微重力通过m6A修饰影响骨骼肌糖原代谢初探

Effects of microgravity on skeletal muscle glucogen metabolism through m6A modification

长期航天飞行会导致骨骼肌萎缩,糖代谢紊乱可能是重要原因之一。N6-甲基腺嘌呤(N6-methyladenosine,m6A)是真核生物最常见和最保守的信使核糖核酸(messenger RNA,mRNA)修饰,通常参与关键基因表达的调控。本研究主要探究微重力对肌细胞糖原代谢的影响及微重力引起的m6A修饰改变与糖原代谢相关酶表达改变的相关性。通过蒽酮法检测骨骼肌细胞糖原含量,通过流式法检测荧光-D-葡萄糖类似物2-(N-7-硝基-2,1,3-苯并恶二唑-4-氨基)-2-脱氧-D-葡萄糖[2-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino-2-deoxyglucose,2-NBDG]标记骨骼肌细胞的葡萄糖摄取,利用点杂交检测信使RNA m6A修饰的变化情况以及通过qPCR、Western blot检测糖原代谢相关酶和m6A修饰相关酶的表达,通过RNA甲基化免疫共沉淀(methylated RNA Immunoprecipitation,meRIP)检测肌细胞糖原代谢相关酶m6A修饰水平,同时通过GEO数据库分析临床及动物实验中糖原代谢相关酶和m6A修饰相关酶的关联。结果显示,模拟微重力条件下,小鼠成肌细胞C2C12糖原含量降低,葡萄糖摄取能力下降,糖原代谢相关酶糖原合成酶激酶3A(glycogen synthase kinase-3a,Gsk3A)、肌糖原磷酸化酶(glycogen phosphorylase-muscle,Pygm)、脑糖原磷酸化酶(glycogen phosphorylase-brain,Pygb)及磷酸葡萄糖变位酶3(phosphoglucomutase 3,Pgm 3)表达发生变化,m 6 A修饰相关酶甲基转移酶样蛋白3(methyltransferase-like protein 3,Mettl3)表达下调,并且Pgm3和Gsk3a的mRNA发生了m6A修饰改变。本研究结果提示,微重力可能通过m6A修饰相关酶改变骨骼肌糖原代谢相关酶的m6A修饰水平,进而引起骨骼肌糖原含量的改变,影响骨骼肌细胞代谢稳态。本研究在分子水平上初步提示了微重力条件下肌细胞能量代谢紊乱、肌肉萎缩、功能减退的成因,为微重力环境下提高骨骼肌细胞供能水平、减缓肌肉萎缩、提高肌群效能提供了新的思路。

Long-term space flight will lead to skeletal muscle atrophy,and the disorder of glucose metabolism may be one of the important reasons.N6-methyladenosine(m6A)is the most common and conserved messenger RNA modification in eukaryotes and is often involved in the regulation of key gene expression.This study aimed to explore the effect of microgravity on glycogen metabolism in muscle cells and the correlation between m6A modification induced by microgravity and glycogen metabolity-related enzyme expression.The content of glycogen in skeletal muscle cells was detected by anthrone method.The glucose uptake of skeletal muscle cells labeled by fluorescent D-glucose analogs[2-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino-2-deoxyglucose,2-NBDG]was detected by flow cytometry.The changes in m6A modification of messenger RNA were detected by Dot Blot,and expressions of glycogen metabolism related enzymes and m6A modification related enzymes were detected by qPCR and Western blot.The level of m6A modification of glycogen metabolism related enzymes in muscle cells was detected by methylated RNA immunoprecipitation(meRIP).The correlation between glycogen metabolism related enzymes and m6A modification related enzymes in clinical and animal experiments was analyzed using GEO database.The results showed that under simulated microgravity conditions,the glycogen content of mouse myoblasts C2C12 decreased,the glucose uptake capacity decreased,and the expression of glycogen metabolism related enzyme:glycogen synthase kinase-3a(Gsk3a),glycogen phosphorylase-muscle(Pygm),glycogen phosphorylase-brain(Pygb)and phosphoglucomutase 3(Pgm3)were changed.The expression of m6A modified related enzyme methyltransferase-like protein 3(Mettl3)is down-regulated,and the mRNA of Pgm3 and Gsk3a was modified by m6A.The results of this study suggest that microgravity may change the m6A modification of glycogen metabolism related enzymes through m6A modification related enzyme expression change,thus cause glycogen content changes in skeletal muscle and affect skeletal muscle cell metabolic homeostasis.This study preliminarily suggests the causes of energy metabolism disorder,muscle atrophy and functional decline of muscle cells under microgravity condition at molecular level,and provided a new idea for improving energy supply level of skeletal muscle cells,slowing muscle atrophy and improving muscle group efficiency under microgravity environment.