| Chronic inflammation induced by immunological abnormalities in local adipose tissue is the main factor of obesity-induced insulin resistance.Macrophage infiltration and polarization is the central part of immunological abnormalities in local adipose tissue.Transcription factor PPARγ is the crucial regulator of macrophage activation.It was reported that adipogenic and adipocyte differentiation-related mi R-27 a was significantly increased in serum of obese individuals.The direct downstream target of mi R-27 a was PPARγ.Therefore,we hypothesize that mi R-27 a could the molecular regulator of macrophage activation by modulating obesity-induced insulin resistance.In this study,obesity-induced insulin resistance mice,mi R-27 a knockdown obese mice,mi R-27 a overexpression mice and cell co-culture models with macrophages and hypertrophic adipocytes were developed.They were used to explore the molecular mechanism of mi R-27 a in the regulation of macrophage infiltration and polarization.These could demonstrate that mi R-27 a,as a novel target in obesity-induced insulin resistance,make great contribution to regulate local adipose tissue specific immunity in cellular and molecular levels.There were three sections to discuss and study this topic.To explore the influence of adipose tissue macrophage activation in obesityinduced insulin resistance in first section,the obesity-induced insulin resistance mice model was established.The results were shown that mice with high-fat diet feeding for 4w,8w and 12 w,body fat percent,fasting blood glucose,fasting insulin,triglyceride,total cholesterol,low density lipoprotein cholesterol were increased constantly,and level of high density lipoprotein cholesterol was reduced.But glucose tolerance were VI decreased and insulin resistance were more apparent increasingly.In the same time,MCP1 and TNFα in serum and adipose tissue were secreted increasingly.For tissue morphology,adipocyte sizes of HFD group were changed in adipose tissue.The volume of adipocyte was increased.A growing number of macrophages were infiltrated into local adipose tissue and were polarized with more classical M1 macrophages.This explained that macrophage activation in adipose tissue might make great contributions to obesity-induced insulin resistance.In second section,the function of mi R-27 a and the relationship between mi R-27 a and macrophage activation in obesity induced insulin resistance was examined.The results showed that mi R-27 a in serum was increased along with the feeding time in obese mice.When mi R-27 a in serum was increased,the levels of biochemical parameters involving fasting blood glucose,fasting insulin,triglyceride and total cholesterols were increased.Insulin resistance was severe.It suggested that mi R-27 a might take part in insulin resistance.To prove this,mi R-27 a overexpression and mi R-27 a knockdown mice model was established.The level of biochemical parameters in mi R-27 a overexpression group were increased,including body weight,calorie of food intake,fat percent,fasting blood glucose,fasting insulin,triglyceride and total cholesterol.Overexpression of mi R-27 a might impair glucose tolerance and insulin tolerance,promote the development of insulin resistance and decrease insulin sensitivity.Likewise,mi R-27 a overexpression might increase the secretion of inflammatory chemokines and cytokines,MCP1 and TNFα.For morphology,overexpression of mi R-27 a could increase the number of M1 in adipose tissue.However,the levels of body weight and calorie of food intake in mi R-27 a knockdown group were similar with those in LFD group.Fasting blood glucose,fasting insulin,triglyceride and total cholesterol were decreased in mi R-27 a knockdown group compared with mi R-27 a overexpression group.Meanwhile,inhibition of mi R-27 a could improve glucose tolerance or insulin sensitivity and alleviate insulin resistance.Downregulation of mi R-27 a could lighten the accumulation of proinflammatory cytokine sand relieve the phenomena of macrophage polarization.This proved that mi R-27 a might play a significant role in obesity-induced insulin resistance.The mechanism of mi R-27 a VII regulation might be associated with chronic low degree inflammation by mediating macrophage activation.In the last section,the cellular mechanism about mi R-27 a mediation of macrophage activation in obesity induced insulin resistance was elucidated via protein expression analysis and cellular experiments.For protein expression and related signaling pathways,insulin metabolism signaling pathway were inhibited significantly with insulin resistance.The expression of PPARγ went down.But macrophage activation related proteins TLR4 and p-NF-κB expressions went up with IκBα expression decreasing.Macrophage infiltration and polarization pathway was activated.Meanwhile,the expression of TNFR1 and p-JNK in inflammatory signaling pathways were increased,which could stimulate the secretion of inflammatory cytokines.It was shown in cellular experiments that Raw264.7 transfected with mi R-27 a overexpression plasmid could suppress the expression of PPARγ,increase the level of MCP1 and TNFα and decrease the level of ARG1 in supernatant.When incubated with rosiglitazone,activator of PPARγ,MCP1 and TNFα could be released less.But ARG1 expression was increased.Additionally,mi R-27 a overexpression could promote macrophage recruitment and infiltration.But the infiltration number of macrophage could be decreased when incubated with rosiglitazone.As well,after hypertrophic adipocytes stimulated by palmitic acid,mi R-27 a was increased and more macrophage would migrate.But when transfected with mi R-27 a inhibitor at the same time,the ratio of macrophage migration would go down.Macrophage infiltration did not occur when there were no adipocytes but with palmitic acid stimulation.This illustrated that the mechanism of mi R-27 a in obesity-induced insulin resistance might be to increase the secretion of mi R-27 a,blocking PPARγ.Macrophage activation was promoted to result in the development of insulin resistance.In summary,this project aimed to elucidate the importance of mi R-27 a in regulation of macrophage activation in obesity induced insulin resistance for the first time.The mechanism of the regulation might be that upregulation of mi R-27 a might block the expression of PPARγ in obesity.And inhibited PPARγ could alleviated the influence of NF-κB.Activated NF-κB might promote the secretion and accumulation of proinflammatory chemokines and cytokines,which could recruit more macrophage into local adipose tissue.So mi R-27 a could activate macrophages for infiltration and polarization,leading to the development of insulin resistance.This regulation could provide prospective intervention targets for obesity-induced insulin resistance and offer the experimental and theoretical basis for prevention and treatment of diabetes. |
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