FIG. 8.

Rapamycin treatment diminishes the amount of rapidly insulin-mobilized GLUT4 in CHO cells. CHO cells expressing the GLUT4 reporter were cultured in DMEM, treated with the indicated concentrations of rapamycin for 36 h, and serum starved during the last 12 h of this period. Cells were stimulated with 160 nM insulin for the indicated times, then chilled, stained for externalized Myc epitope, and subjected to flow cytometry to determine the relative proportion of GLUT4 at the cell surface in each sample. Increasing concentrations of rapamycin caused a progressive diminution of the first phase of GLUT4 externalization (i.e., the overshoot before the steady-state response). Simultaneously, the proportion of GLUT4 at the cell surface in the basal state increased slightly but progressively with increasing rapamycin concentration. Together with the data presented in Fig. 7, these data show that amino acid abundance regulates the amount of rapidly insulin-translocated GLUT4 in CHO cells through a rapamycin-sensitive mechanism.