SREBP-1-independent regulation of lipogenic gene
expression in adipocytes
Motohiro Sekiya,* Naoya Yahagi,*,† Takashi Matsuzaka,§ Yoshinori Takeuchi,§
Yoshimi Nakagawa,§ Haruka Takahashi,* Hiroaki Okazaki,* Yoko Iizuka,* Ken Ohashi,*Takanari Gotoda,* Shun Ishibashi,* Ryozo Nagai,*,† Tsutomu Yamazaki,*,† Takashi Kadowaki,*Nobuhiro Yamada,§ Jun-ichi Osuga,* and Hitoshi Shimano1,§
Departments of Internal Medicine* and Clinical Bioinformatics,† Graduate School of Medicine,
University of Tokyo, Tokyo 113-8655, Japan; and Advanced Biomedical Applications,§ Graduate School ofComprehensive Human Sciences, University of Tsukuba, Ibaraki 305-8575, Japan

Supplementary key words sterol regulatory element-binding protein &lipogenesis & fatty acid synthase

The fatty acid biosynthetic pathway, composed of some
25 enzymes, has been elucidated in detail (1). Amongthese enzymes, FAS, the main synthetic enzyme that catManuscript received 10 August 2006 and in revised form 22 January 2007
and in re-revised form 12 April 2007.
Published, JLR Papers in Press, April 24, 2007.
DOI 10.1194/jlr.M700033-JLR200

alyzes the condensation of malonyl-CoA to produce the
16 carbon saturated fatty acid palmitate, and acetylcoenzyme A carboxylase (ACC), which synthesizes malonylCoA from acetyl-CoA, are of particular importance. The
regulation of these lipogenic enzymes has two remarkablefeatures. First, their overall enzymatic activities largely depend on the amount of expressed protein that is primarily
controlled at the transcriptional level, although regulation
through phosphorylation is also important for some enzymes, such as ACC. Second, their rates of transcription
are coordinately regulated (2). Therefore, it has been presumed that these genes share a regulatory sequence in theirpromoters that interacts with common trans-acting factors.
In the liver, the most likely factor conducting this coordinated transcriptional regulation has been revealed to be
sterol regulatory element-binding protein (SREBP)-1 (3, 4).
SREBPs are transcription factors that belong to the basic
helix-loop-helix leucine zipper family and are considered
to be profoundly involved in the transcriptional regulationof cholesterogenic and lipogenic enzymes (5, 6). Unlike
other members of the basic helix-loop-helix leucine zipper
family, SREBPs are synthesized as precursors bound to theendoplasmic reticulum and nuclear envelope. Upon activation, SREBPs are released from the membrane into the
nucleus as mature protein by a sequential two-step cleavage process. To date, three SREBP isoforms, SREBP-1a,-1c, and -2, have been identified and characterized. SREBP-1aand -1c are transcribed from the same gene, each by a distinct promoter, and the predominant SREBP-1 isoform inliver and adipose tissue is 1c rather than 1a (7).
The role of SREBP-1c for the regulation of lipogenesis
in the liver has been well established by several lines ofAbbreviations: ACC, acetyl-coenzyme A carboxylase; ALLN, N-acetylleucyl-leucyl-norleucinal; ChIP, chromatin immunoprecipitation; LXR,
liver X receptor; RXR, retinoid X receptor; SREBP, sterol regulatory
element-binding protein.
1
To whom correspondence should be addressed.
e-mail: shimano-tky@umin.ac.jp

Copyright D 2007 by the American Society for Biochemistry and Molecular Biology, Inc.
This article is available online at http://www.jlr.org

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Abstract Sterol regulatory element-binding protein(SREBP)-1c is now well established as a key transcription
factor for the regulation of lipogenic enzyme genes such as
FAS in hepatocytes. Meanwhile, the mechanisms of lipogenic gene regulation in adipocytes remain unclear. Here,
we demonstrate that those in adipocytes are independent of
SREBP-1c. In adipocytes, unlike in hepatocytes, the stimulation of SREBP-1c expression by liver X receptor agonist
does not accompany lipogenic gene upregulation, although
nuclear SREBP-1c protein is concomitantly increased, indicating that the activation process of SREBP-1c by the cleavage
system is intact in adipocytes. Supportively, transcriptional
activity of the mature form of SREBP-1c for the FAS promoter was negligible when measured by reporter analysis.
As an underlying mechanism, accessibility of SREBP-1c tothe functional elements was involved, because chromatin
immunoprecipitation assays revealed that SREBP-1c does
not bind to the functional SRE/E-box site on the FAS
promoter in adipocytes. Moreover, genetic disruption of
SREBP-1 did not cause any changes in lipogenic gene expression in adipose tissue. In summary, in adipocytes,
unlike in hepatocytes, increments in nuclear SREBP-1c are
not accompanied by transactivation of lipogenic genes;
thus, SREBP-1c is not committed to the regulation of lipogenesis.—Sekiya, M., N. Yahagi, T. Matsuzaka, Y. Takeuchi,
Y. Nakagawa, H. Takahashi, H. Okazaki, Y. Iizuka, K.
Ohashi, T. Gotoda, S. Ishibashi, R. Nagai, T. Yamazaki, T.
Kadowaki, N. Yamada, J-i. Osuga, and H. Shimano. SREBP-1independent regulation of lipogenic gene expression in
adipocytes. J. Lipid Res. 2007. 48: 1581–1591.

