Screening Of Mitochondrial-related Signaling Molecules That Improve Skeletal Muscle Insulin Resistance Through Exercise Based On Phosphorylation Proteomic

Objective: In this research,proteomics and phosphoproteomics were used to screen differentially expressed proteins between endurance exercise mice and normal mice,and biological information analysis was used to predict the signal pathways that these screened proteins and phosphorylation changes might participate in regulation,and follow-up verification experiments were carried out around mitochondrial quality control in the insulin resistance model.Methods: In the first part,8-week-old C57BL/6 mice were randomly divided into control group(C,n=10)and endurance exercise group(E,n=10).Mice in the control group were naturally reared for 12 weeks,while those in the endurance exercise group were given 12 weeks of moderate-intensity treadmill exercise,five days a week,and samples were taken 24 hours after the last exercise.Protein was extracted from gastrocnemius muscle samples of two groups of mice,and then it was identified by protein omics and phosphorylated proteomics.The differentially expressed proteins and phosphorylated proteins were classified and enriched by bioinformatics analysis,and the differentially expressed proteins and their related mitochondrial steady-state pathway proteins were screened.In the second part,C57BL/6 mice aged 8 weeks were randomly divided into control group(C,n=8),exercise insulin resistance group(E,n=8)and insulin resistance group(IR,n=8).Mice in the control group were fed naturally for 12 weeks,while mice in the exercise insulin resistance group and insulin resistance group were induced by high-fat diet.The success of insulin resistance model was judged by blood sugar and insulin determination experiments.Exercise insulin resistance group used12 weeks of moderate intensity treadmill exercise,5 days a week,and took samples24 hours after the last exercise.Western-blot experiment was used to detect the changes of protein expression levels of related signal pathways,including Rheb,PINK1,AMPKα,p-AMPKα(T172),AKT,p-Akt(S473)and COX Ⅳ.The related data were processed by Graph Pad Prism 5.0,and expressed as mean plus or minus standard deviation.Two-factor analysis of variance was used in the comparison of multiple groups,and independent sample t test was used in the comparison of pairwise groups,with p < 0.05 indicating significant difference;p<0.01 indicates a highly significant difference.Results:(1)According to the criteria of P < 0.05 and variation multiple greater than 1.2 or less than 0.83,compared with the control group,38 differential proteins were screened out in the endurance exercise group,among which Rheb,as one of the differential proteins,increased significantly.The results of bioinformatics analysis show that Rheb,as a signal transduction factor,is related to mitochondrial autophagy,mitochondrial biosynthesis and cell metabolism-related proteins AMPK and Akt.(2)Compared with the control group,the expression of Rheb in insulin resistance group has no significant change;Compared with the insulin resistance group,the expression of Rheb in the insulin resistance+exercise group increased significantly(p<0.05).Compared with the control group,the expression levels of p-AMPKα(T172)and p-Akt(S473)in insulin resistance group were significantly decreased(p<0.05).Compared with the insulin resistance group,the expression levels of p-AMPKα(T172)and p-Akt(S473)in the insulin resistance+exercise group were significantly higher(p<0.01).There was no significant difference in the expression levels of AMPKα and Akt among the groups.Compared with the control group,there was no significant change in insulin resistance group;Compared with the insulin resistance group,the expression level of PINK1 in the insulin resistance+exercise group was significantly higher(p<0.01).Compared with the control group,the expression level of COX Ⅳ in insulin resistance group decreased significantly(p< 0.05).Compared with the insulin resistance group,the expression level of COX Ⅳ in the insulin resistance+exercise group increased significantly(p<0.01).Conclusions:(1)Through the joint analysis of proteomics and phosphoproteomics,Rheb was screened and determined as one of the key proteins for endurance training to improve the mitochondrial function of skeletal muscle.Bioinformatics analysis suggests that Rheb is closely related to Akt signaling pathway,AMPK signaling pathway and mitochondrial biosynthesis function of skeletal muscle.(2)Exercise may induce Rheb to pass through Akt/AMPK pathway,on the one hand,activate mitochondrial autophagy mediated by PINK1,on the other hand,promote mitochondrial biosynthesis,thus improving the mitochondrial homeostasis of insulin-resistant skeletal muscle.