Intracellular cAMP contents regulate NAMPT expression via induction of C/EBPβ in adipocytes

Intracellular cAMP contents regulate NAMPT expression via

induction of C/EBPβ in adipocytes

Takakazu Mitani a, Shun Watanabe a, Kenjiro Wada b, Hiroshi Fujii c,d, Soichiro

Nakamura a, Shigeru Katayama a,d

Graduate School of Science and Technology, Department of Agriculture, Division of

Food Science and Biotechnology, Shinshu University, Kami-ina, Nagano, Japan.

Department of Bioscience and Biotechnology, Shinshu University, Kami-ina, Nagano,

Japan

Graduate School of Science and Technology, Department of Biomedical Engineering,

Shinshu University, Kami-ina, Nagano, Japan.

Department of Biomolecular Innovation, Institute for Biomedical Sciences,

Interdisciplinary Cluster for Cutting Edge Research, Shinshu University, Kami-ina,

Nagano, Japan.

Running title: cAMP induces NAMPT expression

Address for Correspondence: Takakazu Mitani, Ph.D., Department of Bioscience and

Biotechnology, Shinshu University, 8304 Minami-minowa, Kamiina, Nagano, Japan.

399-4598, Tel & FAX: +81-265-77-1608. E-mail: mitani@shinshu-u.ac.jp

Abstract

A decline in intracellular nicotinamide adenine mononucleotide (NAD+) causes

adipose tissue dysfunction. Nicotinamide phosphoribosyltransferase (NAMPT)

the rate-limiting step in the NAD+ biosynthesis pathway. However, the molecular

mechanism that mediates regulation of NAMPT expression in adipocytes is yet to be

elucidated. This study found that intracellular cAMP regulates NAMPT expression and

promoter activity in 3T3-L1 adipocytes. cAMP-mediated Nampt promoter activity was

suppressed by protein kinase A inhibitor H89, whereas AMP-activated protein kinase

inhibitor compound C did not affect cAMP-mediated Nampt promoter activity.

Intracellular cAMP induced CCAAT/enhancer-binding protein β (C/EBPβ) expression.

Knockdown of C/EBPβ suppressed NAMPT expression and promoter activity.

Furthermore, the Nampt promoter was activated by C/EBPβ, while LIP activated the

dominant-negative form of C/EBPβ. Promoter sequence analysis revealed that the

region from −96 to −76 on Nampt was required for C/EBPβ-mediated promoter activity.

Additionally, chromatin immunoprecipitation assay demonstrated that C/EBPβ was

bound to the promoter sequences of Nampt. Finally, NAMPT inhibitor FK866

suppressed adipogenesis in 3T3-L1 cells, and this suppressive effect was restored by

nicotinamide mononucleotide treatment. These findings showed that intracellular cAMP

increased NAMPT levels by induction of C/EBPβ expression and indicated that the

induction of NAMPT expression was important for adipogenesis.

Keywords; adipogenesis; cAMP; CCAAT-enhancer-binding protein β; nicotinamide

adenine dinucleotide; nicotinamide phosphoribosyltransferase

Abbreviations: CCAAT/enhancer-binding protein, C/EBP;

3-isobutyl-1-methylxanthine, IBMX; nicotinamide adenine dinucleotide, NAD+;

nicotinamide phosphoribosyltransferase, NAMPT; peroxisome proliferator-activated

receptor, PPAR.

1. Introduction

Adipocytes store triglycerides as energy and are involved in secretory functions via

cytokines, known as adipokines. Secretory adipokines, such as leptin, adiponectin, and

interleukin-6, are carried to distant organs via the circulatory system; they maintain

metabolic functions, including appetite, body weight, insulin sensitivity, and

inflammation. Therefore, adipocyte dysfunction causes metabolic diseases, such as type

2 diabetes mellitus, cardiovascular disease, and hypertension [1,2].

Adipogenesis is a unique process of adipocyte differentiation, which is regulated by

transcriptional factors such as CCAAT/enhancer-binding proteins (C/EBPs),

peroxisome proliferator-activated receptor γ (PPARγ), and sterol regulatory

element-binding protein 1 (SREBP-1) [3,4]. The second messenger molecule cAMP

increases in the early stage of adipogenesis. Intracellular cAMP stimulates two signal

pathways, namely protein kinase A (PKA) and AMP-activated protein kinase (AMPK)

pathways [5,6]. PKA signaling mediates the induction of C/EBPβ expression [5].

