CTRP6 Regulates Porcine Adipocyte Proliferation ... - ACS Publications

May 23, 2017 - ABSTRACT: Intramuscular fat (IMF) and subcutaneous fat (SCF), which are modulated by adipogenesis of intramuscular and subcutaneous ...
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CTRP6 regulates porcine adipocyte proliferation and differentiation by the AdipoR1/MAPK signaling pathway Wenjing Wu, Jin Zhang, Chen Zhao, Yunmei Sun, Pang Wei-jun, and Gongshe Yang J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 23 May 2017 Downloaded from http://pubs.acs.org on May 30, 2017

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Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Journal of Agricultural and Food Chemistry

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CTRP6

regulates

porcine

adipocyte

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AdipoR1/MAPK signaling pathway

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Wenjing Wua,b, Jin Zhangb, Chen Zhaoa, Yunmei Suna, Weijun Panga & Gongshe Yanga*

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a

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Technology, Northwest A&F University, Yangling Shaanxi, 712100, China

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b

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*Correspondence: Gongshe Yang, Ph.D.

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Address: No. 22 Xinong Road, Yangling, Shaanxi Province 712100, China

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Tel: 86-29-87091017

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Fax: 86-29-87092430

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E-mail: [email protected]

proliferation

and

differentiation

by

the

Laboratory of Animal Fat Deposition & Muscle Development, College of Animal Science and

College of Biological and Chemical Engineering, Jiaxing University, Jiaxing Zhejiang, 314000, China

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ABSTRACT

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Intramuscular fat (IMF) and subcutaneous fat (SCF), which are modulated by adipogenesis of

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intramuscular and subcutaneous adipocytes, play key roles in pork quality. C1q/tumor necrosis

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factor-related protein 6 (CTRP6), an adipokine , plays an important role in the differentiation of

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3T3-L1 cells. However, the effect and regulatory mechanisms of CTRP6 on porcine adipogenesis,

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and whether CTRP6 has the same effect on intramuscular and subcutaneous adipocytes are still

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unknown. Here, we found that CTRP6 significantly inhibited both adipocyte proliferation assessed

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by proliferative marker expression, but CTRP6 decreased the proliferation rate of intramuscular

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adipocytes (IM) to a greater extent than subcutaneous adipocytes (SC). Moreover, CTRP6

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promoted the activity of p38 signaling pathway during the proliferation of both cell

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types. Nevertheless, in subcutaneous adipocytes, CTRP6 also influenced the phosphorylation of

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extracellular regulated protein kinases1/2 (p-Erk1/2), but not in intramuscular adipocytes.

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Additionally, during the differentiation of intramuscular and subcutaneous adipocytes CTRP6

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increased adipogenic genes expression and the level of p-p38, while decreased the activity of

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p-Erk1/2. Interestingly, the effect of CTRP6 shRNA or CTRP6 recombinant protein was

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attenuated by U0126 (a special p-Erk inhibitor) or SB203580 (a special p-p38 inhibitor) in

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adipocytes. By target gene prediction and experimental validation, we demonstrated that CTRP6

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may be a target of miR-29a in porcine adipocytes. Moreover, AdipoR1was identified as a receptor

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of CTRP6 in intramuscular adipocytes, but not in subcutaneous adipocytes. On the basis of the

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above findings, we suggest that CTRP6 was the target gene of miR-29a, inhibited intramuscular

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and subcutaneous adipocyte proliferation but promoted differentiation by mitogen-activated

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protein kinase (MAPK) signaling pathway. These findings indicate that CTRP6 played an

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Journal of Agricultural and Food Chemistry

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essentially regulatory role in fat development.

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Keywords: CTRP6; intramuscular adipocyte; subcutaneous adipocyte; MAPK signaling

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pathway; proliferation; differentiation

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INTRODUCTION

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Current knowledge makes it possible to subdivide porcine carcass fat into at least three separate

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and measurable compartments, namely subcutaneous (SCF), intramuscular (IMF) and visceral fat

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(VAF).1 SCF affects not only the lean percentage of carcass, but also the willingness of consumers

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to purchase the meat.2 Intramuscular fat (IMF) on the other hand is an important factor affecting

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meat quality, showing a close correlation with traits such as flavor, juiciness and tenderness.3 To

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improve the quality of pork, it is necessary to increase IMF while reducing other types of fat, such

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as SCF and perirenal fat (PF) Because of differences in localization and tissue environment, the

