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. 35832–35839, 1999
Printed in U.S.A.

Sterol Regulatory Element-binding Protein-1 as a Key Transcription
Factor for Nutritional Induction of Lipogenic Enzyme Genes*(Received for publication, May 28, 1999, and in revised form, September 7, 1999)

Hitoshi Shimano‡, Naoya Yahagi, Michiyo Amemiya-Kudo, Alyssa H. Hasty§, Jun-ichi Osuga,
Yoshiaki Tamura, Futoshi Shionoiri, Yoko Iizuka, Ken Ohashi, Kenji Harada, Takanari Gotoda,
Shun Ishibashi, and Nobuhiro Yamada¶From the Department of Metabolic Diseases, Faculty of Medicine, University of Tokyo, Tokyo 113-8655, JapanTo elucidate the physiological role of sterol regulatory
element-binding protein-1 (SREBP-1), the hepatic
mRNA levels of genes encoding various lipogenic enzymes were estimated in SREBP-1 gene knockout mice
after a fasting-refeeding treatment, which is an established dietary manipulation for the induction of lipogenic enzymes. In the fasted state, the mRNA levels of all
lipogenic enzymes were consistently low in both wildtype and SREBP-12/2 mice. However, the absence of
SREBP-1 severely impaired the marked induction of hepatic mRNAs of fatty acid synthetic genes, such asacetyl-CoA carboxylase, fatty acid synthase, and stearoyl-CoA desaturase, that was observed upon refeeding in
the wild-type mice. Furthermore, the refeeding responses of other lipogenic enzymes, glycerol-3-phosphate acyltransferase, ATP citrate lyase, malic enzyme,
glucose-6-phosphate dehydrogenase, and S14 mRNAs,
were completely abolished in SREBP-12/2 mice. In contrast, mRNA levels for cholesterol biosynthetic genes
were elevated in the refed SREBP-12/2 livers accompanied by an increase in nuclear SREBP-2 protein. Whenfed a high carbohydrate diet for 14 days, the mRNAlevels for these lipogenic enzymes were also strikingly
lower in SREBP-12/2 mice than those in wild-type mice.
These data demonstrate that SREBP-1 plays a crucialrole in the induction of lipogenesis but not cholesterol
biosynthesis in liver when excess energy by carbohydrates is consumed.

Cholesterol and fatty acids are the primary lipids synthesized in liver. However, biosynthetic pathways for cholesteroland fatty acids are under distinct and separate regulation (fora review, see Ref. 1). In contrast to cholesterol synthesis, which
is tightly regulated by a feedback system to maintain cellularcholesterol levels, fatty acid synthesis is driven primarily by
the availability of carbohydrates and the actions of hormonessuch as insulin. Despite these different patterns of regulation,
recent evidence suggests that both biosynthetic pathways can* 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.
‡ 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.
§ Recipient of a fellowship under the Japan Society for the Promotionof Science Postdoctoral Fellowship Program for Foreign Researchers.
¶ Present address: Division of Endocrinology and Metabolism, Dept.
of Internal Medicine, University of Tsukuba, Ibaraki 305-8575, Japan.

be controlled by a common family of transcription factors designated sterol regulatory element binding proteins (SREBPs)1(reviewed in Ref. 2). SREBPs belong to a large class of transcription factors containing basic helix-loop-helix-leucine zipper. Unlike other members of this class, SREBPs are synthesized as membrane-bound precursors that require cleavage by
a two-step proteolytic process in order to release their aminoterminal basic helix-loop-helix-leucine zipper-containing domain into the nucleus to activate their target genes in a sterolregulated manner (2). Another unique feature of SREBPs isthat they have a dual binding specificity to both classic palindromic E-boxes and nonpalindromic sterol regulatory elements(SREs) (2– 4).
The SREBP target genes include enzymes of cholesterol biosynthesis: HMG-CoA reductase, HMG-CoA synthase, farnesyl
diphosphate synthase, squalene synthase, and SREBP-2 itself,each of which contains a SRE or SRE-like sequence in its
promoter (5–9). The SREBPs also bind to regulatory sequencesin the promoters of the genes involved in the biosynthesis offatty acids: acetyl-CoA carboxylase, fatty acid synthase, andstearoyl-CoA desaturase (3, 10 –12) as well as glycerol-3-phosphate acyltransferase, a gene involved in the production of
triglycerides (13).
To date, three SREBPs have been identified: SREBP-1a and
SREBP-1c, produced from a single gene through the use ofalternate promoters, and SREBP-2 from a separate gene (2).
The rat homologue of SREBP-1c, named ADD1, was cloned
independently as a protein that binds to E-boxes and presumably promotes adipocyte differentiation (14). All actively growing cultured cells so far studied produce predominantly
SREBP-1a and SREBP-2, whereas most organs including liverfrom adult animals predominantly synthesize SREBP-1c and
SREBP-2 (15). All three SREBPs are capable of activating eachof the known target genes, although with differing efficiencies.
SRBP-1c is weaker than SREBP-1a and SREBP-2 due to its
shorter transactivation domain (16, 17).
To gain insight into the distinct roles of each SREBP isoform
in vivo, transgenic mice that overexpress truncated, active
nuclear forms of human SREBP-1a, -1c, or -2 in liver were
produced and characterized (16, 18, 19). The different SREBPoverexpressing transgenic animals showed a different patternof increase in hepatic synthesis and accumulation of cholesterol
and/or fatty acids. These data suggest that the SREBP-1 isoforms are more selective in activating fatty acid biosynthetic
genes, while SREBP-2 is more specific for controlling cholesterol biosynthesis. The mechanism for the relative specificity of
1
The abbreviations used are: SREBP, sterol regulatory elementbinding protein; SRE, sterol regulatory element; ACC, acetyl-CoA carboxylase; FAS, fatty acid synthase; SCD, stearoyl-CoA desaturase;
CYP4A2, cytochrome P-450 4A2; SCAP, SREBP cleavage activating
protein; PPARa, peroxisome proliferator-activated receptor a.

