Insulin regulates two distinct pools of GLUT4-containing vesicles. 3T3-L1 adipocytes were transfected with plasmids encoding TfR-pHluorin or VAMP2-pHluorin, and imaged by TIRFM before and after 100 nM insulin stimulation. (A) Ratios of the fluorescence intensities of fusion pore open and fully fused states, I_d/I_f, were measured using the indicated reporters. Histograms were plotted and fitted with Gaussian distributions, with basal VAMP2-pHluorin is shown as a reference (blue). TfR-pHluorin data were similar for basal and insulin-stimulated cells; insulin data are shown. (B) Cumulative probability plots of data. Significance was assessed using a Kolmogorov-Smirnov test with basal VAMP2-pHluorin data as a reference set. (C) Dual brake–accelerator model of GSV translocation. Insulin acts both to release an intracellular brake (1) and to accelerate docking (2) and full fusion (3) at the PM, and switches vesicle traffic between two cycles (see text).