Involvement of Vesicular H+-ATPase in Insulin-Stimulated Glucose Transport in 3T3-F442A Adipocytes

Abstract

Abstract. 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 H 2 O. 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 insulinstimulated 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 A 1 , 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.

Differential regulation of cardiac glucose and fatty acid uptake by endosomal pH and actin filaments

“…The blockade of insulin/oligomycin-stimulated glucose uptake treatment of cardiomyocytes with endosomal alkalinizing agents is in agreement with earlier studies on insulin-stimulated GLUT4 translocation and glucose uptake in adipocytes (8) and cardiomyocytes (46). Perhaps, the hypothetical GLUT4 transit compartment with budding proteins inhibited by a rise in pH might offer an explanation not only for the incomplete GLUT4 translocation, but also for the inhibition of insulin/ oligomycin-induced GLUT4 translocation upon endosomal alkalinization.…”

“…On the basis of pharmacological agents able to disrupt the pH gradient between endosomes and cytoplasm, as investigated in adipocytes, skeletal muscle, and heart, the role of endosomal pH in regulation of subcellular GLUT4 localization is controversial. For example, it has been reported that bafilomycin-A, a specific inhibitor of the v-ATPase, stimulates (6) or inhibits GLUT4 translocation (8,46). Furthermore, dissipation of the endosomal/cytoplasmic pH gradient with the proton ionophore monensin did not affect insulin-induced GLUT4 translocation (32).…”

Subcellular trafficking of the substrate transporters GLUT4 and CD36 in cardiomyocytes

“…From these data, it was concluded that proper regulation of endosomal pH is important for the formation of small insulin-responsive vesicles [119]. This has also been studied in cardiomyocytes, and in these cells, v-ATPases in the GLUT4-containing vesicles may play a role in insulin-stimulated increase of GLUT4 translocation and glucose uptake [120, 121]. When examining GLUT1 in mouse mammary epithelial cells, it was found that endosomal acidification was important for directed trafficking of GLUT1 [122].…”

SNAREs: Cogs and Coordinators in Signaling and Development

“…Homotypic SNARE fusion in yeast has been associated with vacuole acidification (Ungermann et al, 1999). Similarly, the sensitivity of vesicle traffic to the H 1 -ATPase inhibitor bafilomycin A can be understood in the context of energizing K 1 /H 1 exchange to facilitate vesicle swelling and fusion (Cousin and Nicholls, 1997;Palokangas et al, 1998;Choi et al, 2007). Thus, it is notable that the VHA-a1 protein, a member of the vacuolar V-type H 1 -ATPases, localizes to the Golgi and the TGN in Arabidopsis, consistent with a projected role in vesicle acidification (Dettmer et al, 2006).…”