Green Tea Polyphenol Epigallocatechin Gallate
Inhibits Adipogenesis and Induces Apoptosis in
3T3-L1 Adipocytes
Ji Lin,* Mary Anne Della-Fera,* and Clifton A. Baile*
Abstract
LIN, JI, MARY ANNE DELLA-FERA, AND CLIFTON A.
BAILE. Green tea polyphenol epigallocatechin gallate
inhibits adipogenesis and induces apoptosis in 3T3-L1
adipocytes. Obes Res. 2005;13:982990.
Objective: Green tea catechins have been shown to promote
loss of body fat and to inhibit growth of many cancer cell
types by inducing apoptosis. The objective of this study was
to determine whether epigallocatechin gallate (EGCG), the
primary green tea catechin, could act directly on adipocytes
to inhibit adipogenesis and induce apoptosis.
Research Methods and Procedures: Mouse 3T3-L1 pre-
adipocytes and mature adipocytes were used. To test the
effect of EGCG on viability, cells were incubated for 3, 6,
12, or 24 hours with 0, 50, 100, or 200 M EGCG. Viability
was quantitated by MTS assay. To determine the effect of
EGCG on apoptosis, adipocytes were incubated for 24 hours
with 0 to 200 M EGCG, then stained with annexin V and
propidium iodide and analyzed by laser scanning cytometry.
Both preadipocytes and adipocytes were also analyzed for
apoptosis by terminal deoxynucleotidyl transferase dUTP
nick-end labeling assay. To determine the effect of EGCG
on adipogenesis, maturing preadipocytes were incubated
during the 6-day induction period with 0 to 200 M EGCG,
then stained with Oil-Red-O and analyzed for lipid content.
Results: EGCG had no effect on either viability or apoptosis
of preconfluent preadipocytes. EGCG also did not affect
viability of mature adipocytes; however, EGCG increased
Received for review December 1, 2004.
Accepted in final form March 28, 2005.
The costs of publication of this article were defrayed, in part, by the payment of page
charges. This article must, therefore, be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
Departments of *Animal and Dairy Science and Foods and Nutrition, University of
Georgia, Athens, Georgia.
Address correspondence to Clifton A. Baile, 444 Edgar L. Rhodes Center for Animal and
Dairy Science, University of Georgia, Athens, GA 30602-2771.
E-mail: cbaile@uga.edu
Copyright 2005 NAASO
982 OBESITY RESEARCH Vol. 13 No. 6 June 2005
apoptosis in mature adipocytes, as demonstrated by both
laser scanning cytometry and terminal deoxynucleotidyl
transferase dUTP nick-end labeling assays. Furthermore,
EGCG dose-dependently inhibited lipid accumulation in
maturing preadipocytes.
Discussion: These results demonstrate that EGCG can act
directly to inhibit differentiation of preadipocytes and to
induce apoptosis of mature adipocytes and, thus, could be
an important adjunct in the treatment of obesity.
Key words: green tea catechins, lipid accumulation, la-
ser scanning cytometry, cell death, cell viability
Introduction
The prevalence of overweight and obesity has reached
epidemic proportions in recent years. This growing obesity
trend has been seen in the United States and is steadily
increasing in other western countries as well as in develop-
ing countries (1). Over the past 2 decades, the occurrence of
obesity among U.S. adults has increased by 74%, and about
two-thirds of U.S. adults are currently either overweight or
obese (2). In addition, the incidence of obesity and over-
weight seen in U.S. children is continuing to rise (3).
Evidence indicates that overweight and obesity are strongly
associated with major health risks such as cardiovascular
disease, diabetes, and cancer (4). These obesity-related ad-
verse health consequences have led to an increased number
of preventable deaths (5). Therefore, the prevention and
treatment of obesity are critical to curtail the rising inci-
dence of morbidity and mortality.
Obesity is a complex disorder with multiple causes in-
cluding both genetic and environmental factors. The cause
of obesity is usually attributed to a combination of these
factors, and the disorder itself involves specific biological
factors that regulate energy homeostasis. Recent studies
indicate that adipose tissue plays an important role in energy
homeostasis, and its function is not limited to a site of
energy storage and mobilization but is extended as an active
EGCG Induces Apoptosis of Adipocytes, Lin, Della-Fera, and Baile
endocrine organ (6). Adipose tissue secretes peptides, such
as leptin and tumor necrosis factor-, that are involved in
food intake and energy expenditure. These cytokines are
also linked with conditions such as insulin resistance, char-
acteristic of type 2 diabetes (7). Adipose mass, at any given
time, reflects average adipocyte size and total adipocyte
number. It was once thought that adipocyte acquisition was
permanent and that weight gain/loss caused only variation
in cell size (8); however, it is now believed that adipose tis-
sue undergoes dynamic remodeling throughout adulthood.
