The Role Of Sestrin2/AMPK-mediated Skeletal Muscle Autophagy In Aerobic Exercise Attenuating Insulin Resistance In C57BL/6 Mice

Excessive supply of HFD and physical inactivity might be the major contributors to the development of obesity,consequently obesity-associated diseases,such as IR,T2 DM,cardiovascular disease etc,whereas the precise mechanism of the process is still unknown.Skeletal muscle is a key contributor to insulin-stimulated glucose disposal and defects at different levels of insulin signaling pathway in this tissue will lead to IR and then T2 DM.Aerobic exercise can enhance metabolism of protein,fat and glucose and has been wildly used as a project to treat IR and metabolic dysfunction,however the precise mechanisms still need to be determined underlying the process of exercise improved insulin sensitization.Sesns are evolutionarily conserved stress-inducible proteins whose expression is up-regulated by various environmental insults including genotoxic,oxidative,and nutritional stress.Mammalian and most vertebrates have three Sesns paralogs,while invertebrates have a single ortholog.Of note,activation of Sesns could activate AMPK accompanied by suppression of m TOR,leading ultimately to autophagy induction.As a highly regulated cellular process,autophagy has emerged as an important player in maintaining energy homeostasis in different tissues including skeletal muscle.A deficiency in autophagy causes intercellular accumulation of excess fat,aggregated proteins,and dysfunctional organelles,and such autophagy deficiency cascade potentially results in deterioration of whole-body energy metabolism and development of IR and some other metabolic disorders.Therefore,optimal autophagic flux in skeletal muscle could be important for the physiological and the pathological regulatory processes.Autophagy regulation involves several signaling pathways orchestration,among which AMPK/m TOR signal pathway plays an important role in regulating energy homeostasis and metabolic stress.It is also becoming increasingly evident that autophagy is involved in exercise-induced adaptations through AMPK/m TOR signal.Recent studies proved that exercisemediated autophagy not only improve muscle mass and maintain muscle metabolic homeostasis,but also effectively prevent mice from IR,obesity,T2 DM and other relative metabolic disorders.Further,recent findings established the relationship between Sesn2-regulated AMPK and the improvement of glucose homeostasis by short-term exercise.It is well-established that Sesns are clearly involved in m TOR,AMPK,p53,Fox O,and PRX signaling pathways,although the Sesns-exercise relationship has not been explored so far,the aforementioned pathways are implicated in critial metabolism processes by autophagy regulation,thus suggesting a potential correlation between the autophagy modulation of Sesns and the promoting metabolism of AMPK.Accordingly,loss of Sesns can cause several chronic pathologies phenotypes,which are similar to those associated with obesity,aging,and lack of exercise.Moreover both high-intensity interval running and moderate-intensity continuous running induce activation of AMPK and Sesns upstream effector p53 phosphorylation in human skeletal muscle,whereas mice knocked out for p53 have reduced endurance capacity and muscle performance.In this regard,the previous study led by our colleagues investigated the role of AMPK in the signaling mechanism underlying the effects of exercise on muscle autophagy and insulin sensitivity and demonstrated that a single-bout exercise leads to a significant increase in the expression of Sesns and the physical interaction between Sesns and AMPK,along with autophagy activation and improvement of IR in skeletal muscles.These observations proposed a new hypothesis that Sesns are novel mediators of exercise-induced autophagy signaling in muscle,which is highly linked with insulin sensitivity.However the role of Sesns upregulation in the signaling mechanism underlying the effects of long-term aerobic exercise on muscle autophagy and insulin sensitivity remains unclear.As mentioned above,we speculated that Sesns-mediated autophagy may function as a crucial regulator for cellular energy homeostasis and metabolism.Here we used IR mice induced by high fat diet and AMPK?2 knockout mice to undergo 6-week treadmill training in vivo and induced IR model of C212 myotubes in vitro to investigate the relationship between the chronic aerobic exercise,and autophagy regulation and whether Sesn2/AMPK activation is involved in this process.We wonder if chronic exercise can increase Sesns and induce autophagy in obese mice and if the improvement in metabolic conditions is correlated with the autophagy activity.With this in mind,we also sought to investigate if there is any relationship between induced autophagy and AMPK activity in obese mice.This study aims to delve into the mechanisms of the role Sesn2 regulated AMPK signaling in autophagy activation and contribution to long-term adaptations to aerobic exericise mediated insulin sensitization,aiming to provide some theoretic evendience for prevention and treatment of IR and relative metabolic abnormalities through physical activities.Method:To study the relationship between the chronic aerobic exercise,and autophagy and whether Sesn2/AMPK activation is involved in this process,we used IR mice induced by high fat diet and AMPK?2 knockout mice to undergo 6-week treadmill training in vivo,and then investigated the effects of different intervention on the improvement of mice metabolic conditions and study the role of AMPK in mediating exercise improved insulin sensitivity.In vitro,C2C12 myotubes IR induced by PA were transfected with Ad-Sesn2 and/or Ad-AMPK?2-DN to explore the potential effects of Sesn2/AMPK in skeletal muscle metabolism and autophagy regulation.