THE JOURNAL OF BIOLOGICAL CHEMISTRY© 2000 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 275, No. 40, Issue of October 6, pp. 31069 –31077, 2000
Printed in U.S.A.

Sterol Regulatory Element-binding Protein-1 Is Regulated by
Glucose at the Transcriptional Level*
Received for publication, April 19, 2000, and in revised form, June 20, 2000
Published, JBC Papers in Press, July 25, 2000, DOI 10.1074/jbc.M003335200

Alyssa H. Hasty‡§, Hitoshi Shimano¶储, Naoya Yahagi‡, Michiyo Amemiya-Kudo‡,
Stéphane Perrey‡, Tomohiro Yoshikawa‡, Jun-ichi Osuga‡, Hiroaki Okazaki‡,
Yoshiaki Tamura‡, Yoko Iizuka‡, Futoshi Shionoiri‡, Ken Ohashi‡, Kenji Harada‡,
Takanari Gotoda‡, Ryozo Nagai‡, Shun Ishibashi‡, and Nobuhiro Yamada¶From the ‡Department of Metabolic Diseasese, University of Tokyo, Tokyo 113-8655, Japan and the¶Department of Internal Medicine, Institute of Clinical Medicine, University of Tsukuba, 1-1-1 Tennodai,
Tsukuba, Ibaraki 305-8575, Japan

In vivo studies suggest that sterol regulatory elementbinding protein (SREBP)-1 plays a key role in the upregulation of lipogenic genes in the livers of animals
that have consumed excess amounts of carbohydrates.
In light of this, we sought to use an established mouse
hepatocyte cell line, H2-35, to further define the mechanism by which glucose regulates nuclear SREBP-1 levels. First, we show that these cells transcribe high levelsof SREBP-1c that are increased 4-fold upon differentiation from a prehepatocyte to a hepatocyte phenotype,making them an ideal cell culture model for the study of
SREBP-1c induction. Second, we demonstrate that thepresence of precursor and mature forms of SREBP-1
protein are positively regulated by medium glucose concentrations ranging from 5.5 to 25 mM and are also regulated by insulin, with the amount of insulin in the fetal
bovine serum being sufficient for maximal stimulationof SREBP-1 expression. Third, we show that the increase
in SREBP-1 protein is due to an increase in SREBP-1
mRNA. Reporter gene analysis of the SREBP-1c promoter demonstrated a glucose-dependent induction oftranscription. In contrast, expression of a fixed amountof the precursor form of SREBP-1c protein showed that
glucose does not influence its cleavage. Fourth, we demonstrate that the glucose induction of SREBP could not
be reproduced by fructose, xylose, or galactose nor by
glucose analogs 2-deoxy glucose and 3-O-methyl glucopyranose. These data provide strong evidence for the
induction of SREBP-1c mRNA by glucose leading to increased mature protein in the nucleus, thus providing a
potential mechanism for the up-regulation of lipogenic
genes by glucose in vivo.

Sterol regulatory element-binding proteins (SREBPs)1 are* 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 Japan Society for the Promotionof Science Postdoctoral Fellowship Program for Foreign Researchers.
储 To whom correspondence should be addressed: Dept. of Internal
Medicine, Institute of Clinical Medicine, University of Tsukuba, 1-1-1
Tennodai, Tsukuba, Ibaraki 305-8575, Japan. Fax: 81-298-53-3053;
E-mail: shimano-tky@umin.ac.jp.
1
The abbreviations used are: SREBP, sterol regulatory elementbinding protein; Bt2cAMP, dibutyryl cAMP; azaserine, O-diazoacetyl-LThis paper is available on line at http://www.jbc.org

members of the basic helix-loop-helix leucine zipper family oftranscription factors (1, 2). In contrast to other members of
this, SREBPs are synthesized as ⬃1150-amino acid precursor
proteins that remain bound to the endoplasmic reticulum andthe nuclear envelope in the presence of sufficient sterol concentrations. Upon sterol deprivation, the precursor protein undergoes a sequential two-step cleavage process to release the NH2terminal portion (3). This NH2-terminal, mature SREBP thenenters the nucleus and activates the transcription of genes
involved in cholesterol and fatty acid synthesis by binding tosterol regulatory elements or to palindromic sequences called
E-boxes within their promoter regions (4, 5). Genes involved incholesterol synthesis and regulated by SREBPs include the low
density lipoprotein receptor, 3-hydroxy-3-methylglutaryl-CoA
synthase, 3-hydroxy-3-methylglutaryl-CoA reductase, squalene synthase, and SREBP-2 (6 – 8). Fatty acid synthetic genes
such as acetyl-CoA carboxylase, fatty acid synthase (FAS) (9),stearoyl-CoA desaturase-1 and -2 (SCD) (10), ATP citrate lyase(ACL) (11), S14 (12), and glycerol-3-phosphate acyltransferase(13) are also directly activated by SREBPs.
Currently, there are three forms of SREBP that have beencharacterized. SREBP-1a and -1c are derived from a single
gene through the use of alternate promoters that give rise todifferent first exons (2), and SREBP-1a is the more common
isoform found in cultured cells and is a stronger activator oftranscription than SREBP-1c because of a longer transactivating domain (14, 15). Transgenic mouse studies have shown that
SREBP-1 plays a more active role in regulating the transcription of genes involved in fatty acid synthesis than those involved in cholesterol synthesis (15). SREBP-2 is derived from adifferent gene and is known to be actively involved in thetranscription of cholesterogenic enzymes. It has been shownthat all cultured cells analyzed to date exclusively express
SREBP-2 and the -1a isoform of SREBP-1, whereas most organs, including the liver, express predominantly SREBP-2 andthe -1c isoform of SREBP-1 (14).
It is known that both glucose and insulin are required for the
production of fatty acids via the induction of lipogenic enzymes.
The precise roles of glucose and insulin in this action are notknown; however, it has been shown that glucose must be meserine; FAS, fatty acid synthase; ATP citrate lyase, ACL; stearoyl CoA
desaturase, SCD; GFAT, glutamine:fructose-6-phosphate amidotransferase; PI, phosphoinositide; ALLN, N-acetyl-leucinal-leucinal-norleucinal calpain inhibitor; DLS, delipidated serum; DMEM, Dulbecco’s
minimally modified medium; OH, hydroxycholesterol; SCAP, SREBP
cleavage-activating protein; FBS, fetal bovine serum; HG, high glucose;LG, low glucose; BSA, bovine serum albumin; HSV, herpes simplex
virus.

