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Annales d’Endocrinologie 75 (2014) 1–9

Original article

Obestatin changes proliferation, differentiation and apoptosis of porcinepreadipocytes

L’obestatine influence la prolifération, la différentiation et l’apoptose des cellulespréadipocytaires de porc

Shengqiu Tang a,1, Xiaoying Dong a,1,∗, Wei Zhang b

a College of Yingdong agricultural science and engineering, Shaoguan university, Daxue road, Zhenjiang district, Shaoguan 512005, Chinab Hubei Key laboratory of animal embryo and molecular breeding, Hubei academy of agricultural science, Wuhan 430064, China

bstract

Obestatin, originally identified and purified from rat stomach extracts, was reported to bind to orphan G protein-coupled receptor, GPR39, andnhibit appetite and gastric motility. This study was conducted to investigate the effects of porcine obestatin on proliferation, differentiation andpoptosis of porcine preadipocytes isolated from subcutaneous fat of piglets. At indicated times of culture, morphology of preadipocytes andccumulated lipid droplets within the cells were identified by invert microscope. After treating with obestatin (0, 0.1, 1, 10 and 100 nM), cellroliferation was measured by MTT method and protein expression of CCAAT/enhancer binding protein-� (C/EBP�), peroxisome proliferator-ctivated receptor-� (PPAR�), Caspase-7 and Caspase-9 was determined by Western Blot, mRNA expression of GPR39 and Caspase-3 was

nalyzed by RT-PCR, and the activity of Caspase-3 was measured by spectrophotometric method. The results showed that obestatin had no effectn GPR39 expression, while promotes the optical density (OD) value of cells, enhanced protein expression of PPAR� and C/EBPa, decreasedRNA expression and activity of Caspase-3, and inhibited protein expression of Caspase-7 and Caspase-9 in a dose-dependent manner. These

esults suggested that obestatin enhance proliferation and differentiation of preadipocytes promoting PPAR� and C/EBPa expression, and inhibitingreadipocyte apoptosis by decreasing expression of Caspase-3, Caspase-7 and Caspase-9.

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2013 Elsevier Masson SAS. All rights reserved.

. Introduction

A number of clinical studies have demonstrated that adiposeissue is integrally involved in coordinating a variety of biolog-cal processes including energy metabolism, immune function,nd neuroendocrine function [1]. The important endocrine func-ion of adipose tissue is emphasized by the adverse metaboliconsequences of both adipose tissue excess and deficiency2]. Besides the biological repertoire necessary for storing andeleasing energy, adipose tissue contains the metabolic machin-ry to permit communication with distant organs including theentral nervous system (CNS). It is now clear that adipose tis-ue is a complex and highly active metabolic and endocrine

rgan [3,4]. Besides adipocytes, adipose tissue contains con-ective tissue matrix, nerve tissue, stromovascular cells, andmmune cells [5]. The previous findings suggested that visceral

∗ Corresponding author.E-mail address: dongcathy@163.com (X. Dong).

1 These authors contributed equally to this work.

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003-4266/$ – see front matter © 2013 Elsevier Masson SAS. All rights reserved.ttp://dx.doi.org/10.1016/j.ando.2013.10.003

at accumulation played crucial roles in the development of car-iovascular disease as well as the development of obesity-relatedisorders such as hyperlipidemia, diabetes mellitus and the so-alled metabolic syndrome [6]. Given these clinical findings,dipocytes functions have been intensively investigated in theast 10 years, and have been revealed to act as endocrine cells,hich express and secrete several endocrine hormones such as

eptin and adiponectin [7]. To present, most in vitro studies ondipocyte differentiation and metabolism have been performedsing the murine preadipocyte cell line, 3T3-L1 and bovinentramuscular preadipocytes, which have enabled researcherso establish a great deal of knowledge on adipogenesis [8].

