INCREASED INTRACELLULAR SEQUESTRATION OF THE INSULIN-REGULATED AMINOPEPTIDASE UPON DIFFERENTIATION OF 3T3-L1 CELLS

In fat and muscle cells, the glucose transporter GLUT4 is sequestered in an intracellular compartment under basal conditions and redistribut es markedly to the plasma membrane in response to insulin. Recently, w e characterized a membrane aminopeptidase, designated IRAP (insulin-re gulated aminopeptidase), that colocalizes with intracellular GLUT4 and similarly redistributes markedly to the plasma membrane in response t o insulin in adipocytes. In contrast to GLUT4, IRAP is also expressed in 3T3-L1 fibroblasts, and this finding provided an opportunity to com pare its subcellular distribution in fibroblasts and adipocytes. The r elative amount of IRAP at the cell surface was measured by a cell surf ace biotinylation method. The portion of total IRAP at the cell surfac e in unstimulated adipocytes was 30% of that in unstimulated fibroblas ts. Upon insulin treatment the portion of IRAP at the cell surface was the same in fibroblasts and adipocytes, and was increased 1.8-fold in fibroblasts and 8-fold in adipocytes. A similar analysis of the distr ibution of the transferrin receptor (TfR), the paradigm for recycling plasma membrane receptors, revealed that the portions of the TfR at th e cell surface in both the basal and insulin-treated states were almos t unchanged upon differentiation, and that insulin caused an increase of about 1.6-fold in the amount of TfR at the cell surface. These resu lts show that enhanced intracellular sequestration of IRAP occurs duri ng adipogenesis, and that this effect underlies the larger insulin-eli cited fold increase of IRAP at the cell surface in adipocytes.