THE JOURNAL OF BIOLOGICAL CHEMISTRY© 2004 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 279, No. 13, Issue of March 26, pp. 12027–12035, 2004
Printed in U.S.A.

SREBP-1 Interacts with Hepatocyte Nuclear Factor-4␣ andInterferes with PGC-1 Recruitment to Suppress Hepatic
Gluconeogenic Genes*
Received for publication, September 17, 2003, and in revised form, December 22, 2003
Published, JBC Papers in Press, January 13, 2004, DOI 10.1074/jbc.M310333200

Takashi Yamamoto, Hitoshi Shimano‡, Yoshimi Nakagawa, Tomohiro Ide, Naoya Yahagi§,
Takashi Matsuzaka, Masanori Nakakuki, Akimitsu Takahashi, Hiroaki Suzuki, Hirohito Sone,
Hideo Toyoshima, Ryuichiro Sato¶, and Nobuhiro Yamada
From the Department of Internal Medicine, Institute of Clinical Medicine, University of Tsukuba, 1-1-1 Tennodai,
Tsukuba, Ibaraki 305-8575, the §Department of Internal Medicine, Graduate School of Medicine, and ¶Department ofApplied Biological Chemistry, Graduate School of Agricultural and Life Sciences, the University of Tokyo,
Tokyo 113-8657, JapanThe hepatocyte nuclear factor-4␣ (HNF-4␣)/PGC-1
pathway plays a crucial role in the transcriptional regulation of hepatic gluconeogenic enzymes such as phosphoenolpyruvate carboxykinase (PEPCK) and Glc-6Pase, genes that are activated at fasting and suppressedin a fed state. SREBP-1c dominates the nutritional regulation of lipogenic genes inverse to gluconeogenesis.
Here we show the mechanism by which SREBP-1 suppresses expression of gluconeogenic genes. A series of
luciferase reporter assays demonstrated that SREBP-1aand -1c effectively inhibited the PEPCK promoter activity that was induced by HNF-4␣. The HNF-4␣-bindingsite in the glucocorticoid-response unit was responsible
for the SREBP-1 inhibition, although SREBP-1 did notbind to the PEPCK promoter as demonstrated by electrophoretic mobility shift assays. The inhibitory effectwas more potent in the isoform of SREBP-1a than
SREBP-1c and was eliminated by deletion of the aminoterminal transactivation domain of SREBP-1. Coimmunoprecipitation experiments demonstrated that these
two transcription factors directly interact through thetransactivation domain of SREBP-1 and the ligand binding/AF2 domains of HNF-4␣. Estimation of coactivator
recruitment using HNF-4␣-Gal4DBD fusion assay
showed that SREBP-1 competitively inhibited PGC-1 recruitment, a requirement for HNF-4␣ activation. Consistent with these results, hepatic PEPCK and Glc-6Pase mRNA levels are suppressed by overexpression of
SREBP-1a and -1c in the transgenic mice. Our data indicate that SREBP-1 has a novel role as negative regulator of gluconeogenic genes through a cross-talk with
HNF-4␣ interference with PGC-1 recruitment.

Regulation of gluconeogenesis is crucial to maintain glucose
homeostasis. Gluconeogenic genes such as phosphoenolpyruvate carboxykinase (PEPCK)1 and glucose-6-phosphatase (Glc-* This work was supported by grants-in-aid from the Ministry of
Science, Education, Culture, and Technology of Japan. The costs ofpublication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
‡ To whom correspondence should be addressed: Dept. of Internal
Medicine, Institute of Clinical Medicine, University of Tsukuba, Tennodai, Tsukuba, Ibaraki, 305-8575, Japan. Fax: 81-29-863-2081;
E-mail: shimano-tky@umin.ac.jp.
1
The abbreviations used are: PEPCK, phosphoenolpyruvate carboxykinase; Glc-6-Pase, glucose-6-phosphatase; HNF-4␣, hepatocyte
This paper is available on line at http://www.jbc.org

6-Pase) are nutritionally regulated in the liver at the transcriptional level, are highly activated during fasting or starvation,
and are suppressed in a fed state. PEPCK promoter has been
extensively studied and shown to be induced by several hormonal stimuli (glucocorticoid, glucagons, and thyroid hormone)and transcriptional factors (cAMP-response element-binding
protein, C/EBP␣, HNF-3, HNF-4␣, and PPAR␣) (1). Amongthese, insulin is a dominant negative regulator of PEPCK promoter through several pathways (1). Hepatocyte nuclear factor-4␣ (HNF-4␣) is a highly conserved member of the nuclear
receptor superfamily and was initially identified as a transcriptional factor required for liver-specific gene expression (2), although it is also expressed in kidney, intestine, and pancreas(3). Recently, HNF-4␣ and a coactivator, peroxisome proliferator-activated receptor-␥ coactivator-1 (PGC-1), have beenshown to activate expression of PEPCK (4) and Glc-6-Pase (5)by binding to HNF-4␣-binding cis-elements in their promoters.
Crucial roles of HNF-4␣ and PGC-1 in nutritional regulation ofthese genes, and thus gluconeogenesis, were confirmed by analysis of liver-specific HNF-4␣ knockout mice and by adenovirusmediated overexpression of PGC-1 in mouse livers (6, 7).
The sterol regulatory element-binding protein (SREBP) family has been established as a group of transcription factors
regulating transcription of genes involved in cholesterol andfatty acid synthesis. SREBP proteins are initially bound to therough endoplasmic reticulum membrane and form a complex
with SREBP cleavage-activating protein (SCAP), a sterol-sensing molecule. Upon sterol deprivation, SREBP is cleaved toliberate the amino-terminal portion, containing a basic helixloop-helix leucine zipper domain, and enters the nucleus where
it can bind to specific sterol-response elements (SRE) in the
promoters of target genes (8 –10). Three isoforms of SREBP-1a,-1c, and -2 are known. Although SREBP-2 plays a crucial role
in regulation of cholesterol synthesis, SREBP-1c controls gene
expression of lipogenic enzymes (11–14). Insulin and glucose
have been well known to stimulate lipogenesis, presumably
through induction of SREBP-1c expression of which is highly
nutritionally regulated in the liver and adipose tissue. Recently, we reported (15, 16) that cross-talk between PPARs andnuclear factor-4␣; EMSA, electrophoretic mobility shift assay; PPAR,
peroxisome proliferator-activated receptor; LXR, liver X receptor; aa,amino acids; GST, glutathione S-transferase; DTT, dithiothreitol; LBD,
ligand binding domain; SREBP, sterol regulatory element-binding protein; PBS, phosphate-buffered saline; GRU, glucocorticoid-response
unit; SRE, sterol-response elements; RE, response element; TA, transactivation domain.

