Thesis

Regulation of GLUT4 translocation in adipocytes : the role of SNARE proteins and caveolae

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Awarding institution
  • University of Strathclyde
Date of award
  • 2023
Thesis identifier
  • T16751
Person Identifier (Local)
  • 201983077
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Abstract
  • Background: Following a meal, insulin stimulates the translocation of the glucose transporter GLUT4 to the plasma membrane (PM) of fat and muscle cells, resulting in increased glucose uptake. This process is impaired in individuals with type 2 diabetes and insulin resistance. Fusion of GLUT4 storage vesicles (GSVs) with the PM requires the formation of a SNARE complex containing the t-SNAREs Syntaxin4 and SNAP23, and the v-SNARE VAMP2, along with the regulatory SM-protein Munc18c. Previous work has shown that insulin-stimulated phosphorylation of Sx4 and Munc18c increases SNARE complex formation and subsequent GSV fusion with the PM. In adipocytes, bulb-shaped membrane microdomains known as caveolae play a key role in insulin action in adipocytes. Methods: Caveolae isolation, immunoblotting methods (including with phospho-specific antibodies), glucose transport assays, and co-localisation experiments using immunofluorescence microscopy and Proximity Ligation Assays in 3T3-L1 adipocytes were used to examine the role of Sx4 phosphorylation in GLUT4 trafficking and to investigate caveolae as a potential site of SNARE protein phosphorylation and SNARE complex formation. siRNA knockdown in 3T3-L1 adipocytes and immunoblotting experiments with primary mouse fat lysates were used to explore the role of the caveolae component EHD2 in GLUT4 trafficking and glucose transport. Results: SNARE proteins were found to be enriched in caveolae-enriched cell fractions. Sx4 was found to co-localise and closely interact with the insulin receptor and the caveolae component Caveolin1. Knockdown of EHD2 in 3T3-L1 adipocytes resulted in reduced expression of key glucose transport proteins, reduced maximum glucose transport, and loss of the insulin stimulated increase in interactions between SNARE proteins. Loss of EHD2 in mice resulted in a reduced fold-change in insulin-stimulated Sx4 phosphorylation. Conclusions: These data argue for an important role for caveolae in insulin stimulated glucose uptake, suggesting they act as a focal point for the intersection of the signalling and trafficking machinery in cultured adipocytes.
Advisor / supervisor
  • Gould, Gwyn W.
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