(1)IR mice model and experiment trial4-week old male C57BL/6 mice were divided into normal chow and HFD group at random,which were fed chow and high fat diet respectively for up to 16 weeks.The mice showed IR symptoms in HFD group and in normal chow group were then continued to be fed with high fat diet and normal chow,and divided into high fat diet control(HC),high fat diet exercise group(HE),normal chow control(NC)and normal chow exercise group(NE)respectivly,Meanwhile,the mice from exercise groups were underwent a 6-week treadmill running aerobic exercise.Additionally,6-week old male wild type or AMPK?2 knockout mice were divided into control and exercise group at random respectively,the mice from exercise group were also underwent a 6-week treadmill training.(2)Mice phenotypeOGTT was to detect the level of mice glucose tolerance;insulin ELISA kit was used to determine mice fasting serum insulin level;Lipid profiles were determined by CHOD-PAP method;lipid deposition level in skeletal muscle was detected by oil red O staining.In addition,the body weight and body composition was determined by animal body composition system.(3)Experimental methods and indices determinationThe protein levels of Sesn2,Beclin1,p ULK1-S555,LC3II/I,p62,AMPK?2,p AMPK-T172,p ACC-S79,p IRS1-S636/39,p IRS1-S307,p S6K1-T389,p AKT-S473,p AKT-T308 and ?-actin were detected by proteins Western Blot method;Real-time PCR was used to determine Sesn2 m RNA expression and glucose uptake in myotubes was measured by 2-NBDG.(4)Cell culture and treatmentC2C12 cells were induced by DMEM containing 2% horse serum;The differentiated myotubes were treated with Palmitate at the indicated concentration to induce IR;Adenovirus expressing SESN2 or dominant-negative(K45R)mutation of AMPK?2 were generated;Myotubes were transfected with Ad-Sesn2 and/or Ad-AMPK?2-DN or Ad-GFP respectively;3-MA or AICAR was applied 1hr before treatment with PA and followed by insulin stimulation for autophagy inhibition.Results:(1)The Effects of HFD and aerobic exercise on mice metabolic indexThe mice in HC were showed higher levels in body weight,fat content,FINs,FFAs,serum TC,TG and LDL-c,along with impaired OGTT and decreased HDL-c level;while 6-week aerobic exercise reversed these trends as evidenced by reduced total body weight,FINs level and fat content,improved glucose tolerance and lipid metabolic dysfunction.(2)Role of Sesn2 mediated autophagy and insulin sensitivityIn vivo study,long term of HFD decreased Sesn2 protein expression level,while 6-week aerobic exercise increased Sesn2 protein expression;Sesn2 protein level decreased significantly in palmitate-induced IR myotubes.(3)Ad-Sesn2 co-treatment significantly prevented myotubes from palmitate-mediated reduction of insulin-mediated glucose uptake.Simultaneously,the increased p IRS1-S636/639 and p IRS1-S307 level and decrease of p AKT-S473 and p AKT-T308 in the presence of insulin were significantly alleviated by Ad-Sesn2 under IR condition,suggesting Sesn2 effectively restored the impaired insulin signaling.(4)The expression of p S6K1-T389 significantly decreased after Ad-Sesn2 transfection even in the presence of palmitate.And the reduction of Beclin1,p ULK1-S555,LC3II/I and accumulation of p62 in IR cells were alleviated by Sesn2 overexpression compared to the controls,indicating that Sesn2 reversed the palmitate-suppressed autophagic signaling in this context.(5)Autophagy suppression significantly aggravated increase of p IRS1-S636/639 and decrease of p AKT-S473 in IR myotubes.Correspondingly,Ad-Sesn2-induced decrease of p IRS1-S636/639 and increase of p AKT-S473 in the presence of insulin were remarkably abolished under the condition of autophagy inhibition.Furthermore,palmitate-reduced in glucose uptake was aggravated and the protective effects of Sesn2 against palmitate-induced inhibition of glucose uptake in the presence of insulin were abolished by pretreatment with 3-MA.(6)6-week exercise increased the autophagy activation and AMPK signaling pathway activity in skeletal muscle in vivo;Consistently impaired AMPK and ACC by long term of Palmitate treatment were restored by Sesn2 overexpression in vitro.(7)AICAR promoted autophagy activity;Ad-AMPK?2-DN aggravated reduction of LC3II/I and p62 accumulation and abrogated Sesn2-reduced p62 accumulation in IR myotubes.On the other hand,Ad-Sesn2 co-treatment did not restored p ULK1-S555 after transfect cell with Ad-AMPK?2-DN.Sesn2-induced inhibition of p IRS1-S636/639 and upregulation of p AKT-S473 were abolished in palmitate treated myotubes under condition of Ad-AMPK?2-DN.These data showed that Sesn2-induced autophagy contributed to restore the impaired insulin signaling through activation of AMPK signal.(8)Aerobic exercise can increase p ULK1-S555 and LC3II/I expression and p62 degradation in wild-type mice skeletal muscle;however these indices were similar in AMPK?2-knockout mice.Furthermore,the activity of autophagy was significantly lower in AMPK?2 KO-EX mice than WT-EX mice,indicating that AMPK?2 plays an essential role in exercise induced autophagy acitivation.Conclusion:(1)Long term high-fat feeding induced IR and decreased the level of protein expression of Sesn2;(2)6-weeks aerobic exercise ameliorated long term HFD-induced abnormal glucose and lipid metabolism in C57BL/6 mice,which was associated with the increased the level of Sesn2 protein expression following exercise;(3)Sesn2-induced autophagy contributed to restore the impaired insulin signaling effectively through activation of AMPK signaling pathway;(4)Exercise-upregualed Sesn2 maybe involved in improvement of IR in mice skeletal muscle,which is in an AMPK-mediated autophagy activation manner.