12027

12028SREBP-1 Inhibition of HNF-4␣/PGC-1 Pathway

LXRs could be involved in reciprocal nutritional regulation of
fatty acid metabolism. Considering the physiologically relatedroles of gluconeogenesis and lipogenesis, their transcriptional
regulators HNF-4␣ and SREBP-1c could reciprocally regulate
their target genes. In fact, fasting/feeding response to andinsulin/glucose effect on these factors are completely opposite.
Despite this knowledge, a mutual interaction between thesetwo pathways has never been explored. Here we investigate theeffects of SREBPs on the HNF-4␣/PGC-1 pathway, and we
examine the potential role of SREBPs in the regulation of
gluconeogenesis.
EXPERIMENTAL PROCEDURES

Animals—SREBP transgenic mice used in this study were described
previously (17, 18). Mice were housed in colony cages and maintainedon a 12-h light/12-h dark cycle. Before time of killing, transgenic animals and littermate controls were placed on the low carbohydrate/highprotein diet for 1 week to induce expression of the transgene. Mice were
killed after a 12-h fast.
Plasmid Construction—Plasmid constructs were produced by using
general molecular biology procedures. A series of PEPECK-LUC vectors
were generated by inserting the rat PEPCK promoter region (from ⫺1021
to ⫹278, from ⫺480 to ⫹278, or from ⫺335 to ⫹278) amplified by PCR
into the pGL3-basic vector (Promega). The HNF-4␣ RE LUC vector containing four copies of HNF-4␣-response elements was generated by inserting the double-stranded oligonucleotides of HNF-4␣-response element
upstream of the native PEPCK TATA box linked to a luciferase gene
(pUC8). The expression vectors for human SREBP-1a, -1c, and -2 wereconstructed as described previously (19). Transactivation domain-deleted(⌬TA-SREBP-1) SREBP-1 expression plasmid was constructed by inserting PCR-amplified SREBP-1 fragment (90 – 460 aa) into pcDNA3.1. The
expression vector for mouse PGC-1 was generated by inserting PCRamplified full-length fragment into pcDNA3.1 (Invitrogen). The expression vector for mouse HNF-4␣ was generated by inserting PCR-amplified
full-length fragment into pCMV7. The expression vector for Gal4-HNF-4␣
fusion protein was generated by inserting PCR-amplified HNF-4␣ fragment (115– 455 aa) downstream of Gal4 in the pM vector (Clontech). The
expression vector for FLAG-tagged HNF-4␣ was generated by inserting
full-length HNF-4␣ cDNA fragment downstream of the FLAG tag basedon pcDNA3.1 (Invitrogen). The expression vector for c-Myc-tagged
SREBP-1a was generated by inserting the fragment of the nuclear form of
SREBP-1a into pcDNA3.1 Myc/His (Invitrogen). The expression vectors
for GST-SREBP-1a, GST-SREBP-1c, or GST-SREBP-1a (1–100 aa) weregenerated by inserting the PCR-amplified fragment into pGEX-4T (Amersham Biosciences). The expression vectors for the HNF-4␣ partial fragment were generated by inserting the PCR-amplified fragment into
pcDNA3.1 (Invitrogen). The expression vectors for CBP and p300 (pCMV)
were a kind gift from Dr. T. Nakajima. All PCR-based constructions wereverified by DNA sequencing.
Northern Blotting—Total RNA from mouse livers was prepared with
Trizol reagent (Invitrogen). Equal aliquots of total RNA from mice were
subjected to Northern blot analysis. cDNA probes for mouse PEPCK,
Glc-6-Pase, and acidic ribosomal phosphoprotein PO (36B4) were prepared from reverse transcriptase-PCR of mouse liver total RNA withthe following primers: 5⬘-CACACCATTGCAATTATGCCT-3⬘ and 5⬘-GCGTTGAATGCTTTCTCAAAGT-3⬘ for PEPCK; 5⬘-GCTTGGACTCACTGCACG-3⬘ and 5⬘-GACTCTGCATGCCTTACAAAGA-3⬘ for Glc-6Pase; and 5⬘-ATGATTATCCAAAATGCTTCATTG-3⬘ and 5⬘-AACAGCATATCCCGAATCTCA-3⬘ for 36B4. Full-length cDNA was used as
cDNA probes for PGC-1 and HNF-4␣. Blot hybridization was performed
with the cDNA probes for mouse PEPCK, Glc-6-Pase, HNF-4␣, PGC-1
and 36B4 labeled with [␣-32P]dCTP (6000 Ci/mmol) using the Megaprime DNA Labeling System (Amersham Biosciences). Labeled probes
were incubated with RNA transferred membranes for 2 h at 65 °C. Afterwashing with washing buffer (0.1⫻ SSC, 0.1% SDS) two times, hybridized membranes were visualized by BAS-2500 (Fuji).
Transient Transfection and Luciferase Assay—HepG2 cells weregrown at 37 °C in an atmosphere of 5% CO2 in minimum Eagle’s
medium (Sigma) containing non-essential amino acids for minimum
Eagle’s medium, 1 mM sodium pyruvate, 100 units/ml penicillin, 100
␮g/ml streptomycin sulfate and supplemented with 10% fetal bovine
serum. Before transfection, HepG2 cells were seeded in 24-well plates
at a density of 2.5 ⫻ 104 cells/well or in 12-well plates at a density of
5.0 ⫻ 104 cells/well. After adhesion, cells were transfected with FuGENE 6 (Roche Applied Science) according to the manufacturer’s protocol. The total amount of DNA was adjusted to 0.5 ␮g/well in 24-wellplates or 1.0 ␮g/well in 12-well plates with empty vector DNA. Forskolin (10 –30 ␮M) (a protein kinase A activator) was added to medium after
4 h of transfection. Me2SO was used as a vehicle-only control. After 24