35832

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

SREBP-1 as a Key Regulator of Lipogenic Enzymes
each transcription factor is not currently known.
Liver, the principal organ of lipogenesis as well as cholesterogenesis, is responsible for production of triglycerides fromexcess dietary carbohydrate. A high carbohydrate diet inducesmRNA levels for a group of genes designated lipogenic enzymes(reviewed in Refs. 20 –22). This transcriptional induction of
lipogenic enzymes is most prominently seen during fastingrefeeding treatment to rodents. When mice are fasted, lipogenesis declines. Refeeding fasted animals with a high carbohydrate diet causes marked induction of the mRNA levels of
lipogenic enzymes to levels higher than pretreatment, which isoften referred to as “overshooting.” These lipogenic enzymes
include not only genes for fatty acid biosynthesis, such asacetyl-CoA carboxylase (ACC), fatty acid synthase (FAS), andstearoyl-CoA desaturase (SCD), but also glycerol-3-phosphate
acyltransferase for triglyceride synthesis; glucose-6-phosphate
dehydrogenase and malic enzyme, which provide the NADPH
for reductive biosynthesis; and ATP citrate lyase, which produces acetyl-CoA in the cytosol and liver-type pyruvate kinase
in the glycolytic pathway. Each of these lipogenic genes isknown to be regulated at a transcriptional level and has beenshown to be activated in the livers of SREBP transgenic mice
(16, 18, 23). The distinct regulation of SREBP-1 and -2 has been
observed in the physiological response of mouse liver to nutritional change. The amounts of SREBP-1c protein and mRNA,but not those of SREBP-2, dramatically increased after refeeding fasted mice in a similar manner to lipogenic enzyme
mRNAs (24). Furthermore, transgenic mice overexpressing
SREBP-1c, even when fasted, mimicked the refeeding state of
ACC, FAS, and SCD mRNA levels in wild-type animals (24).
Taken together, the data suggest that a high level expression of
nuclear SREBP-1c in the refed state could be responsible forinduction of lipogenic enzymes. However, due to the artificial
overexpression of SREBPs in the transgenic mice, we cannot
conclude whether SREBP-1 is a physiological regulator fortranscription of lipogenic enzymes. The disruption of the
SREBP-1 gene caused partial embryonic lethality, with only
10 –30% of expected homozygous mice surviving (25). In theinitial analysis of the surviving SREBP-12/2 mice, there wereno significant changes in hepatic mRNA levels of ACC, FAS,
and SCD, while genes in the cholesterol biosynthetic pathway
were activated (25). However, the study was performed in thenonfasted state of a regular diet, which is not a favorable
condition for lipogenesis. In the current study, the SREBP-1
knockout mice were used to re-estimate the effects of the absence of functional SREBP-1 protein on expression of the lipogenic enzymes in an inducible situation (fasting-refeeding
treatment or prolonged high carbohydrate diet). After these
dietary manipulations, wild-type mice showed marked elevation in all lipogenic genes, while those of SREBP-12/2 miceremained suppressed, suggesting that SREBP-1 is crucial for
nutritional induction of hepatic lipogenic enzymes.
EXPERIMENTAL PROCEDURES

Animals and Dietary Manipulation—Mice were housed in colony
cages with a 12-h light/12-h dark cycle. Mice homozygous for disrupted
SREBP-1 gene allele B (SREBP-12/2) were prepared as described previously (25) and used for the current studies with age- (30 –33 weeks)
and sex-matched wild-type animals on the same genetic background(hybrid between C57Black/6J and 129 Sv/Ev). For the fasting andrefeeding study, groups of four female SREBP-12/2 and wild-type mice
were set up. All animals had been fed on a regular chow diet until thefasting and refeeding treatment started. For the refeeding group, animals were fasted 24 h (from 22:00 to 22:00) and then refed with a highcarbohydrate/fat-free diet for 12 h (22:00 –10:00). This fasting andrefeeding cycle was repeated three times at 3-day intervals for the
refeeding group to adapt the animals to the dietary manipulation. The
final fasting and refeeding cycle was performed after a 1-week interval.
The mean percentage increase in the body weights during the refeeding

