Trafficking of Glut4-Green Fluorescent Protein chimaeras in 3T3-L1 adipocytes suggests distinct internalization mechanisms regulating cell surface Glut4 levels

Insulin stimulates glucose transport in adipose and muscle tissue by stimul ating the movement ('translocation') of an intracellular pool of glucose tr ansporters (the Glut4 isoform) to the plasma membrane. We have engineered a series of chimaeras between Glut4 and green fluorescent protein (GFP) from Aequoria victoria and expressed these proteins in 3T3-L1 adipocytes by mic roinjection of plasmid cDNA. In the absence of insulin, GFP-Glut4 is locali zed intracellularly within a perinuclear compartment and multiple intracell ular punctate structures. In response to insulin, chimaeric GFP-Glut4 speci es exhibit a profound redistribution to the cell surface with kinetics comp arable with the endogenous protein. The intracellular localization of GFP-G lut4 overlaps partially with compartments labelled with Texas Red transferr in, but is largely distinct from intracellular structures identified using Lysotracker-Red(R). K+-depletion resulted in the accumulation of GFP-Glut4 at the cell surface, but to an lesser extent than that observed in response to insulin. In contrast with native Glut4 removal of the insulin stimulus or treatment of insulin-stimulated cells with phosphatidylinositol 3'-kinas e inhibitors did not result in re-internalization of the chimaeric GFP-Glut 4 from the plasma membrane, suggesting that the recycling properties of thi s species differ from the native Glut4 molecule. We suggest that the recycl ing pathway utilized by GFP-Glut4 in the absence of insulin is distinct fro m that used to internalize GFP-Glut4 from the plasma membrane after withdra wal of the insulin stimulus, which may reflect distinct pathways for intern alization of endogenous Glut4 in the presence or absence of insulin.