Obestatin, derived from preproghrelin gene and composed of3 acid amino residues, is purified from stomach extracts andetected in peripheral plasma [9]. Obestatin was originally pro-osed to be the ligand for GPR39 [9], a receptor related to thehrelin receptor subfamily, but this remains controversial. This

ovel peptide has shown to be multifunctional, which is involvedn inhibiting thirst [10], regulating gastrointestinal functions,nfluence food intake and body weight gain [9,11], as well asmproving sleeping [12] and memory [13]. Obestatin also plays

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n important role in the regulation of growth hormone release,ppetite, and energy metabolism [14]. Recently, the findingsave demonstrated that obestatin stimulate the proliferation ofuman retinal pigment epithelial and gastric cancer cells [15,16],revents apoptosis in both rodent �-cells and human pancreaticslets by binding to specific obestatin receptors. In rat H9c2, car-iac cells or isolated ventricular myocytes subjected to I/R, 50mol/l obestatin-(1-23) reduced cardiomyocyte apoptosis andeduced Caspase-3 activation [17]. These suggest that obestatinay also play a role in cell survival [18]. However, obestatin

nd its effects on porcine preadipocytes are not fully elucidated.herefore, to gain further insight into the influence of obestatinn porcine preadipocytes, in the present study, we have investi-ated the effects of obestatin on proliferation, differentiation andpoptosis of porcine preadipocytes. It may provide a base knowl-dge for our understanding of obestatin in regulating lipidosisnd lipometabolism.

. Materials and methods

.1. Cell isolation and purification

Cell isolation and purification was done according to theethods described by Akanbi et al. [19]. Adipocytes were

reshly isolated from samples of subcutaneous adipose tissue ofandrace piglets at 7 days old. Briefly, pieces of adipose tissue ofiglets were incubated for 30 min at 37 ◦C with 0.1% (w/v) colla-enase I containing 10 mM HEPES, 118 mM NaCl, 50 mM KCl,

mM CaCl2, l5 mM D-glucose and 1.5% (w/v) BSA (Gibco,SA) in a sterile 50 mL plastic tube. Following digestion at7 ◦C for 60–70 min with gentle shaking in a water bath, theuspension was filtered through sterile nylon mesh with 100ores to remove undigested tissue and mature adipocytes andentrifugated at 800 × g for 5 min. After washing with DMEM,ed cell lysate (154 mM NH4Cl, 5.7 mM K2HPO4 and 0.1 mMDTA) was added to remove red cells. The isolated and puri-ed cells then were numbered and photographed for followingnalyses. All animals received human care in compliance withhe guide for the care and use of experimental animals (Animalare Committee, 2002).

.2. Cell culture

Porcine preadipocytes were plated at a density of 1 × 105

ells/mL and grown in DMEM supplemented with 10% fetalovine serum (FBS), and 100 U/mL penicillin and 100 mg/mLtreptomysin at 37 ◦C under a humidified atmosphere contain-ng 5% CO2. Fresh medium without FBS was replenished every4 h. In following studies, porcine preadipocytes were treatedith 0, 0.1, 1, 10 and 100 nM porcine obestatin peptide (Phoenix,A, USA) to study cell proliferation, differentiation and apop-

osis.

.3. Preadipocyte identification

Porcine preadipocytes in primary cultures were identi-ed through visualizing the morphology and their functions

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crinologie 75 (2014) 1–9

onversing into adipocytes. At 24 h, cells were rinsed three timesn 0.01 mmol/L PBS and then fixed in trypan blue for 10 min.ubsequently, the fixed cells were rapidly rinsed with PBS for

times and visualized. The cells at indicated times in cultureere also visualized by inverted microscope (Olympus, Japan).he preadipocytes and their conversion into adipocytes wereonfirmed by the detection of lipid droplets under microscopysing oil red O staining which was given a detail presentation inection of oil red O staining.

.4. MTT method

The endogenous effect of obestatin peptide on cellroliferation was evaluated by 3-(4,5-dimethylthiazol-2-yl)-,5-diphenyltetrazolium bromide (MTT) method. Briefly, thereadipocytes isolated and identified above were seeded in 96-ell plates at a density of 1.0 × 105/mL in DMEM containing0% FBS for 24 h, then the medium was changed and the cellsere cultured in a serum-free medium. After incubation for 24 h,

ells were incubated with different exogenous concentrations ofhe peptide (0, 0.1, 1, 10 and 100 nM) in fresh DMEM. At 24 h,2 h and 120 h, cell proliferation was evaluated by adding 15 �l

mg/mL methyl thiazolyl tetrazolium (MTT; Sigma-Aldrich,SA) solution to each well of one cell culture plate, and further

ncubating for 4 h. Then the medium was removed, and 150 �limethyl sulphoxide was added to each well, and the plate wasgitated for 10 min on a shaker to dissolve formazan. The 490 nmbsorbance was measured using a microplate reader (Bio-Rad,ercules, CA, USA).

