| Insulin stimulates translocation of the glucose transporter isoform 4 (Glut4) from an intracellular storage compartment to the cell surface in fat and skeletal muscle cells. The nature of the Glut4 storage compartment remains controversial. According to one model, this compartment represents preformed small vesicles that fuse with the plasma membrane in response to insulin stimulation. Alternatively, Glut4 may be retained in large donor membranes, and insulin may stimulate the formation of transport vesicles that deliver Glut4 to the cell surface. In extracts of fat and skeletal muscle cells, Glut4 is predominantly found in small insulin-sensitive 60--70 S vesicles. However, these vesicles might conceivably derive artificially from large donor membranes during cell homogenization. Here, a cell-free reconstitution assay was used to demonstrate that small Glut4-containing vesicles are formed from large donor membranes in a cytosol-, ATP-, time-, and temperature-dependent fashion and, therefore, do not represent an artifact of homogenization. Thus, small insulin-responsive vesicles represent the major form of Glut4 storage in living adipose cells. Fusion of these vesicles with the plasma membrane may be largely responsible for the primary effect of insulin on glucose transport in fat tissue. In addition, our results suggest that insulin may also stimulate the formation of Glut4 vesicles and accelerate Glut4 recycling to the plasma membrane.; Earlier results by other groups demonstrated the presence of phosphatidylinositol 4-kinase (PI4K) in Glut4-vesicles from fat and skeletal muscle cells. However, as it has previously been shown, Glut4-vesicles are not homogeneous and represent a mixture of at least two vesicular populations: cellugyrin-positive and cellugyrin-negative vesicles which are different in size, protein composition and functional properties. It has not been clear which subpopulation of Glut4-vesicles contains (PI4K) activity. In addition, the molecular identity of (PI4K) associated with Glut4-vesicles has not been defined. Sequential immunoadsorption, sucrose gradient analysis, and immunofluorescence were used to show that virtually all (PI4K) activity in the Glut4 pathway belongs to type IIalpha, and that this kinase is primarily associated with cellugyrin-positive rather than cellugyrin-negative Glut4-vesicles. The results imply that PI4KIIalpha in cellugyrin-positive vesicles may play a key role in the biogenesis of the Glut4 storage compartment. |
