Biochemical and Biophysical Research Communications 363 (2007) 329–335
www.elsevier.com/locate/ybbrc

In vivo promoter analysis on refeeding response of hepatic
sterol regulatory element-binding protein-1c expression
Yoshinori Takeuchi a,b, Naoya Yahagi a,c, Yoshimi Nakagawa b,c, Takashi Matsuzaka b,c,
Ritsuko Shimizu c, Motohiro Sekiya a, Yoko Iizuka a, Ken Ohashi a, Takanari Gotoda a,
Masayuki Yamamoto c, Ryozo Nagai a, Takashi Kadowaki a, Nobuhiro Yamada b,
Jun-ichi Osuga a, Hitoshi Shimano b,c,*ab

Department of Internal Medicine, Graduate School of Medicine, University of Tokyo, Tokyo 113-8655, Japan
Advanced Biomedical Applications, Graduate School of Comprehensive Human Sciences, University of Tsukuba, 1-1-1 Tennodai,
Tsukuba, Ibaraki 305-8575, Japanc
Center for Tsukuba Advance Research Alliance, University of Tsukuba, Ibaraki 305-8575, Japan
Received 24 August 2007
Available online 6 September 2007

Abstract
Sterol regulatory element-binding protein (SREBP)-1c is the master regulator of lipogenic gene expression in liver. The mRNA abundance of SREBP-1c is markedly induced when animals are refed after starvation, although the regulatory mechanism is so far unknown.
To investigate the mechanism of refeeding response of SREBP-1c gene expression in vivo, we generated a transgenic mouse model thatcarries 2.2 kb promoter region fused to the luciferase reporter gene. These transgenic mice exhibited refeeding responses of the reporter inliver and adipose tissues with extents essentially identical to those of endogenous SREBP-1c mRNA. The same results were obtained
from experiments using adenovirus-mediated SREBP-1c-promoter-luciferase fusion gene transduction to liver. These data demonstratethat the regulation of SREBP-1c gene expression is at the transcription level, and that the 2.2 kb 5 0 -ﬂanking region is suﬃcient for thisregulation. Moreover, when these transgenic or adenovirus-infected mice were placed on insulin-depleted state by streptozotocin treatment, the reporter expression was upregulated as strongly as in control mice, demonstrating that this regulation is not dominated byserum insulin level. These mice are the ﬁrst models to provide the mechanistic insight into the transcriptional regulation of SREBP1c gene in vivo.
 2007 Elsevier Inc. All rights reserved.
Keywords: Lipogenesis; Promoter analysis; Insulin; Transcription; Liver; Adipose tissue; Transgenic mouse; Adenovirus

The conversion of carbohydrate into fat is known as denovo lipogenesis, which is catalyzed by series of lipogenic
enzymes such as acetyl-CoA carboxylase and fatty acid synthase [1]. The activities of these enzymes are nutritionallyregulated, i.e. downregulated when animals are starved
and upregulated when they are refed. This regulation of*

Corresponding author. Address: Advanced Biomedical Applications,
Graduate School of Comprehensive Human Sciences, University ofTsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan. Fax: +81
29 853 3174.
E-mail address: shimano-tky@umin.ac.jp (H. Shimano).
0006-291X/$ - see front matter  2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.bbrc.2007.08.165

these lipogenic enzymes has two remarkable features. First,
their overall enzymatic activities largely depend on theamount of expressed protein that is primarily controlled at
the transcriptional level. Second, their rates of transcription
are coordinately regulated [2]. Therefore, it has been postulated that these genes share a regulatory sequence in theirpromoters that interacts with common trans-acting factors.
In the liver, the most likely factor conducting this coordinatetranscriptional regulation has been revealed to be sterol regulatory element-binding protein (SREBP)-1 [3,4].
SREBPs are transcription factors that belong to thebasic-helix–loop–helix-leucine zipper (bHLH-Zip) family