.5. Oil red O staining

In order to determine the extent of preadipocyte differen-iation, Oil Red O staining were used in this study [20]. Thereadipocytes were seeded in 96-well plates at a density of.0 × 104 cells per well in DMEM containing 10% FBS for4 h, and 0, 0.1, 1, 10 and 100 nM obestatin was added to theerum-free medium once cells were confluent and had begun toifferentiate. At indicated times, the medium was removed, andells were washed three times with phosphate-buffered salinePBS). After washing three times with PBS, cells were stainedor at least 10 min with 1% filtered oil red O (Amresco, USA).he stain was removed, and the cells were washed twice with0% isopropanol. About 1.5 mL of isopropanol was added tohe stained 96-well culture plates for 5 min after washing withBS, and the plate was agitated for 5 min on a shaker to dis-olve formazan. The 550 nm absorbance was measured using aicroplate reader (Bio-Rad, Hercules, CA, USA).

.6. Analysis of Caspase-3 activity

The activity of Caspase-3, a central component of the pro-eolytic cascade during apoptosis, was assessed in cell lysates

sing a Caspase-3 colorimetric kit (Assay Designs, Bologna,taly). Briefly, preadipocytes were scraped from the plates at4 h after obestatin (0, 0.1, 1, 10 and 100 nM) addition, and theedium with the preadipocytes was centrifugated at 1500 rpm,

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◦C for 15 min. Cell lysis buffer was added after removal ofhe supernatant, and the pellet was resuspended and incubatedor 10 min on ice. After centrifugation by 10,000 × g for 1 min,he supernatant (cytosolic extract) was diluted with cell lysisuffer to the protein concentration of 1 �g/�l. The diluted extract50 �l) was mixed with 50 �l of 2 × reaction buffer contain-ng 10 mM DTT, and 5 �l of 4 mM DEVD-pNA substrate wasdded. After incubation at 37 ◦C for 1 h, absorbance at 405 nmas measured.

.7. RT-PCR

Expression of GPR39 and Caspase-3 in preadipocytes wasetermined by RNA preparation and reverse transcription poly-erase chain reaction (RT-PCR). Briefly, when fibroblast-like

readipocytes were 80–90% confluent, the cells were furtherreated with 2 mL obestatin (0, 0.1, 1, 10 and 100 nM) andultured for 24 h. Total cellular RNA was isolated from cells on-well plates by the guanidine thiocyanate/phenol-chloroformxtraction method using TRIZOL reagent following the manu-acturer’s instructions (Invitrogen, Carlsbad, CA, USA). RNAuality was assessed by agarose gel electrophoresis and comple-entary DNA (cDNA) was synthesized with random hexamer

TaKaRa, Osaka, Japan). Real-time PCR were performed usinghe ABI Prism 7500 Sequence Detector (Applied Biosystems,oster City, CA, USA) according to the manufacturer’s pro-

ocol. The PCR reaction was carried out at 1 cycle of 50 ◦Cor 2 min and 95 ◦C for 10 min, followed by 40 cycles of 95 ◦Cor 15 s, 60 ◦C for 15 s and 72 ◦C for 30s. We used 18SrRNAxpression as an internal control. Specific primer sequencesere synthesized in BIOSUNE Biological Technology Corp

Shanghai, China), and the sequences of the primers were asollows: GPR39 (forward: 5’-ACCACGCCCAGCTACACTCT-’; reverse: 5’-GCAAACAGCAAGGGCAGT-3’), Caspase-3forward: 5’-TGGGATTGAGACGGACAGT-3’; reverse:’- AGTAACCAGGTGCTGTAGAAT-3’) and �-actinforward: 5’-TTCCAGCCCTCCTTCCTG-3’; reverse: 5’-GTCCTTGCGGATGTCG-3’).

