Cell death-inducing DFF45-like effector, a lipid droplet-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 Ye1 and Qing Li1 State Key Laboratory of Cancer Biology, Department of Pathology, Xijing Hospital, Fourth Military Medical University, Xi’an, China State Key Laboratory of Cancer Biology, Xijing Hospital of Digestive Diseases, Fourth Military Medical University, Xi’an, China Department of Vascular and Endocrine Surgery, Xijing Hospital, Fourth Military Medical University, Xi’an, China Department of Plastic and Burns, Tangdu Hospital, Fourth Military Medical University, Xi’an, China 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 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 effector 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 differentiation 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 adipocytic tumors, suggesting that the expression of CIDEC should be positively 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 differentiation 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 Introduction Over the past 50 years, mounting evidence has shown that obesity-related diseases, such as type diabetes and cardiovascular disease, are serious health problems, 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 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 FEBS Journal 277 (2010) 4173–4183 ª 2010 The Authors Journal compilation ª 2010 FEBS 4173 CIDEC in human adipocyte differentiation F Li et al is closely linked to obesity and obesity-related diseases [2] Therefore, to understand obesity in greater detail, it is critical to elucidate the physiological role of adipose tissue and the mechanism of adipocyte differentiation, particularly during embryonic and fetal life [3] Cell death-inducing DFF45-like effector (CIDE) proteins, 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 Fsp27 was depleted during adipogenesis or in differentiated 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 differentiation 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 differentiation of adipocytes Furthermore, stable knockdown of CIDEC during adipogenesis of human primary preadipocytes resulted in differentiation defects 4174 Results CIDEC is increasingly expressed during the differentiation of fetal adipose tissues To investigate the expression of CIDEC in fetal adipose tissues at different stages of development, paraffin-embedded fetal adipose tissue samples were analyzed by immunohistochemistry using an affinitypurified antibody of human CIDEC As shown in Fig 1A, CIDEC was strongly expressed in adipose tissues obtained from third-trimester (week 33 of gestation) 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 maturation 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 differentiation in adipocyte-derived tumors (lipoma and liposarcoma) Thirty normal adipose tissue specimens, 15 lipoma specimens and 30 liposarcoma specimens were collected using routine procedures Immunohistochemical 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 However, CIDEC was undetectable in the 10 myxoid liposarcoma specimens and in the five de-differentiated liposarcoma specimens Figure 2A shows representative FEBS Journal 277 (2010) 4173–4183 ª 2010 The Authors Journal compilation ª 2010 FEBS F Li et al CIDEC in human adipocyte differentiation A a b c B C D Fig 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 relative 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) slides 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 undetectable 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 immunoblotting (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-differentiation 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 containing the fluorescence marker DsRed1 The transfected COS-7 cells were cultured 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 spherical structures of CIDEC surrounded the lipid droplets, as visualized by staining with Bodipy 493 ⁄ 503 Moreover, the plasmid expressing enhanced green fluorescent protein (EGFP)-tagged adipophilin (also named perilipin-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- FEBS Journal 277 (2010) 4173–4183 ª 2010 The Authors Journal compilation ª 2010 FEBS 4175 CIDEC in human adipocyte differentiation F Li et al B A a b Fig Expression of CIDEC in normal adipose tissues, lipomas and liposarcomas (A) immunohistochemical staining for CIDEC showed that it was present in normal adipose tissue (NAT) (a) and lipoma (b) However, 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-differentiated 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 liposarcoma 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) C c D d e tagged 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 droplets, 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-adipocytes 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 phenotype of mature adipocytes (Fig 4A) The neutral 4176 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 and showed an increase during the course of differentiation, reaching a peak on day 14 Furthermore, as a key regulator of adipogenesis, PPARc was also detected in adipocytes on day during differentiation (Fig 4D) Using quantitative PCR, we observed that the mRNA levels of CIDEC and PPARc were significantly increased in adipocytes during the differentiation of pre-adipocytes (Fig 4C), which was consistent with the change of protein levels These data suggest that the expression of CIDEC might be attributable to the differentiation or maturation of adipocytes FEBS Journal 277 (2010) 4173–4183 ª 2010 The Authors Journal compilation ª 2010 FEBS F Li et al CIDEC in human adipocyte differentiation A B Fig 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 lM 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 Fig The expression of CIDEC increased during the differentiation of human primary pre-adipocytes (A) Lipid droplets were detectable in human differentiated preadipocytes 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 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 western blotting (as in D) were carried out Similar 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 differentiation (0 day) was designated as 1.0 **P < 0.01, ***P < 0.001) A B C D E FEBS Journal 277 (2010) 4173–4183 ª 2010 The Authors Journal compilation ª 2010 FEBS 4177 CIDEC in human adipocyte differentiation F Li et al Knockdown 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 promoterdriven CIDEC short hairpin RNA (shRNA) before induction of differentiation Using western blot analysis, 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 B A C D E Fig 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 CIDEC-depleted adipocytes compared with control cells (n = 4, **P < 0.01, ***P < 0.001) (D) Glycerol released from control and CIDECdepleted adipocytes was assessed under basal conditions and after stimulation with isoproterenol for 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) 4178 FEBS Journal 277 (2010) 4173–4183 ª 2010 The Authors Journal compilation ª 2010 FEBS F Li et al molecules 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, compared with control adipocytes (Fig 5E) These data demonstrate that CIDEC can contribute to the accumulation of neutral lipid and the maturation of lipid droplets, which are key features of differentiated adipocytes Loss of CIDEC led to immature morphology, reduction of TG accumulation, increased lipolysis and impeded the maturation of lipid droplets, suggesting important roles of CIDEC during the differentiation of pre-adipocytes Discussion The differentiation of adipocytes is the process of formation of new adipocytes from pre-adipocyte precursors, 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 adipose tissue are detectable between the 14th and 16th weeks of gestation in humans, and that the second trimester 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 samples, 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 differentiation, human primary pre-adipocytes were stimulated to differentiate into mature adipocytes, and the expression of CIDEC gradually increased along with the expression of PPARc and FABP, which are important molecules involved in adipocyte differentiation CIDEC in human 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 reticulum- or lysosome-specific markers showed no overlapping 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 droplet-associated protein and might localize on other subcellular 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 capable of the assimilation of lipid to form a lipid-containing 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 adipocytes 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 proteins (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 adipogenesis At early stages, the droplets are coated with adipophilin; however, during maturation, perilipin displaces adipophilin [22] We found that the mRNA level of adipophilin remained unchanged, while that of perilipin was decreased in CIDEC-silenced adipocytes It can be concluded that knockdown of CIDEC in preadipocytes 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 ciselement on the Fsp27 promoter [23] Recently, it was found that the thiazolidinedione, BRL49653, an agonist of PPARc, increases the abundance of Fsp27 FEBS Journal 277 (2010) 4173–4183 ª 2010 The Authors Journal compilation ª 2010 FEBS 4179 CIDEC in human adipocyte differentiation F Li et al mRNA 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 interactions between CIDEC and other lipid droplet-associated proteins, and to confirm the pathway through which CIDEC affects the expression of PPARc, especially 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 adipose 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 inhibit 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 stillborn 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 consent 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 liposarcomas, 10 myxoid liposarcomas and five de-differentiated liposarcomas) specimens were obtained from Xijing Hospital, the first affiliated hospital of the Fourth Military Medical University (Xi’an, China) The patients (39 men 4180 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 kgỈm)2] undergoing abdominal liposuction treatment at the Department of Plastic