DIABETES-INSULIN-GLUCAGON-GASTROINTESTINAL
Insulin Stimulates the Expression of Carbohydrate
Response Element Binding Protein (ChREBP) by
Attenuating the Repressive Effect of Pit-1, Oct-1/Oct-2,
and Unc-86 Homeodomain Protein Octamer
Transcription Factor-1
Adam S. Sirek, Ling Liu, Mark Naples, Khosrow Adeli, Dominic S. Ng, and Tianru Jin
Departments of Physiology (A.S.S., D.S.N., T.J.), Medicine (D.S.N., T.J.), and Laboratory Medicine and Pathobiology
(D.S.N., T.J.), University of Toronto, Toronto, Canada M5S 3G3; Li Ka Shing Knowledge Institute (A.S.S., D.S.N.), St.
Michael’s Hospital, Toronto, Canada M5B 1W8; Toronto General Research Institute (A.S.S., L.L., T.J.), University Health
Network, Toronto, Canada M5G 1L7; and Department of Molecular Structure and Function (M.N., K.A.), Research
Institute, The Hospital for Sick Children, Toronto, Canada M5G 1X8
The carbohydrate response element binding protein (ChREBP) has been recognized as a key controller of hepatic lipogenesis. Whereas the function of ChREBP has been extensively investigated,
mechanisms underlying its transcription remain largely unknown, although ChREBP production is
elevated in a hyperinsulinemic mouse model. We located a conserved Pit-1, Oct-1/Oct-2, and Unc-86
(POU) protein binding site (ATGCTAAT) within the proximal promoter region of human ChREBP.
This site interacts with the POU homeodomain protein octamer transcription factor-1 (Oct-1), as
detected by gel shift and chromatin immunoprecipitation assays. Oct-1 cotransfection in the human HepG2 cell line repressed ChREBP promoter activity approximately 50 –75% (P ⬍ 0.01 to P ⬍
0.001), and this repression was dependent on the existence of the POU binding site. Furthermore,
overexpression of Oct-1 repressed endogenous ChREBP mRNA and protein expression, whereas
knockdown of Oct-1 expression, using a lentivirus-based small hairpin RNA approach, led to increased ChREBP mRNA and protein expression. In contrast, HepG2 cells treated with 10 or 100 nM
insulin for 4 or 8 h resulted in an approximately 2-fold increase of ChREBP promoter activity (P ⬍
0.05 to P ⬍ 0.01). Insulin (10 nM) also stimulated endogenous ChREBP expression in HepG2 and
primary hamster hepatocytes. More importantly, we found that the stimulatory effect of insulin
on ChREBP promoter activity was dependent on the presence of the POU binding site, and insulin
treatment reduced Oct-1 expression levels. Our observations therefore identify Oct-1 as a transcriptional repressor of ChREBP and suggest that insulin stimulates ChREBP expression via attenuating the repressive effect of Oct-1. (Endocrinology 150: 3483–3492, 2009)
C
onversion of excess dietary carbohydrates into triglycerides
occurs primarily within the liver. An increase in dietary carbohydrates leads to increased expression of several key regulatory enzymes that are involved in lipogenesis, including L-type
pyruvate kinase, acetyl Co-A carboxylase, and fatty acid synthase (FAS). Until recently, insulin was thought to be the main
inducer of lipogenic gene expression, mediated via the sterol
regulatory element binding protein (SREBP)-1c (1). This transcription factor, a member of the basic helix-loop-helixleucine-zipper family, induces lipogenic gene expression
through binding to the sterol regulator element (SRE) within
the promoter sequence of its target genes (2– 4). However, in
SREBP-1c⫺/⫺ mice, there is only about 50% reduction in fatty
acid synthesis (5). Thus, SREBP-1c alone does not account for
ISSN Print 0013-7227 ISSN Online 1945-7170
Printed in U.S.A.
Copyright © 2009 by The Endocrine Society
doi: 10.1210/en.2008-1702 Received December 4, 2008. Accepted March 31, 2009.
First Published Online April 9, 2009
Abbreviations: ChIP, Chromatin immunoprecipitation; ChREBP, carbohydrate response
element binding protein; FAS, fatty acid synthase; FBS, fetal bovine serum; GFP, green
fluorescent protein; GSK, glycogen synthase kinase; hChREBP, human ChREBP; LUC, luciferase; MEK, MAPK kinase; mut, mutant; Oct-1, octamer transcription factor-1; oxLDL,
oxidized low-density lipoprotein; POU, Pit-1, Oct-1/Oct-2, and Unc-86 homeodomain protein family; PPAR, peroxisome proliferator-activated receptor; shRNA, small hairpin RNA;
SRE, sterol regulator element; SREBP, sterol response element binding protein; T2DM, type
2 diabetes mellitus; wt, wild type.
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all lipogenic activity in response to glucose and insulin
signaling.
Studies in recent years identified the carbohydrate response
element binding protein (ChREBP, also known as MLX interacting protein like) as another important activator of lipogenic
gene expression (6, 7). ChREBP, also a member of the basic
helix-loop-helix-leucine-zipper family, is primarily expressed in
the liver and may act in concert with its functional heterodimeric
partner, MLX (8 –11). ChREBP imparts transcriptional control
via binding to the carbohydrate response element of its lipogenic
target gene promoters (8, 11–16), and a high carbohydrate diet
induces the DNA binding activity of ChREBP (7). ChREBP is
required for both basal and carbohydrate-induced expression of
several liver enzymes for coordinated control of glucose metabolism and fatty acid synthesis (7, 15, 17–19). ChREBP⫺/⫺ mice
show reduced expression of mRNA levels encoding malic enzyme and L-type pyruvate kinase along with decreased lipogenesis (20). mRNA that encodes acetyl Co-A carboxylase and FAS
were also shown to be reduced in ChREBP⫺/⫺ mice (18). In a
hyperinsulinemic mouse model, however, liver ChREBP expression levels were increased (21).
