wild-type Mice Livers—To examine the effect of PUFA on
SREBP-1 in mice livers, wild-type mice were fed a high carbohydrate diet supplemented with PUFA (linoleate, EPA, or two
kinds of fish oil rich in EPA or docosahexaenoic acid) for 7 days.
Immunoblot analysis of liver nuclear extracts from these mice

FIG. 1. Immunoblot analysis of SREBP-1 and -2 in nuclear
extracts (a and b) and membrane fractions (c) from livers of
PUFA-fed mice. Mice (three male C57BL/6J, 8 weeks old) were fed ahigh carbohydrate fat-free diet (lane 1) or a high carbohydrate dietsupplemented with 5% EPA ethyl ester (lane 2), 20% sardine fish oil(lane 3), or 20% tuna fish oil (lane 4) for 7 days and sacrificed in a
nonfasted state. Aliquots of nuclear extracts (20 mg of protein) or membrane fractions (30 mg of protein) from pooled livers of each group were
subjected to immunoblot analysis. The primary antibodies used werepolyclonal anti-mouse SREBP-1 and -2.

showed that feeding wild-type mice a diet with EPA for 7 days
resulted in a ;3-fold decrease in the amount of hepatic mature
SREBP-1 protein, compared with feeding with the control diet(high carbohydrate without fat). This suppressive effect of EPA
on the mature SREBP-1 was consistently observed in six independent experiments including Figs. 1, a b, and c, 2, and 7. Fishoil (sardine, rich in EPA, or tuna, rich in docosahexaenoic acid)decreased mature SREBP-1 protein more profoundly, as shownin Figs. 1, b and c, 2, and 7. Dietary linoleate also suppressedmature SREBP-1 to a lesser extent (Fig. 2). In contrast, neither
saturated (tristearin) nor monounsaturated (triolein) fatty acids reduced mature SREBP-1 (Fig. 2). The amount of mature
SREBP-2 protein did not change significantly with any of the
dietary manipulations (Figs. 1, a and b, 2, 6a, and 7).
Posttranslational Regulation of SREBP-1 by PUFA—The
generation of mature SREBP-1 protein requires several steps
including transcription, translation, and a proteolytic cleavage
from microsomal membranes. To clarify the mechanism bywhich PUFA down-regulates mature SREBP-1 protein, theamounts of SREBP-1 (both precursor and mature forms in themembrane and nuclear extracts, respectively; Fig. 1c) as wellas SREBP-1 mRNA levels (Fig. 3) were compared between
control and PUFA (EPA or fish oil)-fed mice. In contrast to themature protein, which was profoundly decreased by PUFA
feeding, no significant reduction was observed in either mRNAor membrane-bound precursor protein levels. This indicatesthat PUFA regulates the abundance of mature SREBP-1 protein mainly at a posttranslational level, presumably throughcleavage and/or degradative processes.
Suppression of Lipogenic Gene Expression by PUFA—We
compared mRNA levels of genes encoding lipogenic enzymes in
the liver of mice fed diets with or without PUFA (linoleate,
EPA, or fish oil) for 7 days as measured by Northern blot
analysis. Consistent with previous reports, dietary PUFA suppressed hepatic expression of lipogenic genes such as ACC,
FAS, stearoyl-CoA desaturase 1, glycerol-3-phosphate acyltransferase, ATP citrate lyase, malic enzyme, glucose-6-phosphate dehydrogenase, PK, and Spot 14 (Fig. 3). This downregulation of lipogenic genes was confined to PUFA (linoleate,
EPA, fish oil)-fed groups of mice and was not observed in mice
fed saturated (i.e. tristearin) or monounsaturated (i.e. triolein)

35842

PUFA Regulation of SREBP-1

FIG. 2. Immunoblot analysis of SREBP-1 and -2 in nuclear
extracts from livers of mice fed a high carbohydrate diet containing various fatty acids. Mice (three male C57BL/6J, 8 weeks old)
were fed a high carbohydrate fat-free diet (lane 1) or a high carbohydrate diet supplemented with 20% tristearin (lane 2), 20% triolein (lane3), 5% linoleate ethyl ester (lane 4), 5% EPA ethyl ester (lane 5), 20%
sardine fish oil (lane 6), or 20% tuna fish oil (lane 7) for 7 days andsacrificed in a nonfasted state. Aliquots of nuclear extracts (20 mg ofprotein) from pooled livers of each group were subjected to immunoblot
with antibody against mouse SREBP-1 or -2.

