Involvement of vesicular H⁺-ATPase in insulin-stimulated glucose transport in 3T3-F442A adipocytes

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.