Although the function or posttranslational regulation of
ChREBP in response to the elevation of glucose levels has been
extensively investigated (22), very little is known about the transcriptional regulation of ChREBP expression. One recent study
has shown the isolation of the rat ChREBP gene promoter and a
search for potential cis elements that may regulate the expression
of this promoter (23). We show here the isolation of the human
ChREBP gene promoter and the location of a Pit-1, Oct-1/
Oct-2, and Unc-86 (POU) homeodomain protein biding motif
(ATGCTAAT) within the proximal promoter region of the
ChREBP gene. We found that ChREBP promoter activity and endogenous ChREBP expression were negatively regulated by the
POU homeodomain protein octamer transcription factor-1 (Oct1). In contrast, insulin was shown to stimulate ChREBP promoter
and endogenous ChREBP expression. More importantly, the stimulatory effect of insulin on ChREBP promoter activity was found to
be dependent on the presence of the POU protein binding site, and
insulin treatment reduced Oct-1 expression level. We suggest that
this represents a novel mechanism by which insulin exerts its physiological and pathological effect on lipogenesis.
hChREBP-LUC [mutant (mut)]. As illustrated (see Fig. 2A), all the fusion
gene constructs contain the wt Oct-binding site (ATGCTAAT) with the
exception of the ⫺123-bp hChREBP-LUC (mut) in which the Oct-binding
site is mutated (ATACGGAT).
Materials and Methods
Cell culture, DNA transfection, and LUC reporter gene
analysis
The HepG2 cell line was grown in ␣-MEM (Invitrogen Life Technologies), enriched with 5% fetal bovine serum (FBS), and cultured without antibiotics for all the experiments. The COS-7 fibroblasts were cultured in DMEM-Low (Invitrogen Life Technologies) containing 5%
FBS. DNA transfection was performed using Lipofectamine 2000 as per
the manufacturer’s instruction (Invitrogen Life Technologies). LUC reporter gene analyses were performed as previously described (24). The
MAPK kinase (MEK) inhibitor PD98059 was purchased from Calbiochem (EMD Biosciences, Inc., San Diego, CA). Methods for isolating and
culturing primary hepatocytes from Syrian golden hamsters have been
reported previously (25, 26). Briefly, livers from anesthetized (isoflurane) male hamsters weighing 100 –120 g (Charles River, Montreal,
Québec, Canada) were perfused and digested using specialized media
(Life Technologies) according to the manufacturer’s instructions. Hepatocytes released from digested liver tissue were washed twice in hepatocyte wash medium, resuspended in attachment media (Williams E containing 5% FBS, 1 ␮g/ml insulin, and antibiotics), and seeded in Primaria
cell culture plates (BD Biosciences) at a density of 1.1 ⫻ 106 cells per
35-mm dish. After a 4-h attachment period, the cells were cultured in
serum-free ␣-MEM for 4 h before insulin treatment. All animal protocols
were approved by the animal ethics committee at the Hospital for Sick
Children (Toronto, Ontario, Canada).
RNA extraction and real-time RT-PCR
RNA was extracted using Trizol reagent (Invitrogen Life Technology) as
per the manufacturer’s instructions. Real-time RT-PCR was conducted with
Rotor-Gene 3000 (Corbett Research, Dorval, Québec, Canada), using the
SYBR Green PCR master mix (Applied Biosystems, Foster City, CA) as
described previously (27). DNA sequences of the primers for real-time
RT-PCR in detecting hChREBP are: forward, 5⬘-GGTCACTTCATGGTGTCGTC-3⬘, and reverse, 5⬘-CACATCTGTAGGCCAGGCT-3⬘.
Expression levels of ChREBP mRNA are presented as relative copy numbers (with the untreated samples defined as 1) and normalized to the
expression of ␤-actin mRNA.
Western blotting and nuclear extraction
Antibodies against Oct-1, glycogen synthase kinase (GSK)-3, phospho-GSK-3, ERK, phospho-ERK, and ␤-actin were purchased from
Santa Cruz Biotechnology Inc. (Santa Cruz, CA). The antibody for
ChREBP was purchased from Novus Biologicals (Novus Biologicals,
Littleton, CO). Methods for nuclear protein extraction, cytosolic content, whole-cell protein preparation, and Western blotting have been
described previously (27).
