The Study Of Ub Mediated HDAC4/5 Degradation In The Process Of Aerobic Exercise Improving Skeletal Muscle Metabolism

Objective:With the economic development and the rising of people's living standards,the incidence of metabolic diseases such as obesity and type 2 diabetes has been rising year by year and the incidence of young people has become higher.This has brought huge economic burdens to families and society.Insulin resistance?IR?is an important pathophysiological basis of the above metabolic diseases.Its main manifestations are peripheral tissues and organs such as skeletal muscle,liver,and adipose tissue.The insulin sensitivity is reduced,resulting in normal levels of insulin unable to maintain normal blood glucose levels.It is well-known that aerobic exercise has an inhibitory effect on the occurrence and development of metabolic diseases such as obesity and IR,and can also significantly increases organize cellular insulin sensitivity and have a significant effect on the prevention of IR.However,the mechanism by which aerobic exercise increases tissue insulin sensitivity and prevents IR is not fully understood.Histone Deacetylase?HDAC?is an important gene transcription regulator in cells,and it maintains the dynamic balance of histone acetylation in the nucleus together with Histone Acetyltransferase?HAT?.In the nucleus,a DNA fragment with a certain length tightly surrounds the histone octamer and forms the basic structural unit of the cell chromatin,the nucleosome.HDAC regulates the deacetylation of histone octamers to loosen the structure of nucleosomes,which in turn dissociates DNA fragments from histone octamers and facilitates DNA specific binding related transcription factors and promoters to initiate the transcriptional process.HAT,on the other hand,plays the opposite role of HDAC.It is due to the existence of HAT and HDAC that the transcription of cell genes is maintained at equilibrium.HDACs fall into four types,including Type I,II,III,and IV HDACs.The results of previous studies showed that HDAC4/5 in type II HDAC can significantly inhibit the expression of genes related to glucose and lipid metabolism and thus affect the sensitivity of peripheral tissues such as the skeletal muscle and liver to insulin.At the same time,aerobic exercise significantly improved the expression of HDAC4/5 in skeletal muscle of insulin resistance mice,and decreased the inhibitory effect of HDAC4/5 on genes related to downstream glucose and lipid metabolism,thereby improving insulin resistance.This is further evidence that the improvement of insulin resistance by aerobic exercise may be achieved by reducing HDAC4/5.However,the mechanism by which aerobic exercise reduces the expression of HDAC4/5 in mouse skeletal muscle remains unclear.Previous studies found that HDAC4/5 protein expression were specific for muscle fiber type and were significantly higher in fast twitch fiber?Type II,glycolytic?fibers than in slow twitch fiber?Type I,oxidized?fibers.Protein degradation in tissue cells is mainly mediated by the Ubiquitin-Proteasome System?UPS?.Since the expression of HDAC4/5 gene were not significantly different in each muscle fiber type,the reason for the difference between different fiber types of skeletal muscle presumably due to different protein degradation rates.Therefore,long-term aerobic exercise reduced the soleus?Type I,Oxidized?of mice.The HDAC4/5 protein content may be related to the rate of protein degradation.Taken together,the present study was designed to investigate whether aerobic exercise reduces the expression of HDAC4/5 in soleus of mice through the ubiquitin-proteasome system and investigates the ubiquitin-proteasome system in aerobic exercise-induced mouse soleus HDAC4/5 The mechanism of action of expression in order to provide experimental basis for revealing the mechanism of aerobic exercise in preventing obesity,type 2 diabetes and other metabolic related diseases.Methods:?1?Animal model:Sixty,4-week-old C57BL/6 male mice,after a week of adaptive feeding,were randomly divided into a quiet group control group?C?,a quiet+MG132 group?C+M?,and exercise group?E?,Exercise+MG132 group?E+M?,after the end of exercise intervention,body composition analysis was performed,followed by death;?2?Exercise program:75%VO₂max intensity of aerobic treadmill exercise,1time/day,1 hour/time,5 times/week,lasting 6 weeks;?3?Dosage regimen:mice were given intraperitoneal injection of 20S proteasome inhibitor MG132 at a dose of 10?g/kg/d,1 time per day,5 times per week for 6 weeks;?4?Rodent Diet:Mice were feed with rodent diet purchased from Beijing Huafukang Company for 6 weeks;?5?Body composition analysis:After the exercise intervention,the body composition index?BMI?,body fat content?FM?,and fat free mass?FFM?of the ImpediVET test animal body were used to perform statistical analysis.;?6?Western blot was used to verify the inhibitory effect of MG132 on 20S proteasome;Myoglobin,troponin I?TNNI?,Cytochrome C?Cyto C?,MuRF1and HDAC4/5 were used to detect the soleus and gastrocnemius of mice.?7?Co-Immuneprecipitation?Co-IP?was used to detect the binding of HDAC4/5and ubiquitin in soleus from each group.Results:?1?Body