3T3-L1 adipocyte apoptosis induced by thiazolidinediones
is peroxisome proliferator-activated receptor-c-dependent
and mediated by the caspase-3-dependent apoptotic
pathway
Yuanyuan Xiao, Taichang Yuan, Wenqi Yao and Kan Liao
State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese
Academy of Sciences, Shanghai, China
Introduction
To date, studies of adipocyte physiology have primarily
focused on adipogenesis [1–3]. The interaction between
extracellular signals and the transcriptional cascade dur-
ing adipogenesis has been well studied [4]. However,
owing to the remarkable ability of adipocytes to resist
apoptosis, adipocyte apoptosis is much less studied and
is poorly understood. Recently, evidence from several
in vivo and in vitro studies has indicated that apoptosis
is a significant factor in adipocyte depletion during
weight reduction [5–8]. In addition, an animal model of
adipocyte apoptosis has been developed as a tool for the
study of obesity-related diseases [9]. Adipocyte apopto-
sis is induced by some adipokines, such as leptin and
tumor necrosis factor-a [10–12]. Additionally, natural
Keywords
3T3-L1 adipocyte; adipocyte apoptosis;
Akt-1; PPARc; thiazolidinediones
Correspondence
K. Liao, Institute of Biochemistry and Cell
Biology, 320 Yueyang Road, Shanghai
200031, China
Fax: +86 21 54921011
Tel: +86 21 54921113

FEBS Journal 277 (2010) 687–696 ª 2009 The Authors Journal compilation ª 2009 FEBS 687
compounds can be screened for their ability to induce
adipocyte apoptosis, allowing the identification of
potential obesity drugs [13–15]. Although the regulation
of adipocyte apoptosis is not fully understood, it pro-
vides a potential target for treatment of obesity and its
related diseases.
Thiazolidinediones (TZDs), such as troglitazone,
rosiglitazone and pioglitazone, are drugs used clinically
for type II diabetes. TZDs activate the adipogenic
transcription factor peroxisome proliferator-activated
receptor-c (PPARc) and induce metabolic changes in
adipose tissue [16]. The mechanism of insulin sensitiza-
tion by TZDs has been explored, but is not fully
understood. In vivo studies have indicated that TZDs
may increase the number of small adipocytes by pro-
moting adipocyte differentiation, and decrease the
number of large adipocytes by inducing adipocyte
apoptosis [17,18].
PPARc is highly expressed in adipose tissue, and is
considered to be the ‘master switch’ of adipocyte dif-
ferentiation. Upon ligand binding, PPARc heterodi-
merizes with the retinoid X receptor, translocates into
the nucleus, and activates multiple genes, especially
adipogenic genes [19]. In the mature adipocyte, PPARc
regulates the expression of genes involved in lipid
metabolism [19]. Besides its function in adipogenesis,
PPARc is also implicated in leptin-induced adipocyte
apoptosis [10]. During leptin-induced adipocyte apop-
tosis, the expression of PPARc is increased by 80%

induced adipocyte apoptosis could be detected by acri-
dine orange (AO) ⁄ ethidium bromide (EB) staining or
Hoechst 33258 staining (Fig. 1B,C). Quantitative anal-
ysis by flow cytometry indicated that adipocyte apop-
tosis increased with prolonged troglitazone treatment
(Fig. 1D). Propidium iodide (PI) staining and terminal
deoxynucleotidyl transferase dUTP nick end labeling
(TUNEL) assay produced similar results (Fig. 1E,F).
Representative fluorescence-activated cell sorting
(FACS) plots of PI staining are shown in Fig. S1.
GW9662 is an antagonist of PPARc, and irrevers-
ibly inhibits PPARc activity [28]. During induction of
3T3-L1 adipocyte differentiation, the presence of
GW9662 completely blocked differentiation (Fig. 2A).
As troglitazone-induced adipocyte apoptosis is proba-
bly mediated by PPARc, GW9662 should have an
inhibitory effect on adipocyte apoptosis. As shown in
Fig. 2B,C, troglitazone-induced adipocyte apoptosis
was, indeed, inhibited in the presence of GW9662. In
addition, pioglitazone, another TZD, and PGJ2, which
directly binds to and activates PPARc [29], also induce
adipocyte apoptosis, and GW9662 treatment blocked
this induction (Fig. 2D–F). Taken together, these
results suggested that TZD-induced adipocyte apopto-
sis is PPAR c-dependent.
Inhibition of Akt-1 in adipocyte apoptosis
The phosphoinositide 3-kinase (PI3K)–Akt-1 signaling
cascade is essential for 3T3-L1 adipocyte differentiation
[30]. In many cells, the PI3K–Akt-1 signaling cascade is
also one of the important signals for cell survival [31].

