| Objectives:As the pathophysiological basis of many metabolic diseases,The pathogenesis of Insulin resistance(IR)is of great significance for the treatment of these diseases.Aerobic exercise has entered the clinical and become an important treatment for metabolic diseases as an effective intervention.However,the mechanism is still unclear.Therefore,exploring the specific mechanism of aerobic exercise to improve the energy metabolism of the body is of great significance for the treatment of metabolic diseases such as type 2 diabetes and obesity.Class IIa histone deacetylases(HDACs)is a subtype of the HDACs family,including HDAC4,HDAC5,HDAC7,HDAC9,in which HDAC4 and HDAC5 are kinematically sensitive,i.e.exercise can alter the amount of protein expression.Meanwhile,they have physiological functions that regulate cardiovascular,musculoskeletal,nervous and immune systems.This is mainly due to their ability to shuttle between the nucleus and the cytoplasm in order to alter the acetylation of histones,thereby altering the structure of chromatin and ultimately affecting the expression of metabolic related genes.In our previous studies,we have found that HDAC4 and HDAC5 phosphorylation can affect their intracellular localization and affect the transcriptional expression of CPT1,PGC-1?,GLUT4,and other metabolic genes,indicating that HDAC4 and HDAC5 play important roles in regulating energy metabolism in the body.In addition,exercise significantly reduced the protein expression of HDAC4 and HDAC5 in the soleus muscle,while the high-fat diet reduced their protein content.Therefore,we propose the following interrogation: In addition to phosphorylation,does the change in HDAC4 and HDAC5 protein content also lead to energy metabolism in the body? How do exercise and high-fat diets alter HDAC4 and HDAC5 protein levels? As a key factor in energy regulation of the body,what role does AMPK play in the regulation of HDAC5 protein content in skeletal muscle of mice?Methods:(1)In order to explore the effect of aerobic exercise on energy metabolism in IR mice,we first constructed IR mice by using an 8-week high-fat diet,and randomly divided IR mice into a sedentary group(HC),an exercise group(HE),a sedentary + MG132 group(HCM),a exercise + MG132 group(HEM).After the completion of the insulin resistance model and the exercise intervention,the body weight,fat mass(FA),free fat mass(FFA),body mass index(BMI)and other indicators were measured by body composition analyzer.Plasma insulin,cholesterol,triglyceride,free fatty acid levels and IR mice were detected by ELISA.Real-time PCR was used to investigate the effects of exercise on the expression of lipid catabolism-related genes in skeletal muscle of IR mice.(2)In order to explore the effect of aerobic exercise on the aerobic oxidative capacity of skeletal muscle in mice,the following exercise program was adopted for the mice: after the IR model was successfully constructed,the mice were subjected to a one-week adaptive treadmill training,and the initial speed was set.Set at 8-10 m / min,without slope,exercise time is 30 minutes;then gradually increase the load and maintain 75% of the maximum oxygen uptake in the mouse,i.e.12 m / min,60 min/time,5 times / Week,the total duration is 6 weeks.Subsequently,Western Blot was used to detect the content of myoglobin,cytochrome(Cytochrome C)and troponin I in the soleus muscle of each group to detect the aerobic oxidation ability of skeletal muscle.(3)In order to clarify the regulation of HDAC4 and HDAC5 protein content in the soleus muscle of IR mice,we used Real-time PCR to detect the mRNA changes of mouse soleus HDAC4 and HDAC5 by using Real-time PCR;and use Western Blot to detect the change of HDAC4 and HDAC5 protein content in mouse.(4)To investigate whether exercise promotes ubiquitination of HDAC4 and HDAC5 in IR mice by the ubiquitin-proteasome system,we injected intraperitoneal injection of 20 S proteasome inhibitor MG132 into IR mice under resting or exercise conditions.Western Blot verified the inhibitory effect and the expression of HDAC4 and HDAC5 in the soleus muscle of mice after inhibition of proteasome;the inhibitor was intraperitoneally injected at a dose of 10?g/kg/d;the non-MG132 group was injected intraperitoneally with drug solvent 0.01% DMSO as a control.The duration of drug intervention was 6 weeks.(5)To further verify the role of ubiquitin-proteasome in aerobic exercise regulation of HDAC4/5 protein content in soleus muscle of IR mice,we used CO-IP to detect changes of HDAC4/5 ubiquitination in soleus muscle of each group and the combination of HDAC4/5,20 S proteasome,and MuRF1.