Subsequently, C/EBPβ induces the expression of PPARγ and C/EBPα to promote

adipogenesis. C/EBPβ mRNA is translated into three different isoforms, full-length

C/EBPβ, LAP, and LIP, by alternative translation of the start codon [7]. LAP provides

transactivation capacity and is associated with adipogenesis, whereas LIP represents a

strongly shortened isoform, which is transcriptionally inactive and supports

proliferation [8].

Nicotinamide adenine dinucleotide (NAD+) plays a vital role in cellular energy

metabolism and homeostasis pathways in multiple organs. In addition, NAD+ acts as a

multifunctional coenzyme by modulating the key NAD+-dependent enzymes, such as

sirtuins and poly (ADP-ribose) polymerases [9]. Through these activities, NAD+ is

involved in cellular processes that regulate signaling and transcriptional events, such as

survival, stress-response, circadian rhythm, and aging. Thus, a decline in NAD+ content

causes cellular dysfunction and is particularly problematic for adipocytes. NAD+ is

synthesized in the de novo and salvage pathways. In the salvage pathway, nicotinamide

(an NAD+ precursor) is metabolized into nicotinamide mononucleotide (NMN) by

nicotinamide phosphoribosyltransferase (NAMPT), which is the rate-limiting enzyme in

the NAD+ salvage pathway. Subsequently, NMN is rapidly converted into NAD+ by

nicotinamide mononucleotide adenylyltransferase [9-11]. NAMPT is also known as

visfatin, and its expression is gradually increased in adipogenesis [12]. NAMPT

expression in adipocytes is regulated by environmental and nutritional conditions. For

example, diet-induced obesity decreases NAMPT expression levels and NAD+ content,

resulting in increased metabolic dysfunction [13,14]. In contrast, calorie restriction

increases NAMPT expression and NAD+ content [15]. However, the molecular

mechanism by which NAMPT expression is regulated in the adipocytes has yet to be

elucidated. In this study, we determined the role of cAMP in the induction of NAMPT

expression in the 3T3-L1 adipocytes.

2. Material and methods

2.1. Cell culture

Murine 3T3-L1 preadipocytes were obtained from the Japanese Collection of

Research Bioresources (IFO050416), and the culture and adipocyte differentiation

methods were described previously [16]. Briefly, for induction of adipocyte

differentiation, confluent cells were treated with a DMI cocktail [1 μM dexamethasone,

0.5 mM 3-isobutyl-1-methylxanthine (IBMX) and 10 μg/mL insulin] in Dulbecco’s

modified Eagle’s medium with high glucose (4.5 g/L glucose), supplemented with 10%

fetal bovine serum for the first 2 days. Then the cells were cultured in the same medium

with insulin for another 5 days. The medium was changed every 2 days.

2.2. siRNA oligonucleotides

Double-stranded siRNA for mouse C/EBPβ (siC/EBPβ) was chemically synthesized

by Sigma-Aldrich, and the sequences for the siRNA duplexes were as follows:

siC/EBPβ 5´-GAGCGACGAGTACAAGAT-3´. The control siRNA (siCTL) was

purchased from Sigma-Aldrich (MISSION siRNA Universal Negative Control#1). The

duplexes (20 nM) were transfected into the 3T3-L1 cells using Lipofectamine

RNAiMAX reagent (Thermo Fisher Scientific) for 24 h, according to the manufacture’s

protocol.

2.3. Plasmids

Myc tag was inserted into a pLVSIN-CMV Pur vector (Takara Bio., Shiga, Japan),

creating pLVSIN-Myc. Then mouse full-length C/EBPβ (1-296 amino acid), splicing

variant C/EBPβ (LIP; 152-296 amino acid) cDNAs were amplified by PCR and

subcloned into the pLVSIN-Myc vector, yielding a C/EBPβ expression vector with

N-terminal Myc tag (pLVSIN-Myc-C/EBPβ and pLVSIN-Myc-LIP). Mouse PPARγ

cDNAs were amplified by PCR and subcloned into a pLVSIN-Myc vector, yielding a

Myc-tag fused PPARγ expression vector (pLVSIN-Myc- PPARγ). The promoter region

of the mouse Nampt gene (−2000/+72) was amplified by PCR using mouse genomic

DNA from adipose tissues as a template. The amplified DNA was subcloned into the

pGL4.14 plasmid (Promega), and termed pGL4-Nampt (−2000/+72). We then generated

other reporter vectors, which were inserted at various lengths into the Nampt promoter