.8. Western blot analysis

For Western blot analysis, six well dishes of cells treatedith obestatin at concentrations of 0, 1, 10 and 100 nM for4 h were washed in PBS and incubated in RIPA buffer contain-ng a protease inhibitor cocktail on ice for 30 minutes. Proteinsere separated by 10% sodium dodecyl sulfate-polyacrylamideel electrophoresis (SDS-PAGE) and transferred electrophoret-cally to PVDF membranes. To block non-specific binding, theembranes were blocked in 5% non-fat milk for 2 hour at

oom temperature and incubated at 4 ◦C overnight with poly-lonal anti-PPAR� (1:2000 diluted; Affinity Bioreagents, Tokyo,apan), polyclonal anti-C/EBP� (1:500 diluted; Santa Cruz, CA,SA), polyclonal anti-Caspase-7 (1:1000 diluted; Santa Cruz,

A, USA) or anti-Caspase-9 (1:1000 diluted; Santa Cruz, CA,SA). Following overnight incubation, the membranes wereashed three times for 10 minutes with PBS containing 0.5%ween-20 and immunoblotted with HRP-conjugated anti-rabbit

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crinologie 75 (2014) 1–9 3

gG antibody (diluted 1:1000; Amersham Biosciences, Tokyo,apan) at 37 ◦C for 1 h. The membranes were then developedith enhanced chemiluminescence (ECL) substrate (Beyotime,hina) and exposed to X-ray film. �-actin (monoclonal anti-�-ctin, 1/1000, Beyotime, China) was used as a control in thistudy. Band density was quantitated using Image J software.

.9. Statistical analysis

Statistical analysis was carried out with one-way Anova andssessed for statistical difference by one-way analysis of vari-nce (Anova) using SPSS15.0 software. Values are expressed aseans ± SE. The mean values and standard deviations were cal-

ulated from three independent experiments. Differences wereonsidered statistically significant at P < 0.05 and P < 0.01.

. Results

.1. Morphology of cultured preadipocytes

Morphology of cultured preadipocytes was determined at dif-erent times of culture. As shown in Fig. 1a, using trypan bluetaining, we found that porcine preadipocytes adhered well to theeiling surface of flasks after 24 h of culture, and more than 90%f these cells on the ceiling were mature adipocytes with lipidroplets. On 48 h of culture, about 30% cells exhibited cellularorphology of mature adipocytes with lipid droplets (Fig. 1b).fter 72 h of culture, multilocular adipocytes underwent cellivision yielding fibroblast-like fat cells containing only a fewipid droplets (Fig. 1c). Finally after approximately 5 days, theells spread and extended further showing fibroblast-like cellhapes with no visible fat droplets (Fig. 1d). To test whether thebroblast-like cells is preadipocytes, and whether these cells canifferentiate into functional adipocytes, cell differentiation wasurther examined using oil red O staining. The results suggestedhat the fibroblast-like cells (Fig. 1e) could redifferentiate into

ature adipocytes and lipid droplets were visualized after oiled O staining (Fig. 1f) at 7 days of culture.

.2. Obestatin had no influence on expression ofreadipocytes

After treatment with obestatin, mRNA expression of GPR39as determined by RT-PCR. As shown in Fig. 2, we verified

hat, compared to the control, mRNA expression of GPR39 wasot significantly changed at different times in cells treated withifferent levels of obestatin (P > 0.05).

.3. Obestatin promotes proliferation of preadipocytes

The MTT activity in each well was used to determine the pro-iferation of preadipocytes following treatment with obestatin.s illustrated in Fig. 3, preadipocytes receiving obestatin treat-

ent showed higher proliferation responses in comparison to

he control (P < 0.05). The proliferation activity of preadipocytesreated with obestatin was significantly increased at all exam-ned obestatin concentrations in a dose-dependent manner. The

4 S. Tang et al. / Annales d’Endocrinologie 75 (2014) 1–9

Fig. 1. Morphological changes of porcine predipocytes during ceiling culture. Mature adipocytes adhered to the ceiling surface of flasks and cell viability wasvisualized after staining with trypan blue (a) or without trypan blue (b). Mature adipocytes underwent cell division yielding fibroblast-like fat cells containing a fewlipid droplets (c). After approximately 5 days of culture, the cells spread and extended further showing fibroblast-like cell shapes with no visible fat droplets (d).After 7 days, the fibroblast-like cells (e) were re-differentiated to mature adipocytes, lipid droplets in adipocytes were visualized after oil red O staining (f).

