Glucose uptake into adipose tissue and skeletal muscle is enhanced by the insulin-mediated translocation of glucose transporters from intracellular membranes to the plasma membrane. Insulin primarily stimulates the redistribution of glucose transporter isoform 4 (GLUT4) from a specialised pool of GLUT4 storage vesicles (GSVs) to the cell surface. In addition, stimulation of cells with insulin initiates a modest translocation of the glucose transporter, GLUT I, from the endocytic network to the plasma membrane. GLUT4 is engaged in a slow but continual cycle between the plasma membrane and various intracellular compartments, including the endosomal network and GSVs. By contrast, despite sharing many sequence similarities with GLUT4, GLUT! recycles at a considerably higher rate, predominantly localising to the endosomes and is excluded from GSVs. Thus, the overall aim of this thesis was to examine the trafficking of GLUT4 in 3T3-L I adipocytes and explore the mechanism ofGLUT4 and GLUTI segregation and the proteins involved in the sorting of GLUT4 into GSVs. A correlative light electron microscopy (CLEM) internalisation assay was developed in order to study the trafficking itinerary of internalising GLUT4 at the ultrastructural level and identify potential proteins involved in the sorting of the transporter. Using this technique, GLUT4 was shown to transit through a variety of different intracellular compartments, before concentrating in a cluster of small insulin-responsive vesicles, approximately 50-100nm in diameter. GLUT4 sorting, translocation and internalisation is mediated by a series of Rab proteins and Rab-binding effector molecules. Previous studies have demonstrated that Rab 14 is a key protein involved in the exocytosis of GLUT4 vesicles in skeletal muscle. Confocal and electron microscopy revealed that over expression of constitutively-active Rab 14 (RabI4Q70L) in 3T3-LI adipocytes resulted in the development of enlarged ' ring-like' early endosomal structures, positive for both endogenous and internalised GLUT4. Furthermore, the CLEM assay demonstrated that knockdown of Rab 14 disrupted GLUT4 transit out of the early endosomes and into the GSVs, thus implicating Rabl4 in the endosomal sorting of GLUT4 prior to the packaging of the transporter into GSVs. The process of GLUT I and GLUT4 segregation was examined using a GLUT4 mutant protein (HA.GLUT4.SQV) containing the C-terminal PDZ ligand of GLUT 1 (DSQV), which has previously been shown to interact with SNX27 in HeLa cells during the recycling of GLUTI back to the cell surface. The HA.GLUT4.SQV mutant trafficked in a manner similar to GLUT I, redistributing to the endosomal network and recycling at an increased rate. Taken together, the data suggests that SNX27 may be responsible for the fast recycling of GLUT 1 in 3T3-LI adipocytes and the interaction of the PDZ ligand located at the C-terminus of GLUT 1 with the PDZ domain ofSNX27 is responsible for the segregation of GLUT 1 away from GLUT4. Finally, the trafficking of the Rab 1 1 effector protein, Rip 11 , which has previously been shown to be important in the insulin-mediated fusion of GLUT4 vesicles, was examined. Rip II predominantly localised to the endocytic network and co-localised with GLUT4 on recycling endosomes. Insulin stimulated the accumulation of Rip II at the cell surface by inhibiting the internalisation of Rip II in a PI 3-kinase dependent and Akt independent manner.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:652040 |
| Date | January 2013 |
| Creators | Hodgson, Lorna Rebecca |
| Publisher | University of Bristol |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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