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Y. Takeuchi et al. / Biochemical and Biophysical Research Communications 363 (2007) 329–335

ered primary at least in regard to refeeding response.
However, the mechanism of this regulation is yet to be clariﬁed, including the point whether this regulation is at thetranscription level or not.
It has been reported that elevated concentrations of glucose and insulin mimic the refeeding state in vivo andupregulate SREBP-1 expression in cultured hepatocytes[12,13]. However, these responses induced by high dosage
of glucose and insulin are within two to three-fold changes
at best, and far weaker than the refeeding response
observed in the in vivo liver. Therefore, to elucidate the precise mechanism of SREBP-1 regulation by nutrient availability, it is inevitable for one to study the in vivo mousemodel.
These situations prompted us to generate transgenic
mice that carry SREBP-1c-promoter-driven reporter gene
cassette to investigate the nutritional regulation of the promoter activities in vivo. Moreover, based on the resultsfrom these experiments, we further assessed the promoter
activities using adenovirus-mediated gene delivery to theliver, which complemented the data from transgenic miceand are considered to be profoundly involved in the transcriptional regulation of cholesterogenic and lipogenic
enzymes [5]. The role of SREBP-1 in the regulation of lipogenesis in the liver has been well established by several lines
of evidence, especially from transgenic and knockout
mouse models [4,6–8]. In these models, hepatic SREBP-1
protein levels dominate the mRNA expression levels for abattery of lipogenic genes. Thus, SREBP-1 is now wellestablished as the key transcription factor for the regulation of lipogenic gene expression and by extension triglyceride storage in liver [9,10].
It has been well known that the mRNA and nuclear protein abundance of SREBP-1c, the main isoform of SREBP1 in lipogenic organs such as liver and adipose tissue, isphysiologically regulated by nutrient availability, i.e. it isdownregulated when animals are starved and upregulated
when they are refed, thereby adjusting lipogenic gene
expression levels to the nutritional conditions [11]. Because
the quantity of mRNA and nuclear protein of SREBP-1cgoes up and down in parallel depending on dietary condition, the control at the mRNA expression level is consid-

A. Transgene constructprobe1probe2

-2.2k

+40

BamHI
NheI

NheI

SmaI

BamHI
SalI

Luciferase genemouse SREBP-1c promoter 2.2kb

SV40 Late Poly(A) Signal

B. Southern blot
probe 1
Line A
WT

Tg

Line B
WT

Tg
3.3kb

probe 2Tg
WTLine ALine B

1.1kb
1.0

2.1

3.9

Fig. 1. Transgene construct and Southern blot. (A) Construct used to generate 2.2 kb-SREBP-1c-promoter-Luc transgenic mice. The mouse SREBP-1c
promoter region spanning 2.2 k to +40 bp was fused to a luciferase reporter gene and SV40 late polyadenylation sequence. (B) Southern blotting ofgenomic DNA from transgenic mice. Genomic DNA prepared from tail samples of wild-type (WT) controls and two lines (line A and B) of transgenic
mice were analyzed by Southern blotting using two distinct probes. For probe 1 (shown in upper panel), a cDNA probe for luciferase gene was used. DNA
samples were digested with BamHI. For probe 2 (shown in lower panel), a cDNA probe for mouse SREBP-1c promoter region was used to determine thecopy number of transgene. DNA samples were digested with NheI.

Y. Takeuchi et al. / Biochemical and Biophysical Research Communications 363 (2007) 329–335

by excluding the potential possibility of positional eﬀects intransgenic mouse model.
Materials and methods
Animals and treatment. Seven- to nine-week-old ICR and C57BL/6J
male mice were purchased from CLEA. All animals were housed in atemperature-controlled environment with a 12 h-light/dark cycle and freeaccess to standard laboratory diet (MF from Oriental Yeast, composed of
60% carbohydrate, 13% fat, and 27% protein on a caloric basis) and water.
For the fasting group, animals were starved 24 h, and for the refeedinggroup, they were refed at 16 h after a 24 h starvation. To make insulindepleted states, streptozotocin (100 mg/kg body weight, Sigma) was
administered by two intraperitoneal injections with 1-day interval following an overnight fasting period as described previously [14]. Mice wereconsidered diabetic when tail vain blood glucose levels exceeded 350 mg/
dl, and euthanized within 8 days from the ﬁrst injection. All the experiments were repeated more than twice and reproducibility was conﬁrmed.
Production of transgenic mice. pBP1c2600Luc plasmid was constructed
into pGL2 basic vector (Promega) as previously described [15]. The SmaI–SalI fragment of this plasmid spanning 2.2 kb to +40 bp of the mouse
SREBP-1c promoter fused to luciferase gene was microinjected into BDF1
eggs. Among the 55 oﬀsprings, eight mice had the integrated transgene.
Among eight founder mice, two were crossbred to C57BL/6J backgroundmore than 5 times, and thus two lines of 2.2 kb-SREBP-1c-promoterLuc transgenic mice were established.
Generation of recombinant adenovirus. The SmaI–BglII fragment of
pBP1c2600bp-Luc containing 2.2 kb to +40 bp of the mouse SREBP-1c
promoter was subcloned into the pGL3 basic vector (Promega) at SmaIand BglII sites. Then the SREBP-1c promoter-luciferase gene cassette wascut out of the vector by NotI and SalI, and after both ends were blunted, it
was subcloned into the pENTR4 vector (Invitrogen) at the EcoRI site(blunted). Then, 2.2 kb-SREBP-1c-promoter-Luc adenoviral plasmid
was generated by homologous recombination between the subcloned