31069

31070Glucose Regulation of SREBP-1 Expression

tabolized for this effect and that insulin is only permissive to
glucose action (16, 17). In support of a role for SREBP-1 in the
induction of lipogenic genes by insulin, several different groups
have shown through various lines of evidence that SREBP-1c is
up-regulated by insulin in vivo and in primary hepatocyte
cultures and that the induction of lipogenic enzymes corresponds to the increased concentration of SREBP-1c within arelatively short time period (approximately 6 h) (18 –22). With
respect to the influence of glucose on SREBP-1 expression, it
has been shown that glucose does not induce SREBP-1 expression after a 6-h incubation (22); however, a later study showedthat two lipogenic enzymes, FAS and S14, can be induced by
glucose even in the absence of insulin after a 16-h incubation(19). Therefore, it remains possible that glucose can increase
SREBP-1 expression, leading to the induction of lipogenic
genes, and that this occurs after a longer period of time than
the induction caused by addition of insulin.
Lipogenic enzymes, including FAS and acetyl-CoA carboxylase, are a group of genes involved in energy storage throughfatty acid and triglyceride synthesis (23, 24). Excess amounts ofcarbohydrates taken up by cells are converted to triglycerides
through these enzymes in lipogenic organs such as liver andadipose tissue. The lipogenic enzymes are coordinately regulated at the transcriptional level during different metabolicstates (23, 24). Recent in vivo studies demonstrated that
SREBP-1 plays a crucial role in the dietary regulation of mosthepatic lipogenic genes. These include studies of the effects of
the absence or over-expression of SREBP-1 on hepatic lipogenic
gene expression (25–27), as well as physiological changes of
SREBP-1 protein in normal mice after dietary manipulationsuch as placement on high carbohydrate diets, polyunsaturated
fatty acid-enriched diets, and fasting-refeeding regimens (28 –32). The fuel metabolism in these nutritionally challenged mice
involves a time-dependent, multi-organ, complex milieu of metabolites and hormones. To dissect the molecular mechanism
by which nutritional change regulates those metabolic genes, it
is important to establish an in vitro cultured cell system tomodel these in vivo conditions.
The mouse hepatic cell line H2-35 was originally developed
to induce liver-specific gene transcription in a temperaturesensitive manner by transformation with a temperature-sensitive strain of SV40 (33). Upon inducing cell differentiation byincreasing the incubation temperature from 33 to 39 °C, mRNAlevels for hepatic genes such as albumin, SCD-1, and hepatocyte nuclear factor 3-␣ were found to be increased by as muchas 100-fold (33–35). Interestingly, SCD levels were also foundto be regulated by both insulin and glucose in this cell line (34).
In the current study, these H2-35 cells were used as an invitro model of differentiated hepatocytes to analyze the glucose
induction of SREBP-1 expression. H2-35 cells were initiallycharacterized to compare expression levels of SREBP-1a and-1c. In contrast to other hepatic cell lines, H2-35 cells expressedsignificant amounts of SREBP-1c. It was found that both
SREBP-1a and SREBP-1c are up-regulated in a dose-dependent manner by the presence of excess glucose in the medium,with the -1c isoform being more strongly up-regulated than the-1a isoform. The induction is primarily at the level of transcription and results in an increase of the membrane and nuclear
forms of SREBP-1. This glucose-induced up-regulation of
SREBP-1 in H2-35 cells cannot be achieved by other monosaccharides or glucose analogs but can be inhibited by both azaserine, a glutamine:fructose-6-phosphate (GFAT) inhibitor,
and the PI 3-kinase inhibitor, Ly294002.
EXPERIMENTAL PROCEDURES

