In the present study, we found that the expression of CIDEC increased during the differentiation of fetal adipose tissues, but decreased during the de-differentiation of adip-ocytic tumo
Trang 1droplet-associated protein, might be involved in the
differentiation of human adipocytes
Fanfan Li1, Yu Gu1, Wenpeng Dong2, Hang Li1, Liying Zhang1, Nanlin Li3, Wangzhou Li4,
Lijun Zhang1, Yue Song1, Lina Jiang1, Jing Ye1and Qing Li1
1 State Key Laboratory of Cancer Biology, Department of Pathology, Xijing Hospital, Fourth Military Medical University, Xi’an, China
2 State Key Laboratory of Cancer Biology, Xijing Hospital of Digestive Diseases, Fourth Military Medical University, Xi’an, China
3 Department of Vascular and Endocrine Surgery, Xijing Hospital, Fourth Military Medical University, Xi’an, China
4 Department of Plastic and Burns, Tangdu Hospital, Fourth Military Medical University, Xi’an, China
Introduction
Over the past 50 years, mounting evidence has shown
that obesity-related diseases, such as type 2 diabetes
and cardiovascular disease, are serious health
prob-lems, which stimulated a surge of interest in the study
of adipocyte biology [1] Owing to an imbalance
between energy intake and expenditure, obesity is often characterized by an increase in both the size and the number of adipocytes Previous data have shown that adipose tissues play crucial roles in the development
of obesity, and that the differentiation of adipocytes
Keywords
adipocyte differentiation; cell death-inducing
DFF45-like effector C (CIDEC); obesity;
peroxisome proliferator-activated receptor-c
(PPARc); RNAi
Correspondence
Qing Li and Jing Ye, State Key Laboratory
of Cancer Biology, Department of
Pathology, Xijing Hospital, Fourth Military
Medical University, 15# Changle West
Road, Xi’an 710032, China
Fax: 86 29 84776793
Tel: 86 29 84774541
E-mail: liqing@fmmu.edu.cn;
yejing@fmmu.edu.cn
(Received 12 March 2010, revised 29 July
2010, accepted 3 August 2010)
doi:10.1111/j.1742-4658.2010.07806.x
Cell death-inducing DFF45-like effector (CIDE) family proteins, including cell death-inducing DFF45-like effector A (CIDEA), cell death-inducing DFF45-like effector B (CIDEB) and cell death-inducing DFF45-like effec-tor C (CIDEC) [fat-specific protein of 27 kDa in rodent (FSP27) in rodents], were originally identified by their sequence homology to the N-terminal region of DNA fragmentation factor DFF40⁄ 45 Recent reports have revealed that CIDE family proteins play important roles in lipid metabolism Several studies involving knockdown mice revealed that FSP27 is a lipid droplet-targeting protein that can promote the formation
of lipid droplets However, the detailed roles of human CIDEC in the dif-ferentiation of human adipocytes remain unknown In the present study,
we found that the expression of CIDEC increased during the differentiation
of fetal adipose tissues, but decreased during the de-differentiation of adip-ocytic tumors, suggesting that the expression of CIDEC should be posi-tively correlated with the differentiation of adipocytes Furthermore, we verified that human CIDEC was localized on the surface of lipid droplets Using human primary pre-adipocytes, we confirmed that the expression of CIDEC was elevated during the differentiation of pre-adipocytes, and knockdown of CIDEC in human primary pre-adipocytes resulted in differ-entiation defects These data demonstrate that CIDEC is essential for the differentiation of adipose tissue Together with regulating adipocyte lipid metabolism, CIDEC should be a potential target for regulating adipocyte differentiation and reducing fat cell mass
Abbreviations
CIDEC, cell death-inducing DFF45-like effector C; EGFP, enhanced green fluorescent protein; FSP27, fat-specific protein of 27 kDa; FABP, fatty acid-binding protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HA, hemagglutinin; PPARc, peroxisome proliferator-activated receptor-c; shRNA, short hairpin RNA; TG, triglyceride.