and Burns, Tangdu Hospital, the second affiliated hospital of the Fourth Military 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 mgỈmL)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 Orbital Tabletop Shaker, 200 rpm, 37 °C, 30 min) The digested adipose tissue was filtered through a 150-lm cell strainer, and the floating adipocytes were separated from the medium containing the stroma-vascular fraction by centrifugation for 10 at 3000 rpm Centrifugalization 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 lgỈmL)1 of transferrin, 33 lm biotin, 0.5 lm insulin, 0.2 nm triiodothyronine, 0.5 mm 3-isobutyl-1-methylxanthine, 0.1 lm hydrocortisone and 17 lm pantothenate) After incubation for a further days, the medium was replaced with the above-mentioned serum-free adipogenic medium minus 3-isobutyl-1-methylxanthine Cells were collected 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 inserting 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 LipofectamineÔ (Invitrogen, USA) and about 10% of the cells were DsRed1positive FEBS Journal 277 (2010) 4173–4183 ª 2010 The Authors Journal compilation ª 2010 FEBS F Li et al CIDEC in human adipocyte differentiation Immunofluorescence assay Immunohistochemistry Immunofluorescence analyses were carried out on cells grown on cover-slips The cells were fixed, for 20 at room temperature in NaCl ⁄ Pi containing 3% paraformaldehyde, permeabilized for 15 in NaCl ⁄ Pi containing 0.1% saponin, then incubated with HA antibody (sc-7392; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for hour at room temperature Intracellular lipids were visualized with lgỈmL)1 of Bodipy 493 ⁄ 503 Fluorescence imaging was assessed using confocal microscopy (Olympus FV1000, Tokyo, Japan) Immunohistochemistry was carried out as previously described [27] Briefly, the deparaffinized and rehydrated slides were blocked with 50 mLỈL)1 of fetal bovine serum for 30 to reduce nonspecific binding Then, incubate slides in a humidified chamber at °C 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 37 °C for 40 min, and stained with 3,3¢-Diaminobenzidine-H2O2 for 5–10 and counterstained with hematoxylin Quantitative PCR Total RNA was extracted from tissues and cells using TRIzol (Invitrogen) cDNA was synthesized from total RNA using the PrimeScriptÔ RT reagent Kit (TAKARA, Dalian, China) The mRNA levels were analyzed by real-time PCR performed with the Bio-Rad iQ4 Multicolor Real-time iCycler (Bio-Rad Laboratories, CA, USA) using SYBRỊ Premix Ex TaqƠ (Takara) The primers (sense and antisense, respectively) were as follows: CIDEC, 5¢-TTGATGTGGCCCGT GTAACGTTTG-3¢ and 5¢-AAGCTTCCTTCATGATGCG CTTGG-3¢; PPARc, 5¢-TGGAATTAGATGACAGCGAC TTGG-3¢ and 5¢-CTGGAGCAGCTTGGCAAACA-3¢; glyceraldehyde-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¢ Depletion of CIDEC in pre-adipocytes The 21-nucleotide shRNA constructs, targeting CIDEC mRNA, were designed using siRNA target finder software (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 carrying CIDEC shRNA were carried out following previously 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 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, mm EDTA, 10% glycerol, 0.5% sodium deoxycholate, 1% Nonidet P-40, 0.1% SDS and protease inhibitor 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–antibody immune complexes were detected with horseradish peroxidase-conjugated secondary antibodies and enhanced chemiluminescence reagents (Pierce Biotechnology, Rockford, 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 bromide-activated Sepharose 4B (Amersham Biosciences Corp., Piscataway, NJ, USA) coupled to the fusion protein Nile Red (Sigma-Aldrich) (1 mgỈmL)1) in acetone was prepared and stored protected from light BODIPY 493 ⁄ 503 (Sigma-Aldrich) was dissolved in ethanol to give a stock of mgỈmL)1 (which can be stored in the dark at )20°C) To stain the neutral lipids, cells in the monolayer were first washed three times with NaCl ⁄ Pi and then fixed in NaCl ⁄ Pi containing 4% formaldehyde After three washes, the fixed cells were stained with Nile Red solution (1 lgỈmL)1) or BODIPY 493 ⁄ 503 (1 lgỈmL)1) for 10 at room temperature, 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 chloroform ⁄ 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% CuSO4 in 10% phosphoric acid and developed by drying in an oven at 150 °C The protein concentration was FEBS Journal 277 (2010) 4173–4183 ª 2010 The Authors Journal compilation ª 2010 FEBS 4181 CIDEC in human adipocyte differentiation F Li et al determined using the Bio-Rad Protein Assay (Bio-Rad #500-0001) and the amount of TG was quantified using bio-rad quantity one Software Lipolysis assay Cells were incubated in DMEM (without phenol red) containing 1% fatty acid-free BSA and with or without 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 manufacturer’s instructions (Sigma) Statistical analysis All values are given as mean ± SE Paired samples were analyzed using the paired-sample ttest, with Bonferroni correction 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 laboratory 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 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