AMPK调控绵羊肌内前体脂肪细胞分化的研究

doi:10.11843/j.issn.0366-6964.2018.08.004

摘要/Abstract

摘要：

旨在研究AMPK在羔羊肌内前体脂肪细胞分化中的作用及机制。本研究采集3日龄羔羊背最长肌，采用差速贴壁法分离肌束膜中原代细胞。改变AMPK与Wnt信号通路的活性并诱导细胞成脂分化，采用qRT-PCR和Western blot方法检测脂肪细胞分化关键基因及蛋白的表达，应用油红O染色观察成熟脂肪细胞的油滴情况。结果表明，AMPK激活明显减少了油红的着色，极显著抑制了PPARγ、C/EBPα和aP2成脂关键基因的mRNA表达及降低了对应的蛋白含量（P<0.01），而抑制AMPK活性则表现出相反的结果。激活Wnt信号通路活性抑制了肌内前体脂肪细胞的分化，极显著降低PPARγ（P<0.01）和C/EBPα（P<0.01）mRNA表达，显著减少aP2的mRNA表达（P<0.05），极显著降低了对应蛋白的含量（P<0.01），而抑制Wnt信号通路活性则促进了细胞的成脂分化。AMPK的活性变化未影响β-catenin的转录，但极显著改变了β-catenin的蛋白表达水平（P<0.01）。进一步研究表明，AMPK活化后极显著增加了GSK3β的磷酸化水平（P<0.01），降低了其活性。结果显示，AMPK通过改变GSK3β的磷酸化水平影响胞内β-catenin稳定性，进而影响羔羊肌内前体脂肪细胞的成脂分化。

Abstract:

The objective of this study was to investigate the roles and mechanisms of AMP-activated protein kinase(AMPK) in sheep muscle derived preadipocytes differentiation. The longissimus dorsi(LD) muscle was sampled from lamb of 3 days old, and the perimysium was isolated and digested for preadipocytes isolation by differential adhesion method. The activity of AMPK and Wnt/β-catenin signaling pathways were altered, and then the cells adipogenic differentiation was induced. qRT-PCR and Western blot were used to detect the expression patterns of key genes and proteins in preadipocytes differentiation. Oil-Red-O staining was used to observe the lipid droplet accumulation in mature adipocytes. The results showed that activating AMPK was associated with less Oil-Red-O staining. Meanwhile, the expressions of adipogenic genes and corresponding proteins including PPARγ, C/EBPα and aP2 were down-regulated at significant level of 0.01 when AMPK was activated, while inhibition of AMPK activity showed opposite effects. The activation of Wnt/β-catenin signaling pathway inhibited preadipocytes adipogenesis, decreased the expressions of PPARγ and C/EBPα mRNA at significant level of 0.01, and down-regulated aP2 gene expression at significant level of 0.05, moreover, the corresponding protein contents were also decreased at significant level of 0.01. Furthermore, inhibition of Wnt/β-catenin signaling pathway promoted adipogenesis. β-catenin transcription was not affected by alteration of AMPK activity but β-catenin protein content significantly altered(P<0.01). Finally, AMPK activation increased GSK3β phosphorylation at significant level of 0.01. The result indicated that AMPK regulated sheep muscle derived preadipocytes differentiation through altering GSK3β phosphorylation and affecting the stability of β-catenin.

中图分类号:

S826.2

李戡, 刘文忠, 张瑞鑫, 李倩, 张婷, 秦旭泽, 张建新, 赵俊星. AMPK调控绵羊肌内前体脂肪细胞分化的研究[J]. 畜牧兽医学报, 2018, 49(8): 1594-1604.

LI Kan, LIU Wen-zhong, ZHANG Rui-xin, LI Qian, ZHANG Ting, QIN Xu-ze, ZHANG Jian-xin, ZHAO Jun-xing. AMPK Regulates Sheep Muscle Derived Preadipocytes Differentiation[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(8): 1594-1604.