Trang 2is closely linked to obesity and obesity-related
dis-eases [2] Therefore, to understand obesity in greater
detail, it is critical to elucidate the physiological role
of adipose tissue and the mechanism of adipocyte
dif-ferentiation, particularly during embryonic and fetal
life [3]
Cell death-inducing DFF45-like effector (CIDE)
pro-teins, including cell death-inducing DFF45-like effector A
(CIDEA), cell death-inducing DFF45-like effector (CIDEB)
and cell death-inducing DFF45-like effector (CIDEC)
[also known as fat-specific protein of 27 kDa (FSP27)
in rodents], were originally identified by their
sequence homology to the N-terminal region of
DNA fragmentation factor DFF40⁄ 45 [4,5] Animals
deficient in CIDEA, CIDEB or FSP27 display lean
phenotypes with higher energy expenditure and are
resistant to diet-induced obesity [6–8], suggesting a
universal role of CIDE proteins in the regulation of
energy homeostasis FSP27, the rodent homolog of
human CIDEC, was first identified in differentiated
TA1 adipocytes Peroxisome proliferator-activated
receptor-c (PPARc) and CCAAT⁄ enhancer-binding
protein, key regulators during adipose differentiation,
play critical roles in regulating the transcription of
Fsp27 [9] Over-expression of FSP27 in 3T3-L1
pre-adipocytes as well as in COS-7 cells markedly
increases the size of lipid droplets and enhances the
accumulation of total neutral lipids [10,11], both of
which are characteristics of mature adipocytes When
Fsp27was depleted during adipogenesis or in
differenti-ated 3T3-L1 cells, the lipid droplets were uniformly
dispersed into smaller structures, and lipolysis was
modestly increased [10,11]
Although CIDEC, is 66% homologus to FSP27, the
functional phenotypes of the two proteins are not fully
consistent For example, there is an obvious difference
of insulin sensitivity between human CIDEC and
mouse FSP27 [12–14] Therefore, it is necessary to
study the function of CIDEC in humans In this
research, we examined the role of CIDEC in human
adipocyte differentiation We observed that CIDEC
was expressed at increasingly higher levels during the
differentiation of fetal adipose tissues and expressed at
decreasingly lower levels during the de-differentiation
of adipocytic tumors, suggesting that the expression of
CIDEC should be positively correlated with the
differ-entiation of adipocytes We also verified that CIDEC
was localized on the surface of lipid droplets In
human primary pre-adipocytes, we confirmed that the
expression of CIDEC was elevated during the
differen-tiation of adipocytes Furthermore, stable knockdown
of CIDEC during adipogenesis of human primary
pre-adipocytes resulted in differentiation defects
Results
CIDEC is increasingly expressed during the differ-entiation of fetal adipose tissues
To investigate the expression of CIDEC in fetal adi-pose tissues at different stages of development, paraf-fin-embedded fetal adipose tissue samples were analyzed by immunohistochemistry using an affinity-purified antibody of human CIDEC As shown in Fig 1A, CIDEC was strongly expressed in adipose tis-sues obtained from third-trimester (week 33 of gesta-tion) fetal samples, which were already composed of mature adipocytes that contained large unilocular lipid droplets occupying most of the cytoplasm However, CIDEC was not readily detected in second-trimester (weeks 18 and 23 of gestation) fetal samples, which contained undifferentiated pre-adipocytes Western blotting and real-time PCR analyses showed that both the protein and the mRNA levels of CIDEC were markedly increased in adipose tissues of third-trimester fetal samples (Fig 1B,C), which was consistent with the immunohistochemistry result
As an important regulator in adipogenesis, PPARc also plays a key role in maintaining the characteristics
of mature adipocytes, and recent reports revealed that PPARc was required for the transcriptional activity of CIDEC during adipogenesis [15] We also observed that the mRNA and protein levels of PPARc were markedly increased during the differentiation of fetal adipose tissues (Fig 1B,C,D), which paralleled the increased expression of CIDEC These results suggest that the expression of CIDEC in fetal adipose tissues should be correlated with the differentiation or matu-ration of adipocytes
Expression of CIDEC decreases with de-differentiation of adipocytic tumors The expression of CIDEC, which increased with the maturation of fetal adipose tissues, prompted us to investigate the relationship between CIDEC and dif-ferentiation in adipocyte-derived tumors (lipoma and liposarcoma) Thirty normal adipose tissue specimens,
15 lipoma specimens and 30 liposarcoma specimens were collected using routine procedures Immunohis-tochemical staining showed that CIDEC was present
in all normal adipose tissue and lipoma specimens Interestingly, lower levels of CIDEC were detected in all 15 well-differentiated liposarcoma specimens How-ever, CIDEC was undetectable in the 10 myxoid lipo-sarcoma specimens and in the five de-differentiated liposarcoma specimens Figure 2A shows representative
Trang 3slides demonstrating the CIDEC-staining patterns in
normal adipose tissues and adipocytic tumors Using
quantitative PCR, lower CIDEC mRNA transcript