A. Luciferase activities
Liver

10

RLU/mg

RLU/mgLine B
300**

pENTR4 plasmid and pAd promoterless vector using Clonase recombinase (Gateway system, Invitrogen). Similarly, SV40-promoter-Luc control
adenoviral plasmid was generated from pGL3 promoter vector (Promega).
After the transfection into 293A cells, adenoviruses were collected by CsCl
gradient centrifugation. Following titer determination by limiting dilution,adenoviruses were injected intravenously into ICR male mice at the doseof 6.0 · 106 P.F.U./body unless otherwise indicated.
Northern blotting. Total RNA from liver and subctaneous fat pad was
isolated with Trizol Reagent (Invitrogen), and 7.5 lg RNA equally pooledamong each group was analyzed. cDNA probes were cloned previously[4,16,17]. Blots were exposed to imaging plate for BAS2000 BIO IMAGING ANALYZER (Fuji Photo Film).
Luciferase assays. Tissue samples were homogenized in Reporter Lysis
Buﬀer (Promega) and after centrifugation, luciferase activity in the
supernatant was measured by standard kits (Promega) on a luminometer.
Adenoviral DNA isolation and quantiﬁcation. The pellets obtained in
the process described above were digested in SNET buﬀer (1% SDS,
400 mM NaCl, 5 mM EDTA, and 20 mM Tris–HCl at pH 8.0) containing
proteinase K (0.01 mg/ml). After DNA extraction with phenol/chloroform
and RNase treatment, the amount of adenovirus DNA was determinedwith Sybr-Green Dye (ABgene)-based real-time PCR with primers 5 0 GTCCGGTTATGTAAACAATCC-3 0and
5 0 -ATGAAGAAGTGT
TCGTCTTCG-3 0 .

Results and discussion
Generation of transgenic mice carrying SREBP-1c
promoter-luciferase fusion gene
To examine the transcriptional regulation of SREBP-1cgene in vivo, we have generated transgenic mice carrying
the luciferase reporter gene driven by the 2.2 kb SREBP1c 5 0 -ﬂanking sequence that we have studied in vitro previ-

B. Intrinsic gene expression

Line A
15

331

Liver
*

Line A WT Line A Tg

Line B WT Line B Tg

FastedFastedRefed

FastedRefed

Refed Fasted

Refed

200

SREBP-1
100

5

36B4
0
Refed

FastedLine A

WAT
10

9.2

Fold(Refed/Fasted)

0
FastedLine B

8.9

10.3

WAT
*

*

8.6

Refed

Line A WT Line A Tg

Line B WT Line B Tg

FastedFasted20

RLU/mg

RLU/mg8
6
4

0

FastedRefed

FastedRefed

Refed Fasted

Refed

10.5

10.3

SREBP-1
10

36B4

5

2

Refed

15

0

Fold(Refed/Fasted)

Fasted

Refed

10.9

8.6

Fig. 2. Refeeding response of transgenic mice. (A) Transgene expression as assessed by luciferase activities of liver and white adipose tissue (WAT) fromtwo lines of transgenic mice. Transgenic mice were either fasted for 24 h or refed for 16 h after a 24 h fast before euthanized. Each group consists of 4–7
male mice. Results are means ± SE. *P < 0.05 and **P < 0.01, respectively. (B) Intrinsic gene expression as assessed by Northern blot analyses of wild type
(WT) control and transgenic (Tg) mice. The quantiﬁcation results were obtained with BAS2000 system and normalized to the signal generated from 36B4
mRNA (used as a loading control), and the fold-changes between refed vs. fasted states are shown.

