Insulin regulates cellular glucose uptake by changing the amount of glucose transporter-4 (GLUT4) in the plasma membrane through stimulation of GLUT4 exocytosis. However, how the particular trafficking, tethering, and fusion steps are regulated by insulin is still debated. In a 3T3-L1 adipocyte cell line, the Exocyst complex and its Exo70 subunit were shown to critically affect GLUT4 exocytosis. Here we investigated the effects of Exo70 on tethering and fusion of GLUT4 vesicles in primary isolated rat adipose cells. We found that Exo70 wild type was sequestered away from the plasma membrane in non-stimulated cells, and its overexpression had no effect on GLUT4 trafficking. The addition of insulin increased the amount of Exo70 in the vicinity of the plasma membrane and stimulated the tethering and fusion of GLUT4 vesicles, but the rates of fusion and GLUT4 exposure were not affected by overexpression of Exo70. Surprisingly, the Exo70-N mutant induced insulin-independent tethering of GLUT4 vesicles, which, however, did not lead to fusion and exposure of GLUT4 at the plasma membrane. Upon insulin stimulation, the stationary pretethered GLUT4 vesicles in Exo70-N mutant cells underwent fusion without relocation. Taken together, our data suggest that fusion of GLUT4 vesicles is the rate-limiting step regulated by insulin downstream of Exo70-mediated tethering. From animal models and cell line experiments it is known that insulin enhances glucose uptake by recruiting GLUT4 from specialized vesicles to the plasma membrane. To date, however, scarce microscopic data exist on the trafficking and recycling of GLUT4 in primary adipose cells. In this study we applied multi-color total-internal reflection fluorescence microscopy to visualize the dynamic recycling of GLUT4 in live adipose cells isolated from rat and human. We introduced a double-labeled red fluorescent HA-GLUT4-mCherry construct that is successfully expressed in primary rat and human adipose cells, and shows correct localization in insulin-responsive vesicles together with endogenous GLUT4. In non-stimulated primary cells, GLUT4 was found to be sequestered in intracellular vesicles densely distributed under PM, but distinct from the perinuclear distribution observed in 3T3-L1 cultured adipocytes. We quantified the dynamic stages of GLUT4 trafficking and identified microtubule-based movement of GLUT4 vesicles, tethering events at PM, and fusion events. We observed two modes of fusion: i) GLUT4 diffusing completely into PM after fusion, or ii) GLUT4 retained as clusters at the site of fusion. Insulin selectively stimulated the fusion events that dispersed GLUT4 molecules into PM, while under non-stimulated conditions, the majority of fusion events led to the formation of clusters. HA-antibody labeling confirmed that GLUT4 molecules in the clusters were exposed at the cell surface and thus constituted functional transporters. We further tested GLUT4 association with Clathrin-coated pits and Caveolae, both of which have been implicated in the recycling of GLUT4. While mobile and stationary GLUT4 vesicles did not show any labeling with Caveolin, we did observe clathrin to accumulate at the GLUT4 clusters. However, no clustering of GLUT4 was observed at pre-existing Clathrin patches. These data indicate that in primary adipose cells, GLUT4 clusters represent the key intermediate step between exocytosis and endocytosis of GLUT4, and thus may be the crucial site for regulation of GLUT4 recycling. 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 |
