While the glucose transporter-4 (GLUT4) is fundamental to insulin-regulated glucose metabolism, its dynamic spatial organization in the plasma membrane (PM) is unclear. Here, using multicolor TIRF microscopy in transfected adipose cells, we demonstrate that insulin regulates not only the exocytosis of GLUT4 storage vesicles, but also the PM distribution of GLUT4 itself. In the basal state, domains (clusters) of GLUT4 molecules in PM are created by an exocytosis that retains GLUT4 at the fusion site. Surprisingly, when insulin induces a burst of GLUT4 exocytosis, it does not merely accelerate this basal exocytosis, but rather stimulates approximately 60-fold another mode of exocytosis that disperses GLUT4 into PM. In contradistinction, internalization of most GLUT4, regardless of insulin, occurs from pre-existing clusters via the subsequent recruitment of clathrin. The data fit a new kinetic model that features multifunctional clusters as intermediates of exocytosis and endocytosis. Upon insulin addition, GLUT4 transport vesicles accumulate and fuse at plasma membrane hot spots, creating clusters. To study the state of the GLUT4 molecules in these PM clusters in primary human adipose cells, we introduced a photo-switchable GLUT4 construct, HA-GLUT4-EOS, and applied a novel photo-activation localization microscopy technique, together with total-internal reflection fluorescence microscopy to track single GLUT4 molecules. We detected two distinct classes of GLUT4 molecule motions: unconstrained lateral diffusion and cluster-confined immobilization. We found that single GLUT4 molecules could get trapped in clusters, severely limiting their diffusion. Conversely, GLUT4 molecules were detected leaving their trapped state in these PM clusters (released) and resuming diffusion at 0.1 m2/s. Double-labeling of insulin-responsive vesicles with GLUT4-mCherry and IRAP-pHluorin was used to detect individual fusion events with PM. GLUT4 clusters were formed through fusion of GLUT4-containing vesicles with PM in an insulin-independent way. GLUT4 molecules were retained within the clusters by an unknown mechanism specific to GLUT4, but not to IRAP. Insulin, on the other hand, enhanced the rate of fusion events that released all GLUT4 into PM. These data provide the first evidence of a dynamic exchange of GLUT4 molecules between clusters and PM, and link insulin-dependent and insulin-independent GLUT4 recycling pathways. Moreover, these findings suggest that the amount of GLUT4 present in PM is not merely defined by the rates of exocytosis and endocytosis, but rather relies on GLUT4-specific molecular interactions at clusters that regulate its recycling. Thus we confirm a non-uniform GLUT4 distribution in the plasma membrane of primary human adipose cells and show the dynamic nature of GLUT4 organization into clusters.
| Lizunov, Vladimir A; Stenkula, Karin; Troy, Aaron et al. (2013) Insulin regulates Glut4 confinement in plasma membrane clusters in adipose cells. PLoS One 8:e57559 |
| Lizunov, Vladimir A; Lee, Jo-Ping; Skarulis, Monica C et al. (2013) Impaired Tethering and Fusion of GLUT4 Vesicles in Insulin-Resistant Human Adipose Cells. Diabetes 62:3114-9 |
| Stenkula, Karin G; Lizunov, Vladimir A; Cushman, Samuel W et al. (2010) Insulin controls the spatial distribution of GLUT4 on the cell surface through regulation of its postfusion dispersal. Cell Metab 12:250-9 |
| Lizunov, Vladimir A; Lisinski, Ivonne; Stenkula, Karin et al. (2009) Insulin regulates fusion of GLUT4 vesicles independent of Exo70-mediated tethering. J Biol Chem 284:7914-9 |
