Analysis of VAMP levels and function in 3T3L1 adipocytes

A major action of the hormone insulin is to increase glucose uptake into muscle and adipose tissues. This occurs through the insulin-regulated translocation of the glucose transporter GLUT4 from intracellular deposits to the cell surface. The dysregulation of this process is a major facet of insulin-resistance and Type-2 diabetes. In the absence of insulin, GLUT4 is sequestered intracellularly in two interlinked pools. These pools are the endosomal recycling compartment and a specialised insulin-sensitive compartment. GLUT4 vesicles within the specialised insulin-sensitive compartment are termed GSVs. GSVs provide a readily available store of GLUT4 that can be rapidly mobilised to the cell surface in response to insulin stimulation. GLUT4 within the cell is continually recycled through multiple compartments, including the endosomal recycling compartment and GSVs. Perturbation of flux through any of these compartments disrupts GLUT4 traffic and thus disrupts the insulin-response. Consequently, understanding these trafficking steps is an important research goal. The formation of GSVs and their insulin-stimulated translocation to, and fusion with, the plasma membrane are examples of regulated and specific membrane trafficking events. As such these events require SNARE proteins. This study has examined the functions of each of the members of the post-Golgi vSNAREs that make up the VAMP subfamily in the trafficking of GLUT4 into the insulin-sensitive compartment, as well as examining the contribution each of these vSNAREs plays in the SNARE complex involved in the insulin-stimulated translocation of GLUT4 to the cell surface. GLUT4 traffics through the endosomal system and the trans Golgi network (TGN) en route into GSVs in a process known to involve the t-SNARE syntaxin 16. In order to dissect the roles VAMP proteins play in the intracellular recycling and trafficking of GLUT4, the interactions of VAMP proteins with syntaxin 16 and its regulatory Sec-1/Munc-18 protein, mVps45 have been examined. Further, the effects of depletion of these VAMPs in GLUT4 distribution has been examined in HeLa cells expressing HA-GLUT4-GFP as a surrogate for adipose and muscle tissue. The fusion of GLUT4-containing vesicles with the cell surface in response to insulin stimulation is a key component of the insulin-response. Although there is consensus that tSNAREs involved in this process are syntaxin 4 and SNAP23, there is discord in the literature regarding the role each vSNARE plays in this fusion event. Many studies support a role for VAMP2, however recently it has been suggested that VAMPs 3 and 8 may also be involved. The roles of these VAMPs, as well as the other post-Golgi vSNAREs expressed in adipocytes, in the fusion of GLUT4-containing vesicles with the cell surface have been examined. This has been achieved through the characterisation of the expression level, subcellular distribution of the VAMPs and determination of associations that take place between each VAMP protein and the tSNARE complex made up of syntaxin 4 and SNAP23. The results from these experiments show that despite all VAMPs being capable of forming SDS-resistant SNARE complexes with either syntaxin 4 and SNAP23 or syntaxin 16 and SNAP23, in the cellular environment only VAMP2 interacts with syntaxin 4 and only VAMP4 interacts with syntaxin 16. This, alongside the finding that each VAMP protein is expressed at varying levels and localises with different pools of GLUT4, suggests that VAMP2, not VAMPs 3 or 8, is the major vSNARE involved in the trafficking of GLUT4 to the cell surface. The finding that VAMP4 is the only VAMP that interacts with syntaxin 16, alongside the results of VAMP depletion in HeLa cells expressing HA-GLUT4-GFP, support the hypothesis that VAMP4 is involved in sorting GLUT4 out of the endosomal pool and into GSVs