The Role Of MG53 In The Effects Of Treadmill Exercise On Insulin Resistance In The Skeletal Muscle Of Diabetic Db/db Mice

China is facing increasing challenges in dealing with the alarming rise of health concerns and chronic diseases such as diabetes mellitus and cardiovascular diseases,which correlate with the rapid lifestyle changes.Epidemiological studies have shown that nearly a third of the world's diabetic adults are from China and one in eleven adults has diabetes worldwide,posing a severe threat to the health of human population.Without drug treatment or non-drug intervention,the short-term diabetes can cause acute complications including diabetic ketoacidosis,diabetic hyperosmolar state etc.,while severe complications of the long-term diabetes such as cardiovascular diseases,chronic kidney diseases and stroke can be induced.Due to the fact that the pathogenesis of diabetes is complicated,which is associated with multiple factors,there has been no effective therapeutic method to cure diabetes so far.Exercise has started to gain attention as a promising therapeutic intervention against human diseases.Basic researches utilizing diet-induced murine models of type 2 diabetes mellitus(T2DM)revealed that exercise could improve glycemic control and insulin resistance.However,the detailed mechanisms of exercise-induced insulin resistance amelioration in the leptin receptor deficiency-induced(db/db)mouse model are poorly elucidated.MG53,also named TRIM72,is a skeletal-and cardiac muscle-specific protein that was initially reported to facilitate membrane repair.On the discovery of its multi-functions,MG53 has been recently shown to act as an E3 ligase to mediate the degradation of insulin receptor(IR)and insulin receptor substrate 1(IRS1)and in turn,to disturb the normal transduction of insulin signaling.Therefore,as a negative regulator of insulin action,MG53 might potentially participate in the pathogenesis of insulin resistance.Future investigations targeting on the role of MG53 in the effects of treadmill exercise on alleviating insulin resistance might be a guidance to the treatment and therapy of diabetes and related metabolic diseases.Objective: In the current study,we aimed to investigate whether 12-week treadmill exercise could alleviate insulin resistance and skeletal muscle atrophy,and to explore whether MG53 along with IRS/AKT/m TOR cascade played a role in the physiopathological changes of db/db mice.Methods: Twenty 6-week-old db/db(BSK.Cg-Dock7 m +/+ Leprdb/JNju)mice and twenty age-matched non-diabetic m/m(C57BLKS/JNju)mice were purchased from Model Animal Research Center of Nanjing University.All mice were housed on a 12:12h lightdark cycle under controlled temperature(22±2?)and humidity(50±10%)and were fed with standard chow and water ad libitum.After one week of acclimatization,mice were randomly assigned to four groups as MC(m/m control,n=10)group,ME(m/m exercise,n=10)group,DC(db/db control,n=10)group and DE(db/db exercise,n=10)group.Exercise training was performed on a treadmill and the detailed intervention regimen(0% grade)were as follows:(1)week 1,the intensity of running was set at 7.0-13.3m/min for 15-20 min per day,4 days a week;(2)week 2,the intensity was increased at 13.3m/min for 30-45 min per day,6 days a week;(3)week 3-12,the intensity was increased and stabilized at 13.3m/min for 1h per day,6 days a week.The body weights and fasting blood glucose levels were measured on a regular basis during the whole experimental process.After treadmill training intervention,glucose and insulin tolerance tests,insulin resistance index(HOMA-IR,homeostasis model assessment of insulin resistance)and lipid metabolic profile were determined using blood samples.Skeletal muscles were utilized for determination of cross-sectional area(CSA),protein level detection of MG53,?-IR,IRS1,AKT,p-?-IR-Tyr1146,p-IRS1-Ser307,p-AKT-Ser473,p-m TOR-Ser2448,p-p70S6k-Thr389 and p-S6-Ser235.Results:(1)Compared with MC mice,the body weight of mice in group ME showed no significant changes,both of which were stabilized at a certain level.However,the body weight of db/db mice increased till mid-term of exercise intervention and then decreased till the end of exercise training.Compared with DC mice,the body weight of DE mice decreased significantly during their age of 11 to 15 weeks old(p<0.05),but there was no significant difference bwteen these two groups at the endpoint of exercise intervention.(2)Compared with m/m mice,the fasting blood glucose(FBG)level of db/db mice increased significantly before exercise intervention.After 12-week treadmill exercise,the FBG of DC mice remained at a significantly higher level when compared with MC mice(p<0.05)and compared with DC mice,the FBG of mice in group DE decreased significantly(p<0.05).