(−1500/+72, −1000/+72, −500/+72, −50/+72, −2000/−501, −2000/−51 and −500/−51),

which was constructed using pGL4-Nampt (−2000/+72) as a template. The introduction

of mutations from −91 to −83 into the promoter region of Nampt was performed by

two-step PCR, and the resultant DNAs were subcloned into pGL4.14 and termed

pGL4-Nampt (−500/+72 Mut). The nucleotide sequence of the Nampt promoter changed

from 5´-TTAAGCAA-3´ to 5´-AATTCGTT-3´. The pGL4-3×PPRE-Luc plasmid was

constructed by the insertion of three tandem repeats of PPARγ responsive elements

(PPREs) in pGL4.14. The resultant sequence of the PPREs oligonucleotide is as

follows:

5´-AGGTCAAAGGTCAGACAGGTCAAAGGTCAGACAGGTCAAAGGTCA-3´.

2.4. Measurement of intracellular lipid and culture adiponectin contents, and cell

survival

Adipocyte differentiation was induced by treatment with a DMI cocktail for 7 days.

The cells were fixed with 4% paraformaldehyde in PBS and incubated with Oil red O

solution (0.5% w/v) for 10 min at 23°C. Following staining, cells were washed, and the

lipid contents were extracted using isopropanol containing 4% (v/v) TritonX-100. The

extracted dye was measured at an absorbance of 492 nm. The culture adiponectin

contents were measured using a commercial ELISA kit (Shibayagi, Gunma, Japan). Cell

survival was determined by crystal violet staining assay, as described previously [16].

Briefly, the cells were incubated with the indicated concentrations of FK866 for 72 h.

The cells were fixed and stained. Then, the dye was extracted, and the absorbance was

measured at 595 nm with a reference wavelength at 630 nm.

2.5. Luciferase reporter assay

The cells were transiently transfected with various reporter vectors [Nampt

promoter-inserted pGL4.14, pGL4-3×PPRE-Luc, and pRL-SV40 (control reporter

vector; Promega)] using Lipofectamine 3000 (Thermo Fisher Scientific). Following 24

h transfection, the medium was changed to fresh medium, and the cells were incubated

10

with or without compounds for 24 h. Luciferase activities were measured using the

dual-luciferase reporter assay kit and GloMax 20/20 Luminometer (Promega).

2.6. Western blotting

Western blotting was performed as described previously [16]. Briefly, the cell lysates

were subjected to SDS-PAGE and analyzed by western blotting using the following

mouse monoclonal antibodies: anti-β-actin (1/10000, clone; C4), anti-PPARγ (1/5000,

clone; E-8, Santa Cruz Biotechnology, Santa Cruz, CA), and anti-Myc (1/3000, clone;

MC045, Nacalai Tesque, Kyoto, Japan); other antibodies used were rabbit polyclonal

anti-NAMPT (1/5000, GeneTex, Irvine, CA) and C/EBPβ (1/5000, Bethyl Laboratories,

Edison, NJ) antibodies. The immunoreactive proteins were visualized using LAS500

(GE healthcare). The intensity of each band was quantified using ImageJ (version

1.44o; NIH, Bethesda, MD), and the ratio of each protein level was normalized to that

of the β-actin (loading control) level.

2.7. Chromatin immunoprecipitation (ChIP)

ChIP was performed as described previously [16]. Briefly, cells were incubated with

11

IBMX for 24 h, followed by fixation and lysis. The cell lysates were incubated with

rabbit polyclonal anti-C/EBPβ IgG or control rabbit IgG overnight at 4ºC. Then

protein-DNA complexes were washed and eluted with the fragments. The promoter

regions of Nampt were amplified by PCR using the following primer sets; P1

(5´-CAAAGGCCTTGAGAACCAGAGC-3´ and

5´-CTTGTGAGACTATGCCGGGG-3´); P2 (5´-CGCGCTCCGTTCCCTGCTCT-3´

and 5´-GCGGCTGCGAGCAAGGAGAAAA-3´).

2.8. Statistical analysis

Data were analyzed by one- or two-way analysis of variance via Tukey’s post hoc or

Dunnett post hoc testing. Statistical analysis was performed with JMP statistical

software version 11.2.0 (SAS Institute. Cary, NC). Data are expressed as means ± SD,

and p < 0.05 was considered statistically significant.

