Biochimica et Biophysica Acta 1820 (2012) 461–468
Contents lists available at SciVerse ScienceDirect
Biochimica et Biophysica Acta
journal homepage: www.elsevier.com/locate/bbagen
Nobiletin suppresses adipocyte differentiation of 3T3-L1 cells by an insulin and IBMX
mixture induction
Kota Kanda a, Kosuke Nishi a, Ayumu Kadota b, Sogo Nishimoto a, Ming-Cheh Liu c, Takuya Sugahara a, d,⁎
a
Faculty of Agriculture, Ehime University, Matsuyama, Ehime 790-8566, Japan
Ikata Service Co. Ltd., Nishiuwa, Ehime 769-0421, Japan
Department of Pharmacology, The University of Toledo, Toledo, OH 43606, USA
d
South Ehime Fisheries Research Center, Ehime University, Ainan, Ehime 798-4292, Japan
b
c
a r t i c l e
i n f o
Article history:
Received 26 May 2011
Received in revised form 25 November 2011
Accepted 28 November 2011
Available online 8 December 2011
Keywords:
Nobiletin
PPARγ
STAT5
3T3-L1
cAMP
CREB
a b s t r a c t
Background: Nobiletin is a citrus flavonoid which possesses the flavone structure with six methoxy groups.
Although nobiletin has been reported to display anti-inflammatory, anti-tumor, and anti-diabetes activities,
its effect on adipocyte differentiation remained unclear. In the present study, we investigated the effect of
nobiletin on the differentiation of 3T3-L1 preadipocytes into adipocytes.
Methods: 3T3-L1 preadipocytes were treated with nobiletin under various differentiation conditions. The effect of nobiletin on adipocyte differentiation was evaluated by oil red O staining, real-time RT-PCR, and Western blotting.
Results: Nobiletin significantly suppressed the differentiation of 3T3-L1 preadipocytes into adipocytes, upon induction with insulin together with a cAMP elevator such as 3-isobutyl-1-methylxanthine (IBMX), by downregulating the expression of the gene encoding peroxisome proliferator-activated receptor (PPAR) γ2. In addition,
nobiletin decreased the phosphorylation of cAMP-response element-binding protein (CREB) and strongly enhanced the phophorylation of signal transducer and activator of transcription (STAT) 5.
General significance: Nobiletin has a suppressive effect on the differentiation of preadipocytes into adipocytes
when cells were induced with a general differentiation cocktail such as insulin, IBMX, and dexamethasone.
© 2011 Elsevier B.V. All rights reserved.
1. Introduction
Obesity is an etiologic factor for type 2 diabetes, cardiovascular
disease, and hypertension. Obesity results from an increase in both
the number and size of adipocytes [1]. Adipocytes represent a major
energy reservoir in the body, storing excess energy as lipids and releasing it on demand. In addition, adipocytes constitute an endocrine
system by secreting adipose-derived hormones known as adipokines,
which regulate tissues such as muscles and adipose tissues [2]. Thus,
the regulation of adipocyte differentiation is crucial for therapies
Abbreviations: cAMP, cyclic adenosine monophosphate; C/EBP, CCAAT/enhancerbinding protein; CREB, cAMP-response element-binding protein; DEX, dexamethasone;
DMEM, Dulbecco's modified Eagle's medium; EDTA, ethylenediaminetetraacetic acid;
FBS, fetal bovine serum; IBMX, 3-isobutyl-1-methylxanthine; HRP, horseradish peroxidase;
MEF, mice embryonic fibroblast; MEK, mitogen-activated protein kinase/extracellular
signaling-regulated protein kinase; PBS, phosphate-buffered saline; PKA, protein kinase A;
PPAR, peroxisome proliferators-activated receptor; SD, standard deviation; STAT, signal
transducer and activator of transcription; TBS-T, Tris-buffered saline with 0.1% Tween 20
⁎ Corresponding author at: Faculty of Agriculture, Ehime University, 3-5-7 Tarumi,
Matsuyama, Ehime 790-8566, Japan. Tel./fax: + 81 89 946 9863.
E-mail address: mars95@agr.ehime-u.ac.jp (T. Sugahara).
0304-4165/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbagen.2011.11.015
aimed to prevent and improve obesity and the related metabolic
syndrome.
