Fig. 2.

Insulin increases superoxide dismutase activity selectively in Akita cardiomyocytes. Cardiomyocytes were isolated from wild-type and Akita mice and cultured for 24 h in the presence or absence of insulin. (A) Superoxide dismutase activity was measured in cardiomyocyte lysates from wild-type (left) and Akita (right) mice after being cultured for 24 h in the presence or absence of insulin. Insulin treatment significantly increased superoxide dismutase activity in Akita cardiomyocytes (***p < .0005; n = 12 or 13 biological replicates for WT and Akita, respectively). (B) Superoxide dismutase activity was measured in cardiomyocyte lysates from wild-type (left) and Akita (right) mice after being cultured for 24 h with insulin and the presence or absence of LY294002. LY294002 treatment significantly decreased superoxide dismutase activity in Akita cardiomyocytes (**p < .01; n = 5 or 6 biological replicates for WT and Akita, respectively). Less than 5% of SOD activity was inhibited by 5 mM KCN, indicating the data primarily represent SOD2 and/or other nonenzymatic scavenging activity. Statistics were performed by Student's t-test. (C) Change in superoxide dismutase 2 (SOD2) levels in Akita primary cardiomyocytes in the presence or absence of insulin overnight were assessed by western blotting. Quantitative SOD2 protein levels in the lack of insulin were statistically insignificant as compared to basal (+insulin) or + insulin with LY294002 supplemented conditions. (D) SOD2 K68 acetylation levels were measured by Western blot in Akita cardiomyocytes cultured either in the presence or absence of insulin overnight treatment. Quantification indicated no statistical significance (n = 5).