or 48 h of transfection, cells were washed with phosphate-bufferedsaline (PBS) and harvested. Luciferase assays were carried out according to the manufacturer’s protocol with Dual Luciferase assay kit (Promega), and luciferase activity was quantified by using Wallac 1420
multilabel counter (PerkinElmer Life Sciences). As the internal standard, SV40 Renilla luciferase control vector was also cotransfected tonormalize for transfection efficiency. All experiments were performed intriplicate (mean ⫾ S.D.)
GST Pull-down Assay—GST and GST fusion proteins were expressed in Escherichia coli (BL21, DE3) and purified using standard
techniques using pGEX-4T according to the manufacturer’s protocol.
Bacteria culture in ampicillin were grown at 37 °C to A600 of 0.6 before
induction with 100 ␮M isopropyl-1-thio-␤-D-galactopyranoside at 25 °C
for 6 h. Cells were pelleted and resuspended in buffer A (25 mM Hepes(pH 7.9), 50 mM KCl, 6% (v/v) glycerol, 5 mM EDTA, 5 mM MgCl2, 1 mM
DTT, 0.05% Triton X-100, protease inhibitor mixture (Roche Applied
Science), (N-acetyl-leucyl-leucyl-norleucinal 15 ␮g/ml). The cells werelysed on ice by sonication and centrifuged at 14,000 ⫻ g for 10 min at
4 °C. The supernatant was mixed for 10 min at 4 °C for 1 h on a rotatorwith glutathione-Sepharose beads that were preswollen in buffer A.
After absorption, beads were wash three times with buffer A. GST or
GST fusion proteins were eluted with elution buffer (10 mM glutathione,
50 mM Tris-HCl (pH 8.0)) at room temperature for 10 min. The concentration and sizes of GST and GST fusion proteins were estimated by
SDS-PAGE and immunoblotting, using a known quantity of molecularweight standards. After dialysis, the purified samples were stored at⫺80 °C until pull-down assays were performed.
35
L-[ S]Methionine-labeled proteins were prepared by using TNT T7
quick-coupled transcription/translation system (Promega). After GST
or GST fusion proteins were incubated with glutathione-Sepharose
beads (Amersham Biosciences) for 1 h at 4 °C and washed extensively,
a 50% slurry of GST or GST fusion protein-coupled beads were incubated with radiolabeled HNF-4␣ or PGC-1 in binding buffer (2 mM
Hepes-KOH (pH 7.8), 10 mM KCl, 0.2 mM EDTA, 1 mM MgCl2, 10%
glycerol, 1 mM DTT) for 2 h at 4 °C or room temperature. After beads
were washed extensively three times with PBS containing 0.1% Triton
X-100 or 0.1% Nonidet P-40, GST or GST fusion proteins were eluted
with elution buffer (10 mM glutathione, 50 mM Tris-HCl (pH 8.0)) atroom temperature for 10 min, and labeled proteins were analyzed by
SDS-PAGE, and gels were dried and visualized with BAS-2500.
Coimmunoprecipitation—293T cells were grown at 37 °C in an atmosphere of 5% CO2 in Dulbecco’s modified Eagle’s medium (Sigma) containing 100 units/ml penicillin, 100 ␮g/ml streptomycin sulfate andsupplemented with 10% fetal bovine serum. 293T cells were transfectedwith the expression vector for FLAG-HNF-4␣ and c-myc-SREBP-1a byusing FuGENE 6 (Roche Applied Science). Before 5 h of harvesting
cells, N-acetyl-leucyl-leucyl-norleucinal (15 ␮g/ml) was added to themedium as a protease inhibitor. After 48 h of transfection, cells were
harvested on ice, and nuclear proteins were prepared as described
previously (19). Nuclear extracts (800 ␮g) were incubated for 1 h at 4 °C
with anti-FLAG antibody (mouse monoclonal antibody, Promega) andincubated overnight at 4 °C with protein G-agarose matrix. After incubation, the beads were washed three times with PBS containing 0.1%
Nonidet P-40 and analyzed on 6% gel by SDS-PAGE. Western blot
analysis was performed by using anti-c-Myc antibody (mouse monoclonal antibody, Roche Applied Science) and anti-HNF-4␣ antibody(goat polyclonal antibody sc-6556, Santa Cruz Biotechnology).
Electrophoresis Mobility Shift Assay—In vitro translated SREBP-1aand HNF-4␣ proteins were produced using the TNT T7 quick-coupledtranscription/translation system (Promega). Double-stranded oligonucleotides were prepared by combining equal amounts of the complementary single-stranded DNA in TE buffer followed by heating to 95 °C for
5 min and then gradually cooling to 37 °C. The annealed oligonucleotides were labeled by using the Klenow element of E. coli DNA polymerase in the presence of [␣-32P]dCTP. The binding reactions (final
volume of 15 ␮l) were carried out in binding buffer (2 mM Hepes-KOH(pH 7.8), 10 mM KCl, 0.2 mM EDTA, 1 mM MgCl2, 10% glycerol, 1 mM
DTT) with labeled probe and 0.4 ␮g of poly(dI-dC) and TNT reaction
lysate. Reaction mixtures were incubated for 1 h at 4 °C and thenanalyzed on 4% polyacrylamide gel in 0.5⫻ TBE at 90 V for 1 h at room
temperature. Supershift reactions were performed by adding antibody
to the reaction mixture. Antibodies used were sc-6556 (Santa Cruz
Biotechnology) for HNF-4␣ and sc-8984 for SREBP-1 (Santa Cruz Biotechnology). After drying, gels were analyzed by BAS-2500 (Fuji). The
probe sequence for EMSA is as follows: PEPCK GRU probe (5⬘-GAAT-SREBP-1 Inhibition of HNF-4␣/PGC-1 Pathway

