title: GLUT-4 translocation in skeletal muscle studied with a cell-free assay: involvement of phospholipase D.
abstract: 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 transferrin receptor proteins from large to small membrane structures. The in vitro GLUT-4 transfer could be blocked by neomycin (a PLD inhibitor), and neomycin also reduced insulin-stimulated glucose transport in intact incubated soleus muscles. Furthermore, protein kinase B(beta) (PKB(beta)) was found to associate with the GLUT-4 protein and was transferred to small vesicles in response to ammonium sulfate in vitro. Finally, addition of cytosolic proteins, prepared from basal skeletal muscle, and GTP nucleotides to an enriched GLUT-4 membrane fraction resulted in in vitro transfer of GLUT-4 to small membranes (6.8-fold vs. unstimulated control). The cytosol and nucleotide-induced 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 vesicles from donor GLUT-4 membranes involving PLD activity and binding of PKB(beta) to GLUT-4.
authors.affiliation: Copenhagen Muscle Research Center, Department of Human Physiology, University of Copenhagen, 13 Universitetsparken, DK-2100 Copenhagen, Denmark.
authors.forename: S
authors.initials: S
authors.lastname: Kristiansen
authors.forename: J N
authors.initials: JN
authors.lastname: Nielsen
authors.forename: S
authors.initials: S
authors.lastname: Bourgoin
authors.forename: A
authors.initials: A
authors.lastname: Klip
authors.forename: M
authors.initials: M
authors.lastname: Franco
authors.forename: E A
authors.initials: EA
authors.lastname: Richter
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000645:Ammonium Sulfate / Q000494:pharmacology; D000818:Animals; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D002474:Cell-Free System; D003600:Cytosol / Q000737:chemistry; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D051275:Glucose Transporter Type 4; D006160:Guanosine Triphosphate / Q000494:pharmacology; D007163:Immunosorbent Techniques; D007328:Insulin / Q000494:pharmacology; D008297:Male; D009004:Monosaccharide Transport Proteins / Q000378:metabolism*; D009124:Muscle Proteins / Q000494:pharmacology; D018482:Muscle, Skeletal / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D009355:Neomycin / Q000494:pharmacology; D010712:Phosphatidic Acids / Q000378:metabolism; D010739:Phospholipase D / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar
chemical_list: D004791:Enzyme Inhibitors; D051275:Glucose Transporter Type 4; D007328:Insulin; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; D010712:Phosphatidic Acids; C495694:Slc2a4 protein, rat; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D010739:Phospholipase D; D009355:Neomycin; D005033:Ethylmaleimide; D000645:Ammonium Sulfate