AMPK调控绵羊肌内前体脂肪细胞分化的研究

AMPK Regulates Sheep Muscle Derived Preadipocytes Differentiation

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。

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/EBPαand 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 PPARγand C/EBPα mRNA at significant level of 0.01,and downregulated aP2 gene expression at significant level of 0.05,moreover,the corresponding proteincontents 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 butβ-catenin protein content significantly altered（P〈0.01）.Finally,AMPK activation increased GSK3βphosphorylation at significant level of 0.01.The result indicated that AMPK regulated sheep muscle derived preadipocytes differentiation through altering GSK3βphosphorylation and affecting the stability ofβ-catenin.