35833
2/2

was 8% for wild-type and SREBP-1
mice. For the fasting group, theanimals were fasted for 24 h (10:00 –10:00). Both groups were sacrificed
between 10:00 and 10:30.
Materials—Regular laboratory diet and high carbohydrate/fat-freediet (70% sucrose and 20% casein) were purchased from Oriental Yeast
Co., Ltd. (Osaka, Japan). Rabbit polyclonal antibody against mouse
upstream stimulatory factor (USF)-1 and USF-2 were purchased fromSanta Cruz Biotechnology, Inc. (Santa Cruz, CA).
Plasma cholesterol, triglycerides, and nonesterified fatty acids were
measured by enzymatic assays using commercial kits (Determiner TC,
TG, and NEFA, respectively, Kyowa Medics, Co., Ltd., Tokyo, Japan).
The content of cholesterol and triglycerides in liver was measured as
described previously (26).
Nuclear Extract Preparation and Immunoblot Analysis—Liver nuclear extracts were prepared as described except for the use of phenylmethylsulfonyl fluoride (1 mM) instead of Pefabloc (27). The samples of
20-mg nuclear protein were subjected to immunoblot analysis withrabbit IgG against mouse SREBP-1 (amino acids 32–250) (18) oragainst mouse SREBP-2 (amino acids 32–250) (25), followed by horseradish peroxidase-linked goat IgG against rabbit IgG and the ECL kit(Amersham Pharmacia Biotech).
Total RNA Preparation and Blot Hybridization with cDNA Probes—
Total RNA was extracted from mouse livers and parametrial adipose
tissues using TRIZOL Reagent (Life Technologies, Inc.). Equal aliquotsof total RNA from mice in each group were pooled (total 15 mg), subjected to formalin-denatured agarose electrophoresis, and transferred
to nylon membrane (Hybond N, Amersham Pharmacia Biotech). Blot
hybridization was performed with the cDNA probes labeled with[a-32P]CTP (6000 Ci/mmol) using the Megaprime DNA Labeling System(Amersham Pharmacia Biotech). The cDNA probes for mouse S14, ATP
citrate lyase, malic enzyme, liver-type pyruvate kinase, PPARa, acylCoA oxidase, cytochrome P-450 4A2 (CYP4A2), and acidic ribosomal
phosphoprotein PO (36B4) were prepared by cloning reverse transcriptase-polymerase chain reaction products from mouse liver RNA
into TA cloning vectors (Invitrogen). The primers used for polymerase
chain reaction were as follows: for rat ATP citrate lyase, 59 primer was
ACATCTGTACCACCTCAGCCATCCAGAA and 39 primer was GCAGTGGCGTCCACCTTGGCCGCCA (28); for rat liver-type pyruvate kinase,59 primer was CCCAGGAGCTGGGCACTGCCTTCTTCCAGC and 39
primer was AGCCCGTCGTCAATGTAGATGCGGCCCCCCAC (29, 30);
for mouse malic enzyme, 59 primer was CCACCAGCGCGGCTACCTGCTGACGCGGGA and 39 primer was CCTCTGACTCGCCGGTGCCGCAGCCCGATG (31); for mouse PPARa, 59 primer was GCCATGGTGGACACGGAAAGCCCACTCTGCCC and 39 primer was AGATCAGTACATGTCTCTGTAGATCTCTTG (32); for mouse acyl CoA oxidase, 59
primer was ATGAATCCCGATCTGCGCAAGGAGC and 39 primer was
AAAGGCATGTAACCCGTAGCACTCC (33); for rat CYP4A2, 59 primer
was CTCTGTATTTAGCCCTACAAGATCCCTGGA and 39 primer was
ATGATAGCCTTGGTGTAGGACCTGGAATTT (34); and for mouse
36B4, 59 primer was ATGATTATCCAAAATGCTTCATTG and 39
primer was AACAGCATATCCCGAATCTCA (35). Other probes were as
described previously (16, 18).
The resulting bands were quantified by exposure of the filters to
BAS2000 with BAStation software (Fuji Photo Film Co., Ltd., Tokyo,
Japan), and the results were normalized to the signal generated from
36B4 mRNA.
Transfections and Luciferase Assays—Human embryonic kidney 293
cells were grown at 37 °C in an atmosphere of 5% CO2 in Dulbecco’s
modified Eagle’s medium containing 100 units/ml penicillin and 100
mg/ml streptomycin sulfate supplemented with 10% fetal calf serum.
Transfection studies were carried out with cells plated on 12-well plates
as described previously (16) except that SuperFect (Qiagen) was used
for transfection according to the manufacturer’s protocol. The indicatedamount of each expression plasmid was transfected simultaneouslywith 0.25 mg of a luciferase reporter plasmid (pFAS-Luc (16)) and 0.5 mgof a control pTKb-gal reference plasmid (Promega). The total amount of
DNA in each transfection was adjusted to 3 mg/well with vector DNA.
The amount of luciferase activity in transfectants was normalized to theamount of b-galactosidase activity as measured by a kit (Promega).
RESULTSTo show that SREBP-1 controls transcriptional regulation ofhepatic lipogenic enzymes, we hypothesized that the absence of
SREBP-1 should cause an impaired response of lipogenic enzyme mRNAs in the liver to refeeding. To test this hypothesis,
we performed a fasting and refeeding treatment on SREBP12/2 and wild-type mice. Table I shows the phenotypic charac-

35834

SREBP-1 as a Key Regulator of Lipogenic Enzymes

TABLE I
Phenotypic characteristics of wild-type and homozygous (SREBP-12/2) mice for the disrupted SREBP-1 gene
Each value represents the mean 6 S.E. Two sets of four female age-matched wild-type and SREBP-12/2 mice on the same genetic background
had been fed on a regular chow diet. One set of animals were fasted 24 h, and the other set were fasted 24 h and refed with high carbohydrate/
fat-free diet 12 h prior to sacrifice at 10:00. *, **, and *** denote significance versus fasted wild type at p , 0.05, p , 0.01, and p , 0.001,
respectively; #, ##, and ### denote significance versus refed wild type at p , 0.05, p , 0.01, and p , 0.001, respectively; and $, $$, and $$$ denotesignificance versus fasted SREBP-12/2 at p , 0.05, p , 0.01, and p , 0.001, respectively.
FastedGenotype

