Apoptosis was induced in mature 3T3-L1 adipocytes by treatment with troglitazone, pioglitazone or prosta-glandin J2, and could be blocked by the PPARc antagonist GW9662.. Adipocyte apopt
Trang 1is 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
E-mail: kliao@sibs.ac.cn
(Received 11 March 2009, revised 16
November 2009, accepted 24 November
2009)
doi:10.1111/j.1742-4658.2009.07514.x
Although thiazolidinediones (TZDs) are potent promoters of adipogenesis
in the preadipocyte, they induce apoptosis in several other cell types, such as cancer cells, endothelial cells and T-lymphocytes In this study,
we investigated the proapoptotic effect of TZDs in mature 3T3-L1 adipocytes, which express high levels of the peroxisome proliferator-acti-vated receptor-c (PPARc) protein Apoptosis was induced in mature 3T3-L1 adipocytes by treatment with troglitazone, pioglitazone or prosta-glandin J2, and could be blocked by the PPARc antagonist GW9662 Treatment with PPARc agonists also decreased Akt-1 protein and phos-phorylation levels without affecting phosphoinositide 3-kinase and PTEN Further analysis indicated that in troglitazone-treated 3T3-L1 adipocytes, Bad phosphorylation and Bcl-2 protein levels were reduced, and Bax translocation to the mitochondria was increased Subsequently, cyto-chrome c release and caspase-3 cleavage were observed TZD-induced adipocyte apoptosis could be blocked by the caspase-3 inhibitor Ac-DEVD-CHO or by overexpression of Bcl2 In cultured rat primary adipocytes, similar apoptosis-inducing effects of troglitazone were also observed Thus, TZDs promote apoptosis in adipocytes through a PPARc-dependent pathway This apoptosis is mediated by the inhibition
of Akt-1, which decreases Bad phosphorylation and activates the mito-chondrial apoptotic pathway
Abbreviations
AO, acridine orange; EB, ethidium bromide; FACS, fluorescence-activated cell sorting; pBad, phosphorylated Bad; pAkt-1, phosphorylated Akt-1; PGJ2, 15-deoxy-D12,14-prostaglandin J; 2PI, propidium iodide; PI3K, phosphoinositide 3-kinase; PPARc, peroxisome proliferator-activated receptor-c; SD, standard deviation; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; TZD, thiazolidinedione.
Trang 2compounds 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%
[10] Furthermore, the activation of PPARc stimulates
apoptosis in a variety of cell types, including cancer
cells, endothelial cells and T-lymphocytes [20–25]
These observations provided the impetus for us to
study the effect of TZDs and PPARc on adipocyte
apoptosis
In comparison with other cell types, adipocytes are
quite resistant to apoptosis The expression of high
level of PPARc protein in adipocytes, however, should
make them more susceptible to PPARc
agonist-induced apoptosis [19–25] Although TZDs are potent
PPARc agonists and inducers of adipocyte
differentia-tion, their function in inducing adipocyte apoptosis
has not been fully explored 3T3-L1 adipocytes
consti-tute a good model system for studying PPARc
ago-nist-induced apoptosis [26,27] In the present study, we
investigated PPARc agonist-induced adipocyte
apopto-sis by using 3T3-L1 adipocytes and rat primary
adipo-cytes Adipocyte apoptosis could be induced by
troglitazone, pioglitazone or
15-deoxy-D12,14-prosta-glandin J2 (PGJ2), and reversed by GW9662, a
PPARc antagonist Further analysis suggests that
TZDs induce the downregulation of Akt-1 and lead to
the activation of caspase-3-dependent apoptosis
Results
