Proc. Natl. Acad. Sci. USA
Vol. 95, pp. 5987–5992, May 1998
Biochemistry

Regulation of sterol regulatory element binding proteins in liversof fasted and refed mice(cholesterol biosynthesisyfatty acid biosynthesisytransgenic miceytranscriptional regulation)

JAY D. HORTON*, YURIY BASHMAKOV, IICHIRO SHIMOMURA, AND HITOSHI SHIMANO
Department of Molecular Genetics, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Boulevard, Room L5-238,
Dallas, TX 75235-9046

Communicated by Michael S. Brown, University of Texas Southwestern Medical Center, Dallas, TX, March 12, 1998 (received for review
February 10, 1998)

cleaved to release the NH2-terminal segment so that it canenter the nucleus. The cleaved form of SREBPs, designatedthe nuclear form, binds to 10-bp sterol regulatory elements inthe enhancer regions of target genes to activate transcription.
The target genes involved in cholesterol metabolism include
the LDL receptor, 3-hydroxy-3-methylglutaryl-CoA (HMGCoA) synthase, HMG-CoA reductase, farnesyl-diphosphate(FPP) synthase, squalene synthase, and SREBP-2 (1, 5–8).
Genes involved in fatty acid and triglyceride synthesis that are
regulated by SREBPs include acetyl-CoA carboxylase (ACC),
fatty acid synthase (FAS), and glycerol-3-phosphate acyltransferase (9–12).
In cultured cells, the cleavage of SREBP-1 and -2 is coordinately regulated (1). When cells are overloaded with sterols,
the proteolytic cleavage is suppressed, thereby decreasing theactive nuclear form of all SREBPs with a consequent decline
in transcription of all target genes. In vivo experiments using
hamsters fed a cholesterol-rich diet also demonstrated decreased nuclear forms of SREBP-1 and -2 in liver (13). When
cultured cells are deprived of sterols by growth in lipoproteindeficient serum and an HMG-CoA reductase inhibitor (compactin), SREBP cleavage is induced and the active nuclear
forms of SREBP-1 and -2 increase (1). Cholesterol depletion
had a different effect in liver. When hamsters were fed acholesterol-depleting diet (lovastatin and colestipol), the nuclear form of SREBP-2 was markedly increased, but thenuclear form of SREBP-1 was decreased (14). A rationale forthe discrepancy between cultured cells and liver was provided
by the discovery that the predominant form of SREBP-1 inhamster, mouse, and human liver is SREBP-1c (13, 15). All
actively growing cell culture lines studied to date have predominantly the SREBP-1a and -2 isoforms (15). We havepreviously shown that SREBP-1c is a much weaker activator oftranscription than is SREBP-1a, presumably because theformer has a much shorter transcriptional activation domain(16). Therefore, when animals are fed cholesterol-depleting
diets, a weak activator of transcription (SREBP-1c) is replaced
by a much stronger activator of transcription (SREBP-2) (1),and this likely explains the increase in mRNAs for cholesterol
biosynthetic enzymes.
In studies with transgenic mice overexpressing the nuclear
form of SREBP-1c in the liver, we observed 2- to 4-foldincreases in mRNAs for genes involved in fatty acid synthesis,
including ACC, FAS, and stearoyl-CoA desaturase 1 (SCD-1).