Downregulation of Akt-1 protein level and inhibition
of its phosphorylation appeared to be common effects
in TZD-induced adipocyte apoptosis.
Troglitazone induces adipocyte apoptosis
through the mitochondrial pathway
Bad is one of the prodeath proteins in the Bcl-2 fam-
ily, and interacts with prosurvival Bcl-2 family mem-
bers to inactivate them [32]. Akt-1 phosphorylates Bad
at Ser136, inactivating Bad and thereby promoting cell
survival [33]. As shown in Fig. 4A, the basal phos-
phorylation level of Bad gradually decreased when the
cells were treated with troglitazone. The decreased
Akt-1 activity in TZD-treated adipocytes might regu-
late adipocyte apoptosis through Bad activity. In addi-
tion, troglitazone treatment decreased the protein level
of prosurvival Bcl-2, but not of prodeath Bax
(Fig. 4A). Consequently, the Bax ⁄ Bcl-2 ratio was
increased, promoting apoptosis.
In the Bcl-2 family, the disequilibrium of prodeath
and prosurvival proteins leads to the translocation of
A
B
C
D
EF
Fig. 1. Troglitazone-induced 3T3-L1 adipocyte apoptosis (A) Oil Red O-stained 3T3-L1 adipocytes 8 days after differentiation induction.
PPARc protein was detected by western blot with an antibody against PPARc. Pre, 3T3-L1 preadipocytes; Adi, differentiated 3T3-L1 adipo-
cytes. (B) AO ⁄ EB staining for apoptotic 3T3-L1 adipocytes. Scale bar: 50 lm. 3T3-L1 adipocytes were treated with 12.5 l
M troglitazone (Tro)
(12.5 l

cytes in vitro (Fig. 6A). In rat primary adipocytes,
troglitazone treatment induced apoptosis, Akt-1
decrease and caspase-3 cleavage (Fig. 6B–D).
Discussion
The function of PPARc in adipogenesis is well estab-
lished [19]. However, in many other types of cell, the
activation of PPARc is linked to the induction of
apoptosis. It has been suggested that PPARc is
involved in the suppression of cell proliferation and
promotion of apoptosis in endothelial and tumor cells
[20–22]. PPARc is also implicated in leptin-induced
adipocyte apoptosis [10]. In the present study, we
found that PPAR c agonists induce adipocyte apoptosis
in a PPARc-dependent manner (Figs 1 and 2). In prea-
dipocytes or small adipocytes, adipogenesis may be the
dominant effect of PPARc agonists, whereas in mature
adipocytes, apoptosis may be induced by PPARc
agonists. These effects may change the balance of adi-
pocyte populations in adipose tissue. The insensitive
A
B
CF
E
D
Fig. 2. GW9662 blocks TZD (troglitazone
and pioglitazone)-induced adipocyte apopto-
sis. (A) Inhibition of 3T3-L1 adipocyte differ-
entiation by GW9662 (GW). GW9662 was
added to the cells during differentiation
induction, and the cells were stained with