TZD-induced apoptosis in 3T3-L1 adipocytes After 3T3-L1 adipocyte differentiation, the expression
of PPARc was greatly increased (Fig 1A) Troglitaz-one, an agonist of PPARc, induced apoptosis in mature 3T3-L1 adipocytes (Fig 1B,C) Troglitazone-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 PPARc-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]
To investigate the function of the PI3K–Akt-1 signaling pathway in troglitazone-induced adipocyte apoptosis, the components of the PI3K–Akt-1 signaling cascade were analyzed As shown in Fig 3A,B, treatment of 3T3-L1 adipocytes with troglitazone reduced the levels
of Akt-1 protein and inhibited its phosphorylation The densitometer scanning data of Akt-1 western blot are shown in Fig S1 However, other components of the PI3K–Akt-1 signaling cascade, such as PI3K and PTEN, were not affected by troglitazone (Fig 3C) Troglitazone activates PPARc, a transcription factor responsible for the expression of many adipogenic genes [19] Akt-1 did not appear to be the target gene
Trang 3of PPARc, because the expression of Akt-1 was only
slightly induced by troglitazone, whereas the
expres-sion levels of two PPARc target genes, adipose fatty
acid-binding protein (aP2) and CD36, were greatly
increased in troglitazone-treated 3T3-L1 adipocytes
(Fig 3D) Thus, the reduction of Akt-1 protein level in
troglitazone-treated adipocytes was most likely due to
decreased Akt-1 protein stability
GW9662 treatment not only blocked
troglitazone-induced adipocyte apoptosis (Fig 2B,C), but also
restored the troglitazone-induced decrease in Akt-1
protein level and its phosphorylation (Fig 3E)
GW9662 treatment also reversed the downregulation
of Akt-1 protein level in pioglitazone-induced or
PGJ2-induced apoptotic adipocytes (Fig 3F,G)
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
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 M ) or not (Control) for 96 h, stained with AO ⁄ EB, and visualized by confocal microscopy (C) Hoechst 33258 staining for apoptotic 3T3-L1 adipocytes Scale bar: 50 lm Hoechst, stained with Hoechst 33258; B field, bright field (D) Troglitazone-induced 3T3-L1 adipocyte apoptosis 3T3-L1 adipocytes treated with troglitazone for the indicated times (0, 24, 48, 72, 96 or 120 h) were stained with PI and analyzed
by FACS The percentage of cells with sub-G 1 DNA content was counted as apoptotic adipocytes The results are means ± SDs of three experiments *P < 0.05; **P < 0.01 (E) 3T3-L1 adipocyte apoptosis induced by troglitazone at different concentrations Cells were treated with troglitazone for 96 h, and analyzed as indicated in (D) (F) TUNEL assay for apoptotic 3T3-L1 adipocytes Cells were treated with troglit-azone at the indicated concentrations for 96 h Adipocyte apoptosis was measured by TUNEL assay.
Trang 4prodeath proteins to the mitochondria, causing
cyto-chrome c release and caspase-3 activation [34,35] In
troglitazone-treated apoptotic adipocytes, the
translo-cation of Bax from the cytoplasm into the
mitochon-dria, cytochrome c release and cleavage of caspase-3
were all observed (Fig 4B–D)
To confirm the involvement of Bcl-2 and caspase-3
in troglitazone-induced adipocyte apoptosis, Bcl-2 was
overexpressed in 3T3-L1 cells that were then
differenti-ated into adipocytes (Fig 5A,B) The overexpression
of Bcl-2 in 3T3-L1 adipocytes greatly inhibited
troglit-azone-induced apoptosis (Fig 5C,D) In addition,
Ac-DEVD-CHO, a caspase-3 inhibitor, inhibited
trog-litazone-induced adipocyte apoptosis and caspase-3