Body weight (g)Liver/body weight
Liver cholesterol (mg/g)
Liver triglyceride (mg/g)
Plasma cholesterol (mg/dl)
Plasma triglyceride (mg/dl)
Plasma NEFAa (mg/dl)
Blood sugar (mg/dl)
a

Refed

Wild type

SREBP-12/2

Wild type

SREBP-12/2

24.8 6 0.90
0.036 6 0.002
2.0 6 0.037
43.5 6 6.5
60.7 6 3.5
59.0 6 4.7
618 6 10072.0 6 9.5

26.6 6 0.81
0.036 6 0.002
3.8 6 0.39*
61.0 6 1.8
41.0 6 4.6*
46.9 6 4.9
534 6 20.0***
84.0 6 4.5

28.5 6 2.6
0.064 6 0.006**
3.4 6 0.39
48.3 6 8.7
63.0 6 7.8
84.4 6 9.1*
192 6 70
146 6 1.7**

22.3 6 0.46#$
0.055 6 0.007$$
8.4 6 0.62###$$
30.0 6 5.1$$
40.3 6 3.3##
36.0 6 2.0###
50.5 6 11$$$145 6 11$$

Nonesterified fatty acid.

teristics of both groups in the study. The mean percentage
increase in body weight during the refeeding was the same (8%)
between refed wild-type and SREBP-12/2 mice. The livers fromboth refed groups were significantly enlarged, which is customarily observed in refed rodents. Cholesterol content in the liver
was significantly higher in SREBP-12/2 mice than in wild-typemice in both fasted and refed states, which is consistent withthe previous report on the nonfasted SREBP-12/2 mice (25).
The fasting-refeeding treatment decreased liver triglyceride
content in SREBP-12/2 mice, while no change was detected in
the livers of wild-type mice. In the wild-type animals, plasma
nonesterified fatty acid levels were elevated by fasting andsuppressed by refeeding. In the SREBP-12/2 mice, the suppression of plasma NEFA by refeeding seemed more pronounced
than that observed in wild-type mice. There was no significant
difference between SREBP-12/2 and wild-type mice in blood
sugar levels in either nutritional state.
Fig. 1 shows immunoblot analysis of SREBPs in liver nuclear
extracts. In the fasted state, the mature form of SREBP-1 inthe liver nuclear extracts pooled from wild-type mice wasbarely detectable, while refeeding dramatically increased themature protein level. The aberrant protein of ;40 kDa derived
from the disrupted SREBP-1 gene in the knockout mice (indicated by an asterisk in Fig. 1) was also markedly enhanced byrefeeding. This aberrant protein consists of the N-terminal partof the native SREBP-1 and lacks the helix-loop-helix region,
leading to a lack of any transactivity for SRE-containing promoters (25). The amount of the SREBP-2 protein in the nuclear
extracts from SREBP-12/2 mice was approximately 2-fold increased compared with that of wild-type mice in both fasted
and refed states.
Figs. 2–5 compare mRNA levels for various genes in thelivers of wild-type and SREBP-12/2 mice that were fasted, or
fasted and refed. Fig. 2 shows the results for SREBP-1 and -2,
SREBP cleavage activating protein (SCAP), and HMG CoA
synthase and reductase. In accordance with the nuclear protein
data, SREBP-1 mRNA level in the fasted wild-type animals
was very low and markedly induced by refeeding. The aberrant
SREBP-1 transcript from the disrupted gene was also elevatedin the refed liver. In contrast, there was no significant change
in mRNA levels of SREBP-2 in the fasted and refed wild-typemice and in fasted SREBP2/2 mice. Refed SREBP-12/2 mice
showed only a slight increase in SREBP-2 mRNA level ascompared with other groups. The mRNA of SCAP did notchange markedly except that refed SREBP-12/2 mice had aslightly decreased level, the biological significance of which iscurrently unknown. The mRNA levels for both hydroxymethylglutaryl-CoA synthase and reductase, rate-limiting enzymes
in the cholesterol synthetic pathway, were significantly in-

FIG. 1. Immunoblot analysis of SREBP-1 and SREBP-2 in nuclear extracts from livers of fasted or refed wild-type (WT) and
SREBP-12/2 (KO) mice. For each group, livers from four mice shownin Table I were pooled, and aliquots (30 mg of protein) of nuclear
extracts were subjected to immunoblot analysis with rabbit anti-mouse
SREBP-1 or SREBP-2 IgG as the primary antibody and horseradish
peroxidase-labeled donkey anti-rabbit IgG (Amersham Pharmacia Biotech) as the secondary antibody. In the nuclear extracts of SREBP-12/2
mice, aberrant SREBP-1 proteins derived from disrupted allele of the
SREBP-1 gene were detected at ;40 kDa (25) as indicated by anasterisk for comparison with a native mature protein of 68 kDa from thewild-type SREBP-1 gene.

creased only in refed SREBP2/2 mice.
Fig. 3 shows changes in mRNA levels in the genes involved infatty acid biosynthesis. The acetyl-CoA carboxylase, fatty acidsynthase, and stearoyl-CoA desaturase 1 are lipogenic enzymes
that play a central role in the production of major long chain
monounsaturated fatty acids in mammals by conversion ofacetyl-CoA to palmitoleate (C16:1) and oleate (C18:1). The
mRNA levels of these enzymes were suppressed in livers offasted wild-type mice and required longer exposure for signal
detection (data not shown; see Table II for reference). They
were markedly elevated in the normal livers by refeeding (Fig.
3). The mRNA levels for acetyl-CoA carboxylase and fatty acidsynthase in the fasted SREBP-12/2 mice were low and essentially similar to those of fasted normal mice. However, the
SREBP-12/2 mice had a blunted induction of these enzymes
upon refeeding compared with wild-type mice (Fig. 3 and Table
II). The absence of SREBP-1 caused significant decreases in
SCD1 mRNA levels in both fasted and refed states. The mostdramatic differences in the hepatic mRNA levels of refedSREBP-1 as a Key Regulator of Lipogenic Enzymes