Plasmid construction
The plasmid constructs pcDNA3.1-Oct-1 and pcDNA3.1-Cdx-2
were generated by inserting a copy of the Oct-1 (NM_002697) or Cdx-2
coding sequence into the pcDNA3.1/Myc/HisA vector (Invitrogen Life
Technology, Burlington, Ontario, Canada). A 1.4-kb human ChREBP
gene 5⬘ flanking sequence was isolated from the human hepatocellular
carcinoma cell line HepG2 (GenBank no. FG480402). Consensus transcription binding sites within this promoter region were identified using
the online transcription element search system (TESS), provided by Computational Biology and Informatics Laboratory, School of Medicine,
University of Pennsylvania (http://www.cbil.upenn.edu/tess). Four
human ChREBP (hChREBP)-luciferase (LUC) fusion gene constructs
were generated and designated as ⫺1.4-kb hChREBP-LUC, ⫺328-bp
hChREBP-LUC, ⫺123-bp hChREBP-LUC [wild type (wt)], and ⫺123-bp
EMSA
Method for EMSA or gel shift assay, with the undenatured PAGE,
have been previously described (28). The POU-binding site containing
probe (ChREBP-POU probe) was made by annealing two complementary oligo nucleotides: ChREBP-POU forward, 5⬘-GATCTGCGTAAGGATTATGCTAATACAAGCCCCGCG-3⬘, and ChREBP-POU reverse,
5⬘-GATCCGCGGGGCTTGTATTAGCATAATCCTTACGCA-3⬘. The
position of the mut POU binding site in the hChREBP promoter is indicated in Fig. 1A. The POU-binding site containing probe (ChREBPPOUmut) was made by annealing the following two complementary
oligo nucleotides: ChREBP-POUmut forward, 5⬘-GATCTGCGTAAGGATTATACGGATACAAGCCCCGCG-3⬘, and ChREBP-POUmut reverse, 5⬘-GATCCGCGGGGCTTGTATCCGTATAATCCTTACGCA-3⬘.
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Chromatin immunoprecipitation (ChIP)
The method of ChIP assay in assessing DNAprotein interactions within the intact living cells
has been previously described (29). Briefly,
HepG2 cells incubated with or without insulin
(10 nM) for 4 h were treated with 1% formaldehyde for 10 min to cross-link chromatin DNA
and nuclear proteins. After a neutralization procedure, chromatin DNA in the harvested samples
was sheared by sonication to an average length of
600 bp. The Oct-1 antibody was then used (final
dilution of 1:200) to precipitate the sheared chromatin. After washing, elution, reverse cross-linking, and purification, approximately one twentieth of the purified DNA (2 ␮l) was taken for
each of the PCR or real-time PCRs. DNA sequences of the primers used in the ChIP assay
are: POU forward, 5⬘-CAGGACTCCAAGGAAAGACG-3⬘, POU reverse, 5⬘-GTCTGTGTCCGAGTCCGAGT-3⬘; intron-I forward (as control), 5⬘-AGGGCATCTAAGGTCCTGGT-3⬘, and
intron-I reverse (as control): 5⬘-CCCCAGCTATCTCTGACTGG-3⬘.
Oct-1 overexpression and lentiviral
Oct-1 small hairpin RNA (shRNA)
knockdown
A pool of HepG2 cell clones that overexpress
Oct-1 were generated by stably expressing
pcDNA3.1-Oct-1 with antibiotic (G418) selection. Lentiviral knockdown of Oct-1 was performed using the pGIPZ lentiviral shRNA mir
system (ThermoFischer Scientific, Huntsville,
AL), in which green fluorescent protein (GFP)
expression served as the indication of virus infection efficiency.
Statistical analysis
Quantitative results are expressed as a
mean ⫾ SD. Statistical analyses were performed
using the Student’s t test or ANOVA. Null hypotheses were rejected at P ⬎ 0.05. All experiments were performed independently as a minimum of triplicates.
Results
FIG. 1. Oct-1 binds to the ChREBP promoter. A, An illustration of the rat, mouse, and human ChREBP proximal
promoter regions. Oct, Evolutionarily conserved POU protein binding site; SP-1, specificity protein-1; SRE,
potential binding site of SREBP; TATA, TATA box; TSS, transcription start site; Y box, consensus binding site for Y
box transcription factors. DNA sequence information was obtained from GenBank (NM_032951, human;
NM_133552, rat; and NM_021455, mouse). B and C, EMSA shows the interaction between HepG2 nuclear
proteins and the POU binding site. Ab, Antibody; C1, complex 1; C2, complex 2; FP, free probe; GATA-1; GATA
binding protein 1; NE, nuclear extract. D, An illustration of the positions of the primers used in the ChIP assay. E,
HepG2 cells were treated with or without insulin (10 nM) for 4 h before harvested for the ChIP assay with the
Oct-1 antibody. IP, Oct-1 precipitated chromatin DNA was used as the targets for PCR using POU or intron-1
control primers; input, PCR was conducted using sonicated chromatin DNA samples. F, Quantitative assessment
of the effect of insulin on the binding of Oct-1 to the POU binding motif by real-time PCR after the ChIP assay in
HepG2 cells. Values are presented as arbitrary copy number, with the untreated sample defined as 1 (n ⫽ 3).
Oct-1 interacts with the POU protein
binding site within the proximal
promoter region of ChREBP
After the isolation of the 1.4-kb human
ChREBP gene 5⬘ flanking sequence, we
identified the existence of a typical POU
protein binding site at the position between
⫺103 and ⫺96 bp. This octamer motif
(ATGCTAAT) and the flanking sequences
were found to be conserved between human
and rodent species (Fig. 1A). The SRE motif
identified recently in the rat ChREBP gene
promoter region, which is also conserved in
the mouse ChREBP gene promoter, how
ever, was not found in the hChREBP pro-
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FIG. 2. Oct-1 cotransfection represses ChREBP promoter activity. A, A schematic illustration of the four
hChREBP-LUC fusion gene constructs. TATA, TATA box; Oct, the POU binding site. B, Cos-7 cells were
cotransfected with 2 ␮g ⫺328 bp hChREBP-LUC along with indicated amount of pcDNA3.1 or pcDNA3.1Oct-1. C, HepG2 cells were cotransfected with 2 ␮g ⫺328 bp hChREBP-LUC along with indicated amount
of pcDNA3.1, pcDNA3.1-Oct-1, or pcDNA3.1-Cdx-2. D, HepG2 cells were cotransfected with 2 ␮g either
the wt or mut ⫺132 bp hChREBP-LUC along with indicated amount of pcDNA3.1 or pcDNA3.1-Oct-1. LUC
reporter gene activity was assessed 18 h after the transfection. Results are presented as relative LUC
activity, with the values obtained from the control vector (pcDNA3.1) cotransfected cells as 1-fold. *, P ⬍
0.05; **, P ⬍ 0.01; †, P ⬍ 0.001.