cleavage (Fig 5E–G)
The Bcl-2-dependent and caspase-3-dependent
apop-tosis induced by troglitazone in 3T3-L1 adipocytes was
verified in cultured rat primary adipocytes Cells
iso-lated from rat fat pads were differentiated into
adipo-cytes in vitro (Fig 6A) In rat primary adipoadipo-cytes,
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 PPARc agonists induce adipocyte apoptosis
in a PPARc-dependent manner (Figs 1 and 2) In prea-dipocytes or small aprea-dipocytes, 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
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 Oil Red O at day 8 (B) Inhibition of troglitaz-one-induced adipocyte apoptosis by GW9662 Scale bar: 50 lm 3T3-L1 adipo-cytes were treated with troglitazone (Tro) (12.5 l M ) and GW9662 (1–10 l M ) for 96 h Apoptosis was analyzed by AO ⁄ EB or Hoe-chst 33258 (Hoe ⁄ B.F) staining (C) Quantita-tive analysis of GW9662 inhibition of adipocyte apoptosis AO ⁄ EB, apoptosis measured by AO ⁄ EB staining; Hoe ⁄ B.F, apoptosis measured by Hoechst 33258 staining Results are means ± SDs of three independent experiments **P < 0.01 (D) Pioglitazone (Pio)-induced 3T3-L1 adipo-cyte apoptosis 3T3-L1 adipoadipo-cytes were treated with 1 l M pioglitazone (1 l M Pio) for
96 h in the presence or absence of 10 l M
GW9662 (GW) (10 l M ) Apoptosis was mea-sured by AO ⁄ EB staining (E) Pioglitazone-induced 3T3-L1 adipocyte apoptosis mea-sured by Hoechst 33258 staining (hoechst) (F) PGJ2-induced 3T3-L1 adipocyte apopto-sis 3T3-L1 adipocytes were treated with 10
or 25 l M PGJ2 for 96 h in the presence or absence of 10 l M GW9662 Apoptosis was measured by AO ⁄ EB staining Results are means ± SDs of three independent experiments *P < 0.05.
Trang 5mature 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 addition, the AMP-activated protein kinase pathway can affect Akt-1 protein levels in the cell [39]
It has been reported that troglitazone induces apopto-sis in prostate cancer cells by directly binding to Bcl-2 and Bcl-xL [40] Owing to its inability to bind to Bcl-2 and Bcl-xL, pioglitazone cannot induce apoptosis in these prostate cancer cells [40] In adipocytes, troglitaz-one, pioglitazone and PGJ2 all induce apoptosis (Fig 2) However, TZDs do not induce adipocyte apop-tosis by directly binding to Bcl-2; rather, they do so by reducing Bcl-2 levels Bcl-2 overexpression blocks trog-litazone-induced adipocyte apoptosis (Fig 5C,D) It is possible that a similar mechanism is involved in the reduction of Akt-1 and Bcl-2 protein levels (Figs 3 and 4) Furthermore, the decrease in Akt-1 activity in apop-totic adipocytes reduces Bad phosphorylation [33] (Fig 4A) The decrease of Bcl-2 protein level and
A
D
E
Fig 3 Akt-1 reduction in troglitazone (Tro)-induced adipocyte apoptosis (A) Akt-1 and pAkt-1 in troglitazone-induced 3T3-L1 adipocytes Cells were treated with troglitazone at the indicated concentrations (0, 3, 6, 12.5, 25 50 l M ) for 96 h Akt-1 and pAkt-1 were detected by western blot (B) Time course of Akt-1 decrease in troglitazone-induced 3T3-L1 adipocytes (C) 3T3-L1 adipocytes were treated with troglitazone (0, 12.5, 25 or 50 l M ) for 96 h, and PI3K p85, PTEN and PPARc were detected by western blot (D) Expression of aP2, CD36 and Akt-1 in troglitazone-treated 3T3-L1 adipocytes 3T3-L1 adipocytes were treated with troglitazone (3, 6 or 12.5 l 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 l M ) (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 l M GW9662).