levels were found in well-differentiated liposarcomas,
and the mRNA levels of CIDEC were almost
unde-tectable in myxoid liposarcomas or de-differentiated
liposarcomas (Fig 2B) Furthermore, the decreased
levels of CIDEC, found in liposarcomas differentiated
to various degrees, were confirmed by
immunoblot-ting (Fig 2C,D) These results indicated that higher
levels of CIDEC are present in normal fat tissue and
in well-differentiated adipocytic tumors than in poorly
differentiated adipocytic tumors, indicating that the
expression of CIDEC decreases along with the
de-dif-ferentiation of adipocytic tumors In summary, these
results imply that CIDEC could be involved in the
differentiation of adipocytes
CIDEC localizes on the surface of lipid droplets
In order to evaluate the subcellular localization of human CIDEC, COS-7 cells were transfected with a vector that expressed a fusion protein of CIDEC con-taining the fluorescence marker DsRed1 The
transfect-ed COS-7 cells were culturtransfect-ed for 24 h in the presence of
100 lm oleic acid to promote the enlargement of lipid droplets As shown in Fig 3A, CIDEC was localized to strikingly different spherical structures, and the spheri-cal structures of CIDEC surrounded the lipid droplets,
as visualized by staining with Bodipy 493⁄ 503 More-over, the plasmid expressing enhanced green fluorescent protein (EGFP)-tagged adipophilin (also named perili-pin-2) was co-transfected into COS-7 cells with a plasmid containing hemagglutinin (HA)-tagged CIDEC We observed that CIDEC could partly overlap with
EGFP-A
B
Fig 1 Expression of CIDEC increased during the differentiation of fetal adipose tissue (A) Immunohistochemical staining was used to determine the expression of CIDEC in fetal adipose tissues obtained at weeks 18 (a), 23 (b) and 33 (c) of gestation (scale bar = 25 lm) The expression of CIDEC increased along with the differentiation of adipose tissue, and the highest level of expression of CIDEC was detected
in mature adipose tissue (B) Real-time PCR analysis of CIDEC and PPARc in different developmental stages of fetal adipose tissue The rela-tive mRNA levels of CIDEC and PPARc in fetal adipose tissue obtained at week 33 of gestation were higher than in that obtained at weeks
18 and 23 of gestation (The relative mRNA level in fetal adipose tissue obtained at week 18 of gestation was designated as 1.0 n = 3,
*P < 0.05, **P < 0.01) (C) Immunoblot analysis of CIDEC and PPARc in fetal adipose tissues showed a higher level of expression of these proteins in mature adipose tissue GAPDH was used as loading control (D) The relative quantity of CIDEC and PPARc protein was analyzed using QUANTITY ONE software (Bio-Rad) (The relative level of protein in the 18th week of gestation was designated as 1.0 n = 3, *P < 0.05,
**P < 0.01).
Trang 4tagged adipophilin, a lipid droplet-targeting protein
(Fig 3B) These data indicate that only a small amount
of CIDEC was localized on the surface of lipid
drop-lets, suggesting that CIDEC is also likely to be localized
on subcellular compartments other than lipid droplets
The expression of CIDEC is elevated during the
differentiation of adipocytes
To gain further insight into the roles of CIDEC in
adipocyte differentiation, human primary
pre-adipo-cytes were successfully isolated and cultured in vitro
The pre-adipocytes were induced using adipogenic
cocktails upon reaching confluence After 14 days of
induction, the majority of the cells displayed a
phe-notype of mature adipocytes (Fig 4A) The neutral
lipids accumulated in the cytoplasm, and a large number of lipid droplets were observed after staining the cells with Oil Red O (Fig 4B) Concurrently, CIDEC was detected in adipocytes from day 3 and showed an increase during the course of differentia-tion, reaching a peak on day 14 Furthermore, as a key regulator of adipogenesis, PPARc was also detected in adipocytes on day 3 during differentia-tion (Fig 4D) Using quantitative PCR, we observed that the mRNA levels of CIDEC and PPARc were significantly increased in adipocytes during the differ-entiation of pre-adipocytes (Fig 4C), which was con-sistent with the change of protein levels These data suggest that the expression of CIDEC might be attributable to the differentiation or maturation of adipocytes
A
a
b
c
d
e
B
C
D
Fig 2 Expression of CIDEC in normal adi-pose tissues, lipomas and liposarcomas (A) immunohistochemical staining for CIDEC showed that it was present in normal adi-pose tissue (NAT) (a) and lipoma (b) How-ever, only low levels of CIDEC could be detected in well-differentiated liposarcomas (WDLPS) (c), and no CIDEC was detectable
in myxoid liposarcomas (MLPS) (d) or de-dif-ferentiated liposarcomas (DDLPS) (e) (scale bar = 50 lm) (B) Real-time PCR revealed that the relative mRNA levels of CIDEC were higher in normal adipose tissue and lipoma than in liposarcoma (The relative mRNA level of NAT was designated as 1.0,
***P < 0.001) (C) Western blot analysis showed that the levels of CIDEC protein were high in normal adipose tissue and lipoma, but lower or negative in
liposarco-ma GAPDH was used as the loading control (D) The relative quantity of CIDEC protein was analyzed using QUANTITY ONE software (The relative protein level of NAT was designated as 1.0, **P < 0.01