(3)The results of IPGTT showed that compared with mice in group MC,the AUC(areas under curve)of IPGTT of mice in group DC increased significantly(p<0.05),while compared with DC mice,the AUC of IPGTT of DE mice showed a significant decrease(p<0.05).The results of IPITT demonstrated that the AUC of DC mice increased significantly compared with MC mice(p<0.05),whilst the AUC of DE mice decreased significantly when compared with DC mice(p<0.05).Compared with mice in group MC,the serum glucose level(p<0.05),serum insulin level(p < 0.05)and HOMA-IR(p < 0.05)of mice in group DC all increased significantly,while compared with mice in group DC,the serum glucose level(p<0.05),serum insulin level(p<0.05)and HOMA-IR(p<0.05)of mice in group DE all exhibited significant decreases.(4)Compared with mice in group MC,the lipid parameters namely serum triglyceride(p<0.05)and total cholesterol(p<0.05)increased significantly in DC group.Compared with mice in group DC,the levels of serum triglyceride(p<0.05)and total cholesterol(p<0.05)in group DE decreased significantly.The weight of epididymal fat(p<0.05),the weight of inguinal fat(p<0.05),the ratio between epididymal fat weight and body weight(p<0.05)and the ratio between inguinal fat weight and body weight(p<0.05)all showed significant increases in DC mice when compared with MC mice,while when compared with DC mice,the weight of epididymal fat(p<0.05),the ratio between epididymal fat weight and body weight(p<0.05)and the ratio between inguinal fat weight and body weight(p<0.05)in DE mice decreased significantly.(5)The overall upregulation of MG53 protein expression in different skeletal muscles namely quadriceps(p<0.05),gastrocnemius(p<0.05)and soleus muscle(p<0.05)could be observed in DC mice when compared with MC mice and there was an overall downregulation of MG53 expression in quadriceps(p < 0.05),gastrocnemius(p<0.05)and soleus muscle(p<0.05)in DE mice after 12-week treadmill exercise.(6)Acute insulin stimulation was performed in the current study.When compared with the protein levels before acute insulin resistance,the phosphorylated protein levels of ?-IR(p<0.05)and AKT(p<0.05)in group MC,ME and DE all increased significantly after acute insulin stimulation.When compared with mice in group MC after acute insulin stimulation,the phosphorylated protein levels of ?-IR(p<0.05)and AKT(p < 0.05)decreased significantly and that of IRS1(p < 0.05)increased significantly in group DC,while the phosphorylated protein levels of ?-IR and AKT(p<0.05)showed significant increases and that of IRS1(p<0.05)showed significant decrease in group DE in comparison with DC mice.No significant difference was found in the total protein expression levels of ?-IR and AKT among four groups except that the total IRS1 protein level of DC mice was downregulated with significance compared with mice in group MC(p<0.05).The expression of MG53 was highly negatively correlated with that of IRS1(r=0.7218,p<0.01).(7)The wet weight of gastrocnemius muscle(p<0.05),the wet weight of anterior tibial muscle(p<0.05),the ratio between gastrocnemius muscle weight and body weight(p<0.05)and the ratio between anterior tibial muscle weight and body weight(p<0.05)all showed significant increases in DC mice when compared with MC mice,while when compared with DC mice,they all decreased significantly(p<0.05)in DE mice.Plantaris muscle size significantly increased in exercise intervention groups(p<0.05),whereas there was no significant difference between MC and DC mice.When compared with the protein levels before acute insulin resistance,the phosphorylated protein levels of m TOR(p < 0.05),p70S6k(p < 0.05)and S6 ribosomal protein(p<0.05)in group MC,ME and DE all increased significantly after acute insulin stimulation.When compared with mice in group MC after acute insulin stimulation,the phosphorylated protein levels of m TOR(p<0.05),p70S6k(p<0.05)and S6 ribosomal protein(p<0.05)decreased significantly in group DC,while they all showed significant increases in group DE in comparison with DC mice.Conclusion:(1)12-week moderate treadmill exercise training could improve the body composition of diabetic db/db mice via decreasing the fat mass index and increasing the skeletal muscle index significantly,which was of benefits to improve the lipid metabolic disorder and diabetic skeletal muscle atrophy.(2)The glucose tolerance and insulin sensitity of diabetic db/db mice could be effectively improved by 12-week moderate treadmill exercise,resulting in a better glucose homeostasis.(3)MG53 protein expression was pathologically upregulated in different skeletal muscles in diabetic db/db mice,which could be reversed by 12-week moderate treadmill exercise.(4)Skeletal muscle insulin resistance in diabetic db/db mice could be effectively attenuated by12-week moderate treadmill exercise through inhibiting MG53-mediated ubiquitin-dependent degradation of IRS1.(5)Insulin-dependent protein synthsis and skeletal muscle atrophy could be effectively improved by 12-week moderate treadmill exercise by regulating MG53/IRS1/AKT/m TOR pathway.