3T3-L1 cells are derived from the 3T3 Swiss albino mouse cell line,
and are used as an in vitro model of adipocytes to study insulin pathways, obesity, and cardiovascular diseases [3]. 3T3-L1 preadipocytes
differentiate upon exposure to inducers such as insulin, 3-isobutyl1-methylxanthine (IBMX), and dexamethasone (DEX) [4]. These inducers activate the signaling cascade, thereby facilitate the expression of peroxisome proliferators-activated receptor (PPAR) γ or the
production of PPARγ ligands. PPARγ is a master regulator of adipocyte differentiation. Forced expression of PPARγ gene is sufficient to
induce the adipogenesis in fibroblasts [5], and no factor has been discovered that promotes adipocyte differentiation in the absence of
PPARγ [6]. In the signaling cascade, the expression of PPARγ gene
is induced by CCAAT/enhancer-binding protein (C/EBP) β [7]. In
addition, cyclic adenosine monophosphate (cAMP)-response elementbinding protein (CREB) is known to be involved in adipocyte differentiation [8,9]. CREB is activated through the phosphorylation by protein
kinase A (PKA), and may subsequently promote the expression of
C/EBPβ gene. Moreover, signal transducer and activator of transcription (STAT) 5 has been shown to regulate the adipogenesis [10]. Activation of STAT5 typically induces adipocyte differentiation [11],
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Committee and were performed in accordance with applicable guidelines and regulations.
while excessive activation of STAT5 has been reported to inhibit the
adipogenesis in 3T3-L1 cells [12].
Nobiletin, a citrus flavonoid which has a typical flavonoid structure with six methoxy groups, is a major component in citrus fruits
[13]. It has been reported that nobiletin displays anti-inflammatory
[14], anti-tumor [15], and anti-diabetes activities [16]. In addition, it
has been reported that nobiletin may stimulate the differentiation
of preadipocytes into adipocytes by the induction with insulin alone
[17], and enhance the expression of adiponectin gene at mRNA and
protein levels [17–19]. On the other hand, Miyamoyo et al. [20]
reported a suppressive effect of nobiletin on adipocyte differentiation
of 3T3-L1 cells and proposed that nobiletin may inhibit the mitogenactivated protein kinase/extracellular signaling-regulated protein kinase (MEK) signaling pathway, thereby leading to the suppression of
adipocyte differentiation. Therefore, the effect of nobiletin on adipocyte
differentiation remained unclear. In this study, we investigated the effect of nobiletin on the differentiation of 3T3-L1 preadipocytes into adipocytes under various inducing conditions.
Oil red O staining was performed using a lipid assay kit (Primary
Cell, Hokkaido, Japan) according to the manufacturer's instructions.
Briefly, cells were washed with phosphate-buffered saline (PBS) and
fixed for at least 15 min with 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4 (Nacalai Tesque, Kyoto, Japan). The cells were
washed twice with PBS, and then stained for 15 min using an oil red
O staining solution. After washing with distilled water, stained cells
were observed under a microscope. Following the microscopic observation, an extraction solution was added to cells and the absorbance
of the extracted dye was spectrophotometrically measured at
540 nm using a microplate reader (SH-9000, Corona Electric, Ibaraki,
Japan).
2. Materials and methods
2.5. Real-time RT-PCR
2.1. Reagents
Total RNA was isolated from 3T3-L1 cells using Sepasol-RNA I Super G
(Nacalai Tesque) according to the manufacturer's instructions. Total RNA
was used as a template for cDNA synthesis performed using MMLVreverse transcriptase (Promega, Madison, WI, USA) and oligo-(dT)20
primer. The real-time PCR mixture, with a final volume of 20 μl, consisted
of Fast SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA,
USA), 1 μM of a forward primer, 1 μM of a reverse primer, and 0.1 μg of a
cDNA sample. The thermal cycling conditions were: 20 s at 95 °C, and 40
cycles of 3 s at 95 °C and 30 s at 60 °C. PCR products were measured with
a StepOnePlus Real-time PCR System (Applied Biosystems), and the relative gene expression was calculated based on the comparative CT method
using a StepOne Software v2.1 (Applied Biosystems). The expression of
β-actin mRNA was used as an endogenous control. Primer pairs used
here for the amplification were: 5′-CATCCGTAAAGACCTCTATGCCAAC-3′
and 5′-ATGGAGCCACCGATCCACA-3′ for mouse β-actin; 5′-AACTCTGGGAGATTCTCCTGTTGA-3′ and 5′-GAAGTGCTCATAGGCAGTGCAT-3′ for
mouse PPARγ2, 5′-CATGGCCAAGCCCAACAT-3′ and 5′-CGCCCAGTTTGAAGGAAATC-3′ for mouse aP2; 5′-GGGGTTGTTGATGTTTTTGG-3′ and
5′-CGAAACGAAAAGGTTCTCA-3′ for mouse C/EBPβ. Data were shown as
the fold difference normalized to the β-actin.