***P < 0.001).
Trang 5B
Fig 3 CIDEC localizes on the surface of lipid droplets in COS-7 (A) COS-7 cells transfected with DsRed1-tagged CIDEC (red, middle panel) were incubated with 100 l M oleic acid for 24 h to enlarge the lipid droplets, which were visualized by Bodipy 493 ⁄ 503 staining (green, left panel) In the merged image (right panel), DsRed1-tagged CIDEC formed annular structures around the lipid droplets, suggesting that CIDEC should localize on the surface of lipid droplets Nuclei were labeled with Hochest 33258 (B) COS-7 cells were co-transfected with HA-tagged CIDEC and EGFP-tagged adipophilin Indirect immunofluorescence showed the co-localization of CIDEC with adipophilin, a lipid droplet-targeting protein Nuclei were stained with Hochest 33258 Scale bar = 10 lm.
A
B
E
Fig 4 The expression of CIDEC increased
during the differentiation of human primary
pre-adipocytes (A) Lipid droplets were
detectable in human differentiated
pre-adipocytes in phase-contrast micrographs
(Scale bar = 10 lm) (B) The lipid droplets in
differentiated pre-adipocytes were visualized
using Bodipy staining (scale bar = 10 lm).
(C) The mRNA levels of CIDEC and PPARc
were assessed using quantitative PCR.
Significantly higher levels of CIDEC and
PPARc were detected on days 7 and 14
during the differentiation of adipocytes (The
relative mRNA level before differentiation
(day 0) was designated as 1.0 *P < 0.05,
**P < 0.01, ***P < 0.001) (D) Immunoblot
analysis revealed that the expression of
CIDEC increased in human pre-adipocytes
during differentiation, and the expression of
PPARc showed a similar pattern FABP was
used as an adipocyte differentiation marker.
(E) Densitometric analyses of the relative
levels of the indicated proteins after
wes-tern blotting (as in D) were carried out
Simi-lar experiments were performed five times
and the intensity of the individual bands in
each western blot was quantified by
QUANTITY ONE software and used for statistical
analysis (The relative protein level before
dif-ferentiation (0 day) was designated as 1.0.
**P < 0.01, ***P < 0.001).
Trang 6Knockdown of CIDEC in pre-adipocytes results in
differentiation defects
To investigate the effects of endogenous CIDEC on
lipid droplet morphology, lipid metabolism and the
maturation of lipid droplets, the pre-adipocytes were
infected with a lentivirus carrying the U6
promoter-driven CIDEC short hairpin RNA (shRNA) before
induction of differentiation Using western blot
analy-sis, we found that the shRNA specific for CIDEC
resulted in the loss of at least 90% of CIDEC in
adipocytes (Fig 5A) This depletion of CIDEC
resulted in the formation of numerous small lipid
droplets in adipocytes during adipogenesis, in contrast
to the fewer and larger lipid droplets present in control cells (Fig 5B) Furthermore, when analyzed using TLC, the triglyceride (TG) content of CIDEC-depleted adipocytes was found to be significantly lower than that of control adipocytes (Fig 5C) To determine the rate of lipolysis, the amount of glycerol released into the medium was measured under basal conditions and after stimulation with isoproterenol, and the results revealed that the rate of lipolysis was significantly increased in CIDEC-depleted adipocytes compared with control adipocytes (Fig 5D) Additionally,
we performed quantitative PCR analysis on several
A
B
E
Fig 5 Depletion of CIDEC could block the differentiation of pre-adipocytes (A) Immunoblot analysis revealed that the expression of CIDEC was significantly reduced (by at least 90%) in differentiated human adipocytes infected with lentivirus-carrying CIDEC shRNA (B) The lipid droplets were fragmentated in the adipocytes with knockdown CIDEC after 14 days of differentiation (right panel) compared with the control group (left panel) The lipid droplets were stained with Nile Red (red stain) Nuclei were stained with Hochest 33258 (blue stain) (scale bar = 10 lm) (C) The amount of TG in differentiated adipocytes was quantified using TLC A lower concentration of TG was found in depleted adipocytes compared with control cells (n = 4, **P < 0.01, ***P < 0.001) (D) Glycerol released from control and CIDEC-depleted adipocytes was assessed under basal conditions and after stimulation with isoproterenol for 1 h (n = 4, **P < 0.01,
***P < 0.001) (E) The mRNA levels of PPARc, adipophilin, FABP and perilipin and were assessed using quantitative PCR The results revealed that the mRNA level of adipophilin was not changed, the mRNA levels of perilipin and FABP were decreased and the mRNA level
of PPARc was increased in CIDEC-silenced adipocytes, compared with mature adipocytes (The relative mRNA level in the control group was designated as 1.0 n = 3, *P < 0.05, **P < 0.01, ***P < 0.001).
