The Mechanism Of Inhibition Of Insulin On Snail1-Mediated Lipolysis

People's living levels were increasing dramatically with the development of society.And there were more patient sufferring from the obesity and diabetes.In order to treat these diseases effectively,researchers have carried out a lot of research in the field of metabolism,such as fat,liver and pancreas.So far,fat related genes,such as adiponectin,leptin and PPARr,have been found,and the important role of them in the lipogenesis has been elucidated.However,the research on lipolysis is not so clearly.In this paper,the role of zinc finger protein snail1 in lipolysis was invetigated.Three cellular model was used: 1)Overexpression of snail1 in mature 3T3L1 adipocytes through adenovirus infection;2)and 3)Mouse ears mesenchymal stem cells(EMSC)and adiposederived stem cells(ADSC)were separated from adipose specific knockout snail1 mice or control group snail1 F/F mice,cre adenovirus was used also.The results showed that overexpression of snail1 could significantly decrease lipolysis activities in 3T3L1 mature fat cells;On the contrary,snail1 knockdown in mature adipocytes(EMSC cell model and ADSC cell model)could significantly increase lipolysis activities.Tissue specific condition knockout mice model was used also,compared with control mice(snail1 F/F),fat specific knockout mice adiponectin-cre(+)snail1 F/F mice could increase lipolyiss under fasting 16 h,or CL316243 injection treatment.Fat pad from the two kind of mice was prepared,compared with the control group(snail1 F/F),the snail1 knockout group(adiponectin-cre(+)snail1 F/F)could increase the lipolysis activities both on basal level and on adrenergic stimulation levels.These above results indicated that snail1 could inhibit lipolysis.Snail1 belong to the nuclear factor families,archieves showed that snail1 could inhibit the expression of some downstream gene,such as PPARr,AdipoQ.In this paper,WesterinBlotting result showed that,compared with control group,knockout of snail1 expression could increase the expression of adipose triglyceride lipase(ATGL)in both adipocytes cells and fat tissues from mice.ATGL is one of the key lipase for fat mobilization.Triglycerides(TAG)was specifically hydrolyzed by ATGL,which was considered to be the rate-limiting enzyme.Then,ATGL-lucifease construction was prepared,and snail1 and ATGL-luciferase plasmids were co-transfected into 293 T cells,the luciferase assay results showed that snail1 could dcrease ATGL-luciferase activities in a dose-dependent manner.Bioinformatic methods was used to analysis the ATGL promoter region,and three snail1 specific binding sites E2-Box sites(CACCTG/CAGGTG)were found in 0-1000 bp area,then three mutant ATGLluciferase(-123,-333,-902)were made.Further mutant luciferase assay results showed that,the first E2-Box binding site(-123)is the snail1 binding site in ATGL promoter area,which could block the inhibition of snail1 on ATGL-luciferase.To further analysis,Chromatin Immunoprecipitation(ChIP)assay was used,and the results showed that snail1 could directly bind to ATGL promoter region(0-200bp),which is consistent with the results of luciferase assay.Under normal physiological conditions,insulin could suppressed the lipolysis by decreasing the expression of ATGL,but the mechanism was not clear.In this paper,after 2h of insulin treatment on 3T3L1 mature fat cells,results showed that the expression of snail1 were significant increase in the RNA levels and protein levels.Expression of snail1 was also increased after insulin treatment in mature adipose cell isolated from c57 wild type mice.Human adipose stem cells(human fat ADSC cell line)from Macdougald lab in University of Michigan Medical School was induce into mature human fat cell,consistent results were found that expression of snial1 was increased under insulin treatment in human adipocyetes.PI3 K / AKT signaling pathway inhibitor and Erk signaling pathway inhibitor was used to prevent the two signal pathways.Results showed insulin could stimulated through PI3 K / AKT signaling pathway,and inhibited GSK3 functions,then increased the expression levels of snail1.Compared with the control group,knockout snail1 could resist the effect of insulin decrease ATGL expression.Based on the results above,it could be concluded that snail mediated insulin's effect on dcreaing ATGL expression on lipolysis.