Adipogenesis occurs when extra fat mass is needed for
significant caloric gain or for cellular homeostasis (9). In
obese or overweight individuals, the increased fat mass can
be a result of either increased cell size (hypertrophy) or
increased fat cell number (hyperplasia) or both. Adipose
tissue hyperplasia is a result of increased adipogenesis,
which includes preadipocyte proliferation and adipocyte
differentiation. Thus, potential therapeutic agents, espe-
cially from low-toxicity natural products, that have the
ability to reduce or inhibit adipogenesis or increase cell
death by apoptosis could have a profound impact as a
strategy for treating and preventing obesity and related
metabolic disorders.
Tea is second only to water as one of the most widely
consumed beverages in the world, and its origins date back
thousands of years. Green tea, which is prepared by drying
fresh tea leaves, contains a number of bioactive compo-
nents, including polyphenols, caffeine, amino acids, and
other trace compounds such as lipids and vitamins. Green
tea polyphenols consist mainly of catechins, including ()-
epigallocatechin gallate (EGCG),1 which is the most abun-
dant (10) and strongest bioactive chemical. Green tea ex-
tracts, specifically those containing a high proportion of
EGCG, have been shown to increase 24-hour energy expen-
diture and fat oxidation (11) and increase weight loss in
humans (12). In addition, EGCG treatment has been shown
to reduce fat depot weight in mice (13,14). Although in-
creased thermogenesis and inhibition of lipase activity have
been suggested to be responsible for EGCG-induced weight
loss, EGCG has been shown to have antiproliferative effects
that may also impact adipose tissue. Consumption of green
tea has been associated with decreased risk of cancer and
atherosclerosis in humans (1517), and studies indicate that
EGCG inhibits tumor cell growth and induces tumor cell
death through an apoptotic mechanism (18,19). In this
study, we used 3T3- L1 adipocytes to investigate the direct
effects of EGCG on adipogenesis and its potential to induce
adipocyte apoptosis.
1
Nonstandard abbreviations: EGCG, ()-epigallocatechin gallate; DMEM, Dulbeccos
modified Eagles medium; DMSO, dimethyl sulfoxide; FBS, fetal bovine serum; LSC, laser
scanning cytometry; PBS, phosphate-buffered saline; AV, annexin V-fluorescein isothio-
cyanate; PI, propidium iodide; TUNEL, terminal deoxynucleotidyl transferase dUTP nick-
end labeling; GTP, green tea polyphenol; MAP, mitogen-activated protein; ERK, extracel-
lular signal-regulated kinase.
Research Methods and Procedures
3T3-L1 Cell Culture
3T3-L1 mouse embryo fibroblasts were obtained from
the American Type Culture Collection (Manassas, VA) and
were cultured as described previously (20). Briefly, the cells
were cultured at 37 C in a humidified 5% CO2 atmosphere
and grown in Dulbeccos modified Eagles medium
(DMEM) with 10% bovine calf serum added. After induc-
ing confluency for 2 days (Day 0), preadipocytes were
cultured in DMEM with 10% fetal bovine serum (FBS/
DMEM) medium, supplemented with 167 nM insulin, 0.5
M isobutylmethylxanthine, and 1 M dexamethasone for
2 days (Day 2). The cells were then maintained in a culture
medium with 167 nM insulin supplement for another 2 days
(Day 4), followed by culturing with 10% FBS/DMEM me-
dium for an additional 4 days, at which time 90% of the
cells were mature adipocytes and had accumulated fat drop-
lets. All media contained 100 U/mL penicillin, 100 g/mL
streptomycin, and 292 g of L-glutamine/mL (Invitrogen,
Carlsbad, CA).
All experiments, except where otherwise indicated, were
performed at least in quadruplicate at concentrations of 0
[1:1000 dimethyl sulfoxide (DMSO)], 50, 100, or 200 M
EGCG (98.1% purity, purchased from LKT Laboratory,
Inc., St. Paul, MN, prepared in 200 mM stock with DMSO)
added to the culture medium for a duration of 3, 6, 12, or 24
hours.