MATERIALS AND METHODS
Materials
The synthetic LXR agonist T0901317 was purchased from
Cayman Chemical (Ann Arbor, MI). The RXR agonist LG100268
was synthesized as described elsewhere (16). Standard laboratory

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chow (MF; composed of 60% carbohydrate, 13% fat, and 27%
protein on a caloric basis) was obtained from Oriental Yeast(Tokyo, Japan). Other materials were purchased from Sigma
unless indicated otherwise.

Animals
Eight week old male Wister rats and 8 week old male C57BL/6J
mice were purchased from CLEA (Tokyo, Japan). All animals
were maintained in a temperature-controlled environment witha 12 h light/dark cycle and were given free access to standard
chow and water. The dosage of T0901317 (suspended with 0.9%
carboxymethylcellulose, 9.95% polyethylene glycol 400, and0.05% Tween 80, administered orally) was 10 mg/kg for rats
and 50 mg/kg for mice. Animals were euthanized at 12 h afteradministration. The protocol of dietary manipulation was as follows: for the fasting group, mice were starved for 24 h and rats
were starved for 48 h; for the refeeding group, they were refed at
12 h or 24 h after a 24 h (for mice) or 48 h (for rats) starvation,
respectively, unless stated otherwise. All groups of animals in one
experiment were euthanized at the same time. SREBP-1-nullmice (16 weeks of age, female) on the C57BL/6J backgroundhave been reported previously (4).

RNA isolation and Northern blotting
Total RNA from liver, epididymal fat pads, and cultured cells
was isolated with Trizol Reagent (Invitrogen), and a 10 mg RNA
sample equally pooled among each group was run on a 1% agarose gel containing formaldehyde and transferred to a nylon
membrane. cDNA probes were cloned as described previously(4, 17). The probes were labeled with [a-32P]dCTP using the
Megaprime DNA Labeling System (Amersham Biosciences).
The membranes were hybridized with the radiolabeled probe
in Rapid-hyb buffer (Amersham Biosciences) at 65jC and washedin 0.13 SSC buffer with 0.1% SDS at 65jC. Blots were exposed toboth Kodak XAR-5 film and the imaging plate for the BAS2000
BIO Imaging Analyzer (Fuji Photo Film, Tokyo, Japan). The
quantification results obtained from the BAS2000 system werenormalized to the signal generated from 36B4 (acidic ribosomal
phosphoprotein P0) mRNA.

Quantitative real-time PCR analysis
Two micrograms of total RNA was reverse-transcribed using
the ThermoScript RT-PCR system (Invitrogen). Quantitative realtime PCR was performed using SYBR Green dye (Applied Biosystems, Foster City, CA) in an ABI Prism 7900 PCR instrument(Applied Biosystems). The relative abundance of each transcript
was calculated by a standard curve of cycle thresholds for serial
dilutions of a cDNA sample and normalized to 36B4. Primer
sequences are available upon request.

Nuclear protein extraction from liver and fat
Nuclear extract protein from mouse or rat liver was prepared
as described previously (18). Briefly, excised livers (0.5 g) were
homogenized in a Polytron in 5 ml of buffer A, which consistedof 10 mM HEPES at pH 7.9, 25 mM KCl, 1 mM EDTA, 2 M sucrose, 10% glycerol, 0.15 mM spermine, and 2 mM spermidine,
supplemented with protease inhibitors [6 mg/ml N -acetyl-leucylleucyl-norleucinal (ALLN; Calbiochem), 2.5 mg/ml pepstatin A,
2 mg/ml leupeptin, 0.1 mM phenylmethylsulfonyl fluoride, and
2.5 mg/ml aprotinin]. Pooled homogenate was then subjected to
one stroke of a Teflon pestle in a Potter-Elvejiem homogenizer,
followed by filtration through two layers of cheesecloth, and layered over 10 ml of buffer A. After centrifugation at 24,000 rpm ona Beckman SW28 rotor for 1 h at 4jC, the resulting nuclear pellet

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evidence, especially from transgenic and knockout mousemodels (4, 8). In these models, mature hepatic SREBP-1c
protein levels determine mRNA expression levels for abattery of lipogenic genes in the liver. Moreover, mature
hepatic SREBP-1c is physiologically regulated by nutrient availability (i.e., it is downregulated when animals
are starved and upregulated when they are refed), thereby
adjusting lipogenic gene expression levels to the nutritional conditions (9). Thus, regarding its role in the liver,
SREBP-1c is now well established as a key transcription
factor for the regulation of lipogenic gene expression andby extension triglyceride storage in liver (10, 11).
Despite the extensive knowledge gained in recent years
regarding the role of SREBP-1 in lipogenesis in liver, its
physiological role in adipocytes remains obscure. Although the mRNA expression of SREBP-1c in adipocytes
is also drastically altered by dietary conditions, we havereported that targeted disruption of the SREBP-1 genescarcely affected the dynamic changes of lipogenic gene
expression in adipose tissue (4). Conversely, the impact of
SREBP-1c overexpression in adipocytes was also evaluatedin transgenic mice; however, it disrupted the differentiation processes of adipocytes, resulting in lipodystrophy.
Hence, the effect of SREBP-1c on lipogenic gene regulation in adipocytes was not able to be evaluated (12),although ectopic overexpression of SREBP-1a drives fatty
acid synthesis in the adipose tissue of transgenic mice (13).
These situations prompted us to investigate the effectsof SREBP-1c activation in adipocytes by stimulating the
SREBP-1c promoter. We and others have found that liver
X receptors (LXRs), nuclear receptor-type transcription
factors, activate the transcription of the SREBP-1c genethrough binding to LXR elements in the promoter, together with retinoid X receptors (RXRs) (14, 15).
Here, we show that the stimulation of the SREBP-1cgene with LXR agonist has negligible effects on the expression of lipogenic enzymes in adipocytes, despite a concomitant increase in nuclear active SREBP-1c, indicatingthat the transcriptional activity of SREBP-1c against lipogenic genes is almost nil in adipocytes in contrast to hepatocytes. In fact, luciferase reporter gene assays demonstrated
that the recombinant nuclear active form of SREBP-1c had
far lower activity for the FAS promoter in 3T3-L1 adipocytes than in HepG2 hepatoma cells. Consistent with theseobservations, chromatin immunoprecipitation (ChIP) assays revealed that SREBP-1 is not recruited to the functional cis element on the FAS promoter in LXR-activatedadipocytes. Therefore, lipogenic genes are controlled almost independently of SREBP-1c in adipocytes, and thetriglyceride biosynthetic pathway is differently regulated
between liver and adipose tissue.