Fig. 2. Effects of obestatin on GPR39 expression of porcine pradipocytes in primary culture. After approximately 5 days of culture, the cells were treated withdifferent levels of obestatin (0, 0.1, 1, 10 or 100 nM). After 24, 72 or 120 h incubation, the cells were harvested and total cellular RNAs were extracted. RT-PCRanalysis was performed to measure mRNA expression of GPR39 and �-actin. Data are expressed as mean ± SE of three independent experiments in triplicate.* P < 0.05 ** P < 0.01, or P > 0.05 vs control.

S. Tang et al. / Annales d’Endocrinologie 75 (2014) 1–9 5

Fig. 3. Effects of obestatin on the proliferation of porcine pradipocytes in primary culture by MTT method. The cells were treated with different doses of obestatin(0, 0.1, 1, 10 or 100 nM), and cells were stained with oil red O-eluted solution, and the OD value of the cells was evaluated by a microplate reader with a 550 nmabsorbance after the incubation time, 24, 72 or 120 h. Data are expressed as mean ± SE of three independent experiments in triplicate. * P < 0.05 ** P < 0.01, orP > 0.05 vs control.

Fig. 4. Effect of obestatin on the differentiation of porcine preadipocytes. (A) After approximately 5 days of culture, the cells beginning to differentiate were treatedwith obestain at concentration of 0, 0.1, 1, 10 or 100 nM, and the cell differentiation was evaluated using the oil red staining after 24 h incubation. (B) In the meantime,extracts of circa 20 �g total cells were prepared and subjected to western blot analysis with antibodies specific for C/EBP� and PPAR�, respectively. Anti-�-actinwas served as an internal control. Band density of C/EBP� (C) and PPAR� (D) and PPAR� g2 (E) was quantitated using Image J software. Data are expressed asmean ± SE of three independent experiments in triplicate. * P < 0.05 ** P < 0.01, or P > 0.05 vs control.

6 Endocrinologie 75 (2014) 1–9

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Fig. 5. Changes of Caspase-3 activity and mRNA expression in porcinepreadipocytes. (A) After approximately 5 days of culture, the cells were treatedwith different levels of obestatin (0, 0.1, 1, 10 or 100 nM). After 48 h incubation,the cells were stained with oil red O-eluted solution, and the OD value of thecells was determined by a microplate reader with a 405 nm absorbance. (B) Atthe same time, the cells were harvested and total cellular RNAs were extracted.RT-PCR analysis was performed to measure mRNA expression of Caspase-3and �-actin. Data are expressed as mean ± SE of three independent experimentsin triplicate. * P < 0.05 ** P < 0.01, or P > 0.05 vs control.

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aximal proliferative concentration of obestatin was 100 nM. Itndicated that obestatin had the ability to promote preadipocyteroliferation.

.4. Obestatin enhances differentiation of preadipocytes

To investigate the effects of obestatin on adipocyte differen-iation, porcine preadipocytes beginning to differentiate werereated with different levels of obestatin (0, 0.1, 1, 10 and00 nM) for 24 h. The results in Fig. 4A demonstrated that ODalue was promoted in cells treated with obestatin and the effectas dose-dependent, indicating obestatin could enhance differ-

ntiation of porcine preadipocytes. Additionally, two markerenes of differentiation, C/EBP� and PPAR�, were determinedy western blot (Fig. 4B). The results indicated that 0.1 nMbestatin could increase protein expression of C/EBP�, but theffect was not significant (P > 0.05). Higher levels of obestatinight stimulate expression of C/EBP� significantly (P < 0.01)

Fig. 4 C). Expression of PPAR� including PPAR�1 (Fig. 4D)nd PPAR�2 (Fig. 4E) was also increased in all obestatin-treatedells in a dose-dependent manner. Taken together, obestatinould enhance the differentiation of preadipocytes.

