AMPK通过Wnt/β-catenin信号通路调控绵羊肌内脂肪前体细胞分化的研究

Muscle-derived preadipocytes differentiation is regulated by multiple transcriptional factors and signaling pathways. AMPK is recognized as energy sensor in cells and more and more evidences suggest its roles in cell proliferation and differentiation. Our previous studies showed that AMPK cross-talked with Wnt/β-catenin signaling pathway to regulate mesenchymal stem cells (MSCs) differentiation in mice. We also observed that the AMPK activity was negatively correlated with IMF in beef cattle. Based on our data, we hypothesize that AMPK regulates preadipocytes differentiation in sheep skeletal muscle. On this study, we will isolate the muscle-derived preadipocytes from the perimysium of LD muscle, and single clone will be selected and induced adipogenesis in adipogenic medium. Those cells possessed adipogenic potential will be used in our study. We will firstly determine the role of AMPK in muscle-derived preadipocytes differentiation, and then explore whether Wnt/β-catenin signaling pathway is involved in this process. In addition, we will measure the IMF contents, AMPK activity and Wnt/β-catenin signaling pathway in different skeletal muscle developmental stages, and analyze their correlation, to further confirm the role of AMPK in sheep muscle-derived pre-adipocyte differentiation. Our study will provide basic data to sheep breeding that aims at improvement of meat quality.

肌内脂肪前体细胞分化受多种信号分子及其通路调控。AMPK是细胞能量代谢的开关；最新研究表明AMPK在调控细胞增殖与分化方面发挥了重要作用。申请者研究结果证明AMPK通过Wnt/β-catenin信号通路抑制小鼠间充质干细胞的成脂分化，并发现AMPK活性与肉牛肌内脂肪含量呈负相关。基于前期研究结果，我们假设AMPK参与绵羊肌内脂肪前体细胞分化。本项目拟从羔羊背最长肌肌束膜中分离脂肪前体细胞，经单克隆筛选后建立细胞系。选取成脂能力明显差异的两组细胞，应用分子生物学、细胞生物学等技术手段，研究AMPK活性与绵羊肌内脂肪前体细胞分化能力的关系，并证明其调控作用是否通过Wnt/β-catenin信号通路。实验拟比较绵羊骨骼肌不同发育期IMF含量、AMPK活性及Wnt/β-catenin信号，并做关联分析，阐明骨骼肌中三者的关系。本研究将为绵羊肉质遗传改良工作提供科学依据。

肌内脂肪（IMF）是决定羊肉品质的关键因素之一，其含量受脂肪细胞数量及脂肪合成能力的影响。增加肌内脂肪前体细胞的成脂潜能将有利于肌内脂肪的沉积。AMPK（AMP-activated protein Kinase）是细胞调节能量代谢最重要的酶之一，参与了糖、脂肪及蛋白质的代谢过程。本项目旨在研究AMPK在羔羊肌内脂肪前体细胞分化中的作用及机制。取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，并成功进行了成脂诱导分化。改变了细胞中AMPK活性，证明了AMPK激活显著减少了油红的着色，抑制了PPARγ、C/EBPα和aP2等关键基因的表达，而抑制AMPK活性则表现出相反的结果。通过改变Wnt信号通路活性，证明了在绵羊肌内脂肪前体细胞中，Wnt通路激活显著抑制了肌内脂肪前体细胞的分化，并下调了PPARγ、C/EBPα和aP2等基因的表达，而抑制Wnt信号通路活性则促进了细胞的成脂分化。进一步研究表明，AMPK的活性变化未影响β-catenin的转录，但显著改变了β-catenin的蛋白含量，并调控了其转录活性。项目证明了HDAC5通过H3K9影响羔羊肌内脂肪前体细胞的分化，但AMPK并未影响其在细胞中的定位。试验证明了AMPK活性改变后影响了Akt及GSK3β的活性，进而提高了β-catenin在细胞内的稳定性。项目测定了不同生长时期的绵羊（初生，1月龄，2月龄，4月龄和6月龄）背最长肌中IMF含量及AMPK活性，发现了AMPK活性与LD中IMF含量呈负相关。此外，项目还探索了在肉羊饲料中添加了一定浓度的酿酒葡萄皮渣改变AMPK活性，以实现对肉品质及肌内脂肪含量等的影响的可能。本研究建立的AMPK与肌内脂肪前体细胞分化的关系可为旨在提高IMF含量的绵羊育种工作提供理论依据。同时，如能在肌内脂肪细胞形成的时间窗口有效的改变羔羊体内AMPK活性，将为实现IMF含量的提高及肉品质的提升提供新的途径。