Insulin signaling alters antioxidant capacity in the diabetic heart

Figures

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  Fig. 1. Akita cardiomyocytes have enhanced ROS production in response to antimycin A and to the lack of insulin. Cardiomyocytes were isolated from wild-type and Akita mice and ROS production was measured by EPR spin trapping with CMH. (A) ROS production was measured basally in cardiomyocytes cultured in standard culture media. (B) ROS production was measured in the presence or absence of 1.0 μM antimycin A and shown as the raw intensities (left) and the % change (right). Antimycin A significantly increased ROS production in Akita cardiomyocytes (*p < .05; **p < .01; n = 6 biological replicates). (C) ROS production was compared when cardiomyocytes isolated from wild-type (left) and Akita (right) mice were cultured for 24 h in the presence or absence of 10 μg/mL insulin. Insulin treatment significantly decreased ROS in Akita cardiomyocytes (***p < .005; n = 9 or 10 biological replicates for WT and Akita, respectively). Statistics were performed by Student's t-test.

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  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…

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  Fig. 3. Insulin increases thioredoxin level in Akita cardiomyocytes. (A)(B) Mitochondrial NADH-supported superoxide production was measured fluorometrically using mitoplast isolated from Akita mice hearts underwent 1-week either vehicle (-ins) or 5U/kg insulin (+ins) treatment. Mitochondria isolated from insulin injected animals exhibited decreased capacity of superoxide production, and that was in part due to increased endogenous antioxidant (*p < .05; n = 6 biological replicates). Statistics were performed by Student's t-test. (C) Quantitative proteomic analysis was performed by selective reactive monitoring mass-spectrometry utilizing whole heart homogenates acquired from Akita hearts underwent 1-week of vehicle or insulin treatment. Among the 29 proteins reported in the antioxidant panel, thioredoxin 1 (txn1) was the only protein exhibiting statistically significant insulin sensitivity (**p < .02; n = 4 biological replicates). (D) Total and oxidized glutathione levels in those homogenates were unchanged. (E)(F) Change in thioredoxin interacting-protein (TXNIP) levels in WT and Akita primary cardiomyocytes by insulin overnight incubation were assessed by western blotting. Quantitative…

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  Fig. 4. Thioredoxin scavenges enzymatically generated superoxide in vitro. The capacity of superoxide scavenging activity of thioredoxin (Trx) and thiol-containing small molecules were quantified using xanthine and xanthine oxidase-generated superoxide and pure proteins. (A) The superoxide scavenging activity required cycling reduced Trx. Representative kinetics traces are from control (no addition; black), with the addition of Trx and thioredoxin reductase (TR) at middle time point (blue), and with the addition of Trx and TR in the presence of NADPH (red). (B) Quantification of the superoxide scavenging activity was assessed with varied Trx concentration, while all other variables were fixed. The capacity of superoxide scavenging were found to be Trx concentration-dependent and can be fit to a classical Hill equation (see text for EC50 value). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)