Studies of the exocytic snares involved in GLUT4 translocation

Brandie, Fiona Marie (2006) Studies of the exocytic snares involved in GLUT4 translocation. PhD thesis, University of Glasgow.

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Abstract

A specialised example of vesicular traffic is the translocation of the glucose transporter Glut4 from intracellular storage compartments to the plasma membrane of fat and muscle cells in response to insulin. In the basal state the majority of Glut4 is held intracellularly, sequestered away from the constitutively recycling endosomal pathway, in a proposed population of specialised Glut4 Storage Vesicles (GSVs). Insulin stimulates glucose transport into adipose cells by promoting the translocation of these GSVs to the plasma membrane where they fuse increasing the Glut4 levels at the cell surface and therefore significantly increasing facilitative glucose transport. The ability of insulin to stimulate glucose transport into muscle and adipose tissue, the main sites of glucose uptake, is central to the ability of insulin to regulate whole body glucose homeostasis, hi individuals with type 2 diabetes this ability of insulin to stimulate glucose transport is impaired. The incidence of type 2 diabetes is increasing rapidly and therefore understanding the molecular basis of insulin-stimulated glucose uptake is of fundamental importance. It has been over 25 years since the first evidence that insulin stimulation led to a translocation of glucose transport form an intracellular site to the plasma membrane of insulin responsive cells. Over this period of time significant advances have been made in the understanding of insulin-stimulated glucose uptake, both in the field of insulin signalling and Glut4 trafficking, however the intersection between these two processes is yet to be established. One major advance in the knowledge of Glut4 trafficking was the identification of the SNARE machinery involved in the fusion of GSVs to the cell surface. In eukaryotes all intracellular trafficking events are facilitated by a family of highly conserved proteins called SNAREs. GLUT4 containing vesicles are enriched in VAMP2, while the plasma membrane of adipocytes is enriched in syntaxin 4 and SNAP23, which together serve as a t-SNARE complex. In vitro these three SNAREs form a highly stable core complex and several studies show that these three proteins mediate the fusion of Glut4-containing vesicles with the plasma membrane. Whether this fusion event is regulated by insulin is yet to be established. The fusion process facilitated by SNARE proteins has been successfully reconstituted in vitro using recombinant proteins expressed in E. coli. Using the exocytic neuronal SNAREs in this assay it was demonstrated that SNAREpins, that is the complex formed between cognate sets of v- and t-SNAREs, are necessary and sufficient to fuse artificial membranes, ha Chapter 3 as the first step towards studying the regulation of fusion facilitated by syntaxin 4, SNAP23 and VAMP2, an in vitro fusion assay using these SNAREs was established. The three SNARE proteins were successfully expressed and purified from E.coli prior to reconstitution into synthetic liposomes that were subsequently analysed for fusion. The results of this assay show that these SNARE proteins in isolation are capable of fusing artificial membranes, a fact previously assumed but never definitively shown. (Abstract shortened by ProQuest.).

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Gwyn Gould
Keywords: Cellular biology
Date of Award: 2006
Depositing User: Enlighten Team
Unique ID: glathesis:2006-71745
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 17 May 2019 09:31
Last Modified: 17 May 2019 09:31
URI: http://theses.gla.ac.uk/id/eprint/71745

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