title: Analysis of the subcellular distribution and trafficking of the insulin-responsive glucose transporter, GLUT4
Creators: name: Millar, Caroline Ann
Publisher: name: University of Glasgow
Year: 1998
Subjects: subject: Molecular biology.

description: The translocation of a facilitative glucose transporter, GLUT4, from an intracellular site to the cell surface accounts for the large insulin-dependent increase in glucose transport observed in muscle and adipose tissue. Recent studies have indicated that a large proportion of intracellular GLUT4 is segregated from the recycling endosomal system in a post-endocytic storage compartment in 3T3-L1 adipocytes. However, the nature of this GLUT4 storage compartment, and the mechanism by which GLUT4 reaches the cell surface upon insulin stimulation are poorly understood. Here, I have used Tf-HRP-mediated endosomal ablation analysis to examine the protein composition of the post-endocytic compartment with the aim of further defining the relationship of this compartment to other, well characterised, intracellular membrane structures. The results provide further evidence for the existence of a GLUT4 compartment segregated from the endosomal system, which is devoid of cell surface to endosome recycling markers including the TfR and SCAMPs. The observation that within this compartment, GLUT4 co-localises with TGN marker proteins, gamma-adaptin and the CD-M6PR, suggests that GLUT4 traffics through the TGN as part of its recycling itinerary. I further exploited this technique to examine the translocation of GLUT4 to the cell surface in response to insulin and GTPgammaS. The results indicate that insulin is still capable of stimulating GLUT4 translocation following endosomal ablation under conditions where GTPgammaS-stimulated GLUT4 translocation was effectively inhibited. In addition, both insulin- and GTPgammaS-stimulated GLUT1 translocation were blocked following ablation of the recycling endosomal system. On the basis of these and other data, I propose that insulin stimulates the exocytosis of two distinct compartments, one being the post-endocytic storage compartment and the other being the endosomal system, which may also be stimulated by GTPgammaS. Recent studies have implicated ARF proteins to function as regulators of regulated exocytosis. To investigate the role of ARF proteins in insulin-stimulated GLUT4 translocation, we examined the effect of myristoylated ARF peptides on insulin-stimulated GLUT4 translocation and glucose transport in permeabilised 3T3-L1 adipocytes. A myristoylated peptide corresponding to the N-terminus of ARF6 markedly inhibited insulin-stimulated GLUT4 translocation and 2-deoxy-D-glucose transport, whereas ARF5 and ARF1 peptides were without effect. In addition, an effective inhibitor of PLD, butan-1-ol was observed to have no effect on insulin-stimulated GLUT4 translocation and glucose transport. These results suggest that ARF6 plays a crucial role in insulin-stimulated GLUT4 translocation. However, PLD does not appear to function as a downstream effector of ARF in this event. Finally, I have shown that a chimeric protein comprising the signal sequence of the human growth hormone and HRP (ssHRP) may be targeted to the exocytic pathway and secreted from 3T3-L1 adipocytes. However, due to the time constraints of this study, I was unable to use this HRP probe to ablate the TGN, thus enabling me to investigate the role of this sorting compartment in GLUT4 trafficking.