MTS Cell Viability Assay
Tests were performed in 96-well plates. For mature adi-
pocytes, the seeding density was 5000 cells/well, and cells
underwent induction until matured. For preadipocytes, seed-
ing density of 2500 cells/well was used, and cells were
cultured overnight before treatment (doubling time approx-
imately 18 hours). Cells were incubated with either DMSO
(1:1000) or increasing concentrations of EGCG for up to 24
hours. The medium was then changed and replaced with 100
L of fresh 10% FBS and 20 L of MTS solution (Pro-
mega, Madison, WI). Cells were then returned to the incu-
bator for an additional 2 hours before 25 L of 10% sodium
dodecyl sulfate was added to stop the reaction. The plate
was analyzed by spectrometry at a wavelength of 490 nm.
Apoptosis Assay
Laser Scanning Cytometry (LSC). Mature adipocytes
were incubated with either DMSO (1:1000) or increasing
concentrations of EGCG for 24 hours. Monolayer cells from
each treatment were washed twice with cold phosphate-
buffered saline (PBS) and once in binding buffer and then
incubated in the dark with gentle agitation for 10 minutes
with 5 L of annexin V-fluorescein isothiocyanate (AV)
and 5 L of propidium iodide (PI) in 450 L of binding
buffer (BD Biosciences, San Diego, CA) at ambient tem-
perature. LSC was carried out as previously described (21).
OBESITY RESEARCH Vol. 13 No. 6 June 2005 983
EGCG Induces Apoptosis of Adipocytes, Lin, Della-Fera, and Baile
Figure 1: EGCG effect on preadipocyte (A) and mature adipocyte (B) viability measured by MTS as percentage of controls at different
incubation times from 3 to 24 hours.
Data were analyzed by WinCyte and CompuSort software
for sorting and morphological relocation. Scanning the user-
defined region of interest took 30 minutes to complete or
stopped automatically when 5000 events were collected.
Terminal Deoxynucleotidyl Transferase dUTP Nick-End
Labeling (TUNEL) Image for Apoptosis Detection. Both
preadipocytes and mature adipocytes were incubated with 0
(1:1000 DMSO) or 200 M EGCG for 24 hours. In addi-
tion, cells undergoing diphenylmethane-4,4-diisocyanate
induction for adipogenesis were coincubated with 0 or 200
M EGCG for 6 days before TUNEL assay (Day 6). An
APO-BrdU TUNEL kit was obtained from Molecular
Probes (Eugene, OR). Monolayer cells were fixed with 10%
formalin (PBS buffered) and stored in 70% ethanol at
20 C for 24 hours. Cells were washed twice in the
washing buffer, followed by 1-hour incubation in a humid-
ity box at 37 C with gentle agitation in 150 L of DNA-
labeling solution (containing 30 L of reaction buffer, 2.25
L of terminal deoxynucleotidyl transferase, 24 L of
BrdUTP, and 93.75 L of dH2O). After a brief wash, the
cells were then incubated for 30 minutes with Alexa Fluor
488 dye-labeled anti-BrdU antibody (1:20 dilution), fol-
lowed by an additional 30 minutes with PI/RNase buffer to
stain the nucleus at room temperature in the dark, and
viewed with a fluorescent microscope.
Oil-Red-O Staining for the Effect of EGCG on
Adipogenesis
Three concentrations of EGCG (50, 100, and 200 M)
along with 1:1000 DMSO control were added with the
induction medium from Days 0 to 6 of adipogenesis in
addition to the no-addition normal controls. Medium was
changed every 2 days. On Day 6, cells were stained with
Oil-Red-O. In brief, dishes were washed with cold PBS and
fixed in 10% neutral formalin. After two changes of pro-
pylene glycol, Oil-Red-O was added with agitation for 7
minutes, followed by washing in 85% propylene glycol. The
dishes were then rinsed in distilled water and counterstained
984 OBESITY RESEARCH Vol. 13 No. 6 June 2005
with hematoxylin. For each dish, three images were taken
and analyzed for average lipid droplet size, percentage lipid
area, and total droplet number with ImagePro Plus 5.0
software (MediaCybernetics, Silver Spring, MD).
Data Analysis
Data are presented as mean SEM and analyzed by
two-way ANOVA according to the general linear model
procedure. Significance of differences between groups was
established using Tukeys test; p 0.05 was considered
significant.
Results
Preadipocyte viability was not affected by any concen-
tration of EGCG during the 24-hour incubation period (Fig-
ure 1A). Likewise, viability of mature adipocytes was not
affected by EGCG, as measured by the MTS assay (Figure
1B).
