The role of synaptotagmins in insulin-stimulated glucose uptake in the 3T3-L1 adipocyte

Miller, Steven Charles Maclean (2006) The role of synaptotagmins in insulin-stimulated glucose uptake in the 3T3-L1 adipocyte. PhD thesis, University of Glasgow.

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Within the adipocyte, skeletal myocyte and cardiac myocyte the sugar transporter GLUT4 exists in numerous distinct subcellular membrane compartments, and a complex series of trafficking steps are known to regulate its distribution. When insulin binds to its receptor at the cell surface, the ensuing cascade of protein-protein and protein-lipid interactions leads to the translocation of a GLUT4-containing vesicle (GSV) to the plasmalemma (PM), where it docks, binds and fuses in a SNARE-dependent manner. In this way GLUT4 is delivered to its intended site of action, and glucose can enter the cell. The precise steps which regulate intracellular GLUT4 trafficking and those which govern GSV-PM fusion are hitherto unknown. GSV-PM fusion is known to disrupted in insulin resistant states such as Type-2 Diabetes, which is a growing global healthcare concern. Such SNARE-mediated fusion events are best understood in neurons, where membrane fusion occurs in a calcium-dependent manner. The calcium-sensing species is an isoform of the 15-member Synaptotagmin (SYT) family, which binds the SNARE complex and the target and vesicle membranes. As a result, otherwise opposing thermodynamic forces between adjacent lipid bilayers are overcome and fusion can occur. In this thesis experiments designed to investigate the role of SYTs in insulin action in the 3T3-L1 adipocyte are described. Using RTPCR, the expression SYT isoforms in 3T3-L1 ceils was demonstrated. SYT7 (calcium-dependent) and SYT11 (calcium- independent) were shown to be the predominant isoforms, and SYT11 expression was seen to be upregulated as cells acquire insulin sensitivity during differentiation. Following their generation, novel affinity-purified SYT7- and SYT11- specific antisera were used to investigate the subcellular localisation of each SYT protein, and both were found to occupy intracellular membrane compartments in common with GLUT4. siRNA-mediated knockdown of SYT7 potentiates glucose uptake, and SYT11 knockdown leads to abrogated insulin-stimulated glucose uptake and reduced intracellular GLUT4. Finally, SYT7 can be seen to bind the Syntaxin4 / SNAP23 / VAMP2 SNARE complex in vitro. Together these data point to an important role for SYT7 and SYT11 in the 3T3-L1 adipocyte, which may represent control of GSV-PM fusion, the regulation of intracellular GLUT4 trafficking or both.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Physiology
Colleges/Schools: College of Medical Veterinary and Life Sciences
Supervisor's Name: Connell, Prof. John and Gould, Prof. Gwyn
Date of Award: 2006
Depositing User: Enlighten Team
Unique ID: glathesis:2006-71428
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 10 May 2019 10:49
Last Modified: 21 May 2021 09:56

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