Primary culture of rat preadipocytes
Fibroblastic preadipocytes were isolated from epididymal fat
pads of Wister rats by collagenase digestion as described previously (20). In brief, the epididymal fat pads from male Wister
rat were removed and minced in KRBH buffer (130 mM NaCl,
5.2 mM KCl, 1.3 mM KH2PO4, 2.7 mM CaCl2, 1.3 mM MgSO4,
24.8 mM NaHCO3, and 10 mM HEPES at pH 7.4) supplemented
with 3% (w/v) BSA, 2 mM glucose, and 200 nM adenosine. After
digestion by collagenase (type II; 1.5 mg/ml) at 37jC for 1 h in ashaking water bath, the digest was filtered through sterile 250 mm
nylon mesh. The adipocytes were allowed to float to the top of the
tube and the infranatant was collected, passed through a 25 mmstainless steel filter, and centrifuged at 250 g for 10 min. The pellet was resuspended in MEM-a medium (Invitrogen) with 10%
FBS supplemented with penicillin and streptomycin (100 U/ml
and 100 mg/ml, respectively; Invitrogen). Red blood cells werelysed by hypotonic shock (21). The cells were plated on 60 mm
culture dishes at a density of 6 3 105 cells/well. Medium waschanged every 2 days. After the cells reached confluence, differentiation was induced in MEM-a medium with 10% FBS by theaddition of 0.5 mM isobutylmethylxanthine (Wako), 0.25 mM
dexamethasone, 5 mg/ml bovine insulin, and 1 mM pioglitazone(provided by Takeda Pharmaceutical). The differentiation ofcells was morphologically confirmed. Differentiated adipocytes
were treated with either vehicle (ethanol) or 10 mM T0901317 for
12 h in MEM-a medium containing the indicated amounts of
glucose and insulin.

Primary culture of rat hepatocytes
Hepatocytes were isolated from nonfasted 4 week old Wisterrats by the collagenase perfusion method as described previously(22). Cells were resuspended in DMEM containing 25 mM glucose supplemented with 5% FBS and seeded on collagen-coated
100 mm dishes at a final density of 4 3 104 cells/cm2. After 4 h,
attached cells were cultured with Medium 199 containing Earle’s
salts (Invitrogen) and 5% FBS. After incubation for 20 h, cells
were treated with either vehicle (ethanol) or 10 mM T0901317 for
12 h in similar medium containing the indicated amounts of
glucose and insulin.

Preparation of nuclear extracts from cultured cells
Nuclear proteins from cultured cells were extracted as described previously (23). In brief, 2 h before collection, ALLN
(6 mg/ml) was added to the medium. After collection, cells were
resuspended in buffer A (10 mM HEPES at pH 7.6, 1 mM KCl,
1.5 mM MgCl2, 1 mM EDTA, and 1 mM EGTA) and passed
through a 26 gauge needle 20 times and then briefly centrifuged.
The pellet, containing the nuclei, was resuspended in buffer B(20 mM HEPES at pH 7.6, 25% glycerol, 0.42 M NaCl, 1.5 mM
MgCl2, 1 mM EDTA, 1 mM EGTA, and 0.5 mM dithiothreitol)and rotated at 4jC for 15 min and then centrifuged at 15,000 g
for 20 min. The supernatant was collected as nuclear extract.
Whole cell lysates were harvested with a buffer (20 mM Tris-HCl
at pH 7.4) containing detergents (1% Triton X-100, 0.1% SDS,
and 1% sodium deoxycholate) and protease inhibitors according
to a standard protocol.

Immunoblotting of SREBP proteins
Membrane fractions from livers and epididymal fat pads
were prepared as described previously (18). Aliquots of nuclear
extract (20 mg) and membrane fraction (50 mg) proteins were
subjected to SDS-PAGE. Immunoblot analysis was performed
using the ECL Western Blotting Detection System (Amersham
Biosciences) and exposed to Kodak XAR-5 film. The primary
antibodies (rabbit polyclonal; No. 931 for mouse SREBP-1a and-1c, No. 772 for SREBP-1c, which does not bind to SREBP-1a, andNo. 528 for SREBP-2) were used as described previously (11, 17,24). The precursor and mature SREBP-1 bands are ?125 and65 kDa, respectively.

