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.