ABSTRACTHepatic lipid synthesis is known to be regulated by food consumption. In rodents fasting decreases thesynthesis of cholesterol as well as fatty acids. Refeeding a highcarbohydrateylow fat diet enhances fatty acid synthesis by 5to 20-fold above the fed state, whereas cholesterol synthesis
returns only to the prefasted level. Sterol regulatory elementbinding proteins (SREBPs) are transcription factors that
regulate genes involved in cholesterol and fatty acid synthesis.
Here, we show that fasting markedly reduces the amounts of
SREBP-1 and -2 in mouse liver nuclei, with corresponding
decreases in the mRNAs for SREBP-activated target genes.
Refeeding a high carbohydrateylow fat diet resulted in a 4- to
5-fold increase of nuclear SREBP-1 above nonfasted levels,
whereas nuclear SREBP-2 protein returned only to the nonfasted level. The hepatic mRNAs for fatty acid biosynthetic
enzymes increased 5- to 10-fold above nonfasted levels, apattern that paralleled the changes in nuclear SREBP-1. The
hepatic mRNAs for enzymes involved in cholesterol synthesis
returned to the nonfasted level, closely following the patternof nuclear SREBP-2 regulation. Transgenic mice that overproduce nuclear SREBP-1c failed to show the normal decreasein hepatic mRNA levels for cholesterol and fatty acid synthetic
enzymes upon fasting. We conclude that SREBPs are regulated by food consumption in the mouse liver and that the
decline in nuclear SREBP-1c upon fasting may explain in partthe decrease in mRNAs encoding enzymes of the fatty acidbiosynthetic pathway.
Sterol regulatory element binding proteins (SREBPs) are
membrane-bound transcription factors of the basic-helix–
loop–helix–leucine zipper (bHLH-Zip) family that have beenshown to regulate enzymes responsible for the synthesis ofcholesterol, fatty acids, and the low density lipoprotein (LDL)
receptor (see ref. 1 for review). Three members of the SREBP
family have been identified and characterized (1). SREBP-1aand -1c are derived from a single gene through the use ofalternative transcription start sites that produce alternate
forms of exon 1 (2). The rat homologue of SREBP-1c (adipocyte determination differentiation-dependent factor 1;
ADD1) was independently cloned by Tontonoz, et al. (3) as atranscription factor that bound to E-box sequences and may be
involved in adipocyte differentiation. The third protein,
SREBP-2, is derived from a separate gene (4).
All SREBPs are synthesized as '1150-amino acid precursors bound to the endoplasmic reticulum and nuclear envelope(1). The NH2-terminal segment of '500 amino acids contains
all of the sequences necessary to activate transcription. To be
active, the membrane-bound SREBP must be proteolytically

Abbreviations: ACC, acetyl-CoA carboxylase; ADD, adipocyte determination differentiation-dependent factor; FAS, fatty acid synthase;
FPP, farnesyl diphosphate; HMG-CoA, 3-hydroxy-3-methylglutarylCoA; LDL, low density lipoprotein; PEPCK, phosphoenolpyruvatecarboxykinase; SCD-1, stearoyl-CoA desaturase 1; SREBP, sterol
regulatory element binding protein.
*To whom reprint requests should be addressed at: University of Texas
Southwestern Medical Center, 5323 Harry Hines Blvd., Room L5238, Dallas, TX 75235-9046. e-mail: horton06@utsw.swmed.edu.

The publication costs of this article were defrayed in part by page charge
payment. This article must therefore be hereby marked ‘‘advertisement’’ inaccordance with 18 U.S.C. §1734 solely to indicate this fact.
© 1998 by The National Academy of Sciences 0027-8424y98y955987-6$2.00y0
PNAS is available online at http:yywww.pnas.org.

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In contrast, there were no significant changes in any mRNAsfor genes involved in cholesterol synthesis (16).
Physiological regulation of hepatic lipogenic genes is mostdramatic in animal studies using fastingyrefeeding protocols.
Fasting markedly reduces the substrate flux through the fatty
acid synthesis pathway (see ref. 17 for review). Similar changes
occur in cholesterol synthesis (18). Refeeding a fat-free, highcarbohydrate diet induces the synthesis of fatty acids to levels
significantly higher than the normal fed state (often referred
to as ‘‘overshoot’’), whereas the synthesis of cholesterol returns
only to the prefasted level (17). In white fat, Kim et al. (19)have shown that the mRNA for SREBP-1cyADD1 is markedly
reduced by fasting and elevated upon refeeding. They also
showed that SREBP-1cyADD1 binds to the previously defined
insulin response element in the promoter of FAS, suggestingthat SREBP-1cyADD1 could be responsible for feedinginduced changes observed in genes that regulate fatty acidsynthesis in adipose tissue.
In the current studies, we employ the fastingyrefeeding
protocol in wild-type mice to determine if nuclear SREBP-1a,-1c, and -2 are regulated in liver under these metabolic
conditions. To further study the effects of SREBP-1c on
potential target genes, we take advantage of the rat phosphoenolpyruvate carboxykinase (PEPCK) promoter used tooverexpress the dominant-positive form of human SREBP-1c
in transgenic mice (16). Fasted SREBP-1c transgenic mice
maintain high levels of hepatic SREBP-1c expression, thus
allowing the examination of its effects on potential target genes
in the fasted liver.

