In secretory cells, osmotic swelling of secretory granules is proposed to be an intermediate step in exocytic fusion of the granules with the plasma membrane. For osmotic swelling of the granule, a H+ gradient generated by vacuolar-type H+-ATPase (V-ATPase) may be a driving force for accumulation of K+ via its exchange with H+, concurrent with accumulation of Cl- and H2O. Here, we investigated whether a similar chemiosmotic mechanism is involved in the insulin-stimulated recruitment of GLUT4 to the plasma membrane in 3T3-F442A adipocytes. Incubating cells in a hypoosmotic medium significantly increased 2-deoxy glucose (2-DG) uptake and the plasma membrane GLUT4 content (possibly via induction of osmotic swelling of GLUT4-containing vesicles (G4V)) and also potentiated the insulin-stimulated 2-DG uptake. Promotion of the G4V membrane ionic permeability using nigericin, an electroneutral K+/H + exchange ionophore, increased 2-DG uptake and the plasma membrane GLUT4 content. However, co-treatment with nigericin and insulin did not show an additive effect. Bafilomycin A1, a diagnostically specific inhibitor of V-ATPase, inhibited insulin- and nigericin-stimulated 2-DG uptake. Immunoadsorption plus immunoblotting demonstrated that GLUT4 and V-ATPase co-localize in the same intracellular membranes. Together, these results indicate that V-ATPases in the G4V membrane may play an important role in the insulin-stimulated exocytic fusion of G4V with the plasma membrane via its participation in osmotic swelling of the vesicle.
- Chemiosmotic mechanism
- Vacuolar-type H-ATPase
ASJC Scopus subject areas
- Endocrinology, Diabetes and Metabolism