FIG. 2. Quantification of mRNA levels of SREBP-1 and -2,
SCAP, hydroxymethylglutaryl-CoA synthase, and reductase inlivers from fasted or refed wild-type (WT) and SREBP-12/2 (KO)mice as measured by Northern blot analysis. Total RNA wasextracted from the livers of the mice in each treatment group (Table I).
Equal aliquots of 15 mg were pooled and subjected to electrophoresisand blot hybridization with the indicated 32P-labeled cDNA probe. The
shorter band observed in SREBP-12/2 mice with SREBP-1 probe is theaberrant transcript derived from the disrupted SREBP-1 gene (25). The
relative ratio of the signal from each group versus fasted wild-type micewas calculated after correction by the corresponding signal from 36B4
using the BAS system (Fuji) and is shown in Table II.

FIG. 3. Quantification of mRNA levels of the genes involved infatty acid synthesis in livers from fasted or refed wild-type (WT)and SREBP-12/2 (KO) mice as measured by Northern blot analysis. The RNA filters prepared as described in the Fig. 2 legend were
hybridized with 32P-labeled cDNA probe of mouse acetyl-CoA carboxylase, fatty acid synthase, or stearoyl-CoA desaturase. The results ofquantification by the BAS system were shown in Table II.

SREBP-12/2 mice were observed in other lipogenic enzymes.
As depicted in Fig. 4, the genes for glycerol-3-phosphate acyltransferase, ATP citrate lyase, glucose-6-phosphate dehydrogenase, and S14 were markedly activated by refeeding in thewild-type mice (Fig. 4 and Table II). In contrast, these refeeding responses were severely reduced in SREBP-12/2 mice. Notably, the mRNA levels of glucose-6-phosphate dehydrogenase,glycerol-3-phosphate acyltransferase, and S14 in the refed
SREBP-12/2 mice remained barely detectable, similar to theirfasting level. The mRNA level of ATP citrate lyase in the refed
SREBP-12/2 livers was also markedly lower than that in refedwild-type mice. The refeeding response of malic enzyme in wild
type mice was relatively small (6-fold) as compared with otherlipogenic enzymes. This induction of malic enzyme was also
attenuated in SREBP-12/2 mice (Fig. 4 and Table II). These
data suggested that SREBP-1 dominates transcriptional regulation for those lipogenic enzymes in the liver.
To confirm the validity of the fasting-refeeding protocol in
our studies, we measured mRNA levels for genes that were
known to be regulated by fasting and refeeding but in a different fashion from lipogenic enzymes. Acyl-CoA oxidase andCYP4A2 are involved in peroxisomal and microsomal oxidationof fatty acids, respectively. Induction of these enzymes has
been shown to be mediated through PPARa and its ligands (36)and has also been reported to be induced by fasting and suppressed by refeeding (37). As shown in Fig. 5, both the acyl-CoA

35835

oxidase and CYP4A2 mRNAs were 4-fold higher in fasted livers
than in refed livers from both wild-type and SREBP-12/2 mice,
confirming the previous report (37). These data indicate that,as expected, SREBP-1 has no effect on the message levels of
fatty acid oxidation genes during a fasting-refeeding cycle. The
mRNAs of PPARa were also found to be regulated in a similarmanner to its down stream genes, suggesting that the transcriptional change of PPARa could partially contribute to thenutritional regulation of acyl-CoA oxidase and CYP4A2 genes.
In addition to liver, adipose tissue is another organ in which
lipogenic enzymes are thought to respond to fasting-refeedingat the transcription level. As shown in Fig. 6, fasting-refeedingchanges in mRNA levels of lipogenic enzymes from adipose
tissues of wild-type mice were less dramatic than those observed in the liver. Notably, stearoyl-CoA desaturase, glucose6-P dehydrogenase, and pyruvate kinase had little response torefeeding. SREBP-12/2 adipose tissues exhibited impaired induction of other lipogenic enzymes, although the reduction was
generally less pronounced than that observed in the livers withexception of malic enzyme in which the refeeding pattern was
similar in both tissues. Quantification of the relative amount ofmRNA is summarized in Table II.
Lipogenic enzymes in the liver were also induced when animals were put on a high carbohydrate diet for a prolonged
period of a time. The promoter regions of lipogenic enzymes
such as fatty acid synthase, liver-type pyruvate kinase, andS14 have been extensively analyzed and shown to containcis-acting elements, identified as carbohydrate (glucose or insulin)-response elements (38 – 40). To determine the long term
effect of a high carbohydrate diet on hepatic mRNA levels of
lipogenic enzymes, two sets of wild-type and SREBP-12/2 mice
were fed a high sucrose diet and a normal chow diet for 2
weeks. The mice in this series of experiments were sacrificed ina nonfasted state. The hepatic mRNA levels for lipogenic genes
in the four groups of animals are shown in Fig. 7. All of thelipogenic genes were robustly induced by a high carbohydrate
diet in livers of wild type mice as compared with those on anormal diet. The carbohydrate induction of acetyl-CoA carboxylase, fatty acid synthase, stearoyl-CoA desaturase, ATP citrate lyase, and pyruvate kinase was partially blunted in
SREBP2/2 mice. In contrast, it was almost abolished in glycerol-3-phosphate acyltransferase, glucose-6-phosphate dehydrogenase, malic enzyme, and S14. Therefore, the amounts ofthese lipogenic enzyme mRNAs after a high carbohydrate dietwere profoundly lower in SREBP-12/2 mice compared with
controls, while differences between groups on a normal chowdiet were not striking for any of lipogenic enzymes. This pattern is very similar to that observed in the refeeding response.
The USFs have been shown to play an important role in thetranscription of fatty acid synthase, S14, and liver pyruvatekinase. To examine the roles of USFs in conjunction with thedeletion of the SREBP-1 gene, we determined the amounts of
USF-1 and -2 in wild-type and SREBP-12/2 mice by immunoblot analysis (Fig. 8). There was no significant difference in theamount of USF-1 or -2 protein in the liver nuclear extracts fromSREBP-12/2 and wild-type mice in either fasted or refed state.
The SREBP-12/2 mice produce an aberrant mRNA and theresulting shortened protein from the disrupted SREBP-1 gene,which has been shown to be inactive for SRE-containing promoters (25). To exclude a minor possibility that this aberrant
protein might have dominant negative effects on transactivityof lipogenic enzymes, causing reduction in their mRNA levels,
we investigated the effect of overexpression of the aberrant
SREBP-1 protein on fatty acid synthase promoter activity by
luciferase reporter assays (Fig. 9). When the expression vector
containing the cDNA encoding aberrant SREBP-1 protein (25)35836SREBP-1 as a Key Regulator of Lipogenic Enzymes