Materials—Calpain inhibitor (ALLN) and Ly294002 were purchasedfrom Calbiochem (La Jolla, CA). BSA, protease inhibitors (phenylmeth-ylsulfonyl fluoride, pepstatin A, leupeptin, and aprotinin), L-glucose,fructose, 2-deoxy glucose, 3-O-methyl glucopyranose, mannitol, galactose, xylose, dibutyryl cAMP (Bt2cAMP), forskolin, and azaserine werepurchased from Sigma. Fetal bovine serum (FBS), low glucose (5.5 mM,
LG), and high glucose (25 mM, HG) media were purchased from Life
Technologies, Inc. Both rabbit and mouse secondary antibodies for
immunoblotting were purchased from Amersham Pharmacia Biotech.
Delipidated serum (DLS) was prepared from fetal calf serum as described previously (36).
Preparation of Nuclear Extracts—Nuclear extracts were performed
as described (37). In brief, cells were regularly passaged in high glucose
Dulbecco’s minimally modified medium (DMEM) with dexamethasone
at 33 °C. 24 h prior to treatment, the cells were split into new dishes
and placed at 39 °C to initiate differentiation. Cells were washed withprewarmed phosphate-buffered saline and then placed in treatment
medium for 3–24 h (all experiments were performed after a 24-h incubation with the exception of the time course experiments). 2 h prior tocollection, ALLN (1.25 ␮g/ml) was added to the cells. After collection,
cells were washed in phosphate-buffered saline containing ALLN andresuspended in buffer A (10 mM HEPES, pH 7.6, 1 mM KCl, 1.5 mM
MgCl2, 1 mM EDTA, 1 mM EGTA). Pellets were passed through a 22
gauge needle 10 times and then briefly centrifuged. The pellet, containing the nuclei, was resuspended in buffer C (20 mM HEPES, pH 7.6,
25% glycerol, 0.42 M NaCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA)
rotated at 4 °C and then prepared for electrophoresis by addition of SDS
loading buffer and boiling. The supernatant was centrifuged at 55,000
rpm, and the remaining membrane-containing pellets were resuspended in a buffer containing 50 mM Tris HCl, 2 mM CaCl2, 80 mM NaCl,
1% Triton X-100. All buffers used contained a mixture of proteaseinhibitors containing phenylmethylsulfonyl fluoride, pepstatin A, leupeptin, and aprotinin. A small portion of each sample was removed andanalyzed for protein content by a BCA reaction (Pierce). Samples (50 ␮gof protein) were electrophoresed on 10% SDS-polyacrylamide gels andtransferred to Hybond ECL membranes (Amersham Pharmacia
Biotech).
Immunoblot Assays—Prior to immunoblotting, membranes were
stained with Ponceau S stain (Sigma) to verify equal loading of proteins.
Monoclonal antibody for mouse SREBP-1, 2A4, was prepared from a
hybrid cell line purchased from American Type Culture Collection.
Polyclonal antibody specific for mouse SREBP-1c and SREBP-2 wereprepared as described in Refs. 31 and 25, respectively. All other blots
were probed with the monoclonal 2A4 antibody for discrimination between the 1a and 1c isoforms unless otherwise indicated. Secondary
antibodies were horseradish peroxidase-conjugated goat anti-mouse or-rabbit, and detection of immunoreactive bands was performed using
the ECL kit by Amersham Pharmacia Biotech.
RNA Preparation and Northern Blotting—Total RNA was preparedfrom the cells with TRIzol Reagent (Life Technologies, Inc.). RNA waselectrophoresed through formalin-denatured gels and transferred to
Hybond-N membranes (Amersham Pharmacia Biotech). The cDNA
probes for SREBP-1, FAS, SCD, ACL, and 36B4 were prepared as
described previously (25, 27). Probes were labeled with [␣-32P]dCTP
using Megaprime DNA Labeling System kit (Amersham Pharmacia
Biotech). Membranes were hybridized with the radiolabeled probes in
Rapid-hyb Buffer (Amersham Pharmacia Biotech) and washed in 0.1⫻
SSC, 0.1% SDS at 65 °C. Membranes were exposed to Kodak XAR-5 film
for 2–5 h at ⫺80 °C.
RNase Protection Assays—RNA was isolated from H2-35 and HepG2
cells cultured in either low glucose or high glucose DMEM using the
TRIzol reagent by Life Technologies, Inc. RNase protection assays were
performed with the same probes and conditions as described previously(14).
Establishment of Stable Transfectancts—A reporter gene containing
mouse SREBP-1c promoter (a 2.6-kilobase BamHI-AvrII fragment fromthe mouse SREBP-1 genomic clone) (26) was prepared by insertion into
the pGL2 luciferase construct (Promega). These constructs (purified
with Qiagen maxi DNA preparation kit) were individually co-transfected with a neomycin resistance-containing vector (pMAMneo, from
CLONTECH) into H2-35 cells using Superfect reagent (Qiagen). Empty
pGL2 vector was also co-transfected with pMAMneo to be used ascontrols for the luciferase assays. TK-HSV-SREBP-1c, an expression
vector of human SREBP-1c precursor with an HSV-Tag at the amino
terminus under the control of the thymidine kinase promoter, wasconstructed as described previously (37). Transfected cell clones wereselected with 500 ␮g/ml G418 (Life Technologies, Inc.).
Luciferase Assay—Luciferase assay reagent (Promega) was added tolysed cell samples and luciferase activity was detected using the Micro-

Glucose Regulation of SREBP-1 Expression

31071

FIG. 1. Western blot for SREBP-1a and -1c Isoforms in H2-35
Cells. H2-35 cells were cultured for 24 h in media containing 25 mM
glucose. Nuclear extracts were prepared and subject to 10% SDS-PAGE.
After electrophoresis, proteins were transferred to nitrocellulose membranes and blotted with either a polyclonal antibody specific for
SREBP-1c (left-hand panel) or a monoclonal antibody that recognizes
SREBP-1c, as well as the -1a isoform (right-hand panel). *, nonspecificband that binds to both antibodies.
plate Luminometer LB 96V by EG&G Berthold (Germany) and analyzed with Winglow software.
RESULTS

Characterization of the H2-35 Cell Line—All cultured cells
analyzed to date express almost exclusively the 1a isoform of
SREBP-1, whereas most organs, including liver, predominantly express the 1c isoform (14). In the current studies,H2-35 cells, a well differentiated mouse hepatocyte cell line(33), were evaluated to determine levels of SREBP-1c expression by RNase protection assays. Interestingly, when H2-35
cells were cultured at the differentiation temperature of 39 °C
in high glucose (25 mM) media, they expressed high levels of the
1c isoform as well as the 1a isoform, whereas barely detectablelevels of the 1c isoform were found in HepG2 cells (data notshown). In addition, the use of a polyclonal antibody that specifically recognizes SREBP-1c and a monoclonal antibody forboth the 1c and 1a isoforms demonstrated that the 1c isoform ishighly expressed at the protein level as well (Fig. 1). This isthen the first report of a liver cell line in which SREBP-1c isexpressed at levels comparable with SREBP-1a and is therefore an appropriate cell culture model for the study of
SREBP-1c regulation in the liver.
Sterol Regulation of SREBP-1 and SREBP-2 Mature
Forms—To determine the pattern of sterol regulation of
SREBP-1 and -2 in H2-35 cells, both H2-35 and 293 cells(nonhepatocytes) were depleted of cholesterol and/or fatty acids
and assayed for SREBP levels. When cells were provided withcholesterol and 25-hydroxycholesterol (OH) (20 and 2 ␮g/ml,
respectively), the mature form of SREBP-2 was nearly abolished in both H2-35 and 293 cells. In comparison, SREBP-1 wasonly moderately reduced (Fig. 2, compare lanes 1 and 2). Interestingly, SREBP-1 seemed to be regulated by sterols to a lesser
extent in the H2-35 liver cell line than it was in the 293 cells.
When cells were depleted of cholesterol and fatty acids bytreatment with DLS, they exhibited an increase in SREBP-1
and -2 expression (Fig. 2, lane 3). The DLS induced SREBP-2
cleavage to the nucleus was completely abolished by the addition of cholesterol and 25-OH, whereas SREBP-1 cleavage wasonly moderately reduced (Fig. 2, lane 4). In contrast, whenoleate was added to the DLS containing media, mature
SREBP-1 levels were decreased, whereas SREBP-2 levels remained relatively unchanged (lane 5). These data suggest that
although mature SREBP-2 can be completely controlled bycholesterol at the level of cleavage, SREBP-1 is only partially
responsive to sterol regulation and is also controlled by fatty
acid levels.
Glucose Regulation of Precursor and Mature Forms of
SREBP-1—Because H2-35 cells are known to differentiate at
39 °C, initial studies on the glucose regulation of SREBP-1
focused on measuring the levels of precursor and mature
SREBP proteins from nuclear extracts of H2-35 cells cultured
in different concentrations of glucose for 24 h at either 33 or
39 °C. It was noted that when cells were cultured for 24 h in

