BBRC
Biochemical and Biophysical Research Communications 338 (2005) 694 699
www.elsevier.com/locate/ybbrc
Genistein, EGCG, and capsaicin inhibit adipocyte differentiation
process via activating AMP-activated protein kinase
a b b b c
Jin-Taek Hwang , In-Ja Park , Jang-In Shin , Yun Kyoung Lee , Seong Kyu Lee ,
c a b,*
Haing Woon Baik , Joohun Ha , Ock Jin Park
a
Department of Biochemistry and Molecular Biology, Medical Research Center for Bioreaction to Reactive Oxygen Species,
Kyung Hee University College of Medicine, Seoul 130-701, Republic of Korea
b
Department of Food and Nutrition, Hannam University, Daejeon 306-791, Republic of Korea
c
Department of Biochemistry, College of Medicine, Eulji University, Daejeon 301-832, Republic of Korea
Received 20 September 2005
Available online 11 October 2005
Abstract
Phytochemicals such as soy isoflavone genistein have been reported to possess therapeutic effects for obesity, diabetes, and cardiovas-
cular diseases. In the present study, the molecular basis of selective phytochemicals with emphasis on their ability to control intracellular
signaling cascades of AMP-activated kinase (AMPK) responsible for the inhibition of adipogenesis was investigated. Recently, the
evolutionarily conserved serine/threonine kinase, AMPK, emerges as a possible target molecule of anti-obesity. Hypothalamic AMPK
was found to integrate nutritional and hormonal signals modulating feeding behavior and energy expenditure. We have investigated the
effects of genistein, EGCG, and capsaicin on adipocyte differentiation in relation to AMPK activation in 3T3-L1 cells. Genistein
(20 200 lM) significantly inhibited the process of adipocyte differentiation and led to apoptosis of mature adipocytes. Genistein, EGCG,
and capsaicin stimulated the intracellular ROS release, which activated AMPK rapidly. We suggest that AMPK is a novel and critical
component of both inhibition of adipocyte differentiation and apoptosis of mature adipocytes by genistein or EGCG or capsaicin further
implying AMPK as a prime target of obesity control.
Ó 2005 Elsevier Inc. All rights reserved.
Keywords: AMP-activated protein kinase; Obesity; Genistein; EGCG; Capsaicin; Apoptosis
Obesity is a complex multifactorial chronic disease that Genistein, a soybean derived bioactive polyphenol, has
increases the risk for developing hypertension, type 2 dia- been the subject of numerous researches as a chemothera-
betes, and coronary heart disease, and remains a major peutic agent [2]. This phytoestrogen has been implicated
health obstacle in the industrialized world. Obesity arises in cancer control primarily because of its strong anti-prolif-
from the imbalance between energy intake and energy erative and apoptotic potential [3]. Also, it has been report-
expenditure that may lead to a pathologic growth of adipo- ed that genistein exhibits anti-adipogenic effects in several
cytes. It is known that the amount of adipose tissue can be adipocytes, although its precise mechanism of action is
regulated by the inhibition of adipogenesis from precursor not known [4].
cells as well as the control of adipocyte size. Obesity is in- We have investigated the molecular events leading to the
duced by the hypertrophy of adipocytes and to the recruit- inhibition of adipogenesis by genistein, specially focusing
ment of new adipocytes from precursor cells and these two on the role of AMP-activated protein kinase (AMPK).
processes are dependent on the regulation of adipocyte dif- AMPK represents a metabolite-sensing protein kinase that
ferentiation [1]. shares amino acid sequence homology with yeast SNF1 [5].
AMPK is known to play a major role in energy homeostasis
* by coordinating a number of adaptive responses in ATP-de-
Corresponding author. Fax: [phone].
pleting metabolic states such as ischemia/reperfusion,
E-mail address: [e-mail] (O.J. Park).
0006-291X/$ - see front matter Ó 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.bbrc.2005.09.195
J.-T. Hwang et al. / Biochemical and Biophysical Research Communications 338 (2005) 694 699 695
Cell proliferation by MTT assay. Cells were seeded on 96-well micro-
hypoxia, heat shock, oxidative stress, and exercise [6].
plates at 4000 cells/well and incubated with each test compound for the
AMPK is sensitively regulated by allosteric binding of
indicated time period. Supernatant was discarded and then cells were
AMP under pathological or physiological conditions of
incubated with 100 ll MTT solution (2 mg/ml MTT in PBS) for 4 h.
