Effect Of Exercise On PI3K/AKt/mTor And MAPK Signal Transduction In Rat Skeletal Muscle

ObjectiveThe uptake of glucose in skeletal muscle is a dissipation energy and voluntaryprocedure, and the glucose transporter on the membrane is necessary for the entry intomuscle cell of glucose. Most of the GLUT-4 is in the cytoplasm, but exercise or insulincan make a translocation of GLUT-4 from an intracellular location to the cell surface.Then it takes glucose into the cell, and enhances the utilization and uptake of glucose,meanwhile decreasing the level of blood glucose to maintaine it's balance. However,the GLUT-4mRNA in the cell of diabetes rat skeletal muscle is markedly lower than thenormal. Under the glycemia condition, the decreased synthesis of GLUT-4 proteinsuggests that the decreased transport and uptake of glucose which leads toinsulin-postreceptor resistance, results from the inhibited expression of GLUT-4 geneand the reduced transcription. Exercise can ameliorate the insulin-resistant, enhance thesensitivity of skeletal muscle to insulin, and improve the synthesis of GLUT-4 protein.Equally important, exercise can enhance the sensitivity of rat skeletal muscle toinsulin, increase the combination of insulin with it's receptor, and then exert it'sbiological effects through the insulin-signaling pathway. The signal of insulin receptormainly includes PI3-K/PKB/mTOR and MAPK. Studies show that exercise effects onthe two pathways above, improves the metabolism of glucose and enhances theutilization and ingestion of glucose. Furthermore exercise can decrease the level ofblood glucose after training, and maintain it's balance, which has applied to clinic. Butthe molecular mechanism of the two pathways is not the same, the effects of exerciseon PI3-K/PKB/mTOR is by increasing the sensibility of skeletal muscle to insulin.Exercise can activate PI3-K by insulin, then the activated production of PI3-K can leadto the translocation of PKB from cytoplasm to plasma membrane, and phosphorylateit's Ser473 and Thy308. And the activation of PKB is an important requirement for cell survival. The activated PKB mainly acts on it's downstream substrates like mTOR,GSK-3 and so on,to exert it's biology effects widely. Additionally, it can launch thetranslation by altering the state of phosphorylation of P70^S6K. The phosphorylatedP70^S6K can urge the phosphorylation of 40s ribosomal protein S6 to launch thetranslation. Nevertheless, there is a dual effects on ERK1/2 and P38 responding toexercise. It can both enhance the sensibility of skeletal muscle to insulin and activateERK1/2 and P³⁸ directly (ameliorating the level of it's phosphorylation,the expressionof MAPK gene and increasing ERK1/2 and P³⁸ protein) to exert correspondingbiology effects.In summary, exercise can directly or indirectly act on PI3-K/PKB/mTOR andMAPK, and regulate the metabolic level of glucose by ameliorating the level of it'sphosphorylation and the expression of the protein and gene. However, because the aimof studies and the means of exercise were different, such as the diverse intensity,duration and frequency of exercise, the results were not the same. This study useddiverse intensity and recovery time of long-term endurance trained rats, to analyze theresponse of exercised rats to glucose tolerance; and observe the alteration ofPI3-K/PKB/mTOR and ERK1/2 and the level of phosphorylation,the gene and proteinof P³⁸. Moreover, we compared the feature and the rules of protein kinase signalingcascade among the rats of different intensity-exercise and recovery time. The objectiveis to provide the theoretical reference of molecular mechanism for the clinic therapy fordiabetes, and offer the experiential foundation of the exercise intensity selection for theprevention and clinic subsidiary therapy. And what's more, we made a furtherdiscussion about the exercise therapy, which cooperates with medicine, to determinethe basis of reasonable drug screen.Methods1.Glucose tolerance was analysed by using glucose tolerance test in each group ofrats. And the relation between the trained and rest control rats was also observed.2.Western blot was used to determine the effects of different exercise intensity andrecovery time on the expression of GLUT-4 protein and the phosphorylation of PI3-K,PKB,mTOR,ERK1/2,P³⁸,besides the change of those non-phosphorylation protein.Additionally, we analysed the characteristics of the phosphorylation level and the alteration of protein among each group of diverse exercise.3.RT-PCR was used to examine the effects of different intensity- exercise andrecovery time on the expression of GLUT-4,PI3-K,PKB,P70^S6K and P³⁸. And weobtained the rule of the expression alteration of each group of rats responding todifferent exercise.Results一,Glucose tolerance.Glucose tolerance test analyses indicated that serum levels of insulin in trainedrats were higher than those in the control rats (p＜0.05å’Œp＜0.01).Regular endurancetraining is associated with decreased blood sugar content in limosis condition. But itwas not significant.