Changes in the signalling status of the small GTP-binding proteins Rac and Rho do not influence insulin-stimulated hexose transport

Summary

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 Rac^N17 using vaccinia virus-mediated gene transfer. The expression levels of the Rac^N17 protein were monitored by Western blotting. The abrogation of endogenous Rac signalling by expression of Rac ^N17 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 Rac^N17 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 pro- found effect of LPA on the structure of the cytoskeleton and on the phosphorylation of Focal Adhesion Kinase (pl25^FAK), 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, down-stream of PI3kinase to this process.

Key words

Insulin - Rac - Rho - hexose transport