FIGURE 1.

NO enhances insulin responsiveness. A, COS-7 cells ectopically expressing human eNOS were stimulated with insulin (20 nm). Aliquots of total lysates (25 μg) were subjected to immunoblotting with antibodies as indicated. B, COS-7 transfectants were treated with insulin for 5 min. Insulin-induced NO production was measured by the assay detecting the level of NO (represented by the concentration of nitrite) released to culture media. The data are presented as the means ± S.E. (n = 3). *, p < 0.05 versus mock transfectants. C, COS-7 transfectants were stimulated with insulin. Insulin receptor β-subunit (InRβ) was immunoprecipitated (IP) and then subjected to immunoblotting with antibodies as indicated. Aliquots of total lysate were also subjected to immunoblotting. The right panels show a densitometric analysis of the gel image as a ratio of phosphorylated InRβ relative to total InRβ. A.U., arbitrary unit. D, eNOS transfectants were treated with dimethyl sulfoxide (DMSO) or 300 μm c-PTIO for 30 min prior to insulin stimulation. The immunoprecipitated InRβ or aliquots of total lysates were subjected to immunoblotting with antibodies as indicated (left panel). The ratio of phosphorylated InRβ relative to total InRβ is shown in the right panels. Similar results (C and D) were observed in two independent experiments.