3. Results

3.1. cAMP stimulates Nampt promoter activity in 3T3-L1 cells

We examined the effect of differentiation inducers on the promoter activity of Nampt

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in 3T3-L1 cells using luciferase reporter assay. Although insulin and dexamethasone did

not stimulate Nampt promoter activity, IBMX increased its promoter activity in a

dose-dependent manner (Fig. 1A and 1B). Adipocyte differentiation increased NAMPT

protein levels, and IBMX enhanced NAMPT protein levels in the differentiated 3T3-L1

adipocytes (Fig. 1C), while IBMX did not affect the protein levels of PPARγ.

Phosphodiesterase (PDE) is an enzyme that breaks cAMP into AMP, and IBMX

increases intracellular cAMP contents by inhibiting PDE function. Thus, to determine

whether increases in cAMP contents could activate Nampt promoter, 3T3-L1 cells were

incubated in the presence of other cAMP inducers. Forskolin increased intracellular

cAMP contents by stimulation of adenylate cyclase. Forskolin and cAMP analog

bucladesine increase the Nampt promoter activity and NAMPT protein levels (Fig. 1D

and 1E). IBMX-mediated promoter activity suppresses H89, an inhibitor of PKA

signaling, but did not suppress the AMPK inhibitor compound C (Fig. 1F). These results

indicated that the Nampt promoter activity was enhanced by cAMP-PKA signaling in

the 3T3-L1 adipocytes.

3.2. C/EBPβ is involved in Nampt promoter activity in the adipocytes

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IBMX induced C/EBPβ expression in the 3T3-L1 cells [17]. We determined whether

the involvement of the C/EBPβ protein-induced NAMPT expression using

C/EBPβ-specific siRNA. NAMPT protein levels increased significantly in the control

siRNA-treated 3T3-L1 cells at 5-7 days following the start of adipocyte differentiation

(Fig. 2A). While C/EBPβ knockdown was suppressed, an increase in adipocyte

differentiation-induced NAMPT protein levels. The knockdown of C/EBPβ also

suppresses IBMX-stimulated Nampt promoter activity (Fig. 2B). Furthermore, when the

C/EBPβ expression vector is transfected into 3T3-L1 cells, Nampt promoter activity is

induced by exogenous C/EBPβ (Fig. 2C). However, exogenous C/EBPβ did not

influence the empty luciferase vector. Cebpb-encoded protein isoforms, LIP (152-296)

were examined for their effect on the Nampt promoter activity. The results showed that

the C/EBPβ full length and LIP isoforms increased promoter activity, and LIP was more

potent than C/EBP full length in the upregulation of the Nampt promoter activity (Fig.

2D). We determined whether C/EBPβ-induced PPARγ expression was involved in

Nampt promoter activity. Rosiglitazone, a PPARγ ligand, stimulated the transcriptional

activity of the 3×PPRE-Luc but did not influence the Nampt promoter activity (Fig. 2E).

These results indicated that the Nampt promoter activity was regulated by the C/EBPβ

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protein, but not by PPARγ.

3.3. Located from −96 to −76 bp is required for the C/EBPβ-induced Nampt promoter

activity

To identify the C/EBPβ responsive region of the Nampt promoter, we generated

luciferase reporter constructs, which contained six series of deletion constructs of the

Nampt promoter (Fig. 3A, left panels). The promoter activity was driven by −2000/+72,

−1500/+72, −500/+72, and −2000/−51 Nampt promoters (Fig. 3A, right panels),

induced by IBMX, while deletion of the sequence from −500 to −51 bp abolished

IBMX responsiveness. Multi-genome analysis using the database showed that the

regions located at −1934/–1925 and −96/–76 in the Nampt promoter region contained

the C/EBPβ-binding element candidate, and the region from −96 to –76 bp was highly

conserved among mammalians, such as mice, humans, chimpanzee, rats, cattle, and pigs

(Fig. 3B). Therefore, we generated mutant constructs located from −96 to –76 bp in the

Nampt promoter region. In the mutant construct, IBMX, and exogenous C/EBPβ did not

influence the Nampt promoter activity (Fig. 3C). Furthermore, we determined whether

C/EBPβ could bind to the promoter region of Nampt using a ChIP assay with

15

specific-primer sets, as depicted in Fig. 3D (upper panels). IBMX treatment increased

the levels of C/EBPβ on the DNA, which were located at −138/–41 of the Nampt

promoter region. While IBMX treatment did not affect the interaction between C/EBPβ

and the DNA in the Nampt promoter region from −2144 to –1828 (Fig. 3D, bottom

panels). These results indicated that C/EBPβ induced NAMPT expression via binding to

the promoter region (−97/−76) on Nampt in 3T3-L1 cells.

