The Effect And Mechanism Of Exercise On Autophagy And Inflammation In Adipose Tissue In Obesity Mice

Autophagy as an intracellular metabolic waste clearance and recycling mechanism play an important role in lipid metabolism in skeletal muscle,pancreas,liver and other metabolically active organ cells in obesity and insulin resistance.But little research was found about the effect of autophagy in adipose tissue,especially the role of autophagy in lipid metabolism of adipose tissue after exercise.Even in a limited number of research reports,there are still some controversial results,such as how the activity of adipose tissue autophagy changes? And are there any site specific changes in the autophagic activity of adipose tissue? What are the effects of exercise on the activity of basal autophagy in adipose tissue and in obese subjects? Whether autophagy by improving inflammation in adipose tissue of obese to defat after exercise? Meanwhile,long time aerobic exercise as the essential contents of the traditional exercise prescription has become the main form of sport which are researched by scholars in autophagy mediated mechanism of weight loss.It worthy to note that resistance exercise has gradually been recognized in recent years,but its effect on autophagy in adipose tissue has not been reported.Therefore,the purpose of this study is to investigate the changes of obese adipose tissue autophagy and inflammation,and to observe there is fat specific difference in autophagic activity changes in obesity or not.And then to compare the effectiveness of endurance exercise and resistance exercise in fat reduction,then to examine the impact of exercise on autophagy activity in visceral adipose tissue in obesity,finally to explore the relationship between autophagy and inflammation in adipose tissue after exercise.Thus this study can provide theoretical support and practical strategies for anti-obesity and related metabolic diseases.1 Establishment of animal model of obesityObjective: To establish the animal model of obesity.Methods: A total of 170 male C57BL/6 mice were randomly divided into normal diet group(NFD: n=16)and high fat diet group(HFD: n=154),and were fed with normal diet and high fat diet respectively.After eight weeks of different dietary intervention,according to the theory of susceptibility to obesity,the one third weight increment of body weight was screened from HFD group,and the weight of screened mice must increase more than 20% compared with NFD group.And then NC group(n=8)and HC group(n=8)were randomly selected from the NFD group and the HFD group respectively to measure body weight,length,fat mass and blood lipid to evaluate the model of obesity.Results: Body weight in both group were significantly increased(p<0.05)after 8wks intervention,and body weight,Lee's index,visceral fat mass and body fat index of HC group were significantly higher than NC group(p<0.05).TC and LDL-TC were significantly increased(p<0.05),HDL-TC decreased significantly(p<0.05)compared with NC group.At the same time we found that visceral fat cell diameter became longer,and the volume became bigger,and had a large cell number in NC group compared with HC group.Suggestion: Through 8wks of high fat diet C57BL/6 mice were successfully induced as animal models of obesity.2 Specific changes of autophagy activity in subcutaneous and visceral adipose tissue of obesityObjective: To detect the specific changes of autophagy activity in subcutaneous and visceral adipose tissue of obesity,and to explore the correlated relationship between autophagy and inflammation of obesityMethods: Lean group and Obesity group were composed by selecting 8 mice respectively from NFD group and the screened obesity model group,then subcutaneous adipose tissue(Sc)and epididymal fat pad(Vis)were collected to be detected autophagy and inflammatory marker genes and proteins expression using RT-PCR and WB.Results:(1)Autophagy activity.The gene expression data was handled by two way ANOVA and post hoc test showed that compared with Lean-Sc group,Obesity-Sc group p62 mRNA expression was significantly increased(p<0.05);The same method was used in protein expression data showed that compared with the Lean-Visc group,Obesity-Visc group Becn1 protein expression and the ratio of LC3?/? was increased significantly(p<0.05),and compared with the Lean-Sc group,Obesity-Sc group p62 protein expression was decreased significantly(p<0.05).