Biochem. J. (2010) 430, 245–254 (Printed in Great Britain)
245
doi:10.1042/BJ20100701
Sterol-regulatory-element-binding protein 1c mediates the effect of insulin
on the expression of Cidea in mouse hepatocytes
Rui WANG*, Xingxing KONG*, Anfang CUI*, Xiaojun LIU*, Ruolan XIANG†, Yanli YANG‡, Youfei GUAN†, Fude FANG*1 and
Yongsheng CHANG*1
*National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100005,
China, †Department of Physiology and Pathophysiology, Beijing University Health Science Center, Beijing 100191, China, and ‡Deparment of Internal Medicine, Peking Union Medical
College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100730, China
Members of the Cide [cell death-inducing DFFA (DNA
fragmentation factor-α)-like effector] gene family have been
reported to be associated with lipid metabolism. In the
present study, we show that Cidea mRNA levels are markedly
reduced by fasting and are restored upon refeeding in
mouse livers. To elucidate the molecular mechanism, the
promoter region of the mouse Cidea gene was analysed
and a putative SRE (sterol-regulatory element) was identified.
Studies using luciferase reporter constructs together with electrophoretic mobility-shift assays and chromatin immunoprecipitation confirmed the binding of SREBP-1c (SRE-binding protein
1c) to the putative SRE. Furthermore, adenovirus-mediated
overexpression of SREBP-1c led to a dramatic increase in Cidea
mRNA. In contrast with the induction of Cidea expression
by insulin and TO901317 in wild-type mouse hepatocytes,
the stimulatory effects were lost in hepatocytes prepared
from SREBP-1c-null mice. Adenovirus-mediated overexpression
of Cidea in hepatocytes promoted lipid accumulation and
triacylglycerol (triglyceride) storage; however, knockdown of
Cidea compromised the ability of SREBP-1c to stimulate lipid
accumulation. Taken together, these results suggest that SREBP1c directly mediates the effect of insulin on Cidea in hepatocytes
and that Cidea, at least in part, mediates SREBP-1c-dependent
lipid accumulation.
INTRODUCTION
along with SCAP (SREBP-cleavage-activating protein). Upon
activation, the SREBP-1c and SCAP complex cluster into COPII
(coatamer protein II) vesicles and translocate to the Golgi, where
SREBPs are processed into their mature forms [15,16]. The
mature SREBP-1c enters the nucleus to activate numerous target
genes.
Studies on lipid metabolism have shed light on a new
family of proteins, the Cide (cell death-inducing DFFA-like
effector) family, composed of three members, Cidea, Cideb and
Cidec/FSP27 (fat-specific protein 27). Of these three members,
Cidea is highly expressed in human WAT (white adipose tissue)
[17]. A V115F polymorphism in the human CIDEA gene has been
shown to be associated with obesity [18]. Furthermore, CIDEA
mRNA levels are significantly increased in human adipose tissue
and the lipolytic rate in WAT is reduced by 40 % following weight
loss [19,20]. Cidea is also reported to be highly expressed in
mouse BAT (brown adipose tissue) [17,21]. A study on Cideanull mice showed that these mice exhibit a lean phenotype,
increased lipolysis, a heightened metabolic rate and increased
thermogenesis, and that they are resistant to high-fat-diet-induced
obesity and diabetes, which suggests that Cidea plays an important
role in the regulation of energy homoeostasis [21]. Ectopic
overexpression of Cidea in preadipocytes greatly enhances
lipid droplet size and regulates triacylglycerol deposition [19].
Interestingly, increased Cidea expression is observed in Type 2
diabetic mouse livers, and the elevated Cidea expression could
be reversed by weight loss and normalization of plasma insulin
Insulin controls not only glucose, but also lipid and protein
metabolism in mammalian tissues. In Type 2 diabetes and
obesity, hyperinsulinaemia is associated with elevated hepatic
synthesis of fatty acids and triacylglycerols (triglycerides) [1].
Critical enzymes involved in lipogenesis, such as FAS (fatty acid
synthase), ACC (acetyl-CoA carboxylase) and SCD-1 (sterolCoA desaturase 1), are insulin targets and are regulated by
SREBP-1c [SRE (sterol-regulatory element)-binding protein-1c]
[2–5]. SREBP-1c has been identified as an important transcription
factor in insulin-mediated lipogenesis signalling in liver, adipose
tissue and muscle [3,6–8]. Chen et al. [7] demonstrated that the
LXR (liver X receptor)–RXR (retinoid X receptor) heterodimer
mediates the insulin-induced transcriptional activation of SREBP1c. Studies of TO901317, a highly selective LXR agonist, have
shown that it can strongly stimulate SREBP-1c expression in
mouse livers and can increase lipogenesis [9,10]. SREBP-1c
abundance is closely related to the nutritional state of the liver
and adipose tissue [2,11,12].
SREBP-1c belongs to the SREBP family, which is composed of
three members, SREBP-1a, SREBP-1c and SREBP-2. SREBP1a and SREBP-1c are produced from a single gene through
the use of alternate promoters and different first exons, whereas
SREBP-2 is produced from a separate gene [13,14]. SREBP-1c
is synthesized as a 125 kDa precursor, which, in the presence
of sterol, is bound to the endoplasmic reticulum membrane
Key words: Cidea, fasting and refeeding, insulin, lipid accumulation, sterol-regulatory-element-binding protein 1c (SREBP-1c),
TO901317.