To measure apoptosis, a combination of AV/PI dual
staining was used. AV detects externalized cell membrane
phosphatidylserine, which is one characteristic of apoptosis;
and PI, normally impenetrable through the intact cell mem-
brane, is able to stain the nucleus during middle or late stage
apoptosis when membrane integrity is lost. By measuring
the AV/PI intensity over time, the LSC system can provide
progressive information of the relative changes of cell pop-
ulation on apoptotic changes from early stage to end stage
apoptosis, as shown in Figure 3. In this sample scatter
graph, based on fluorescent intensity of each channel, the
cell populations were segmented into three regions: viable
(Box 1, low AV and PI intensity), apoptotic (Box 2, high
AV with low PI intensity), and dead (Box 3, high PI
intensity). Cells within Box 2 were stained primarily with
AV, indicating that cells were undergoing early apoptosis;
cells within Box 3 that had higher PI intensity combined
with either higher or lower AV intensity were at a later stage
of apoptosis (secondary necrosis). By calculating the per-
EGCG Induces Apoptosis of Adipocytes, Lin, Della-Fera, and Baile
Figure 2: Twenty-four hour (A) and time course (B) EGCG effect on apoptosis of mature adipocytes as measured by LSC. Means with
different letters are different, p 0.05.
centage of cells within each box, a relative apoptotic rate
could be determined. The overall effect of 24-hour EGCG
treatment on mature adipocyte apoptosis is shown in Figure
2A. In DMSO-treated adipocytes, total apoptotic rate was
low; EGCG markedly increased apoptosis at the 50 and 200
M concentrations (p 0.05), although the increase with
100 M EGCG was not significant. Figure 2B shows the
time course of the apoptotic-inducing effect of EGCG in
mature adipocytes. Apoptosis was significantly increased at
12 hours of incubation by 100 M EGCG; by 24 hours, the
percentage of total apoptotic cells was significantly in-
creased by 50 and 200 M EGCG (p 0.05).
Figure 3: Sample scatter graph generated by LSC with three boxes
indicating viable (Box 1), early apoptotic (Box 2), and late apo-
ptotic (Box 3) based on fluorescent intensity of AV/PI.
Longer incubation with EGCG resulted in a higher pro-
portion of dead cells, as shown in Figure 4. Three- or 4-day
incubation resulted in 50% dead cells on LSC analysis;
however, we did not include this set of data into statistical
analysis because many of the dead cells were lost during
preparation before scanning on the LSC, so we were not
able to determine the actual percentages from each popula-
tion.
The TUNEL staining of EGCG-treated adipocytes was
consistent with LSC data. DMSO control did not show any
TUNEL-positive nuclei (Figure 5A), but there was an in-
crease in TUNEL staining in EGCG-treated cells (Figure
5B). For preadipocytes, 24-hour EGCG treatment did not
result in any TUNEL-positive cells (Figure 5C).
Oil-Red-O staining showed that 6-day incubation of
EGCG during the differentiation period significantly inhib-
ited 3T3-L1 adipogenesis (Figures 6 and 7). We selected
three parameters from ImagePro software to analyze adipo-
genesis: average size of lipid droplet, ratio of lipid area to
total area analyzed (percentage lipid), and total number of
lipid droplets counted. The average size of lipid droplet in
untreated and DMSO controls was 915.19 122 and
1149 267 pixels2, respectively, with no statistical differ-
ence. However, EGCG treatment resulted in a dose-depen-
dent decrease in lipid droplet size, with 200 M EGCG
having the greatest effect (Figure 7): the average lipid
droplet sizes for adipocytes treated with EGCG concentra-
tions of 50, 100, and 200 M were 618.14 62, 539.64
67, and 277.7 39 pixels2, respectively. The percentage
lipid and total lipid droplet number were also reduced
markedly by EGCG treatment. In addition, in EGCG treat-
ment groups, not only was lipogenesis reduced, but total cell
counts were also decreased, especially in cells treated with
200 M EGCG. TUNEL assay indicated that apoptosis was
occurring with EGCG treatment during these periods (Fig-
ure 5D).
OBESITY RESEARCH Vol. 13 No. 6 June 2005 985
EGCG Induces Apoptosis of Adipocytes, Lin, Della-Fera, and Baile

Figure 4: LSC scatter graph set of AV/PI intensity from mature adipocytes treated for 3 or 4 days with 0, 50, 100, or 200 M EGCG. The
high proportion of dead cells with these prolonged incubation periods prevented collection of accurate quantitative data.