FIG. 4. Immunoblot analysis of SREBP-1 (a) and -2 (b) in nuclear extracts from livers of wild-type (WT) or TgSREBP-1c (Tg)mice on PUFA diets. Wild-type mice were fed a high carbohydrate
fat-free diet (lane 1), and homozygous TgSREBP-1c mice were fed ahigh protein diet with 20% triolein (lane 2), 5% EPA ethyl ester plus
20% triolein (lane 3), or 20% sardine fish oil (lane 4) for 7 days andsacrificed in a nonfasted state. Aliquots of nuclear extracts (20 mg ofprotein) from pooled livers of each group were subjected to immunoblotting with polyclonal antibody against SREBP-1c (a) or SREBP-2 (b).

FIG. 3. Northern blot analysis of lipogenic and glycolytic enzymes from livers of mice fed a diet containing various fatty
acids. Mice (three male C57BL/6J, 8 weeks old) were fed the indicateddiet for 7 days and sacrificed in a nonfasted state. Diets were as follows.
Lane 1, a high carbohydrate fat-free diet; lane 2, a high carbohydrate
diet with 20% tristearin (18:0); lane 3, 20% triolein (18:1); lane 4, 5%
linoleate ethyl ester (18:2); lane 5, 5% EPA ethyl ester (20:5); lane 6,
20% sardine fish oil; lane 7, 20% tuna fish oil. Total RNA (10 mg) pooled
equally from three mice was subjected to Northern blotting, followed byhybridization with the indicated cDNA probes. A cDNA probe for 36B4
(acidic ribosomal phosphoprotein P0) was used to confirm equal loading. SCD1, stearoyl-CoA desaturase 1; GPAT, glycerol-3-phosphate
acyltransferase; ACL, ATP citrate lyase; ME, malic enzyme; G6PD,
glucose-6-phosphate dehydrogenase; S14, Spot 14.

fatty acids. This specificity to PUFA corresponded to the pattern of suppression observed with mature SREBP-1 protein(Fig. 2). These findings are in support of the hypothesis that
PUFA decreases mRNA levels of lipogenic genes through the
suppression of mature SREBP-1.
The Effects of PUFA on the Mature Form of SREBP-1 and
Lipogenic Gene Expression in TgSREBP-1c Mice Livers—To
examine whether the PUFA suppression of lipogenic genes was
ascribed to the decrease in mature SREBP-1, a transgenic
mouse model that forcibly expresses a mature form of SREBP-1
protein in the liver (TgSREBP-1c) was used. In these transgenic mice, the expression of a nuclear form of SREBP-1c thatis active without cleavage is under control of the rat PEPCK
promoter and is induced when animals are fed a high protein,
low carbohydrate diet (9). As shown in Fig. 4a, the mature

SREBP-1 in the liver of TgSREBP-1c mice fed a high protein
diet is dominated by the transgene product, the amount of
which was comparable with that of intrinsic SREBP-1 matureform in wild-type mice on a high carbohydrate diet (Fig. 4a,lanes 1 and 2). The hepatic mRNA levels of lipogenic genes inthese TgSREBP-1c mice were also similar to those in wild-typemice on a high carbohydrate diet (Fig. 5; lanes 1 and 2).
The effects of PUFA on the amount of mature SREBP-1 andthe mRNA levels of lipogenic genes were examined in thesetransgenic mice. It was found that the amount of the transgene
product was not affected by dietary PUFA (EPA or fish oil) (Fig.
4a). Furthermore, dietary PUFA did not change the mRNAlevels of lipogenic genes such as ACC, FAS, stearoyl-CoA desaturase 1, glycerol-3-phosphate acyltransferase, ATP citratelyase, malic enzyme, glucose-6-phosphate dehydrogenase, Spot
14, or PK in the liver of TgSREBP-1c mice (Fig. 5). These
results demonstrate that the suppressive effect of PUFA on
lipogenic genes is primarily mediated through the decrease in
the amount of mature SREBP-1.
The Comparison of Protein and Carbohydrate-based Diets—To confirm that dietary PUFA suppresses mature
SREBP-1 and lipogenic genes in mice fed protein-based diets inthe same way as in mice placed on carbohydrate-based diets,
wild-type mice were fed a high protein diet supplemented withtriolein or fish oil for 7 days, and the amount of mature
SREBP-1 (Fig. 6a) and the mRNA levels of lipogenic genes (Fig.
6b) in the liver were evaluated by immunoblot and Northernblotting analyses, respectively. As expected, dietary PUFA exerted the same suppressive effect on the background of highprotein diets as was observed on high carbohydrate diets. It
was also revealed that a high carbohydrate diet elevated mature SREBP-1 as well as mRNAs for lipogenic genes morestrongly than a high protein diet. These results provide further
evidence for the dependence of lipogenic gene transcription on
mature SREBP-1.

