| Objective: The long-term nutrient overload and physically inactivity lead to obesity,type 2 diabetes and a series of metabolic disease. Collectively, some data from epidemiologically research suggest that individuals with obesity,type 2 diabetes display a varying of degree of IR. Accumulating evidences also have revealed that increased dysfunction in glucose uptake is frequently considered to be the major cause of IR at least partially, thus the ways to enhance glucose transport became urgent issues to be solved. Moreover recent studies indicate that AS160, a Rabase-binding protein, which is a downstream substrate of Akt, plays an important role in mediating GLUT4 trafficking during Insulin-PI3K-Akt/AS160 signaling, however it is impaired in some individuals with obesity or type 2 diabetes. Of particular interest, besides insulin signal, the role of AS160 activation involving in GLUT4 trafficking is increased by many stimuli. It is well known that aerobic training can attenuate IR, whereas the underlying mechanism is not clear. Considering the potential role of AS160 on GLUT4 trafficking, here we studied the effect of AS160 on aerobic training improving the glucose transport by observing changes in expression of genes and proteins in normal and IR mice skeletal mucsle involving in Akt/AS160/GLUT4 trafficking signaling, aiming to provide some theoretic evendience for prevention and treatment of IR and relative metabolic abnormalities through activity.Methods: (1) Creating mouse model of insulin resistance: First, 80 male, 4-week old C57BL/6 mice were divided into normal chow group and high-fat diet group randomly, each group has 40 animals fed by normal chow and high fat diet respectively. 10 weeks later, The normal chow control group was randomly divided into normal chow diet control (NC) and normal chow exercise group (NE), meanwhile mice from high fat diet group which have insulin resistance symptoms continued to be fed with high diet and were randomly allocated to one of the following groups: high-fat diet control (HC) and high-fat diet exercise training (HE) during the subsequent 6-week period. (2) Exercise protocol: Exercise training consisted of motorized treadmill running for 1h/day, 5 days/week at an intensity of 75%VO2max. (3) Measurement of fasting serum insulin level: After experiment period, animals were fasted for 16 hours before undergoing bleeding for the measurement of fasting serum insulin level. Mice were anesthetized by surgically perfusion with chloral hydrate. Blood sample was draw by removing eye ball, centrifuged to gain the extract of serum. Insulin concentration was measured by ELISA. (4) Oral glucose tolerance test (OGTT): Mice were fasted for 12 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, T120 and T180 Min were measured respectively. (5) RT-PCR technology was used to detect Akt,AS160 and GLUT4 mRNA expression in skeletal muscle. (6) Western blot was used to detect Akt,pAkt-Ser473,AS160,pAS160-Thr642 and GLUT4 protein expression in skeletal muscle. (7) Immunofluorescence to detect Akt,pAkt-Ser473,AS160,pAS160-Thr642 and GLUT4 : 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. Cofocal microscope was used to observe the expression of each protein in skeletal muscle sections. Results: (1) Change of body weight: Body weight was increased by high-fat feeding (p<0.01) but was decreased by exercise training (p<0.05). Comparing to NC group, the body weight of the mice in HC group was increased by 21.99% (P<0.05), while it was decreased by 22.75%(P<0.01) in HE . (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 elevated 77.19% (P<0.01) in HC(P<0.001vs NC group ) but decreased 25.33% in HE group((P<0.05 vs 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 Min was still higher than that of fasting value. (4) The expression of Akt2 mRNA and protein and Akt phosphorylation protein at Ser473 in skeletal muscle: Our results showed the expression of Akt2 mRNA (79.49±4.95 vs 100.00±0.00, P<0.01) and protein (84.77±4.94 vs 100.00±0.00, P<0.01) as well as pAkt-Ser473 protein level (81.53±6.36 vs 100.00±0.00, P<0.01) were significantly decreased in HC group comparing with NC group, but increased respectively in Akt2 mRNA(92.20±4.49 vs 79.49±4.95, P<0.01)and protein(94.63±4.67 vs 84.77±4.94, P<0.05)and phosphorylation expression(98.82±4.80 vs 81.53±6.36, P<0.05) in HE. The same result has been proved by Immunofluorescence staining. (5) The expression of total AS160 mRNA and protein and AS160 phosphorylation protein at Thr642: we found a significant decrease in PAS160-Thr642 protein level ((69.62±7.44 vs 100.00±0.00, P<0.001), while total AS160 mRNA (107.89±9.44 vs 100.00±0.00, P>0.05) and AS160 protein(109.25±8.00 vs 100.00±0.00, P>0.05)level were similar between HC group and NC group, however increased in PAS160-Thr642 protein(85.18±6.94 vs 69.62±7.44, P<0.01)through total AS160mRNA (105.53±7.59 vs 107.89±9.44, P>0.05) as well as AS160 protein (102.22±3.36 vs 109.25±8.00, P>0.05)expression of HE group was the same compared with HC group were observed as well. The results of the expression of total AS160 protein and the pAS160-Thr642 protein by Immunofluorescence staining also support our result from Western Blot. (6) The expression of GLUT4 mRNA and protein in skeletal muscle: The expression of GLUT4 mRNA(84.21±5.20 vs 100.00, P<0.01)and protein(80.40±3.90 vs 100.00, P<0.01)were significantly decreased in HC group compared to NC group but increased in GLUT4 mRNA(95.20±7.19 vs 84.21±5.20, P<0.05)and protein(94.04±8.34 vs 80.40±3.90, P<0.05)expression in HE group were shown respectively. This result also further confirmed by Immunofluorescence.Conclusion:1. The dysfunction of Akt/AS160 signaling pathway contributes to high-fat diet induced IR in C57BL/6 mice.2. Aerobic training can promote the activity of Akt/AS160 signaling pathway in skeletal muscle of both normal and IR mice by increaring phosphorylation expression of Akt/AS160, whereas the expression of translation of GLUT4 is elevated. Our results revealed that 10-week high-fat diet induced IR in C57BL/6 mice,while 6-week treadmill training could improve skeletal muscle glucose metabolism in mice, which attenuating insulin resistance.3. 6-week aerobic training on mice could significantly promote skeletal muscle insulin sensitivity, accordingly aerobic training may act as a complementary therapy for the treatment of IR. |
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