Trang 6binding 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,
horseradish peroxidase-conjugated secondary antibodies,
dexamethasone, 1-methyl-3-isobutylxanthine, DMEM⁄ F12 and insulin were purchased from Sigma (St Louis, MO, USA) Pioglitazone, PGJ2 and Ac-DEVD-CHO were from Cayman Chemical Company (Ann Arbor, MI, USA) Anti-bodies against Akt-1, phosphorylated Akt-1 (pAkt-1) (Ser473), Bcl2, Bax and Hsp90 were from Santa Cruz Bio-technology, Inc (Santa Cruz, CA, USA) Antibodies against PPARc, caspase-3 and PI3K p85 were from Cell Signaling Technology, Inc (Beverly, MA, USA) APO-DIRECT kit and antibody against caspase-8 were from BD Pharmingen (San Jose, CA, USA) Antibodies against Bad and phosphorylated Bad (pBad) (Ser136) were from Assay Designs (Ann Arbor, MI, USA) Antibody against cyto-chrome c was from BioVision, Inc (Palo Alto, CA, USA) Hoechst 33258 and antibody against OxPhos complex V subunit a were from Molecular Probes (Invitrogen, Carls-bad, CA, USA) SYBR Green real-time PCR master mix was from Toyobo (Shanghai, China) TRIzol reagent, Lipo-fectamine 2000 and DMEM were from Invitrogen Collage-nase type I was from Worthington Biochemical Corp (Lakewood, NJ, USA) Hygromycin B was from Amresco Inc (Solon, OH, USA)
Cell culture and Oil Red O staining
3T3-L1 preadipocytes were cultured and induced to differ-entiate as described previously [41] On day 8, 3T3-L1 adipocytes were fixed and stained with Oil Red O to reveal triglyceride droplets [41] For inhibition of adipocyte differ-entiation by GW9662, GW9662 was added to 3T3-L1 prea-dipocytes with the differentiation inducers
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 lgÆmL)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⁄ Piand 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 Na2HPO4) for 1 h, treated with RNase (200 lgÆmL)1) for 1 h, stained with 30 lgÆmL)1 PI in
C
D
Fig 4 Troglitazone (Tro) induces adipocyte apoptosis through the
mitochondrial pathway (A) 3T3-L1 adipocytes were treated with
12.5 l M troglitazone for the indicated time (0, 12, 24, 48, 72 and
96 h) Proteins were detected by western blot with the appropriate
antibodies (B) Translocation of Bax into mitochondria Bax in
cyto-solic (Cyto.) and mitochondrial (Mito.) fractions from 3T3-L1
adipo-cytes treated with troglitazone (0, 3, 6, 12.5, 25 and 50 l M ) for
96 h were detected by western blot Total, whole cell extract.
(C) Cytochrome c (Cyto C) release in troglitazone-treated 3T3-L1
adipocytes The cells were treated as described in (B) Hsp90
(Hsp90), OxPhos complex V subunit a (Cox Va) and cytochrome c
were detected by western blot (D) Caspase-3 (Casp3) cleavage in
troglitazone-treated 3T3-L1 adipocytes The cells were treated with
troglitazone for 96 h, and the cleaved (clved) caspase-3 was
detected by western blot.