FIG. 2. Sterol regulation of SREBP-1 and SREBP-2 matureform in 293 and H2-35 cell lines. Following a 24-h incubation in theindicated culture media conditions, nuclear extracts were preparedfrom H2-35 and 293 cells and used for Western blot analysis as described under “Experimental Procedures.” A, representative blots for
SREBP-1 and SREBP-2 of nuclear extracts from 293 cells. B, blots for
SREBP-1a and -1c as well as SREBP-2 of nuclear extracts from H2-35
cells. Cells in all lanes were cultured in 25 mM glucose DMEM with thefollowing additions: lane 1, 10% FCS, cholesterol (20 ␮g/ml), and 25-OH(2 ␮g/ml); lane 2, 10% FCS; lane 3, 10% DLS; lane 4, 10% DLS withcholesterol and 25-OH; lane 5, 10% DLS plus oleate (50 ␮M); lane 6, 10%
DLS, cholesterol, 25-OH, and oleate. The asterisks indicate an unrelated protein that cross-reacts with the respective antibodies.

2.75 mM glucose media, nuclear SREBP levels were almost
undetectable, regardless of the differentiation state of the cells(Fig. 3A). Glucose levels in spent media from these cells were
too low to detect. However, when media glucose concentrations
were higher (5.5–25 mM), there was a notable difference in thepattern of expression of SREBP-1 as the cells converted froman undifferentiated to a differentiated phenotype. Upon differentiation at 39 °C, the levels of the precursor and mature forms
of SREBP-1 were increased by glucose in a dose-dependent
manner (Fig. 3A); however, when H2-35 cells were maintained
at 33 °C, they expressed only moderate levels of the precursor
and mature forms of SREBP-1, and these levels were not significantly influenced by media glucose concentrations. These
data demonstrate that glucose responsiveness of SREBP-1 requires H2-35 cells to be in a differentiated state. The additionof mannitol to low glucose DMEM to bring the final molar
concentration to 25 mM did not have any effect on SREBP-1
expression levels (data not shown), indicating that the glucose
effect is not related to osmolarity. Because the antibody used
for these studies is able to distinguish between the 1a and 1c
isoforms, it is also of importance to note the difference in
up-regulation of these two isoforms by glucose. Densitometricanalysis of band intensity revealed that, although SREBP-1a(the upper band) was increased 1.9- and 2.3-fold in media
containing 12.5 and 25 mM glucose compared with 5.5 mM,
respectively, SREBP-1c was increased by 2.8- and 3.6-fold.
These data indicate the possibility that SREBP-1c is moresensitive to glucose regulation than is SREBP-1a.
To see the effects of insulin on SREBP-1 expression, H2-35
cells were cultured in serum-free media containing BSA. Importantly, SREBP-1 was induced by HG conditions in media
lacking insulin (Fig. 3B, lanes 1 and 3). The addition of 1 ng/mlof insulin to HG media increased SREBP-1 expression slightly

31072

Glucose Regulation of SREBP-1 Expression

FIG. 3. Glucose stimulation of SREBP-1 precursor and matureforms at 39 °C. H2-35 cells were grown in media containing increasing
concentrations of glucose for 24 h at either 33 °C or the differentiation
temperature of 39 °C. Nuclear extracts and membranes were prepared
and assayed for the presence of precursor and mature forms (top andbottom panels of A, respectively) of SREBP-1 by immunoblotting, according to “Experimental Procedures.” Lanes 1– 4, extracts from cells
cultured in increasing amounts of glucose (as indicated above the lanes)at 39 °C; lanes 5– 8, extracts from cells cultured at 33 °C. B, cells weregrown in LG and HG media containing either FBS or BSA, as indicated.
The effects of the addition of insulin can be noted when as little as 1
ng/ml was added to serum-free media. C, mature SREBP-1 from cells
grown in HG FBS-containing media and treated with increasing levelsof glucose and insulin (100 –500 ng/ml).

(Fig. 3B, lanes 5 and 6); however, additional increases in insulin concentration did not further induce SREBP-1 expression(data not shown). Cells cultured in media containing FBS(which contains low levels of insulin) showed a substantial
induction of SREBP-1 expression compared with those cultured
in BSA in both LG and HG conditions (Fig. 3B). In contrast to
the serum-free media results, when insulin was added to HG
media containing FBS, even at very high doses (100 –500 ng/ml) there was no change in expression levels of SREBP-1 (Fig.
3C). We conclude then that insulin mildly stimulates SREBP
expression in differentiated H2-35 cells when added to serumfree media; however, high glucose media with FBS (and theinsulin it contains) provides maximal stimulation for SREBP-1
expression. It is also important to note that a change from LG
to HG, both in the complete absence of insulin (serum-free
media), as well as in the presence of saturating concentrationsof insulin (FBS media), resulted in the induction of SREBP-1
nuclear protein.
Time Course of SREBP-1 Regulation—To evaluate the timing of the effects of glucose on precursor and mature forms of
SREBP-1, nuclear extracts from mouse liver H2-35 cells cultured in various concentrations of glucose were analyzed at 3,
6, 12, and 24 h after treatment (Fig. 4). It was found that
glucose had an appreciable dose-dependent effect on both precursor and nuclear forms of SREBP starting at 12 h, with nodetectable effects at earlier times. Even after 48 h, the effects of
HG on SREBP-1 expression could still be detected (data not

