Changes in the signalling status of the small GTP-binding proteins Rac and Rho do not influence insulin-stimulated hexose transport.
Post-receptor signalling molecules that convey the signal from the activated insulin receptor to the actual process of Glut4 translocation and hexose uptake are poorly understood. Various studies have suggested a requirement of the lipid kinase phosphatidylinositol-3 kinase (PI3-kinase) in this process. PI3kinase regulates the activation status of the small GTP-binding protein Rac which, in turn, is able to activate another G-protein Rho. Rac and Rho are known to regulate the structure of the membrane- and cytoplasmic actin-cytoskeleton. We have examined whether Rac and Rho transfer the signals generated by PI3kinase towards insulin-stimulated hexose uptake. For that purpose, we expressed in 3T3-L1 adipocytes the dominant-negative mutant of RacN17 using vaccinia virus-mediated gene transfer. The expression levels of the RacN17 protein were monitored by Western blotting. The abrogation of endogenous Rac signalling by expression of RacN17 was inferred from the observed loss of arachidonic acid release in response to insulin. Basal and insulin-stimulated hexose transport were not affected by expression of the RacN17 mutant. A possible contribution of Rho.GTP to stimulation of hexose uptake was examined by pre-incubation of adipocytes with lysophosphatidic acid (LPA). We observed a profound effect of LPA on the structure of the cytoskeleton and on the phosphorylation of Focal Adhesion Kinase (p125FAK), indicating that 3T3-L1 adipocytes respond to LPA and that Rho was activated by LPA. However, no effect was detected on the basal or on the insulin-stimulated hexose transport. We conclude that Rac and Rho are unlikely to be involved in insulin-stimulated hexose transport, suggesting a possible contribution of other signalling pathways, downstream of PI3kinase to this process.
Dorrestijn
J
J
Department of Medical Biochemistry, Leiden University, The Netherlands.
Bos
J L
JL
Van der Zon
G C
GC
Maassen
J A
JA
D016475:3T3 Cells; D017667:Adipocytes / Q000187:drug effects; D000818:Animals; D001692:Biological Transport; D019204:GTP-Binding Proteins / Q000502:physiology*; D006601:Hexoses / Q000493:pharmacokinetics*; D007004:Hypoglycemic Agents / Q000494:pharmacology*; D007328:Insulin / Q000494:pharmacology*; D051379:Mice; D008970:Molecular Weight; D015398:Signal Transduction / Q000502:physiology*; D013268:Stimulation, Chemical; D020744:rac GTP-Binding Proteins
D006601:Hexoses; D007004:Hypoglycemic Agents; D007328:Insulin; D019204:GTP-Binding Proteins; D020744:rac GTP-Binding Proteins