PUFA Regulation of SREBP-1

FIG. 5. Northern blot analysis of lipogenic and glycolytic enzymes from livers of wild-type or TgSREBP-1c mice on PUFA
diets. Wild-type mice were fed a high carbohydrate fat-free diet (lane1), and homozygous TgSREBP-1c mice were fed a high protein diet with
20% triolein (lane 2), 5% EPA ethyl ester plus 20% triolein (lane 3) or
20% sardine fish oil (lane 4) for 7 days and sacrificed in a nonfasted
state. Total RNA (10 mg) pooled equally from livers of each group was
subjected to Northern blotting, followed by hybridization with the indicated cDNA probes. A cDNA probe for 36B4 (acidic ribosomal phosphoprotein P0) was used to confirm equal loading. SCD1, stearoyl-CoA
desaturase 1; GPAT, glycerol-3-phosphate acyltransferase; ACL, ATP
citrate lyase; ME, malic enzyme; G6PD, glucose-6-phosphate dehydrogenase; S14, Spot 14.

The Effect of Peroxisome Proliferator-activated Receptor a(PPARa) Ligand on Mature SREBP-1—It is well known that
PUFA are ligands for PPARa (16, 17), and some biologicaleffects of PUFA such as induction of peroxisomal and microsomal fatty acid oxidation are mediated by PPARa (18). To determine whether PPARa is involved in the suppression of mature SREBP-1 by PUFA, the amount of mature SREBP-1 inwild-type mice fed a diet containing fenofibrate, a ligand for
PPARa, for 7 days was examined. Immunoblot analysis of liver
nuclear extracts showed that neither fenofibrate nor troglitazone, a ligand for PPARg, affected the amount of mature
SREBP-1 in the liver (Fig. 7). These data suggest that the
suppressive effect of PUFA on mature SREBP-1 is not mediated by PPARa or -g. The expected effects of PPARa wereconfirmed by the observation of increased mRNAs of acyl-CoAoxidase and cytochrome P-450 4A2 (data not shown).
DISCUSSION

In the present study, we clearly showed that the suppressionof lipogenic gene expression by PUFA in the liver was primarily
due to decreases in the mature form of SREBP-1. We first
demonstrated that the abundance of mature SREBP-1 in theliver was decreased by dietary PUFA. A similar observation
has been reported in a study in cultured cells (19). However, it
was reported that mature SREBP-1 was also decreased byoleate, a monounsaturated fatty acid, which exhibited no significant effect on hepatic SREBP-1 in our in vivo study. Extensive in vivo studies using mouse or rat liver have demonstrated
that an antilipogenic effect is confined to PUFA and that this
response appears to be specific to liver (20). Our results describing the effects of different fatty acids on SREBP-1 nuclear
protein in the liver are consistent with these previous reports.
The cause for the discrepancy in the effect of oleate between

35843

FIG. 6. Immunoblot analysis of SREBP-1 and -2 in nuclear
extracts (a) and Northern blot analysis of lipogenic enzymes (b)from livers of wild-type mice fed a high protein or high carbohydrate diet with or without PUFA. Mice (three male C57BL/6J, 8
weeks old) were fed a high protein diet mixed with 20% triolein (lane 1),
a high protein diet with 20% sardine fish oil (lane 2), a high carbohydrate diet with 20% triolein (lane 3), or a high carbohydrate diet with
20% sardine fish oil (lane 4) for 7 days and sacrificed in a nonfasted
state. For immunoblot analysis, aliquots of nuclear extracts (20 mg ofprotein) from pooled livers of each group were used. For Northernblotting, 10 mg of total RNA was pooled equally from livers of each groupand blotted to a nylon membrane, followed by hybridization with theindicated cDNA probes. SCD1, stearoyl-CoA desaturase 1.