12029

FIG. 1. PEPCK promoter activity is induced by HNF-4␣ and forskolin and is repressed by SREBP-1a, -1c, and -2. A, schematic
representation of glucocorticoid-response unit in PEPCK promoter. B, schematic representation of the isoforms and the transactivation domaindeleted mutant form of nuclear SREBP-1 (⌬TA-SREBP-1). C, regulation of PEPCK promoter by HNF-4␣ and SREBPs. HepG2 cells in a 12-wellplate were cotransfected with PEPCK-LUC (⫺480 to ⫹278) vector (400 ng/well), expression vectors for HNF-4␣ (300 ng/well), SREBP, or⌬TA-mutant SREBP-1 (300 ng/well). After 4 h of transfection, forskolin (30 ␮M) or vehicle (Me2SO) was added to medium. Cells were harvested
after 24 h of transfection. The luciferase activity was measured and normalized by Renilla luciferase activity. D, dose-dependent suppression of
HNF-4␣-induced PEPCK promoter by SREBPs. HepG2 cells in a 24-well plate were cotransfected with PEPCK-LUC (⫺480 to ⫹278) vector (100ng/well) and expression vector for HNF4␣ (200 ng/well), together with expression vector for SREBP or mutant SREBP-1 (0, 5, 20, 60, and 200
ng/well). Cells were harvested after 24 h of transfection. The luciferase activity was measured and normalized by Renilla luciferase activity.
HNF-4␣-induced PEPCK promoter activity without SREBPs was set at 100%. The basal promoter activity with empty vector is indicated by ⫻. All
experiments were performed in triplicate.
TCCCTTCTCATGACCTTTGGCCGTGGGAGTGACACCTCACAGCTGTGGTGTTTTGACAAC-3⬘), AF1 site probe (5⬘-GAATTCCTTCTCATGACCTTTGGCCGTGGGAGTGA-3⬘), and LDL-R SRE probe (5⬘-GAAAATCACCCCACTGCAAA-3⬘).
RESULTS

SREBP Inhibits the Transcriptional Activity of HNF-4␣ Induced by Forskolin—In the process of seeking genes downregulated by SREBPs, we identified the PEPCK gene by DNA
microarray analysis of liver RNAs from SREBP-1a transgenic
and wild type mice. The hepatic expression level of PEPCK was
5.3-fold decreased by SREBP-1a overexpression. This result
was consistent with the previous report that adenovirus-mediated overexpression of SREBP-1c in primary hepatocytes represses mRNA of PEPCK gene induced by cAMP and dexamethasone (20). The glucocorticoid-response unit (GRU) has
been designated as a functional region to regulate PEPCK genetranscription by cooperation between glucocorticoid receptor
and other transcriptional factors in the liver (Fig. 1A). It encompasses the glucocorticoid-response element, AF1, AF2, andAF3 sites. AF1 is the binding site of HNF-4␣ and is responsiblefor HNF-4␣ activation of the PEPCK promoter (4). To investigate the role of SREBP as a regulator of PEPCK promoter
activity, we tested whether SREBP could affect the PEPCK
promoter in HepG2 cells by using a luciferase reporter genelinked to the rat PEPCK promoter region containing GRU. As
shown in Fig. 1C, HNF-4␣ expression caused induction of
PEPCK promoter activity. The HNF-4␣ transactivation of the
PEPCK promoter was markedly enhanced by addition of fors-

kolin, a protein kinase A activator (Fig. 1C), presumably due toprotein kinase A phosphorylation of cAMP-response elementbinding protein, another PEPCK transactivator through thecAMP-response element site (Fig. 1A). Addition of SREBPs
resulted in a marked inhibition of the PEPCK promoter activity induced by HNF-4␣ and forskolin (Fig. 1C). Among theisoforms of SREBPs, SREBP-1a and -2 had the stronger inhibitory effect than SREBP-1c. The difference is likely due to theshorter amino-terminal transactivation domain of SREBP-1c
as compared with that of SREBP-1a (Fig. 1B). When the aminoterminal transactivation domain (TA) of SREBP-1 was deleted(⌬TA-SREBP-1, Fig. 1B), the SREBP-1 inhibitory effect on the
PEPCK promoter was completely abolished, although this mutant still possessed binding activity to the sterol regulatory
element (SRE). These results suggest that the amino-terminaltransactivation domain of SREBP is essential for the suppression of HNF-4␣/forskolin-induced PEPCK promoter activity. As
shown Fig. 1D, the PEPCK promoter activity induced solely by
HNF-4␣ was also repressed by SREBPs in a dose-dependent
manner (Fig. 1C). These data indicated that inhibitory action of
SREBPs on PEPCK expression is transcriptional repression
mediated through interference with HNF-4␣ activation.
Suppression of PEPCK Promoter by SREBPs Was Mediatedthrough GRU—To identify the cis-acting element responsible
for the inhibitory effect of SREBP on HNF-4␣, a series ofdeleted PEPCK promoter LUC plasmids was constructed fortransfection studies in HepG2 cells (Fig. 2). In the presence of
GRU, luciferase activity was increased greater than 6-fold by12030SREBP-1 Inhibition of HNF-4␣/PGC-1 Pathway