moter region (Fig. 1A). Except for the ubiquitously expressed
Oct-1, no other POU homeodomain proteins are abundantly
expressed in the hepatocytes. To examine whether Oct-1 interacts with this binding site, we conducted EMSA using nuclear proteins isolated from HepG2 cells. As shown in lane 2
of Fig. 1B, HepG2 nuclear proteins formed two complexes
with the ChREBP-POU probe: a major complex C1 and a
minor complex C2. To investigate whether one of them represents the interaction between Oct-1 and the ChREBP-POU
probe, Oct-1 antibody and a control GATA-1 antibody were
used in the EMSA. The preincubation of nuclear proteins with
the Oct-1 antibody (lane 3) but not the control GATA-1 antibody (lane 4) resulted in a retarded migration of C1, suggesting that the major complex C1 contains Oct-1.
The retardation of C1 in the presence of Oct-1 antibody in the
undenatured PAGE was minimal (Fig. 1B). To further verify that
this does represent a supershifting event, we extended the separation time by the PAGE and observed with two different dosages
of Oct-1 antibody a clear retardation in the migration of the C1
complex (Fig. 1C, comparing lanes 3 and 4 with lane 2). Furthermore, the addition of excess amount of unlabeled ChREBPPOU probe (lanes 7 and 8), but not the mut one (lanes 5 and 6),
completely blocked the formation of the C1 complex, indicating
that the ATGCTAAT motif is essential for the formation of C1.
We therefore conclude that Oct-1 binds to the evolutionarily
conserved POU protein binding site within the human ChREBP
promoter region.
To determine that the interaction between Oct-1 and the POU binding motif
within the proximal region of the hChREBP
promoter occurs in the living hepatocytes,
we conducted an investigation using the
ChIP approach. Figure 1D shows the positions of the POU binding site, TATA box,
and the testing as well as the control primers
used in the ChIP assay. Figure 1E shows that
Oct-1 antibody precipitated the POU binding site containing chromatin DNA but not
the control DNA within the Intron I region
of the hChREBP gene. After regular PCR
for detection, we also found that 4 h insulin
treatment substantially reduced the amount
of POU binding site containing chromatin
DNA precipitated by the Oct-1 antibody
(Fig. 1E). Quantitatively analysis by realtime PCR demonstrated that insulin treatment resulted in an approximately 70% reduction (P ⬍ 0.001, Fig. 1F). We then
assessed Oct-1 content in ChIP samples by
Western blotting after the reverse crosslinking procedure. The Oct-1 content in
the HepG2 cells treated with insulin was
significantly decreased (supplemental
Fig. 1, published as supplemental data on
The Endocrine Society’s Journals Online
web site at http://endo.endojournals.org).
Oct-1 functions as a transcriptional repressor of ChREBP
To investigate whether the binding of Oct-1 to the hChREBP
promoter affects its gene transcription, LUC reporter gene analyses were performed using four hChREBP-LUC reporter gene
constructs (Fig. 2A). When the COS-7 naïve cell system was
examined, pcDNA3.1-Oct-1 cotransfection was shown to repress the activity of ⫺1.4 kb hChREBP-LUC (supplemental Fig.
2) and ⫺328 bp hChREBP-LUC in a dose-dependent manner
(Fig. 2B). Cotransfection with 4 or 6 ␮g pcDNA3.1-Oct-1 led to
a 65 and 90% repression, respectively. Similar experiments
were carried out in the ChREBP-expressing HepG2 cell line. As
shown in Fig. 2C, 4 or 6 ␮g Oct-1 cotransfection resulted in
approximately 50% (P ⬍ 0.01) and 75% (P ⬍ 0.01) reduction
of the reporter activity of ⫺328 bp hChREBP-LUC, respectively.
Similar results were observed for ⫺1.4 kb hChREBP-LUC (supplemental Fig. 3). Cotransfection with 4 or 6 ␮g Cdx-2, an unrelated homeobox gene, generated no appreciable repression,
indicating the specificity of the repressive effect of Oct-1 (Fig.
2C). To confirm the involvement of the POU binding site in
Oct-1-mediated repression, HepG2 cells were cotransfected with
Oct-1 and ⫺123 bp hChREBP-LUC (wt) or Oct-1 and ⫺123 bp
hChREBP-LUC (mut). Approximately 45% (P ⬍ 0.001) and
50% (P ⬍ 0.001) repression on the activity of ⫺123 bp
hChREBP-LUC (wt) were observed when 4 or 6 ␮g Oct-1 were
applied in the cotransfection, respectively (Fig. 2D). The repressive effect of Oct-1, however, was not observed for the mut counterpart (Fig. 2D).