Nobiletin (5,6,7,8,3′,4′-hexamethoxyflavone; >95% purity) was purchased from Wako Pure Chemical Industries (Osaka, Japan). Nobiletin
was dissolved in DMSO to make a 0.1 M solution and used for the experiments described below. Insulin, DEX, and IBMX were products of Sigma
(St. Louis, MO, USA). Antibodies against β-actin (sc-1616), PPARγ (sc7196), and STAT5 (sc-835) were from Santa Cruz Biotechnology
(Santa Cruz, CA, USA). Antibodies against CREB (#9197), phosphorylated CREB (#9198), and phosphorylated STAT5 (#9351) were purchased
from Cell Signaling Technology (Danvers, MA, USA).
2.2. Cells and cell culture
3T3-L1 preadipocytes obtained from American Type Culture
Collection (Rockville, MD, USA) were cultured in Dulbecco's modified
Eagle's medium (DMEM; Sigma) containing 10% fetal bovine serum
(FBS; SAFC Biosciences, Lenexa, KS, USA) at 37 °C in a humidified atmosphere of 5% CO2. Differentiation of 3T3-L1 preadipocytes into mature adipocytes was conducted as reported previously [21]. Briefly,
2 days after reaching confluency, 3T3-L1 preadipocytes grown in individual wells of collagen-coated 24-well cell culture plates (BD Falcon,
Franklin Lakes, NJ, USA) were subjected to induction by replacing the
culture medium with the induction DMEM, and the induction proceeded for 2 days. Subsequently, culture media were changed to
DMEM containing 10% FBS and 5 μg/ml of insulin every 2 days unless
otherwise noted. The complete induction medium consisted of
DMEM medium containing 10% FBS, 5 μg/ml of insulin, 0.5 mM
IBMX, and 10 μM DEX. Incomplete induction media lacking one or
two of the inducing reagents from the complete induction medium
were indicated in the “Results” section and in the figure legends.
2.3. Isolation and cell culture of mouse embryonic fibroblasts (MEFs)
MEFs were isolated from BALB/c mouse embryos on day 14.5 of
gestation. Briefly, embryos were minced and treated with trypsin
and ethylenediaminetetraacetic acid (EDTA) at 37 °C for 10 min, and
were then added DNase I (Takara Bio, Shiga, Japan) and incubated
for 5 min. Cells were dispersed by pipetting, passed through a
40 μm cell strainer (BD Falcon), and washed with DMEM containing
10% FBS. Methods for cell culture and the induction into adipocytes
were the same as those described above for 3T3-L1 cells. All animal
experiments described here were carried out in accordance with a
protocol approved by the Ehime University Animal Care and Use
2.4. Oil red O staining
2.6. Western blot analysis
Total cell lysate was prepared using a lysis buffer consisting of
50 mM Tris, 150 mM NaCl, 1 mM EDTA, 50 mM NaF, 30 mM Na4P2O7,
2 μg/ml of aprotinin (Roche Applied Science, Basel, Switzerland), pefabloc SC (Roche Applied Science), and a protease inhibitor cocktail
(Roche Applied Science). Proteins in the total cell lysate were separated
by a polyacrylamide gel electrophoresis and transferred onto a PVDF
membrane (Hybond-P, GE Healthcare, Buckinghamshire, UK). The
membrane was incubated with Tris-buffered saline containing 0.1%
Tween 20 (TBS-T) and 5% skim milk for 1 h at room temperature.
After washing with TBS-T, the membrane was incubated with a primary
antibody in 5% bovine serum albumin-TBS-T at 4 °C overnight. After
washing with TBS-T, the membrane was incubated with a horseradish
peroxidase (HRP)-conjugated anti-rabbit immunoglobulin antibody
(DakoCytomation, Glostrup, Denmark) or HRP-conjugated anti-goat
IgG antibody (Santa Cruz Biotechnology) in 5% skim milk-TBS-T at
room temperature for 1 h. After washing with TBS-T, blots were developed by an ECL Western Blotting Detection Reagent (GE Healthcare) or
ImmunoStar LD (Wako Pure Chemical Industries). Bands were visualized using a ChemiDoc XRS Plus apparatus (Bio-Rad Laboratories,
Hercules, CA, USA), and the chemiluminescent intensity was quantified
using the Quantity One software (Bio-Rad Laboratories).
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2.7. Statistical analysis
Data obtained were expressed as the mean ± standard deviation
(SD). The student's t test was used to assess the statistical significance
of the difference against the control. Values with *p b 0.05 or **p b 0.01
were considered to be statistically significant.
