| Objective:Insulin resistance(IR)is important pathophysiological basis of type 2 diabetes mellitus(T2DM).Aerobic exercise has been used in clinical treatment of obesity and T2 DM due to its improvement on energy metabolism.However,the exact mechanism of aerobic exercise improving the homeostasis of energy metabolism is not clear.Histone deacetylase(HDAC)plays key roles in regulating cellular genes’ transcription balance.Studies showed that HDAC 4/5 could significantly regulate the skeletal muscle energy metabolism.Furthermore,aerobic exercise could significantly reduce the expression of HDAC 4/5 in mice.Scriptaid,as a type II HDAC inhibitor,has been found to have the effect of simulating exercise,which may be related to its inhibition of HDAC and to its regulation of acetylation to intracellular proteins,but it is still not clear which proteins may Scriptaid specifically affects.Compared with phosphorylation and methylation modification,the research on acetylation remains stagnant.With the development of high-throughput technology,there is no technical barrier for large-scale acetylation modification nowadays.In the last 10 years,the research results show that acetylation plays an important role in the regulation of metabolism,it regulates various metabolic enzymes’ activity.There are gathering evidences show that acetylation generally modifies metabolic enzymes’ activity and then regulate the energy metabolism pathway.In conclusion,this study explored the effect of scriptaid on acetylation modification of key enzymes of glucose and lipid metabolism during the process of aerobic exercise improving skeletal muscle energy metabolism,in order to reveal that acetylation modification can improve energy metabolism of the body and provide new targets for the prevention and treatment of metabolic diseases such as IR and T2 DM.Methods:(1)Animal model: 27 C57BL/6 male mice of 5 weeks old were randomly divided into quiet control group(C),exercise group(E)and scriptaid group(S)after one week of adaptive feeding.After the exercise intervention and drug administration intervention,the aerobic capacity test,muscle strength test and body composition analysis were carried out,and then the mice were killed.(2)Exercise protocol: the mice in the exercise group took 6 weeks of aerobic running exercise with 0 gradient,the exercise intensity was 75% VO2 max,60 min/time,1 time/day,5 times/ week.(3)Administration protocol: the mice in the scriptaid group were intervened for 6 weeks,and were intraperitoneally injected with scriptaid(dissolved in normal saline containing 5% DMSO)at a dose of 1 mg/kg,1 time/day,5 times/ week.(4)Aerobic capacity test: randomly select three mice from the quiet control group and scriptaid administration group to carry out the continuous treadmill exercise with a gradient of0 and a speed of 12 m/min,record the time when the mice can no longer exercise from exhaustion to dehydration.(5)Muscle strength test: place the mice on the test platform of yls-13 a mice muscle strength tester,after the mice hold the fixed bar on the test platform by the four limbs,pull the tail of mice backward to drive the test platform backward,record the muscle strength reading,and repeat 4 times.(6)Body composition test: after the exercise intervention,we used the "Impedi Vet experimental animal composition analyzer" to measure the body fat content,free fat body weight,body mass index and other indicators of mice,and then statistical analysis was carried out.(7)OGTT test: the mice were forbidden to eat at 7:00 p.m.the day before test,but they could drink freely.The tail vein blood was taken at 8:00a.m.the next day to measure fasting blood glucose;20% glucose normal saline was used to gavage through intragastric administration at a dose of 1 ml/kg body weight,and the blood glucose values at the time points of 15 minutes,30 minutes,60 minutes and 120 minutes after gavage were recorded and analyzed statistically.(8)Determination of acetylated protein: protein extraction,trypsin digestion,TMT Labeling,HPLC fractionation,affinity enrichment,LC-MS / MS analysis and database retrieval.(9)Computer molecular docking simulation: We use autodock v4.2.6 software to simulate the molecular docking of the target protein and its small molecular substrate,record and analyze the docking results.Results:(1)6-week aerobic exercise and scriptaid administration can enhance the muscle strength and the activity of citrate synthetase in skeletal muscle of mice,and scriptaid administration can significantly prolong the exhausted running time;6-week aerobic exercise can significantly reduce the body weight,body fat content and body mass index of mice and increase the feeding efficiency of mice,but scriptaid has no effect on the body weight,body fat content and body mass index of mice.(2)6-week aerobic exercise significantly reduced the content of total cholesterol,triglyceride,insulin and free fatty acid in serum of mice,and the OGTT curve was significantly lower than that of the control group at 15,30 and 60 minutes,the area under the OGTT curve was significantly reduced in the exercise group,and exercise significantly improved the glucose tolerance of mice;Scriptaid did not affect serum cholesterol and triglyceride,but significantly reduced serum insulin and free fatty acid content,and OGTT curve was significantly lower than that of the quiet control group at 15,30 and 60 minutes,the area under OGTT curve was significantly reduced,scriptaid significantly improved glucose tolerance in mice.(3)Exercise and scriptaidadministration caused different changes in acetylation modification of a large number of proteins in skeletal muscle of mice: exercise significantly changed the key enzymes of glucose metabolism,such as Pfkm,Pkm,Pgk1,Pdha1,Idh1 and Ogdh.Exercise also changed the acetylation of Acadm,Hadha,Hadh and Acaa2,the key enzymes of lipid metabolism;acetylation of Pfkm,Pkm,Pgk1,Pdha1,Idh1,Ogdh,Acadm,Hadha,Hadh and Acaa2 were significantly changed by Scriptaid.(4)The results of computer molecular docking simulation experiment: Pfkm,144 K acetylation blocked its binding with substrate frutose 6-phosphate;Pgk1,139 k acetylation blocked its binding with substrate 1,3-diphosphoglycerate,275 k acetylation did not affect its binding with substrate 1,3-diphosphoglycerate;Pdha1,147 k acetylation blocked its binding with substrate pyruvate;Pkm,206 K acetylation enhanced its binding with substrate ADP,and its binding with substrate phosphoenolpyruvate was unchanged;IDH1,its binding to NADP + was strengthened after acetylation of 81 k,;Ogdh,564 k acetylation enhanced its binding to2-oxoglutarate,while 389 k acetylation had no effect on its binding to 2-oxoglutarate;Acadm,179 K acetylation weakened its binding to hexanoyl Co A;Hadha,728 K acetylation hindered its binding to substrate 2-trans-hexenoyl Co A;Hadh,81 K acetylation blocked its binding to 3S-hydroxyhexanoyl Co A;Acaa2,137 K acetylation blocked its binding to substrate 3-oxohexanoyl Co A.Conclusions:(1)Long term aerobic exercise and scriptaid administration can significantly enhance aerobic exercise capacity,improve skeletal muscle glucose tolerance and relieve insulin resistance.(2)Long term aerobic exercise and scriptaid significantly enhanced the acetylation of titin protein in sarcomere and affected the acetylation of titin interacting protein in skeletal muscle.(3)Long term aerobic exercise and scriptaid administration can significantly affect the acetylation modification level of some key enzymes in the pathway of glucose and lipid metabolism of skeletal muscle,and change their enzyme activity through acetylation modification,so as to affect the metabolism process catalyzed by the enzyme and improve the balance of glucose and lipid metabolism. |
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