TABLE II
Quantification of lipogenic enzyme mRNAs in livers and adipose tissues from fasted or refed wild-type and SREBP-12/2 mice
Values are the relative ratio of each signal versus the corresponding fasted wild type corrected for the signal from 36B4 as loading control.
Liver
Genea

Adipose tissue

FastedWild-type

SREBP-1
1.0
SREBP-2
1.0
Lipogenic enzymes
ACC
1.0
FAS
1.0
SCD
1.0
GPAT
1.0
ACL
1.0
G6PD
1.0
ME
1.0
LPK
1.0
S14
1.0
Cholesterogenic enzymes
HMG CoA Syn
1.0
HMG CoA Red
1.0
PPARa-inducible genes
PPARa
1.0
AOX
1.0
CYP4A2
1.0

Refed

FastedRefed

SREBP-12/2

Wild-type

SREBP-12/2

Wild-type

SREBP-12/2

Wild-type

SREBP-12/2

0.6
0.9

8.5
0.9

5.3b
1.4

1.0
1.0

0.50
1.0

4.2
0.9

9.6b
1.0

1.5
2.0
0.26
0.93
3.7
0.4
1.3
2.0
2.2

20
90
16
8.0
103
14
5.9
19
150

9.3
21
3.8
0.53
17
1.6
1.6
14
13

1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0

0.50
0.37
0.61
1.2
0.58
0.85
0.53
0.68
0.66

2.3
2.5
1.5
3.6
29
1.8
5.8
1.3
8.5

1.7
1.3
0.7
2.4
9.5
0.72
1.2
0.9
5.1

0.92
0.7

0.80
1.8

4.0
4.1

1.0
1.0

1.2
0.5

1.7
1.7

3.4
3.8

1.4
0.9
1.1

0.3
0.4
0.22

0.3
0.2
0.16

NDc
ND
ND

ND
ND
ND

ND
ND
ND

ND
ND
ND

a
GPAT, glycerol-3-phosphate acyltransferase; ACL, ATP citrate lyase; ME, malic enzyme; Syn, synthase; Red, reductase; AOX, acyl-CoAoxidase.
b
The signal of aberrant SREBP-1 mRNA derived from the disrupted SREBP-1 gene.
c
ND, not determined.

FIG. 4. Quantification of mRNA levels of various genes supporting lipogenesis in livers from fasted or refed wild-type (WT)and SREBP-12/2 (KO) mice as measured by Northern blot analysis. The RNA filters prepared as described in the Fig. 2 legend were
hybridized with the indicated 32P-labeled cDNA probe. The results ofquantification by the BAS system were shown in Table II.

FIG. 5. Quantification of mRNA levels of PPARa, acyl-CoA oxidase, and CYP4A2 genes in livers from fasted or refed wild-type(WT) and SREBP-12/2 (KO) mice as measured by Northern blot
analysis. The RNA filters prepared as described in the Fig. 2 legend
were hybridized with the indicated 32P-labeled cDNA probe. The resultsof quantification on the BAS imaging plate were shown in Table II.

was transfected into 293 cells, there was no significant change
in the luciferase activity derived from the reporter gene towhich the promoter of fatty acid synthase was fused (pFASLuc) (25). Meanwhile, an expression vector for an authentic
dominant negative version of SREBP-1 that lacks its N-terminal transactivation domain (41) suppressed the activity of FAS-

FIG. 6. Quantification of mRNA levels of various genes in adipose tissue from fasted or refed wild-type (WT) and SREBP-12/2(KO) mice as measured by Northern blot analysis. Total RNA was
isolated from parametrial adipose tissue of each treatment group (TableI). The RNA filters were prepared as described for the livers in the Fig. 2
legend and hybridized with the indicated 32P-labeled cDNA probes. The
shorter band observed in SREBP-12/2 mice with SREBP-1 probe was theaberrant transcript derived from the disrupted SREBP-1 gene (25). The
results of quantification by the BAS system were shown in Table II.

luc gene in a dose-dependent manner. These data suggest thatthe aberrant SREBP-1 protein does not influence the promoterof the fatty acid synthase gene in either a negative or positive
fashion.

