GLUT-4 translocation in skeletal muscle studied with a cell-free assay: involvement of phospholipase D

GLUT-4-containing membranes immunoprecipitated from insulin-stimulated rat skeletal muscle produce the phospholipase D (PLD) product phosphatidic acid . In vitro stimulation of PLD in crude membrane with ammonium sulfate (5 mM ) resulted in transfer of GLUT-4 (3.0-fold vs. control) as well as transfer rin receptor proteins from large to small membrane structures. The in vitro GLUT-4 transfer could be blocked by neomycin (a PLD inhibitor), and neomyc in also reduced insulin-stimulated glucose transport in intact incubated so leus muscles. Furthermore, protein kinase B-beta (PKBbeta) was found to ass ociate with the GLUT-4 protein and was transferred to small vesicles in res ponse to ammonium sulfate in vitro. Finally, addition of cytosolic proteins , prepared from basal skeletal muscle, and GTP nucleotides to an enriched G LLTT-4 membrane fraction resulted in in vitro transfer of GLUT-4 to small m embranes (6.8-fold vs. unstimulated control). The cytosol and nucleotide-in duced GLUT-4 transfer could be blocked by neomycin and N-ethylmaleimide. In conclusion, we have developed a cell-free assay that demonstrates in vitro GLUT-4 transfer. This transfer may suggest release of GLUT-4-containing ve sicles from donor GLUT-4 membranes involving PLD activity and binding of PK Bbeta to GLUT-4.