Transfection and luciferase assays
An expression plasmid for the human nuclear form of SREBP1c constructed in pcDNA3.1(1) (Invitrogen) was described previously (25). Luciferase reporter plasmids for the SRE promoter(SRE-Luc) and the fatty acid synthase gene promoter (FAS-Luc)
were prepared as described previously (8, 24). Human hepatoma
HepG2 cells were cultured in DMEM containing 25 mM glucose,
100 U/ml penicillin, and 100 mg/ml streptomycin sulfate supplemented with 10% FBS. On day 0, cells were plated on a 12-wellplate at 4.5 3 104 cells/well. On day 2, the indicated amountsof mature SREBP-1c expression plasmids, mock plasmids [empty
pcDNA3.1(1)] to adjust total DNA amount, and luciferase reporter plasmids (FAS-Luc or SRE-Luc; 0.25 mg each) mixed withan SV-b-galactosidase reference plasmid (0.1 mg; p-SV-b-gal;
Promega) were cotransfected into HepG2 cells using SuperFect
Transfection Reagent (Qiagen) according to the manufacturer’s
protocol. The luciferase activity in transfectants was measured ona luminometer and normalized to b-galactosidase activity measured by standard kits (Promega).
3T3-L1 adipocytes were transfected by electroporation as described previously (26, 27). In brief, after cells became confluent(day 0), adipose conversion was induced in DMEM containing 25 mM glucose, 10% FBS, 0.5 mM isobutylmethylxanthine, 5 mg/ml bovine insulin, 0.25 mM dexamethasone, 1 mM
pioglitazone, 33 mM biotin (Wako), 17 mM pantothenate (Wako),
and antibiotics. After 48 h, the induction medium was removed
and replaced by DMEM containing 25 mM glucose, 10% FBS,
insulin, pioglitazone, biotin, and pantothenate. This medium waschanged every 2 days. The adipocytes at day 8 of differentiation
grown on 60 mm dishes were detached from dishes with 0.25%
trypsin and 0.5 mg/ml collagenase (Wako) in PBS, washed twice,and resuspended in PBS. The indicated amounts of SREBP-1c
expression plasmids, mock plasmids to adjust total DNA amount,luciferase reporter plasmids (FAS-Luc or SRE-Luc; 15 mg each),and control plasmids (pSV-b-gal; 10 mg) were cotransfected to

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was resuspended in a buffer containing 10 mM HEPES at pH 7.9,
100 mM KCl, 2 mM MgCl2, 1 mM EDTA, 1 mM dithiothreitol,and 10% glycerol, supplemented with protease inhibitors, after
which 0.1 volume of 5 M NaCl was added. Each mixture was agitated gently for 30 min at 4jC and then centrifuged at 89,000 rpm
on a Himac S120AT2 rotor (Hitachi, Tokyo, Japan) for 30 min
at 4jC. The supernatant was used as nuclear extract.
Nuclear protein from white adipose tissue was prepared as
described previously (19). In brief, fresh epididymal fat pads(?3 g) were rinsed in ice-cold PBS, minced, and homogenizedwith 10 strokes of a Teflon homogenizer in 15 ml of NDS buffer
at 4jC (10 mM Tris-HCl at pH 7.5, 10 mM NaCl, 60 mM KCl,
0.15 mM spermine, 0.5 mM spermidine, 14 mM mercaptoethanol, 0.5 mM EGTA, 2 mM EDTA, 0.5% Nonidet P-40, and 1 mM
dithiothreitol) supplemented with protease inhibitors (6 mg/ml
ALLN, 2 mg/ml leupeptin, 2.5 mg/ml aprotinin, 2.5 mg/ml
pepstatin A, and 0.1 mM phenylmethylsulfonyl fluoride). The
Nonidet P-40 concentration was increased to 1%, and nuclei werepelleted at 700 g for 10 min, washed once with 25 ml of NDS
buffer (1% Nonidet P-40), filtered through 70 mm mesh, pelleted
at 500 g for 10 min, resuspended in 1 volume of 1% citric acid,lysed by the addition of 2.5 volumes of 0.1 M Tris-HCl, 2.5% SDS,
and 0.1 M dithiothreitol, sonicated briefly, and heated to 90jC
for 5 min. Aliquots of nuclear protein (20 mg) were subjected to
SDS-PAGE.

the cells by a pulse current generated from the electroporator(Cell-Porator; Invitrogen) at 160 V with 880 mF capacitance anda low ohm setting. After electroporation, cells were immediately mixed with fresh medium for 10 min before being reseeded onto 24-well collagen-coated plates and assayed at 40 hafter transfection.

ChIP assays

Fig. 1. Sterol regulatory element-binding protein (SREBP)-1c activation by liver X receptor (LXR) agonists does not increase lipogenic
gene expression in mouse adipose tissue. Northern blot (A) and real-time PCR (B) analyses of liver and adipose tissue of mice treatedwith LXR agonist are shown. C57BL/6J mice (8 weeks old, male, four animals for each group) were treated with the LXR agonist T0901317(50 mg/kg) as indicated and euthanized at 12 h after treatment in a 24 h fasted state. Refed control mice were refed for 12 h after 24 hof starvation. A: Total RNA (10 mg) pooled equally was subjected to Northern blotting. A cDNA probe for 36B4 (acidic ribosomal
phosphoprotein P0) was used to confirm equal loading. B: Real-time PCR analysis of RNA isolated from liver and adipose tissue. mRNAlevels of target genes were normalized to 36B4. Values are means 6 SEM presented as fold changes relative to liver controls. ACC, acetylcoenzyme A carboxylase; FACE, fatty acyl-coenzyme A elongase; SCD1, stearoyl-coenzyme A desaturase 1; WAT, white adipose tissue. * P ,0.05, ** P , 0.01 versus controls.

