Characterize The Skeletal Muscle MTOR Signal Pathway After Exercise And Its Roles In The Regulation Of Protein Synthesis

Aim:In the current study,we investigated the alternation of skeletal muscle mTOR signal pathway after either exercise(various strength or length) or injection of rapamycin and further characterized the mechansim of roles of exercise in the regulation of mTOR pathway protein synthesis.Methods:Experiment 1:The following exercises were applied to male SD mice:three different strength(10m/min,18 m/min and 26 m/min at 10%slope) and fixed time(60 mins),or fixed strength(18m/min at 10%slope) and various time(30,60 and 90 mins).Gastrocnemius was isolated 1,6,12 and 18 hrs, respectively,after exercise.Experiment 2:The male SD mice were divided into the following four groups:control(C),exercise(E),injection of rapamycin(R), and combination of exercise and injection of rapamycin(ER).For the mice in either R or ER group,rapamycin was injected 2 hrs before exercise.The following strength of exercise,18m/min at 10%slope for 60 mins,was applied to the mice in either E or ER groups.After either 1 day treatment or 2 week's continued treatment,Gastrocnemius was isolated 1,6,12 and 24 hrs,respectively, after exercise.The protein levels of both endogenesour and phosphorylated forms of Akt,mTOR,S6K and 4E-BP1 were determined by western blot analysis. BCA assay was used to measure the protein concentrations of muscle.Histological experiments were performed to observe the morphology.Results:1) Both 60 mins low strength exercise and 30 min medium level of exercise had no effect on the protein levels of either endogenesour or phosphorylated forms of Akt and mTOR.2) Exercise 60 mins at either medium or high strength increased the protein levels of both endogenesour and phosphorylated forms of Akt and mTOR.The increase in phosphorylated form of Akt was corresponded to the strength of exercise,with the highest level at the strongest strength of exercise.No noticable difference in the phosphorylated form of mTOR was observed with either strength of exercise.The alteration of Akt protein level happened earlier than that of mTOR.3) The phosphoryaltion of mTOR happened earlier in 90 mins medium strength exercise,in comparison to 60 mins exercise.4) The phosphorylated state of S6K lasted longer than that of 4E-BP1 after 60 mins medium strength of exercise.5) Exercise increased mTOR protein level,which is not affected by the injection of rapamycin.6) Two week exercise attenuated the inhibiton of mTOR phospharylation by rapamycin.7) Morphological changes were observed in muscles with injection of rapamycin for 2 weeks.Conclusions:1) Strength of exercise can alter the phosphorylated level of Akt,but has no significant effect on the mTOR phosphorylation.2) There is a lag phase between mTOR and Akt phosphorylation,with mTOR phosphorylation happened later.3) The length of exercise also affects the phosphorylated levels of Akt and mTOR.No obvious changes in the phosphorylated levels of Akt and mTOR were observed with too long or too short exercises at fixed strength.4) There is a correlation between mTOR phosphorylation and S6K/4E-BP1 phosphorylation.5) Rapamycin inhibits the phosphorylation of both mTOR and its downstream signal molecules,whild has no effect on the total level of mTOR.