Abbreviations used: ACC, acetyl-CoA carboxylase; APOAII, apolipoprotein AII; ChIP, chromatin immunoprecipitation; Cide, cell death-inducing DFFA
(DNA fragmentation factor-α)-like effector; EMSA, electrophoretic mobility-shift assay; FAS, fatty acid synthase; FBS, fetal bovine serum; FoxA2, forkhead
box A2; GFP, green fluorescent protein; HA, haemagglutinin; HBSS, Hanks balanced salt solution; HEK, human embryonic kidney; LXR, liver X receptor;
PPAR, peroxisome-proliferator-activated receptor; SCD-1, sterol-CoA desaturase 1; siRNA, small interfering RNA; SRE, sterol-regulatory element; SREBP,
SRE-binding protein; SCAP, SREBP-cleavage-activating protein; WAT, white adipose tissue.
1
Correspondence may be addressed to either of these authors (email [email protected] or [email protected])
c The Authors Journal compilation c 2010 Biochemical Society
246
R. Wang and others
[22]. Cidea is expressed at low levels in normal mouse livers.
However, its expression is markedly up-regulated in ob/ob mouse
livers [23]. A further study demonstrated that Cidea is activated
by PPARα (peroxisome-proliferator-activated receptor α) and
PPARγ ligands, and by PPARα and PPARγ overexpression [24].
The above studies have demonstrated that Cidea is a critical factor
involved in lipid metabolism. However, the exact function of
Cidea and the link between insulin and Cidea are still not clear.
In the present study, we investigated the association between
insulin, SREBP-1c and Cidea expression. Our data show that the
induction of Cidea gene expression by insulin and TO901317
is mediated through SREBP-1c, and that Cidea, at least in
part, mediates the SREBP-1c-dependent stimulation of lipid
accumulation in hepatocytes.
MATERIALS AND METHODS
Mouse treatments
C57BL/6 mice (male, 8 weeks old) were purchased from the
Institute of Laboratory Animal Sciences, Chinese Academy of
Medical Sciences and Peking Union Medical College. Mice were
kept in a 12 h light/12 h dark cycle and fed a normal chow diet.
The mice were anaesthetized with an intraperitoneal injection of
bromethol. Livers were perfused with PBS in order to remove
blood cells before collection. The mouse protocol was approved
by the Animal Research Committee at the Institute of Laboratory
Animal Sciences, Chinese Academy of Medical Sciences and
Peking Union Medical College. For the fasting and refeeding
experiments, three male mice were included in each treatment
group. In the fasting groups, mice were fasted for 48 h before
being killed. In the refeeding groups, mice fed a normal chow diet
were fasted for 48 h and then were refed a high-carbohydrate/lowfat diet for 12, 24 or 48 h before being killed. Livers were collected
and immediately used for nuclear protein preparation and ChIP
(chromatin immunoprecipitation) assays. Part of the tissue was
frozen in liquid nitrogen for mRNA quantification.
Plasmid construction and recombinant adenovirus preparation
The 3 kb mouse Cidea promoter plasmid pL-Cid1 was a gift from
Dr Janardan K. Reddy (Department of Pathology, Feinberg School
of Medicine, Northwestern University, Chicago, IL, U.S.A.)
[24]. Different deletion fragments were generated by PCR using
pL-Cid1 as the template and then cloned into the NheI/HindIII
site of the pGL3-basic vector. The primers used were the
following: Cidea promoter + 98 bp, 5 -CCCAAGCTTTCCCTGGCGGTCTCCATTGG-3 (reverse); − 532 bp, 5 -CTAGCTAGCACTAGCAACTCCTAATTCCA-3 (forward); − 250 bp,
5 -CTAGCTAGCACGCGTGTCCCCAGCAACTT-3 (forward);
and − 100 bp, 5 -CTAGCTAGCCACGCACACCTGCTTCTCTA3 (forward). The putative SREBP-binding site was
mutated by PCR. The primers used were the following: 5 ATACATCAGCAAGCGGAGTGTCCTAACAGGC-3 (reverse);
and 5 -TGCTGATGTATCACCTGCTTCTCTACTGCCAAGCCAT-3 (forward). Mouse Cidea cDNA was cloned by PCR
amplification from mouse liver cDNA and inserted into the
Adtrack vector. pcDNA3.1-hSREBP-1a and pcDNA-hSREBP-1c,
containing cDNAs encoding the active amino terminal fragments
of human SREBP-1a (amino acids 1– 460) and human SREBP-1c
(amino acids 1– 436), were provided by Professor Hitoshi
Shimano (Department of Internal Medicine, Institute of Clinical
Medicine, University of Tsukuba, Ibaraki, Japan) [25]. HA
(haemagglutinin) and FLAG tags were added to the C-termini
of mature SREBP-1a and SREBP-1c respectively by PCR. All
c The Authors Journal compilation c 2010 Biochemical Society
clones were verified by sequencing. Recombinant adenoviruses
containing Cidea, mature SREBP-1a and SREBP-1c cDNAs
were generated as previously described [26].
Preparation of primary hepatocytes
SREBP-1c−/− or SREBP-1c+/+ mice (3 months old, male,
C57BL/6J; 129S6/SvEv mixed background) [27] were kept
in a 12 h light/12 h dark cycle and fed a normal chow diet.