(2)Inflammatory states.The gene expression data was handled by two way ANOVA and post hoc test showed that compared with Lean-Sc group,the IL-6 mRNA expression of Obesity-Sc group and Lean-Visc group were both significantly increased(p<0.05).Lean-Sc and Obesity-Sc group,Lean-Visc and Obesity-Visc group were compared respectively and found that the IL-10 mRNA expression were significantly decreased(p<0.05).The same method was used in protein expression data showed that compared with the Lean-Sc group,Obesity-Sc and Lean-Visc group IL-6?IL-1? protein expression were increased significantly(p<0.05).Compared with Lean-Visc group,Obesity-Visc group IL-6?IL-1? protein expression were increased significantly(p<0.05),and IL-10 protein expression decreased obviously(p<0.05).Compared with Obesity-Sc group,Obesity-Visc IL-6 protein expression was significantly increased(p<0.05),and the IL-1? decreased significantly(p<0.05).(3)The relationship between autophagy activity and inflammatory states under obesity.Becn1 protein expression was positively correlated with the expression of IL-6(r=0.29,p=0.05),the gene and protein expression between p62 and IL-6 showed a positive correlation(r=0.30,p>0.05;r=0.51,p<0.05)respectively,and showed a weak positive correlation between p62 and IL-10(r=0.28,p>0.05;r=0.25,p<0.05)respectively.IL-10 protein expression was positively correlated with LC3(r=0.52,p<0.05);IL-1? showed no any correlation with autophagy related proteins.Suggestion: Activity of autophagy in adipose tissue of obese was increased,and there are no differences between subcutaneous and visceral fat.Inflammation response was also increased in adipose tissue of obese,and visceral fat has a higher inflammation response.Autophagy and inflammation had the same trend under obesity in adipose tissue,the activation of autophagy under obesity in adipose tissue may be compensatory mechanism to limit excessive inflammatory reaction.3 Effects of different exercise on autophagy and inflammation in visceral adipose tissue of obeseObjective: to compare the effectiveness of endurance exercise and resistance exercise in fat reduction,then to examine the impact of exercise on autophagy activity and inflammation response in visceral adipose tissue in obesity,finally to explore the relationship between autophagy and inflammation in adipose tissue after exercise.Methods: First we selected 8 mice from NFD group as normal diet control group(NNC),then we selected 42 mice from the obesity model and divided randomly into high fat diet group(HHC,n=9),normal diet intervention group(HNC,n=9),normal diet combined with endurance exercise intervention group(HNC,n=12),and normal diet combined with resistance exercise intervention group(HNR,n=12).HNE and HNR group respectively conducted treadmill and ladder climbing exercise respectively for 8 weeks among them.The mice were fed with the corresponding diet in control group.Then to detect the gene and protein expression of autophagy inflammation,ER stress and antioxidant markers using RT-PCR and WB,in addition,TEM and IHC were used to observe the autophagosome in visceral adipose tissue.Results:(1)Obesity characteristics.Compared with HHC,the body weight,Lee's index,visceral fat mass and body fat index were significantly decreased(p<0.05)in HNC group and exercise group,and the exercise group decreases was higher than HNC group,but there is no difference(p>0.05)between the two exercise group.(2)Autophagy activity.Compared with HHC group: Atg5 mRNA expression and LC3II/I were decreased significantly(p<0.05)in HNC group.ULK1,Becn1,Atg5 mRNA expression were decreased significantly(p<0.05)and Atg7,p62 mRNA expression were significantly increased(p<0.05)in HNE group,meanwhile the expression of Becn1 protein and LC3II/I significantly decreased(p<0.05)and the p62 protein expression was significantly increased(p<0.05)in HNE group.Atg5 mRNA expression decreased significantly(p<0.05)and Atg7,p62 mRNA increased significantly(p<0.05)in HNR group,meanwhile the Becn1 protein expression and LC3II/I decreased significantly(p<0.05)in HNR group.Compared with HNC group: the gene expression of Atg7 and p62 increased significantly(p<0.05)in HNE group accompanied by the significant increasing of p62 protein expression.Atg7 mRNA expression increased significantly(p<0.05)in HNR group.Compared with HNE group: Becn1 mRNA expression increased significantly(p<0.05)and p62 protein expression decreased significantly(p<0.05)in HNR group.