measured by AO ⁄ EB staining. Results are
means ± SDs of three independent
experiments. *P < 0.05.
Apoptosis induced by TZDs in 3T3-L1 adipocyte Y. Xiao et al.
690 FEBS Journal 277 (2010) 687–696 ª 2009 The Authors Journal compilation ª 2009 FEBS
mature adipocytes may be replaced by sensitive new
adipocytes after treatment with TZDs. This is consis-
tent with the ability of troglitazone to reduce the num-
ber of large adipocytes and improve insulin sensitivity
in obese Zucker rats [17,18].
Akt-1 is one of the key regulators of cell survival,
and many studies have demonstrated that activated
Akt-1 blocks cellular apoptosis [36]. In human lung
carcinoma cells, rosiglitazone suppresses cellular
proliferation via a PPARc-dependent Akt-1 signaling
pathway, as well as by a PPARc-independent AMP-
activated protein kinase pathway [28]. Only the
inhibitory effect through the PPARc-dependent Akt-1
signaling pathway is reversed by GW9662 [28]. Simi-
larly, adipocyte apoptosis is also induced by troglitaz-
one through a PPARc-dependent Akt-1 signaling
pathway (Fig. 3A,B). However, the mechanism by
which PPARc agonists reduce Akt-1 levels is not fully
understood. It is not by suppression of Akt-1 tran-
scription, as Akt-1 transcription is independent of
PPARc (Fig. 3D). The inhibition or reduction of
Akt-1 activity by PPARc agonists has been observed
in many cell types [28,37,38]. It is possible that the
expression of some PPARc target genes affects the
protein degradation, destabilizing the Akt-1 protein. In

M) for 48 h, and mRNA levels were
determined by real-time PCR. Results are means ± SDs of three independent experiments. *P < 0.05; **P < 0.01. (E) Akt-1 activation by
GW6992. 3T3-L1 adipocytes were treated with 12.5 l
M troglitazone for 96 h in the presence or absence of GW9662 (5 or 10 lM). (F) Akt-1
in pioglitazone (Pio)-induced 3T3-L1 adipocytes. Cells were treated with pioglitazone at the indicated concentrations (0, 0.5, 1 or 5 l
M) for
96 h in the presence or absence of 10 l
M GW9662. (G) Akt-1 in PGJ2-induced 3T3-L1 adipocytes. Cells were treated with PGJ2 at the indi-
cated concentrations (0, 10 or 25 l
M) for 96 h in the presence or absence of GW9662 (10 lM GW9662).
Y. Xiao et al. Apoptosis induced by TZDs in 3T3-L1 adipocyte
FEBS Journal 277 (2010) 687–696 ª 2009 The Authors Journal compilation ª 2009 FEBS 691
binding by nonphosphorylated Bad causes the release of
Bax, which is translocated into the mitochondria, where
it activates the mitochondrial pathway (Fig. 4A,B).
Thus, the intrinsic apoptotic pathway (regulation of
Bcl-2 family, cytochrome c release, and caspase-3 cleav-
age) is involved in TZD-induced adipocyte apoptosis.
Our current results suggest that the decreases in lev-
els of several important apoptosis proteins, e.g. Akt-1
and Bcl-2, is the important event in TZD-induced adi-
pocyte apoptosis. The correlation between PPARc
activation and protein degradation requires further
investigation. In light of the apoptotic effect of PPARc
in adipocytes, we conclude that PPARc is one of the
key regulators involved in the physiology of the adipo-
cyte, from its birth to its death.
Experimental procedures
Materials
GW9662, troglitazone, antibodies against PTEN and actin,

3T3-L1 adipocyte apoptosis
The mature 3T3-L1 adipocytes were treated with troglitaz-
one at the indicated concentrations for 96 h or as indicated
in the figure legends. For the evaluation of apoptosis by
AO ⁄ EB staining, the treated 3T3-L1 adipocytes were
stained with 10 lgÆmL
)1
AO ⁄ EB in culture medium and
visualized by confocal microscope (Leica TCS SP2 Confo-
cal Microscope System, Leica microsystems, Wetzlar,
Germany). Ten microscopic fields were captured for each
sample by fluorescence microscopy, and the average apop-
totic rate was determined using totallab software v2.01
(Nonlinear Dynamics, Newcastle, UK). For Hoechst 33258
staining, the cells were stained with 10 lg ÆmL
)1
Hoe-
chst 33258 for 30 min. The stained cells were visualized and
analyzed as described for AO ⁄ EB staining.
For evaluation of apoptotic cells by flow cytometry [42],
PI staining or TUNEL assay was conducted. For the PI
staining, cells were washed with NaCl ⁄ P
i
and fixed in 70%
(v ⁄ v) ice-cold ethanol overnight. The fixed cells were then
washed with phosphate ⁄ citrate buffer (4 mm citric acid,
pH 7.8, 192 mm Na
2
HPO
4

