CTRP6 Regulates Porcine Adipocyte Proliferation ... - ACS Publications

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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

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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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ACS Paragon Plus Environment


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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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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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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

CTRP6 Regulates Porcine Adipocyte Proliferation ... - ACS Publications

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