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development and metabolism of intramuscular adipocytes (IM) are different from subcutaneous

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adipocytes (SC).4, 5 It has been reported that IMF grows more slowly than SCF, and has the lowest

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lipid content compared to SCF and PF.6,7 Moreover, in intramuscular adipocytes, expression of

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genes involved in lipolysis and lipogenesis , such as lipoprotein lipase (LPL) and fatty acid

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synthase (FAS) was lower, whereas genes that participate in cell proliferation, such as insulin-like

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growth factor II (IGF-II) and prohibitin-1, were higher relative to subcutaneous adipocytes.8,

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Similarly, with a primary porcine cell culture system, Zhou et al. (2007) discovered that

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conjugated linoleic acid increases adipogenesis and lipid content in IMF- but not SCF-derived

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adipocytes by differentially regulating adipocyte-specific gene expression.10

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The above reports indicate that the regulation of subcutaneous and intramuscular adipocyte

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adipogenesis is different, and certain genes play an important role in this process. C1qTNF-related

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protein 6 (CTRP6) is an adipokine, including four domains: the C-terminal C1q globular,

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collagen-like, short variable region, and N-terminal signal peptide.11 A recent study revealed that

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CTRP6 levels in serum and fat tissues were enhanced in ob/ob, obese and adiponectin null-mice.12

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As reported, the expression of CTRP6 was downregulated by rosiglitazone in adipose tissue.13

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Further, we previously showed that preventing CTRP6 expression and secretion by siRNA

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knockdown inhibited differentiation of mouse adipocytes.14 However, the role of CTRP6 on

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adipogenesis of porcine adipocytes, the regulatory mechanisms, and whether this differs between

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intramuscular and subcutaneous adipocytes, remain unclear.

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Mitogen-activated protein kinases (MAPK), such as ,extracellular signal-regulated kinase

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(Erk), p38 and c-Jun NH2-terminal kinase (JNK) play pivotal roles in many important cellular

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processes such as cell proliferation and differentiation.15, 16 It has been shown that JNK inhibition

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protects against adipose tissue expansion.17-19 Moreover, p38 inhibitors could inhibit the

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differentiation of 3T3-L1 adipocytes.20,

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stimulation by growth factors correlates with activation of Erk1/2, and that Erk1/2 can activate cell

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cycle regulatory proteins.22 Notably, the adipocyte-specific transcription factor PPARγ can be

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phosphorylated by Erk1/2 to decrease its transcriptional activity and inhibit adipocyte

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differentiation.23 Previously, we have reported that knockdown of CTRP6 could regulate the

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activation of the Erk1/2 signaling pathway to inhibit lipogenesis both in 3T3-L1 adipocytes and

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C2C12 myoblasts.24

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Recent evidence supports the notion that mitogenic

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In this study, we detected the involvement of CTRP6 in intramuscular and subcutaneous

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adipocyte formation, the underlying cellular mechanisms, and whether the target site of CTRP6 is

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species-specific. The results indicate that CTRP6 is a target gene of miR-29a that regulates

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proliferation and differentiation of intramuscular and subcutaneous adipocytes through the

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AdipoR1(Adiponectin Receptor 1)/MAPK pathway. Our findings will give us an insight into the

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role of CTRP6 in porcine intramuscular and subcutaneous fat deposition, which may provide a

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prospective direction and solid foundation for promoting porcine meat quality.

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METHODS

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Experimental animals and reagents

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Crossbred pigs (Duroc×Yorkshire×Landrace, male, normal diet) were purchased from the

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experimental farm of Northwest Agriculture and Forestry University (Yangling, Shaanxi, China).

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The pigs were executed in conformity with Northwest Agriculture and Forestry University Animal

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Care Committee guidelines. Tissues were collected from three 3-day-old pigs (1-2kg) and three

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180-day-old pigs (90-100kg), respectively.

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Cell culture and adipocyte differentiation

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Porcine IM and SC were isolated from longissimus dorsi and neck subcutaneous depots of

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piglings (3-5d, male) under aseptic environments. The isolated adipose tissue and muscle tissue

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were washed 3 times in phosphate buffered saline (PBS). Then the tissues were minced and

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digested with collagenase type I (Invitrogen, Carlsbad, CA, USA) for 1 hour at 37°C, passed

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through 200-mesh sieve. The adipocytes were collected with centrifugation at 1360 x g for 15 min,

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seeded in culture flasks. Cells were cultured to confluence (day 0) in growth medium

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(DMEM/F12), then induced to differentiate using differentiation cocktail (DMEM/F12 added with

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10% FBS, 0.5 mM isobutylmethylxanthine (IBMX), 20 nM insulin, 0.5 mM dexamethasone) for 2

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days. Then the cells were cultured in DMEM/F12 with 10% FBS and 20 nM insulin for another 6

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days.