3.4. NAMPT promotes adipogenesis in 3T3-L1 cells.

High-fat diet-induced adipose expansion was suppressed in the NAMPT knockout

mice [18]. We observed that NAMPT inhibitor FK866 decreased the intracellular lipid

contents in 3T3-L1 cells (Fig. 4A). In addition, the treatment of NMN restored the

FK866-decreased lipid contents. By contrast, FK866 did not influence cell survival in

the 3T3-L1 cells (Fig. 4B). Interestingly, NAMPT inhibition decreased the production

of adiponectin that was an insulin-sensitizing adipokine, and this effect was canceled by

the NMN treatment (Fig. 4C). These results indicated that NAMPT-mediated NAD+

synthesis was involved in adipogenesis of 3T3-L1 cells.

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4. Discussion

NAMPT is a rate-limiting enzyme in the NAD+ biosynthesis pathway, and a decline

in NAMPT could cause a decrease in intracellular NAD+. NAMPT expression and

NAD+ were decreased in the diet-induced obesity and aging mice and resulted in the

development of type 2 diabetes mellitus [19,13]. Therefore, supplementation with

NAD+ or increasing the expression of NAMPT could lead to the prevention of obesityor aging-associated diseases. A previous report has indicated that NAMPT expression

was induced during adipogenesis in the adipocytes [12]. However, it was unclear how

NAMPT affected adipogenesis in the adipocytes, as the regulatory mechanism of

NAMPT expression has not yet been elucidated. In this study, we revealed that NAMPT

was involved in the adipogenesis of the 3T3-L1 cells. In addition, our report was the

first to show that cAMP-induced C/EBPβ was the primary regulator of Nampt

expression during adipogenesis.

NAMPT inhibition suppressed adipogenesis and adiponectin production in the

3T3-L1 cells. In the fat-specific NAMPT knockout mice, the high-fat diet interfered

with the healthy expansion of the adipose tissue mass, and adipose deposits resulted in

adipose tissue fibrosis [18]. Adipocyte hypertrophy induced insulin resistance via the

17

secretion of pro-inflammatory adipokines from the adipocytes [9]. By contrast, healthy

adipocyte differentiation is associated with improved insulin resistance through the

secretion of insulin-sensitizing adipokine, adiponectin in the adipocytes [20]. The

knockout of NAMPT reduced adiponectin expression in the adipose tissue, and adipose

tissue expression was restored by NMN administration [19]. Thus, these results

indicated that NAMPT contributed to the healthy functions of the adipocyte as well as

adipogenesis.

Nampt expression was stimulated by the full-length C/EBPβ and LIP. C/EBPβ plays a

pivotal role in adipogenesis and induces the expression of PPARγ [21]. The LIP isoform

is regarded as a dominant-negative inhibitor for the full-length C/EBPβ, and

overexpression of LIP resulted in anti-adipogenic activity [8]. However, Bégay et al.

(2018) reported that the LIP isoform was sufficient to function in the development of

adipose in the Cebpb knockout mice, and proposed that the LIP isoform likely had more

physiological functions than its known role as a dominant-negative inhibitor [22]. These

results indicated that C/EBPβ promoted adipogenesis through two independent

pathways, such as the C/EBPβ-mediated PPARγ or NAMPT induction pathways.

There are multiple cAMP-binding proteins in all cells. cAMP-bound Epac1 enhanced

18

phosphorylation of AMPK, an intracellular energy sensor, as it increased the

intracellular calcium concentration [23]. In the skeletal muscle, exercise-mediated

AMPK activation increased the Nampt expression [24]. However, our data showed that

the AMPK inhibitor compound C did not influence the IBMX-induced Nampt promoter

activity (Fig. 1F). cAMP-PKA signaling-activated CREB positively controls the

C/EBPβ expression [25]. In addition, CREB improved the protein stability of C/EBPβ

as it induced SUMO-specific protease [26]. Therefore, our results indicated that cAMP

signaling contributed to Nampt promoter activity through increased C/EBPβ expression

levels and protein stability in the adipocytes.