each group were prepared for immunoblotting as described(11). The starting times for the experiments were staggered sothat all mice were sacrificed at the same time, which was 10 h
into the light cycle.
For all other fastingyrefeeding experiments, 8-wk-old SJL
male mice were divided into three treatment groups. Five mice
were maintained on Teklad 4% laboratory chow, five were
fasted for 24 h, and five were fasted for 24 h then refed the highcarbohydrateylow fat diet for 12 h prior to sacrifice as described above. Pooled liver membranes and nuclear extracts
were prepared as described above. The characteristics of themice in this study are shown in Table 1.
SREBP-1c Transgenic Mice. A transgene construct encoding amino acids 1–436 of human SREBP-1c under control of
the rat PEPCK promoter was made as previously describedand used to generate mice overexpressing the nuclear form of
SREBP-1c (16). The line used in all transgenic animal studies
was TgSREBP-1c436, and the mice were sacrificed at 12 wk ofage. Two weeks before sacrifice, six transgenic and six littermate controls were placed on a low carbohydrateyhigh protein
diet (no. 5789C-3) from Purina Mills (St. Louis) containing
71% (wtywt) casein and 4.25% (wtywt) sucrose. This diet wasused to induce maximal expression of the transgene (16). Prior
to sacrifice, three TgSREBP-1c436 and three littermate control mice were fasted for 6 h to further induce the transgene,while the remaining three TgSREBP-1c436 and three control
mice were maintained on the low carbohydrateyhigh protein
diet. The phenotypic characteristics of the mice used for thisexperiment are presented in Table 2.

MATERIALS AND METHODSRESULTSRNA Analysis. Northern blots (11) and RNase protection
assays (15) of total RNA were carried out as described in theindicated references. A 32P-labeled 25-bp oligonucleotide
probe for the 28S ribosomal RNA was used as a loading control(20).
Immunoblotting. Immunoblot analysis were carried out with
the Enhanced Chemiluminesence Western Blotting Detection
System kit (Amersham) as described (11). The primary antibodies were as previously described (11, 21).
Wild-Type Mice. All mice were housed in colony cages witha 14-h lighty10-h dark cycle. Six-week-old SJL male mice werepurchased from The Jackson Laboratory and adapted to theenvironment for 2 wk prior to study. For the fasting andrefeeding time course experiments, three male mice were
included in each treatment group. The treatment groups in thefasting time course consisted of mice fed ad libitum with
Teklad 4% (wtywt) MouseyRat Diet 7001 from Harlan TekladPremier Laboratory Diets (Madison, WI) or mice fasted for 1,
3, 6, 9, 12, or 24 h before sacrifice. For the refeeding timecourse, mice were fed ad libitum; fasted for 24 h; or fasted for
24 h then refed a high carbohydrateylow fat diet (catalog no.
TD 88122; Harlan Teklad Diets) for 1, 3, 6, 9, 12, or 24 h before
sacrifice. Pooled liver membranes and nuclear extracts from

Fig. 1 shows an experiment in which mice were fasted for 24 h
and refed a high carbohydrateylow fat diet for 12 h, after whichliver extracts were assayed by immunoblotting for the membrane-bound precursor forms and mature nuclear forms of
SREBP-1 and -2. Inasmuch as our antibody does not distinguish between SREBP-1a and -1c, we use the noncommittal
term SREBP-1. The phenotypic characteristics of the miceused in this study are shown in Table 1. Nonfasted mice showed
the precursor forms of both SREBPs in membranes and thecleaved forms in nuclear extracts (Fig. 1, lanes 1 and 4). In thisexperiment and others, we noted that the SREBP bands often
appeared as closely spaced doublets and that the migration of
upper and lower bands varied between preparations with noconsistent relation to the physiological state of the animal. For
purposes of the present study, we consider both bands together
to represent either the precursor or nuclear forms of SREBPs.
When the mice were fasted for 24 h, there was a marked
decline in the nuclear forms of both SREBPs (Fig. 1B, lanes
2 and 5). The precursor form of SREBP-1 also declined (lane2), but the precursor form of SREBP-2 did not change (lane5). When the mice were refed for 12 h, the amount of the
precursor form of SREBP-1 returned to normal, and there wasa marked increase in the nuclear form, which reached a level

Table 1.