For hepatocyte preparation, mice were anaesthetized with an
intraperitoneal injection of bromethol. Livers were subjected
to two-step collagenase perfusion through the portal vein as
previously described [28]. First, 50 ml of Ca2+ -free/Mg2+ -free
HBSS (Hanks balanced salt solution) supplemented with 2 mM
EGTA, 20 mM Hepes and 10 mM NaHCO3 , pH 7.4, was infused
with a pump at a flow rate of 7 ml/min for 6 min. Then the livers
were perfused with HBSS containing 0.05 % (w/v) collagenase
type I (Sigma) at the same flow rate for 5 min. The HBSS was
pre-infused with 95 % O2 and 5 % CO2 and kept in a 37 ◦C
water bath. After the two-step perfusion, livers were excised and
hepatocytes were flushed out and filtered through a 250 μm pore
size mesh nylon filter. Cells were washed three times with ice-cold
HBSS and centrifuged at 50 g for 2 min at 4 ◦C. The cells were
more than 80 % viable, as evaluated by Trypan Blue exclusion.
Hepatocytes were seeded at 107 cells/10 cm2 on rat tail collagencoated plates and cultured with RPMI 1640 medium (H10394;
Invitrogen) containing 10 % (v/v) FBS (fetal bovine serum),
2.5 nM insulin, 1 nM dexamethasone, 50 units/ml penicillin and
50 μg/ml streptomycin. After 2 h, unattached cells were washed
away with PBS and fresh medium was added for an overnight
incubation.
Transient transfection and luciferase assays
HepG2 cells were grown in high-glucose DMEM (Dulbecco’s
modified Eagle’s medium) (H10391; Invitrogen) containing
10 % (v/v) FBS in 24-well plates. When the cells reached
80 % confluency, they were co-transfected with Cidea promoter
plasmids and pcDNA3.1-hSREBP-1a, pcDNA3.1-hSREBP-1c or
with the vehicle pcDNA3.1 plasmid using LipofectamineTM 2000
(11668027; Invitrogen). A Renilla luciferase expression vector,
pCMV-RL-TK, was used as an internal control to adjust for
transfection efficiency. Luciferase activity was measured 48 h
later with the Dual Luciferase Reporter Assay System (E1980;
Promega).
Cell culture and treatment
Mouse primary hepatocytes were kept in RPMI 1640 medium
containing 10 % (v/v) FBS, 50 units/ml of penicillin and 50 μg/ml
of streptomycin. For adenovirus infection, the cells were infected
at a multiplicity of infection sufficient to infect >95 % of the cells,
as determined by GFP (green fluorescent protein) fluorescence.
The final concentrations of insulin and TO901317 used to treat
the cells were 80 nM and 5 μM respectively. Cells were harvested
48 h after treatment for quantitative real-time PCR and Western
blot analysis.
EMSA (electrophoretic mobility-shift assay)
The plasmids pCMV6-XL5-hSREBP-1a/HA or pCMV6-XL5hSREBP-1c/FLAG were used to transfect HEK (human
embryonic kidney)-293A cells. Nuclear extracts were prepared
using the NE-PER Nuclear and Cytoplasmic extraction reagent
(78833; Pierce). The EMSA experiment was performed
using the Chemiluminescent Nucleic Acid Detection Module
kit (89880; Pierce). Synthetic oligomers were prepared
SREBP-1c up-regulates Cidea in mouse hepatocytes
commercially by Invitrogen Biotechnology. The biotinlabelled oligonucleotide containing the Cidea promoter SRE
was: 5 -TTAGGACACTCCGCTCGCCCCACGCACACCTGCTTCTCTACTGCCAAGCC-3 and the SRE-mutated oligonucleotide was: 5 -TTAGGACACTCCGCTAGATGATGGCACACCTGCTTCTCTACTGCCAAGCC-3 . These oligonucleotides
annealed with their respective reverse complementary sequences
to form double-stranded oligonucleotides.
ChIP assays
Mouse livers were homogenized and treated for 10 min with
1 % (v/v) formaldehyde at room temperature (25 ◦C). The crosslinking reaction was stopped by the addition of glycine at
a final concentration of 125 mM. Chromatin was extracted
and fragmented further to an average length of 500 bp by
sonication. After pre-clearing with Protein G beads, the chromatin
was immunoprecipitated with antibodies against SREBP-1 or
FoxA2 (forkhead box A2) (sc-13551; Santa Cruz Biotechnology
and 07-633; Millipore), or with a control IgG at 4 ◦C
overnight. Then antibody–protein–DNA complexes were isolated
by immunoprecipitation with Protein G beads. After extensive
washing, the complexes were eluted and incubated in a 65 ◦C water
bath to reverse the formaldehyde cross-links. DNA was extracted
by phenol/chloroform/isoamyl alcohol (25:24:1) and precipitated
with ethanol. Samples were analysed by PCR. The primers were
designed to amplify a region of the mouse Cidea gene promoter
that contains the SREBP-1-binding site. The forward and
reverse primers used were 5 -CTTTGGAGACTCCAGCATGC3 and 5 -TCTCTTCTGGAGACTGGGGT-3 respectively. The
CYP3A11 promoter was used as a positive control for the FoxA2
antibody [29].
247
used were the following: monoclonal mouse anti-SREBP-1
antibody (sc-13551; Santa Cruz Biotechnology), rabbit anti-Cidea
antibody (provided by Dr Peng Li, Department of Biological
Science and Biotechnology, Tsing Hua University, Beijing,
China) [30], monoclonal mouse anti-β-actin antibody (A5441;
Sigma), monoclonal mouse anti-HA antibody (H9658; Sigma)
and monoclonal mouse anti-FLAG antibody (F3165; Sigma).
