Change Of AMPK In Skeletal Muscle During Different Exercise And Regulation MTOR And Its Downstream Signaling

IntroductionAMPK was an intracellular energy sensor in skeletal muscle,which could activate by exercise intensity and time,but the change feature was not clear under different intensity and time.Strength was the base to all physical quality,which depend on protein synthesis in skeletal muscle.AMPK could depress protein synthesis,but the molecular mechanism was not clear.So,the two questions maybe elucidate through animal experiment in this research.MethodsTreadmill exercise and AICAR injection models with rat were used in Expâ… andâ…¡. Expâ… was made up by different intensity,time and 2 weeks exercise.In different intensity part,used low(L),moderate(M),and high intensity(H) which speed was 10m/min,18m/min and 26m/min respectively,10%slope and 60min time.In different time part,used respectively 30,60 and 90min time,18m/min speed,10%slope.In 2 weeks training part,used 18m/min speed,10%slope,60min/d.After 2 weeks training, rats completed a bout exercise as 26m/min,10%and 60 times.In Expâ…¡,rats was divided into rest,a bout exercise group,with high intensity,a bout AICAR injection,with a subcutaneous injection(0.5mg/g),or an equivalent volume of normal saline as control.In Expâ… andâ…¡,white gastrocnemius samples were collected at 0h,1h and 6h after exercise or at 1h,2h and 6h after AICAR injection.AMP,ATP contents were determined by HPLC.AMPK activity was determined by[γ-³²P]ATP as P donor and SAMS peptide as substrate.Phosphorylation mTOR(Ser2448) was determined by western blot,as well as 4E-BP1(Thr37/46),P70S6K(Thr389).Resultsa.Comparing with rest,The AMPK activity was not changed in L,but increased 50%(P＜0.01) and 1.8 fold(P＜0.01) at 0 of M and H exercise,respectively,then retrieved to rest level at 6h.;AMP contents were significantly changed at 0 of M and H exercise (16%,P＜0.05;62%,P＜0.01,respectively).AMP:ATP ratio was increased at 0 of M and H exercise(33%,P＜0.05;89%,P＜0.01,respectively).AMPK activity was significant positive correlation to AMP:ATP ratio at 0(r=0.89).b.AMP content of 60 and 90 min was significantly increased at 0 h(0.183,0.212 Vs 0.158,P＜0.05),then returned to basal at 1h as well as AMP:ATP ratio(0.048,0.053 Vs 0.037,P＜0.05,0.01,respectively).ATP content had no change in all groups.AMPK activity of all groups were significantly increased at 0h(4.16,4.79,5.26 Vs 3.18,P＜0.05,0.01,0.01,respectively),and kept increasing at 1h,then returned to basal at 6h.c.Comparing with rest,AMP,AMP:ATP ratio and AMPK activity of 2 weeks training group was increased 42%,80%,80%, respectively,P＜0.01.AMP and AMP:ATP ratio returned to rest in 1h but AMPK in 6h. Comparing with no training group,the amplification of AMP and AMPK of training group decreased 20%(P＜0.05) and 100%(P＜0.01),respectively,d.A bout of exercise and AICAR injection could activate AMPK activity in skeletal muscle.The recovery time was 2h for injection groups and 6h for exercise groups,e.At post-exercise 0h or post-inject 1h,mTOR Ser2448 and 4E-BP1 Thr37/46 phosphorylation were significantly decreased,and increased at 1h or 2h,then to the peaking at 6h.P70S6K Thr389 phosphorylation was not change in injection groups but increased in exercise groups at all times.Conclusionsa.The AMPK activity was increased as intensity and time-dependent in exercise;it was regulated mainly by AMP:ATP ratio increasing which depended rather increasing AMP than decreasing ATP.b.Short-term training attenuated AMPK signaling in skeletal muscle during acute exercise through attenuating the amplification of AMP and AMP: ATP ratio,c.A bout of exercise and AICAR injection could activate AMPK activity in skeletal muscle.The mechanism was related to dephosphorylation mTOR Ser2448 and 4E-BP1 Thr37/46,but not to dephosphorylation S6K1 Thr389.d.The mechanism which the rate of protein synthesis increased was related to up regulation mTOR Ser2448 and 4E-BP1 Thr37/46 after exercise or AICAR injection.On S6K1 Thr389 by AICAR was different to exercise,e.The time course of down regulating AMPK signaling was not full synchronization with up regulating mTOR signaling path,to indicate that it could involved in other mechanisms activating mTOR for protein synthesis after exercise.