ATP depletion [7]. The persistent activation of AMPK
Absorbance was measured using an autoreader (Spectra Max 360,
showed to be connected to p53-dependent cellular senes- Molecular Device, Minnesota, USA).
cence suggesting its role as an intrinsic regulator of the cell
cycle in mammalian cells [8]. Moreover, AMPK cascades Results
have emerged as novel targets for the treatment of obesity
and type 2 diabetes [9]. AMPK is known to be activated Genistein inhibits adipocyte differentiation
with 5-amino-imidazole-4-carboxamide riboside (AICAR),
which is converted to a nucleotide that mimics the effect of We first examined anti-obesity potential of genistein by
AMP, and the long-term treatment with AICAR has pre- determining pre-adipocyte differentiation into adipocytes.
vented the development of diabetes in animal models [10]. Cultured 3T3-L1 adipocytes were exposed to genistein at
Also the pro-apoptotic potential of the activated AMPK different doses (at day 0), and cell differentiation was
was observed in the AMPK over-expressed conditions of performed with a differentiation medium. At day 8, differ-
various cells [11]. entiations were terminated and fat drops were detected by
We have hypothesized that genistein mediates the inhibi- Oil-Red O staining. As shown Fig. 1A, treatment of 3T3-
tion of adipocyte differentiation and induces apoptosis of L1 cells with genistein markedly inhibited adipocyte differ-
mature adipocyte through the activation of AMPK signal- entiation dose-dependently, and genistein (100 lM) also
ing. Our results show that genistein activates AMPK, abrogated adipocyte differentiation in a time-dependent
blocks adipocyte differentiation comparable to AICAR, manner. These results indicated that genistein may have
and induces apoptosis of adipocytes through the genera- been efficiently blocking adipocyte differentiation and have
tion of ROS. potential of anti-obesity effects in 3T3-L1 cells.
Genistein also induces apoptosis of mature adipocyte
Materials and methods
Cell culture and reagents. The 3T3-L1 pre-adipocyte was purchased Several reports indicated that certain naturally occur-
from ATCC (Gaithersburg, MD). Cells were cultured in DulbeccoÕs
ring compounds have been shown to promote loss of body
modified EagleÕs medium (DMEM) containing 10% FBS under normoxic
fat by inducing apoptosis [12]. Thus, inducing apoptosis of
conditions (20% O2, 5% CO2, and 75% N2) in a CO2 incubator at 37 °C.
mature adipocytes can be important for the treatment of
Insulin was obtained from Eli Lilly (Indianapolis, IN, USA). IBMX and
obesity with the naturally occurring compounds. There-
dexamethasone were purchased from Sigma (St. Louis, MO, USA).
Hoechst 33342 and AICAR (5-aminoimidazole-4-carboxamide-ribose) fore, we next examined the apoptotic possibility of geni-
were also purchased from Sigma. The anti-phospho-specific antibodies
stein in mature adipocyte. 3T3-L1 cells were fully
that recognize phosphorylated ACC-Ser79 and AMPK antibodies were
differentiated at day 8, and matured adipocyte was exposed
from Cell Signaling Technology. Antibodies for b-actin were purchased
to genistein for indicated concentrations. After stimulation,
from Santa Cruz Biotechnology (Santa Cruz, CA).
cell apoptosis was detected by either MTT assay or Hoe-
Adipocyte differentiation. The cells were plated in 6-well plates, and at
day 0 (usually 2 days after the cells had achieved confluence), and adi- chst33342 dye. These results indicated that genistein effec-
pocyte differentiation was induced with hormone cocktail containing 1 lM
tively induced apoptosis in mature 3T3-L1 adipocyte
dexamethasone, 5 lg/ml insulin, and 0.5 mM IBMX for 2 days. After 2
(Figs. 2A and B).
days, the medium was changed with the regular medium, and at day 8, the
cells were treated with various stimuli.