Trang 7molecules involved in adipocyte differentiation and
lipid droplet formation on day 14 after induction As
lipid droplet-targeting proteins, perilipin and fatty
acid-binding protein (FABP) are expressed at late and
mature stages of lipid droplet formation Interestingly,
we found that the mRNA level of adipophilin was not
changed, whereas the mRNA levels of perilipin and
FABP decreased, and the mRNA level of PPARc
increased, in CIDEC knockdown adipocytes,
com-pared with control adipocytes (Fig 5E) These data
demonstrate that CIDEC can contribute to the
accu-mulation of neutral lipid and the maturation of lipid
droplets, which are key features of differentiated
adipocytes Loss of CIDEC led to immature
morphol-ogy, reduction of TG accumulation, increased lipolysis
and impeded the maturation of lipid droplets,
suggest-ing important roles of CIDEC dursuggest-ing the
differentia-tion of pre-adipocytes
Discussion
The differentiation of adipocytes is the process of
for-mation of new adipocytes from pre-adipocyte
precur-sors, and is accompanied by the up-regulation of genes
encoding proteins critical for lipid synthesis, lipolysis,
lipid transport, insulin sensitivity and other adipocyte
functions These proteins include PPARc, FABP, fatty
acid synthetase, fatty acid transporter and hormone-sensitive
lipase [16] PPARc has been identified as an important
adipogenic regulator⁄ switch and provides dynamic
and specific regulation during the differentiation of
pre-adipocytes into mature adipocytes [17]
With regard to embryonic development of adipose
tissue, studies have shown that the first traces of
adi-pose tissue are detectable between the 14th and 16th
weeks of gestation in humans, and that the second
tri-mester of gestation is the critical period in adipogenesis
After the 23rd week of gestation, although the number
of fat cells remains constant, the size of the lobules
grows and then multilocular adipocytes appear [18,19]
To our knowledge, we are the first group to present
data on the role of CIDEC in adipocyte differentiation
in vivo, which is evidenced by the increased expression
of CIDEC in third-trimester fetal adipose tissue
sam-ples, as well as the decreased expression of CIDEC in
conjunction with the de-differentiation of adipocytic
tumors To further confirm that the expression of
CIDEC correlates positively with adipocyte
differentia-tion, human primary pre-adipocytes were stimulated to
differentiate into mature adipocytes, and the expression
of CIDEC gradually increased along with the
expres-sion of PPARc and FABP, which are important
molecules involved in adipocyte differentiation
Although Liang et al [5] have reported finding CIDEC in an aggregated form near some mitochondria, and staining of CIDEC with Golgi-, endoplasmic retic-ulum- or lysosome-specific markers showed no over-lapping staining, the exact localization of CIDEC still remains to be clarified In this study, we observed that CIDEC was present on the surface of lipid droplets as well as diffuse within COS-7 cells, which is similar to the results observed in 3T3-L1 pre-adipocytes [14] Notably, a recent study has demonstrated that FSP27 co-localizes with the endoplasmic reticulum-specific protein CB5 in 3T3-L1 adipocytes [20] Consequently, these results suggest that CIDEC may be a lipid drop-let-associated protein and might localize on other sub-cellular compartments besides lipid droplets
Adipogenesis, a component of morphogenesis, may
be defined in general terms as the proliferation and subsequent differentiation of the fat-cell lineage capa-ble of the assimilation of lipid to form a lipid-contain-ing adipocyte [21] Our results revealed that the depletion of CIDEC resulted in increased lipolysis and decreased consumption of TG in adipocytes during adipogenesis Furthermore, morphological observation revealed that the depletion of CIDEC resulted in the formation of numerous smaller lipid droplets in adipo-cytes during adipogenesis Thus, we speculated that CIDEC is essential for the formation and maturation
of lipid droplets in adipocytes