(6)To explore the role of AMPK in the ubiquitination and degradation of HDAC5 in the soleus muscle of exercise-regulated mice,we first constructed a normal resting and long-term aerobic exercise model of AMPK knockout mice and detected the expression of related proteins according to the above methods,to explore the role of AMPK in the ubiquitination of HDAC5.Results:(1)Analysis of body composition and plasma biochemical results in mice: 8-week high-fat diet can cause insulin resistance in mice,mainly including increase of plasma insulin levels,impair of glucose tolerance,and significantly increase of plasma free fatty acids,cholesterol,and triglycerides with loss of demineral body weight and increase of body fat content and BMI.6 weeks of aerobic exercise can significantly reverse the above changes,that is,reduce IR plasma insulin,fasting blood glucose and postprandial blood glucose,plasma free fatty acids,cholesterol,glycerol Ester,increase fat loss,reduce body fat and BMI.(2)Effect of aerobic exercise on aerobic oxidative capacity of soleus muscle in IR mice:high-fat diet has no significantly effect on mouse skeletal muscle aerobic oxidation protein Myoglobin(myoglobin),cytochrome(Cytochrome C),troponin(Troponin I),but 6 weeks of aerobic exercise significantly increased the expression of the above-mentioned related proteins in skeletal muscle of IR mice,indicating that aerobic exercise can significantly increase the aerobic oxidation ability of mice.(3)Effect of aerobic exercise on HDAC4/5 mRNA and protein expression in soleus muscle of IR mice: At the genetic level,exercise has no significant effect on the mRNA of HDAC4 in the soleus muscle of IR mice,but can significantly increase the expression of HDAC5 mRNA.However,at the protein level,the HDAC4 and HDAC5 protein content was significantly increased in the HC group,and significantly decreased the HE group,indicating that the gene and protein levels were not consistent.Therefore,changes in the regulation of HDAC4 and HDAC5 protein content may due to ubiquitin proteasome systems through increasing their ubiquitination degradation.(4)Effect of inhibition of proteasome on HDAC4 and HDAC5 protein content: intraperitoneal injection of MG132 significantly inhibited the expression of 20 S proteasome.From the protein level,after inhibition of the 20 S proteasome,HDAC4 and HDAC5 expression showed a significant increase in both the quiet group and the exercise group,indicating that HDAC4 and HDAC5 can be degraded by the ubiquitin-proteasome system.But compared with the HCM group,in the HEM group,there are still differences,indicating that the regulation of HDAC4 and HDAC5 degradation by exercise is partially dependent on the ubiquitin proteasome.(5)Inhibition of the effect of proteasome on HDAC4/5 ubiquitination,skeletal muscle E3 ligase,and 20 S proteasome: a.After inhibition of 20 S proteasome,whether in silence or in motion,the ubiquitination of HDAC4 ubiquitin Significantly increased and the ubiquitination of HDAC5 significantly decreased,indicating that HDAC5 may be regulated by deubiquitinating enzymes and HDAC4 may cause accumulation of ubiquitination marks due to inability to pass proteasome degradation;b.with MuRF1 Binding: Compared with the HC group,the binding of HDAC5 to MuRF1 in the HE group was significantly increased;compared with the HC group,the binding of skeletal muscle-specific E3 ligase MuRF1 was significantly decrease in HDAC5 and MuRF1 further indicated that there may be deubiquitinating enzyme involvement in HDAC5 ubiquitination modification;c.Binding to the 20 S proteasome: Compared with the HC group,the binding of HDAC4 to the 20 S proteasome in the HE group increased,while the HDAC5 did not change significantly,which may be due to no significant change in the protein content of the 20 S proteasome after exercise;In the HCM group,HDAC4 and 20 S proteasome binding were significantly increased,while HDAC5 binding was significantly decreased,which may be due to the increased ubiquitination-labeled HDAC4 ubiquitination label and the decrease in HDAC5 ubiquitination label;(6)The role of AMPK?2 in the reguation of ubiquitination and degradation of skeletal muscle HDAC5 by exercise: Comparing the HDAC5 protein content of the wild-type and knockout mouse soleus muscle,it can be seen that AMPK?2 knockdown can significantly increase the HDAC5 protein content in both quiet and exercise states.According to CO-IP results,after AMPK?2 was knocked out,HDAC5 was ubiquitinated,and the binding amount to MuRF1 and 20 S proteasomes was significantly decreased,indicating that AMPK?2 knockdown significantly inhibited HDAC5 ubiquitination labeling,thereby inhibiting its degradation by proteasome. |
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