Genistein significantly activates AMP-activated protein
Protein extract and Western blotting. Cells were rinsed twice with ice-
cold PBS and scraped with lysis buffer (50 mM Tris HCl, pH 7.4, 1% NP- kinase in 3T3-L1 cells via ROS generation
40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM
PMSF, 1 mM sodium orthovanadate, 1 mM NaF, 1 lg/ml aprotinin,
Recent report shows that hypoxic suppressions of adipo-
1 lg/ml leupeptin, and 1 lg/ml pepstatin) and subjected to Western blot
genesis are associated with AMPK activation and can in-
analysis.
duce the failure of mitotic clonal expansion at the early
Oil-Red O staining. On day 8 of adipocyte differentiation induction, the
cells were stained with Oil-Red O dye. The cells were fixed with 70% phase of adipogenesis [13]. We next tested whether there is
ethanol and dehydrated with 100% propylene glycol. The cells were
AMPK activation in the inhibitory process of adipocyte dif-
stained with Oil-Red O and with HarriÕs hematoxylin. Fat droplets in
ferentiation by treating genistein. 3T3-L1 cells were cultured
adipocytes were stained red.
with normal medium and then exposed to genistein
Chromatin staining with Hoechst 33342. Apoptosis was observed by
(100 lM) and differentiation medium for the indicated time
chromatin staining with Hoechst 33342, as previously described [21].
Cells were incubated with each stimulus. Termination of incubation, periods. AMPK activation and its substrate acetyl-CoA car-
the supernatant was discarded and cells were fixed with 3.5% form-
boxylase (ACC) phosphorylation were detected by Western
aldehyde in PBS for 30min at room temperature, washed four times
blot analysis. As shown in Fig. 3A, AMPK phosphorylation
with PBS, and exposed to Hoechst 33342 (10 lM) for 30 min at room
increased 2.4-fold in a time-dependent manner, and its sub-
temperature. Stained cell preparations were examined under ultraviolet
strates, ACC-Ser79 phosphorylation showed enhancement.
illumination with a fluorescence microscope (Olympus Optical, Tokyo,
Japan). Also AMPK and ACC were significantly activated by
696 J.-T. Hwang et al. / Biochemical and Biophysical Research Communications 338 (2005) 694 699
Fig. 2. Genistein also induced apoptosis of mature 3T3-L1 cells. Cells
were fully differentiated at day 8 and exposed to genistein for indicated
concentrations. After inducing apoptosis by genistein, apoptosis was
detected either by MTT assay or Hoechst33342 dye.
and these effects were abolished by NAC (5 mM) treatment.
These results indicate that ROS is necessary for the AMPK
activation in the inhibitory process of adipocyte differentia-
tion by genistein in 3T3-L1 cells.
AICAR, an AMPK activator, also inhibits adipocyte
differentiation and induces apoptosis of mature adipocyte
To evaluate the involvement of AMPK in lipogenesis
accurately, we next tested the effects of AMPK activation
with AICAR (an AMPK activator) on adipocyte differen-
tiation. Treatment with 500 lM 2 mM AICAR significant-
ly phosphorylated either AMPK or ACC, and at the same
Fig. 1. Effects of genistein on adipocyte differentiation in 3T3-L1 cells.
time, adipocyte differentiations were inhibited (Fig. 4).
Confluent 3T3-L1 cells were exposed to differentiation cocktail (1 lM
These results indicate that AMPK plays a critical role in
dexamethasone, 5 lg/ml insulin, and 0.5 mM IBMX) for 2 days, and cells
were changed into normal DMEM in the presence or absence of genistein
adipogenesis and is essential for the blocking process of
for the indicated concentrations (A) or cells exposed to genistein for the
adipocyte differentiation blocking.
indicated time period (B). These cells were fixed with 3.5% formaldehyde
in PBS and fat drops were stained by Oil-Red O dye staining.
Other naturally occurring compounds also activate AMPK
and inhibit adipocyte differentiation
genistein in a concentration-dependent manner (Fig. 3B).