3. Results
3.1. Effect of nobiletin on lipid accumulation in 3T3-L1 cells
In an initial study, 3T3-L1 preadipocytes were treated with the
complete induction medium containing insulin, IBMX, and DEX to induce their differentiation into mature adipocytes. Nobiletin was
added to the complete induction medium for 2 days. On day 2 after
induction, culture medium was changed to DMEM supplemented
with 10% FBS and 5 μg/ml of insulin, and the cells were cultured for
another 5 days. Lipid accumulation in the adipocytes was measured
by oil red O-staining on day 7. As shown in Fig. 1A and B, nobiletin
significantly suppressed the lipid accumulation in 3T3-L1 adipocytes
in a dose-dependent manner with statistical significances, suggesting
that nobiletin inhibits adipogenesis in 3T3-L1 cells when the adipocyte differentiation was induced with insulin, IBMX, and DEX.
and DEX. In the experiments described below, we used 3T3-L1 cells
to analyze further the mechanism of the suppressive effect of nobiletin on adipogenesis, because nobiletin exerted a stronger effect on
3T3-L1 cells than on MEFs.
3.3. Evaluation of the optimal effect of nobiletin in suppressing the lipid
accumulation
To clarify the mechanism of the suppressive effect of nobiletin on
adipogenesis, we first examined the time course of the effect of nobiletin on adipocyte differentiation in regard to the suppression of lipid accumulation. 3T3-L1 cells were treated with nobiletin at different times
(from 0 to 6 days) following induction, and thereafter were monitored
for different durations (from 1 to 7 days). Results shown in Fig. 2A indicated that when nobiletin treatment was administered between day
0 and day 2 upon induction, the lipid accumulation was significantly
suppressed (Fig. 2B). Interestingly, treatment of nobiletin after 2 days
of induction exerted virtually no effect on lipid accumulation in differentiated 3T3-L1 adipocytes. It was also observed that no differences in
lipid accumulation were observed irrespective of the duration of treatment with nobiletin. These results indicated that nobiletin can affect
only 3T3-L1 preadipocytes within the first 2 days following the induction treatment to suppress the lipid accumulation.
3.2. Effect of nobiletin on lipid accumulation in MEFs
3.4. Effect of nobiletin on the translational level of PPARγ
To investigate whether the suppressive effect of nobiletin on lipid
accumulation is specific only for 3T3-L1 cells, primary preadipocytes,
MEFs were used. Adipocyte differentiation of MEFs was induced with
the complete induction medium supplemented with different concentrations of nobiletin. On day 2 after induction, culture media
were changed to DMEM supplemented with 10% FBS and insulin,
and the cells were cultured for another 5 days. Subsequent oil red
O-staining revealed that nobiletin significantly suppressed lipid accumulation in MEFs (Fig. 1C, D), indicating that nobiletin inhibits the
adipogenesis not only in 3T3-L1 cells, but also in primary preadipocytes when their differentiation was induced with insulin, IBMX,
A
The adipogenesis is known to be promoted by increased expression of PPARγ gene. Therefore, we decided to investigate PPARγ expression level in 3T3-L1 cells by Western blot analysis. Nobiletin did
not affect the protein expression of PPARγ1 in 3T3-L1 cells (Fig. 3A,
B), but significantly suppressed the PPARγ2 protein expression
(Fig. 3A, C). It has been reported that the expression of PPARγ2 promotes the adipogenesis in 3T3-L1 cells, whereas PPARγ1 may not
[22]. These results therefore suggest that nobiletin may inhibit the adipocyte differentiation by down-regulating PPARγ2 expression level
in 3T3-L1 cells.
C
Pre
0
1
10
100
Pre
0
Nobiletin (µM)
10
100
D
B
1.0
0.8
0.8
**
**
0.6
**
0.4
0.2
0.0
Pre
0
1
10
Nobiletin (µM)
100
Absorbance (540 nm)
Absorbance (540 nm)
1
Nobiletin (µM)
**
*
1
10
0.6
**
0.4
0.2
0.0
Pre
0
100
Nobiletin (µM)
Fig. 1. Effect of nobiletin on lipid accumulation in 3T3-L1 cells and MEFs. (A) Micrographs of 3T3-L1 cells. (B) Lipid accumulation in 3T3-L1 cells assessed by the spectrophotometric
measurement of oil red O-stained 3T3-L1 cells. (C) Micrographs of MEFs. (D) Lipid accumulation in MEFs was assessed by the spectrophotometric measurement of oil red O-stained
cells. 3T3-L1 cells and MEFs were induced to differentiate with 10% FBS-DMEM medium containing insulin, DEX, IBMX, and 0, 1, 10, or 100 μM of nobiletin for 2 days, and then
culture media were replaced with 10% FBS-DMEM medium containing insulin for the following 5 days. Control cells were treated with DMSO instead of nobiletin. Preadipocytes
were cultured in 10% FBS-DMEM medium. Data were represented as the mean ± SD (n = 3). *p b 0.05 and **p b 0.01 against control.
