Role Of AMPK/mTOR Signaling In Mediating The Physiological Process Of Exercise Induced Insulin Sensitization

Objective: Aerobic exercise has been wildly used as a method to treat IR and metabolic syndrome. While the precise mechanisms still need to be determined underlying the process of exercise mediated insulin sensitization. AMPK and m TOR signaling is an essential signaling pathway involved in nutrients and energy regulation, and plays an important role in development of IR and obesity. Recent studies proved that AMPK/m TOR signaling is also an exercise sensitive pathway. This study aims to figure out the role of AMPK/m TOR signaling in aerobic exericise mediated insulin sensitization and its underlying mechanism. m TOR functions in celluar metabolism by regulating fatty acid anabolic metabolism. On the other hand, AMPK/ m TOR also mediated autophagy regulation of several factors, like growth factors, nutrients, stress, and energy status. Therefore, our study focused on the role and underlying mechanisms of AMPK/m TOR in lipid metabolism and autophagy regulation in mediating exercise induced insulin sensitization.Methods: In vivo and in vitro studies were performed to explore the role of AMPK/m TOR/S6K1 in skeletal muscle lipid metabolism and autophagy regulation and its role in mediating exercise induced insulin sensitization. 1. Animal Model 5 weeks old C57BL/6 mice were divided into control group and HFD-feeding group randomly, and were fed normal chow and high fat diet respectively for up to 10 weeks. Then the mice showed IR symptoms in IR model group were divided into high fat diet control(HC) and high fat diet exercise group(HE), both of which continued to be fed with high fat diet. 2. Mice phenotype Fasting serum insulin level was determined by insulin ELISA kit; Serum total cholesterol and triglyceride level were determined by CHOD-PAP method; 16-hour fasted mice were given 20% glucose by gavage through a gastric tube, which was inserted in the stomach. Blood samples were measured by One Touch at 15, 30, 60and 120 min after glucose administration. 3. Expression profile of skeletal muscle by using microarray analysis Total RNA was extracted with TRIZOL reagent, and puri?ed with RNeasy Mini Kit. The 32 K Mouse Genome Array was constructed by Capital-Bio Corporation. Statistical Analysis of Microarray was then performed. Oligonucleotide microarray was applied to analyze the effect of aerobic exercise and HFD at the transcriptional level, and selected genes were confirmed by real-time PCR. 4. Tissue culture and treatment Myotubes were induced by treating cells with 2% equine serum DMEM. si RNA was used to knockdown S6K1 expression; Rapamycin was used to inhibit m TOR activity; Autophagy level was measured by transfecting cells with GFP-LC3 plasmid; 3-MA was used to inhibit autophagy acitivity; Palmitate was used to saturate C2C12. 5. Experimental methods Real-Time PCR was used to measure gene expression; Western Blot was used to detect protein expression; 2-NBDG method was used to measure glucose uptake; IP was used to measure the bingding rate between proteins.Results: 1. Effects of long term HFD and exercise on mice metabolism Body weight, FIN, serum TC and TG level were higher in HC mice, while 6 weeks of aerobic exercise reversed this trend. OGTT showed exercise also reversed long term HFD-feeding induced glucose intolerance. 40 differently expressed genes that involved in exercise induced insulin sensitization by analyzing microarray data. Fold-change of the RT-PCR results correlated to the fold-change reported by the microarray. 2. Role of AMPK/m TOR mediated lipid metabolism in exercise induced insulin Sensitization Palmitate increased acitivity of m TORC1 and AKT signaling in C2C12 cells, while IRS1 activity significantly decreased; IRS1/AKT signaling significantly increased in by knocking down S6K1. While in palmitate pretreated C2C12 cells, knocking down of S6K1 doesn’t affect activity of AKT, indicating that m TORC1 and IRS1 signaling are not involved in the process of palmitate inducecd AKT acitivation.Palmitate inhibis the acitivity of AMPK; In palmitate treated C2C12 cells, activation of AMPK by using AICAR caused decreased activity of m TORC1 and AKT signaling; Inhibition of AMPK by treating cells with Compound C increased acitivity of m TORC1 and Akt signaling. While, knocking down of S6K1 doesn’t affect activity of AKT in response to Compound C treatment, indicating that m TORC1 is not involved in AMPK induced AKT inhibition. Long term saturation of palmitate caused increased SREBP-1 cleavage and activation, while acitivation of AMPK inhibits activity of SREBP-1. On the other hand, knocking down of S6K1 significantly decreased expression of SREBP-1c, and blocked the inhibition of AMPK induced SREBP-1c activation. In vivo study, we found that long term of HFD-feeding increased acitivity of SREBP-1c, while 6 weeks of exercise reversed this trend. Activation of AMPK increased expression of CPT-1 in palmitate pretreated C2C12 cells, and knocking down of S6K1 doesn’t affect the protein expression of CPT-1; Knockdown of S6K1 caused decreased expression of ACC protein, one of SREBP-1 downstream targets. 3. Role of AMPK/m TOR mediated autophagy regulation in exercise induced insulin Sensitization Palmitate treatment promote the activity of autophagy, and glucose uptake in C2C12 cells, whild addition of autophagy inhibitor 3-MA caused significantly decreased glucose uptake. Inhibition of AMPK by using Compound C caused decreased activity of autophagy, while on the other hand, 3-MA also decreased activity of AMPK. 1 hour of exercise increased the acitivity of autophagy and AMPK signaling in skeletal muscle. While the activity of autophagy was significantly lower in AMPKα2-/- mice than wild type mice, indicating that AMPKα2 plays an essential role in exercise induced autophagy acitivation. Long term of aerobic exercise also increased expression of SESN2 and SESN3, and a single cute of exercise increased the binding rate between AMPK and SESN2/3.Conclusion: 1. 6 weeks of aerobic exercise ameliorated long term HFD-feeding induced insulin resistance in C57/BL6 mice. 2. AMPK inhibits lipogenesis by inhibiting the cleavage and activation of transcriptional factor SREBP-1 through m TORC1/S6K1 signaling. 3. Exercise induced binding between AMPK and Sestrins maybe involved in AMPK mediated metabolism adaption and autophagy activation in skeletal muscle.