The Role of Rab11-Interacting Proteins in Insulin-stimulated GLUT4 translocation

GLUT4 translocation to the plasma membrane underlies the ability of insulin to stimulate glucose uptake yet the complete signal transduction pathway and the molecular mechanisms underlying this event remain incompletely defined. In recent years, the family of small Rab GTPases have emerged as key players in all aspects of membrane trafficking and have therefore become of increasing interest to the GLUT4 trafficking field. The importance of Rab GTPases in the regulation of insulin-stimulated GLUT4 translocation recently gained further support following the identification of AS160, aRab GTPase Activating Protein, as a downstream target of the insulin-activated kinase, PKB. The regulation of AS160 by insulin signalling appears to mediate both the intracellular retention of GLUT4 and its insulinstimulated translocation to the cell surface. AS160 has therefore emerged as a critical intermediate in the insulin signal transduction pathway leading to GLUT4 translocation and has provided significant insight into the molecular machinery governing this process. This thesis has focused largely on the role of Rip11, a Rab11 effector protein, in the regulation of insulin-stimulated GLUT4 translocation. Data presented within the study demonstrates that Rip11 displays many trafficking properties which are highly reminiscent of GLUT4. These include: a pronounced redistribution of Rip11 to the cell surface follOWing insulin treatment; a dependence on PI 3-Kinase activity for insulin-stimulated Rip11 translocation; the induction of Rip11 translocation in the absence of insulin through the expression of constitutively active PKB; and the inability of PDGF to recapitulate the effects of insulin and constitutively active PKB on Rip11 localisation. A combination of over-expression studies and siRNA-mediated knockdown were employed to further evaluate the functional role of Rip11 in 3T3-L1 adipocytes. The over-expression of Rip11 was found to block insulin-stimulated GLUT4 fusion but not translocation to the cell surface whilst the ablation of Rip11 from 3T3-L1 adipocytes was found to inhibit insulinstimulated glucose uptake in a dose-dependent manner. Furthermore, Rip11 was found to interact with the PKB substrate AS160 in a manner which is regulated by insulin yet was independent of the Rip11 binding partner, Rab11. Taken together the data presented within this thesis suggest that Rip11 is required for insulinstimulated glucose uptake and possibly functions at the cell surface to coordinate the formation of a GLUT4 vesicle fusion complex. Rip11 may also play an additional role in the regulation of GLUT4 retention and/or the initiation of GLUT4 translocation by virtue of its interaction with AS160. Rip11 therefore represents a novel insulin-regulated protein, which may potentially function as a scaffolding protein to coordinate the protein kinase signalling and trafficking machinery required to stimulate glucose uptake in response to insulin.EThOS - Electronic Theses Online ServiceGBUnited Kingdo