FIG. 2. GRU is responsible for the inhibitory effect of SREBP-1a on PEPCK promoter activity. HepG2 cells in a 24-well plate were
cotransfected with sequentially deleted PEPCK-LUC vectors as indicated in the left panel (100 ng/well), expression vectors for HNF-4␣ (200ng/well) and SREBP-1a (200 ng/well). Cells were harvested after 24 h of transfection. The luciferase activity was measured and normalized by
Renilla luciferase activity. All experiments were completed in triplicate.

HNF-4␣, and the absence of the GRU (from ⫺335 to ⫹278)
considerably decreased the HNF-4␣ induction, which is consistent with the previous report that the effective cis-acting element for HNF-4␣ is the AF1 site in GRU. However, there still
remained a slight HNF-4␣ induction (2-fold) in this construct,which is presumably due to another DR-1 site (AF3). The
HNF-4␣ induction, as observed in GRU-containing luciferase
constructs, was equally suppressed by SREBP-1a. Deletion of
GRU completely abolished this SREBP suppression. These results demonstrate that GRU is essential for the inhibitory
effect of SREBP on HNF-4␣ activation of the PEPCK promoter.
SRE-like Sequences in GRU Are Not Responsible for the
Inhibitory Effect of SREBP on HNF-4␣—SREBP could function
as a negative regulator of transcription by direct binding to the
SRE as reported for the promoter of microsomal triglyceridetransfer protein (21). As depicted in Fig. 3A, the SRE-likesequences were identified in the GRU of the PEPCK promoter
region. However, EMSAs demonstrated that SREBP-1a did notbind to the GRU of the PEPCK promoter, whereas HNF-4␣
specifically bound to this region (Fig. 3B), excluding the possibility that SREBPs might suppress PEPCK promoter throughtheir direct binding.
SREBP Is a Negative Regulator of HNF-4␣ via PGC-1—
Next, we focused on the HNF-4␣-response element (HNF-4␣RE) in GRU as an SREBP suppression site. The enhancer
construct containing HNF-4␣-RE (AF1 site) was transfected
into HepG2 cells for these studies (Fig. 4A). Activation of
HNF-4␣ RE-LUC by HNF-4␣ was markedly enhanced by
PGC-1 coexpression in a dose-dependent and saturable manner(Fig. 4B). This result is consistent with the previous report that
PGC-1 is an essential coactivator of HNF-4␣ (7). As shown in
Fig. 4C, both SREBP-1a and SREBP-1c efficiently repressed
this PGC-1-mediated HNF-4␣ activity. These data demonstrate the potential for a direct interaction between HNF-4␣
and SREBP in this inhibition.
SREBP Physically Interacts with HNF-4␣ in Vitro and in
Vivo —To test this possibility, we used GST pull-down assays
with GST-SREBP-1 fusion proteins and in vitro translated
HNF-4␣ as the target. As shown Fig. 5A, both GST-SREBP-1aand GST-SREBP-1c, but not GST, were able to pull-down HNF4␣. These results indicate that SREBP-1 could bind HNF-4␣ invitro. The amino-terminal region of SREBP-1a GST-SREBP-1a(1–100 aa) was also able to pull down HNF-4␣. These data
support the concept that the amino-terminal region of
SREBP-1 is responsible for its inhibitory effect on HNF-4␣.
Additionally, under the same conditions, the interaction between SREBP-1a and PGC-1 could not be detected, as shown in
Fig. 5A. Lack of SREBP-1/PGC-1 interaction precludes thepossibility of competition between HNF-4␣ and SREBP-1 in
PGC-1 recruitment as a plausible mechanism for SREBP-1
inhibition of HNF-4␣ activity. Next, to determine the binding

