Mechanistic Study Of The Role Of Sestrins In The Process Of Aerobic Exercise Ameliorating Energy Metabolism In The Skeletal Muscle Of C57BL/6 Mice

Aim:It is widely accepted that exercise training is beneficial for the prevention and treatment of IR.However,the underlying mechanism by which exercise training improving skeletal muscle energy metabolism is still not fully described.Recent studies have found that Sestrins(SESNs),a stress-responsive protein family,may play an important role in aerobic exercise to improve insulin sensitivity in skeletal muscle.SESNs(SESN1-3)are highly conserved stress-inducible protein.Concomitant ablation of SESN2 and SESN3 has been reported to provoke hepatic m TORC1/S6K1 activation and insulin resistance even without nutritional overload and obesity,implicating that SESN2 and SESN3 have an important homeostatic function in the control of mammalian glucose and lipid metabolism,while the role of SESNS in regulating metabolism remains unknown.Our previous results demonstrated that physical exercise increased SESN2 and SESN3 expression in murine skeletal muscle and both of them Co-immunoprecipitated with AMPK?2increased after 1 h of treadmill exercise,while the relationship between SESNs and AMPK in response to exercise remains unknown.Together,the purpose of the study is:1.to determine whether aerobic exercise regulates the amount of SESNs in skeletal muscle;2.to determine whether AMPK?2 involves in the regulation of SESNs expression in response to aerobic exercise;3.to determine the underlying mechanism of SESNs regulationg glucose and lipid metabolism.Our project provided the theoretical basis for the mechanism that aerobic exercise improving IR.Method:Male wild type,AMPK?2^+/-and AMPK?2^-/-C57BL/6 mice C57BL/6 mice were divided into normal and exercise,the exercise groups were underwent 6-week treadmill training.The mice from the exercise group were exercised on a motor-driven rodent treadmill for 5 days a week for a total of 6 weeks.The running intensity and time were increased to 75%VO₂max(12 m/min)for 60 min/day.Citrate synthase activity was determined by enzyme kinetics.The protein levels of SESN1,SESN2,SESN3,AMPK?2,p AMPK-Thr172,AKT,p AKT-Thr308,p AKT-Ser473,GLUT4,COX4,Cytochrome C,LC3 and p62 were determined by western blot.C2C12 cells were treated by SESN2/DN-AMPK adenovirus,SESN3 expression vector,si RNA-SESN2/SESN3 or 3MA.The protein levels of SESN2,SESN3,AKT,p AKT-Thr308,p AKT-Ser473,GLUT4,p AMPK-Thr172,p ACC-Ser79,LC3 and p62were determined by western blot.Glucose uptake was measured using a nonradioactive fluorescent glucose(2-NBDG)method.Male wild type and SESN2^-/-mice were divided into normal chow(NC)and high-fat diet(HFD)groups to create insulin resistance mice model.After 8 weeks the IR model group was then divided into HFD sedentary control and HFD exercise groups(HE).Mice in HE group underwent 6-week treadmill exercise.Lipid profiles were detected;skeletal muscle and liver lipid ectopic deposition was detected by oil red O staining.The protein levels of SESN2,p JNK-Thr183/Tyr185,ACC,p ACC-Ser79,FASN,SERBP-1 and CPT-1 were determined by western blot.C2C12 cells were treated by SESN2 adenovirus,si RNA-SESN2,SP600125 or PA.The protein levels of SESN2,p JNK-Thr183/Tyr185,ACC,p ACC-Ser79,FASN,SERBP-1 and CPT-1were determined by western blot.Lipid ectopic deposition was detected by oil red O staining.Results:1.6-week exercise increased mitochondrial biogenesis,citrate synthase,and activated insulin signaling pathway in skeletal muscle.Exercise increased AMPK?2,p AMPK-Thr172,SESN2 and SESN3 expression significantly in comparison with control.AMPK?2 mediated the increasing of SESN2/SESN3 protein content after6-week exercise training,and even without exercise,AMPK?2 could regulate SESN3content.Exercise failed to stimulate the autophagy activity in AMPK?2^-/-mice.2.Overexpression of SESN3 or SESN2 and SESN3 together increased glucose uptake,activated the insulin signaling pathway,and promoted GLUT4 translocation in myotubes.However,inhibition of SESNs had no effect on glucose uptake.SESNs could reverse reduced glucose uptake following autophagy inhibition.Co-transfection of flag-SESN3 and DN-AMPK promoted glucose uptake when compared with DN-AMPK,as did co-transfection of Ad-SESN2,flag-SESN3,and DN-AMPK,which indicated that SESNs regulated glucose metabolism through an AMPK-independent pathway.3.Absence of SESN2 exacerbated phenotypes caused by high-fat diets.Exercise ameliorated phenotype caused by HFD.In WT mice,high-fat diet intervention can significantly increase the content of SESN2 and inhibit the expression of p ACC-Ser79,FASN and SREBP-1;aerobic exercise can significantly increase the content of p ACC-Ser79 and SESN2 in control and obese mice,the expression of murine FASN in control mice,the content of SREBP-1 in high-fat mice and the reduction of p JNK-Thr183/Tyr185 in obese mice.In SESN2^-/-mice,high-fat diet intervention can increase the content of ACC,CPT-1 and p JNK-Thr183/Tyr185,and reduce the expression of FASN and SREBP-1.Aerobic exercise can significantly increase the CPT-1 and FASN content,reduced the content of SREBP-1 in control mice,reduced expression of ACC,SREBP-1 and p JNK-Thr183/Tyr185 in obese mice.4.Palmitate intervention can significantly increase lipid deposition in myotubes of all groups,especially in si-SESN2-treated myotubes.Inhibition of SESN2 can increase the content of CPT-1,p JNK-Thr183/Tyr185.Inhibiting JNK can reduce the protein content of ACC,p ACC-Ser79,SREBP-1 and FASN;activating JNK can increase the expression of ACC,p ACC-Ser79 and FANS,and reduce the expression of CPT-1.Conculision:Exercise training regulated SESNs via AMPK and AMPK was essential to induce SESN3 even in sedentary states.SESN3 was a critical factor in promoting glucose uptake,and SESN2 synergized with SESN3 in regulating glucose metabolism.SESN2 alone however,was not effective in this regard.SESNs induced glucose uptake via the promotion of GLUT4 translocation.In addition,SESNs regulated skeletal muscle glucose metabolism independent of autophagy.It was likely that SESNs,particularly SESN3,played a key role in exercise training-mediated skeletal muscle glucose metabolism.SESN2 may regulate ACC,CPT-1,SREBP-1 and FASN by affecting JNK activity,thereby affecting lipid metabolism.