FIGURE 1.

ROS mediates the hypertrophic signal of insulin. A, NAC reduces ROS levels upon treatment with insulin. The neonatal rat cardiomyocytes were incubated with 5 μm DCFH-DA for 30 min at 37 °C, and then treated with 20 μg/ml insulin. ROS was analyzed 1 h after insulin treatment. *, p < 0.05 versus control; ^#, p < 0.05 versus insulin alone. B, insulin-induced ROS elevation is attenuated by catalase. Cardiomyocytes were infected with Ad-β-galactosidase or Ad-catalase at an m.o.i. of 50. 24 h after infection cells were incubated with 5 μm DCFH-DA for 30 min at 37 °C, and then treated with 20 μg/ml insulin. ROS was analyzed 1 h after insulin treatment. *, p < 0.05 versus control; ^#, p < 0.05 versus insulin alone. C, NAC attenuates insulin-induced hypertrophic responses analyzed by cell surface area measurement. Cardiomyocytes were treated as described for A. 48 h after treatment cell surface area was measured. *, p < 0.05 versus control; ^#, p < 0.05 versus insulin alone. D, NAC attenuates insulin-induced hypertrophic responses revealed by protein/DNA ratio. Cardiomyocytes were treated as described for C. 48 h after treatment cells were harvested for the detection of protein/DNA ratio. *, p < 0.05 versus control; ^#, p < 0.05 versus insulin alone. E and F, catalase inhibits hypertrophy induced by insulin. Cardiomyocytes were treated as described for B. 48 h after treatment cells were collected for the analysis of cell surface area (E) and protein/DNA ratio (F). *, p < 0.05 versus control; ^#, p < 0.05 versus insulin alone. G, insulin treatment led to sarcomere organization blocked by NAC and catalase. Representative photos show sarcomere organization. The neonatal rat cardiomyocytes were treated as described for A and B. 48 h after treatment cells were collected for the staining with TRITC-conjugated phalloidin. The nuclei were stained with 4′,6-diamidino-2-phenylindole. Bar = 10 μm. Data in Fig. 1 are expressed as the mean ± S.E. of three independent experiments.