Arrest of endosome acidification by bafilomycin A(1) mimics insulin actionon GLUT4 translocation in 3T3-L1 adipocytes

In insulin-sensitive fat and muscle cells, the major glucose transporter GL UT4 is constitutively sequestered in endosomal tubulovesicular membranes, a nd moves to the cell surface in response to insulin. While sequence informa tion within GLUT4 appears to be responsible for its constitutive intracellu lar sequestration, the regulatory elements and mechanisms that enable this protein to achieve its unique sorting pattern under basal and insulin-stimu lated conditions are poorly understood. We show here that arrest of endosom e acidification in insulin-sensitive 3T3-L1 adipocytes by bafilomycin A(1), a specific inhibitor of the vacuolar proton pump, results in the rapid and dose-dependent translocation of GLUT4 from the cell interior to the membra ne surface; the effects of maximally stimulatory concentrations of bafilomy cin A(1) (400-800 nM) were equivalent to 50-65 % of the effects of acute in sulin treatment. Like insulin, bafilomycin A(1) induced the redistribution of GLUT1 and Rab4, but not that of other proteins whose membrane localizati on has been shown to be insulin-insensitive. Studies to address the mechani sm of this effect demonstrated that neither autophosphorylation nor interna lization of the insulin receptor was altered by bafilomycin A(1) treatment. Bafilomycin-induced GLUT4 translocation was not blocked by cell pretreatme nt with wortmannin. Taken together, these data indicate that arrest of endo some acidification mimics insulin action on GLUT4 and GLUT1 translocation b y a mechanism distal to insulin receptor and phosphatidylinositol 3-kinase activation, and suggest an important role for endosomal pH in the membrane dynamics of the glucose transporters.