Insulin Signal Affects The Browning Of Mouse And Human Adipose Stem Cells And Its Mechanism

Obesity is due to over-hypertrophy and abnormal proliferation and differentiation of white adipocytes,which have the main function of storing energy in animals.Brown adipose tissue(BAT)in mammals acts as a heat-generating tissue,which helps the body to increase energy consumption and maintain the balance of energy metabolism.BAT is scattered in the human body,its content is only less than 50 grams.If we can make fully use of white adipocytes that are rich in content and strong in plasticity,induce white adipocytes into brown-like fat cells(white fat cells browning)to increase the body's energy consumption and improving insulin resistance,will provide new ideas for the treatment of obesity.Therefore,firstly,it is necessary to clarify the factors affecting the browning of white fat cells and their regulatory mechanisms.Domestic and foreign research have shown that insulin signaling pathways play important regulatory roles in adipogenic differentiation of white preadipocytes and dedifferentiation of mature adipocytes.So,does the insulin signal also play a role in the browning of white adipocytes? What is the degree to which it affects the browning of white adipocytes and its molecular mechanism? This thesis aims at these issues,designed related experiments,in order to initially clarify the role of insulin signaling in the process of white adipocyte browning and its molecular mechanism.Mouse and human primary adipose stem cells(ASCs)were selected for this experiment.In this experiment,the subcutaneous ASCs(msASCs),epididymal ASCs(esASCs)and human subcutaneous ASCs(hsASCs)were used as experimental materials for wild-type(WT)and leptin receptor gene(db)spontaneous point mutations in db/db obese mice.Insulin signal specific inhibitor(Iinstinib,OSI-906)was used to suppress the insulin signaling pathway,and the correlation between insulin signal and browning in animal white adipocytes was explored.Set 1)induction group and 2)insulin signal suppression group,analysis of insulin signaling and browning of white fat cells by morphological and the transcription and translation levels express of browning markers,key molecules of the insulin signaling pathway and key adipogenesis molecules to preliminary exploration of its molecular mechanism.1.Determine the browning induction method.Refer to the previous browning induction method and improve it.Browning induction of WT msASCs,meASCs,and hsASCs,detection of their browning marker molecule expression level(WB).The rusults indicates that improved browning induction method can make these browning.The browning degree of msASCs were higher than meASCs,so we chose msASCs as follow-up experimental materials.To further increase the degree of browning,low temperature treatment at 26°C was performed at different time points in the induction of msASCs and hsASCs browning.The results showed that low temperature can significantly promote the browning of msASCs and hsASCs,and the degree of browning of msASCs and hsASCs increases with the prolonged treatment of low temperature.Therefore,the subsequent experiments will use the joint induction method optimized by this experiment.2.Using msASCs and hsASCs as experimental materials to select the optimal concentration of insulin signal inhibitor OSI-906,and set gradients of 0,0.5,1 and 1.5 ?M,respectively.After the induction,Nile red staining was performed to observe adipogenesis.Phosphorylation of AKT,a key marker in the insulin signaling pathway,was used as a measure of the extent of inhibition of insulin signaling.The results showed that was inhibition AKT phosphorylation OSI-906 dose-dependent.When 1.5 ?M OSI-906 was added,the inhibition of p-AKT in msASCs and hsASCs was the highest(91% and 87%).Considering the toxic side effects of high concentrations,the 1.5 ?M OSI-906 was used as final concentration.3.To investigate the correlation between insulin signal and browning,WT,db/db msASCs,and hsASCs were used as experimental materials.Browning induction was using the joint induction method.Simple induction group and insulin signal suppression group were set.The results showed that: after blocking the insulin signal,the adipogenesis was significantly inhibited,and the adipogenic rate decreased from 60±5.5% to 5±2.5%;the expression of the key lipid-forming molecules PPAR? and C/EBP?,and the browning-related molecules UCP1,PGC-1? and PRDM16 were significantly down-regulated,which significantly up-regulated the phosphorylation level of AMPK and the expression of SIRT1.Uur experimental results show that the insulin signal positively regulates the browning of white adipocytes,which promotes the browning adipogenisis process and the browning level.However,the insulin signalling inhibition group still retained a low degree of browning.In addition to the reason that the insulin signal has not been completely blocked,it may indicated that there are other signaling pathways involved in the regulation of white adipocyte browning.We analysis AMP activated kinase(AMPK)and sirtuin1 type 1(SIRT1)which are related to the insulin signaling pathway and involved in the browning of white adipocytes.Inhibition of insulin signaling pathway can significantly promote the phosphorylation of AMPK and upregulate SIRT1 expression,maintaining its browning level.Based on WT,db/db msASCs and hsASCs,the expression levels of these key adipogenic and browning-related factors were consistent,indicating that the correlation between insulin signal and browning of WAC was not affected by genotype and species.