A novel method to monitor insulin-stimulated GTP-loading of Rab11a in cardiomyocytes.

As a member of the Rab small GTPase family, Rab11a has been shown to be involved in different vesicle trafficking processes. In earlier work we identified Rab11a to be present in GLUT4-containing vesicles after insulin stimulation and showed its involvement in insulin-dependent glucose uptake. However, it remained elusive if Rab11a is directly activated by the insulin signalling cascade and at which step a potential activation occurs. To examine the GTP-loading of Rab11a, we introduced a biotinylated GTP-analog into H9c2-hIR cells, transiently overexpressing HA-tagged Rab11a, and measured its binding to the GTPase after insulin stimulation. We observed that Rab11a is transiently GTP-loaded after insulin stimulation with a 2.3 (+/-0.3) fold activation (n=5), reaching its maximum after 4 min and declining back to basal after additional 2 min. The activation of Rab11a is phosphatidylinositol 3-kinase (PI3-kinase) dependent and downstream of Akt, as shown by in vitro knockdown of this kinase. These data show that Rab11a is directly activated by insulin and represents an element of the GLUT4 trafficking machinery.