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Lentiviral mediated knockdown of
Oct-1 results in increased ChREBP
expression
To further test the hypothesis that Oct-1
functions as a transcriptional repressor, we
examined ChREBP expression in response
to Oct-1 knockdown by lentivirus-mediated shRNA in HepG2 cells. In both the
control (scrambled shRNA) and the Oct-1
shRNA-containing lentivirus system, the
expression of GFP served as the indicator of
lentivirus infection. Figure 4A shows the
nucleotide sequences of three shRNA constructs against human Oct-1. After the infection with either control or Oct-1
shRNA-containing lentivirus, the infection
efficiency was monitored by detecting GFP
expression (Fig. 4B). Figure 4C shows that,
compared with mock infected and control
virus infected HepG2 cells, Oct-1 protein
expression in the HepG2 cells that were inFIG. 3. Oct-1 overexpression represses endogenous ChREBP expression. A, HepG2 cells were transiently
fected with Oct-1 shRNA dropped to 49%
transfected with indicated amount of Myc-tagged Oct-1. The expression of Oct-1, Myc-tagged Oct-1,
(P ⬍ 0.05). The expression of ChREBP proChREBP, and ␤-actin (loading control) were detected by Western blotting (left panel). Right panel,
Densitometric analysis of the results from left panel (n ⫽ 3). B, HepG2 cells were stably transfected with
tein in those cells, however, increased 2.3Myc-tagged Oct-1 (Myc-Tag). The expression of Oct-1, Myc-tagged Oct-1, ChREBP, and ␤-actin
fold (P ⬍ 0.01). The reporter gene activity
(loading control) were detected by Western blotting (left panel). wt, wt HepG2; S-Oct, HepG2 cells
of ⫺328 bp hChREBP-LUC in the mock
that stably express Myc-tagged Oct-1. Right panel, Densitometric analysis of the results from the
left panel (n ⫽ 3). C, wt (⫺) or Oct-1 overexpressing HepG2 cells were transfected with ⫺328 bp
infected HepG2 cells and the scrambled
hChREBP-LUC. Cells were harvested for LUC reporter analysis. Data are presented as relative LUC
shRNA-expressing HepG2 cells were comactivity, with the value in the insulin untreated wt HepG2 cells defined as 1-fold (n ⫽ 3). D, Detection
parable, whereas the reporter gene activity
of ChREBP expression by real-time RT-PCR in the presence and absence of insulin (10 nM). *, P ⬍ 0.05;
**, P ⬍ 0.01.
of this fusion gene construct was 103%
higher (P ⬍ 0.001) in the HepG2 cells expressing the Oct-1 shRNA (Fig. 4D). FiTo examine whether Oct-1 affects endogenous ChREBP
nally, we assessed ChREBP mRNA expression in the three cell
expression, we first transiently transfected HepG2 cells with
populations by real-time RT-PCR. ChREBP expression level
4 or 6 ␮g of Myc-tagged Oct-1. The expression of exogenous
in the cells that express Oct-1 shRNA was 126% (P ⬍ 0.01)
Myc-tagged Oct-1 was detected by the anti-Myc-tag antibody,
higher than that of the mock infected or control shRNA-inand the overall increase of Oct-1 expression was detected by
fected HepG2 cells (Fig. 4E). In all three cell populations,
the Oct-1 antibody (Fig. 3A). Compared with the mock transinsulin treatment significantly stimulated ChREBP mRNA exfected cells, Oct-1 transfection moderately (less than 30%)
pression (Fig. 4E).
but significantly repressed endogenous ChREBP protein expression, as detected by Western blotting (Fig. 3A, left panel),
Insulin stimulates ChREBP expression
followed by a densitometric analysis (Fig. 3A, right panel).
As shown in Figs. 3D and 4E, insulin treatment stimulated
The lack of a robust repressive effect of Oct-1 on endogendogenous ChREBP mRNA expression. To further assess the
enous ChREBP protein expression in Oct-1 transient transstimulatory effect of insulin, we transfected HepG2 cells with the
fection could be due to the low transfection efficiency of this
hChREBP-LUC reporter gene. Eighteen hours after the transfeccell line. To circumvent this limitation, we generated HepG2
tion, cells were washed with serum-free medium and incubated
cells that stably express Myc-tagged Oct-1 and assessed the
with or without insulin for 4 or 8 h. As shown in Fig. 5, A and
effect of Oct-1 overexpression on ChREBP expression. ComB, insulin at the concentration of 10 or 100 nM stimulated the
pared with the control, ChREBP protein (Fig. 3B), promoter
activity of ⫺328 bp hChREBP-LUC approximately 2-fold. Sim(Fig. 3C), and mRNA (Fig. 3D) expression in Oct-1 stably
ilar observation was made when the ⫺1.4 kb hChREBP-LUC
transfected HepG2 cells was reduced 50 –70%. We also found
was examined (supplemental Fig. 4). No response was observed
that insulin stimulates ChREBP protein and mRNA (Fig. 3D)
with 1 nM of insulin treatment (data not shown). The lack of
expression (discussed below), consistent with the in vivo
dosage-dependent activation may suggest that the stimulation by
observation that ChREBP production is increased in the
insulin at 10 nM reaches the maximum in our experimental conhyperinsulinemic mice (21). In the HepG2 cells that overexditions. When ⫺123 bp hChREBP-LUC (wt) was examined, 2,
press Oct-1, the stimulatory effect of insulin was attenuated
4, and 8 h insulin treatment significantly stimulated its activity
(Fig. 5C). The fusion gene ⫺123 bp hChREBP-LUC (mut)
(Fig. 3D).
