CTRP6 Regulates Porcine Adipocyte Proliferation and Differentiation

Subscriber access provided by UNIV OF ARIZONA

Article

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

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

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.

Page 1 of 35

Journal of Agricultural and Food Chemistry

1

CTRP6

regulates

porcine

adipocyte

2

AdipoR1/MAPK signaling pathway

3

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

4

a

5

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

6

b

7

*Correspondence: Gongshe Yang, Ph.D.

8

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

9

Tel: 86-29-87091017

10

Fax: 86-29-87092430

11

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

1

ACS Paragon Plus Environment


12

ABSTRACT

13

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

14

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

15

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

16

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

17

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

18

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

19

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

20

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

21

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

22

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

23

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

24

Additionally, during the differentiation of intramuscular and subcutaneous adipocytes CTRP6

25

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

26

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

27

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

28

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

29

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

30

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

31

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

32

and subcutaneous adipocyte proliferation but promoted differentiation by mitogen-activated

33

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

2


Page 2 of 35

Page 3 of 35


34

essentially regulatory role in fat development.

35

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

36

pathway; proliferation; differentiation

37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55

3



56

Page 4 of 35

INTRODUCTION

57

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

58

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

59

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

60

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

61

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

62

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

63

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

64

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

65

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

66

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

67

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

68

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

69

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

70

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

71

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

72

adipocytes by differentially regulating adipocyte-specific gene expression.10

9

73

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

74

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

75

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

76

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

77

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

4


Page 5 of 35


78

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

79

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

80

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

81

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

82

intramuscular and subcutaneous adipocytes, remain unclear.

83

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

84

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

85

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

86

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

87

differentiation of 3T3-L1 adipocytes.20,

88

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

89

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

90

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

91

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

92

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

93

C2C12 myoblasts.24

21

Recent evidence supports the notion that mitogenic

94

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

95

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

96

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

97

proliferation and differentiation of intramuscular and subcutaneous adipocytes through the

98

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

99

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

5



100

prospective direction and solid foundation for promoting porcine meat quality.

101

METHODS

102

Experimental animals and reagents

103

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

104

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

105

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

106

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

107

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

108

Cell culture and adipocyte differentiation

109

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

110

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

111

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

112

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

113

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

114

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

115

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

116

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

117

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

118

days.

119

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

120

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

121

Flow Cytometry

6


Page 6 of 35

Page 7 of 35

122 123 124


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

125

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

126

CCK-8 detection

127

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

128

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

129

manufacturer’s instructions.

130

Oil Red O staining

131

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

132

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

133

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

134

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

135

fluorescence microscopy (Nikon, Tokyo, Japan).

136

RNA isolation and Quantitative real-time PCR

137

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

138

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

139

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

140

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

141

gene was analyzed by 2-ΔΔCT method. .

142

ELISA detection of CTRP6 level

143

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

7



Page 8 of 35

144

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

145

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

146

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

147

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

148

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

149

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

150

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

151

Immunofluorescence

152

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

153

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

154

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

155

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

156

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

157

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

158

Western blot analysis

159

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

160

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

161

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

162

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

163

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

164

difluoride

165

chemiluminescence reagents (Millipore, Massachusetts, USA).

membrane

(CST,

Danvers,

Massachusetts,

USA)

and

visualized

with

Image-J Software was used for

8


Page 9 of 35


166

analysis and quantification.

167

Statistical analysis.

168

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

169

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

CTRP6 Regulates Porcine Adipocyte Proliferation and Differentiation

Recommend Documents