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Title: Insulin signalling and membrane fusion in adipose cells
Author: Ribé, David Raymond
ISNI:       0000 0001 3515 6986
Awarding Body: University of Bath
Current Institution: University of Bath
Date of Award: 2005
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Insulin regulates glucose uptake in responsive cells by trafficking GLUcose Transporter 4 (GLUT4) molecules to the plasma membranes of these cells. This thesis focuses on events at the plasma membrane, including membrane fusion and activation of GLUT4. Wortmannin inhibition experiments in rat adipocytes did not show the trafficking-independent inhibition of insulin-stimulated glucose transport activity at low wortmannin concentrations seen in other cell types. The p38 MAP kinase inhibitor SB203580 inhibited labelling of cell surface GLUT4 as well as glucose transport activity. Purified recombinant protein standards were used to quantify SyNaptosomeAssociated Protein (SNAP) REceptor (SNARE) proteins in rat adipocytes. There were approximately 2,500,000 copies of syntaxin4, 8,900,000 copies of SNAP23, 1,900,000 copies of VAMP2 and 1,000,000 copies of Muncl8c in each adipose cell. Syntaxin4, SNAP23 and Muncl8 were predominantly localised to the plasma membrane. SNAP23 was in large excess compared to the other proteins in plasma membranes, suggesting that its availability was tightly regulated. VAMP2 was distributed between the plasma membrane and intracellular membranes. Insulin caused a two-fold increase in VAMP2 at the plasma membrane. The small amounts of syntaxin4 in intracellular membranes were in large excess over Muncl8c in the same membranes. These results suggest that Muncl8c is unlikely to play a major role in trafficking syntaxin4 to the plasma membrane as has been suggested. A number of approaches were applied to expressing and purifying recombinant SNAREs. The purified proteins were reconstituted into liposomes. This system is intended to be the future basis for experiments on SNARE function and their regulation by insulin.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available