Effects of fasting and refeeding on physiologic parameters of wild-type SJL miceParameterNonfasted

FastedRefed

No. and sex
Body weight, gLiver weight, gLiver weightybody weight
Liver cholesterol content, mgyg
Liver triglyceride content, mgyg
Total plasma cholesterol, mgydl
Total plasma triglycerides, mgydl

5?
21 6 0.30
1.2 6 0.02
0.058 6 0.001
2.2 6 0.07
9.5 6 1.3
100 6 6.4
159 6 165?
17 6 0.17
0.80 6 0.02
0.047 6 0.001
2.5 6 0.04
16 6 2.5
115 6 2.3
128 6 5.4

5?
19 6 0.30
1.5 6 0.08
0.079 6 0.003
1.5 6 0.08
14 6 1.9
78 6 4.7
369 6 34

Each value represents the mean 6 SEM. All of the animals were 8 wk old at the time of sacrifice andwere fed chow ad libitum, fasted for 24 h, or fasted for 24 h and refed a high carbohydrateylow fat dietfor 12 h.

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Table 2.

Proc. Natl. Acad. Sci. USA 95 (1998)

5989

Phenotypic comparison of wild-type and TgSREBP-1c436 mice in the fed and fasted state
Wild-typeTgSREBP-1c436

Parameter

Fed

FastedFed

FastedNo. and sex
Body weight, gLiver weight, gLiver weightybody weight
Liver cholesterol content, mgyg
Liver triglyceride content, mgyg
Total plasma cholesterol, mgydl
Total plasma triglycerides, mgydl

2 ?, 1 /28.0 6 0.5
1.47 6 0.13
0.05 6 0.004
2.21 6 0.12
6.3 6 2.5
93 6 19130 6 30

1 ?, 2 /25.4 6 2.8
1.29 6 0.14
0.05 6 0.003
2.45 6 0.04
12 6 4.9
86 6 18
93 6 7.2

2 ?, 1 /25.0 6 3.1
1.44 6 0.24
0.06 6 0.003
2.41 6 0.14
24 6 2.9
99 6 14100 6 14

2 ?, 1 /25.4 6 2.1
1.39 6 0.19
0.05 6 0.003
2.84 6 0.21
21 6 4.0
111 6 12107 6 14Each value represents the mean 6 SEM. All of the mice were 12 wk old at the time of sacrifice andwere fed the high proteinylow carbohydrate diet for 2 wk before study. Food was removed from fasted
mice 6 h before sacrifice.

that was approximately 4- to 5-fold higher than that seen in
nonfasted animals (compare lanes 3 and 1). Refeeding caused
no change in the amount of SREBP-2 precursor, and it
restored the mature nuclear SREBP-2 to the same levelmeasured in nonfasted animals (compare lanes 6 and 4).
To study the fasting-induced decrease and the refeedinginduced overshoot of nuclear SREBP-1 in more detail, we
performed a time-course experiment (Fig. 2). The nuclear
form of SREBP-1 declined perceptibly after a 3-h fast, and it
was barely detectable after 6 h. At this time point, theprecursor form also declined. When the animals had been
fasted for 24 h and then refed, the amount of nuclear SREBP-1
reached prefasting levels at 3 h and exceeded this level at 6–24
h.
Fig. 3 shows the levels of various lipid-related mRNAs inlivers of nonfasted mice, mice fasted for 24 h, and mice fasted
for 24 h and refed for 12 h. The mRNAs were assessed by
Northern blotting. Fasting reduced the levels of mRNA for
SREBP-1 and -2 by 40% and 30%, respectively. Refeeding led