In addition, the roles of proteins that associate with lipid droplets during adipogenesis, such as PAT pro-teins (named after the founding members of the family: perilipin, adipophilin⁄ adipocyte differentiation-related protein and TIP47), are of great interest It is believed that different lipid droplet-targeting proteins are coated on lipid droplets at different stages of adipo-genesis At early stages, the droplets are coated with adipophilin; however, during maturation, perilipin dis-places adipophilin [22] We found that the mRNA level
of adipophilin remained unchanged, while that of per-ilipin was decreased in CIDEC-silenced adipocytes It can be concluded that knockdown of CIDEC in pre-adipocytes results in defects of the maturation of lipid droplets and impedes adipocyte differentiation because maturation of lipid droplets is an important phenotype
of differentiated adipocytes
It is noteworthy that we observed an up-regulation
of PPARc in human primary pre-adipocytes with CIDEC knockdown A previous study showed that Fsp27 might be a direct mediator of PPARc-dependent hepatic steatosis and identified a PPARc-specific cis-element on the Fsp27 promoter [23] Recently, it was found that the thiazolidinedione, BRL49653, an ago-nist of PPARc, increases the abundance of Fsp27
Trang 8mRNA in 3T3-L1 adipocytes, whereas the expression
of a dominant-negative mutant of PPARc results in a
decrease in the amount of Fsp27 mRNA in 3T3-L1
adipocytes [24] Furthermore, our study found that
CIDEC and PPARc are expressed at increased levels
during the differentiation of fetal adipose tissues and
with the maturation of human primary adipocytes,
suggesting that CIDEC might be a target of PPARc
transactivation These data suggest that CIDEC may not
only be a downstream target of PPARc transactivation,
but is also likely to be involved in a feedback-sensing
pathway The results obtained using the Fsp27-knockout
mice also revealed that the expression of PPARc was
significantly increased [13], as was multilocular lipid
droplet formation, enhanced mitochondrial biogenesis
and glucose and free fatty acids oxidation, in white
adipose tissue [24] The increased levels of intracellular
fatty acids may stimulate the expression of PPARc in
white adipose tissue and thereby induce the secondary
mitochondrial biogenesis [24,25] Therefore, further
studies are necessary to characterize the physical
inter-actions between CIDEC and other lipid
droplet-associ-ated proteins, and to confirm the pathway through
which CIDEC affects the expression of PPARc,
espe-cially in humans
In summary, we found that CIDEC was expressed
at increased levels in mature and differentiated adipose
tissues, but at decreased levels in de-differentiated
adi-pose tumors It was demonstrated that CIDEC plays
important roles in the differentiation of adipose tissue
and in the regulation of adipocyte lipid metabolism,
indicating the potential of CIDEC as a target to
inhi-bit adipocyte differentiation, reduce fat cell mass and
improve insulin sensitivity
Materials and methods
Samples of human tissues and cell lines
Fetal adipose tissue samples were obtained from nine
still-born fetuses at weeks 18, 23 and 33 of gestation (three
samples at each time-point) under the agreement of the
local Ethics Committee and after obtaining informed
con-sent The causes of death were fetal distress caused by
eclampsia in four cases, and congenital heart disease in
five cases The fetal body weights were, respectively, 537,
490, 598, 780, 860, 1028, 1432, 1350 and 1890 g Samples
of normal adipose tissue (n = 30), and of lipoma
(n = 15) and liposarcoma (15 well-differentiated
liposarco-mas, 10 myxoid liposarcomas and five de-differentiated
liposarcomas) specimens were obtained from Xijing
Hospi-tal, the first affiliated hospital of the Fourth Military
Medical University (Xi’an, China) The patients (39 men
and 36 women) had a mean age of 38 (range: 17-69) years The human pre-adipocytes were isolated from human subcutaneous adipose tissue obtained from five patients [35.4 ± 2.2 years of age, body mass index (BMI): 27.2 ± 1.4 kgÆm)2] undergoing abdominal liposuction treatment at the Department of Plastic and Burns, Tangdu Hospital, the second affiliated hospital of the Fourth Mili-tary Medical University The Ethics Committee of the hospital approved this study, and informed consent was obtained from the patient The COS-7 and 293T cells were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China)