Then the membrane was incubated with secondary antibodies
labelled with HRP (horseradish peroxidase) at room temperature
for 2 h and detected with ECL (enhanced chemiluminescence)
reagents (RPN2106; GE Company).
RNA interference and lipid droplet analysis
Short hairpin RNA-coding DNAs were synthesized by Invitrogen
and inserted into adenovirus plasmids. Adenoviruses were
prepared according to previously described procedures [26,31].
siRNAs (small interfering RNAs) transcribed from the above
adenoviruses included the following: scrambled siRNA,
5 -GGA5 -CUUACGCUGAGUACUUCGA-3 ; siCidea1,
CACCGGGUAGUAAGUA-3 [19]; and siCidea2, 5 -AGAUGUACUCCGUGUCCUA-3 . Mouse primary hepatocytes were
infected with Ad-GFP, Ad-SREBP-1c, Ad-Cidea, Adscrambled siRNA (control), Ad-siCidea1 and Ad-siCidea2.
Gene knockdown efficiency was examined by real-time PCR.
For Oil Red O staining, cells were fixed with paraformaldehyde
first and then stained with Oil Red O. After three washes, the
cells were viewed under a microscope.
Hepatocyte triacylglycerol content
RNA extraction, reverse transcription and real-time PCR
Total RNA was extracted from mouse livers and mouse primary
hepatocytes using the Invitrogen TRIzol® reagent and reverse
transcribed into cDNA using Invitrogen SuperScript® III Reverse
Transcriptase. Quantitative real-time PCR was performed using
the SYBR Green I Q-PCR kit (Transgen) using the above
cDNA as the template. A melting curve analysis was performed
after the PCR. Primers used for the real-time PCR were the
following: mouse β-actin, 5 -CCAGCCTTCCTTCTTGGGTAT-3 (forward) and 5 -TGCTGGAAGGTGGACAGTGAG-3
(reverse); mouse Cidea, 5 -TCCTCGGCTGTCTCAATG-3
(forward) and 5 -TGGCTGCTCTTCTGTATCG-3 (reverse);
mouse
SREBP-1c,
5 -CTTCTGGAGACATCGCAAAC-3
(forward) and 5 -GGTAGACAACAGCCGCATC-3 (reverse);
mouse FAS, 5 -CTTGGGTGCTGACTACAACC-3 (forward)
and 5 -GCCCTCCCGTACACTCACTC-3 (reverse); mouse
ACC-1, 5 -AGGAAGATGGCGTCCGCTCTG-3 (forward) and
5 -GGTGAGATGTGCTGGGTCAT-3 (reverse); and mouse
SCD-1, 5 -TGGGTTGGCTGCTTGTG-3 (forward) and 5 -GCGTGGGCAGGATGAAG-3 (reverse).
Western blot analysis
Cell lysates were centrifuged at 12 000 g at 4 ◦C for 15 min,
and the supernatants were kept for Western blot analysis.
Protein concentrations were determined using the Pierce BCA
(bicinchoninic acid) kit. Equal amounts of protein were separated
by SDS/PAGE (10 % gel) and transferred onto 0.22 μM PVDF
membranes (Millipore) after electrophoresis. Blots were probed
with primary antibodies at 4 ◦C overnight. The primary antibodies
Primary hepatocytes were washed three times with PBS before
the cells were disrupted with cell lysis buffer (20 mM Tris/HCl,
pH 7.4, 1 mM EDTA and 1 % Triton X-100). After being
centrifuged at 12 000 g for 10 min, supernatants were collected
for an enzymatic assay using a triacylglycerol assay kit (Sigma).
Statistical analysis
Quantitative data are represented as the means +
− S.E.M. For
statistical analysis, the differences between groups were examined
using a paired Student’s t test, and P 0.05 was considered
statistically significant.
RESULTS
Cidea gene expression in mouse livers decreased during fasting
and was restored upon refeeding
mRNA was extracted from mouse livers and quantified by realtime PCR. Fasting led to a decrease in the mRNA levels of SREBP1c, Cidea, FAS and ACC-1 compared with the mRNA levels
in mice fed with normal diet (Figure 1), and refeeding caused
these levels to increase from the fasting levels. The regulatory
trends of SREBP-1c, ACC-1 and FAS under fasting–refeeding
conditions were consistent with previous reports [12,32]. Cidea
showed an expression pattern that was similar to those of FAS and
ACC-1 under these conditions. Our Western blot analysis result
showed that fasting led to a decrease in nuclear SREBP-1c protein
levels and refeeding restored SREBP-1c expression in mice liver
c The Authors Journal compilation c 2010 Biochemical Society
248
Figure 1
R. Wang and others
Changes in mRNA levels in mouse livers following fasting and refeeding treatments
C57BL/6 mice (male, 8 weeks old) were divided into 5 groups: control (normal diet), fasted and fasted and refed for 12 h, refed for 24 h and refed for 48 h before being killed. Livers were perfused
with PBS to remove blood cells. Total liver RNA was extracted using the Invitrogen TRIzol® reagent; SREBP-1c (A), Cidea (B), FAS (C) and ACC-1 (D) mRNA levels were quantified by real-time
PCR. The expression of β-actin was used as a reference. Values were normalized (=1.0) to the expression levels of the normal diet group, *P 0.05 and **P 0.01. (E). Nuclear proteins were
extracted from livers and subjected to Western blot analysis to evaluate nuclear mature SREBP-1c.