FIG. 1. Immunoblot analysis of SREBP-1 (lanes 1–3) and
SREBP-2 (lanes 4–6) in membranes (A) and nuclear extracts (B) fromlivers of mice in a nonfasted state (lanes 1 and 4), fasted for 24 h (lanes
2 and 5), or fasted for 24 h and refed a high carbohydrateylow fat dietfor 12 h (lanes 3 and 6). For each group, livers from five mice shownin Table 1 were pooled, and aliquots (30 mg) of membranes and nuclear
extracts were subjected to SDSyPAGE and electrophoretically transferred to a nitrocellulose filter. Immunoblot analysis was carried out
with 5 mgyml rabbit anti-mouse SREBP-1 IgG (lanes 1–3) or SREBP-2
IgG (lanes 4–6). Bound antibodies were visualized as described in the
text. Filters were exposed to film for 60 s for SREBP-1 (lanes 1–3) and90 s for SREBP-2 (lanes 4–6). P and N denote the precursor andcleaved nuclear forms of SREBP-1 or SREBP-2, respectively.

to a 5-fold overshoot in the mRNA for SREBP-1, but not
SREBP-2. These results paralleled those seen at the nuclear
protein level. The mRNAs for some of the cholesterol-supply
genes (LDL receptor, HMG-CoA synthase, and squalene
synthase) declined by 40–70% upon fasting and returned
either to basal levels or to slightly elevated levels after refeeding. The mRNAs for the fatty acid synthetic enzymes (ACC,
FAS, and SCD-1) also declined by 60–80% after fasting, andthey rose markedly by 3.4- to 14-fold after refeeding. HMGCoA reductase mRNAs showed little change on fasting but
increased on refeeding. The mRNAs for apoproteins B and E
showed only minor changes after fasting and refeeding. The
hepatic mRNA changes in fasted and refed mice were similarin three additional independent studies (data not shown).
The SREBP-1 gene uses two different promoters to produce
two different transcripts, -1a and -1c, that differ only in the firstexon (15, 22). To determine whether the fasting–refeeding
protocol affected the amounts of one or both transcripts, we
performed an RNase protection experiment (Fig. 4). As noted
previously, in nonfasted mice the SREBP-1c transcript in theliver was considerably more abundant than the SREBP-1atranscript (15). Fasting reduced the amount of the SREBP-1ctranscript by 60%, and refeeding raised the level by 3.8-fold. In
contrast, the fasting–refeeding protocol produced only minorchanges in the amount of the SREBP-1a transcript. Under all
conditions, the SREBP-1c transcript was more abundant thanthe SREBP-1a transcript, suggesting that the -1c isoform
accounted for the bulk of SREBP-1 observed on the immunoblots.
To determine the effects of SREBP-1c expression in thefasted state, we used the previously described transgenic micethat overexpress the nuclear form of human SREBP-1c (16).

FIG. 2. Changes in SREBP-1 protein in the livers of mice at various
times after fasting (A) and refeeding a high carbohydrateylow fat dietafter 24-h fast (B). Livers from three 8-wk-old SJL male mice werepooled for each treatment group, and aliquots (30 mg) of membranes(upper gels) and nuclear extracts (lower gels) were subjected to
SDSyPAGE and electrophoretically transferred to a nitrocellulose
filter. Immunoblot analysis was carried out with 5 mgyml rabbit
anti-mouse SREBP-1 IgG. Bound antibodies were visualized as described in the text. Filters were exposed to film for 60 s. P and N denotethe precursor and nuclear cleaved forms of SREBP-1, respectively.

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FIG. 3. Amounts of various mRNAs in livers from nonfasted mice (N), mice fasted for 24 h (F), or mice fasted for 24 h and refed a highcarbohydrateylow fat diet for 12 h (R) as measured by Northern blot hybridization. The mice are described in Table 1. Total RNA was isolated
from five mice in each treatment group, pooled, and subjected to electrophoresis and blot hybridization with the indicated 32P-labeled probe. The
amount of radioactivity in each band was quantified by exposure of the filters to a Bio-Imaging Analyzer with BAS1000 MACBUS software (Fuji
Medical Systems). The fold change in each mRNA relative to that of nonfasted mice was calculated after correction for loading differences by using
the 28S ribosomal RNA as a loading control. These values are shown below each blot.