One of the AMPK activation mechanisms was suspected Several reported suggest that several naturally occurring
to be ROS, since it was recently reported that various ther- compounds have potential of anti-obesity effects, and
apeutic effects of natural occurring compounds involve re- therefore we tested the effects of EGCG or capsaicin on
lease of ROS [14]. We tested the activation of AMPK via AMPK activation as well as adipocyte differentiation pro-
ROS release in inhibition of genistein-inhibited adipocyte cess. 3T3-L1 pre-adipocyte was pretreated with EGCG or
differentiation. As shown in Fig. 3C, genistein significantly capsaicin and then immediately incubated with differentia-
induced ROS generation, which led to AMPK activation, tion medium. After each time period, AMPK activation
J.-T. Hwang et al. / Biochemical and Biophysical Research Communications 338 (2005) 694 699 697
Fig. 3. The effects of AMPK activation by genistein. 3T3-L1 cells were fully differentiated by differentiation cocktail, cells were exposed to genistein
(100 lM) for the indicated time periods (A) or various concentrations, respectively, (B) and AMPK activation and its substrate acetyl-CoA carboxylase
(ACC) phosphorylation were detected by Western blot analysis. Also differentiated cells were exposed to genistein for 12 h in the presence or absence of
NAC (5 mM). After additional incubation for 30 min in the presence of 10 lM DCFH-DA the changes in fluorescence intensity were measured by
fluorescence-activated cell scanning analysis. Under the same conditions, the phosphorylation level of ACC-Ser79 (P-ACC) were examined (C).
and adipocyte differentiation were detected either with pounds have drawn attention because of their relative safe-
Western blot or Oil-Red O staining. As shown Fig. 5, either ness and accumulated evidence of anti-obesity and anti-
EGCG or capsaicin can activate AMPK and also inhibit diabetic effects in animals and humans [15]. We report
adipocyte differentiation in 3T3-L1 cells. These results the evidence that genistein exerts the inhibition of adipo-
strongly indicate that AMPK activation is necessary for cyte differentiation and the induction of adipocyte apopto-
inhibition effect of adipocyte differentiation by EGCG sis through the activation of AMPK paralleled with the
and capsaicin. generation of ROS. We also confirmed that green tea pol-
yphenol EGCG and red pepper polyphenol capsaicin
Discussion blocked the adipocyte differentiation in 3T3-L1 adipocyte
cultures. The anti-proliferatory and lipolytic effects of these
A variety of naturally occurring flavonoids have been phytochemicals have been attributed to their ability to
found to possess beneficial effects on health, and these com- modulate various signaling pathways, specially, the control
698 J.-T. Hwang et al. / Biochemical and Biophysical Research Communications 338 (2005) 694 699
dent manner. The mechanism by which affects AMPK reg-
ulation with physiological stimuli or anti-obesity agents
might present a promising target for the development of
strategies for the treatment of obesity. AMPK cascades
have been postulated to respond to the intracellular level
of AMP or AMP:ATP ratio [7] and to be highly sensitive
to the oxidative stress. ROS have been suggested to be up-
stream molecules of AMPK activated signals. We suggest
that the generation of ROS generated by the phytochemi-
cals is one of the responsible elements for the activation
Fig. 4. Effects of AICAR on adipocyte differentiation and apoptosis of
of AMP kinase.
mature adipocytes. Confluent 3T3-L1 cells were exposed to differentiation
The exact mechanism to stimulate pre-adipocyte mito-
cocktail (1 lM dexamethasone, 5 lg/ml insulin, and 0.5 mM IBMX) for 2
sis and differentiation in vivo remains exclusive. Howev-
days, and cells were changed into normal DMEM in the presence or
er, it is proposed that hypertrophy of fat cells grown
absence of AICAR for the indicated concentrations (A). These cells were
fixed with 3.5% formaldehyde in PBS and fat drops were stained by Oil- beyond a certain size might propagate to differentiate
Red O dye staining or apoptosis was detected with Hoechst33342 dye.
by sending specific signals [20]. Adipocyte inducers stim-
ulate pre-adipocytes to undergo mitotic clonal expansion
before transcriptional activation of adipocyte genes be-
fore anchoring adipocyte phenotypes. The balance be-
tween positive and negative signals of adipogenesis
determines the fate of differentiation of pre-adipocytes.
It is not clear from the present study whether the activa-
tion of AMPK is mediated by one of the negative signals
or acting directly on adipocyte differentiation as a nega-
tive signal.
We have tested whether AICAR has similar effect on
adipocyte differentiation and AMPK activation in compar-
ison with genistein. Both AICAR and genistein similarly
blocked the differentiation and the early clonal expansion
of pre-adipocytes. The present study strongly suggests that
the activation of AMPK is necessary for the inhibition of
adipogenesis in 3T3-L1 cells by phytochemicals such as
genistein, EGCG, and capsaicin, and AMPK as a primary
target of adipogenesis control.
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