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A
B
Day
0
1
2
3
a:
Preadipocyte
b:
Control
4
5
6
7
Absorbance (540 nm)
0.0
0.4
0.8
*
c:
1.2
d:
f:
**
Nobiletin (100 µM)
**
e:
g:
h:
**
i:
Fig. 2. Suppressive effect of nobiletin on the lipid accumulation by a various treating period. (A) 3T3-L1 preadipocytes were induced to differentiate with 10% FBS-DMEM medium
containing insulin, DEX, and IBMX for 2 days (from day 0 to day 2), and then culture media were replaced with 10% FBS-DMEM medium containing insulin for the following 5 days.
Nobiletin (100 μM) was added during the period indicated as an arrow, and oil red O-staining was performed on day 7 from initiating the differentiation. Preadipocytes were cultured in 10% FBS-DMEM medium. Control cells were treated with DMSO instead of nobiletin. (B) Quantitative analysis of lipid accumulation was assessed by the spectrophotometric
measurement of oil red O-stained adipocytes. Data were represented as the mean ± SD (n = 2). *p b 0.05 and **p b 0.01 against control.
expression at the protein level as shown in Fig. 3C, the transcripts of
PPARγ2 and aP2, a target gene of PPARγ, were significantly decreased
following treatment with nobiletin on day 1 (Fig. 4A) and on day 3
(Fig. 4B), respectively. Moreover, nobiletin significantly suppressed
C/EBPβ expression on day 1 (Fig. 4C). It is well known that in the
3.5. Effect of nobiletin on adipogenic gene expression
To clarify the mechanism underlying the regulation of PPARγ2 expression, we investigated the expression of adipogenic genes by realtime RT-PCR. Consistent with the observation of increased PPARγ2
A
2
PPARγ 1
Actin
(day)
0
1
2
3
4
Control
B
3
4
C
PPARγ2
*
10
5
Preadipocytes
Relative expression
Relative expression
2
Nobiletin
PPARγ1
Control
4
Nobiletin
3
2
1
0
1
0
1
2
3
Days after induction
4
**
5
**
0
0
1
2
3
4
Days after induction
Fig. 3. Western blot analysis of PPARγ protein in 3T3-L1 cells treated with nobiletin. (A) A representative image from two independent experiments. (B) PPARγ1 expression level in
each cell sample relative to that in preadipocytes compensated with actin as an internal control. (C) PPARγ2 expression level in each cell sample relative to that in preadipocytes
compensated with actin as an internal control. 3T3-L1 cells were induced to differentiate with 10% FBS-DMEM medium containing insulin, DEX, IBMX, and 100 μM of nobiletin for
2 days, and then culture media were replaced with 10% FBS-DMEM medium containing insulin for the following 2 days. Preadipocytes were cultured in 10% FBS-DMEM medium.
Control cells were treated with DMSO instead of nobiletin. Cell lysates were prepared at the indicated day after the differentiation induction, and Western blotting was performed.
Data were represented as the mean ± SD (n = 2). *p b 0.05 and **p b 0.01 against control.
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A
Relative expression
B
PPARγ2
Preadipocytes
Control
Nobiletin
50
40
30
**
C
aP2
1000
60
C/EBPβ
10
*
800
8
600
6
400
4
200
2
*
20
10
0
0
1
2
3
4
Days after induction
0
0
1
2
3
0
4
0
1
2
3
4
Days after induction
Days after induction
Fig. 4. Effect of nobiletin on the expression of adipogenic genes in 3T3-L1 cells. (A) PPARγ2; (B) aP2; (C) C/EBPβ. 3T3-L1 cells were induced to differentiate with 10% FBS-DMEM
medium containing insulin, DEX, IBMX, and 100 μM of nobiletin for 2 days, and then culture media were replaced with 10% FBS-DMEM medium containing insulin for the following
2 days. Preadipocytes were cultured in 10% FBS-DMEM medium. Control cells were treated with DMSO instead of nobiletin. Total RNA was prepared at the indicated day after the
differentiation induction and analyzed for the indicated genes. β-Actin mRNA was analyzed as an internal control. Data were represented as a relative expression level ± SD against
preadipocytes (n = 3). *p b 0.05 and **p b 0.01 against control.
first 2 days during the adipocyte differentiation, the expression of
C/EBPβ gene is up-regulated and that C/EBPβ promotes the PPARγ
gene expression [23]. Taken together, these findings suggest that nobiletin may suppress the adipocyte differentiation by down-regulating
PPARγ2 gene expression via decreased C/EBPβ expression.