Discussion Green tea polyphenols (GTPs) constitute a large part of

In our study, the green tea compound EGCG inhibited
3T3-L1 adipogenesis and resulted in adipocyte apoptosis.
Average size of lipid droplets and total lipid droplet number
were decreased dose-dependently by EGCG treatment. Few
mature adipocytes with normal morphology could be found
with 200 M EGCG treatment (Figures 6 and 7).
green tea leaf (up to 20%) (10) or 36% of the dry weight
composition (22). Most GTPs are catechins that belong to
the family of flavonoids, which are comprised of two aro-
matic rings with a C6-C3-C6 carbon structure. Five main
catechins that compose 90% of GTP are ()-gallocat-
echin, ()-epigallocatechin, ()-epicatechin, ()-EGCG,

986 OBESITY RESEARCH Vol. 13 No. 6 June 2005

EGCG Induces Apoptosis of Adipocytes, Lin, Della-Fera, and Baile

Figure 5: TUNEL images. (A) Control (DMSO) treatment showing no TUNEL signal but only PI staining for normal intact nuclei. (B)
EGCG treatment (200 M) for 6 days during adipogenesis showing bright condensed nuclei, as indicated by arrows for apoptotic cells. (C)
Preadipocytes treated for 24 hours with 200 M EGCG showing no TUNEL-positive staining; PI staining for intact nuclei only. (D) Mature
adipocytes treated with 200 M EGCG for 24 hours; arrows indicate apoptotic cells (400).

and ()-epicatechin gallate. EGCG, which is an esterified
or galloyl catechin, is the most abundant, accounting for
54% to 59% of total green tea catechins (10). Although tea
is consumed worldwide and has been used in traditional
Chinese medicine (10), its health benefits are receiving
renewed attention. EGCG has been shown to have antioxi-
dative and radical scavenging actions (23,24) and anticar-
cinogenic or cancer-preventing actions (17,25). EGCG
treatment has been shown to induce cancer cell apoptosis
(26). EGCG treatment also has been shown to suppress
high-fat diet-induced increase of plasma lipids (27) and to
reduce food intake and decrease fat deposition in rats (13).
Possible mechanisms of action for EGCG-induced inhi-
bition of adipogenesis could involve mitogen-activated pro-
tein (MAP) kinase, especially the extracellular signal-regu-
lated kinases (ERKs) that are stimulated by growth-related
signals. Although contradictory reports exist (28), Prusty et
al. (29) recently showed that ERK1/2 phosphorylation pro-
moted CCAAT/enhancer binding protein- and peroxisome
proliferator-activated receptor- expression, both key me-
diators of adipogenesis, in 3T3-L1 cells, and inhibition of
ERK1/2 significantly attenuated expression of CCAAT/en-
hancer binding protein- and peroxisome proliferator-acti-
vated receptor-. EGCG has been shown to inhibit the
phosphorylation of ERK1/2 and AKT kinases in epidermal
and mast cell lines (30,31); recently, Hung et al. (32)
showed that EGCG reduced phospho-ERK1 and phospho-
ERK2 levels in 3T3-L1 preadipocytes. Thus, EGCG may
act to inhibit the ERK signaling pathway in adipocytes,
resulting in inhibition of lipogenesis.
In addition to the inhibitory effect on differentiation,
apoptosis was also enhanced by incubation with EGCG
during differentiation, suggesting that EGCG can reduce
adipose tissue mass both by inhibiting maturation and by
increasing cell death. Because cells were treated with
EGCG for 6 days during preadipocyte differentiation, apo-
ptotic cells were more apparent. In mature adipocytes incu-
bated for 24 hours, TUNEL staining showed positive apo-
ptotic cells, which was consistent with the LSC assay.
However, we did not find TUNEL-positive cells in preadi-
pocytes after 24-hour EGCG incubation, suggesting that the
sensitivity to EGCG-induced apoptosis develops as cells
mature.
