GLUT4 trafficking in muscle cells has been studied to determine how distinct signalling pathways induce GLUT4 translocation. Two different cell models were adopted for these investigations; cardiomyocytes isolated from a transgenic mouse line expressing HA-GLUT4-GFP in muscle and L6 myotubes retrovirally expressing HA-GLUT4. The GLUT4 constructs were largely excluded from the external membrane under basal conditions in both cell models. GLUT4 was trafficked to the external membrane in to response all stimuli studied in cardiomyocytes (insulin, contraction and hypoxia) and L6 myotubes (insulin, AICAR and A-769662). By comparing the anti-HA and GFP signals at the sarcolemma and transverse tubules in cardiomyocytes, it has also be possible to observe an enhancement of GSV fusion with the sarcolemma following stimulation with insulin and contraction. This effect was specific to these stimuli and to the sarcolemma. Insulin-stimulation of GLUT4 exocytosis was not detected under steady-state conditions in L6 myotubes. Here, the major effect of insulin-stimulation and AMPK-activation was on GLUT4 internalisation. The rate constant for GLUT4 internalisation was very rapid in basal cells and was decreased during the steady-state responses to insulin and the AMPK-activators AICAR and A-769662. In cardiomyocytes, internalising GLUT4 colocalised with clathrin at puncta at the sarcolemma. This indicates that GLUT4 is internalised via a clathrin-mediated route. Investigations into the amount of GLUT4 recycling in L6 cells under steady-state conditions revealed that a large proportion of cellular GLUT4 recycles with the cell surface under basal conditions. Insulin-stimulation and AMPK-activation additively mobilised GLUT4 in L6 cells. This implies a non-convergent mobilisation of GLUT4 in response to activation of the PKB/Akt and AMPK signalling pathways. Data obtained from an in vitro kinase assay confirmed that serine 237 of TBC1D1 is a bone fide AMPK phosphorylation site. Furthermore, phosphorylation of this site in L6 myotubes incubated with AMPK activators has been confirmed using a novel antibody specific to TBC1D1 phosphorylated at serine 237. This thesis discusses the consequences and importance of multiple controls impinging on GLUT4 traffic and highlights the advantages and limitations of kinetic studies of these processes.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:518302 |
| Date | January 2010 |
| Creators | Fazakerley, Daniel John |
| Contributors | Holman, Geoffrey ; Koumanov, Francoise |
| Publisher | University of Bath |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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