C/EBPβ was bound to the −138/−42 in the Nampt promoter region of the 3T3-L1

cells. There were two candidate C/EBPs-binding sequences in the −2000/+72 Nampt

promoter region. Specifically, the −96/–76 region showed high homology between a

mouse and human, which indicated that the regulation of Nampt expression in the

human adipocytes was by the same mechanism characterized in this study. Furthermore,

another research group has found a transcription factor that binds to DNA near the

transcription start site of Nampt, which was similar to C/EBPβ [27]. Yoon et al. (2017)

found that SREBP-1c stimulated Nampt promoter activity in the murine pancreatic

19

islets, and the region at −450/−455 of Nampt was important for SREBP-1c as it

stimulated promoter activity [27]. SREBP-1c expression increased during

differentiation in the adipocytes [28]. These results suggested that Nampt expression

was additively or synergistically regulated by transcriptional factors, which could bind

near the transcription start site, such as C/EBPβ and SREBP-1c during adipogenesis.

In summary, we showed that cAMP contents regulated Nampt expression in

adipocytes and NAMPT contributed to adipogenesis. Selective PDE inhibitors have

been identified as therapeutic agents for hypertension and coronary heart disease to

increase the intracellular cAMP contents [29]. Thus, these selective PDE inhibitors

might contribute to healthy adipogenesis and the maintenance of adipocyte function

through the regulation of NAMPT levels.

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Acknowledgements

We would like to thank Editage (www.editage.jp) for English language editing (JOB

CODE; ZUMIT_3)

Conflict of Interest

The authors declare that there are no conflicts of interest.

Role of the Funding Source

This work was supported by JSPS KAKENHI (grant number: 18K14405).

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Figure legends

Fig. 1. Effect of cAMP contents on the promoter activity of Nampt in 3T3-L1 cells. (A)

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Nampt promoter activity in 3T3-L1 cells transiently transfected with pGL4-Nampt

(−2000/+72) vector, then incubated with insulin (10 μg/mL), DEX (1 μM), or IBMX

(0.5 mM) for 24 h. (B) Luciferase reporter assay in 3T3-L1 cells is transiently

transfected with pGL4-Nampt (−2000) vector, then incubated with various

concentrations of IBMX for 24 h. (C) NAMPT and PPARγ protein levels in 3T3-L1

cells are treated with IBMX (0.5 mM) for 24 h. (D) Nampt promoter activity in 3T3-L1

cells treated with cAMP inducers, such as bucladesine (cAMP analogue; 100 μM) and

forskolin (adenylyl cyclase activator; 10 μM). (E) NAMPT protein levels in 3T3-L1

cells treated with bucladesine (100 μM) and forskolin (10 μM). *p < 0.05 versus vehicle

groups. (F) Nampt promoter activity in 3T3-L1 cells treated with H89 (1 μM) or

compound C (5 μM) in the presence of IBMX (0.5 mM) for 24 h. Error bars represent

the mean ± SD (n = 4). In (C) and (E), the band intensities of NAMPT or PPARγ are

normalized to those of β-actin, and the relative values for the vehicle are indicated

under the lower panels. Significant differences (p < 0.05) are indicated by the

corresponding letters. All data shown are representative of triplicate independent

experiments.

27

Fig. 2. Involvement of C/EBPβ in Nampt promoter activity. (A) NAMPT protein levels

in 3T3-L1 cells transfected with the control siRNA (siCTL) or C/EBPβ-specific siRNA

(siC/EBPβ). Following siRNA transfection, adipogenesis is induced for 7 days. The

band intensities of NAMPT and C/EBPβ are normalized to those of β-actin, and the

relative values to Day 0 are indicated under the lower panels. n.d. (not detected) (B)

Nampt promoter activity in 3T3-L1 cells treated with siRNA. Following transfection

with siCTL or siC/EBPβ, the cells are transiently transfected with pGL4-Nampt

(−2000/+72) vector, followed by incubation with or without IBMX (0.5 mM) for 24 h.

(C) Nampt promoter activity (upper panel) and Myc-C/EBPβ protein levels (bottom

panel) in the 3T3-L1 cells. The cells are transiently transfected with luciferase reporter

vectors [pGL4.14 (open bars) or pGL4-Nampt (−2000/+72) (closed bars)] and

Myc-C/EBPβ expression vector, and incubated for 24 h. (D) Nampt promoter activity in

full-length C/EBPβ ...