3.6. Influence of different combinations of differentiation inducers on the
suppressive effect of nobiletin
To reveal the interaction of nobiletin with the adipocyte differentiation inducers, the effect of nobiletin on the adipogenesis in 3T3-L1
preadipocytes upon induction with insulin alone, insulin plus IBMX,
or insulin plus DEX was examined. As shown in Fig. 5, nobiletin enhanced PPARγ2 expression when 3T3-L1 preadipocytes were induced
with insulin alone. In contrast, nobiletin significantly suppressed
PPARγ2 expression when differentiation of 3T3-L1 preadipocytes
was induced by insulin plus IBMX. The PPARγ2 gene expression was
unaffected by inducing when the cells were induced by insulin plus
DEX. These results suggest that nobiletin may down-regulate the
signaling pathways mediated by IBMX.
3.7. Effect of nobiletin on the phosphorylation of CREB
IBMX is known to increase the intracellular cAMP level by inhibiting phosphodiesterase. Accumulated cAMP may activate PKA, which
in turn activates CREB through its phosphorylation. Phosphorylated
CREB then participates in inducing C/EBPβ expression, and C/EBPβ
in turn induces the expression of both PPARγ and C/EBPα. As
shown in Fig. 4C, nobiletin reduced C/EBPβ expression. To clarify
whether nobiletin exerts its effect upstream of the C/EBPβ signaling
pathway, the activation of CREB involved in the signaling pathway
stimulated by IBMX was examined. As shown in Fig. 6A, CREB was
A
p-CREB
CREB
(min)
0
5
15
30
60
5
Control
15
30
60
Nobiletin
B
10
Control
Nobiletin
Ratio of p-CREB/CREB
Relative PPARγ2 expression
4
3
2
*
1
5
0
0
Insulin
Insulin
Insulin
+
IBMX
+
DEX
Fig. 5. Effect of nobiletin on the differentiation by various inducer combinations in 3T3L1 cells. 3T3-L1 cells were induced to differentiate with 10% FBS-DMEM medium with
insulin only, with insulin and DEX, or with insulin and IBMX and treated with 100 μM
of nobiletin. Control cells were treated with DMSO instead of nobiletin. Forty-eight
hours after the induction, total RNA samples were prepared and analyzed for PPARγ2
gene expression. β-Actin mRNA was analyzed as an internal control. Results were
expressed as the PPARγ2 expression level relative to control. Data were represented
as the mean ± SD (n = 3). *p b 0.05 against control.
Preadipocytes
Control
Nobiletin
0
5
15
30
60
Time after induction (min)
Fig. 6. Effect of nobiletin on the phosphorylation of CREB in 3T3-L1 cells. (A) A representative image from two independent experiments. (B) The scanned bar graph shows fold the
induction of phosphorylated CREB over CREB. 3T3-L1 cells were induced to differentiate
with 10% FBS-DMEM medium containing insulin, DEX, and IBMX and treated with 100 μM
of nobiletin. Preadipocytes were cultured in 10% FBS-DMEM medium. Control cells were
treated with DMSO instead of nobiletin. Cell lysates were prepared at indicated time after
the differentiation induction, and Western blotting was performed. p-CREB represents the
phosphorylated CREB. Results were expressed as the ratio of p-CREB/CREB in each cell sample relative to that in preadipocytes. Data were represented as the mean±SD (n=2).
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K. Kanda et al. / Biochimica et Biophysica Acta 1820 (2012) 461–468
A
p-STAT5
STAT5
B
4
Ratio of p-STAT5/STAT5
strongly phosphorylated upon treatment with adipocyte differentiation inducers. Interestingly, nobiletin suppressed the phosphorylation
of CREB at 60 min after initiation of the induction, although the phosphorylated amount of CREB was not significantly different from that
of the control (Fig. 6B). The phosphorylation of CREB has been
reported to be induced by the insulin signaling pathway [24,25].
Saito et al. [17] reported that the phosphorylation of CREB was enhanced by nobiletin in 3T3-L1 preadipocytes 1 h following induction
of differentiation when the cells were induced to differentiate with
insulin only. Thus, decreased phosphorylation of CREB by treating
with nobiletin may be neutralized by the activation of the insulin signaling pathway by nobiletin. Therefore, it is possible that there may
be another signaling pathway involved in the suppression of
PPARγ2 gene expression by nobiletin.