FIG. 4. Time course of glucose stimulation of SREBP-1. H2-35
cells were incubated for 3, 6, 12, or 24 h under the following conditions:lane 1, 5.5 mM glucose; lane 2, 25 mM glucose; and lane 3, 25 mM glucose⫹ 100 ng/ml insulin. Cells were cultured in high glucose media for 24 h
at 39 °C prior to beginning of time course study. Nuclear extracts andmembranes were prepared and immunoblotted for SREBP-1 as described under “Experimental Procedures.” The left-hand blots containthe precursor protein, and the right-hand blots contain the mature
protein.

shown). The addition of insulin to the FBS-containing media
did not result in any detectable changes in mature or precursor
SREBP levels at any time point (Fig. 4).
SREBP mRNA Levels after Treatment by Glucose—Previous
studies have focused primarily on insulin-induced transcription of SREBP-1 (18 –21). The data presented in Fig. 3 clearly
demonstrate that both the membrane-bound precursor form
and the nuclear mature form of SREBP-1 are induced by glucose after 12 h; however, further studies were performed todetermine whether the effect of glucose on SREBP-1 was at thetranscriptional level. Northern blot analysis of total RNA from
cells treated with increasing levels of glucose for either 4 or
24 h revealed that although there were no changes in SREBP-1
after a 4-h incubation (data not shown), there was an appreciable glucose dose-dependent increase in SREBP-1 mRNA
when cells were incubated for 24 h (Fig. 5A). The mRNAs forlipogenic enzymes: FAS, ACL, and SCD were also regulated ina similar manner as SREBP-1, with the exception that FAS
message was slightly increased by the addition of insulin. A
probe for ribosomal protein 36B4 mRNA was used as a control
for equal loading. In support of this data, RNase protection
assays also showed that both the 1a and 1c isoforms were
up-regulated when the glucose concentration in the media wasincreased from 5.5 to 25 mM (Fig. 5B), and in line with theprotein data, the 1c isoform RNA was increased 4-fold, whereas
the quantity of SREBP-1a RNA was only doubled.
Luciferase Assays—To further analyze the glucose effect on
SREBP-1c transcription, a luciferase reporter gene was linked
to the mouse SREBP-1c promoter and stably transfected with aneomycin resistance vector into H2-35 cells. Control cells weretransfected with an empty luciferase vector and the neomycin
vector. Glucose increased luciferase activity in a dose-dependent manner (Fig. 6), up to 3.2-fold from 5.5 to 25 mM glucose, alevel similar to the SREBP Northern blot data (Fig. 5) as wellas the protein data (Fig. 3). Together, these studies indicatethat the majority of the glucose-regulatable SREBP expressionis due to an increase in transcription.
Alternate Energy Sources—The time course of glucose-induc-

Glucose Regulation of SREBP-1 Expression

31073

FIG. 6. Glucose effects on SREBP-1c promoter reporter construct. Stable cells lines transfected with a luciferase vector containing
the SREBP-1c promoter or control empty vector were established as
described under “Experimental Procedures.” These stable cell lines
were then grown in increasing concentrations of glucose for 24 h. Cells
were lysed, and the lysate was analyzed for luciferin emission. Control
bars (solid bars) are the average of three samples. The SREBP-1c
luciferase promoter cells (hatched bars) are the averages of five samples
from two separate experiments. Error bars represent standard error. *,p ⬍ 0.05, 3.2-fold increase over 5.5 mM glucose-treated cells. **, p ⬍0.01, 2.4 fold increase over 5.5 mM glucose-treated cells.

FIG. 5. SREBP-1 and FAS mRNA levels in H2-35 cells. A, Northern blot analysis: H2-35 cells were cultured in media containing increasing amounts of glucose. After 24 h, total RNA was extracted from
cells according “Experimental Procedures.” Aliquots of RNA (15 ␮g)were electrophoresed through formalin-denatured agarose gels andthen transferred to nylon membranes. Membranes were probed with
32
P-labeled cDNA for SREBP-1, FAS, ACL, SYN, SCD, and 36B4 (asindicated). B, RNase protection assay. RNase protection assays on thesame RNA samples were performed to distinguish between the 1a and1c isoforms (as indicated).

tion of SREBP-1 indicates that it maybe necessary for glucose
to be metabolized for its effects. Further analysis of glucosestimulated expression of SREBP focused on this issue. Two
glucose analogs, 3-O-methylglucose, which cannot be metabolized, and 2-deoxyglucose, which can be metabolized only as far
as glucose-6-phosphate, as well as L-glucose, were unable toincrease nuclear SREBP-1 levels (data not shown). Other natural monosaccharides were also used in place of glucose todetermine whether they could have a similar effect on SREBP
expression. Fructose, xylose, and galactose were able to upregulate SREBP-1 only slightly compared with glucose; however, when fructose was used at a 10-fold higher concentration
than glucose, comparable levels of nuclear SREBP-1 protein
were detected (Fig. 7). These data demonstrate that glucose
must be metabolized further than glucose-6-phsophate for
SREBP-1 levels to be increased.
Metabolic Pathways Potentially Involved in SREBP-1 Regulation—To determine the role the hexosamine pathway plays
in SREBP expression, H2-35 cells were treated with azaserine,
a drug that irreversibly inhibits GFAT, the first enzyme in thispathway, which transfers the amide group from glutamine tofructose-6-phosphate to form glucosamine-6-phosphate. Interestingly, azaserine decreased the glucose-induced up-regulation of both precursor and mature forms of SREBP-1 in adose-dependent manner (Fig. 8), but its effects could be detected only after 12 h of incubation (data not shown).

FIG. 7. SREBP nuclear protein in cells treated with alternatemonosaccharide sources. H2-35 cells were cultured for 24 h in glucose-free DMEM containing 5.5, 12.5, or 25 mM glucose, or 5.5 and 25
mM of fructose, xylose, and galactose, as indicated. Nuclear extracts
were prepared and SDS-PAGE was performed as described under “Experimental Procedures.” The bottom panel contains the addition of asample that was culture in 250 mM fructose (lane 3).