Isolation and induction of pre-adipocytes Pieces of adipose tissue were immediately digested with
1 mgÆmL)1 of collagenase (Sigma-Aldrich, St Louis, MO, USA) in D-Hank’s solution and incubated in 500 mL flask
on a shaker (Thermo⁄ Forma Scientific 420 Incubator Orbi-tal Tabletop Shaker, 200 rpm, 37C, 30 min) The digested adipose tissue was filtered through a 150-lm cell strainer, and the floating adipocytes were separated from the med-ium containing the stroma-vascular fraction by centrifuga-tion for 10 min at 3000 rpm Centrifugalizacentrifuga-tion separated adipocytes from the stroma-vascular fraction that contained pre-adipocytes (pellet) The stromal vascular pellets were incubated with DMEM⁄ F12 (Invitrogen/Gibco, Carlsbad,
CA, USA) supplemented with 10% fetal bovine serum (Invitrogen/Gibco, Carlsbad, CA, USA) Two days after reaching confluence, the medium was replaced with a serum-free adipogenic medium (DMEM⁄ F12 supplemented with 10 lgÆmL)1 of transferrin, 33 lm biotin, 0.5 lm insu-lin, 0.2 nm triiodothyronine, 0.5 mm 3-isobutyl-1-methyl-xanthine, 0.1 lm hydrocortisone and 17 lm pantothenate) After incubation for a further 2 days, the medium was replaced with the above-mentioned serum-free adipogenic medium minus 3-isobutyl-1-methylxanthine Cells were col-lected at the indicated days of differentiation and used for further experiments
Plasmids and transfections CIDEC plasmid DNA was amplified from the HepG2 cell line using the RT-PCR After cutting with the enzymes (NdeI and BamHI), the purified PCR fragment was cloned into the vector pCMV5-HA (a gift from Dr Peng Li, Tsinghua University) The recombinant vector pShuttle-CMV-DsRed1-CIDEC was constructed by insert-ing the DsRed1 and CIDEC DNA fragments into the vector pShuttle-CMV The plasmid pGFP-adipophilin was also a kind gift from Dr Peng Li The plasmids were transfected into COS-7 cells using Lipofectamine (Invi-trogen, USA) and about 10% of the cells were DsRed1-positive
Trang 9Immunofluorescence assay
Immunofluorescence analyses were carried out on cells
grown on cover-slips The cells were fixed, for 20 min at
room temperature in NaCl⁄ Picontaining 3%
paraformalde-hyde, permeabilized for 15 min in NaCl⁄ Picontaining 0.1%
saponin, then incubated with HA antibody (sc-7392; Santa
Cruz Biotechnology, Santa Cruz, CA, USA) for 1 hour at
room temperature Intracellular lipids were visualized with
1 lgÆmL)1 of Bodipy 493⁄ 503 Fluorescence imaging was
assessed using confocal microscopy (Olympus FV1000,
Tokyo, Japan)
Quantitative PCR
Total RNA was extracted from tissues and cells using TRIzol
(Invitrogen) cDNA was synthesized from total RNA using
the PrimeScript RT reagent Kit (TAKARA, Dalian,
China) The mRNA levels were analyzed by real-time PCR
performed with the Bio-Rad iQ4 Multicolor Real-time
iCy-cler (Bio-Rad Laboratories, CA, USA) using SYBRPremix
Ex Taq (Takara) The primers (sense and antisense,
respec-tively) were as follows: CIDEC, 5¢-TTGATGTGGCCCGT
GTAACGTTTG-3¢ and 5¢-AAGCTTCCTTCATGATGCG
CTTGG-3¢; PPARc, 5¢-TGGAATTAGATGACAGCGAC
TTGG-3¢ and 5¢-CTGGAGCAGCTTGGCAAACA-3¢;
glyc-eraldehyde-3-phosphate dehydrogenase (GAPDH), 5¢-AAC
ATCATCCCTGCCTCTAC-3¢ and 5¢-CTGCTTCACCACC
TTCTTG-3¢; perilipin, 5¢-CCTGCCTTACATGGCTTGTT-3¢
and 5¢-CCTTTGTTGACTGCCATCCT-3¢; and adipophilin,
5¢-CTGAGCACATCGAGTCACATACTCT-3¢ and 5¢-GGA
GCGTCTGGCATGTAGTGT-3¢
Western blot analysis
Total protein lysate from frozen tissues or cultured cells was
prepared in ice-cold RIPA buffer (20 mm Hepes pH 7.5,
150 mm NaCl, 1 mm EDTA, 10% glycerol, 0.5% sodium
de-oxycholate, 1% Nonidet P-40, 0.1% SDS and protease
inhib-itor cocktails) Protein samples were immunoblotted with
antibodies to CIDEC, PPARc (Santa Cruz Biotechnology,
Santa Cruz, CA, USA), perilipin (Sigma-Aldrich, St Louis,
MO, USA), adipophilin (Progen, Heidelberg, Germany),
FABP (Alpha Diagnostic, San Antonio, TX, USA) and
GAPDH (Abcam, Cambridge, UK), and the
protein–anti-body immune complexes were detected with horseradish
per-oxidase-conjugated secondary antibodies and enhanced