SREBP-1 as a Key Regulator of Lipogenic Enzymes

35837

FIG. 7. Amounts of various mRNAs
in livers from wild-type (WT) and
SREBP-12/2 (KO) mice fed a normalchow (Normal) and a high carbohydrate/fat-free (Carbo) diet for 2
weeks. Four male wild-type and SREBP12/2 mice were fed a high carbohydrate/
fat-free diet or a normal regular diet for 2
weeks and sacrificed in a nonfasted state.
Total RNA was prepared from the liversof each group. Equal aliquots of 15 mg
were subjected to hybridization with the indicated 32P-labeled cDNA probes. GPAT,
glycerol-3-phosphate acyltransferase; ACL,
ATP citrate lyase; G6PD, glucose-6-phosphate dehydrogenase; 6PG, 6-phosphogluconate dehydrogenase; ME, malic enzyme;
PK, liver pyruvate kinase.

FIG. 8. Immunoblot analysis of USF-1 and -2 in nuclear extracts from livers of wild-type (WT) and SREBP-12/2 (KO) micethat were fasted and refed. The filter of nuclear extract proteins was
prepared as described in Fig. 1 legend. Immunoblot analysis was carried out with rabbit anti-mouse USF-1 or -2 IgG as the primary
antibody.
DISCUSSION

The current study clearly demonstrates that SREBP-1 plays
a key role in hepatic transcriptional regulation of lipogenic
enzymes. Deletion of the SREBP-1 gene markedly suppressed
expression of an entire class of lipogenic enzymes in the refedstate where lipogenesis should be fully induced. The similar
results from SREBP-12/2 mice fed a high carbohydrate diet for
2 weeks suggest that SREBP-1 is important for long term as
well as short term induction of lipogenic enzymes. In contrast,
in a fasted state in which lipogenesis should be suppressed, theamount of SREBP-1 in wild-type liver was substantially low at
both mRNA and protein levels. Consequently, there was nomarked difference in the fasting mRNA level of each lipogenic
enzyme between wild-type and SREBP-12/2 mice. Thus,
SREBP-1 might not be involved in maintaining the fastinglevel of minimal transcription for lipogenic genes. This also
partially explains the lack of a clear reduction in hepatic
mRNA levels of fatty acid synthetic genes in SREBP-12/2 micein the initial study where the animals were sacrificed in apartially fasted state due to their normal feeding pattern.
The intensity of impairment in refeeding response due to thedeletion of SREBP-1 varies among lipogenic genes in the liver.

FIG. 9. Luciferase reporter gene assay to determine the effectof the aberrant SREBP-1 protein from SREBP-12/2 mice on fatty
acid synthase promoter activities. The 293 cells were transfectedwith the indicated amount of pTK-aberrant SREBP-1c (25) (open or
closed circle) or pTK-SREBP-1DN (dominant negative version of
SREBP-1 under the thymidine kinase promoter) (open or closed triangle) with or without 0.5 mg of pTK-SREBP-1c463 (expression plasmid
for a truncated version of SREBP-1c (25)). A reporter plasmid, the fatty
acid synthase promoter fused to the luciferase gene (FAS-luc (25)), anda reference plasmid of TK-b-galactosidase gene were cotransfected.
After transfection, the cells were cultured for 16 h in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum, 1 mg/ml
25-hydroxycholesterol, and 10 mg/ml cholesterol to suppress endogenous SREBP activity in the presence of TK-SREBP-1c463 (closed circle
or triangle) or supplemented with 10% lipoprotein-deficient serum and
10 mM pravastatin to induce endogenous SREBP activities in the absence of TK-SREBP-1c463 (open circle or triangle). Luciferase activity
was measured and normalized to b-galactosidase activity. The values
were plotted as percentages of the endogenous activity from the cells in
an induced condition. Expression of the truncated (dominant positive)
and aberrant SREBP-1 proteins in the nuclei of transfected cells was
confirmed by immunoblot analysis (insets A and B, respectively).