(Figure 1E). Previous studies have demonstrated that SREBP-1c
is the major mediator of insulin’s effect on FAS and other lipogenic
genes in adipose tissue and in the liver [32]. Thus we speculated
that SREBP-1c may also regulate Cidea gene expression.
SREBP-1c stimulated the transcription of Cidea by directly binding
to the SRE identified in the Cidea gene promoter
We first searched for a sequence motif in the mouse Cidea gene
promoter region. A screen of the sequence from − 600 bp to
+ 98 bp relative to the transcription start site revealed a putative
SRE located from − 134 bp to − 126 bp (Figure 2A). A series
of luciferase reporter constructs under the control of different
deletion fragments of the Cidea promoter was prepared and
transfected into HepG2 cells together with SREBP-1 expression
plasmids or the vehicle pcDNA3.1 plasmid. Dual-luciferase
reporter assay results showed that the overexpression of both
SREBP-1a and SREBP-1c led to the considerable activation of
the reporter gene containing the − 532/+ 98 bp region of the
Cidea promoter (Figure 2B). This activation remained when
the promoter region was truncated to − 250/+ 98 bp, which
still contained the putative SREBP-1-binding site. However,
when the reporter gene was further truncated to − 100/+ 98 bp
(completely deleting the putative SRE), the activation was almost
completely lost. To further confirm that this SRE was functional,
mutation studies were performed. When the putative SRE element
(− 134 bp to − 126 bp) was mutated, the activation of the Cidea
gene promoter by SREBP-1 was lost (Figure 2B), suggesting that
the SRE (− 134/− 126 bp) identified in the Cidea promoter was a
functional SREBP-1 cis-element.
c The Authors Journal compilation c 2010 Biochemical Society
Figure 2
SREBP-1 proteins activate the Cidea promoter in HepG2 cells
(A) Alignment of the consensus SRE sequence with the potential SRE sequence
[− 134 bp/− 126 bp relative to the transcription start site (TSS)] in the Cidea gene promter.
(B) HepG2 cells were co-transfected with plasmids harbouring a 5 -deletion series of the Cidea
promoter fused to a luciferase reporter (including the wild-type Cidea gene promoter sequence
and its mutant) and SREBP-1 protein expression plasmids or pcDNA3.1 (control). Cells were
lysed 48 h after transfection and luciferase activity was measured as described in the Materials
and methods section. The graph depicts relative luciferase activity (RLA) corrected for Renilla
luciferase activity and normalized to the control activity of Cidea (transfected with pcDNA3.1). All
values represent at least three independent transfections, each conducted in triplicate. *P 0.05
compared with control group (transfected with pcDNA3.1).
To determine whether SREBP-1 proteins were physically
bound to the Cidea SRE, an EMSA was performed using nuclear
extracts and the synthetic double-stranded oligonucleotides.
SREBP-1c up-regulates Cidea in mouse hepatocytes
249
and then restore liver SREBP-1c protein levels. Liver chromatin
was precipitated with an anti-SREBP-1 antibody or with an antiFoxA2 antibody as a negative control. As expected, SREBP1 occupied the promoter of the Cidea gene under the normal
diet condition, and this binding was almost undetectable during
fasting. However, occupancy of SREBP-1 at the Cidea gene
promoter was restored in the livers after refeeding (Figure 3B).
In addition, FoxA2 did not bind to the SRE region of the Cidea
gene promoter, although it did occupy the CYP3A11 promoter
(Figure 3B), which is a well-defined FoxA2 downstream target
gene [29].
Adenovirus-mediated overexpression of mature SREBP-1
stimulates Cidea expression
Because the SREBP-1 proteins activate the mouse Cidea promoter
by directly binding to the Cidea SRE element, we reasoned that
the SREBP-1 proteins could affect Cidea at the mRNA level. We
used mouse primary hepatocytes as a model system. Hepatocytes
were infected with adenoviruses expressing mature SREBP-1a,
SREBP-1c or GFP as a negative control. Western blotting
results indicated that the adenoviruses effectively mediated the
expression of the mature active SREBP-1 proteins (Figure 4D).
Total RNA was extracted from these cells and the levels were
quantified by real-time PCR. As expected, overexpression of
mature SREBP-1a and SREBP-1c led to a dramatic induction
of Cidea, FAS and SCD-1 (Figures 4A–4C). Additionally,
overexpressing SREBP-1c also increases Cidea mRNA levels in
3T3-L1 and C2C12 cells (results not shown).
Figure 3 In vitro and in vivo binding of SREBP-1 proteins to the SRE of the
Cidea gene promoter
(A) EMSA was performed as described in the Materials and methods section. The unlabelled
wild-type and mutant probes were used as competitors at a 200-fold molar excess relative to the
biotin-labelled Cidea SRE probe. The biotin-labelled probes were incubated with nuclear extracts
from HEK-293A cells transfected with SREBP-1 expression vectors in the presence or absence
of antibody or in the presence of a 200-fold molar excess of a competitor probe, as indicated.
The resulting DNA–protein complexes were separated from the free probes by SDS/PAGE. Lane
10: binding of SREBP-1c-(1–436) to the biotin-labelled probes corresponding to the previously
identified SRE of the human APOAII gene (positive control). (B) ChIP assays were performed
using fasted and refed mouse livers. After cross-linking, the nuclei were isolated and sonicated,
and protein–DNA complexes were immunoprecipitated with anti-SREBP-1, anti-FoxA2 (negative
control) or normal IgG antibodies (negative control). The resultant DNA was analysed by PCR
with primers amplifying the promoter region flanking Cidea SRE. FoxA2 can specifically bind to
the CYP3A11 gene promoter (positive control), which is consistent with a previous study [29].
