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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