The Mechanistic Study Of GAcrp30/AMPKα2 In The Activation Of Skeletal Muscle Autophagy By HIIT

Objective:In recent years,high intensity interval training(HIIT)has become a new forms of movement emerging into people’s lives,which can quickly improve the heart rate in short term,and increased the demand of oxygen,manufactured of anaerobic condition,resulting that the body still need more oxygen in the recovery period,thus consuming more calories.It is precisely because of this,most of the studies about HIIT focus on the cardiovascular-related clinical manifestations,and for endocrine and metabolic related signal pathway research are more limited.Adipose tissue as endocrine organs,in recent years has been gradually taken seriously,and the strong lipid-lowering effect of HIIT,also makes people began to speculate on some adipokines such as adiponectin(Acrp30).Most studies have shown that HIIT significantly increases the expression of Acrp30,but some studies are on the contrary.The different basic health status and the detection of the form of Acrp30 may play a role.Autophagy is one of the hotspots in biomedical field,and is widely involved in various physiological and pathological processes.Autophagy is the physiological process for deliver fuel for body rapidly,and the response of skeletal muscle autophagy to "hunger" is still blank when the cellular energy is rapidly consumed by HIIT.Moreover,it is known that AMPK plays an important role in exercise-induced autophagy in skeletal muscle,and whether it plays an equally important role in the process of HIIT-induced autophagy of skeletal muscle remains to be further explored.In this study,we selected healthy individuals as the research object to ensure that the basic health status of the subjects was same,and the maximum oxygen uptake(VO2max)was more than 80% VO2 max,while the moderate intensity continuous training(MICT)was used as another control group.Globular adiponectin(g Acrp30)which was the main active mode of Acrp30 used for detection.At the same time,the skeletal muscle autophagic activity was detected and an attempt was made to reveal the possible mechanisms underlying the excellent health effects of HIIT.Methods:(1)Animal model: Four weeks old mice(C57BL/6,male)including AMPKα2+/+(n=12),AMPKα2+/-(n=12),AMPKα2-/-(n=12),and 1 week for adaptive feeding.Then randomly assigned to three groups: the control group(Control),MICT group(MICT) and HIIT group(HIIT).(2)Exercise program: Exercise intervention time is 6 weeks,MICT/HIIT group movement form is 75% or 85% VO2 max aerobic continuous treadmill training,60 minutes/times,5 times / week,once a day(Table 1),then begin to body composition analysis;(3)Dietary formula: All groups was fed with Fu Kang1022 fodder;(4)Body composition analysis: Animals were weighed and recorded after 6 weeks intervention.The body mass index(BMI),total body water and fat mass were measured by Impedi VET experimental animal body composition analyzer.(5)Serum g Acrp30 test: Mice were sacrificed after analysis,and the levels of serum g Acrp30 were detected by enzyme linked immunosorbent assay(ELISA).(6)Cell culture: Induced C2C12 myocytes into myotube cells and then intervented with g Acrp30 and DN-AMPK(K45R);(7)Western blot was used to detect the expression of AMPKα2,p AMPK-Thr172,p ACC-Ser79,LC3II/I,Beclin1 and p62 protein in skeletal muscle tissue or C2C12 myotubes.Results:(1)Body composition: a.Weight: body weight were not significantly different before exercise,after intervention,body weight of MICT and HIIT mice were decreased compared with the same genotype in Control group(P<0.05 or P<0.01);b.BMI:Compared with the same genotype in the control group,BMI of the HIIT group had a significant difference(P>0.05).The level of BMI in AMPKα2+/+ and AMPKα2+/-mice was significantly lower than control in HIIT group(P<0.01 or P<0.01),but there was no significant difference in MICT group(P>0.05)or AMPKα2-/-mice in HIIT(P>0.05);c.Total body water: There were no significant statistical difference in three groups in total body water;d.Fat mass: Compared with the control group of the same genotype mice,MICT group showed a decline in body fat content,but only AMPKα2+/+ mice act out differences(P>0.05),while in HIIT group,AMPKα2+/+ and AMPKα2+/-were shown a significant decrease(P<0.01 or P<0.05),but the content of body fat in AMPKα2-/-was not significant(P>0.05).(2)Serum gAcrp30 level: The serum gAcrp30 was significantly higher than that in the control group in the HIIT group(P<0.05)but not MICT group.(3)Effect of gAcrp30 on C2C12: The expression of pAMPK-Thr172,pACC-Ser79,LC3II/I,Beclin1 and p62 expression were increased under different concentration(0,0.1,0.25,0.5,0.75,1μg/ml)and time gradient(0,3,8,16,24h).(4)Western-blot detection result: The cell autophagy was induced by gAcrp30 at a concentration of 1μg/ml for 24 h,and the induction effect of g Acrp30 was impaired but not blocked by DN-AMPK(K45R).Furthermore,the expression of p AMPK-Thr172,LC3II/I and Beclin1 were increased in the skeletal muscle of AMPK+/+ and AMPK+/-mice after HIIT intervention while the expression of p62 was decreased,and this effect was not significant in AMPK-/-mice.Conclusions:(1)Compared with control group,body weight,fat mass and BMI were decreased in MICT and HIIT,and the decrease in HIIT was more obvious.Furthermore,AMPKα2 played an important role in it;(2)Compared with control group,serum g Acrp30 level was increased after 6weeks of HIIT intervention,and the effect of MICT on g Acrp30 level was not significant;(3)In vitro experiments,the autophagy activity of C2C12 myotubes was increased under g Acrp30 with AMPKα2-independent mechanism;(4)Compared with control group,skeletal muscle autophagy was increased after6 weeks HIIT,and this activation depends on AMPKα2.