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ChIP assays were performed as described by Boyd andFarnham (28) with minor modifications. The supernatant ofsoluble chromatin derived from 1 3 107 cells was used. Briefly, rat
primary hepatocytes and adipocytes were treated with 1% formaldehyde for 10 min. The cross-linking reaction was stopped
by the addition of glycine to a final concentration of 0.125 M for
5 min. After washing with cold PBS, the cells were suspended
in cell lysis buffer containing 10 mM HEPES at pH 7.9, 10 mM
KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 0.5% Nonidet
P-40, and protease inhibitors. After incubation for 30 min on ice,the cells were passed through a 26 gauge needle 20 times on ice.
The nuclei were collected by microcentrifugation at 5,000 rpm
and resuspended in nuclear lysis buffer (50 mM Tris-HCl at pH
8.1, 1% SDS, 10 mM EDTA, and protease inhibitors) on ice for10 min. Samples were sonicated with a Branson sonifier at power
2 for six 10 s pulses to an average DNA length of ,2 kb and thenmicrocentrifuged at 15,000 rpm for 10 min. The supernatant was

diluted 10-fold with buffer containing 0.01% SDS, 16.7 mM TrisHCl at pH 8.1, 1.1% Triton X-100, 1.2 mM EDTA, 167 mM NaCl,and protease inhibitors and precleared for 1.5 h with Protein
G-Sepharose. The supernatant was incubated with normal rabbit IgG or anti-SREBP-1 antibody overnight at 4jC.
Samples were subsequently washed four times using wash
buffer A (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 150 mM
NaCl, and 20 mM Tris-HCl at pH 8.1), wash buffer B (0.1% SDS,
1% Triton X-100, 2 mM EDTA, 500 mM NaCl, and 20 mM TrisHCl at pH 8.1), wash buffer C (0.25 M LiCl, 1% Nonidet P-40,
1% deoxycholate, 1 mM EDTA, and 10 mM Tris-HCl at pH 8.1),
and wash buffer D (1 mM EDTA and 10 mM Tris-HCl at pH 8.0)
and were eluted by 30 min of incubation with 100 ml of elution buffer (1% SDS, 50 mM NaHCO3, and 10 mM DTT). Then,
NaCl was added at a final concentration of 0.3 M and RNA wasremoved by the addition of RNase A, and the samples were incubated at 65jC for 4 h to reverse the formaldehyde-induced crosslinking. Protein was digested by proteinase K in 23 PK buffer(20 mM Tris-HCl at pH 7.5, 10 mM EDTA, and 1% SDS) at 45jC
for 2 h. The resulting chromatin DNA was further purified on
silica beads (Wizard DNA Clean-Up System; Promega) and used
as a template for PCR. Primers used to amplify FAS promoter
regions were as follows: for the SRE/E-box site at 265 (2109 to
163 was amplified), 5¶-GACGCTCATTGGCCTGG-3¶ and5¶-CTCTGGAGGCAGACGACAAG-3¶; for the SRE-1 site at 2150
(294 to 2299 was amplified), 5¶-AGGACAGAGATGAGGGCGTC-3¶

and 5¶-CCAGGCCAATGAGCGTC-3¶; for the nonspecific site
(21,076 to 2904), 5¶-AAGCCACTGCCCATAAGGTT-3¶ and 5¶-TTAAAGGGAGGGAGGGTGAG-3¶. For the SRE/E-box and nonspecific sites, PCR was performed using AmpliTaq Gold (Applied
Biosystems) under the following reaction conditions: after 9 min
at 95jC, 35 cycles of 1 min at 95jC, 30 s at 62jC, and 1 min at 72jC.
For the SRE-1 site, PCR was performed using Advantage-GC Genomic Polymerase Mix (Clontech) under the following reaction
conditions: after 1 min at 95jC, 35 cycles of 30 s at 94jC, 30 s at
62jC, and 2.5 min at 68jC. After amplification, PCR products were
electrophoresed on a 3% NuSieve agarose gel and visualized byethidium bromide staining. These experiments were performedat least three times.

RESULTS

Fig. 2. SREBP-1c activation by LXR agonist does not induce lipogenic gene expression in rat adipose tissue. Northern blot analysis
of liver and adipose tissue of rats treated with LXR agonist is
shown. Wister rats (8 weeks old, male, three animals for eachgroup) were administered the LXR agonist T0901317 (10 mg/kg)
or vehicle alone and euthanized at 12 h after treatment in a fasted
state. Total RNA (10 mg) pooled equally was subjected to Northernblotting. A cDNA probe for 36B4 was used to confirm equal loading. The quantification results were obtained with the BAS2000
system and normalized to the signal generated from 36B4 mRNA,and the fold changes against control are displayed above eachblot. S14, Spot 14; WAT, white adipose tissue.

Posttranslational activation of SREBP-1 is intact, andmature protein is generated in adipose tissue
SREBP-1 is synthesized as a precursor bound to the endoplasmic reticulum and nuclear envelope and is released
from the membrane into the nucleus as a mature protein
by a cleavage process (6). Therefore, it is possible that
defective posttranslational processing of SREBP-1 in adipocytes might result in this blunted response of lipogenic
genes to SREBP-1 overexpression. To test this possibility, we separated nuclei from liver and adipose tissue andquantified the amount of mature SREBP-1 by immunoblot
analysis. As shown in Fig. 3, nuclear contents of SREBP-1
were increased similarly by LXR agonist in both liver andadipose tissue, indicating that posttranslational processing of SREBP-1 is intact in adipose tissue (Fig. 3A, mice;
Fig. 3B, rats). It was further confirmed by SREBP-1cspecific antibody that isoform 1c was increased in the nuclei of adipose tissue. These findings demonstrate that thedefective response of lipogenic genes to SREBP-1 overexpression in adipose tissue is not attributable to an impairment of the SREBP-1c cleavage process.
To deny the possibility that the expression levels of thenuclear form of SREBP-1 protein in adipose tissue stimulated by LXR agonist are too low to increase lipogenic
gene expression, the effect of T0901317 was comparedwith that of refeeding. As shown in Fig. 3C, the matureform of SREBP-1 in the nuclei of LXR-stimulated adipocytes is more abundant than that observed in adipocytes
from refed animals. However, this abundant SREBP-1 protein of the active form did not induce FAS expression at all(Fig. 3D), demonstrating that the amount of SREBP-1 protein is not the cause of the inability of SREBP-1 to upregulate FAS expression in adipocytes.
SREBP-1 and lipogenic gene expression in primarycultured cells
LXR widely modifies lipid and glucose metabolism,
affecting serum levels of insulin and fatty acids (31), which
are known to influence the SREBP-1 pathway (32). To