Although EGCG has been shown to be growth inhibitory
in several tumor cell lines, we did not detect a change in cell
viability within 24 hours of EGCG incubation using the
MTS method. This cell viability assay is based on the
reduction of the tetrazolium salt, MTS, to a colored
formazan compound in viable cells, even in early apoptot-
ing cells (33); thus, extended incubation time may be

OBESITY RESEARCH Vol. 13 No. 6 June 2005 987

EGCG Induces Apoptosis of Adipocytes, Lin, Della-Fera, and Baile
Figure 6: Oil-Red-O staining. Images were obtained at Day 6 of differentiation with EGCG treatment. (A) DMSO-treated control. (B) 50
M EGCG. (C) 100M EGCG. (D) 200 M EGCG (400).
needed to observe an EGCG-reduction of cell viability
using the MTS assay. Hung et al. (32) recently reported that
concentrations of 100 M EGCG and higher reduced via-
bility of 3T3-L1 preadipocytes, as determined by trypan
blue dye exclusion. They also found that the sensitivity of
preadipocytes depended on the growth phase during which
they were tested, as well as the dose and length of the
incubation period.
LSC is a microscope-based cytofluorometer capable of
measuring stoichiometric quantitative and morphological
features of cells. Threshold and contour feature are set by
the user, and cell population information based on fluores-
cence intensity can be sorted, visualized, and stored for later
visualization after scanning. Thus, LSC allows the analysis
of a cell population both quantitatively and with morpho-
logical evidence, which is an advantage over flow cytoflu-
orometry. The AV/PI method is widely used in flow cyt-
ofluorometry to detect apoptosis. Annexin V is a Ca2-
dependent phospholipid binding protein that has a high
affinity for phosphatidylserine, a membrane phospholipid
that is distributed toward the intracellular leaflet of the cell
membrane in normal cells but translocated to the outer
leaflet of the membrane during apoptosis. The exposed
988 OBESITY RESEARCH Vol. 13 No. 6 June 2005
phosphatidylserine is, thus, accessible for AV detection. PI,
a fluorescent dye that binds to DNA, is not able to permeate
normal live cells with intact cell membranes. Thus, it is
used to distinguish dead cells from normal viable cells in
live cell culture. We have used this procedure success-
fully to demonstrate apoptotic changes induced by tumor
necrosis factor- in adipocytes (21). By applying LSC in
conjunction with AV/PI, we observed that apoptotic cells
increased with EGCG treatment. In our study, both AV
and PI intensity were low in DMSO-treated controls, but
EGCG incubation in mature adipocytes resulted in in-
creased AV intensity and late increase in PI staining inten-
sity over time, indicating that an increased number of cells
were undergoing apoptosis. Increased PI intensity alone
does not differentiate apoptotic from necrotic cells; how-
ever, together with the temporal shift and the early increase
in AV intensity, it was clear that apoptosis, followed by
secondary necrosis, was involved in the death process trig-
gered by EGCG.
The mechanism of EGCG in inducing apoptosis seems to
be complicated. Weinreb et al. (34) reported that treatment
of 50 M EGCG in a neuroblastoma cell line increased
mRNA expression of Bax and Bad, members of the pro-
EGCG Induces Apoptosis of Adipocytes, Lin, Della-Fera, and Baile
Figure 7: EGCG effect on lipid droplet size, percentage lipid area,
and total lipid droplet number. Means with different letters are
different, p 0.05.
apoptotic Bcl-2 family; in addition, Fas mRNA expression
was also increased. EGCG also decreased the antiapoptotic
Bcl-2 and Bcl-xL (34,35) and increased Fas and Fas-ligand
(36,37). In addition, EGCG treatment triggered c-Jun N-
terminal kinase and p38 MAP kinase of the MAP kinase
family to induce apoptosis in tumor cells (38). Hung et al.
(32) showed that treatment of 3T3-L1 preadipocytes with
EGCG for 48 hours reduced the level and activity of Cdk2
and decreased protein expression of cyclin D1 in a dose-
dependent manner. Furthermore, EGCG significantly in-
creased protein levels of the cyclin kinase inhibitors p21 and
p27 and increased binding of p21 and p27 to Cdk2. Al-
though we did not find evidence that EGCG induced apo-
ptosis in preadipocytes, if these changes also occur in ma-
ture adipocytes, they may be part of the mechanism
involved in induction of apoptosis. In fact, Gupta et al. (39)
showed that up-regulation of p21 and p27, along with down-
regulation of Cdk2 and cyclin D1, led to cell cycle arrest
and apoptotic cell death in prostate cancer cells.
Our study clearly identified the inhibitory effect of
EGCG on differentiation and the induction of apoptosis in
adipocytes in vitro. Thus, EGCG may prove to be a valuable
natural product in the treatment of obesity.
Acknowledgment
This work was supported, in part, by the Georgia Re-
search Alliance Eminent Scholar endowment (to C.A.B.).
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