The aims of this thesis were to investigate the effects of insulin on the cellular distribution and activity of the Na+/H+ exchanger (NHE1) and vacuolar proton pumps (V-ATPases) in cardiomyocytes and to evaluate their association with GLUT4 vesicles. To address these issues, a highly specific anti-NHE1 antibody was raised, which enabled immunofluorescence, immunoprecipitation and co-immunoprecipitation studies into the effects of insulin on NHE1 distribution to be carried out. These studies provided evidence for an insulin-stimulated increase in NHE1 at the plasma membrane and a decrease in intracellular NHE1, even though the overall amount cellular NHE1 remained constant.
A cell free acidity assay (CFAA) was developed to measure NHE1 activity within subcellular fractions. An insulin-stimulated increase in plasma membrane NHE1 activity was identified in accordance with the increase in plasma membrane NHE1. The CFAA also identified an inhibitable portion of V-ATPase activity within the low density microsomes (LDM) from basal cardiomyocytes, which was absent from LDM of insulin-stimulated cells. The insulin-stimulated decrease in LDM V-ATPase activity implied that in the presence of insulin, V-ATPases translocate, as do GLUT4 vesicles.
Structural associations between two complexes of V-ATPase (namely complexes V0 and V1) with GLUT4 vesicles were identified by co-immunoprecipitation of GLUT4 vesicles and subsequent Western blotting for the V-ATPase protein. The studies generated data that was consistent with the hypothesis that the insulin-stimulated change in V-ATPase activity in LDM samples may partly occur as a result of the insulin-stimulated decrease in general vesicle trafficking and fusion and in part to GLUT4 vesicle translocation and fusion.
The thesis identifies further investigations that are needed to determine whether (a) V-ATPases are necessary for vesicle acidification to occur prior to fusion; (b) whether V-ATPases are involved in the formation of a V0–V0 transcomplex necessary for docking of the GLUT4 vesicles with the PM and finally (c) whether insulin affects the distribution of NHE1 either by stimulating NHE1 trafficking or their turnover.
|Date of Award
|21 May 2008
|Geoff Holman (Supervisor)