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FIG. 4. Knockdown of Oct-1 expression leads to increased ChREBP expression. A, Nucleotide sequences of three shRNA against human Oct-1. hs, mm, Nucleotide
sequence that matches Oct-1 cDNA in both humans and mice; hs, nucleotide sequence that matches Oct-1 cDNA in humans only. B, Detection of GFP expression in the
control (scrambled) and Oct-1 shRNA-expressing HepG2 cells. DIC, Differential Interference Contrast. C, Detection of Oct-1, ChREBP, and ␤-actin (loading control)
expression in mock infected (⫺), scramble (Scr), and Oct-1 shRNA (Oct)-expressing HepG2 cells by Western blotting (left panel). Right panel, Densitometric analysis of
the results from left panel (n ⫽ 3). D, wt (⫺), control shRNA (scrambled, Scr), or Oct-1 shRNA-expressing HepG2 cells were transfected with ⫺328 bp hChREBP-LUC.
Cells were harvested for LUC reporter gene analysis. Data are presented as relative LUC activity, with the value in the insulin untreated wt HepG2 cells defined as 1-fold
(n ⫽ 3). E, Detection of ChREBP mRNA expression in wt (⫺), scramble (Scr), and Oct-1 shRNA-expressing HepG2 cells in the presence or absence of insulin (10 nM), by
real-time RT-PCR. *, P ⬍ 0.05; **, P ⬍ 0.01; †, P ⬍ 0.001.
showed significantly higher basal activity, compared with that of
the wt counterpart. Although 8 h insulin treatment significantly
stimulated the activity of ⫺123 bp hChREBP-LUC (mut), the
stimulation level (1.7-fold) was lower than that in the wt counterpart (2.5-fold) (Fig. 5C). Nevertheless, 2 and 4 h insulin treatment generated no significant stimulation on the activity of
⫺123 bp hChREBP-LUC (mut) (Fig. 5C). These observations
suggest that the stimulatory effect of insulin on hChREBP promoter is at least partially dependent on the POU protein binding
site.
Insulin treatment attenuates Oct-1 expression level
To investigate how insulin treatment reverses the repressive
effect of Oct-1, we assessed the effect of insulin on the expression
of Oct-1 and the known insulin downstream target GSK-3. As
shown in supplemental Fig. 5, insulin treatment stimulated
GSK-3 phosphorylation in the HepG2 cells within the 2-h experimental period. Interestingly, nuclear Oct-1 content reduced,
starting after 30 min of insulin treatment (Fig. 5D, top panel).
We, however, were not able to detect Oct-1 in the cytosolic content of the HepG2 cells (Fig. 5D, bottom panel). This could be
due to the fact that the amount of Oct-1 in the cytosol is beyond
the detection by Western blotting. Alternatively, this may be due
to the potential activation of Oct-1 degradation pathways after
insulin stimulation. We then assessed the effect of insulin on
Oct-1 expression levels in the whole-cell lysates. As shown in Fig.
5E, in both the wt and Oct-1-overexpressing HepG2 cells, 4 and
8 h insulin treatment reduced Oct-1 content, associated with
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FIG. 5. Insulin stimulates ChREBP promoter and endogenous ChREBP expression. A and B, HepG2 cells were transfection with 2 ␮g ⫺328 bp hChREBP-LUC for 18 h.
The cells were then incubated with serum-free medium containing 10 nM (A) or 100 nM (B) insulin for indicated hours before harvested for LUC reporter analysis. C,
HepG2 cells were transfected with 2 ␮g ⫺123 bp ChREBP-LUC (wt) or its mut counterpart for 18 h before the incubation with insulin (10 nM) for indicated hours. Cells
were then harvested for LUC reporter analysis. D, HepG2 cells were treated with insulin (10 nM) for indicated time. Nuclear and cytosolic contents were extracted and
subject to Western blotting for the detection of Oct-1. Histone H3 and ␤-actin, Loading controls. E, F, and G, wt HepG2 and HepG2 cells stably transfected with Oct-1
(S-Oct) (E), scrambled shRNA (Scr), and Oct-1 shRNA (Oct)-expressing HepG2 cells (F), and hamster primary hepatocyte cultures (G), were treated with or without insulin
(10 nM) for indicated hours. Whole-cell lysates were prepared and the expression of ChREBP, Oct-1, phosphorylated ERK (pERK), total ERK, and ␤-actin (loading control)
were examined by Western blotting (representative blots, n ⫽ 3). H, HepG2 cells were transfected with ⫺328 bp hChREBP-LUC for 18 h. The cells were treated with or
without insulin (10 nM) in the presence or absence of the MEK inhibitor PD98059 (pd). DMSO, Dimethyl sulfoxide. For A, B, C, and H, data are presented as relative
LUC activity, with the values in the insulin-untreated cells defined as 1-fold (n ⫽ 3). *, P ⬍ 0.05; **, P ⬍ 0.01; †, P ⬍ 0.001.
increased ChREBP expression and the phosphorylation of ERK,
a downstream target of insulin. Similar observations were then
made in both the control shRNA- and Oct-1 shRNA-expressing
HepG2 cells (Fig. 5F). We then extended this observation in the
primary hepatocyte cultures from Syrian golden hamsters. We
found that indeed 4 and 8 h insulin treatment stimulated
ChREBP protein expression along with the concomitant decrease of Oct-1 expression (Fig. 5G). Using the MEK inhibitor
PD98059, we found that the stimulatory effect of insulin on the
hChREBP promoter was blocked by MEK inhibition (Fig. 5H).