Y. Takeuchi et al. / Biochemical and Biophysical Research Communications 363 (2007) 329–335

bility of mRNA, which is reported to be involved in the regulation by polyunsaturated fatty acids [21].

ously [15,18]. The genomic structure of SREBP-1 gene has
been previously clariﬁed in detail [19], and the SREBP-1c
promoter is located at 10 kb downstream of the SREBP1a promoter. Because refeeding manipulation selectively
upregulates SREBP-1c rather than SREBP-1a [20], theresponsible element was expected to be within the
SREBP-1c 5 0 -ﬂanking region. Since expression of reporter
genes in transgenic animals might be aﬀected by the location of genomic integration, we analyzed two independent
founder lines, designated as line A and B. From Southernblotting analyses of genomic DNA (Fig. 1B), line A andB mice were estimated to harbor 2 and 6 copies of transgene per allele, respectively.

SREBP-1c induction by refeeding without insulin
We have previously reported that the refeeding responseof SREBP-1c does not require insulin [14]. To examine therole of insulin in this transcriptional upregulation of the
SREBP-1c by refeeding, we placed the transgenic mice on
insulin-depleted state with streptozotocin treatment. As
shown in Fig. 3, increase in plasma insulin by refeeding
was essentially abolished, but the SREBP-1c promoter
activities as assessed by luciferase reporter expression werevigorously induced in the liver accompanied by elevation inblood glucose levels. This result indicates that insulin is notinevitable for the upregulation of SREBP-1c gene transcription by refeeding, consistent with our previous report [14].

Transcriptional activation of the SREBP-1c promoter-driven
transgene by refeeding
For these lines of transgenic mice, the expression ofreporter gene in liver and white adipose tissue was assessedby luciferase activity. As shown in Fig. 2, the luciferase activities in the livers of both lines were markedly upregulated byrefeeding manipulation, which paralleled the responses ofendogenous SREBP-1c expression. This result means that
the SREBP-1c expression is regulated at the transcription
level in this situation, rather than through the variable sta-

Adenovirus-mediated transduction of SREBP-1c promoterluciferase fusion gene into the mouse liver and assessment ofrefeeding response
We further took another strategy for in vivo assay of
SREBP-1c promoter activity using an adenovirus vector
for introduction of the reporter gene into the liver. We gen-A. Luciferase activities

C. Blood parametersLine ALine BLine A

Insulin
10**

25

RLU/mg20
RLU/mgPlasma Insulin (ng/ml)

*
60

15
10

40

20
5
0
Refed
STZ

2

STZ

Con STZ Con STZ

STZ

Con

FastedRefed

Con STZ Con STZ

SREBP-1
36B4
4.6

8
6
4
2
Con

STZ

Fasted

Con

STZ

Refed

600

500
400
300
200

0

10.1

10

Refed

100

Fold(Refed/Fasted)

12

0

STZ

600
Blood Glucose (mg/dl)

Refed

Con

14

GlucoseLine BLine A
Fasted4

FastedB. Intrinsic gene expression
Liver

6

Refed

Fasted16

800
FastedLine B

Plasma Insulin (ng/ml)

Liver

Blood Glucose (mg/dl)

332

11.2

4.9

Con

STZ

Fasted

Con

STZ

Refed

500
400
300
200
100
0

Con

STZ

Fasted

Con

STZ

Refed

Fig. 3. Refeeding response of streptozotocin-treated transgenic mice. (A) Transgene expression in streptozotocin-treated mouse liver as assessed byluciferase activity. At two days after second administration of streptozotocin, transgenic mice were either fasted for 24 h or refed at 16 h after a 24 h
starvation, and euthanized to obtain liver samples. Results are means ± SE of 4–6 male mice. *P < 0.05 and **P < 0.01, respectively. (B) Northern blot
analyses visualizing endogenous SREBP-1 mRNA expression pattern in streptozotocin-treated mouse liver. (C) Blood insulin and glucose levels of
streptozotocin-treated transgenic mice.