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https://www.xmsyxb.com/CN/Y2018/V49/I8/1594

参考文献

[1] WOOD J D,ENSER M,FISHER A V,et al.Fat deposition,fatty acid composition and meat quality:A review[J].Meat Sci,2008,78(4):343-358.
[2] 郑程莉,徐刚毅,汪代华.羊肉肌内脂肪及影响因素的研究进展[C]//中国畜牧兽医学会养羊学分会全国养羊生产与学术研讨会.银川:中国畜牧兽医学会养羊学分会,2010:71-73.
ZHENG C L,XU G Y,WANG D H.Research progress on intramuscular fat and influencing factors of mutton[C]//Proceedings of the National Sheep Production and Academic Conference.Yinchuan:Animal husbandry and Veterinary Association of China,2010:71-73.(in Chinese)
[3] WATKINS P J,FRANK D,SINGH T K,et al.Sheepmeat flavor and the effect of different feeding systems:A review[J].J Agric Food Chem,2013,61(15):3561-3579.
[4] 刘建华,乔利英,郭云雁,等.绵羊UCP2基因的克隆及其在脂肪组织中的季节性差异表达[J].畜牧兽医学报,2015,46(7):1114-1121.
LIU J H,QIAO L Y,GUO Y Y,et al.Cloning of ovine UCP2 gene and its seasonally differential expression in adipose tissues[J].Acta Veterinaria et Zootechnica Sinica,2015,46(7):1114-1121. (in Chinese)
[5] 师涛,闫晓茹,潘洋洋,等.miR-132-3p靶向UCP2调节绵羊前体脂肪细胞分化的研究[J].畜牧兽医学报,2017,48(11):2059-2067.
SHI T,YAN X R,PAN Y Y,et al.Study on the regulation of miR-132-3p in ovine preadipocyte differentiation by targeting UCP2[J].Acta Veterinaria et Zootechnica Sinica,2017,48(11):2059-2067. (in Chinese)
[6] 杜琛,李金泉,陈秀娟.白绒山羊PPARγ基因RNA干扰慢病毒载体的构建及对肌内脂肪细胞增殖分化的影响[J].畜牧兽医学报,2016,47(4):671-678.
DU C,LI J Q,CHEN X J.Construction of lentiviral RNAi vector ofPPARγ gene in cashmere goat and its effect on proliferation and differentiation of intramuscular adipocytes[J].Acta Veterinaria et Zootechnica Sinica,2016,47(4):671-678. (in Chinese)
[7] 杜琛,付绍印,高鸿雁,等.绒山羊肌内脂肪细胞成熟前后比较转录组分析[J].畜牧兽医学报,2014,45(5):714-721.
DU C,FU S Y,GAO H Y,et al.Transcriptome analysis of intramuscular preadipocytes and matureadipocyte in cashmere goats[J].Acta Veterinaria et Zootechnica Sinica,2014,45(5):714-721. (in Chinese)
[8] 郑竹清,杜琛,付绍印,等.山羊肌内脂肪细胞lncRNA鉴别及特征分析[J].畜牧兽医学报,2014,45(12):1924-1931.
ZHENG Z Q,DU C,FU S Y,et al.Identification and characterization analysis of long Non-coding RNA from RNA-seq data of intramuscular adipocytes in goats[J].Acta Veterinaria et Zootechnica Sinica,2014,45(12):1924-1931.(in Chinese)
[9] 栾兆进,刘开东,贺建宁,等.绵羊FAM134B、PPARγ、HSL和FAS基因表达量及与肌内脂肪含量的关系[J].畜牧兽医学报,2016,47(12):2379-2389.
LUAN Z J,LIU K D,HE J N,et al.The FAM134B,PPARγ,HSL and FAS gene expression patterns and their association with intramuscular fat content in sheep[J].Acta Veterinaria et Zootechnica Sinica,2016,47(12):2379-2389. (in Chinese)
[10] ZHANG W Y,ZHANG X L,WANG H,et al.AMP-activated protein kinase α1 protects against diet-induced insulin resistance and obesity[J].Diabetes,2013,61(12):3114-3125.
[11] VILLENA J A,VIOLLET B,ANDREELLI F,et al.Induced adiposity and adipocyte hypertrophy in mice lacking the AMP-activated protein kinase-α2 subunit[J].Diabetes,2004,53(9):2242-2249.
[12] HABINOWSKI S A,WITTERS L A.The effects of AICAR on adipocyte differentiation of 3T3-L1 cells[J].Biochem Biophys Res Commun,2001,286(5):852-856.
[13] BIJLAND S,MANCINI S J,SALT I P.Role of AMP-activated protein kinase in adipose tissue metabolism and inflammation[J].Clin Sci,2013,124(8):491-507.
[14] ZHAN T,RINDTORFF N,BOUTROS M.Wnt signaling in cancer[J].Oncogene,2017,36(11):1461-1473.
[15] OKAMURA M,KUDO H,WAKABAYASHI K I,et al.COUP-TFⅡ acts downstream of Wnt/β-catenin signal to silence PPARγ gene expression and repress adipogenesis[J].Proc Natl Acad Sci U S A,2009,106(14):5819-5824.
