The Transcriptional Regulation Mechanism Of Insulin On GABARAPL1 Of Hepatic Cells

Objective To detect the effects of insulin on GABARAPL1 of hepatic cells and to analyse the transcriptional regulation mechanism.Methods HL-7702 cells were cultured and divided into 5 groups: Group A(control group),Group B(autophagy promoted group),Group C(insulin group),Group D(insulin blocked group1),Group E(insulin blocked group2).The detailed treatment of cell groups is as follows: Each group were cultured with DMEM medium,and 100 nM rapamycin were added into 5 groups except the control group.Six hours later,1μM insulin were added into Group C,D and E,Group D were added 50μM LY294002,and Group E were added 100 nM Wortmannin a half hour before the moment when adding insulin.All the treatment of cells was completed after 4 hours.The protein of all groups were extracted.Western Blotting to identify each group’s expression of autophagy related gene(GABARAPAL1,LC3,Belin1),the transmission electron microscope to observe the number of autophagosomes at the same time.We had successfully constructed the excision mutations and point mutations of luciferase reporter vector to inactivate the insulin response elements of GABARAPL1 promoter,and established the reporter including GABARAPL1 promoter sequence.Then the changes of transcriptional activity of GABARAP1 promoter were detected by Luciferase assay.The transcriptional regulation model were further confirmed by electrophoretic mobility shift assay.Results 1.Western Blotting showed that compared with Group B,the protein level of GABARAPL1,LC3 and Beclin1 was decreased in Group C(P<0.05),compared with Group C,the protein level of GABARAPL1,LC3 and Beclin1 was increased in Group D(P<0.01).2.The changes of autophagosomes were observed by transmission electron microscope showed that the number of autophagosomes in insulin group was decreased compared with group B,D and E.3.Luciferase reporter detected that the GABARAPL1 promoter transcriptional activity was obviously reduced when the IRE1 was inactivated(P<0.01).4.EMSA showed that autophagy promoted group and insulin blocked group combined with the probe(biotin-Fox1),the combination of autophagy promoted group,probe and the antibody of FoxO1 was more obvious and laggard,while the combination of insulin group was decreased.Conclusions 1.Insulin may inhibit the expression of GABARAPL1 of hepatic cells.2.Insulin can regulate the transcriptional activity of GABARAPL1 of hepatic cells by breaking FoxO1 away from IRE1 in GABARAPL1 promoter.