As shown in Figure 3(A), the nuclear proteins extracted from
HEK-293A cells overexpressing SREBP-1a–HA and SREBP1c–FLAG reacted with the labelled oligonucleotide probes and
formed shifted bands. The addition of a 200-fold excess of
an unlabelled Cidea SRE competitor abolished the formation
of the protein–DNA complex, leading to the disappearance of
the band-shift. In sharp contrast, the addition of a 200-fold
excess of the mutant unlabelled competitor did not affect the
formation of this complex. When HA and FLAG antibodies were
added to the reaction system individually, super-shift bands
were observed. A well-defined SRE element (AIICD) in the
human APOAII (apolipoprotein AII) promoter [33] was used as a
positive control (Figure 3A). Taken together, these results indicate
that SREBP-1 is specifically involved in this interaction and that
SREBP-1 is physically bound to Cidea SRE in vitro.
In addition, ChIP assays were carried out with mouse livers
to investigate whether SREBP-1 can be recruited to the Cidea
promoter in vivo. Primers were designed to amplify a fragment
flanking the SRE region of the Cidea gene promoter. Mice were
fasted and refed for 48 h before being killed in order to decrease
Insulin and TO901317 induced Cidea expression via SREBP-1c
In order to study further whether SREBP-1c mediates the effect
of insulin on Cidea gene expression, hepatocytes prepared from
SREBP-1c-null mice were used. The SREBP-1c-null mice exhibit
lower total plasma triacylglycerols and a reduction in multiple
mRNAs encoding enzymes for fatty acid synthesis, such as FAS
and ACC. In this model system, the first exon of the SREBP-1c
gene is deleted, thus SREBP-1c transcription is abolished, whereas
the SREBP-1a transcript is left intact [27]. Primary hepatocytes prepared from wild-type mouse livers (wild-type
hepatocytes) as well as SREBP-1c-null mouse livers (knockout
hepatocytes) were treated with insulin or TO901317 for 48 h.
As shown in Figure 5(A), in the wild-type hepatocytes, SREBP1c mRNA increased by approx. 4.6-fold upon insulin treatment,
compared with the control group, which is consistent with
previous studies [2,34]. In accordance, an approx. 4.5-fold
increase in the mRNA levels of Cidea was observed in hepatocytes
following insulin and TO901317 treatment. Our Western blot
results indicate that the protein levels of mature SREBP-1c and
Cidea were also markedly increased in the presence of insulin
and TO901317 (Figure 5B). In addition, adenovirus-mediated
overexpression of SREBP-1c also caused a dramatic increase
in Cidea protein levels. SREBP-1c is the dominant form of
SREBP family proteins in the liver and is selectively stimulated by
insulin in the lipid metabolism pathway [6,34–36]. Because both
Cidea and SREBP-1c gene expression are induced by insulin and
TO901317, and SREBP-1c stimulates Cidea at both the mRNA
and protein levels, it is reasonable to hypothesize that SREBP-1c
mediates the activity of insulin and TO901317 on Cidea gene
expression. Our results confirmed this notion (Figures 5C and
5D). Insulin and TO901317 cannot stimulate Cidea at either the
mRNA level or protein level in primary hepatocytes prepared
from SREBP-1c-null mice, which is in sharp contrast with
what was observed in wild-type mouse hepatocytes. In addition,
c The Authors Journal compilation c 2010 Biochemical Society
250
Figure 4
R. Wang and others
Induction of Cidea by SREBP-1a and SREBP-1c in mouse primary hepatocytes
Primary hepatocytes were isolated and infected with adenoviruses expressing GFP, mature SREBP-1a or mature SREBP-1c, then cells were harvested and RNA was extracted. The mRNA levels of
Cidea (A), FAS (B) and SCD-1 (C) were measured by quantitative real-time PCR. The values were normalized to the β-actin mRNA levels and are relative to the Ad-GFP infected control cells.
*P 0.001 compared with the GFP-infected group. (D) Representative Western blotting analysis of the expression of mature SREBP-1 proteins using protein extracted from cells treated as described
above.
adenovirus-mediated restoration of SREBP-1c in the SREBP-1cnull mouse hepatocytes led to an increase in Cidea expression
(Figure 5D). Taken together, these results strongly suggest that
insulin and TO901317 stimulate Cidea gene expression in an
SREBP-1c-dependent manner.
Cidea increases lipid droplet size and partially mediates SREBP-1c
stimulation of lipid accumulation in hepatocytes
To study the physiological role of the SREBP-1c-dependent
activation of Cidea, adenoviruses expressing siRNA targeting
Cidea were constructed. The siRNA were effectively expressed
under the control of a H1 RNA promoter. Two efficient siRNA
sequences for silencing Cidea expression were selected from
several candidates. Our results indicate that Cidea mRNA was
decreased by siCidea1 and siCidea2, compared with the scrambled
siRNA control group (Figure 6A).
We examined whether Cidea could affect lipid droplet
formation and triacylglycerol content in mouse hepatocytes.
