Dual-mode of insulin action controls GLUT4 vesicle exocytosis

Department of Cell Biology, Yale University School of Medicine, New Haven, CT 06520, USA.
The Journal of Cell Biology (Impact Factor: 9.83). 05/2011; 193(4):643-53. DOI: 10.1083/jcb.201008135
Source: PubMed


Insulin stimulates translocation of GLUT4 storage vesicles (GSVs) to the surface of adipocytes, but precisely where insulin acts is controversial. Here we quantify the size, dynamics, and frequency of single vesicle exocytosis in 3T3-L1 adipocytes. We use a new GSV reporter, VAMP2-pHluorin, and bypass insulin signaling by disrupting the GLUT4-retention protein TUG. Remarkably, in unstimulated TUG-depleted cells, the exocytic rate is similar to that in insulin-stimulated control cells. In TUG-depleted cells, insulin triggers a transient, twofold burst of exocytosis. Surprisingly, insulin promotes fusion pore expansion, blocked by acute perturbation of phospholipase D, which reflects both properties intrinsic to the mobilized vesicles and a novel regulatory site at the fusion pore itself. Prolonged stimulation causes cargo to switch from approximately 60 nm GSVs to larger exocytic vesicles characteristic of endosomes. Our results support a model whereby insulin promotes exocytic flux primarily by releasing an intracellular brake, but also by accelerating plasma membrane fusion and switching vesicle traffic between two distinct circuits.

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    • "Recent studies in primary myotubes and cardiomyocytes [2] [3], however, link the input of PLC to Ca 2+ channels as discussed later in this chapter. Emerging studies in adipocytes suggest that insulin also regulates pore expansion of the inserted GLUT4 vesicle through the action of phospholipase D [32] [44]. Surprisingly, in spite of the prominent regulation of Ca 2+ in cardiomyocyte function [45], its participation in insulin-dependent glucose transport and metabolic actions in the heart is less understood . "
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    • "The number of immuno-gold particles in the endosomes is reduced to 25 % after stimulation with insulin, suggesting a movement of GLUT4 towards the plasma membrane from this compartment . Intriguingly, prolonged insulin stimulation was found to cause fusing cargo to switch from 60 nm GSVs to larger exocytic vesicles, characteristic of endosomes, therefore suggesting an additional role of the endosomes and endosomederived structures in GLUT4 translocation (Xu et al. 2011). Furthermore, transferrin and Rab14-positive endosomal vesicles have been shown to fuse with the plasma membrane in response to insulin stimulation, albeit at a slower rate than GSV fusion. "
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    • "To further investigate the degranulation process, we visualized and compared the fusion processes of individual granules in the WT and Stk24 h/h neutrophils using live-cell TIRF microscopy. We have previously used the fusion protein of the v-SNARE vesicleassociated membrane protein 2 (VAMP2) with a pH-sensitive fluorescent protein, pHluorin, to monitor the exocytosis of GLUT4 storage vesicles (Xu et al., 2011). The fluorescence of pHluorin becomes much brighter when transferred from an acidic (inside vesicles) to a neutral environment (after vesicle fusion with the plasma membrane) (Miesenbö ck et al., 1998; Jankowski et al., 2001; Sankaranarayanan and Ryan, 2001). "
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