Demonstration of insulin-responsive trafficking of GLUT4 and vpTR in fibroblasts

Insulin-responsive trafficking of the GLUT4 glucose transporter and the ins ulin-regulated aminopeptidase (IRAP) in adipose and muscle cells is well es tablished. Insulin regulation of GLUT4 trafficking in these cells underlies the role that adipose tissue and muscle play in the maintenance of whole b ody glucose homeostasis. GLUT4 is expressed in a very limited number of tis sues, most highly in adipose and muscle, while IRAP is expressed in many ti ssues. IRAP's physiological role in any of the tissues in which it is expre ssed, however, is unknown. The fact that IRAP, which traffics by the same i nsulin-regulated pathway as GLUT4, is expressed in 'non-insulin responsive' tissues raises the question of whether these other cell types also have a specialized insulin-regulated trafficking pathway, The existence of an insu lin-responsive pathway in other cell types would allow regulation of IRAP a ctivity at the plasma membrane as a potentially important physiological fun ction of insulin. To address this question we use reporter molecules for bo th GLUT4 and IRAP trafficking to measure insulin-stimulated translocation i n undifferentiated cells by quantitative fluorescence microscopy, One repor ter (vpTR), a chimera between the intracellular domain of IRAP and the extr acellular and transmembrane domains of the transferrin receptor, has been p reviously characterized. The other is a GLUT4 construct with an exofacial H A epitope and a C-terminal GFP, By comparing these reporters to the transfe rrin receptor, a marker for general endocytic trafficking, we demonstrate t he existence of a specialized, insulin-regulated trafficking pathway in two undifferentiated cell types, neither of which normally express GLUT4, The magnitude of translocation in these undifferentiated cells (approximately t hreefold) is similar to that reported for the translocation of GLUT4 in mus cle cells. Thus, undifferentiated cells have the necessary retention and tr anslocation machinery for an insulin response that is large enough to be ph ysiologically important.