As shown in Figures 6(B) and 6(C), adenovirus-mediated
overexpression of Cidea in mouse hepatocytes led to lipid droplet
enlargement and stimulated triacylglycerol storage. Cidea has
been previously reported to stimulate lipid accumulation in
adipocytes [19], and these results suggest that Cidea has the
same affect in hepatocytes. A previous study demonstrates that the
overexpression of mature SREBP-1c in hepatocytes induces lipid
accumulation [8]. Our result confirmed this report (Figures 6B
and 6C). However, when hepatocytes were co-infected with AdSREBP-1c and Ad-siCidea1/2, the specific knockdown of Cidea
by siRNA suppressed the ability of SREBP-1c to stimulate lipid
accumulation (Figures 6B and 6C), suggesting that Cidea is a
c The Authors Journal compilation c 2010 Biochemical Society
direct mediator of SREBP-1c-dependent lipid accumulation in
hepatocytes.
DISCUSSION
It is well established that the activity of insulin on fatty acid
synthesis in the liver is mediated by SREBP-1c [13]. Our present
study on the livers of mice that were fasted and refed revealed
that Cidea mRNA fluctuates in response to dietary changes. The
amount of liver Cidea mRNA declined dramatically in response to
fasting and increased gradually upon refeeding. Cidea showed a
similar expression pattern as SREBP-1c downstream genes under
fasting and refeeding conditions [12]. This implies that Cidea
expression may be regulated by SREBP-1c, and that SREBP-1c
may mediate the effects of insulin on Cidea expression following
a fasting–refeeding treatment.
The main goal of the present study was to elucidate the molecular mechanism of insulin-mediated Cidea stimulation. A screen of
the mouse Cidea promoter region revealed the presence of several
putative transcription-factor-binding sites, including SRE-,
GATA-, HNF4 (hepatocyte nuclear factor 4)- and PPRE (PPARresponsive element)-binding sites, the last of which has been
shown to be functional for PPARα and PPARγ ligands [19,24].
However, no LXRE (LXR-response element) was found. Because
both insulin and TO901317 induce SREBP-1c gene expression
in hepatocytes [34,37], and a potential SRE was identified in
the mouse Cidea promoter region, we speculated that this SRE
may be functional. Indeed, the present studies using EMSA
and ChIP assays demonstrate that the SRE element, which is
located between base pairs − 134 and − 126 from the transcription
initiation site of Cidea, directly interacts with SREBP-1a and
SREBP-1c up-regulates Cidea in mouse hepatocytes
Figure 5
251
SREBP-1c mediates insulin- and LXR-induced Cidea expression
(A) Wild-type primary hepatocytes were isolated and treated with DMSO (control), insulin (80 nM) or the LXR agonist TO901317 (5 μM) for 48 h. Total RNA was extracted and the mRNA levels of
SREBP-1c and Cidea were measured by quantitative real-time PCR. Values were normalized to β-actin mRNA levels and are relative to the DMSO-treated cells. *P 0.05. (B) Wild-type primary
hepatocytes were isolated and infected with Ad-GFP (as control) or Ad-SREBP-1c, then treated with insulin (80 nM), the LXR agonist TO901317 (5 μM) or DMSO (control) as indicated. After 48 h, the
cells were harvested for Western blot analysis. (C) Primary hepatocytes prepared from SREBP-1c -null mice were treated with DMSO (control), insulin (80 nM) or the LXR agonist TO901317 (5 μM)
for 48 h. Total RNA was extracted and the Cidea mRNA levels were measured by quantitative real-time PCR. The values were normalized to β-actin mRNA levels and are relative to the DMSO-treated
cells. ND, not significantly different. (D) Primary hepatocytes prepared from SREBP-1c -null mice were treated as described in (B). Protein levels of SREBP-1c and Cidea were determined by Western
blot analysis.
SREBP-1c both in vitro and in vivo. Our mutation studies using
luciferase reporter constructs confirmed this conclusion.
In the liver, LXR mediates insulin’s induction of SREBP-1c
expression [7]. The resultant SREBP-1c is a general mediator of
insulin’s effects. Our results from the present study indicate that
the reduction in Cidea mRNA in the livers of fasting mice (low
plasma insulin levels) can be gradually restored after refeeding
with a high-carbohydrate/low-fat diet (high plasma insulin levels).
Moreover, overexpression of SREBP-1c leads to a dramatic
increase in Cidea mRNA and protein in mouse hepatocytes. These
results from the present study, in combination with a previous
study [7], imply that the effect of insulin on Cidea expression may
occur through a SREBP-1c-dependent pathway. Further studies
using primary hepatocytes prepared from SREBP-1c-knockout
mouse livers confirm this suggestion. The present study shows that
neither insulin nor TO901317 has a stimulatory effect on Cidea in
SREBP-null hepatocytes. In liver, SREBP-1c is the predominant
isoform of SREBP-1 (SREBP-1c mRNA is 9-fold more abundant
than SREBP-1a) [35]. Although overexpression of SREBP-1a
can also stimulate Cidea, it is not likely that SREBP-1a mediates
the effect of insulin on Cidea since insulin fails to induce the
expression of Cidea in hepatocytes isolated from the SREBP-1cnull mouse liver, even though expression of SREBP-1a remains
intact [27]. These results strongly indicate that SREBP-1c, but
not SREBP-1a or other transcription factors, mediates the effects
of insulin on Cidea gene expression.