It has recently been shown that different signal transduction
pathways are involved in the regulation of expression of metabolic genes. Of particular interest is PI 3-kinase, which has
been shown to be a key switch in the insulin signaling of
metabolic enzymes such as FAS, glycogen synthase, ACL, glucose-6-phosphatase, and phosphoenolpyruvate carboxykinase(reviewed in Ref. 38). To determine the contribution of the PI
3-kinase cascade to SREBP expression, H2-35 cells weretreated with Ly294002, a drug that potently inhibits PI 3-kinase. Ly294002 was demonstrated to have a strong dose-dependent inhibitory effect on SREBP-1 expression at a concentration as low as 10 ␮M, (data not shown) implicating a

31074

Glucose Regulation of SREBP-1 Expression

FIG. 8. Effects of GFAT inhibition on SREBP expression. H2-35
cells were cultured for 24 h in low glucose or high glucose DMEM (lanes
1 and 2, respectively). Lanes 3–5, nuclear protein from cells that were
cultured in high glucose media with the addition of 1, 5, or 25 ␮Mazaserine, respectively. The top panel is of the precursor form, and thebottom panel is the mature form of SREBP-1. Nuclear extracts andmembranes were prepared, electrophoresed, and blotted as describedunder “Experimental Procedures.”potential role for the PI 3-kinase cascade in the expression of
SREBP. Although the glucose induction of SREBP expression
could not be detected until after 12 h of incubation, Ly294002
caused a considerable reduction in basal levels of SREBP precursor and nuclear forms as early as 3 h and was maintained
for up to 24 h after addition to media (Fig. 9A).
Observations from in vivo experiments show that fasting, acondition that promotes gluconeogenesis through increasingplasma glucagon concentrations, completely suppresses
SREBP-1 expression (32). Because glucagon exerts its effects
primarily through the activation of cAMP, cAMP is likely to be
involved in the fasting suppression of SREBP-1. In light of this,the effects of cAMP activation on SREBP-1 expression were
examined. Forskolin, which activates adenylate cyclase leading
to increased cAMP levels, and Bt2cAMP, a cAMP analog, wereadded to H2-35 cells in a time course experiment. The induction of cAMP by the addition of 100 ␮M of each drug led to adecrease in SREBP-1 expression at 3 h after incubation; however, in contrast to the Ly294002 effects, SREBP-1 reverted tonearly base-line levels by 24 h after incubation with thesedrugs (Fig. 9B).
Glucose and Drug Effects on Cells Stably Transfected with
HSV-tagged TK-SREBP-1c—To estimate the effect of glucose
concentration on cleavage of SREBP-1c precursor into the nucleus, a construct containing an HSV-tagged SREBP-1c under
the thymidine kinase promoter (37) was stably transfected into
H2-35 cells. Expression of the precursor form of the transfected
SREBP-1c detected by epitope (HSV) tag antibody in these cells
is not regulated, allowing for the analysis of cleavage of SREBP
without the added component of transcriptional variations. To
estimate the sterol regulated cleavage of the tagged SREBP-1c,
the cells were first cultured for 24 h with cholesterol and25-OH, pravastatin, or oleate. It was demonstrated that
SREBP-1c was cleaved to the mature form, but the addition ofsterols only partially affected the cleavage of SREBP-1 in asimilar manner to the endogenous protein (Fig. 10A). The
membrane-bound SREBP-1c remained unchanged under these
conditions (data not shown). These cells were then cultured for
24 h in media with increasing levels of glucose as well as withthe addition of azaserine or Ly294002. There was very little
variation in the level of precursor protein expressed between
all samples (Fig. 10B). In comparison with previous experiments on endogenous SREBP-1 levels, when the amount of the
precursor protein was stable, there was no change in amountsof the mature form of the protein upon increasing glucose
concentrations. Therefore, there was little, if any, effect of
glucose concentration on the cleavage of SREBP-1 precursor. In

FIG. 9. Effect of Ly294002, forskolin, and Bt2cAMP on SREBP-1
expression. Nuclear extracts were prepared from cells cultured for 3,
6, or 24 h in 25 mM glucose DMEM with the addition of 15 ␮M Ly294002,
100 ␮M forskolin, or 100 ␮M Bt2cAMP, as indicated. Nuclear extracts
were prepared and SREBP-1 Western blotting performed as describedunder “Experimental Procedures.”contrast, both azaserine and Ly294002 caused a reduction in
the level of mature SREBP-1 without changing expression of
the precursor form. These data indicate the potential action ofazaserine and Ly294002 at the cleavage level for the regulationof nuclear concentrations of mature SREBP-1c.
DISCUSSION

In this study, we demonstrate that both precursor and nuclear forms of SREBP-1 are up-regulated by glucose in a dosedependent fashion. Based upon Northern blot analysis, RNase
protection assay, and reporter gene assays, this up-regulation
appears to be almost entirely due to an increase in SREBP-1
transcription. Time course studies demonstrated that thechange in SREBP-1 concentrations occurs only after 12 h ofincubation in the corresponding media, suggesting that theinduction might be mediated through some metabolite of glucose and that it takes some time to have an effect.
SREBP-1 Is Not Tightly Regulated by Sterols in H2-35
Cells—It has been reported that SREBP-1a and -2 are tightly
regulated by intracellular sterol concentrations at the cleavage
level in cultured cells (2), as we observed in the 293 cells in thecurrent study. Addition of cholesterol and 25-OH cholesterol, amore strongly suppressive oxysterol, abolished the cleavage of
SREBP-2 almost completely, resulting in the disappearance ofits mature form. These data correspond with previous data
indicating that sterol regulation is the primary mechanismthat dominates cleavage of SREBP-2 precursor, which, in complex with the putative cholesterol sensor, SREBP cleavageactivating protein (SCAP), translocates to the Golgi in theabsence of sterols, where SREBP is cleaved by site-1 protease(1, 3). In contrast, endogenous and tagged SREBP-1 were only
partially suppressed by sterols in the H2-35 liver cell line. This
relatively low sensitivity to cholesterol in a hepatic cell line
correlates with the observations from in vivo studies in mouse
liver. In livers of cholesterol fed mice, the amount of nuclear
SREBP-1 protein is decreased but is not completely abolishedin the way that SREBP-2 is.2 Together, these data indicate that
there may be a difference in sensitivity to sterol suppression
between cultured cells and tissues and between SREBP-1 and
SREBP-2, possibly through a variance in interaction with
SCAP, suggesting that SREBP-1 has the capability to enter the
nucleus even in the presence of cholesterol. More resistance ofhepatic SREBP-1c to sterol suppression is important for its
regulation of hepatic lipogenesis. The ability of hepatocytes toremove cholesterol via the bile acid synthetic pathway may
2