Y. Takeuchi et al. / Biochemical and Biophysical Research Communications 363 (2007) 329–335

mouse and luciferase activity was adjusted based on thisquantiﬁcation (Fig. 4B). (2) Liver samples on fasted andrefed states were taken from the same mouse by biopsy
under brief anesthesia and data of fasting and refeeding
conditions from one mouse are analyzed consecutively(Fig. 4C). Both approaches produced essentially the same
results, and refeeding responses of 20- to 30-fold increasein luciferase activities were consistently observed in adose-independent way, which paralleled the increases inthe endogenous SREBP-1c mRNA (Fig. 4E and F). In contrast, when SV40-promoter-Luc adenovirus was used as anegative control instead of 2.2 kb-SREBP-1c-promoterLuc adenovirus, no refeeding responses were observed(Fig. 4D).
These results are in complete accordance with thosefrom transgenic mouse model described above. Moreover,
the adenoviral approach can avoid the potential involve-

erated an adenovirus harboring the 2.2 kb promoter regionof SREBP-1c fused to the luciferase reporter gene, and validated that intravenously administered adenoviruses are
uniformly introduced into the mouse liver as shown in
Fig. 4A. Next we examined the refeeding responses of theseadenovirus-infected mice expressing SREBP-1c-promoterluciferase fusion gene in the liver. At the initial estimation,
the absolute luciferase activities observed varied amongindividual mice in a wide range despite the same numberof adenovirus particles for injection. As shown in
Fig. 4A, it was indicated that the variation among diﬀerent
portions of one liver was negligibly small, thus most of thevariation in measured luciferase activities was considered
to originate from inter-mouse variation in viral transduction eﬃciency. Two approaches were taken to cancel thisdispersion: (1) the amount of transducted adenoviral
DNA was quantiﬁed by real-time PCR technique on each

A

B

333

C

D

E

40
20
0

1 2 3

4 5

average

**

12000
10000
8000
6000
4000

30

Fold (Refed/Fasted)

60**
Fold (Refed/Fasted)

RLU/mg/Adenoviral dosage

RLU/mg80

14000

Fold (Refed/Fasted)

40
100

20

10

30

20

30
20
10

10

0

2000
0

F R F R F R F R F R

2FastedRefed

0

Fasted0

Refed

FastedF

Refed

20 60 200 x106 P.F.U./body

6

6

x10 P.F.U./body

0
F

2
R

F

6
R

F

20
R

F

R

60
F

R

200
F

R

SREBP-1

G

H

36B4

I

Fold(Refed/Fasted)

Insulin

6

Refed

Con STZ

Con STZ

SREBP-1

4

36B4
2
Fold(Refed/Fasted)

0

Fasted

Refed

6.2

2.8

6.7

7.9

7.8

7.2

7.3

Glucose16

600
Blood Glucose (mg/dl)

8

FastedPlasma Insulin (ng/ml)

Fold (Refed/Fasted)

