FIG. 7.

Amino acid concentrations regulate the amount of rapidly insulin-mobilized GLUT4 in CHO cells. (a) CHO cells expressing the reporter were cultured in the indicated media for 36 h and serum starved during the last 12 h of this period. Cells were stimulated with 160 nM insulin for various amounts of time, then chilled, stained for externalized Myc epitope tag, and analyzed by FACS as described in the text to determine the relative proportion of GLUT4 at the cell surface in each sample. Compared to the amino acid concentrations in standard MEM (defined as 1× amino acids), concentrations of most amino acids in DMEM are twofold higher, and concentrations of most amino acids in F12 are only 0.08 to 0.5 times as high (depending on the particular amino acid; see Materials and Methods). All media contained 2 mM glutamine. The degree to which insulin stimulates a transient overshoot of the final, steady-state proportion of GLUT4 at the plasma membrane correlates well with the concentrations of essential amino acids in the various media. The data shown are from two separate experiments (mean ± standard deviation) and are normalized to the steady-state response in the presence of insulin (30-min time point). (b) A similar experiment was performed with cells cultured in MEM containing various concentrations of essential amino acids except for glutamine, which was held constant (see text). Higher amino acid concentrations cause a greater overshoot of the final, steady-state fraction of GLUT4 at the plasma membrane after insulin addition.