H. Shimano and N. Yahagi, unpublished data.

Glucose Regulation of SREBP-1 Expression

31075

FIG. 10. Stable cell line expressing
SREBP-1c linked to the TK promoter
and tagged with HSV. The stable cellline TK-SREBP-1c-HSV was prepared as
described under “Experimental Procedures.” Cells were cultured for 24 h under
the indicated conditions, nuclear extracts
and membranes were prepared, andWestern blotting was performed in a similar manner to all other experiments withthe exception that an antibody to the HSV
tag was used for recognition of the recombinant protein. A, cells in all lanes were
cultured in HG DMEM with the following
additions: lane 1, 10% FCS, cholesterol(20 ␮g/ml), and 25-OH (2 ␮g/ml); lane 2,
5% FCS; lane 3, 5% DLS; lane 4, 5% DLS
with 10 ␮M pravastatin; lane 5, 5% DLS
with cholesterol and 25-OH; lane 6, 5%
DLS plus oleate (50 ␮M); lane 7, 5% DLS,
cholesterol, 25-OH, and oleate. B, the top
blot contains the membranes, and the bottom blot contains the nuclear extracts.
Lanes 1–3, extracts from cells grown inincreasing levels of glucose. Lanes 4 and5, azaserine was added to the cells at 5
and 25 ␮M. Lanes 6 and 7, Ly294002 wasadded to the cells at 10 and 15 ␮M.

partially explain why they are relatively more resistant tocholesterol suppression of SREBPs than other nonhepatic cells.
To date, any condition that regulates SREBP-1c expression has
been found to be transcriptional (12, 19, 22, 27–29, 31, 32,
39 – 41). However, neither SREBP-1c nor SREBP-2 can be active in regulating the transcription of target genes, lipogenic
and cholesterogenic, respectively, until their mature forms arecleaved into the nucleus. Sterol-resistant cleavage of SREBP-1cin the liver might be useful to ensure that SREBP-1c canregulate its target lipogenic genes by changing the amount of
SREBP-1c precursor protein. The mechanism for the differences in sterol regulation between SREBP-2 and -1c is currently unknown; however, the relative low sequence homology
between their cholesterol regulatory carboxyl-terminals might
be involved (32).
Glucose Dose Dependence—The current studies clearly showthat glucose is a main source and key signal for SREBP-1c
induction. We compared SREBP-1 expression in cells cultured
in either low (5.5 mM) or high (25 mM) glucose-containing media. These glucose concentrations are commonly used to estimate the effect of glucose in cell culture studies and also reflect
physiological and diabetic glucose levels in human plasma.
H2-35 cells cultured in low glucose media consistently showed
low expression of SREBP-1 mRNA as well as precursor andmature forms of the protein. SREBP-1 expression from cells
cultured at a range of glucose concentrations from 5.5 to 25 mM
demonstrated that the glucose effect is dose-dependent andthat there is no threshold level for the induction of SREBP-1
expression. According to the original report on the establishment of H2-35 cells, they can be maintained in low glucose
DMEM without any impairment of growth (33). In addition, it
has recently been reported that there was a partial stimulationof the S14 gene in low glucose conditions, and the authors

concluded that this glucose concentration is higher than thebasal level for glucose signaling (42). Thus, the reduced level of
SREBP-1 at low glucose concentrations is not due to a generalpoor condition of the cells, and indeed, the cells appeared
healthy at the time of collection. It is likely that the glucose
dose dependence is representative of in vivo hepatic SREBP-1c
expression during fasting and refeeding. When cells were cultured in media initially containing only 2.75 mM glucose, the24 h conditioned media was depleted of glucose (data notshown), and nuclear SREBP-1 was barely detectable, whichmight be comparable with fasting liver conditions. In contrast,
when cells were cultured in high glucose concentrations,
SREBP-1c levels increased by over 3-fold, which might beequivalent to a refed state or a situation of high carbohydrate
feeding. In agreement with this, it has been shown that a highsucrose diet increases SREBP-1 mature form in mouse liver(31), and the current studies support the likelihood that theexcess glucose in the diet is responsible for this effect.
The Effects of Insulin versus Glucose—The current study
clearly demonstrates that insulin mildly stimulates SREBP-1
expression in H2-35 cells, as would be expected, that the FBS
contains enough insulin for maximal SREBP-1 expression inhigh glucose media, and, finally, that supplementation of FBScontaining media with additional insulin cannot boost
SREBP-1 expression levels any higher. Importantly, they also
show that glucose regulates SREBP-1 expression both in thepresence and in the absence of insulin, making this cell line
useful for differentiating between glucose and insulin effects on
SREBP-1 expression. These data also correspond with a previous study in H2-35 cells, in which it was shown that SCD, atarget gene of SREBP-1, can be induced by both glucose andinsulin (in serum-free media) (34), an effect that may well be
due to increased SREBP-1 expression.