**

9.4

14
12
10
8
6
4
2
0

Con STZ Con STZ
Fasted

Refed

500
400
300
200
100
0

Con STZ Con STZ
FastedRefed

Fig. 4. Adenovirus-mediated transduction of 2.2 kb-SREBP-1c-promoter-Luc into liver and assessment of refeeding response. (A) Validation ofhomogenous transfer of reporter gene into various portions of liver. Variation within a mouse liver was estimated by sampling 5 pieces of liver fromdistinct lobes after three days of 2.2 kb-SREBP-1c-promoter-Luc adenovirus injection (6.0 · 106 P.F.U./body). (B) Estimation of refeeding response of
luciferase activity adjusted by real-time PCR. Three days after the administration of 2.2 kb-SREBP-1c-promoter-Luc adenovirus, luciferase activities
were measured and at the same time, the amount of transducted adenoviral DNA was quantiﬁed by real-time PCR technique on each mouse of fasting andrefeeding group and luciferase activities per transducted adenoviral DNA were calculated based on this quantiﬁcation. Results are means ± SE of 4–5
mice. **P < 0.01. (C) Pairwise estimation of refeeding response using biopsy method. 2.2 kb-SREBP-1c-promoter-Luc adenovirus-administered mice
were fasted for 24 h and then liver samples were taken by biopsy under inhalation anesthesia. After refed for 16 h, mice were euthanized to obtain refedliver samples. Luciferase activities were determined and fold changes of luciferase activities were calculated by dividing data on a refed state by those of thesame mouse on a fasted state. (D) Negative control for data shown in (C). SV40-promoter-Luc control adenovirus was used instead of 2.2 kb-SREBP-1cpromoter-Luc adenovirus. (E) Dose independence of refeeding response. Indicated amounts of 2.2 kb-SREBP-1c-promoter-Luc adenoviruses were
injected and refeeding responses were assessed by pairwise method. F, fasted; R, refed. Results are means ± SE of three mice. (F) Northern blot analysis
indicating that endogenous SREBP-1 expression is not aﬀected by adenovirus administration. (G) Refeeding response of luciferase reporter activity inadenovirus-infected and streptozotocin-treated mouse liver. At two days after second administration of streptozotocin, 2.2 kb-SREBP-1c-promoter-Luc
adenovirus (6.0 · 106 P.F.U./body) was intravenously injected into ICR male mice. Three days thereafter, mice were fasted for 24 h and then liver biopsies
were performed under inhalation anesthesia. After refed for 16 h, mice were euthanized to obtain liver sample. Luciferase activities were measured andrefeeding responses were determined pairwise. (H) Refeeding responses of endogenous SREBP-1 mRNA expression in streptozotocin-treated mice asassessed by Northern blot. (I) Blood insulin and glucose levels of the control and streptozotocin-administered mice.

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Y. Takeuchi et al. / Biochemical and Biophysical Research Communications 363 (2007) 329–335

ment of positional eﬀects that might inﬂuence transgenic
mouse data. It is intriguing that whether the promoterreporter fusion gene is inserted in the genome (i.e. in thecase of transgenic mouse) or not (i.e. in the case of adenovirus-mediated gene transfer) does not aﬀect the regulationof the SREBP-1c promoter activity.

Evaluation of insulin involvement based on adenovirusmediated reporter gene transfer technique
The involvement of insulin was also evaluated using thisadenovirus-mediated introduction of SREBP-1c-promoterluciferase fusion gene into the liver. As shown in Fig. 4G,
streptozotocin-treated diabetic mice exhibited strong
refeeding responses of luciferase reporter activities in a similar manner to the responses of intrinsic SREBP-1c
mRNA, fully consistent with the data from transgenic
mice.
Regarding the role of insulin in the upregulation of lipogenesis, many previous reports indicate that insulin
increases the expression of SREBP-1c and thereby upregulates lipogenesis [22–24]. However, our present methods
clearly support that insulin signaling pathway does notdirectly control SREBP-1c expression. This suggests thatthe insulin response in vitro might not be identical to therefeeding response in vivo, thus previous knowledge on
in vitro promoter analyses of SREBP-1c regulation by insulin might not fully hold true of in vivo regulation. For better
understanding of the in vivo physiology, further studies ofin vivo promoter analyses based on our reporter gene transfer technique utilizing adenovirus vector are currently
ongoing.
In summary, we generated two in vivo systems to estimate the SREBP-1c promoter activity. It was demonstrated that the fasting-refeeding regulation of SREBP-1c
expression in the liver is exerted at the transcription level,
and that 2.2 kb of the 5 0 -ﬂanking sequence is suﬃcientfor this regulation. Our adenoviral strategy provides a feasible approach to identify the promoter regions responsible
for refeeding response of SREBP-1c expression, which iscurrently under going.

Acknowledgments
This work was supported by Grants-in-Aid and the
21st Century COE Program from the Ministry of Science, Education, Culture and Technology of Japan. It
was also supported by research grants from the Uehara
Memorial Foundation, ONO Medical Research Foundation, Takeda Science Foundation, Suzuken Memorial
Foundation, Japan Heart Foundation, Kanae Foundation for the Promotion of Medical Science, Senri Life
Science Foundation, and Okinaka Memorial Institutefor Medical Research.

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