.5. Obestatin inhibits apoptosis of porcine preadipocytes

To characterize the effects of obestatin on adipocyte apopto-is, we examined mRNA expression and activation of Caspase-3.s shown in Fig. 5A, compared to the control, mRNA expressionf Caspase-3 was significantly decreased at 48 h in cells treatedith different levels of obestatin, and 100 NM p obestatin-treated

ells exhibited significantly down-regulation (P < 0.01). Fur-hermore, OD values of the oil red O-eluted solution in the 1,0 and 100 nM obestatin-treated groups decreased significantlyompared with the control (P < 0.05) and the effect was moreignificant in 100 nM obestatin-treated group (Fig. 5B).

Additionally, the Caspase-7 and Caspase-9 activation waslso investigated by western blot, the results were shown inig. 6. During cell apoptosis, Caspase-7 and Caspase-9 pro-eeded through proteolytic cleavage of inactive procaspaseo active forms Caspase-9 (39/37 kDa), or active Caspase-720 kDa). It revealed that there was no detectable changesn active forms Caspase-9 (39/37 kDa), or active Caspase-720 kDa) treated with 0.1 nM obestatin (P > 0.05), while 1, 10nd 100 nM obestatin inhibited expression of these proteinsP < 0.01). It demonstrated that treatment with obestatin showednhibitory effects on protein expression of active Caspase-7 andaspase-9.

The results above suggested that treatment of porcinereadipocytes with obestatin significantly inhibited cell apopto-is in a dose-dependent manner by down-regulating expressionf Caspase-3, Caspase-7 and Caspase-9.

. Discussion

Adipose tissue plays an essential role in energy homeostasiss a metabolic and endocrine organ, and dysfunction of adiposeissue is associated with metabolic syndrome. The number of

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dipocytes present in an organism is determined by the capacityf the adipogenic process composed of the preadipocyte pro-iferation and their differentiation into mature adipocytes. Thedipose tissue mass can be reduced by the inhibition of adi-ogenesis from preadipocytes to mature adipocytes, preventionf lipid accumulation in adipocytes, and induction of apoptosisn adipose cells [21,22]. Therefore, proliferation, differentiationnd apoptosis of preadipocytes play an important role in lipidccumulation. Obestatin is a multifunctional peptide, in additiono affecting weight gain, feeding, and gastrointestinal motility,lso showing the influence on water intake, memory, anxiety, andleep via central actions [14]. According to the results of others,t indicated that obestatin could promote adipogenesis in 3T3-1 cells, being a regulator of adipocyte metabolism [23–25].

n the present study, we found that obestatin had no effect onxpression of GPR39, while it promoted the proliferation andifferentiation of preadipocytes, and reduced the apoptosis ofhese cells, confirming the regulation of obestatin in adipocyte

etabolism.

The orphan G protein-coupled receptor GPR39 was identi-

ed as the ligand of obestatin firstly [9]. However, now, it haseen usually accepted that GPR39 is probably not the obestatin

S. Tang et al. / Annales d’Endo

Fig. 6. Effects of obestatin on Caspase-7 and Caspase-9 protein expression inporcine preadipocytes. At 48 h post-treatment with 0, 0.1, 1, 10 or 100 nMobestatin, extracts of circa 20 �g total cells were prepared and subjected towestern blot analysis with antibodies specific for Caspase-7 and Caspase-9(A), respectively. Anti-�-actin was served as an internal control. Band densityof Caspase-9 (B) and Caspase-7 (C) was quantitated using Image J software.Data are expressed as mean ± SE of three independent experiments in triplicate.*

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P < 0.05 ** P < 0.01, or P > 0.05 vs control.

eceptor [26,27]. GPR39 is expressed in human adiposeissue [28] and primary rat adipocytes [29]. A study in isolateddipocytes suggested that obestatin influenced lipid accumula-ion, glucose uptake and lipolysis via the GPR39 receptor [29].he present study demonstrated that obestatin had no effect onPR39 expression, indicating obestatin affected the prolifera-

ion, differentiation and apoptosis of cultured preadipocytes not

hrough GPR39 receptor.