31076

Glucose Regulation of SREBP-1 Expression

The glucose-dependent induction of SREBP-1 suggests that
glucose can be taken up by the cells in a dose-dependent manner, which might be through an insulin-independent mechanism. As other studies have alluded to, it is likely that although
glucose is necessary for SREBP-1c and therefore lipogenic geneinduction, insulin is merely permissive for this induction, probably through the induction of glucokinase gene expression (asdemonstrated in Ref. 19) and/or the translocation of glucose
transporters. It is also possible that insulin might have someeffect on SREBP-1 cleavage, possibly through another insulin
signaling pathway such as activation of MAP kinase (43).
Transcription versus Cleavage—Previous reports on the insulin induction of SREBP-1, as well as our current findings on
its induction by glucose, have all demonstrated the induction to
be at the level of transcription (19, 21, 22, 41). In our study wealso show that SREBP-1c nuclear protein is induced by glucose.
These data indicate that there must also be a mechanism inplace for a corresponding increase in the cleavage of SREBP-1
precursor protein; otherwise the increase in transcription of
SREBP would result only in an up-regulation of the precursor,
membrane-bound form of the protein. To date, the sterol-sensing SCAP and site-1 protease is the only known system forcleavage of SREBPs in physiological metabolism (44), with the
exception of caspase 3 cleavage during apoptosis (45). However,our data indicate that SREBP-1 is only under partial sterol
regulation in differentiated liver cells. This observation points
to the probability of some nonsterol-dependent, residual cleavage activity of site-1 protease for SCAP/SREBP-1c in the liver,
ensuring the regulation of lipogenic genes even in the presenceof excess cholesterol. This residual activity is likely related toprecursor levels of SREBP-1c, because levels of the nuclear
mature form of the protein were reflective of precursor protein
concentrations in both endogenous SREBP-1 studies and in thetagged SREBP-1c studies. A consequence of this would be apartial overlap of sterol and glucose regulation on lipogenesis,
which is reasonable as ACL provides acetyl-CoA as a substrate
for both lipogenesis and cholesterogenesis. The mechanism
involved in the glucose-regulated cleavage of SREBP-1 remains
unclear; however, it is clear that it is not sterol-regulated.
Future studies in this area will be necessary to distinguishbetween the classically understood mechanism of sterol-regulated cleavage of SREBP and this new insulin/glucose regulated or residual cleavage. Another possible explanation for the
nonsterol-dependent cleavage is the presence of an unknown
alternate cleavage system for SREBP-1c, and although the idea
is intriguing, there is no evidence for this.
Contribution of Glucose Analogs and Alternate Monosaccharides Sources—H2-35 cells take 12–24 h to achieve a maximal
effect of high glucose on SREBP-1 induction. This suggests thatthe glucose effect is not mediated through a direct signal transduction pathway. It is likely that it is necessary for glucose to
be metabolized and that it is the accumulation of one of thesemetabolites that is responsible for the induction of SREBP-1.
The experiments with glucose analogs such as 2-deoxy glucose
and 3-O-methyglucopyranose indicate that it is some metabolite following glucose-6-phosphate. The strong effect of azaserine in reducing the glucose-induction of SREBP-1 suggests thepossibility that some metabolite in the hexosamine pathway
might be involved in maintaining cleavage of SREBP-1 precursor. Further study is needed to analyze all of the pathways
from glucose-6-phosphate (the glycolytic, glycogen synthetic,
pentose, and hexosamine pathways) to determine what thismetabolite might be. Our data do not support the notion that
other monosaccharides are involved in the induction of
SREBP-1; however, these data need to be interpreted withcaution, because H2-35 cells have not been analyzed for theirability to utilize these other sugars. In fact, a previous study
demonstrated that galactose cannot be used as a primary carbon source by H2-35 cells because of a deficiency in galactose1-phosphate uridyltransferase (46).
Metabolic Pathways Potentially Involved in SREBP-1 Regulation—Recent studies have shown that in certain circumstances, glucosamine, which enters the hexosamine pathway
downstream from fructose-6 phosphate, has a greater effectthan glucose on processes such as the production of transforming growth factor ␤1 (47), osteopontin (48), and leptin (49). In
each of these studies the GFAT inhibitor, azaserine, decreased
the glucose induced production of these proteins. The results ofthese experiments lead to some interesting possibilities concerning the contribution of the hexosamine pathway to theeffects of glucose on SREBP-1 induction. It was been estimatedthat approximately 2– 4% of glucose that is taken up by cells isshunted to the hexosamine pathway by GFAT (50). In addition
to glucose, other nutrients such as free fatty acids and uridine
can activate the hexosamine pathway. Therefore, it has been
proposed that this pathway can serve as a general nutrient
sensing mechanism by cells (Refs. 49 and 51). In light of this, it
is logical that cells respond to azaserine-induced blocking of thehexosamine pathway by reducing SREBP-1c, the signal for
lipogenesis.
The PI 3-kinase pathway has been shown to be involved in
the induction of many different lipogenic enzymes, includingthat of FAS and acetyl-CoA carboxylase. In addition, PI 3-kinase appears to play a role in insulin-stimulated glucose uptake (52). We demonstrate here a clear effect of the PI 3-kinase
inhibitor, Ly294002, on mature SREBP-1 induction. Because
SREBP-1 has been shown to be upstream from genes such as
FAS (53), the effect of PI 3-kinase inhibition of FAS may be asa secondary effect of its effect on SREBP-1. Of interest is thefact that Ly294002 reduced levels of RNA and precursor forms
of SREBP-1 (seen upon collection and assay of membranes andRNA from H2-35 cells; data not shown) as well as the cleavage ofthe precursor form (Fig. 10) in as little as 3 h. These data provide
evidence that PI 3-kinase signal transduction may be involved in
the activation of SREBP-1c and that blocking of this signal caninterfere with the induction signals provided by glucose.
In contrast, the inhibitory effect of cAMP generation by addition of forskolin and Bt2cAMP on SREBP-1 expression wastransient. This is most likely due to a more rapid degradationof these drugs than Ly294002; however, it may have somebiological significance as to the respective roles of PI 3-kinaseand cAMP in stimulating lipogenesis. More studies will beneeded to determine whether the effects of these drugs ininhibiting SREBP-1 expression are due to their effects on a
glucose metabolite or on insulin signaling pathways.
The current studies demonstrate that well differentiatedH2-35 cells express high levels of SREBP-1c compared withother hepatic cells lines and are therefore a good model to study
the regulation of SREBP-1c. Although previous studies havehighlighted the importance of insulin in the induction of
SREBP-1 leading to regulation of downstream lipogenic enzymes, we show that glucose also plays an important role in
SREBP-1 expression. Further, the SREBP-1 induction by glucose is dose- and time-dependent and is at the level of transcription. Future studies of the SREBP-1c promoter are necessary to understand the mechanism of this regulation and thecorresponding effects on lipogenesis. Other studies focusing on
the glucose metabolism pathways responsible for the carbohydrate response of SREBP-1 and lipogenic genes as well as
possible control of cleavage by different signal transduction
pathways are also of interest.

Glucose Regulation of SREBP-1 Expression
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