Effects Of Exercise And Hydrogen Sulfide On Insulin Resistance Induced By CSE Deficiency In Skeletal Muscle And Its Mechanism

BackgroundDiabetes is one of the top ten causes of death in the world.With the increase of obesity rate,the incidence of diabetes is increasing year by year,and its medical care has increased the family burden and socio-economic costs.Diabetes is characterized by hyperglycemia and insulin resistance.Glucose homeostasis is very important for maintaining normal body function.In the postprandial state,skeletal muscle absorbs about 80%glucose.As one of the main organs of insulin response,skeletal muscle regulates the overall glucose metabolism.The study found that the content of hydrogen sulfide in plasma of diabetes patients was significantly reduced.Cystine-y-Lyase-y-Lysase(CSE)is one of the major hydrogen sulfide synthetases in skeletal muscle.The CSE/hydrogen sulfide system plays an important role in promoting muscle glucose uptake and regulating skeletal muscle insulin sensitivity.However,the current research on the mechanism of hydrogen sulfide in regulating glucose homeostasis is still limited.The role and possible mechanism of hydrogen sulfide in skeletal muscle insulin resistance have not been reported.Therefore,this study focuses on the role of CSE/hydrogen sulfide in regulating glucose homeostasis in skeletal muscle.Exercise is a routine prescription for the prevention and treatment of metabolic disorders such as insulin resistance.Exercise regulates insulin sensitivity of skeletal muscle.Does exercise play an important role in improving insulin resistance caused by muscle CSE/hydrogen sulfide deficiency?This hypothesis needs more research evidence to support.Purposes1.To verify the effect of CSE/hydrogen sulfide deficiency on the overall physiological function of mouse skeletal muscle;2.To verify the role and possible mechanism of exogenous hydrogen sulfide supplementation in improving insulin resistance induced by CSE deficiency in skeletal muscle;3.To verify the role and possible mechanism of exercise in improving insulin resistance induced by CSE deficiency in skeletal muscle.MethodsExperiment 1:The skeletal muscle conditional knockout CSE mice(MCSEKO mice)were established by Cre-LoxP technology.Q-PCR and Western blotting were used to determine the expression of CSE at mRNA and protein levels in skeletal muscle of MCSEKO andFlox mice,respectively.This study monitored the body weight and body composition development of MCSEKO mice and Flox mice aged 3-20 weeks;Food intake and body metabolism under normal diet and high-fat diet;And the muscle mass,muscle morphology,muscle fiber classification,exhausted exercise ability on the treadmill,grip strength,glucose tolerance and insulin tolerance of mice after 20 weeks of age.RNA-seq method was used to identify the molecular biological indicators of skeletal muscle changes in MCSEKO mice,and GO analysis and KEGG analysis were used to enrich the signal pathways that produced significant changes.Experiment 2:In this study,exogenous hydrogen sulfide donor GYY4137(50 mg/kg/day,intraperitoneal injection,lasting for 6 weeks)was used to supplement 14-20 week-old MCSEKO mice and Flox mice.The glucose tolerance and insulin tolerance of 20-weekold mice were monitored.Western blotting was used to determine the expression of insulin receptor substrate 1(IRS 1),p-IRS1,phosphoinositide-3 kinase(PI3K),p-PI3K,protein kinase B(Akt),p-Akt,glucose transporter 4(GLUT4),Protein Kinase G-1(PKG-1),mammalian target of rapamycin(mTOR),p-mTOR,Ribosome S6 protein kinase(S6K),pS6K,ribosomal protein S6(S6),and p-S6 at the protein levels in mouse skeletal muscle.Experiment 3:In this study,14-20 week-old MCSEKO mice and Flox mice were given aerobic treadmill training(speed:12 meters/minute,duration:50 minutes,five times a week,lasting for 6 weeks).The glucose tolerance and insulin tolerance of 20-week-old mice were monitored.Western blotting was used to determine the expression of p-IRS1,IRS1,p-PI3K,PI3K,p-Akt,Akt,GLUT4,PKG-1,p-mTOR,mTOR,p-S6K,S6K,p-S6,S6 in protein level in mouse skeletal muscle.Statistical analysis:All data are expressed as mean±deviation.The normal distribution was evaluated by Shapiro-Wilk test.The statistical significance is determined according to the sample distribution and variance uniformity.The two groups were compared by unpaired two-tailed Student t test or nonparametric test.For more than two groups,single factor analysis of variance(ANOVA)was performed first,and then multiple comparisons were performed afterwards.For a certain index that determines the subjects