The previous studies observed that obestatin stimulatedhe proliferation of human gastric cancer cells by PKC, e

crinologie 75 (2014) 1–9 7

hosphoinositide 3-kinase (PI3 K)/Akt, and ERK1/2 activation15]. Additionally, obestatin induced, in a dose-dependent man-er, cell proliferation by MEK/ERK 1/2 phosphorylation [16] inrimary cultures of human retinal epithelial cells (hRPE cells).ell proliferation was also found after treating with different

evels of obestatin in preadipocytes in this study.The differentiation of preadipocytes into adipocytes is a

omplex process involving coordinated changes in morphol-gy, hormone sensitivity, and gene expression, and is regulatedy a balance of endocrine factors [30,31]. During the termi-al phase of differentiation, activation of the transcriptionalascade leads to increased activity, protein, and mRNA levelsor enzymes involved in triacylglycerol synthesis and degra-ation [21]. Recent findings indicate that these events are keyor regulating adipogenesis as they may promote expressionf critical adipogenic transcription factors, including C/EBP�nd/or PPAR� [32] which have been reported to play an impor-ant role during adipocytic differentiation [22,33]. As illustratedy previous reports, preadipocytes 3T3-L1 can be differentiatednto adipocytes by exposure to a mixture of insulin, isobutyl-ethylxanthine (IBM) and dexamethasone [34] and 3T3-L1

ells have been used as the in vitro experimental model to elu-idate the mechanism of adipocytic differentiation as well asuppressive effects of antiadipocytic agents [33]. Our study indi-ated that obestatin could promote the differentiation of porcinereadipocytes through activating C/EBP� and PPAR� expres-ion.

Apoptosis is a fundamental and complex biological processhat enables an organism to kill and remove unwanted cells dur-ng animal development, normal homeostasis and disease [35].f all the proteins implicated in the activation and execution of

poptosis, caspases stand out as being crucial for this process iniverse metazoan organisms [36]. Although there are at least 14aspases in humans, only a subset of these enzymes is detectablyroteolytically activated by various distinct death stimuli in dif-erent cell types [37,38]. In mammals, principally Caspase-3ppear to be activated in a protease cascade that leads to inappro-riate activation or rapid disablement of key structural proteinsnd important signaling, homeostatic and repair enzymes [39].herefore, Caspase-3 is regarded as a crucial mediator of apop-

osis. A study found that, by acting on specific receptors,bestatin-(1-23) activated PI3 K, PKC-�, PKC-�, and ERK1/2ignaling and protected cardiac cells against myocardial injurynd apoptosis induced by I/R. Furthermore, 50 nmol/L obestatin-1-23) reduced cardiomyocyte apoptosis and reduced Caspase-3ctivation [16]. Recently, Granata et al. (2008) demonstrated thatbestatin prevents apoptosis in both rodent �-cells and humanancreatic islets by binding to specific obestatin receptors andctivation of PI3 K/Akt and ERK1/2. This suggests that obestatinay also play a role in cell survival [18]. Our findings also

uggested obestatin could inhibit apoptosis of preadipocytes byeducing activation of Caspase-3, Caspase-7 and Caspase-9.

In conclusion, our results presented here verified that differ-nt concentration of obestatin in culture medium had no effect on

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PR39 expression, while promoted proliferation and differenti-tion mediated by the promotion of PPAR� and C/EBPa mRNAxpression, and inhibited apoptosis of porcine preadipocytesy the reduction of caspase-3, Caspase-7 and Caspase-9. Itndicated that obestatin was involved in the porcine adipocyte

etabolism.

isclosure of interest

The authors declare that they have no conflicts of interestoncerning this article.

cknowledgement

This work was supported by the grant from the Nationalatural Science Foundation of China (No 31372394), the foun-ation for Distinguished Young Talents in Higher Education ofuangdong, China (No. 2009-400) and Hubei Key Laboratoryf Animal Embryo and Molecular Breeding (2012ZD202).

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[2] Iannucci CV, Capoccia D, Calabria M, Leonetti F. Metabolic syndrome andadipose tissue: new clinical aspects and therapeutic targets. Curr Pharm Des2007;13:2148–68.

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