(3)Inflammatory response state.Compared with HHC group: IL-1? protein expression were decreased significantly(p<0.05)in HNC group.The IL-6 and IL-10 protein expression significantly increased(p<0.05)in HNE group accompanied by the significant increasing of IL-10 gene expression,meanwhile IL-1? protein expression were increased significantly(p<0.05)in HNE group.MCP-1,IL-6,IL-10 mRNA expression were significantly increased(p<0.05)in HNR group accompanied by IL-6?IL-10 protein expression were increased significantly(p<0.05),meanwhile IL-1? protein expression were decreased significantly(p<0.05)in HNR group.Compared with HNC group: the IL-6 and IL-10 protein expression significantly increased(p<0.05)in HNE group accompanied by the significant increasing of IL-10 gene expression,meanwhile IL-1? protein expression was increased significantly(p<0.05)in HNE group.The IL-6,IL-10 mRNA expression increased significantly(p<0.05)and the IL-10 protein expression increased significantly(p<0.05)in HNR group,meanwhile IL-1? protein expression were decreased significantly(p<0.05)in HNR group.Compared with HNE group: the gene expression of IL-6 increased significantly(p<0.05)and IL-10 decreased significantly(p<0.05).(4)The regulator of autophagy.Compared with HHC group: mTORC1 gene and protein expression were significantly increased(p<0.05)in HNE group.mTORC1 mRNA expression was significantly increased(p<0.05)in HNR group.Compared with HNC group: the same change happened in HNE and HNR group.Compared with HNE group: mTORC1 gene expression increased significantly(p<0.05)and protein expression decreased significantly(p<0.05)in HNR group.(5)The relationship between autophagy and inflammation.p62 gene expression showed a moderate correlation with IL-10 and IL-1?(r=0.57;-0.69)(p<0.05).While the protein of p62 expression showed a moderate correlation with IL-6 and IL-10(r=0.51;0.64)(p<0.05)respectively.The protein expression of LC3 and IL-10 showed a significant correlation(r=0.55,p<0.05),and the gene expression of ULK1,Atg5,Atg7 also showed well correlation with IL-10(r=-0.43,-0.67,-0.62 respectively).(6)ER stress.Compared with HHC group: GRP78,CHOP protein expression were significantly decreased in both HNE and HNR group(p<0.05).Compared with the HNC group: GRP78 protein expression decreased significantly(p<0.05)in both HNE and HNR group,CHOP protein expression decreased significantly(p<0.05)in HNR group only.(7)Antioxidant status.Compared with HHC group: SOD1 protein expression increased significantly(p<0.05)in both HNC and HNE group.Compared with HNC group: SOD1 protein expression decreased significantly(p<0.05)in both HNE and HNR group.Suggestion:(1)Normal diet combined with exercise intervention can significantly reduce visceral fat mass,there is no difference between the two exercise protocol in weight loss.(2)Autophagic activity of visceral adipose tissue was inhibited after exercise and the aerobic exercise had the stronger inhibition.In addition,mTORC1 mediated the inhibition of autophagy after aerobic exercise,and the inhibition of resistance exercise on autophagy may not dependent on mTORC1.(3)Normal diet combined with aerobic or resistance exercise has a similar effect in improving chronic inflammatory state in visceral adipose tissue of obese,and the effect of resistance exercise may be weaker than aerobic exercise at the transcriptional level.(4)Autophagy and inflammation showed the same decreasing after normal diet combined with exercise intervention,and the IL-10 is the most sensitive factor in reflecting the relationship between autophagy and inflammation,and the underling mechanisms may include the changes of ER stress and antioxidant status.Conclusion(1)Autophagy was activated in adipose tissue in obesity,and there was no significant difference between different fat depot.But there was a significant difference in the increased inflammatory response between different fat depot,which showed that the inflammation of visceral adipose tissue was stronger than that of subcutaneous adipose tissue.(2)The effectiveness of 8wks normal diet combined with different types of exercise intervention had no difference in weight loss,the autophagic activity of visceral adipose tissue was inhibited after exercise,and compared with resistance exercise,aerobic exercise had a stronger inhibition in autophagic activity.In addition,mTORC1 mediated the inhibition of autophagy after aerobic exercise,and the inhibition of resistance exercise on autophagy may not dependent on mTORC1.(3)Autophagy and inflammation enjoy the same trend before and after exercise intervention in visceral adipose tissue in obesity,and the IL-10 is the most sensitive factor in reflecting the relationship between autophagy and inflammation,and the underling mechanisms may include the changes of adipocyte size,ER stress and antioxidant status.