| Objective: To investigate the role of mTOR/S6K1 signaling pathway in the process of insulin resistance, high fat diet were used to induce insulin resistance in C57BL/6 mice. The effects of aerobic exercise on insulin resistant mice were studied by observing the mRNA and protein expression of mTOR, S6K1, a downstream effector of mTOR, and PGC-1αin skeletal muscle of normal control and insulin resistance C57BL/6 mice. The possible mechanisms of aerobic exercise improving insulin resistance are discussed.Methods: (1) Creating mouse model of insulin resistance: First, 40 male, 8-week old, C57BL/6 mice were divided into two groups: which are normal chow and high-fat diet groups, each group has 20 animals feeding by normal chow and high fat diet respectively. 8-week later, mice from high fat diet group were proved to have insulin resistance symptoms and after that time point the mice were regrouped. The normal chow group was randomly divided into normal chow diet control group (NC) and normal chow exercise group (NE), and the high fat diet group was randomly divided to high fat diet control group (HC) and high fat diet exercise group (HE). (2) Exercise protocol: mice were acclimatized to the motorized treadmill by running 60 min per day at the intensity of 75%VO2max five days per week for 6 weeks. (3) Measurement of fasting serum insulin level: Mice were fasted for 12 hours before bleeding. Mice were anestheszed by ether inhalation. Blood sample was draw from inner canthus, centrifuged to extract serum. Insulin concentration was measured by ELISA. (4) Oral glucose tolerance test (OGTT): Mice were fasted for 14 to 16 hours before experiment. The body weight of each mouse was measured and documented. The serum fasting glucose concentration was measured by using One Touch meter at the time point of T0. Immediately 20% glucose solution was rapidly perfused via mouth based on body weight at 10μl/g, Blood glucose concentration at T15, T30, T60, T90, T150 and T180 min were measured respectively. (5) Morphology of pancreatic islet: The pancreas of each mouse was removed and fixed in 10% formalin buffer. Paraffin block and sections were made. H&E staining was performed and the morphological changes were evaluated under microscope. (6) Northern blot to detect mTOR,S6K1 and PGC-1αmRNA expression in skeletal muscle. (7) Western blot to detect mTOR,S6K1,pS6K1-Thr389 and PGC-1αin skeletal muscle. (8) Immunofluorescence to detect mTOR,S6K1,pS6K1-Thr389: a square of skeletal muscle from each mouse was fixed by 4% paraformadehyde, dehydrated with 30% sucrose and embedded by OCT. Frozen sections were cut and stained by blotting with specific antibody. Co focal microscope was used to observe the expression of each protein in skeletal muscle sections.Results: (1) Changes of body weight: compare to NC group, the body weight of the mice in HC group was increased by 21.99% (P<0.05), while mice in HE group was decreased by 22.75%(P<0.01)comparing with that of HC group. (2) Fasting serum insulin level:The serum fasting insulin level was altered significantly by either high fat diet or aerobic exercise and was also affected by high fat diet plus exercise. Fasting serum insulin level was 181.81% (P<0.01) higher in HC group comparing with NC group and 41.9% (P<0.05) lower in HE group comparing with HC group. (3) OGTT: The peak value of OGTT curve is higher in HC group than NC group, and the time point of peak value shifted back, and blood glucose level decreased slowly after 30 minutes. Furthermore the glucose level at T180 min was still significantly higher than that of fasting level. However, by comparing with HC group, we found that the OGTT peak value of HE group was decreased significantly and the time point of OGTT peak shifted forward, at mean time the blood glucose level decreased at all time points. However, the blood glucose value at T180 was still higher than that of fasting value. (4) The morphology changes of pancreatic islet: the percent of pancreatic islet mass area was increased by 85.71% (P<0.001) in the HC group compared with NC group and decreased by 30.77% (P<0.05) in the HE group compared with HC group. Moreover, the inflammatory reaction could be seen around pancreatic islet sections of the mice from HC group, while the degree of inflammatory reaction was decreased after 6-week aerobic exercise as seen in HE group. (5)The expression of mTOR mRNA and protein in skeletal muscle: Our results showed the expression of mTOR mRNA (125.61±10.43 vs 100.00, P<0.05) and protein (137.41±7.86 vs 100.00, P<0.01) were significantly increased in HC group comparing with NC group and decrease could be seen on mTOR mRNA(105.17±4.10 vs 125.61±10.43, P<0.05)and protein(111.00±7.33 vs 137.41±7.86, P<0.05 in HE group when comparing with HC group. Immunofluorescence staining has also proved the same result. (6) The expression of total S6K1 mRNA and protein and S6K1 phosphorylation protein at Thr389: we found an significant increase in total S6K1 mRNA (154.98±16.26 vs 100.00, P<0.01) and protein (137.36±3.08 vs 100.00,P<0.01) as well as pS6K1-Thr389 protein (390.15±69.62 vs 50.59±16.65,P<0.001)expression of HC group comparing with NC group and decrease in S6K1 mRNA(120.85±2.42 vs 154.98±16.26, P<0.05)and protein (102.51±19.77 vs 137.36±3.0, P<0.05) as well as pS6K1-Thr389 (179.55±35.70 vs 390.15±69.62, P<0.05)expression of HE group compared with HC group were observed as well. The results of the expression of total S6K1 and the pS6K1-Thr389 by immunostaining also support our result from Western Blot. (7) The expression of PGC-1αmRNA and protein in skeletal muscle: expression of PGC-1αmRNA(27.99±2.45 vs 100.00, P<0.001) and protein(52.02±2.13 vs 100.00, P<0.01)were significant increased in HC group comparing with NC group and decreased in PGC-1αmRNA(63.90±7.80 vs 27.99±2..45, P<0.01)and protein(91.97±6.24 vs 52.02±2.13, P<0.05)expression of HE group were observed by comparing with HC group. Our immunostaining discovery further confirmed our results. 2. Aerobic exercise inhibits the activity of skeletal muscle mTOR/S6K1 signaling pathway in IR mice by decreasing the expression of mTOR and S6K1 mRNA and protein expression, whereas the expression of PGC-1αis elevated. Our results implicate that 6-week aerobic exercise on mice could improve skeletal muscle energy catabolism and inhibit anabolism.3. 6-week aerobic exercise on mice could significantly enhance insulin sensitivity supporting the hypothesis that aerobic exercise could be used as the means to prevent the development of insulin resistance.
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