FIG. 7. Immunoblot analysis of SREBP-1 and -2 in nuclear
extracts from livers of mice administered fenofibrate or troglitazone. Diets were as follows. Lane 1, a high carbohydrate diet; lane 2,
a high carbohydrate diet with 5% EPA ethyl ester; lane 3, 20% sardine
fish oil; lane 4, 0.5% fenofibrate; lane 5, 0.1% troglitazone. Mice (threemale C57BL/6J, 8 weeks old) were fed the diet for 7 days and sacrificed
in a nonfasted state. Aliquots of nuclear extracts (20 mg protein) frompooled livers of each group were subjected to immunoblotting with
antibody against mouse SREBP-1 or -2.

liver and cultured cells is currently unknown.
As shown in Fig. 1c, we demonstrate that PUFA regulates
the abundance of mature SREBP-1 protein primarily at the
posttranslational level. Moreover, we show that PUFA did notdecrease the amount of mature SREBP-1 directly expressedfrom the transgene, indicating that PUFA does not accelerate
the degradation of SREBP-1 mature protein. These data suggest that the regulation of SREBP-1 by PUFA occurs at thestep of cleavage of the precursor protein from membranes.
If the suppression of lipogenic gene expression by PUFA iscaused by the decrease in mature SREBP-1 protein, sustained
SREBP-1 expression should abolish the effect of PUFA. To
verify this hypothesis, homozygous TgSREBP-1c mice fed ahigh protein diet were used. As described previously, the rat
PEPCK promoter used for the transgene is activated by feedinga high protein, low carbohydrate diet (21). Consequently, thehepatic expression of mature SREBP-1 in these mice was approximately equal to that of carbohydrate-stimulated wild-typemice and was not affected by PUFA. In the presence of sus-

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PUFA Regulation of SREBP-1

tained SREBP-1 mature protein levels, the expression of lipogenic genes in the liver of TgSREBP-1c mice was maintained ashigh as that of wild-type mice on a high carbohydrate dietirrespective of PUFA ingestion. This indicates that the matureform of SREBP-1 is a determining factor in the suppressive
effect of dietary PUFA on lipogenic gene expression.
It should be noted that the mRNA of PK was expressed morestrongly in wild-type mice fed a high carbohydrate diet than in
TgSREBP-1c mice fed a high protein diet, although similar
amounts of mature SREBP-1 were present. This observationsuggests the possibility that the transcriptional regulation of
the PK gene is more strongly controlled by transcription factor(s) other than SREBP-1. This notion could explain theweaker effect of PUFA on the mRNA level of PK. It is also
consistent with the previous observation that SREBP-1 disruption had a smaller influence on mRNA expression of PK incomparison with those of other lipogenic enzymes whose induction by a high carbohydrate diet was completely abolished in
the SREBP-1 knockout mice.
The cis-acting elements in the promoter region for carbohydrate stimulation and PUFA suppression of lipogenic genes
have been analyzed by many investigators. In the case of enzymes such as FAS (22), ATP citrate lyase (23), and PK (24),glucose/insulin response elements overlap with PUFA response
regions. Especially, the glucose/insulin and PUFA response
element in the FAS promoter has been shown to contain an
SREBP-binding site (25). The PUFA response region in themouse stearoyl-CoA desaturase 1 promoter is also reported tohave an SREBP-binding site (26, 27). These data are supportive of our finding that carbohydrate stimulation and PUFA
suppression are mediated by a common molecule, SREBP-1. In
contrast, promoter analyses of the Spot 14 gene previously
revealed that the PUFA-regulatory region was located separately from the region for dietary carbohydrate and insulin
responses (5).
Since PUFAs are known as activators of PPARa (16, 17), theeffects of PUFA through PPARa were also examined. The finding that fenofibrate did not affect the amount of mature
SREBP-1 in the liver indicates that the suppressive effect of
PUFA on mature SREBP-1 is not mediated by PPARa. Thisfinding is compatible with the previous study using PPARanull mice, which showed that PPARa was not required for the
PUFA-mediated inhibition of either FAS or Spot 14 gene
expression (18).
It has now been established that SREBP-1 and SREBP-2
function to regulate fatty acid synthesis and cholesterol synthesis, respectively, with some overlap in function (9, 10). Since
SREBP-2 is a regulator of the cholesterol biosynthetic pathway, it is itself highly controlled by cellular sterol levels. This
regulation, including the actions of SREBP cleavage-activating
protein (SCAP) and the site 1 and 2 proteases, has been extensively studied (8, 28, 29). However, the more fine tuned regulation of SREBP-1 has yet to be determined. The data pre-sented here indicate for the first time that the cleavage of
SREBP-1 in the liver could be regulated by a mechanism otherthan sterol concentrations and presumably in a fashion related
to fatty acid metabolism. Further studies are needed to clarify
the mechanism by which PUFA modulates the cleavage of
SREBP-1 precursor protein in terms of the regulation of hepatic lipogenesis.
In summary, we have demonstrated that dietary PUFA decreased the amount of hepatic SREBP-1 mature protein by areduction in the cleavage of SREBP-1 precursor protein, causing the suppression of lipogenic gene expression in the liver.
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