Dataset Information

Direct quantification of fusion rate reveals a distal role for AS160 in insulin-stimulated fusion of GLUT4 storage vesicles.

ABSTRACT: Insulin-stimulated GLUT4 translocation to the plasma membrane constitutes a key process for blood glucose control. However, convenient and robust assays to monitor this dynamic process in real time are lacking, which hinders current progress toward elucidation of the underlying molecular events as well as screens for drugs targeting this particular pathway. Here, we have developed a novel dual colored probe to monitor the translocation process of GLUT4 based on dual color fluorescence measurement. We demonstrate that this probe is more than an order of magnitude more sensitive than the current technology for detecting fusion events from single GLUT4 storage vesicles (GSVs). A small fraction of fusion events were found to be of the "kiss-and-run" type. For the first time, we show that insulin stimulation evokes a approximately 40-fold increase in the fusion of GSVs in 3T3-L1 adipocytes, compared with basal conditions. The probe can also be used to monitor the prefusion behavior of GSVs. By quantifying both the docking and fusion rates simultaneously, we demonstrate a proportional inhibition in both docking and fusion of GSVs by a dominant negative mutant of AS160, indicating a role for AS160 in the docking of GSVs but not in the regulation of GSV fusion after docking.

SUBMITTER: Jiang L

PROVIDER: S-EPMC2417169 | biostudies-literature | 2008 Mar

REPOSITORIES: biostudies-literature

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Direct quantification of fusion rate reveals a distal role for AS160 in insulin-stimulated fusion of GLUT4 storage vesicles.

Jiang Li L Fan Junmei J Bai Li L Wang Yan Y Chen Yu Y Yang Lu L Chen Liangyi L Xu Tao T

The Journal of biological chemistry 20071206 13

Insulin-stimulated GLUT4 translocation to the plasma membrane constitutes a key process for blood glucose control. However, convenient and robust assays to monitor this dynamic process in real time are lacking, which hinders current progress toward elucidation of the underlying molecular events as well as screens for drugs targeting this particular pathway. Here, we have developed a novel dual colored probe to monitor the translocation process of GLUT4 based on dual color fluorescence measuremen ...[more]

PMID: 18063571

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Project description:Agents that activate the G-protein G(i) (e.g. adenosine) increase, and agents that activate G(s) [e.g. isoprenaline (isoproterenol)] decrease, steady-state insulin-stimulated glucose transport activity and cell-surface GLUT4 in isolated rat adipose cells without changing plasma membrane GLUT4 content. Here we have further examined the effects of R(s)G(s) and R(i)G(i) ligands (in which R(s) and R(i) are G(s)- and G(i)-coupled receptors respectively) on insulin-stimulated cell-surface GLUT4 and the kinetics of GLUT4 trafficking in these same cells. Rat adipose cells were preincubated for 2 min with or without isoprenaline (200 nM) and adenosine deaminase (1 unit/ml), to stimulate G(s) and decrease the stimulation of G(i) respectively, followed by 0-20 min with insulin (670 nM). Treatment with isoprenaline and adenosine deaminase decreased insulin-stimulated glucose transport activity by 58%. Treatment with isoprenaline and adenosine deaminase also resulted in similar decreases in insulin-stimulated cell-surface GLUT4 as assessed by both bis-mannose photolabelling of the substrate-binding site and biotinylation of the extracellular carbohydrate moiety when evaluated under similar experimental conditions. After stimulation with insulin in the absence of G(s) and the presence of G(i) agents, a distinct sequence of plasma membrane events took place, starting with an increase in immunodetectable GLUT4, then an increase in the accessibility of GLUT4 to bis-mannose photolabel, and finally an increase in glucose transport activity. Pretreatment with isoprenaline and adenosine deaminase before stimulation with insulin did not affect the time course of the increase in immunodetectable GLUT4 in the plasma membrane, but did delay both the increase in accessibility of GLUT4 to photolabel and the increase in glucose transport activity. These results suggest that R(s)G(s) and R(i)G(i) modulate insulin-stimulated glucose transport by influencing the extent to which GLUT4 is associated with occluded vesicles attached to the plasma membrane during exocytosis, perhaps by regulating the fusion process through which the GLUT4 in docked vesicles becomes exposed on the cell surface.

Dataset Information

Direct quantification of fusion rate reveals a distal role for AS160 in insulin-stimulated fusion of GLUT4 storage vesicles.

Publications

Direct quantification of fusion rate reveals a distal role for AS160 in insulin-stimulated fusion of GLUT4 storage vesicles.

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