region in HNF-4␣ to SREBP, we constructed expression plasmids for different domains of HNF-4␣. As shown in Fig. 5B,
SREBP-1a-GST protein pulled down both the ligand binding
domain (LBD) (115–359 aa) and AF2 of HNF-4␣ (342– 455 aa)
with slightly less efficiency, whereas the amino-terminal AF1
and DNA binding domains of HNF-4␣ (1–130 aa) did not bind
to SREBP-1a. The LBD/AF2 part (115– 455 aa) showed a comparable signal to the whole molecule of HNF-4␣. These data
indicated that the binding of these two molecules is mediatedthrough interaction between the TA domain of SREBP and
LBD/AF-2 domains of HNF-4␣. Finally, the direct interaction
between SREBP-1a and HNF-4␣ was confirmed in vivo; asshown in Fig. 5C, FLAG-HNF-4␣ was able to coimmunoprecipitate myc-SREBP-1a in 293T cells.
SREBP Competes with PGC-1 Recruitment for Activation of
HNF-4␣—Based upon the direct interaction between SREBP-1
and HNF-4␣, two hypotheses for the mechanism of the SREBP
inhibitory effect on the HNF-4␣-induced PEPCK promoter
could be considered: 1) SREBP-HNF-4␣ interaction impairs theaffinity of HNF-4␣ to bind to HNF4␣-RE, or 2) it interfereswith the recruitment of PGC-1 by HNF-4␣. To examine the first
hypothesis, we performed EMSA by using AF1 (HNF-4␣-RE) in
the PEPCK promoter as a probe. As shown in Fig. 6, A and B,
the binding of HNF-4␣ to HNF-4␣-RE was not influenced byaddition of either SREBP-1a or PGC-1 at the concentrations
tested here. Next, to determine whether SREBP-1 has an inhibitory effect on the interaction between HNF-4␣ and PGC-1,
a Gal4-luciferase assay system for evaluation of the recruitment of PGC-1 by HNF-4␣ was established. HNF-4␣ LBD and
AF2 domain (115– 455 aa) were fused to the carboxyl terminusof Gal4 (Fig. 7A). This assessment of coactivator recruitment
clearly highlighted PGC-1 and not CBP or p300 as an important coactivator for HNF-4␣ (Fig. 7B). Fig. 7C shows that
PGC-1 dose-dependently increased HNF-4␣ signaling and thatboth SREBP-1a and SREBP-1c competitively repressed the
PGC-1-dependent luciferase activities. In contrast, the aminoterminal TA-deleted SREBP mutant had no inhibitory effect on
this activity. These results indicate that SREBP-1 effectively
inhibits the interaction between HNF-4␣ and PGC-1 throughthe amino-terminal TA region of SREBP-1. In this inhibition,
SREBP-1a was more effective than SREBP-1c.
SREBP-1 Competes with PGC-1 in Activation of HNF4␣—As observed above, SREBP-1a repressed the HNF-4␣ activity more efficiently than SREBP-1c. When dose-dependent
inhibition curves for the PGC-1 recruitment were compared
between SREBP-1a and -1c in HepG2 cells, the difference wascancelled at high doses of SREBP-1, suggesting that SREBP-1a
has a higher affinity in the inhibition than SREBP-1c (Fig. 8A).
To investigate further the kinetics between SREBP-1 and
PGC-1 on competition of HNF-4␣ activation, the dose-dependent inhibition by SREBP-1a was also compared with different

SREBP-1 Inhibition of HNF-4␣/PGC-1 Pathway

12031

FIG. 3. SREBP-1a does not bind to
GRU in PEPCK promoter. A, schematic representation of glucocorticoid-response unit in the PEPCK promoter. Two
SRE-like sequences are shown in the
GRU. The HNF-4␣-binding site in AF1 isindicated. B, EMSA with radiolabeledprobes of PEPCK GRU (indicated in A)
and SRE from low density lipoprotein
LDL receptor gene promoter (LDL-R
SRE). In vitro translated HNF-4␣ and
SREBP-1a were incubated with theseprobes at 4 °C for 1 h, and the DNA-protein complex was run on a 4% polyacrylamide gel at 90 V for 1 h. Specific binding
was confirmed by supershift by the respective antibody (ab). NS represents
nonspecific binding of a reticulocyte lysate protein to GRU.

FIG. 4. SREBP-1 suppresses the activity of HNF-4␣ induced by PGC-1. A, schematic representation of HNF-4␣-responsive element
enhancer plasmid (HNF-4␣ RE-LUC vector). Four tandem copies of AF1 containing HNF-4␣-RE (see Fig. 3A) were fused to the native TATA boxof the PEPCK promoter. B, HepG2 cells in a 24-well plate were cotransfected with HNF-4␣ RE-LUC vector (200 ng/well), expression vector for
PGC-1 (0, 30, 100, and 300 ng/well). Cells were harvested after 24 h of transfection. The luciferase activity was measured and normalized by Renillaluciferase activity. C, HepG2 cells in a 24-well plate were transfected with HNF-4␣ RE-LUC vector (200 ng/well), expression vectors for PGC-1 (100ng/well), and SREBP-1a and -1c (100 ng/well). Cells were harvested after 24 h of transfection. The luciferase activity was measured and normalized
by Renilla luciferase activity. All experiments were performed in triplicate.

amounts of PGC-1 in HepG2 cells. As shown Fig. 8B, PGC-1
competitively restored HNF-4␣ activation repressed by
SREBP-1a in a dose-dependent manner. Thus, it could be concluded that SREBP-1 can compete with PGC-1 in its HNF-4␣
activation through SREBP-1 and HNF-4␣ interaction.
Overexpression of SREBP-1 Repressed Expression of Gluconeogenic Enzyme Genes in the Liver—The series of in vitro
and cell culture experiments shown above strongly suggestthat SREBP negatively regulates the PEPCK promoter. To
investigate the influence of SREBP-1 on PEPCK expressionin vivo, livers of transgenic mice overexpressing SREBP-1aand -1c were used. Northern blot analysis demonstrated thatthe high hepatic PEPCK mRNA level as observed in wild typemice at fasting was reduced by overexpression of SREBP-1aand -1c (Fig. 9). Moreover, the mRNA level of Glc-6-Pase,
another HNF-4␣ target gene involved in hepatic glucose output, was also decreased in these transgenic mice. Expressionof HNF-4␣ and Pgc-1 genes was not affected by SREBP-1
overproduction. These results support the hypothesis that
SREBP-1 negatively regulates gluconeogenic genes in vivo,
presumably through interference with PGC-1 recruitment to
HNF-4␣.