The transgene was constructed by fusing the PEPCK promoter
to a cDNA encoding amino acids 1–436 of human SREBP-1c.
Inasmuch as the PEPCK promoter is more active in the fasted
state than in the fed state, expression of nuclear SREBP-1c wasmaintained in livers of fasted TgSREBP-1c436 mice. Table 2
compares quantitative parameters of wild-type and TgSREBP-1c436 littermate mice sacrificed in the fed state orafter a 6-h fast. Fig. 5 shows immunoblots of the membranebound precursor and nuclear forms of human SREBP-1c(transgene) and endogenous mouse SREBP-1 and -2 in pooled
samples from the same livers. Expression of the human
SREBP-1c transgene protein is similar in the fasted andnonfasted states (lanes 3 and 4). The nuclear forms of endogenous SREBP-1 and -2 were again decreased in livers fromfasted wild-type mice compared with nonfasted mice (lanes 5
vs. 6 and lanes 9 vs. 10). The nuclear forms of endogenous
SREBP-1 and -2 in the SREBP-1c transgenic mice were
essentially suppressed in both the fasted and nonfasted states(lanes 7 vs. 8 and lanes 11 vs. 12).
Fig. 6 shows Northern blots of various mRNAs involved incholesterol and fatty acid metabolism in livers from wild-type

FIG. 4. RNase protection assay for SREBP-1a and SREBP-1c
mRNAs from livers of nonfasted mice (lanes 1 and 4), mice fasted for
24 h (lanes 2 and 5), or mice fasted for 24 h and refed a highcarbohydrateylow fat diet for 12 h (lanes 3 and 6). The animals aredescribed in Table 1. Pooled total RNA was isolated from five mice ineach treatment group, and a 15-mg aliquot was hybridized to 32Plabeled cRNA probes for mouse SREBP-1a, SREBP-1c, and b-actinfor 10 min at 68°C. After RNase digestion, the protected fragments
were separated by gel electrophoresis and exposed to film with anintensifying screen for 8 h at 280°C. The gels were also quantified ina PhosphorImager as described in the text. The fold change in eachmRNA relative to that of the nonfasted mice was calculated after
correction for loading differences, using b-actin mRNA as a loading
control.

and TgSREBP-1c436 mice that were either fed ad libitum(nonfasted) or fasted for 6 h. The mRNAs involved in cholesterol synthesis and regulation (SREBP-1, SREBP-2, LDL
receptor, HMG-CoA synthase, HMG-CoA reductase, FPP
synthase, and squalene synthase) were decreased by 30–50% in
the livers of fasted wild-type mice. However, in livers fromfasted TgSREBP-1c436 mice, the cholesterologenic mRNAs
did not decrease but either were maintained or were slightly
greater than the levels measured in livers from nonfasted
TgSREBP-1c436 mice. The hepatic mRNAs for the fatty acidsynthetic enzymes (ACC, FAS, and SCD-1) were decreased in
the fasted wild-type mice. The fold decrease was less dramaticthan the fold decreases shown in Fig. 3, possibly owing to theshorter fasting period. mRNAs encoding fatty acid synthetic
enzymes were 2- to 4-fold higher in nonfasted TgSREBP1c436 than in nonfasted wild-type mice. These changes are
consistent with previously published results (16). In the liversof fasted TgSREBP-1c436 mice, the fatty acid synthetic enzyme mRNAs were further increased by 1.4- to 2-fold above
nonfasted levels. The 28S ribosome RNA and mRNAs forapoprotein E were essentially unchanged in all groups. The
hepatic mRNA changes for wild-type and TgSREBP-1c436