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When the adipocyte density reached 90 % , the viral suspension of scrambled shRNA or

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pLentiHI-CTRP6 shRNAs, was added for 8 h, then were exchanged for DMEM/F12.

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Flow Cytometry

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Journal of Agricultural and Food Chemistry

Flow cytometry was performed according to a previously published method.25 EdU detection EdU detection was conducted according to a previously published method.25 The cells were

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visualized by a fluorescence microscope (Nikon, Tokyo, Japan).

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CCK-8 detection

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At 48 h after treatment with CTRP6 shRNA or CTRP6 recombinant protein, cell

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proliferation was detected by the CCK-8 kit (Beyotime, Shanghai, China) according to the

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manufacturer’s instructions.

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Oil Red O staining

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The scramble-shRNA, CTRP6-shRNA lentivirus, phosphate buffered saline (PBS) or

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CTRP6-protein treated cells were matured for 8 days, then washed with PBS, fixed with 4%

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paraformaldehyde (PFA) for half an hour at room temperature, and washed again 3 times with

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PBS. After staining with Oil Red O solution for half an hour, the results were visualized on a

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fluorescence microscopy (Nikon, Tokyo, Japan).

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RNA isolation and Quantitative real-time PCR

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Total RNA was extracted by Trizol (Invitrogen, Carlsbad, CA, USA), then reverse transcribed

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into cDNA by the use of the PrimeScriptTM RT reagent Kit (Takara, Kusatsu, Japan). Real-time

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PCR was carried out by the use of SYBR Green master mix and specific primers (Table S1) on a

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BioRad iQ5 system (Bio-Rad, Hercules, California, USA). The relative mRNA abundance of each

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gene was analyzed by 2-ΔΔCT method. .

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ELISA detection of CTRP6 level

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CTRP6 ELISA kits were purchased from Nan Jing Jian Cheng Bioengineering Institute of

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China. Secreted CTRP6 protein levels were detected according to the manufacturer’s

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recommendations. Firstly, cell culture media or standards were added into 96 well antibody-coated

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plate, incubated at 37°C for 30 min, washed 5 times, then incubated with enzyme conjugate

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solution at 37°C for 30 min. Cells were washed 5 times, and incubated with 50 µl Chromogenic

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agent A and Chromogenic agent B in the dark for 15 min at 37°C. 50 μl stop solution was then

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added, and OD values were tested at 450 nm by using a Microplate Reader (Perkinelmer,

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Massachusetts, USA). CTRP6 levels were quantified by standard curve.

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Immunofluorescence

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Porcine adipocytes were fixed with 4% PFA for 1 hour at 37°C, permeabilized with 0.1%

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Triton X-100, blocked with 1% bovine serum albumin (BSA), and incubated overnight with

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CTRP6 antibody (CTRP6 antibody:block buffer was 1:40). Then the adipocytes were washed 5

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times with PBS, incubated for 1 hour with fluorescent secondary antibodies, and then incubated

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for 5 min with DAPI (5g/ml). The adipocytes were washed again with PBS 3 times. Images were

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obtained using laser scanning confocal microscopy (Nikon, Tokyo, Japan).

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Western blot analysis

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Western blot was performed according to a previously published method. Briefly, the total

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protein was iosolated from target tissues with RIPA buffer (Beyotime, Shanghai, China) with

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protease inhibitor (Pierce, Bradenton, Florida, USA) . After centrifugation, the supernatant was

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boiled in loading buffer (Beyotime, Shanghai, China). 12% SDS polyacrylamide gel

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electrophoresis was used to separate proteins, the bands were transferred onto the polyvinylidene

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difluoride

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chemiluminescence reagents (Millipore, Massachusetts, USA).

membrane

(CST,

Danvers,

Massachusetts,

USA)

and

visualized

with

Image-J Software was used for

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analysis and quantification.

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Statistical analysis.

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The experimental data were got from at least three independent experiments and expressed as

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mean ± SEM. Individual comparisons were assessed by Student’s two-tailed t-test. P-values