composition analysis results for each group of mice:a.Body Weight:Compared with group C mice,body weight of group E was significantly decreased?P<0.05?,whereas body weight of mice in group C+M only increased.No statistical significance was found?P>0.05?;Compared with C+M mice,body weight of E+M mice was significantly decreased?P<0.05?,and E+M mice were compared with E mice.Body weight was significantly increased?P<0.05?;b.Fat Mass:Compared with mice in group C,fat mass in group E was significantly decreased after exercise?P<0.05?;compared with C+M group mice,fat mass of E+M mice was significantly lower?P<0.05?.Compared with E group,fat mass of E+M mice was also significantly lower?P<0.05?;c.Free Fat Mass:and C Compared with mice in the group,free fat mass of the mice in the E group was significantly increased after exercise?P<0.05?;compared with the C+M group mice,free fat mass of the E+M group mice was significantly increased?P<0.05?.Compared with group E,free fat mass of E+M group mice also increased significantly?P<0.05?;d.BMI:compared with group C mice,group B mice showed a decreasing trend,but no significant?P>0.05?;Compared with C+M mice,BMI in E+M mice was significantly lower?P<0.05?.Compared with E group,the BMI of E+M mice was also significantly lower?P<0.05?.?2?Western Blot detected markers associated with aerobic metabolism and HDAC4/5:The expression of Cytochrome C in the soleus of group E was significantly higher than that of group C?P<0.05?,but the Myoglobin and Troponin expression of soleus in group E was only comparable to that of group C.There was an increasing trend,but no significant?P>0.05?.In addition,the expression of Myoglobin,Troponin,and Cytochrome C in the gastrocnemius of E mice showed an upward trend,but no significant?P>0.05?.Compared with mice in group C,the expression of Troponin,Myoglobin,and Cytochrome C in the soleus of C+M mice was significantly decreased?P<0.05 or P<0.01?,but the C+M group of gastrocnemius only the expression of cytochrome C was significantly decreased?P<0.05?,while the expression of Troponin and Myoglobin showed only a downward trend,but no significant?P>0.05?;compared with E mice,the expression of Troponin,Myoglobin,and Cytochrome C in the soleus of the E+M group showed a downward trend,but there was no significant difference?P>0.05?.There was no significant change in protein and Cytochrome C expression;mice in the C+M group compared to the C group mice the expression of 20S proteasome in soleus was significantly decreased?P<0.05?.The expression of 20S proteasome in E group mice was significantly increased?P<0.05?;the expression of 20S proteasome in E+M group mice was significantly reduced compared with E group mice.?P<0.05?;compared with C mice,the expression of HDAC4/5 in the soleus muscles of E mice was significantly decreased?P<0.05?,and HDAC4/5 expression in the soleus muscles of C+M mice was significantly increased?P<0.05?;compared with E mice,the expression of HDAC4/5 in the soleus of E+M mice was also significantly increased?P<0.05?.However,there was no significant difference in the expression of HDAC4/5 in gastrocnemius of mice.The expression of MuRF1 in soleus was significantly increased in C+M and E groups compared with C group mice?P<0.05?.Compared to E group mice,MuRF1 was found in soleus of E+M mice.The expression was significantly increased?P<0.05?.?3?Co-IP detected the ubiquitination level of HDAC4/5:Compared with C group,the ubiquitination level of HDAC4/5 in the soleus of group E was significantly increased.In addition,the ubiquitination level of the HDAC4/5 in the soleus muscle of the C+M group was also significantly increased compared with the C group.;the ubiquitination level of the soleus muscle HDAC4/5 was significantly increased in the E+M group compared with the E group.?4?Real-time PCR detection of Glut4/Cpt1 expression in soleus muscle of mice:Compared with mice in group C,the expression levels of Glut4 and Cpt1 genes in the soleus of group E were significantly increased?P<0.05?,while C+M group only Glut4 gene expression was significantly reduced in soleus?P<0.05?,but the expression level of Cpt1 gene was only down-regulated,and there was no significant difference?P>0.05?.In addition,compared with E mice,the expression levels of Glut4 and Cpt1 genes in the soleus of E+M group were significantly lower?P<0.05?.Conclusions:?1?In soleus muscles mainly composed of oxidized muscle fibers,aerobic exercise enhances muscle fiber aerobic metabolism,and the ubiquitin-proteasome system can affect the aerobic metabolism of mouse skeletal muscles;?2?Ubiquitin-proteasome mediated aerobic exercise reduced HDAC4/5expression mainly in type I oxidized myofibers?soleus?,but had no significant effect on type II glycolytic myofibers?gastrocnemius?;?3?Aerobic exercise increased the degradation of HDAC4/5 in the soleus of mice by ubiquitin-proteasome,leading to a significant decrease in Glut4 gene expression;aerobic exercise combined with proteasome inhibitor had no significant effect on the expression of Cpt1 gene in skeletal muscle,suggesting that in addition to HDAC4/5,aerobic exercise may also regulate Cpt1 gene transcription through other pathways.