of the two groups,with the change of different time points,the two-factor repeated measurement analysis of variance is used for the inter-group comparison,and then the post-event multiple comparison is carried out.P<0.05 was considered statistically significant.ResultsExperiment 11.CSE was expressed in skeletal muscle of mice.Compared with Flox mice,the mRNA and protein levels of CSE in skeletal muscle of MCSEKO mice decreased significantly(p<0.05).2.Compared with Flox mice,MCSEKO mice did not have significant changes in body weight,body composition development,food intake and body metabolism,grip strength and treadmill exhausted exercise ability,muscle weight,muscle morphology,and muscle fiber classification under normal diet feeding conditions.3.A total of 275 significantly different genes were detected by RNA-seq assay.GO analysis significantly enriched signal pathways:selective autophagy signal pathway,insulin response signal pathway,insulin stimulated glucose uptake response signal pathway,oxidative stress signal pathway.KEGG analysis significantly enriched signal pathways:cGMP/PKG signal pathway,insulin signal pathway,PI3K-Akt signal pathway and mTOR signal pathway(p<0.05).4.The glucose tolerance test showed that compared with Flox mice,MCSEKO mice had no significant difference in glucose tolerance at the age of 10 and 15 weeks,but glucose tolerance was significantly weakened at the age of 20 weeks(p<0.05).Insulin tolerance test showed that compared with Flox mice,MCSEKO mice showed no significant difference in insulin tolerance at the age of 11 and 16 weeks,but showed significant insulin resistance at the age of 21 weeks(p<0.05).5.Under the condition of high-fat diet,compared with Flox mice,MCSEKO mice had no significant changes in food intake and body metabolism,but their body weight and fat weight were significantly increased,and showed significant impaired glucose tolerance and insulin resistance after 20 weeks of age(p<0.05).Experiment 21.The supplementation of exogenous hydrogen sulfide donor GYY4137 for six weeks significantly improved the glucose tolerance and insulin tolerance of 20-week-old MCSEKO mice(p<0.05).2.Compared with Flox mice,the insulin signal pathway proteins p-IRS1/IRS1,pPI3K/PI3K and p-Akt/Akt in skeletal muscle of MCSEKO mice were significantly downregulated,the protein expression levels of GLUT4 and PKG-1 decreased significantly,and the expression of p-mTOR/mTOR,p-S6K/S6K,and p-S6/S6 protein was significantly upregulated.The 6-week supplementation of exogenous hydrogen sulfide donor GYY4137 significantly improved the activation of insulin signaling pathway IRS1/PI3K/Akt in skeletal muscle of 20-week-old MCSEKO mice.At the same time,it increased the protein expression levels of GLUT4 and PKG-1 in skeletal muscle.Moreover,it alleviates the abnormal activation of mTOR/S6K/S6 signal(p<0.05).Experiment 31.The six-week aerobic treadmill training significantly improved the glucose tolerance and insulin tolerance of 20-week-old MCSEKO mice(p<0.05).2.The six-week aerobic treadmill training significantly improved the activation of insulin signal pathway IRS1/PI3K/Akt in skeletal muscle of 20-week-old MCSEKO mice,increased the protein expression levels of GLUT4 and PKG-1 in skeletal muscle,and alleviated the abnormal activation of mTOR/S6K/S6 signal(p<0.05).Conclusions:1.CSE was expressed in skeletal muscle of mice.The body weight,body composition,muscle morphology,exercise ability and body metabolism of MCSEKO mice were not significantly changed under normal diet.However,under the high-fat diet,or with the increase of age,MCSEKO mice showed obvious glucose tolerance and insulin resistance.Part of the mechanism may be that the lack of CSE in mouse skeletal muscle affects the normal transduction of skeletal muscle glucose uptake and insulin related signaling pathways.2.Exogenous hydrogen sulfide supplementation improved glucose tolerance and insulin resistance in 20-week-old MCSEKO mice.Part of the mechanism may be by upregulating the skeletal muscle insulin signaling pathway,increasing GLUT4 and PKG-1 protein levels,inhibiting abnormal activation of mTOR/S6K/S6 signaling,and thereby improving skeletal muscle insulin sensitivity and glucose uptake.3.6-week treadmill training can also alleviate insulin resistance in 20-week-old MCSEKO mice.Its mechanism of action may be partially through upregulating the IRS/PI3K/Akt signaling pathway,increasing GLUT4 and PKG-1 protein expression,and downregulating mTOR/S6K/S6 signaling transduction,thereby alleviating glucose intolerance and insulin resistance in 20-week-old MCSEKO mice.