title: Studies of post-Golgi syntaxins in Glut4 trafficking of 3T3-L1 adipocytes
Creators: name: Perera, Handunge Kumudu Irani
Publisher: name: University of Glasgow
Year: 2002
Subjects: subject: Physiology, GLUT4, insulin.

description: Insulin stimulates glucose transport in fat and muscle tissues by increasing the translocation of the glucose transporter 4 (GLUT4) from intracellular storage compartments to the plasma membrane. In the basal state, GLUT4 is sequestered away from the constitutively recycling endosomal pathway into a specialised GLUT4 storage vesicle compartment (GSV). This sequestration is at the heart of the ability of insulin to increase plasma membrane GLUT4 levels by such large magnitudes. Studies have shown that the SNARE proteins syntaxin (STX) 4, VAMP2 and SNAP-23 mediate the fusion of GLUT4-containing vesicles with the plasma membrane. However, there are no reported studies of the role of SNAREs in intracellular GLUT4 trafficking pathway. The aim of this work was to characterise and identify the role of post-Golgi syntaxins in GLUT4 trafficking in 3T3-L1 adipocytes. As the first step towards understanding post-Golgi syntaxins in GLUT4 trafficking, the extent of colocalisation of STX 6, 8, 12 and 16 with GLUT4 was determined. The results showed that STX 6 and STX 16 are extensively localised in immuno-purified GLUT4 vesicles and that they translocate to the plasma membrane in response to insulin, resulting in a large increase similar to GLUT4. lodixanol gradient analysis showed that both STX 6 and 16 are present in the two major pools of intracellular GLUT4, GSV and endosomal/TGN pool. In contrast, STX 8 and 12 are localised in GLUT4 vesicles into a significantly lesser degree. Endosomal ablation studies demonstrated that about 50% of the STX 6, 8 and 12 are present in the early or recycling endosomal compartment. Furthermore, in vivo immunoprecipitation studies showed that STX 16 is a SNARE partner of STX 6 in 3T3-L1 adipocytes. Recombinant adenovirus (AdV) over-expressing the cytosolic domains of STX 6, 8 and 12 were produced in order to study the function of these syntaxins in 3T3-L1 adipocytes. Overexpression of the STX 6 cytosolic domain significantly slowed down the resequestration of GLUT4 into intracellular compartments after insulin withdrawal. Furthermore, iodixanol gradient analysis revealed that STX 6 function is required to deliver GLUT4 into the GSV during this process. By contrast, expression of STX 8 and 12 cytosolic domains had no effect on the trafficking events examined. The findings of this study strongly suggest that STX 6 is involved in trafficking of GLUT4 from the recycling pathway to the GSVs. Further studies are required to identify the precise intracellular location where STX 6 functions to move GLUT4 into the GSV, and to identify the other SNARE proteins involved. Data of this study suggests that STX 16 could be the Qa-SNARE of this event. Experiments were performed to understand the biogenesis of the GSV in 3T3-L1 cells. The data suggests that an insulin-responsive compartment which sequesters insulin responsive aminopeptidase, exists by day 4 post-differentiation of 3T3-L1 fibroblasts into adipocytes. The expression pattern of syntaxins during differentiation showed that STX 6 expression followed a very similar pattern to GLUT4.