The Effects And Its Transcriptional Regulation Mechanism Of Insulin On Autophagy Related Gene GABARAPL1

Objective: To investigate the effects and its transcriptional regulation mechanismof insulin on autophagy related gene GABARAPL1.Methods: This experiment intended to observe the changes of autophagy,MDCstaining was applied to the human normal liver cells which were treated by differentinsulin level. In addition, this experiment also wanted to probe into the effects ofinsulin on the autophagy-related genes such as GABARAPL1/Beclin1inthe protein level through western blotting technique. Moerover, to realize thetranscriptional regulation mechanism of insulin on autophagy related geneGABARAPL1,we constructed the luciferase reporter gene vector containingGABARAPL1promoter sequence. Furtheremore, we established the excisionmutations and point mutations of luciferase reporter vector to inactivate theinsulin response elements of GABARAPL1promoter, then transfered these luciferasereporter gene vectors into human normal liver cells which were treated with insulin.Changes of transcriptional activity of GABARAPL1promoter and its mutants weredetected by luciferase assay.Results:1. The autophagy activity of experimental groups (1nM，10nM,100nM group) wasreduced compared with the control group,and the difference was significant instatistic analysis (p<0.01). In addition, there was a certain degree of concentration-response relationship in the range of0nM to100nM.2. Compared with0nM insulin treatment group, the level of GABARAPL1proteinexpression was decreased in the three experimental groups that insulinconcentration was1nM,10nM and100nM respectively,and the expression of Beclin1was the same as GABARAPL1.What,s more, as the concentration of insulin increased,the protein expression of above two kinds of autophagy related gene was also reduced in the range of1nM to100nM.The difference was significant too (p<0.01).3. The GABARAPL1promoter transcriptional activity was declined when the insulinresponse element (IRE1, IRE2or IRE3) was inactivated,and the downtrend was moreobvious in the inactivated IRE1or IRE2locus (p<0.01).4. Compared with control group, the GABARAPL1promoter activity was reduced inthe experimental groups, and the downtrend in the100nM insulin treatment groupwas the most obvious.Conclusions:1. Insulin may inhibit the protein expression and gene transcription of GABARAPL1in human normal liver cells.2. The basal transcriptional activity of GABARAPL1promoter mainly depends on theintact IRE1and IRE2.