We therefore suggest that insulin treatment attenuates the repressive effect of Oct-1 by reducing Oct-1 content, possibly involving ERK activation.
Discussion
Dysregulation of lipogenesis plays an important role in the pathology of many metabolic diseases, including obesity, type 2
diabetes mellitus (T2DM), and the metabolic syndrome (30).
Subjects with these disorders are known to be at risk for a variety
of complications, including nonalcoholic fatty liver disease,
atherosclerosis, and other cardiovascular diseases (30 –34).
Additionally, subjects with T2DM and its cardiovascular
complications are often accompanied with hyperinsulinemia,
hypertriglyceridemia, obesity, and insulin resistance. Hence,
many T2DM patients die as the result of liver, cardiovascular,
and other complications (34, 35).
The identification of ChREBP as a key hepatic glucose sensor
involved in channeling carbohydrates into lipogenic pathways
explained why in vitro high ambient glucose alone was sufficient
in regulating a panel of downstream lipogenic genes, independent of the well-characterized SREBP-1c (6, 19, 22, 30, 36 –38).
Recent genome-wide association studies have linked ChREBP
polymorphisms with low ratios of high-density lipoprotein to
hypertriglyceridemia levels, characteristic of subjects at risk for
liver and cardiovascular diseases (39 – 41). In addition, the assessment of ChREBP⫺/⫺ mice showed that despite normal
SREBP-1c activation, the expression of mRNA that encode enzymes required for lipogenesis were reduced (8, 18, 20). Recent
studies also implicated the involvement of ChREBP-mediated
lipogenesis in the development of fatty liver (38, 42, 43). Fur-
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Sirek et al.
Insulin Activates ChREBP via Attenuating Oct-1
Endocrinology, August 2009, 150(8):3483–3492
thermore, leptin-deficient (ob/ob) mice show increased hepatic
lipogenesis and hepatic steatosis (42, 43). Although this was
previously attributed to SREBP-1c and peroxisome proliferatoractivated receptor (PPAR)-␥ (44 – 46), studies in SREBP-1c⫺/⫺
mice and PPAR␥⫺/⫺ mice showed only partial improvement in
the development of hepatic steatosis (31, 45).
Although the stimulatory effect of glucose on the function of
ChREBP in activating lipogenic gene expression has been extensively investigated (31), we know very little about mechanisms
underlying the expression of this master controller of lipogenesis.
Several recent studies suggested that high ambient glucose, insulin, PPAR␥ agonist, liver X receptor and polyunsaturated fatty
acids may affect ChREBP mRNA expression levels (22, 30, 31,
47– 49). Interestingly, liver X receptors have been shown to directly stimulate ChREBP mRNA expression in addition to their
known regulation of lipogenesis through SREBP-1c and FAS (46).
Furthermore, in ob/ob mice, a well-characterized genetic model of
obesity, insulin resistance, and hyperinsulinemia, ChREBP expression level is substantially higher, suggesting a potential role of insulin in stimulating ChREBP production (42, 43).
A recent study examined the cis-regulatory elements of the rat
ChREBP gene promoter (23). Although this study revealed a few
nuclear protein binding sites, including specificity protein-1 and
nuclear factor Y, on the rat ChREBP promoter, how these and
other transcription factors mediate physiological and pathological signals in regulating ChREBP transcription was not examined. Furthermore, we are not aware of any publication regarding mechanisms underlying the expression of the hChREBP
promoter. In the current study, we show the existence of an
evolutionarily conserved POU protein binding site within the
proximal region of ChREBP promoter and demonstrated by
both EMSA and ChIP that the ubiquitously expressed POU protein Oct-1 interacts with the proximal ChREBP promoter region.
Extensive examinations have shown that Oct-1 may function
as a transcriptional activator (50, 51) or a repressor (52–54) for
a large profile of genes. Whereas Oct-1⫺/⫺ mice are embryonically lethal (55), studies on fibroblasts isolated from Oct-1⫺/⫺
mice demonstrated that this POU protein plays an important role
in regulating the expression of genes that mediate the cellular
stress response (56 –58). More recently Oct-1 was shown to mediate the effect of oxidized low-density lipoprotein (oxLDL) in
repressing the expression of vascular cytochrome P450 monooxygenases (59). In human coronary arterial endothelial cells,
knockdown of Oct-1 expression prevented oxLDL-mediated silencing of cytochrome P450 expression (59). Furthermore, inhibition of oxLDL receptor attenuated oxLDL-mediated endothelial DNA damage and Oct-1 DNA binding and reversed
impaired production of endothelial-derived hyperpolarization
factor (59). Therefore, Oct-1 activation in response to oxidative
stress is among the pathological responses in metabolic dysfunction of coronary arterial endothelium (59).
Our observations suggest that Oct-1 acts as a transcriptional
repressor of ChREBP. We have not only demonstrated that
Oct-1 represses the activity of the ChREBP promoter and that
the repression is dependent on the POU protein binding site
(ATGCTAAT) but also that Oct-1 overexpression leads to reduced ChREBP expression at both mRNA and protein levels.