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SREBP-1c activation by LXR agonist does not induce
lipogenic gene expression in adipose tissue
In our first series of experiments, we examined the effects of SREBP-1c overexpression on lipogenic gene
expression in liver and adipose tissue. As expected, administration of synthetic LXR agonist T0901317 to animals(mice in Fig. 1 and rats in Fig. 2) upregulated SREBP-1
expression and thereby the downstream lipogenic enzyme

genes, such as FAS, ACC, and stearoyl-coenzyme A desaturase 1, in their livers. In contrast, quite unexpectedly,
lipogenic genes in their adipose tissues were not influenced by the treatment, although SREBP-1 was markedly
upregulated in the same way as in liver. These results from
Northern blotting were confirmed by quantitative realtime PCR analyses (Fig. 1B). It is notable that SREBP-1
mRNA levels quantified by real-time PCR are equivalent inboth tissues, as shown in Fig. 1B, denying the possibility
that a lack of lipogenic gene induction in adipose tissue
is a quantitative effect. The effect of LXR activation was
further verified by the marked increase of ABCA1 andABCG1, representatives of LXR-activated genes (29, 30),
which were strongly induced in both tissues. These results
were not altered by combined administration of both LXR
agonist and the RXR agonist LG100268 (30 mg/kg),
which produced additive effects on the SREBP-1 pathway
in the liver but did not influence lipogenic gene expression in adipose tissue (data not shown).

eliminate these indirect effects through systemic factors
and reinforce the in vivo results, we also examined theeffects of SREBP-1c activation in primary cultured hepatocytes and adipocytes in vitro, whose isoform patterns of
SREBP-1 are 1c-predominant as assessed by quantitative
real-time PCR assays (data not shown). In these primarymodels, it was demonstrated that the overexpression of
SREBP-1 mRNA and thereby precursor proteins induced
by LXR agonist led to the increase of mature SREBP-1
proteins in both hepatocytes and adipocytes (Fig. 4). However, the resulting activation of SREBP-1 produced totallydifferent effects on lipogenic gene expression between
hepatocytes and adipocytes; consistent with the in vivo
results described above, FAS mRNA expression was upregulated by SREBP-1c in hepatocytes but was not induced
in adipocytes.
Transcriptional activity of SREBP-1c for the FAS promoter
is negligible in adipocytes
As described above, the contribution of SREBP-1 to lipogenic gene regulation was demonstrated to be different
between hepatocytes and adipocytes. Because SREBP-1c
activation processes are revealed to be equally intact inboth cell types, it is presumed that nuclear SREBP-1 would
have different transcriptional activity in hepatocytes and
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adipocytes. To test this hypothesis, we performed luciferase reporter gene assays and compared transcriptional
activities of nuclear SREBP-1c between HepG2 hepatoma cells and 3T3-L1 adipocytes. We estimated the transcriptional activity of nuclear SREBP-1c against the FAS
promoter by transfecting cells with FAS-Luc reporter plasmids along with mature SREBP-1c expression plasmids.
Transfection efficiency was evaluated using an optimum
SRE-Luc as a control. As shown in Fig. 5, in HepG2 cells,
FAS-Luc and SRE-Luc were vigorously activated by SREBP1c. In contrast, activation of FAS-Luc by the active form of
SREBP-1c was faint in 3T3-L1 adipocytes, whereas SRE-Luc
was highly activated. These data demonstrate that the transcriptional activity of SREBP-1c against the FAS promoter
is far lower in adipocytes compared with hepatocytes.
SREBP-1 does not bind to the functional cis element on the
FAS promoter in adipocytes
To further investigate the molecular mechanism against
the inability of SREBP-1 to activate transcription from the
FAS gene in adipocytes, we evaluated the direct bindingof SREBP-1 to the FAS promoter using ChIP assays. In the
FAS promoter, two potential binding sites for SREBP-1
have been identified: one is 265 SRE/E-box, which contains two tandem copies of SREs and the E-box; the other

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Fig. 3. Posttranslational activation of SREBP-1 is intact and mature protein is generated in adipose tissue. Immunoblot analyses of
SREBPs in nuclear proteins from liver (A, B) and adipose tissue (B, C) are shown. A: Mature forms of SREBP-1 and -2 in the liver. C57BL/6J
mice (8 weeks old, male, four mice for each group) were administered the LXR agonist T0901317 (50 mg/kg) and/or the retinoid
X receptor (RXR) agonist LG100268 (30 mg/kg) as indicated and euthanized at 12 h after treatment in a 24 h fasted state. Refed control
mice were refed for 12 h after 24 h of starvation. B: Mature forms of SREBP-1 and -2 in liver and adipose tissue were quantified from
Wister rats (8 weeks old, male) administered T0901317 (10 mg/kg) or vehicle alone and euthanized at 12 h after treatment in a fasted
state. Nuclei were isolated from liver (pooled within groups) and adipose tissue (individually), and aliquots of nuclear proteins (20 mg) were
subjected to immunoblot analysis. SREBP-1c-specific antibody was also used. C, D: Effect of administering LXR agonist compared withthat of refeeding rats. C: Precursor and mature forms of SREBP-1 in adipose tissue were visualized by immunoblots. D: Northern blottingof SREBP-1 and FAS. Wister rats (8 weeks old, male, three animals for each group) were administered T0901317 (10 mg/kg) or vehicle
alone at 12 h before euthanization. WAT, white adipose tissue.