FIG. 5. Immunoblot analysis of transgenic human SREBP-1c436(lanes 1–4), endogenous mouse SREBP-1 (lanes 5–8), and endogenous mouse SREBP-2 (lanes 9–12) in membranes (A) and nuclear
extracts (B) from livers of wild-type (WT) and TgSREBP-1c436 (Tg)mice in the nonfasted state or after fasting for 6 h as indicated. For
each group, livers from mice shown in Table 2 were pooled, andaliquots (30 mg) of membranes and nuclear extracts were subjected to
SDSyPAGE and electrophoretically transferred to a nitrocellulose
filter. Immunoblot analysis was carried out using 5 mgyml rabbit
anti-human SREBP-1 IgG (lanes 1–4), anti-mouse SREBP-1 IgG(lanes 5–8), or anti-mouse SREBP-2 IgG (lanes 9–12) as the primary
antibody. The anti-mouse SREBP-1 IgG antibody is specific for mouse
SREBP-1 and does not cross-react with the human SREBP-1 transgene product. Bound antibodies were visualized as described in the
text. Filters were exposed to film for 15 s for the truncated human
SREBP-1, 25 s for mouse SREBP-1, and 90 s for mouse SREBP-2.

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Proc. Natl. Acad. Sci. USA 95 (1998)

5991

FIG. 6. Amounts of various mRNAs in livers from wild-type (WT) and TgSREBP-1c436 (Tg) mice in the nonfasted state (N) or fasted (F) for
6 h as measured by blot hybridization. The mice used in this experiment are described in Table 2. Total RNA was isolated, pooled, and subjected
to electrophoresis and blot hybridization with the indicated 32P-labeled probe. The amount of radioactivity in each band was quantified as describedin the text. The fold change in each mRNA relative to that of wild-type nonfasted mice was calculated after correction for loading differences byusing the 28S ribosomal RNA as a loading control. These values are shown below each blot.

were similar in two additional independent studies (data notshown).

DISCUSSION
In the current experiments with SJL mice, the amount of
hepatic nuclear SREBP-1 declined after fasting, and it increased approximately 4-fold above nonfasted levels when themice were refed a high carbohydrateylow fat diet (Fig. 1). Thechanges in SREBP-1 protein were accompanied by a corresponding change in the amount of SREBP-1c mRNA, suggesting that the fasting–refeeding protocol changed the synthesis rate of the full-length SREBP-1c precursor (Figs. 3 and
4). Because the amount of SREBP-1c precursor protein in liver
membranes from refed animals was not increased comparedwith nonfasted animals, the finding of an increase in the
nuclear form of SREBP-1c after refeeding suggests thatrefeeding also increased the cleavage of the SREBP-1c precursor. However, it is also possible there is a concomitant
reduction in the rate of SREBP-1c degradation.
The nuclear form of SREBP-2 decreased after fasting, but
it returned only to basal levels after refeeding. The changes innuclear SREBP-2 coincided with changes in its mRNA level.
Despite the fall in mRNA during fasting, the amount ofprecursor in the fasted state did not fall. This result suggeststhat fasting reduces both the synthesis and the cleavage of
SREBP-2.
Considered together, the data suggest that the changes
observed in nuclear levels of SREBP-1 and -2 with fasting andrefeeding are a result of both transcriptional and posttranscriptional mechanisms. We have also observed changes in
the nuclear forms of SREBP-1 and -2 in epididymal fat pads
from fasted and refed mice identical to those observed in theliver (unpublished observations). The changes that we observed in nuclear SREBP-1 in white fat are similar to thechanges in SREBP-1cyADD1 mRNA that were recently reported by Kim et al. (19).
The fall in nuclear SREBP-1 and -2 during fasting correlatedwith reductions in the levels of mRNAs encoding cholesteroland fatty acid biosynthetic enzymes as well as the LDL
receptor. Upon refeeding the large overshoot in nuclear
SREBP-1 paralleled the overshoot in the levels of mRNA forthe lipogenic enzymes, such as ACC and FAS (Figs. 1 and 3).
These mRNAs increased to levels more than 10-fold above
nonfasted levels after refeeding. In contrast, the mRNAs forcholesterologenic enzymes (HMG-CoA synthase, HMG-CoA
reductase, and squalene synthase) and the LDL receptor rose
only to basal levels during refeeding. This correlated with thechanges in nuclear SREBP-2. These data are consistent withother accumulating data suggesting that SREBP-2 may be