12032SREBP-1 Inhibition of HNF-4␣/PGC-1 Pathway

FIG. 5. SREBP physically interacts with HNF4␣ in vivo and in vitro. A, E. coli expressed GST-SREBP-1a, -1c, -1a(1–100 aa), or GST wereincubated at 4 °C for 2 h with in vitro translated 35S-radiolabeled HNF-4␣. E. coli expressed GST-SREBP-1a was incubated in binding buffer at
4 °C for 2 h with in vitro translated 35S-radiolabeled PGC-1. After washing three times with PBS containing 0.1% Triton X-100, the protein-boundbeads were eluted and analyzed on 10% gel by SDS-PAGE and visualized by autoradiography. B, in vitro translated 35S-radiolabeled HNF-4␣
domains were incubated in binding buffer containing BSA (0.1 ␮g/␮l) with GST-SREBP-1a at room temperature for 2 h. After washing three times
with PBS containing 0.1% Nonidet P-40, the protein-bound beads were eluted and analyzed on 12% gel by SDS-PAGE and visualized byautoradiography. C, 293T cells were cotransfected with expression vectors for Myc-tagged SREBP-1a and FLAG-HNF-4␣. After 48 h of transfection, nuclear extracts were prepared and subjected to coimmunoprecipitation (IP) with anti-FLAG antibody. After incubation at 4 °C overnight andwashing three times with PBS containing 0.1% Nonidet P-40, the samples were analyzed on 6% gel by SDS-PAGE and Western blot with
anti-c-Myc antibody (ab) and anti-HNF-4␣ antibody.

FIG. 6. SREBP-1a and PGC-1 do not affect DNA binding activity of HNF-4␣. EMSA with radiolabeled HNF-4␣-binding site (AF1,
HNF-4␣-RE) probe in PEPCK promoter (A and B) was performed. Indicated amounts of in vitro translated HNF-4␣ protein and SREBP-1a protein(A) or PGC-1 protein (B) were pre-incubated with this radiolabeled probe at 4 °C for 1 h, and the DNA-protein complex was run on a 4%
polyacrylamide gel at 90 V for 1 h.
DISCUSSION

This study clearly demonstrates that SREBP inhibits
HNF-4␣ signaling in the induction of PEPCK promoter activity. A series of reporter assays, EMSA, coimmunoprecipitation,and coactivator recruitment assays, indicate that the SREBP
inhibition is mediated through its direct interaction with HNF4␣, leading to impaired recruitment of PGC-1. The direct interaction is through the TA of SREBP and the LBD/AF2 site of
HNF-4␣. It has been shown that the TA of SREBPs determines
transcriptional activity of the SREBP family; SREBP-1c withthe shortest TA has a lower transactivation potential for many
SREBP target genes as compared with SREBP-1a and -2 withlong TA (17, 18). It is plausible that SREBP-1c was less effective in inhibition of HNF-4␣ signaling due to its weaker affinity
for HNF-4. The kinetics in dose-dependent inhibition curves for
SREBP-1a and -1c and lack of suppressive activity in TAdeleted SREBP mutant all support this hypothesis.
Our data indicate that the protein interaction between
HNF-4␣ and SREBP impairs PGC-1 recruitment for HNF-4␣
activation and does not inhibit HNF-4␣ binding to its ciselement. Considering that PGC-1 has been reported to bind to
the AF2 domain of HNF-4␣ (7), identification of LBD/AF2 andnot LBD of HNF-4␣ as an SREBP-binding site can explain themechanism of inhibition. The mode of inhibition is likely to beSREBP-1 Inhibition of HNF-4␣/PGC-1 Pathway

12033

FIG. 7. PGC-1 recruitment and coactivation of HNF-4␣ was inhibited by SREBPs. A, schematic representation of Gal4-HNF-4␣ fusion
protein expression vector and UAS-LUC vector containing eight copies of UAS, Gal4-binding site. Carboxyl-terminal region (115– 455 aa) of
HNF-4␣ protein was fused to Gal4-DNA binding domain for estimation of PGC-1 recruitment and coactivation of HNF-4␣. B, coactivation of
HNF-4␣ by PGC-1. HepG2 cells in 12-well plates were cotransfected with UAS-LUC vector (300 ng/well), expression vectors for Gal4-HNF-4␣
fusion protein (300 ng/well), and PGC-1, CBP, or p300 (400 ng/well). Cells were harvested after 24 h of transfection. Luciferase activity wasmeasured and normalized by Renilla luciferase activity. C, SREBP suppression of PGC-1 coactivation. HepG2 cells in 24-well plates were
cotransfected with UAS-LUC vector (150 ng/well), expression vectors for Gal4-HNF4-␣ fusion protein (150 ng/well), PGC-1 (0, 10, 30, and 100
ng/well), and SREBP-1a, SREBP-1c, or ⌬TA-SREBP-1 (30 ng/well). Cells were harvested after 48 h of transfection. Luciferase activity wasmeasured and normalized by Renilla luciferase activity. All experiments were performed in triplicate.

FIG. 8. SREBP-1a and -1c competes with PGC-1 in coactivation of HNF-4␣. A, dose-dependent inhibition of SREBP-1a and -1c with PGC-1
recruitment to HNF-4␣. HepG2 cells in 24-well plates were cotransfected with UAS-LUC vector (50 ng/well), expression vectors for Gal4-HNF-4␣
fusion protein (50 ng/well), PGC-1(100 ng/well), and SREBP-1a or SREBP-1c (0, 3, 10, 30, 100, and 300 ng/well). Cells were harvested after 48 hof transfection. PGC-1-induced relative luciferase activity was set at 100%. B, competition between SREBP-1a and PGC-1 in PGC-1 recruitment
to HNF-4␣. HepG2 cells in 24-well plates were cotransfected with UAS-LUC vector (50 ng/well), expression vectors for Gal4-HNF-4␣ fusion protein(50 ng/well), PGC-1 (10, 100, and 200 ng/well), and SREBP-1a (0, 10, 30, and 100 ng/well). Cells were harvested after 48 h of transfection.
PGC-1-induced relative luciferase activity is set at 100%. Luciferase activity was measured and normalized by Renilla luciferase activity. All of theexperiments were performed in triplicate.

