The Influence Of Chrono On Skeletal Muscle Carbohydrate Metabolism And Aerobic Exercise Capacity

Objective: The purpose of this study was to screen skeletal muscle genes in response to the effects of aerobic exercise on glucose tolerance in Apelin knockout mice and to investigate the relationship between this gene and skeletal muscle carbohydrate metabolism.(1)In this study,glucose tolerance test(GTT)and eukaryotic transcriptome sequencing were used to observe the effect of aerobic exercise on glucose tolerance of Apelin KO mice and to screen relevant skeletal muscle genes.(2)Through the cell experiments,it researched the relationship of the selected gene--Chrono(computationally highlighted repressor of the network oscillator,Chrono)and carbohydrate metabolism in myoblast.(3)Through the animal experiments,it was investigated whether Chrono could affect the carbohydrate metabolism,glucose tolerance and aerobic exercise capacity of skeletal muscle of mice.Methods:(1)8-week-old C57BL/6J wild(WT)mice and Apelin KO mice were used,which were further divided into wild control group(WC),wild exercise group(WE),knockout control group(KC)and knockout exercise group(KE),with 20 mice in each group.In the exercise group,aerobic exercise with intensity of 70-75% VO2 max was performed 6 days a week,60 minutes a day.After 4 weeks of aerobic exercise,10 subjects in each group were randomly for the glucose tolerance test(GTT)and insulin tolerance test(ITT).The remaining 10 subjects were used only for the measurement of skeletal muscle Apelin m RNA and protein expression,as well as skeletal muscle transcriptome sequencing for screen and validation of key genes.(2)C2C12 cells were selected and transfected with Chrono plasmid to improve the expression of Chrono,and then divided into Chrono overexpression group(Chrono OE)and control group(Control).The glucose absorption assay measured the glucose absorption capacity of C2C12 cells.The relative expression of GLUT4,TBC1D1,PHKA1,GBE1,HK2,PFKM and PDP1 m RNA were measured by real-time quantitative PCR(RT-q PCR).(3)skeletal muscle-specific Chrono transgenic mice were made by Cre/Lox P technology.Chrono transgenic mice and C67BL/6N mice was further divided into Chrono transgenic group(Chrono TG)and wild-type group(WT).GTT was used to measure mice glucose tolerance.The multi-channel mouse spontaneous activity wheel system was used to measure the spontaneous activity of mice.The aerobic exercise ability of mice was measured by monitoring system of gas metabolism.The lactic acid analyzer measured the lactic acid value of the tail vein before and after the exercise with increasing load.Expression of GLUT4,TBC1D1,GYS1,PHKA1,GBE1,HK2,PFKM,PDP1,PDK4,MCT1,MCT4 and Chrono m RNA in skeletal muscles were measured by RT-q PCR.Glycogen kit,hexokinase(HK)activity kit and glycogen synthase(GYS)activity kit measured glycogen content,HK and GYS activity.Immunoprecipitation was used to measure the binding amount of Chrono and Bmal1 in mice.The contents of LDH-A and LDH-B proteins in mice were measured by western blotting.Results:(1)Compared with WC,the area under curve(AUC)of GTT of KC significantly increased and insulin tolerance decreased.After four weeks of aerobic exercise,KE showed significantly improved glucose tolerance and improved insulin tolerance compared with KC.And Chrono was selected as the key gene related to previous changes by eukaryotic transcriptome sequencing.(2)Compared with Control,the expression of GLUT4,TBC1D1,PHKA1,GBE1,HK2,PFKM and PDP1 m RNA and the glucose absorption capacity were significantly reduced of the presence/absence of insulin in Chrono OE.(3)The body weight and food intake of mice did not change between Chrono TG and WT.Compared with Control,the total counts and night state counts of cycling per day,the AUC of GTT,the speed,time and distance of exhaustion treadmill were significantly decreased in Chrono OE.Three minutes after the exercise of increasing load to exhaustion,the blood lactic acid content of Chrono TG was significantly higher than WT.Compared with WT,in the non-fasting condition,Chrono TG significantly increased blood glucose,while there was no significant change in plasma insulin.The amount of Chrono protein binding to Bmal1,muscle glycogen level,skeletal muscle PDK4 m RNA and LDH-A protein expressions in Chrono TG group were significantly higher than that in WT group.The skeletal muscle's m RNA expressions of GLUT4,TBC1D1,PHKA1,GBE1,HK2,PFKM,PDP1,MCT1 and MCT4,HK activity and LDH-B protein expressions were significantly lower than that of WT group.There was no difference in GYS1 m RNA expression and GYS activity between Chrono TG and WT mice.Conclusions:(1)4-week aerobic exercise improved glucose tolerance and insulin tolerance in Apelin KO mice.Chrono,a key skeletal muscle gene related to its changes,was screened by eukaryotic transcriptome sequencing.(2)Overexpression of Chrono gene can significantly reduce the m RNA expression of genes related to carbohydrate metabolism and the ability of C2C12 cells to absorb glucose.(3)Skeletal musclespecific Chrono overexpression may reduce the transcriptional activity of Bmal1 by increasing Chrono binding to Bmal1 protein,and inhibit the expression and activity of m RNA of genes related to glycogen decomposition,glucose aerobic metabolism and lactic acid transport,thereby causing skeletal muscle glycogen decomposition disorder,impaired glucose tolerance and decreased aerobic exercise capacity.