二,Western blot analyses1,Relative to the control rats, After 48 hours recovery, with injection of trypsinGLUT-4 protein expression in skeletal muscle of one-hour trained rats was significantlyincreased (p＜0.05), as well as after 24 hours recovery (p＜0.01). Similar to theone-hour trained rats, GLUT-4 protein expression of one and half-hour trained rats wasalso significantly increased (p＜0.05). After 48 hours recovery, GLUT-4 proteinexpression in skeletal muscle of one and half-hour trained rats was markedly higherthan that of one-hour trained rats.2,Compared with the control rats, the level ofphosphorylated PI3-K in skeletalmuscle of the one-hour trained rats after 24 hours recovery was significantly increased(p＜0.01), and the level of phosphorylated PI3-K in skeletal muscle of the one andhalf-hour trained rats after 48 hours recovery was significantly increased(p＜0.01) too.However, the level of phosphorylated PI3-K trended downward markedly in skeletalmuscle of one-hour trained rats after 48 hours recovery(p＜0.05). Respectively, inskeletal muscle of the one-hour trained rats after 24 hours recovery and the onehalf-hour trained rats after 24 hours recovery, total contents of PI3K protein wassignificantly higher than that of the control rats(p＜0.01, p＜0.05). In skeletal muscleof the one-hour trained rats, the difference of the phosphorylated PI3-K level betweenafter 24 hours recovery and after 48 hours recovery was significant (p＜0.05). And inskeletal muscle of the one and h alf-hour trained rats, the phosphorylater PI3-K level after 48 hours recovery was greater compared with after 24 hours recovery (p＜0.05).In trained rats, the phosphorylater PI3-K protein expression in skeletal muscle after 24hours recovery was higher than after 48 hours (p＜0.05, p＜0.01), and that of the oneand half-hour trained rats after 24 hours recovery was higher than that of the one-hourtrained rats after 24 hours recovery(p＜0.05).3,The level ofphosphorylated PKB was increased in all trained rats(p＜0.05,p＜0.01). PKB protein content analyse indicated that PKB protein content in the one andhalf-hour trained rats after 24 hours recovery was markedly greater compared with thecontrol rats, as well as after 48 hours recovery (p＜0.01,p＜0.05). In trained rats, PKBprotein content in the one and half-hour trained rats was higher than in the one-hourtrained rats (p＜0.05). At the same time, in one and half-hour trained rats, thedifference of PKB protein content between after 24 hours recovery and after 48 hoursrecovery was significant (p＜0.05).After 24 hours recovery, PKB protein content in theone and half-hour trained rats was significantly higher than that in the one-hour trainedrats(p＜0.01), similar to after 48 hours recovery(p＜0.01).4,In spite of exercise duration and recovery time, exercise training leaded tosignificantly increased level of phosphorylated mTOR(p＜0.01,p＜0.05). The analyseresult of the protein content of mTOR was similar to the level of phosphorylatedmTOR(p＜0.01,p＜0.05). In trained rats, after 24 hours recovery, the level ofphosphorylated mTOR was greater than after 48 hours recovery(p＜0.05). In one-hourtrained rats, the protein content of mTOR after 24 hours recovery was higher than after48 hours recovery. At the same time, after 48 hours recovery, the protein content ofmTOR in one and half-hour trained rats was different from that in one-hour trainedrats(p＜0.05).5,After 24 hour recovery, the level of phosphorylated P70^S6K in the one-hourtrained rats was different from that in the control rats(p＜0.01), similar to after 48 hourrecovery (p＜0.05). We found the similar result in the one half-hour trained rats (p＜0.01). In one-hour trained rats, the level of phosphorylated P70^S6K after 24 hoursrecovery was different from after 48 hours recovery (p＜0.05). After 48 hours recovery,the level of phosphorylated P70^S6K in one and half-hour trained rats was greater thanthat in one-hour trained rats (p＜0.05). 6,In spite of recovery time, the level of phosphorylated ERK 1/2 in theone-hour trained rats was different from that in the control rats (p＜0.05), as well as inthe one and half-hour trained rats (p＜0.01). In one and half-hour trained rats, theprotein content of ERK 1/2 after 24 hours recovery was different from that in thecontrol rats (p＜0.05), as well as after 48 hours recovery (p＜0.01). In the one-hourtrained rats, the level of phosphorylated ERK 1/2 after 24 hours recovery was higherthan after 48 hours recovery (p＜0.05), and after 48 hours recovery, that in one andhalf-hour trained rats was higher than that in one-hour trained rats (p＜0.01). In oneand half-hour trained rats, the protein contents of ERK 1/2 had difference between after24 hours recovery and 48 hours recovery (p＜0.01), and that in one and half-hourtrained rats was higher than in one-hour trained rats (p＜0.01).7,Compared with the control rats, after 24 hours recovery, the level ofphosphorylated P³⁸ in the one-hour trained rats was increased (p＜0.05). Regardless ofrecovery time, the level of phosphorylated P³⁸ in one and half-hour trained rats washigher than that in the control rats(p＜0.01,p＜0.05). After 24 hours recovery, P³⁸protein expression in one and half-hour trained rats was lower than that in the controlrats (p＜0.05). The level of phosphorylated P³⁸ of the one-hour trained rats after 24hours recovery was higher than that after 48 hours recovery(p＜0.05). The level ofphosphorylated P³⁸ of the one and half-hour trained rats after 24 hours recovery washigher than that after 48 hours recovery(p＜0.01). However, after 24 hours recovery,the levels of phosphorylated P³⁸ had difference between the one-hour trained rats andthe one and half-hour trained rats(p＜0.01). After 48 hours recovery, the level ofphosphorylated P³⁸ in one and half-hour trained rats was higher than that in one-hourtrained rats. In one and half-hour trained rats, P³⁸ protein expression after 24 hoursrecovery was reduced, and it recovered after 48 hours recovery.三,RT-PCR analysis1,After 24 hours recovery, the expression of GLUT-4 mRNA in one-hourtrained rats was significantly different from that in the control rats(p＜0.01). In the oneand half-hour trained rats, the expression of GLUT-4 mRNA after 24 hours recovery was significantly increased(p＜0.01), as well as after 48 hours recovery(p＜0.05). In allthe trained rats, regardless of exercise duration, the expression of GLUT-4 mRNA after24 hours recovery was markedly higher than after 48 hours recovery (p＜0.05, p＜0.01). At the same tine, regardless of recovery time, that in one and half-hour trainedrats was higher than that in one-hour trained rats (p＜0.05).2,After 24 hours recovery, the expression of PI3-k mRNA in trained rats wasincreased (p＜0.05). In trained rats, in spite of exercise duration, the expression ofPI3-k mRNA after 24 hours recovery was higher than after 48 hours recovery (p＜0.05).3,Compared with the control rats, the expression of PKB mRNA of theone-hour trained rats and the one and half-hour trained rats after 24 hours recovery wassignificantly increased(p＜0.01). In one-hour trained rats, the expression of PKBmRNA after 24 hours recovery was higher than after 48 hours recovery(p＜0.05), andin one and half-hour trained rats, we found the similar results (p＜0.01).4,After 24 hours recovery, the expression of P70^S6K mRNA in the one-hourtrained rats was significantly different from that in the control rats (p＜0.05). In the oneand half-hour trained rats, the expression of P70^S6K mRNA after 24 hours recovery wasincreased(p＜0.01), as well as after 48 hours recovery(p＜0.01). Regardless of exerciseduration, the expression of P70^S6K mRNA after 24 hours recovery was markedly higherthan after 48 hours recovery(p＜0.05). After 24 hours recovery, the expression ofP70^S6K mRNA in the one and half-hour trained rats was higher than that in the one-hourtrained rats, as well as after 48 hours recovery(p＜0.01).5,One and half-hour exercise leaded to the increased expression of ERK2mRNA not only after 24 hours recovery (p＜0.01) but also after 48 hours recovery (p＜0.05). After 24 hours recovery, the expression of ERK2 mRNA in the one-hour trainedrats was increased too(p＜0.05). In one-hour trained rats, the expression of ERK2mRNA after 24 hours recovery was higher than after 48 hours recovery(p＜0.05). Onthe contrary, in one and half-hour trained rats, the expression of ERK2 mRNA after 48hours recovery was higher than after 24 hours recovery(p＜0.05). After 48 hours recovery, the expression of ERK2 mRNA in the one and half-hour trained rats washigher than that in the one-hour trained rats(p＜0.05).6,After 24 hours recovery, the expression of P³⁸ mRNA in one-hour trained ratswas significantly different from that in the control rats(p＜0.01), and we found similarresult in one and half-hour trained rats. In one-hour trained rats, the expression of P³⁸mRNA after 24 hours recovery was higher than after 48 hours recovery(p＜0.05), aswell as in one and half-hour trained rats(p＜0.05).Conclusion1.Exercise can ameliorate the sensitivity to insulin and increase the expression ofmRNA and GLUT-4 protein in rats.2.Exercise can increase the sensitivity of skeletal muscle to insulin in rats, andenhance the level of phosphorylation of P70^S6K and PI3-K/Akt/mTOR signalingpathway, promoting the expression of the protein and gene.3.Exercise can increase the level of phosphorylation of ERK1/2 and the expressionof the protein, and increase the expression of ERK2 mRNA; and can also increase thelevel of the phosphorylation and expression of P³⁸, but high-intensity exercise canreduce the expression of P³⁸ protein.4.Exercise can increase GLUT-4 protein, improve glycometabolism and regulatethe balance of blood glucose, through the insulin receptor PI3-K/Akt/mTOR andMAPK signaling pathway.