is 2150 SRE-1, which is not functional because of the lackof cofactor binding sites nearby, although the sequence
itself is a complete SRE (5¶-TCACCCCAC-3¶) (33, 34). In our
ChIP assays, SREBP-1 activation augmented its binding
to 265 SRE/E-box in rat primary hepatocytes, as expected(Fig. 6). In sharp contrast to this, SREBP-1 activation did
not result in its recruitment to 265 SRE/E-box in adipocytes, demonstrating that SREBP-1 does not bind to thefunctional cis element on the FAS promoter in adipocytes. Meanwhile, SREBP-1 activation led to its binding to2150 SRE-1 in both hepatocytes and adipocytes. These
results were also confirmed using mouse primary cells(data not shown).

FAS expression in SREBP-1 knockout mice with or without
LXR stimulation. As shown in Fig. 7, genetic deletion of
SREBP-1 impaired the transactivation of FAS in response
to LXR activation in liver, whereas in adipose tissue, LXR
activation did not alter the expression levels of these lipogenic genes in either SREBP-1 knockout or wild-type mice.
These results demonstrate that SREBP-1 does not substantially contribute to the regulation of FAS expression in
adipose tissue, whereas in the liver, SREBP-1 actually regulates the expression level of FAS.

Genetic disruption of SREBP-1 decreases lipogenic gene
expression as well as the responses to LXR activation in theliver, but not in adipose tissue
To further validate that SREBP-1 is not involved in theregulation of lipogenic genes in adipocytes, we evaluatedThis study has clearly demonstrated that lipogenic gene
regulation is primarily independent of SREBP-1c in adipocytes. Hence, the involvement of SREBP-1c in fatty acidsynthesis differs fundamentally between liver and adipose
tissue, the two major lipogenic organs.

DISCUSSIONSREBP-1-independent regulation of lipogenic genes in adipocytes

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Fig. 4. Northern and Western blot analyses of SREBP-1 in primary hepatocytes and adipocytes. A, B: mRNA
quantification of SREBP-1 and lipogenic genes in primary hepatocytes (A) and adipocytes (B). Hepatocytes isolated from rat livers (A) and adipocytes differentiated from rat preadipocytes (B) were incubatedfor 12 h with or without T0901317 (10 mM) and insulin (100 nM). Incubation medium contained either
5 mM glucose (LG; low glucose) or 25 mM glucose (HG; high glucose). Total RNA (10 mg) was subjected to
Northern blotting. A cDNA probe for 36B4 was used to confirm equal loading. The quantification results
were obtained with the BAS2000 system and normalized to the signal generated from 36B4 mRNA, and foldchanges against control are displayed above each blot. C, D: Precursor and mature forms of SREBP-1
proteins from primary hepatocytes (C) and adipocytes (D) are shown. Cells were stimulated as describedabove, whole cell lysates (50 mg of protein) were subjected to immunoblot analysis, and the bands corresponding to precursor and mature SREBP-1 proteins are presented as indicated. These experiments wererepeated twice, and reproducibility was confirmed.

Recently, a microarray analysis of mouse liver and adipose tissue stimulated by LXR agonist was reported byothers (35). In that study, SREBP-1 mRNA expression was
induced in both liver and adipose tissue by LXR agonist,
whereas lipogenic genes such as fatty acid synthase, malic
enzyme, and Spot 14 were upregulated only in liver andnot in adipose tissue, supporting our data. We further
proceeded to explore the underlying mechanism for thisdiscrepancy and have revealed that the active form of
SREBP-1 is definitely generated but does not transactivate lipogenic genes in adipocytes, as a result of defective
binding to functional SRE in promoter regions in vivo.
In our first series of experiments, we demonstrated thatin both adipocytes and hepatocytes the nuclear content
of mature SREBP-1 protein parallels the amount of the
precursor form of SREBP-1, whether its expression is increased by LXR activation or by refeeding. This observation indicates that the proteolytic activation of SREBP-1
precursor protein is intact in adipocytes and that SREBP-1
is basically activated when its mRNA expression is upregulated. This feature of SREBP-1 activation provides a sharp
contrast to that of SREBP-2, which is tightly regulated
through the cleavage processes as a pivotal part of thenegative feedback system to maintain cholesterol homeostasis (6). In fact, SREBP-1 and -2 are reported to be regulated distinctly under some specific conditions (18, 36).
It is intriguing that there is so much difference in theregulation of the proteolytic activation of SREBP-1 and -2,
although they are both subject to the same cleavage machineries, consisting of SREBP cleavage-activating protein
and site-1/2 proteases (37).
Nevertheless, in contrast to hepatocytes, increases of
the active form of SREBP-1c protein failed to activate
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lipogenic genes in adipocytes. This ineffectiveness of
SREBP-1c in adipocytes was validated by four lines of evidence: 1) overexpression of active SREBP-1c had little effect on mRNA levels of lipogenic genes; 2) transcriptional
activity of SREBP-1c against the FAS promoter measured
by reporter analysis was faint; 3) nuclear SREBP-1c failed
to bind to the functional cis element of the FAS promoter
in adipocytes; and 4) genetic disruption of SREBP-1 hardly
affected mRNA levels of lipogenic genes. These results
are further supported by the recent report claimingthat mRNA levels of SREBP-1c do not coincide with thechanges in adipose lipogenic gene expression (38, 39).
Regarding why mature SREBP-1c poorly transactivates
lipogenic genes in adipocytes, there are two possibilities:
1) SREBP-1c cannot reach its binding sites on the promoters; and 2) SREBP-1c properly binds to its cognatesites, but the absence of required coactivator(s) or thepresence of corepressor(s) specific to adipocytes prevents
the genes from being transcribed. Our results from ChIP
assays favor the former hypothesis. SREBP-1 was able toaccess both functional 265 SRE/E-box and nonfunctional
2150 SRE-1 site in hepatocytes. In contrast, SREBP-1
could not bind to 265 SRE/E-box in adipocytes, althoughbinding to 2150 SRE-1 site was intact and similar to thatobserved in hepatocytes.
Another question our studies bring up is what transcription factors then regulate lipogenic gene expression in adipocytes. Unfortunately, we have few clues
on this issue, except that this factor should be functionally activated by refeeding. Upstream stimulatory
factors-1/2 (40–42), carbohydrate response element binding protein (43), and CCAAT/enhancer binding proteina (44) are potential candidates, but an as yet unknown