more specific than SREBP-1c in regulating cholesterologenic
genes.
To test the hypothesis that the fall in SREBP-1 is responsible
for the fall in fatty acid synthesis in fasted mice, we studied thisregulation in transgenic mice that overproduce SREBP-1c ina nonsuppressible fashion. In SREBP-1c transgenic mice thelevel of mRNAs for cholesterologenic enzymes, fatty acidsynthase and the LDL receptor, failed to decline during fasting.
This result suggests that SREBP-1c, though a weaker activatorof transcription, can support basal mRNA levels of target
enzymes in both the fatty acid and cholesterol synthesis
pathways during fasting.
The conclusion that SREBP-1c is important in maintaining
the basal level of transcription of ACC, FAS, and SCD-1 is
supported by recently published data obtained from the
SREBP-1 knockout mouse (21). Mice homozygous for the
disrupted SREBP-1 allele had decreased basal hepatic mRNAlevels of ACC, FAS, and SCD-1 and had significantly reduced
rates of hepatic fatty acid synthesis as measured by [3H]water
incorporation (21).
The lipogenic response to refeeding in liver is generally
thought to reflect the reciprocal effects of insulin and glucagon
which rise and fall, respectively, after refeeding a high carbohydrate diet (23). The molecular mechanisms included in therefeeding response are complex, but reportedly involve transcriptional activation, mRNA stability, and catalytic activationof several key lipogenic enzymes (24). The transcriptional
activation of these lipogenic genes occurs through promoter
elements that are often referred to as carbohydrate or insulin
response elements. Insulin response elements have been localized in the promoters of several genes involved in fatty acidand triglyceride synthesis (see ref. 23 for review), although noclear consensus sequence has been identified. The specifictranscription factors involved in the insulin response have beendifficult to identify and characterize.
The insulin response element in the promoter of the FAS
gene has been intensively studied. Recent work has localized
this element to an E-box sequence at position 264 to 259 (19,25). This region has also been shown to be critical for thetranscriptional activation of FAS by SREBPs (10, 19). Magañaand Osborne (10) reported SREBP-1a and -2 bind to two
separate sites, each of which contains one half of the E-box at264. However, Kim et al. (19) have shown that SREBP-1cy
ADD1 can bind to E-box promoter sequences (CANNTG) as
well as sterol regulatory elements (ATCACCCCAC) associated with genes involved in cholesterol metabolism (26, 27).
They conclude that SREBP-1cyADD1 binds to the E-box at
264 in the FAS promoter and that SREBP-1cyADD1 is theinsulin response factor responsible for the changes in FAS
mRNA observed in adipose tissue from fasted and refed rats.

5992

Biochemistry: Horton et al.

On the other hand, Wang and Sul (25) have published evidence
that upstream stimulatory factors (USF)-1 and -2 bind to thesame E-box at 264 in the FAS promoter and mediate theinsulin response independent of SREBPs. The current datawith the SREBP-1c transgenic mice support the idea thatforced expression of nuclear SREBP-1c raises the mRNAs forlipogenic enzymes even during fasting. These data indicatethat SREBP-1c is sufficient to drive transcription of thesegenes, but they do not address the question of whether the
USFs would also be sufficient under the same circumstances.
More detailed promoter studies will be required to determine
the relative contributions of SREBP-1c andyor USFs to theinsulin response for each particular gene known to have such
an element.

Proc. Natl. Acad. Sci. USA 95 (1998)
8.
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We thank Drs. Michael S. Brown and Joseph L. Goldstein for theircontinued encouragement and critical reading of the manuscript. We
also thank Richard Gibson for invaluable help with the animals andScott Clark and Robin Craddock for excellent technical assistance.
This work was supported by research funds from the National Institutes of Health (HL20948), the Moss Heart Foundation, and the Perot
Family Foundation. J.D.H. is the recipient of a Postdoctoral Fellowship for Physicians from the Howard Hughes Medical Institute. I.S. isthe recipient of a postdoctoral fellowship from the Manpei Suzuki
Diabetes Foundation of Tokyo, Japan.

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