Effects of Mitochondrial-Targeted Antioxidants on Real-Time Blood Nitric Oxide and Hydrogen Peroxide Release in Acute Hyperglycemic Rats

Abstract

We hypothesized that acute hyperglycemia (200 mg/dL) would increase H2O2 and decrease NO levels in blood relative to saline group. By contrast, MitoQ and the SS-31 peptide would attenuate acute hyperglycemiainduced oxidative stress (e.g., H2O2) and improve vascular function (e.g., increase NO production) under acute hyperglycemia. We found that acute hyperglycemia significantly reduced blood NO levels compared to saline group. The administration of MitoQ or SS-31 during hyperglycemia significantly improved blood NO levels, similar to saline control. Meanwhile we found acute hyperglycemia maintained a higher level of H2O2 in blood compared to saline group. By contrast, MitoQ or SS-31 during hyperglycemia significantly reduced blood H2O2 levels compared to those under hyperglycemia. Moreover, SS-31 treatment showed a trend to reduce blood H2O2 levels more than those in MitoQ treatment, but was not significant. These results suggest that mitochondrial derived SO is a significant source of oxidative stress and vascular endothelial dysfunction under acute hyperglycemic conditions. Moreover, treatment with mitochondrial-targeted antioxidants, MitoQ or SS-31, may be beneficial to attenuate hyperglycemia induced oxidative stress and vascular endothelial dysfunction. Male Sprague-Dawley rats (275 to 325g, Charles River, Springfield, MA) were anesthetized with 60 mg/kg of pentobarbital sodium with 1000 unit heparin via intraperitoneal (i.p.) injections. The jugular vein was catheterized to allow for the infusion of saline, 20% D-glucose, or 20% D-glucose with 1.86 mg/kg MitoQ (MW=600 g/mol; complexed with cyclodextrin to improve water solubility, total MW=1714 g/mol) or with 2.7 mg/kg SS-31 (MW=640 g/mol, Genemed Synthesis, Inc., San Antonio, TX). The continuous infusion of 20% D-glucose solution was to maintain hyperglycemia around 200 mg/dL for about 180 min. MitoQ or SS-31 was added to 20% glucose to reach approximately 13 μM and 50 μM in blood, respectively. Both femoral veins will be exposed and catheterized in order to place the calibrated NO and H2O2 microsensors (100 μm, WPI Inc., Sarasota, FL) at random into each femoral vein. These microsensors were then connected to the Apollo 4000 free radical analyzer (WPI Inc., Sarasota, FL) to measure for blood NO and H2O2 levels in real-time. NO, H2O2, and glucose levels will then be recorded at baseline and at 20 minute intervals throughout the 180 minute infusion period [2]. The changes of blood NO (nM) and H2O2 (M) levels were expressed as the relative change to the baseline or to saline group, respectively. All the data was represented as a mean ± SEM. The data were then analyzed by ANOVA using post hoc analysis with the Student Newman Keuls. p<0.05 was considered as significant.