35838SREBP-1 as a Key Regulator of Lipogenic Enzymes

The most dramatic suppression in the refed liver was observedin glucose-6-phosphate dehydrogenase, glycerol-3-phosphate
acyltransferase, and malic enzyme, for which induction ofmRNA levels by the refeeding was completely abolished.
SREBP-1 seems to dominate transcriptional regulation for
those enzymes in the liver. The other hepatic lipogenic enzymes
were also grossly suppressed, but they partially retained arefeeding response in SREBP-12/2 mice. This residual refeeding response in SREBP-12/2 mice was more prominent in the
adipose tissue. These results suggest that other factors, presumably helix-loop-helix proteins, could also be involved innormal refeeding response.
The USFs have been extensively explored as possible transcriptional factors that could be responsible for the nutritional
regulation of lipogenic enzymes such as fatty acid synthase,
S14, and L-pyruvate kinase through binding to the carbohydrate or insulin-response elements. Both USF-1 and USF-2
homodimers and the heterodimer of the two, which is presumably the physiological form, have been shown to bind to andactivate the promoter of those genes (40, 42– 44). However, therole of USFs in nutritional responsiveness of lipogenic enzymes
has been controversial (45, 46). It should be noted that USFs
are relatively abundant proteins and are able to bind the c-Myc
E-box. This binding has been thought to contribute to thedifficulties encountered in analyzing DNA binding of Myc/Max/
Mad network complexes (47). Recently, gene knockout mice for
USF-1 and USF-2 have been generated and reported (48, 49).
The mRNA levels for fatty acid synthase in the livers of refedmice deficient for USF-1 or -2 were markedly reduced, suggesting that USFs are required for fatty acid synthase gene expression (50). In our study, we showed that the amounts of USF
proteins in the nuclear extracts were not changed during fasting and refeeding, whereas the nuclear active mature form of
SREBP-1 was markedly suppressed in fasting and induced
upon refeeding. It is likely that USFs are essential factors thatare required to maintain transcriptional levels of fatty acidsynthase irrespective of nutritional state, while SREBP-1 isnutritionally regulatable and is responsible for induced production of fatty acids when excess energy is available. USFs and
SREBPs do not appear to compensate for each other in theknockout studies. In addition, in vitro data including gel shift
assay have implicated that both USFs and SREBPs bind to thefatty acid synthase promoter independently, and there is noevidence for synergistic action (51). However, these observations supporting mutual independence of USFs from SREBP-1
cannot explain the fact that deficiency of either USFs or
SREBP-1 caused profound suppression (70 – 80%) of fatty acidsynthase mRNA upon refeeding. Further investigation of possible mutual interaction of SREBP-1 and USFs is needed toelucidate the molecular mechanism by which SREBP-1 and
USFs are involved in activation of the fatty acid synthase gene.
Hasegawa and Uyeda et al. (52) recently identified a novel
factor designated glucose response element-binding proteinfrom rat liver nuclear extracts, which binds to glucose response
elements of liver pyruvate kinase and fatty acid synthasegenes. Estimation of the physiological significance for glucose
response element-binding protein awaits its molecular cloning.
Another interesting observation was the regulation of
SREBP-1 mRNA itself in the fasting-refeeding treatment.
SREBP-1 mRNA of wild-type mice was decreased during thefasting and markedly induced during refeeding in the samemanner as other lipogenic enzymes. Therefore, SREBP-1 isregulated in a lipogenic fashion at both mRNA and protein
levels and could belong to the same family of lipogenic enzymes. The data from transgenic mice demonstrated that
SREBP-1 could transactivate the SREBP-1 gene itself, whichmight explain the overshooting phenomenon of lipogenesis atrefeeding by a positive feedback system in SREBP-1 transcriptional regulation. However, in the refed SREBP-12/2 mice, anaberrant transcript derived from the intact promoter of disrupted SREBP-1 gene was also induced in the same way asnative SREBP-1 in the wild-type mice, while the downstream
lipogenic mRNAs were entirely suppressed in the absence offunctional SREBP-1. These data implicate the following intriguing possibility. Although SREBP-1 directly regulates andits absence impairs lipogenic enzyme transcription, there may
be an upstream factor or mechanism which controls transcription of SREBP-1. The primary response to refeeding at thislevel was normal as demonstrated by the induction of theaberrant SREBP-1 mRNA.
It is surprising that a single transcriptional factor, SREBP-1,
as observed in the current study, can induce such a wide range
of genes. Some of the genes analyzed in the current study suchas acetyl-CoA carboxylase, fatty acid synthase, stearoyl-CoA
desaturase, and glycerol-3-phosphate acyltransferase, havebeen proven to be a direct target of SREBPs by promoter
analysis (3, 10 –13). The direct binding of SREBP-1 has yet tobe shown for other lipogenic enzyme genes, such as ATP citratelyase, glucose-6-phosphate dehydrogenase, malic enzyme,
pyruvate kinase, and S14. An extensive promoter analysis ofthese genes might unveil a previously undefined mechanism
for the dual binding specificity of SREBPs to SRE and E-box
sequences (3) or lead to the discovery of new versions of SRE- orE-box-related sequences.
Impaired nutritional induction of lipogenesis in SREBP-12/2
mice provides further evidence that SREBP-1 is more specificto fatty acid synthesis and that SREBP-2 is more specific tocholesterol synthesis. This is demonstrated by the fact that
activated SREBP-2 in the refed SREBP-12/2 mice overshot
cholesterogenic genes but was not sufficient to compensate forthe lack of SREBP-1 in the induction of lipogenic enzymes. As
expected from the high degree of homology between SREBP-1
and -2, both molecules have the ability to bind to and activate
the promoters of all cholesterogenic and lipogenic enzymes
studied to date (17–19, 23). The distinct specificities of
SREBP-1 and -2 for lipogenic and cholesterogenic genes in vivo
might be reflective of nutritional activation of SREBP-1 and -2
at different levels. SREBP-2 regulates cholesterol synthetic
genes through the cleavage of its precursor form to an active
nuclear form in a process of interaction with SCAP and proteases in a sterol-dependent manner. Although it still requires
the cleavage steps, SREBP-1 seems to regulate lipogenesis
through its own mRNA level. Further studies are needed toclarify the nutritional regulation of glucose and fatty acid metabolism in lipogenic organs and to understand how insulin
and/or glucose or its metabolites could signal to the transcriptional regulation of lipogenic genes presumably through
SREBP-1. This information is clinically relevant to understanding the link between glucose and fatty acid metabolism,
because SREBP-1 seems to be involved in insulin resistance aswell as differentiation of adipocytes (53, 54).
Acknowledgments—We are grateful to Drs. Michael S. Brown, Joseph L. Goldstein, and Jay D Horton for continuous support for the
project. We thank Nobuyoshi Machiyama and Naomi Shimano for preparation of figures.
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