3.8. Effect of nobiletin on the phosphorylation of STAT5
To investigate another suppressive mechanism of adipocyte differentiation by nobiletin, the phosphorylation of STAT5 was examined.
STAT5 is known to be involved in the early phase of adipogenesis
[11]. Excessive activation of STAT5, however, has been shown to inhibit adipocyte differentiation [12]. As shown in Fig. 7A and B, nobiletin strongly enhanced the phosphorylation of STAT5, suggesting that
the suppressive effect of nobiletin on adipocyte differentiation is involved in the excessive activation of STAT5. The effect of nobiletin
on STAT5 phosphorylation in the presence or absence of IBMX in
3T3-L1 cells was also examined. Result revealed that the phosphorylation of STAT5 by nobiletin was further enhanced with concomitant
treatment with IBMX (Fig. 8A), though there was no statistical significance (Fig. 8B). Taken together, these findings indicate that the excessive activation of STAT5 might have occurred due to the synergic
effect of nobiletin and IBMX.
A
p-STAT5
STAT5
(min) 0
5
15
30
60
5
Control
Ratio of p-STAT5/STAT5
B 20
15
30
60
Nobiletin
Preadipocytes
Control
Nobiletin
15
10
5
0
0
5
15
30
60
Time after induction (min)
Fig. 7. Effect of nobletin on the phosphorylation of STAT5 in 3T3-L1 cells. (A) A representative image from two independent experiments. (B) The scanned bar graph shows
the fold induction of phosphorylated STAT5 over STAT5. 3T3-L1 cells were induced to
differentiate with 10% FBS-DMEM medium containing insulin, DEX, and IBMX and treated with 100 μM of nobiletin. Control cells were treated with DMSO instead of nobiletin. Cell lysates were prepared at indicated time after the differentiation induction, and
Western blotting was performed. p-STAT5 represents the phosphorylated STAT5. Results were expressed as the ratio of p-STAT5/STAT5 in each cell sample relative to
that in preadipocytes. Data were represented as the mean ± SD (n = 2).
3
2
1
0
Nobiletin
IBMX
–
–
–
+
+
–
+
+
Fig. 8. Effect of nobletin and IBMX on the phosphorylation of STAT5 in 3T3-L1 cells.
(A) A representative image from two independent experiments. (B) The scanned bar
graph shows the fold induction of phosphorylated STAT5 over STAT5. 3T3-L1 cells
were induced to differentiate with 10% FBS-DMEM medium with insulin only or with
insulin and IBMX, and treated with or without 100 μM nobiletin. Control cells were induced with insulin only and treated with DMSO instead of nobiletin. Cell lysates were
prepared 15 min after the differentiation induction, and Western blotting was performed. p-STAT5 represents the phosphorylated STAT5. Results were expressed as
the ratio of p-STAT5/STAT5 in each cell sample relative to that in the control. Data
were represented as the mean ± SD (n = 2).
4. Discussion
In this study, we found that nobiletin suppressed the lipid accumulation in both of 3T3-L1 cells and MEFs. The suppressive effect of
nobiletin on MEFs was milder compared with that on 3T3-L1 cells
(Fig. 1B, D). The absorbance determined for the control MEFs in the
oil red O staining experiment was lower than that determined for
3T3-L1 cells, implying that the induction for adipocyte differentiation
with insulin, IBMX, and DEX might be insufficient for MEFs or might
take longer time than for 3T3-L1 cells.
PPARγ is a member of the nuclear receptor superfamily, and is the
master regulator of the adipogenesis. In the present study, nobiletin
was shown to suppress PPARγ2 expression at both protein and
mRNA levels 3T3-L1 cells (Figs. 3C and 4A). It is possible that the suppressive effect of nobiletin on adipocyte differentiation may be due to
the down-regulation of PPARγ2 expression. This latter speculation
was supported by the observation of the decreased expression of
aP2, a target of PPARγ (Fig. 4B).
C/EBPβ is known to induce PPARγ expression during adipogenic differentiation [23]. In this study, it was demonstrated that the expression
of C/EBPβ gene in 3T3-L1 cells was suppressed by treatment with nobiletin (Fig. 4C). This finding implies that the decreased PPARγ2 expression was caused by the down-regulation of C/EBPβ expression. We
also examined the effect of nobiletin on the phosphorylation of CREB
in 3T3-L1 cells. Results showed that the phosphorylation of CREB was
decreased by treatment with nobiletin 60 min after the induction
(Fig. 6A). Because it is well known that phosphorylated CREB regulates
C/EBPβ expression [26], down-regulation of the expression of C/EBPβ
might be due to the decreased phosphorylation of CREB. On the other
hand, it has been reported that nobiletin stimulates the insulin signaling
to induce the differentiation of 3T3-L1 preadipocytes into adipocytes
[17] and that the insulin signaling also participates in the phosphorylation of CREB [24,25]. Studies have also demonstrated that IBMX may
accelerate the phosphorylation of CREB by activating the cAMPdependent PKA signaling pathway [27]. Moreover, nobiletin has been
K. Kanda et al. / Biochimica et Biophysica Acta 1820 (2012) 461–468
reported to enhance the activation of PKA [28]. Based on these findings,
it is possible that the suppressive effect of nobiletin on the phosphorylation of CREB may be limited.