12034

SREBP-1 Inhibition of HNF-4␣/PGC-1 Pathway

FIG. 9. Expression of gluconeogenic genes was repressed inlivers of SREBP-1 transgenic mouse. Total RNA (10 ␮g/lane) extracted from wild type or transgenic mouse liver was subjected toelectrophoresis and Northern blot hybridization with the indicatedradiolabeled probe.

a competition between SREBP-1a and PGC-1, based upon thedose-dependent kinetic study of SREBP-1 and PGC-1. Meanwhile, it is also possible that binding of SREBP to the LBD of
HNF-4␣ could affect the access of endogenous ligands to HNF4␣, resulting in impairment of PGC-1 recruitment. Physiological HNF-4␣ ligand and the molecular mechanism for HNF-4␣
activation by the ligands are currently an enigma, althoughsome studies including analysis of the three-dimensional structure of HNF-4␣ LBD indicate that the endogenous HNF-4␣
ligand could be palmitate (22). Polyunsaturated fatty acids
have been reported to regulate the transcription of Glc-6-Pasethrough the repression of HNF-4␣ activity in hepatoma cell (5).
Therefore, it is possible that SREBP binding to HNF-4␣ LBD
might affect HNF-4␣ signaling through accessibility of thesepotential ligands or antagonists.
In the livers of SREBP-1a and -1c transgenic mice, mRNAlevels of gluconeogenic enzymes, PEPCK and Glc-6-Pase, weredecreased. Gene expression of Glc-6-Pase as well as PEPCK
seem to be regulated by the HNF-4␣/PGC-1 pathway based
upon recent studies (6, 7) with adenovirus-mediated overexpression of PGC-1 and liver-specific HNF-4␣ knockout mice.
Taken together with our findings on SREBP inhibition of the
HNF-4␣/PGC-1 pathway, SREBPs could be involved in regulation of gluconeogenesis through inhibition of gluconeogenic
genes. From a physiological standpoint, SREBPs, in particular
SREBP-1c, could play a role in regulation of gluconeogenesis,
because it is highly induced in a fed condition for lipogenesis
after the ingestion of excessive carbohydrates as shown in Fig.
10 (23). SREBP-1c inhibition of gluconeogenesis could minimize hepatic glucose output, saving hepatic glucose for lipogenesis. Conversely, in a fasted condition, SREBP-1c expression
was completely suppressed (24, 25) allowing gluconeogenesis tobe sustained for the maintenance of glucose homeostasis. Recently, synthesis of LXR ligand improved glucose tolerance in amodel of diet-induced obesity and insulin resistance. In micetreated with LXR ligand, mRNAs of PEPCK and Glc-6-Pase
were decreased as compared with that of control mice. Since weand other groups (16, 26) demonstrated previously that thetranscription of SREBP-1c is strongly induced through LXR invitro and in vivo, it could be speculated that suppression of
gluconeogenic genes by the LXR ligand could be mediatedthrough a concomitant increase in SREBP-1c.
Recently we reported that cross-talk between PPARs and
LXRs could be involved in reciprocal nutritional regulation of
fatty acid metabolism (15, 16). Our current finding that theprotein interaction between HNF-4␣ and SREBP-1c affects
HNF-4␣ signaling suggests that transcription factors involved
in glucose and lipid metabolism are highly related in mutual
regulation, and provides another example for cross-talk in a

FIG. 10. Role of SREBP in the transcriptional regulation of
gluconeogenic genes. Molecular mechanism in which SREBP inhibits the HNF-4␣-dependent PEPCK promoter activity. SREBP competition with PGC-1 recruitment to HNF-4␣ could be either due to physicalhindrance or impaired accessibility of HNF-4 ligands.

network of nutritional transcription factors. Although the roleof PGC-1 in HNF-4␣ regulation of gluconeogenic genes has
been established, the mechanism for HNF-4␣ signaling in other
HNF-4␣ target genes is not fully understood. Modes of SREBP
effects on other HNF-4␣ signaling could be different fromSREBP inhibition of HNF-4␣ signaling in gluconeogenesis. In
fact, SREBP overexpression did not affect hepatic expression of
apolipoprotein genes that have been shown to be HNF-4␣ targets in HNF-4␣ knockout mice (27). Conversely and very intriguingly, in the light of SREBP signaling, HNF-4␣/SREBPinteraction enhances SREBP transactivation of sterol regulatory genes in some conditions (33). The physiological consequences of HNF-4␣/SREBP interaction could depend upon target genes, coactivators, and cell types.
Insulin is the most potent negative regulator of PEPCK gene
expression. SREBP-1c has been thought to be induced as consequences of insulin/glucose effects in the liver (28, 29). Thus, it
is tempting to speculate that insulin suppression of PEPCK
expression could be at least partly mediated through SREBP
inhibition of PEPCK expression. However, this concept awaits
clarification of the precise molecular mechanism for nutritional
induction of SREBP-1c. The HNF-4␣ and SREBP-1c interaction in hepatic glucose output should be estimated in relevanceto the pathophysiology of hepatic insulin resistance and diabetes. Considering a role of HNF-4␣ as a causative gene formaturity onset diabetes of young (MODY1) (30 –32), it would
also be interesting to investigate roles of SREBP and HNF-4␣
in pancreatic ␤ cells in terms of insulin expression, secretion,and diabetes.
The current studies propose a novel role of SREBP in thetranscriptional regulation of gluconeogenesis and suggest that
the SREBP-1 functions not only as a lipid regulator but also asa regulator of glucose metabolism via the transcriptional regulation of gluconeogenesis.

SREBP-1 Inhibition of HNF-4␣/PGC-1 Pathway
Acknowledgments—We thank A. H. Hasty for critical reading of themanuscript. We also thank T. Yokoo for technical advice and helpful
discussions.
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