chemiluminescence reagents (Pierce Biotechnology,
Rock-ford, IL, USA) The polyclonal antibodies against CIDEC
were generated by injection of rabbits with purified CIDEC
proteins (amino acids 1–172), as previously described [26]
The antibodies generated in response to the fusion protein
were purified by affinity chromatography with cyanogen
bro-mide-activated Sepharose 4B (Amersham Biosciences Corp.,
Piscataway, NJ, USA) coupled to the fusion protein
Immunohistochemistry Immunohistochemistry was carried out as previously described [27] Briefly, the deparaffinized and rehydrated slides were blocked with 50 mLÆL)1 of fetal bovine serum for 30 min to reduce nonspecific binding Then, incubate slides in a humidified chamber at 4C overnight with CIDEC antibody (1:200) Negative controls were obtained
by replacing the primary antibody with nonimmune rabbit serum The sections were subsequently incubated with the second antibody (Dako, Copenhagen, Denmark) at 37C for 40 min, and stained with 3,3¢-Diaminobenzidine-H2O2
for 5–10 min and counterstained with hematoxylin
Depletion of CIDEC in pre-adipocytes The 21-nucleotide shRNA constructs, targeting CIDEC mRNA, were designed using siRNA target finder soft-ware (http://www4.appliedbiosystems.com/techlib/misc/siRNA_ finder.html) The sense oligonucleotides were as follows: CIDEC, 5¢-AACTGTAGAGACAGAAGAGTA-3¢; and scrambled, 5¢-AAGAAGATTGATGTGGCCCGT-3¢ The plasmids pHCMV-VSV-G, pMDLg⁄ pRRE, pRSV Rev and FG12 (kindly provided by Dr Zilong Wen, IMCB, Singapore) were used to generate recombinant lentiviruses The production, purification and titration of lentivirus car-rying CIDEC shRNA were carried out following previ-ously described procedures [28] Before induction of differentiation, pre-adipocytes were infected with lentivirus carrying CIDEC shRNA Then, pre-adipocytes were induced into adipocytes as described above
Staining with Nile Red and BODIPY 493/503 Nile Red (Sigma-Aldrich) (1 mgÆmL)1) in acetone was pre-pared and stored protected from light BODIPY 493⁄ 503 (Sigma-Aldrich) was dissolved in ethanol to give a stock of
1 mgÆmL)1(which can be stored in the dark at)20C) To stain the neutral lipids, cells in the monolayer were first washed three times with NaCl⁄ Piand then fixed in NaCl⁄ Pi
containing 4% formaldehyde After three washes, the fixed cells were stained with Nile Red solution (1 lgÆmL)1) or BODIPY 493⁄ 503 (1 lgÆmL)1) for 10 min at room temper-ature, followed by three washes with water
Lipid extraction and TLC assay Total lipid was extracted from tissue or cells as previously described [29] Dried lipids were reconstituted in chloro-form⁄ methanol (2:1, v ⁄ v) and loaded onto a TLC plate (Sigma) Lipids were resolved in hexane⁄ diethyl ether ⁄ acetic acid (70 : 30 : 1, v⁄ v ⁄ v) The TLC plates were sprayed with 10% CuSO4in 10% phosphoric acid and developed by dry-ing in an oven at 150C The protein concentration was
Trang 10determined using the Bio-Rad Protein Assay (Bio-Rad
#500-0001) and the amount of TG was quantified using
bio-rad quantity oneSoftware
Lipolysis assay
Cells were incubated in DMEM (without phenol red)
con-taining 1% fatty acid-free BSA and with or without 1 mm
isoproterenol, as indicated A 100-lL sample of the medium
was withdrawn at the indicated time-points and used for
the lipolysis assay The glycerol level was determined using
a free-glycerol determination kit, according to the
manufac-turer’s instructions (Sigma)
Statistical analysis
All values are given as mean ± SE Paired samples were
analyzed using the paired-sample ttest, with Bonferroni
cor-rection and Dunnett’s post hoc test for comparisons of
multiple groups All statistical analyses were performed
using spss version 11.0 (SPSS Inc., Chicago, IL, USA)
A probability level of 0.05 was considered significant
Acknowledgements
We would like to thank members in Qing Li’s
labora-tory in the Fourth Military Medical University for
technical assistance and helpful discussion and Dr
Peng Li for critical editing of the manuscript This
work was supported by grants (30671087, 30772261
and 30700268) from the National Natural Science
Foundation of China
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