Furthermore, using a shRNA knockdown approach, we have
shown that reduced Oct-1 expression is associated with increased ChREBP promoter activity as well as elevated endogenous ChREBP expression. Recent studies have shown that Oct-1
is able to recruit nuclear corepressors, including histone deacetylase 1 and silencing mediator of retinoic acid and thyroid hormone receptor (53, 60). Whether in the hepatocytes Oct-1 represses ChREBP gene transcription via recruiting these and other
nuclear corepressors deserves further investigation.
It is well established that the function of SREBP-1c is mainly
regulated by insulin, whereas the function of ChREBP is mainly
regulated by glucose, via posttranslational modification (22).
Physiologically, these two master controllers of lipogenesis regulate lipogenic gene expression in response to acute elevation of
insulin and glucose. We suggest that pathologically, elevated
glucose and insulin levels may lead to increased lipogenesis. For
the following reasons, we examined the effect of insulin on the
expression of ChREBP in hepatocytes. First, ChREBP mRNA
and protein levels were found to be markedly elevated in the liver
of hyperinsulinemic ob/ob mice in both the fasted and fed states
(42). Second, in the 3T3-L1 adipocytes and rat adipose tissue,
both insulin and the high level of glucose (25 mM) were demonstrated to stimulate ChREBP mRNA expression (61). Finally, a
very recent study shows that the expression of the transcription
factor chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) is negatively regulated by insulin and glucose,
possibly using ChREBP as the mediator (62). In the current
study, we found that in the human HepG2 cell line, insulin treatment stimulates both the activity of ChREBP promoter and the
expression of endogenous ChREBP. In the hamster primary
hepatocytes, insulin was also shown to stimulate ChREBP protein expression. We did not observe a substantial stimulatory
effect of 25 mM glucose on ChREBP promoter expression (data
not shown). Possible explanations include: 1) high levels of glucose may not stimulate ChREBP transcription, and 2) the
hChREBP-LUC fusion gene constructs used in this study do not
contain the regulatory elements in response to high levels of glucose.
An important observation of this study is that the stimulatory
effect of insulin on ChREBP promoter activity is strongly dependent on the POU protein binding site. The hChREBP-LUC
fusion gene construct with mutated POU binding site showed
attenuated response to insulin treatment (Fig. 5C). At the endogenous gene expression level, insulin treatment stimulated
ChREBP expression in both the wt HepG2 cells and the HepG2
cells in which Oct-1 expression is reduced about 50% by shRNAmediated knockdown (Figs. 4C and 5F). Insulin was also demonstrated to stimulate ChREBP protein expression in the hamster primary hepatocytes along with reduced Oct-1 expression
(Fig. 5G). These observations collectively suggest that activation
of ChREBP expression by insulin acts, at least in part, via attenuating the repressive effect of Oct-1. In Oct-1-overexpressing
HepG2 cells, however, the stimulatory effect of insulin was substantially attenuated (Figs. 3D and 5E), suggesting that the attenuation by insulin can only reach a certain maximum.
How insulin treatment results in reduced Oct-1 expression is
yet to be investigated. One may speculate that insulin treatment
leads to Oct-1 phosphorylation and nuclear exclusion, followed
Endocrinology, August 2009, 150(8):3483–3492
by accelerated degradation. Indeed, we were unable to detect
Oct-1 in the cytosolic fraction (Fig. 5D, bottom panel). Potential
effects of protein kinase pathways, including protein kinase A,
protein kinase C, and casein kinase 2, on Oct-1 phosphorylation,
and therefore its function has been suggested previously (50,
63– 67). However, how Oct-1 is phosphorylated by these protein
kinases has not been investigated, and there is currently no antibody that recognizes a phosphorylated Oct-1. We found that in
hepatocytes, insulin treatment leads to the activation of GSK-3
and ERK. Because MEK inhibition was shown to block the stimulatory effect of insulin (Fig. 5H), we suggest that insulin may use
ERK as an effector in attenuating the repressive effect of Oct-1.
In summary, we show for the first time an investigation of
the activity of the human ChREBP gene promoter, presenting the
interaction between Oct-1 and the hChREBP promoter and the
profound repressive effect of Oct-1. We demonstrate that Oct-1
and insulin exert opposite effects on ChREBP expression and
conclude that insulin stimulates ChREBP expression, at least
partially, via attenuating the repressive effect of Oct-1. Furthermore, we suggest that Oct-1 should not be simply considered as
a repressor of ChREBP. It is a sensor for both insulin (and possibly other) signaling and various types of stress. Oct-1 may
mediate the stimulatory effect of insulin and the repressive effect
of stress in regulating ChREBP expression and thereby lipogenesis. Finally, whether this novel signaling pathway of insulin
and Oct-1 is also involved in regulating the expression of other
target genes deserves further investigations as this pathway
may underlie a variety of pathologies involving dysregulated
lipogenesis.
Acknowledgments
We thank Ms. Jane Sun for technical assistance as well as Drs. Allen
Volchuk and Jonathan Rocheleau for critical reading of the manuscript.
Address all correspondence and requests for reprints to: Tianru
Jin, Toronto General Research Institute, University Health Network,
MaRS, Toronto Medical Discovery Tower, 101 College Street, 10-354,
Toronto, Ontario, M5G 1L7, Canada. E-mail: [email protected].
This work was supported by a pilot grant from the Department of
Medicine, University of Toronto and an operating grant by the Canadian
Institutes of Health Research (94078, to T.J. and D.S.N.). A.S.S. is a
recipient of Banting and Best Diabetes Centre Novo Nordisk graduate
studentship.
Disclosure Summary: The authors have nothing to disclose.
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