Study On The Regulatory Role Of Phosphatidylinositol Signaling PI?4,5?P₂ In GLUT4 Vesicles Transport In 3T3-L1 Adipocytes

Objective:Our study aims to establish the rapidly PI?4,5?P₂decreasing model of mature 3T3-L1 adipocytes by optogenetics,and investigate the effects of PI?4,5?P₂ on glucose transporter 4?GLUT4?transport in mature 3T3-L1 adipocytes and possible mechanisms.Methods:3T3-L1 preadipocytes were induced differentiation into mature3T3-L1adipocyteswithinsulin,dexamethasoneand3-isobutyl-1-methylxanthine.Mature 3T3-L1 adipocytes were transfected by electroporation with CRY2/CIBN,which is adjustable optogenetic element,organelle localization sequence and 5'phosphatase OCRL to establish the rapidly PI?4,5?P₂ decreasing model of mature 3T3-L1adipocytes.Indirect immunofluorescence staining technique was used to determine the insulin sensitivity of 3T3-L1 adipocytes,and observe the effect of PI?4,5?P₂ on the GLUT4 transport efficiency in different organelles.By the probe of IRAP-pHluorin or Myc-GLUT4-mCherry with the live-cell imaging technique,the effect of rapid,cell-wide PI?4,5?P₂decreases by optogenetics control upon GLUT4 fusion and docking in3T3-L1 adipocytes was observed under insulin stimulation,and the effect of rapid,subcellular PI?4,5?P₂ reduction selectively at GLUT4 Vesicles upon GLUT4 docking was also observed under insulin stimulation.Indirect immunofluorescence staining technique and Western blot were carried out to study the effects of PI?4,5?P₂ on the relative phosphorylation of Akt protein at the threonine 308 and serine 473 site in the 3T3-L1 adipocytesResults:?1?With the rapidly PI?4,5?P₂decreasing model of mature3T3-L1 adipocytes,the 5'phosphatase OCRL was rapidly transferred to the corresponding organelles after the blue light stimulation,thus PI?4,5?P₂ was dephosphorylated;OCRL was slowly leaving the corresponding organelle after the blue light stimulation was stopped,so that PI?4?P was phosphorylated to PI?4,5?P₂.?2?Upon insulin stimulation,the level of PI?4,5?P₂ on the PM was lowered,and the content of GLUT4 on the PM was decreased;when the level of PI?4,5?P₂ on the mitochondria or the trans Golgi network was lowered,the content of GLUT4 on the PM had no significant changes;Without insulin stimulation,along with lowered level of PI?4,5?P₂ on the three sites,the content of GLUT4 on the PM had no significant changes.?3?With insulin stimulation,along with reduced PI?4,5?P₂ on the PM,the docking and fusion level of GLUT vesicles and PM was decreased;?4?Without insulin stimulation,the reduction of PI?4,5?P₂ at the GLUT4 vesicles docking sites undocks vesicles from the PM;?5?With insulin stimulation,along with reduction of PI?4,5?P₂ on the PM,the level of endogenous Akt phosphorylation was inhibited.Conclusion:GLUT4 vesicle docking is a key target of local PI?4,5?P₂metabolism in the secretory pathway of 3T3-L1 adipocytes.Without insulin stimulation,reduction of PI?4,5?P₂ at the GLUT4 vesicles docking sites undocks vesicles from the PM.With insulin stimulation,the PI?4,5?P₂ on the PM promote the docking and fusion of GLUT4 vesicle with PM,probably by activation of PI3K/Akt signal pathway.