[16] XU Z,YU S T,HSU C H,et al.The orphan nuclear receptor chicken ovalbumin upstream promoter-transcription factor Ⅱ is a critical regulator of adipogenesis[J].Proc Natl Acad Sci U S A,2008, 105(7):2421-2426.
[17] ZHAO J X,LI Q,ZHANG R X,et al.Effect of dietary grape pomace on growth performance,meat quality and antioxidant activity in ram lambs[J].Anim Feed Sci Technol,2018,236:76-85.
[18] PARK A,KIM W K,BAE K H.Distinction of white,beige and brown adipocytes derived from mesenchymal stem cells[J].World J Stem Cells,2014,6(1):33-42.
[19] DESAI M,BEALL M,ROSS M G.Developmental origins of obesity:Programmed adipogenesis[J].Curr Diab Rep,2013,13(1):27-33.
[20] TANG Q Q,LANE M D.Adipogenesis:from stem cell to adipocyte[J].Annu Rev Biochem,2012,81:715-736.
[21] HAUSMAN G J,BASU U,DU M,et al.Intermuscular and intramuscular adipose tissues:Bad vs. good adipose tissues[J].Adipocyte,2014,3(4):242-255.
[22] GENTILE P,ORLANDI A,SCIOLI M G,et al.Concise review:Adipose-derived stromal vascular fraction cells and platelet-rich plasma:Basic and clinical implications for tissue engineering therapies in regenerative surgery[J].Stem Cells Trans Med,2012,1(3):230-236.
[23] HAUSMAN G J,POULOS S P,PRINGLE T D,et al.The influence of thiazolidinediones on adipogenesis in vitro and in vivo:Potential modifiers of intramuscular adipose tissue deposition in meat animals[J].J Anim Sci,2008,86(S14):E236-E243.
[24] NISHIMURA T.The role of intramuscular connective tissue in meat texture[J].Anim Sci J,2010,81(1):21-27.
[25] MIHAYLOVA M M,SHAW R J.The AMPK signalling pathway coordinates cell growth,autophagy and metabolism[J].Nat Cell Biol,2011,13(9):1016-1023.
[26] CARLING D,THORNTON C,WOODS A,et al.AMP-activated protein kinase:new regulation,new roles?[J].Biochem J,2012,445(1):11-27.
[27] JEON S M,CHANDEL N S,HAY N.AMPK regulates NADPH homeostasis to promote tumour cell survival during energy stress[J].Nature,2012,485(7400):661-665.
[28] NAKANO A,TAKASHIMA S.LKB1 and AMP-activated protein kinase:Regulators of cell polarity[J].Genes Cells,2012,17(9):737-747.
[29] YANG Y Y,YANG X D,XU B,et al.Chemical constituents of Morus alba L. and their inhibitory effect on 3T3-L1 preadipocyte proliferation and differentiation[J].Fitoterapia,2014,98:222-227.
[30] FIGAROLA J L,RAHBAR S.Small-molecule COH-SR4 inhibits adipocyte differentiation via AMPK activation[J].Int J Mol Med,2013,31(5):1166-1176.
[31] ZHANG W C,WANG Q L,SONG P,et al.Liver kinase B1 is required for white adipose tissue growth and differentiation[J].Diabetes,2013,62(7):2347-2358.
[32] LI H X,LUO X,LIU R X,et al.Roles of Wnt/β-catenin signaling in adipogenic differentiation potential of adipose-derived mesenchymal stem cells[J].Mol Cell Endocrinol,2008,291(1-2):116-124.
[33] 苏尚,吴畏.Wnt/β-catenin信号通路对靶基因转录的调控[J].中国科学:生命科学,2014, 44(10):1029-1042.
SU S,WU W.Regulation of target gene transcription by Wnt/β-catenin signaling[J].Scientia Sinica Vitae,2014,44(10):1029-1042.(in Chinese)
[34] HORIKE N,SAKODA H,KUSHIYAMA A,et al.AMP-activated protein kinase activation increases phosphorylation of glycogen synthase kinase 3β and thereby reduces cAMP-responsive element transcriptional activity and phosphoenolpyruvate carboxykinase C gene expression in the liver[J].J Biol Chem,2008,283(49):33902-33910.
[35] LANZI C R,PERDICARO D,BOTTINI R,et al.Grape pomace extract,rich in polyphenols,stimulate the emergence of brown-like cells in white adipose tissue in spontaneously hypertensive rats and in 3T3-L1 adipocytes[J]. Free Radic Biol Med,2017,108(S1):S83.