Investigating the roles of the cardiomyocyte V-ATPase and NHE1, and their response to insulin stimulation

  • Amelia Louise Preedy

Student thesis: Doctoral ThesisPhD


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 Award21 May 2008
Original languageEnglish
Awarding Institution
  • University of Bath
SupervisorGeoff Holman (Supervisor)

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