TBC1D1 and TBC1D4 are Rab-GTPase Activating Proteins (Rab-GAPs) expressed in insulin-responsive tissues. Both proteins are involved in mechanisms which regulate basal levels of glucose transport and have been identified as targets of insulin and AMP-dependant kinase (AMPK) signalling pathways, which regulate GLUT4 translocation to the plasma membrane in muscle. We have characterised the C2C12 muscle cell model retrovirally expressing HA-epitope tagged GLUT4 in order to investigate how distinct signalling pathways regulate GLUT4 trafficking. Insulin-stimulation and treatment with the AMPK-activator (AICAR) increased the levels of GLUT4 at the plasma membrane by two-fold in C2C12 myotubes. Insulin-stimulation and activation of AMPK mobilised GLUT4 in to the actively cycling pool. However, our data revealed that insulin-stimulation or AMPK activation resulted in distinct effects on GLUT4 trafficking parameters at steady-state. Insulin increased GLUT4 exocytosis (kex) of this cycling pool. Activation of AMPK inhibited GLUT4 internalisation (ken). The combined effect of insulin-stimulation and AMPK-activation was synergistic and led to increased GLUT4 cell surface levels above those obtained with either treatment alone. Insulin-stimulation and AMPK activation in combination resulted in a partially additive effect on the size of the actively recycling GLUT4 pool and further enhanced kex of this cycling pool.Kinetic studies were performed to measure the effect of TBC1D1 and TBC1D4 knockdown on GLUT4 trafficking in C2C12 myotubes. siRNA-mediated knockdown of TBC1D4 did not affect the basal levels of cell surface GLUT4. Knockdown of TBC1D1 increased cell surface levels of GLUT4 in basal and in insulin-stimulated C2C12 myotubes. The knockdown increased the release of GLUT4 in to the actively recycling pool. By contrast TBC1D1 knockdown did not change the levels of GLUT4 at the plasma membrane that occur in the presence of the AMPK-activator (AICAR). Our results support a model whereby TBC1D1 inactivation by signalling-dependant phosphorylation is required for GLUT4 translocation, but with insulin and AICAR having separate and distinguishable effects on the released GLUT4.
|Date of Award||16 Nov 2013|
|Supervisor||Geoff Holman (Supervisor) & Francoise Koumanov (Supervisor)|
Investigation of Rab-GAPs as links between insulin signalling and GLUT4 translocation
Roche, L. (Author). 16 Nov 2013
Student thesis: Doctoral Thesis › PhD