Previous reports suggest that overexpression of SREBP-1c in
the liver leads to a considerable increase in lipid synthesis [8,38],
and our studies indicate that Cidea is a direct target of SREBP1c. Thus we speculated that Cidea may mediate the effect of
SREBP-1c on lipid accumulation. We demonstrate that
the overexpression of Cidea in hepatocytes promotes lipid
accumulation. Similar results were also observed in adipocytes
[19]. However, it is noteworthy that Cidea has a much smaller
effect on lipid droplets than SREBP-1c does. Furthermore,
a knockdown of Cidea expression by adenovirus-mediated
siRNA suppressed the SREBP-1c-mediated stimulation of lipid
accumulation in hepatocytes. This suggests that Cidea, at least
in part, mediates the effect of SREBP-1c on lipid accumulation.
However, we have to point out that knockdown of Cidea was
not performed in hepatocytes treated with insulin or TO901317
since insulin or TO901317 compound alone can not powerfully
stimulate lipogenesis in hepatocytes as overexpression of SREBP1c mediated by adenovirus does, which was evaluated with Oil
Red O staining (results not shown).
Cidea and Cidec are both reported to be lipid droplet proteins
[19,39] and these two proteins are both elevated in ob/ob
mouse steatotic liver [22,23]. One study suggests that Cidec
c The Authors Journal compilation c 2010 Biochemical Society
252
Figure 6
R. Wang and others
Knockdown of Cidea compromises the ability of SREBP-1c to stimulate lipid accumulation
(A) Primary hepatocytes were isolated and infected by Ad-SREBP-1c, Ad-scrambled siRNA (control), Ad-siCidea1 or Ad-siCidea2, as indicated. Cidea mRNA levels were measured by real-time
PCR. *P < 0.05. (B) Wild-type mouse hepatocytes were infected by Ad-GFP (control), Ad-SREBP-1c or Ad-Cidea in the absence or presence of Ad-scrambled siRNA (control), Ad-siCidea1 or
Ad-siCidea2, as indicated. At 24 h later, cells were stained with Oil Red O. (C) Primary hepatocytes were treated as described in (B) and the cells were washed three times with PBS before performing
an enzymatic assay to measure cytoplasmic triacylglycerol levels. Triacylglycerol levels are shown as mg/mg of protein. *P 0.05.
c The Authors Journal compilation c 2010 Biochemical Society
SREBP-1c up-regulates Cidea in mouse hepatocytes
overexpression in hepatocytes causes a greater stimulation of lipid
accumulation than Cidea does [23]. However, we found that Cidec
is only modestly induced in primary hepatocytes by SREBP-1c
as compared with Cidea (results not shown).
In summary, the results of the present study indicates that
changes in Cidea expression follow the fluctuations of SREBP-1c
in the livers of fasted and refed mice. SREBP-1c significantly
stimulates Cidea at both the mRNA and protein levels in primary
hepatocytes. In the liver, SREBP-1c mediates the induction
of Cidea gene expression by insulin and an LXR agonist.
Furthermore, overexpression of Cidea in hepatocytes promotes
lipid droplet formation, whereas knockdown of Cidea expression
partially suppresses the SREBP-1c-mediated effect on lipid
accumulation.
In obese rodents and humans, the up-regulation of SREBP-1c
expression may result in excesive lipogenesis and insulin
resistance, further causing liver steatosis [40–43]. Given the
dramatic induction of Cidea by SREBP-1c and its specific
expression pattern in ob/ob mice [23], Cidea may participate in the
SREBP-1c-dependent liver steatosis pathway. Hyperinsulinaemia
is often associated with hyperlipidaemia in livers through the
induction of lipogenesis-related genes. In obese and Type 2
diabetic subjects, hyperinsulinaemia may result in elevated
SREBP-1c levels in the liver [33,41], which in turn up-regulates
Cidea gene expression, causing lipid droplet accumulation in
hepatocytes and the development of liver steatosis. Further studies
are needed to elucidate the exact function of Cidea in lipid
metabolism in order to fully understand the pathology of liver
steatosis in insulin resistance and obesity.
AUTHOR CONTRIBUTION
Rui Wang performed most of the experiments with the necessary assistance of Xingxing
Kong, Anfang Cui and Xiaojun Liu, who helped Rui Wang with the plasmid construction
and animal treatment. Ruolan Xiang and Yanli Yang helped with hepatocyte triacylglycerol
analysis. Youfei Guan helped with the SREBP-1c-null mouse experiment and gave
instructions on the design of the SREBP-1c-null mouse experiment. Rui Wang wrote
the paper. Yongsheng Chang and Fude Fang conceptualized the project, directed the
experiments, contributed to the calculation of the statistics, helped to finalize the paper
and wrote the grants to apply for funding.
ACKNOWLEDGEMENTS
We thank Dr Janardan K. Reddy for providing the Cidea promoter plasmid, Dr Hitoshi
Shimano for providing SREBP-1a and SREBP-1c plasmids and Dr Peng Li for providing
the anti-Cidea antibody. We also thank Ting Song, Yuan Xue, Di Shao Liuluan Zhu, Xueyu
Dong and Zhuqin Zhang for helpful discussions, technical assistance and assistance with
the mouse treatments.
FUNDING
This work was supported by collective grants from the Major State Basic Research
Development Program of China [973 Program 2006CB503909], the National High
Technology Research and Development Program of China [863 Program 2006AA02Z192
and 2006AA02A409], the National Natural Science Foundation of China [grant numbers
30700386, 30721063, 30800389 and 90919019], the Project for Extramural Scientists
of State Key Laboratory of Agrobiotechnology [grant number 2010SKLAB07-11] and the
Natural Science Foundation of Beijing [grant number 7082056].
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