, and then analyzed with a FACScan flow cytome-
ter (Becton Dickinson FACS Calibur, BD Biosciences, San
Jose, CA, USA). For the TUNEL assay, the fixed cells
were stained with an APO-DIRECT Kit (BD Pharmingen),
following the manufacturer’s protocol. Fluorescein isothio-
cyanate-labeled cells were counted as apoptotic cells.
Western blot
3T3-L1 adipocytes were washed with ice-cold NaCl ⁄ P
i
, and
lysed directly in boiling 1 · Laemmli SDS sample buffer
with 20 mm dithiothreitol. The cell extracts were heated to
100 °C for 10 min, and then subjected to SDS ⁄ PAGE and
western blot [41]. Mitochondria and cytosol of 3T3-L1 adi-
pocyte were isolated following the protocol developed by
Piper et al. [43].
Real-time PCR
Total RNA was extracted with TRIzol reagent (Invitrogen),
following the protocol provided by the manufacturer. RNA
(2 lg) was reverse-transcribed with oligodT primer. The
cDNA samples were then diluted to appropriate concentra-
tions for real-time PCR analysis (MJ Opticon 2; Bio-Rad
Laboratories, Hercules, CA, USA). Actin, a constitutively
expressed gene, was used as an internal control. The target
mRNA was normalized against actin in the same sample.
The PCR primers were as follows: actin forward, 5¢-GA
AATCGTGCGTGACATCAAAG-3¢; actin reverse, 5¢-TG
TAGTTTCATGGA TGCCACAG-3¢; Akt-1 forward, 5¢-A
ACGGACTTCGGGCTGTG-3¢; Akt-1 reverse, 5¢-TTGTC
CTCCAGCACCTCAGG-3¢; CD36 forward, 5¢-TCCAGC

epididymal, inguinal, omental and scapular fat pads were
isolated and washed with NaCl ⁄ P
i
. The adipose tissues were
then cut into fine pieces and incubated in collagenase solution
(1 mgÆmL
)1
collagenase type I, 2% BSA in NaCl ⁄ P
i
). The
cells were incubated for 1.5 h, and separated by centrifuga-
tion at 200 g for 10 min. The pellet was resuspended in eryth-
rocyte lysis buffer (155 mm NH
4
Cl, 5.7 mm K
2
HPO
4
, 0.1 mm
EDTA, pH 7.3) and incubated for 10 min. The suspension
was filtered through a size 200 filter and centrifuged at 200 g
for 10 min. The cells were resuspended in DMEM ⁄ F12, and
the medium was changed 3 h after inoculation. Two days
after inoculation (designated as day 0), cells were induced to
differentiate with 1 lgÆmL
)1
insulin, 1 lm dexamethasone,
and 0.5 mm 1-methyl-3-isobutylxanthine. The medium was
replaced with medium containing 1 lgÆmL
)1

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A
C
D
E
B
Fig. 6. Troglitazone (Tro)-induced rat adipocyte apoptosis (A) Differ-
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Supporting information
The following supplementary material is available:
Fig. S1. Densitometer scanning for western blot and
representative FACS plots for PI staining.
This supplementary material can be found in the
online version of this article.
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Apoptosis induced by TZDs in 3T3-L1 adipocyte Y. Xiao et al.
696 FEBS Journal 277 (2010) 687–696 ª 2009 The Authors Journal compilation ª 2009 FEBS