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Fig. 5. Transcriptional activity of SREBP-1c against
the FAS promoter is negligible in adipocytes. Transcriptional activities of the mature form of SREBP-1c in
HepG2 hepatoma cells (left panels) and 3T3-L1 adipocytes (right panels) were measured by luciferase reporter gene assay. Both types of cells were transfectedwith the indicated amounts of mature SREBP-1c expression plasmids and mock plasmids [pcDNA3.1(1)] toadjust total DNA amount in combination with FAS-Luc
or SRE-Luc and pSV-b-gal (note that transfection methods and scales are different between HepG2 and 3T3L1 and that the amounts of transfected DNA cannot
be compared between them). Luciferase activities were
measured and normalized to b-galactosidase activities.
In upper panels, values are expressed as fold changes
against control (i.e., no SREBP-1c). Error bars represent SEM. These experiments were repeated twice, andreproducibility was confirmed.

factor might be the answer. Further studies in adipocytes
are imperative.
It has been reported by others that fatty acid synthase gene expression is increased by SREBP-1c throughadenovirus-mediated gene transduction to 3T3-L1 adipocytes (45). In our experiments, FAS-Luc reporter activity
was not increased by SREBP-1c transfection (Fig. 5). The
cause of this discrepancy is not clear, but it is possible that theconditions of our transfection by electroporation method

resulted in a more physiological level of SREBP-1c increase
compared with adenovirus-mediated overexpression.
The importance of the LXR pathway in the regulation of
lipogenesis and adipogenesis in adipocytes has been controversial; some reports have concluded that LXR stimulation leads to lipogenic gene activation (46, 47), whereas
others have presented opposing data (35, 48). The current
data support no impact of the LXR ligand on the gene
expression of lipogenic enzymes, but the discrepancy

Fig. 7. Effects of the genetic disruption of SREBP-1. SREBP-1 disruption decreased lipogenic gene
expression as well as the responses to LXR activation in the liver (left panel), whereas it did not do so in
adipose tissue (right panel). SREBP-1 knockout mice in the C57BL/6J background (16 weeks old, female,three mice for each group) and their control littermates were treated with T0901317 (50 mg/kg) andanalyzed by Northern blot. Mice were euthanized at 12 h after treatment in a 24 h fasted state. WAT, white
adipose tissue.

SREBP-1-independent regulation of lipogenic genes in adipocytes

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Fig. 6. SREBP-1 does not bind to the functional cis element on the FAS promoter in adipocytes. A:
Chromatin immunoprecipitation (ChIP) assays using antibody to SREBP-1 and amplifying DNA fragments from the FAS promoter. Primary hepatocytes and adipocytes differentiated from preadipocytes wereincubated for 12 h with or without T0901317 (10 mM). After cross-linking, the nuclei were isolated
and sonicated, and chromatin protein-DNA complexes were immunoprecipitated with control rabbit IgGor anti-SREBP-1 antibodies. The resultant DNA was analyzed by PCR with primers amplifying 2150 SRE-1,
265 SRE/E-box, and a nonspecific region (from 21,076 to 2904) on the FAS promoter. The results shown
here are representative of three independent experiments. B: Schematic representations of the FAS
promoter. The FAS promoter contains two SREBP-1 recognition sites: 2150 SRE-1 and 265 SRE/E-box.
ChIP assays revealed tissue-dependent recruitment of SREBP-1; SREBP-1 can bind to both functional
SRE/E-box and nonfunctional SRE-1 in hepatocytes, whereas the binding to SRE/E-box is prevented
in adipocytes.

might originate from differences in experimental conditions, such as variable glucose availability, that might indirectly influence lipogenesis, considering that the LXR
pathway is reported to upregulate glucose transport (49).
Our main conclusion here is that adipocyte lipogenesis
is independent of SREBP-1c even if upregulated by LXR
activation, consistent with our previous and current datafrom SREBP-1-null mice.
In summary, we have found that lipogenic genes inadipocytes are primarily regulated by factor(s) other than
SREBP-1. Although proteolytic activation of SREBP-1
is intact and the resulting active form of SREBP-1 is upregulated whenever the SREBP-1 gene is stimulated, it
does not function as a transactivator for lipogenic genes
in adipocytes.

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The authors thank Alyssa H. Hasty for critical reading of themanuscript. This work was supported by grants-in-aid fromthe Japan Health Sciences Foundation (to M.S.) and from
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