Treatment of 3T3-L1 preadipocytes with nobiletin suppressed adipocyte differentiation when the cells were induced with insulin plus
IBMX. The differentiation induced with insulin alone or with insulin
and DEX, however, was not inhibited (Fig. 5), suggesting that nobiletin affects the signaling pathways mediated by IBMX. When 3T3-L1
preadipocytes were induced with insulin, DEX, and instead of IBMX,
forskolin, an alternative cAMP elevator, PPARγ2 expression was suppressed by nobiletin as well (data not shown), suggesting that the
suppressive effect of nobiletin on PPARγ2 expression may involve
the cAMP-dependent signaling pathways. Matsuzaki et al. [28] previously reported that nobiletin activates PKA, indicating that the mode
of action of nobiletin in adipocyte differentiation resembles that of a
cAMP elevator. It was observed that the adipocyte differentiation
was enhanced by nobiletin when 3T3-L1 preadipocytes were induced
with insulin alone (Fig. 5), implying that, like IBMX, nobiletin facilitates adipocyte differentiation by stimulating the cAMP-dependent
signaling pathways. Saito et al. [17] also reported that nobiletin enhanced adipocyte differentiation of 3T3-L1 cells when the cells were
induced with insulin alone. As mentioned earlier, cAMP activates
PKA, and the resulting phosphorylated PKA activates CREB. If nobiletin
indeed accelerates the cAMP/PKA/CREB pathway, adipocyte differentiation should be enhanced. In our study, however, nobiletin depressed adipocyte differentiation when preadipocytes were induced with insulin
and a cAMP elevator (IBMX or forskolin). Therefore, it assumes speculated that another key player in the cAMP-dependent adipocyte differentiation may be involved in the suppression of PPARγ2 expression by
nobiletin.
STAT5 is activated by the growth hormone-mediated signaling
and has been shown to participate in the early phase of the adipogenesis by regulating the PPARγ activity [10,11]. STAT5 is activated
in multiple pathways, including a cAMP-activated PKA pathway
[29]. However, excessive activation of STAT5 may inhibit the adipogenesis through down-regulating PPARγ expression [12]. Therefore,
it is possible that STAT5 may be excessively activated by PKA, which
is activated by nobiletin and by IBMX-induced cAMP accumulation.
Therefore, we decided to examine the effect of nobiletin on the
phosphorylation of STAT5 in adipocyte differentiation of 3T3-L1
cells. As expected, the phosphorylation of STAT5 was extensively
stimulated, compared with the control (Fig. 7A, B). In addition,
the extent of phosphorylation of STAT5 was highest when both
nobiletin and IBMX were both used (Fig. 8A, B), indicating that the
suppressive effect of nobiletin on PPARγ2 expression in adipocyte
differentiation is caused by an excessive activation of STAT5,
which controls the PPARγ expression. It can therefore be proposed
that STATs regulate signaling pathways in both positive and negative manners to maintain homeostasis [30]. Excessive activation of
STAT5 may exert a feedback effect on the signaling system to
suppress the adipogenesis through decreased PPARγ2 gene expression. The suppressive effect of nobiletin was partially decreased
when H-89, a PKA inhibitor, was co-administered (data not shown),
supporting the hypothesis.
5. Conclusion
We demonstrated that nobiletin suppresses adipocyte differentiation in 3T3-L1 preadipocytes and in MEFs when these cells were induced with a general inducer cocktail containing insulin, DEX, and
IBMX. The results are consistent with a recent report showing that nobiletin decreased the intracellular triglyceride accumulation in 3T3-L1
cells [19]. Nobiletin exerted a suppressive effect within the first 2 days
after induction of differentiation through the down-regulation of
PPARγ2 expression. The suppressive effect of nobiletin on PPARγ2 expression appeared to be due to decreased phosphorylation of CREB
467
and excessive activation of STAT5 through the cAMP-dependent PKA
signaling induced by both IBMX and nobiletin. More work is warranted
in order to fully elucidate the mechanism underlying the suppression of
adipocyte differentiation by nobiletin.
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