Applied Mathematics Seminar

The insulin-responsive glucose transporter GLUT4 plays an essential role in blood glucose homeostasis by allowing adipose tissue and skeletal muscle to take up glucose after a meal and in the case of skeletal muscle also during exercise. To gain insight into intracellular GLUT4 trafficking, which is largely responsible for the magnitude of GLUT4-mediated glucose uptake, a novel 96 well fluorescence assay was designed based on the retroviral expression of exofacially tagged GLUT4 in 3T3-L1 adipocytes. Immunolabeling of these cells with an anti-tag antibody and a fluorescent secondary antibody respectively after fixation of the cells in the absence or presence of a cell-permeabilizing agent allows for a quantitative analysis of the amount of GLUT4 at the cell surface during insulin stimulation. This enables us to track the transition of the system from a steady state in the absence of insulin to a steady state with both an excess of external insulin and intermediate levels. A model of the kinetics involved in this transition was developed and a time constant for the transition determined as a function of applied insulin. Moreover, incubation of live cells with the anti-tag antibody in the absence or presence of insulin, followed by post-fixation immunolabeling with the fluorescent secondary antibody in the presence of the cell-permeabilizing agent demonstrates the kinetics of GLUT4 trafficking via the plasma membrane and the percentage of total intracellular GLUT4 that is involved in this process. It was found that a significant amount of GLUT4 is excluded from traversing the plasma membrane, suggesting the existence of a static insulin-insensitive GLUT4 pool (silent pool). Modelling this process we were able to estimate both the internalization and exocytosis rate constants for the GLUT4 expression at the plasma membrane, and found that whilst the internalization rate constant remains largely unaffected by insulin, the exocytosis rate constant is altered. Significantly we found that the total amount of GLUT4 involved in the cycling process in steady state changed with changing insulin levels. Some of the possible mechanisms for these effects will be discussed.

(This is joint work with R. Govers and D.E. James of the Garvan Institute of Medical Research, 384 Victoria St, Darlinghurst NSW 2010 )