Trang 7NaCl⁄ Pi, 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⁄ Pi, and
lysed directly in boiling 1· Laemmli SDS sample buffer
with 20 mm dithiothreitol The cell extracts were heated to
100C 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 TAGTTTCATGGATGCCACAG-3¢; Akt-1 forward, 5¢-A ACGGACTTCGGGCTGTG-3¢; Akt-1 reverse, 5¢-TTGTC CTCCAGCACCTCAGG-3¢; CD36 forward, 5¢-TCCAGC CAATGCCTTTGC-3¢; CD36 reverse, 5¢-TGGAGATTAC
A
B
G
Fig 5 Inhibition of troglitazone (Tro)-induced 3T3-L1 adipocyte apoptosis by Bcl-2 overexpression and the caspase-3 inhibitor Ac-DEVD-CHO (A) Adipocyte differentiation of 3T3-L1 cells overexpressing Bcl-2 Vector, control virus-infected cell; Bcl2, Bcl-2 virus-infected cell (B) Bcl-2 overexpression in differentiated adipocytes (C) Troglitazone-induced apoptosis in Bcl-2-overexpressing adipocytes Cells were treated with 12.5 l M troglitazone for 96 h, and apoptosis was measured by AO ⁄ EB staining (D) Expression of Bcl-2 and Akt-1 in Bcl-2-overexpressing 3T3-L1 adipocytes The treatment was the same as in (C) Akt-1 and Bcl-2 were detected by western blot (E) AO ⁄ EB stain-ing for apoptotic 3T3-L1 adipocytes Scale bar: 50 lm 3T3-L1 adipocytes were treated with 12.5 l M troglitazone in the presence of Ac-DEVD-CHO (10 or 20 l M ) for 96 h (F) Inhibition of apoptosis by Ac-DEVD-CHO AO ⁄ EB, apoptosis measured by AO ⁄ EB staining; Hoe-chst, apoptosis measured by Hoechst 33258 staining Results are means ± SDs of three independent experiments **P < 0.01 (G) Inhibi-tion of caspase-3 cleavage by Ac-DEVD-CHO 3T3-L1 adipocytes were treated with 12.5 l M troglitazone in the presence or absence of Ac-DEVD-CHO for 72 or 96 h Cleaved caspase-3 (Casp3 clved) and total caspase-3 (Casp3) were detected by western blot.
Trang 8TTTTTCAGTGCAGAA-3¢; aP2 forward, 5¢-AAAGACA
GCTCCTCCTCGAAGGTT-3¢; and aP2 reverse, 5¢-TGA
CCAAATCCCCATTTACGC-3¢ Standard curves were
gen-erated with 10-fold serial dilutions ranging from 1⁄ 10 to
1⁄ 10 000 of the reverse transcription mixture
Rat primary preadipocyte isolation, culture and
differentiation
Rat (normal male Zucker rat) white adipose tissues from the
epididymal, inguinal, omental and scapular fat pads were
isolated and washed with NaCl⁄ Pi The adipose tissues were
then cut into fine pieces and incubated in collagenase solution
(1 mgÆmL)1 collagenase type I, 2% BSA in NaCl⁄ Pi) 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 NH4Cl, 5.7 mm K2HPO4, 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 insulin after
72 h, and then changed every other day
Retroviral expression of Bcl-2 in 3T3-L1 cells
The mouse Bcl-2 cDNA was inserted into a pMSCVhyg vector 293T cells were cotransfected with pMSCVhyg–Bcl2 and PCL-10A1 to generate the retrovirus 3T3-L1 preadipo-cytes were plated, infected with Bcl-2 retrovirus or control virus, and selected with hygromycin B The infected 3T3-L1 cells were then differentiated into adipocytes, following the standard protocol
Statistical analysis
All experiments were performed at least three times, and data are expressed as means ± standard deviations (SDs) Differences were analyzed by Student’s t-test between trea-ted samples vs control samples P < 0.05 was considered
to be statistically significant
Acknowledgements
This work was supported by grants 30821065 and
30870559 from the China National Nature Sciences Foundation, 2006CB910703 from the Ministry of Sciences and Technology of China, and 07dz05907 from the Committee of Sciences and Technology of Shanghai
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A
C
D
E B
Fig 6 Troglitazone (Tro)-induced rat adipocyte apoptosis (A)
Differ-entiation of cultured rat primary fat pad cells Rat cells from fat
pads, cultured cells isolated from rat fat pads; Rat cells after diff.,
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Apopto-tic rat adipocytes Differentiated rat adipocytes were treated with
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adipocytes The adipocytes were treated with troglitazone at the
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experiments **P < 0.01 (E) Rat adipocytes were treated with
troglitazone for 96 h at the indicated concentrations, and proteins
were detected by western blot Casp3 clved, cleaved caspase-3.
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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
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