Study On Dynamic Translocation Process Of GLUT4 Vesicle And Its Translocation Mechanism In Living Cells

Diabetes mellitus is a metabolic disease with complex pathological mechanism. It is associated with many risk factors which including obesity,hypertension, dyslipidemia and atherosclerosis,but insulin resistant is the most important element that contributes to diabetes.It has been shown that insulin resistant is caused by dysfunction of insulin signaling and intracellular GLUT4 translocation.As the primarily glucose transporter isoform in adipocytes and skeletal muscle cells,GLUT4 is responsive to insulin stimulation and insulin induces translocation of GLUT4 from intracellular compartments to the plasma membrane(PM).But to date,the dynamic translocation process of GLUT4 vesicle and its translocation mechanism are still remaining unknown.In the present study,we employed diverse biomedical techniques like GFP labeling,single cell Confocal and TIRF imaging and quantitative image analyzing to study the dynamic translocation process of GLUT4 vesicles in primary rat adipocytes and C2C12 myoblasts,to investigate the mechanism that insulin and cytoskeleton function on intracellular GLUT4 trafficking.Furthermore,our research provides considerable knowledge for the further studies on spatial and temporal regulation of GLUT4 vesicle trafficking and the researches on identifying the cellular and molecular mechanism of diabetes.The model of primary rat adipocytes culture and efficient transfection was founded.Histological and immunofluorescent means were used to study the physical and biochemical characters of rat adipocytes.By optimizing the conditions of electroporation,high transfection efficiency of rat adipocytes was reached,which makes the studies on GLUT4 vesicle in single living cells possible.The function and mechanism of insulin and microtubule network on intracellular GLUT4 trafficking in primary rat adipocytes were studied.Our results showed that the primary mechanism of insulin action in GLUT4 transiocation is to stimulate the trafficking of GLUT4 vesicles to the PM,reduce their mobility and make them tightly docking and fusion with the PM.The microtubule-depolymerizing reagent nocodazole could destroy the microtubule network in a dose-dependent manner.Completely destroyed microtubule network abolished the long-range movement of GLUT4 vesicles in rat adipocytes but had little effect on the density of vesicles,which indicated that long-range movement of GLUT4 vesicles was essentially microtubule-dependent and microtubule network was not obligatory for GLUT4 distribution in rat adipocytes.We suggested that actin network could also function on intracellular GLUT4 distribution.In addition,long-range bi-directional transportation of GLUT4 vesicles was found and quantitative analysis and reasonable hypothesis were made regarding to these novel movement in our study.By real-time imaging of the dynamic trafficking of EGFP-IRAP,biophysical characters and intracellular localization of IRAP and GLUT4 were studied in rat adipocytes.Our results showed that IRAP had co-localization with endogenous GLUT4 and insulin stimulation induced the translocation of IRAP from perinuclear area to the PM.TIRFM study revealed that most of the EGFP-IRAP were docked to the PM and long-range moving vesicles were seldom observed,which suggested that over-expression of IRAP destroyed the trafficking of GLUT4 vesicles and demonstrated that IRAP could play a role on intracellular localization and translocation of GLUT4 vesicle.The roles of insulin and microtubule cytoskeleton play on distribution of GLUT4 and IRAP and their intracellular trafficking were studied in C2C12 myoblasts.Our results revealed that the expressed IRAP had no co-localization with the endogenous GLUT4 in C2C12 cells,which suggested that biogenesis and maturation of GLUT4 vesicles may connect with the differentiation of cells.In C2C12 cells,insulin greatly enhanced the mobility of GLUT4 and IRAP,but depolymerization of microtubule network by nocodazole reduced the mobility of GLUT4,which revealed their physiological function on translocation of GLUT4 vesicles.In conclusion,our study achieved the following novel findings:1)By establishing the effective gene transfection system,study on the regulation of intracellular GLUT4 translocation was carried out in primary rat adipocytes,which was more close to the physiological conditions when compared with the studies worked on immortalized cell lines.2)Real-time TIRF imaging of the dynamic translocation of GLUT4 vesicles and investigation the regulatory mechanism of insulin and microtubule cytoskeleton on the GLUT4 were studied in C2C12 myoblasts.3)Two labeling systems,GFP tagged GLUT4 and IRAP were employed in this study to visualize the intracellular distribution of GLUT4 vesicles.Integrated the GFP labeling with real-time living cells TIRF imaging,dynamic biophysical characters of GLUT4 vesicles which including heterogeneous distribution of GLUT4 vesicles,their characters of translocation and exocytosis,etc.were quantitatively analyzed,and the physiological mechanism of insulin and microtubule network action in GLUT4 translocation was also investigated.