doi:10.1016/j.freeradbiomed.2011.10.158 132 (2-succinyl)-cysteine (2SC): a Biomarker of Mitochondrial Stress in Type 2 Diabetes Norma Frizzell1, Sonia a Thomas1, and John W Baynes1 1University of South Carolina (2-succinyl)-cysteine (2SC) is a chemical modification of proteins formed by the Michael addition of fumarate to cysteine residues in proteins. the formation of 2SC, termed succination of proteins, increases by ~10-fold in adipocytes grown in high glucose medium and in adipose tissues of Type 2 diabetic mice. in order to determine the mechanisms which contribute to increased fumarate levels and protein succination, we investigated how glucotoxicity affects energy status and mitochondrial metabolism in the 3T3 adipocyte. Growth of 3T3 cells in high glucose medium (30 mM vs. 5 mM) significantly increased the cellular ATP/ADP ratio, NADH/NAD+ratio,and the mitochondrial membrane potential (Δψm), indicating that fuel supply exceeded demand in the adipocyte. the increase in NADH/NAD+ inhibits of Krebs cycle NAD+-dependent dehydrogenases, leading to an increase in Krebs cycle intermediates, including fumarate, resulting in increased succination of proteins. Chemical uncouplers, which dissipate Δψm, lowered the NADH/NAD+ratio, decreased fumarate concentration and reduced protein succination, confirming that nutrient excess (glucotoxicity), in the absence of increased ATP demand, creates a ‘pseudohypoxic’ environment (high NADH/NAD+) which drives the increase in fumarate and protein succination. in support of this hypothesis, metformin (1 mM), a weak inhibitor of Complex I, also caused an increase in fumarate concentration and increased succination of proteins. Although markers of oxidative stress were elevated in the adipocytes in association with this mitochondrial stress, there was no effect of antioxidant treatment on protein succination, indicating that succination and oxidative stress are parallel, but independent processes. We conclude that increased succination of proteins is an early biomarker of glucotoxicity and mitochondrial stress in diabetes.

doi:10.1016/j.freeradbiomed.2011.10.159 133 Racial Differences in Urinary F2-isoprostanes May Reflect Weak Metabolic Adaptation in African Americans Dora Il'yasova1, Ivan Spasojevic1, Karel Base1, Frances Wang1, Ralph B D’Agostino Jr2, and Lynne E Wagenknecht2 1Duke University Medical Center, 2Wake Forest Medical Center We examined levels of four urinary F2-isoprostanes (F2-IsoPs) in a large sample of the Insulin Resistance Atherosclerosis Study (IRAS) multi-ethnic cohort: 237 African Americans (AAs) and 617 participants of the non-AA decent, 342 Non-Hispanic Whites (NHW) and 275 Hispanics. Four F2-IsoP isomers were measured in 854 urine samples using liquid chromatography with tandem mass spectrometry detection: iPF2a-III , 2,3-dinor-iPF2a-III, iPF2a-VI, and 8,12-iso-iPF2a-VI. in AAs, the levels of all four F2-IsoPs were lower compared to NHWs and Hispanics: the adjusted mean differences and their 95% confidence intervals (CIs) were -0.084 (-0.11,-0.06), -1.10 (-1.46,-0.74), -2.07 (-2.64,-1.49), and -1.36 (-1.76,-0.95) for iPF2a-III , 2,3-dinor-iPF2a-III, iPF2a-VI, and 8,12-iso-iPF2a-VI, respectively. Surprisingly, F2-IsoPs did not increase with BMI among AAs, whereas such increase was clearly detected among non-AAs participants. the adjusted beta

coefficient for the association between F2-IsoPs and BMI among AAs varied around the null: the beta coefficients and their 95% CIs were -0.0160 (-0.0418, 0.0099), -0.0115 (-0.3426, 0.3195), 0.0685 (-0.4703, 0.6073), and -0.1241 (-0.5029, 0.2547) for the four urinary F2-IsoPs, as mentioned above respectively. in contrast, the adjusted beta coefficient for this association among NHW and Hispanics were all above the null: 0.0195 (0.0039, 0.0353), 0.8335 (0.6329, 1.0342), 0.5564 (0.2299, 0.8830), and 0.7188 (0.4892, 0.9483) for the four isomers as mentioned above, respectively. In 1999, Weyer C. et al.[1] hypothesized that slow fat oxidation may predispose AAs to obesity and therefore, to type 2 diabetes. Based on our hypothesis published in 2005 that urinary F2-IsoPs reflect the intensity of fat oxidation (Il’yasova et al. Obes Res 2005;13:1638), we explain lower levels of F2-IsoPs among AAs as lower rates of oxidative metabolism, specifically fat oxidation. It is known that fat oxidation increases in response to weight gain. Therefore, lack of the positive association between urinary F2-IsoP levels and BMI among AAs is explained as a weak long-term metabolic adaption to higher adiposity via increase in fat oxidation. [1] Weyer C et al. Am J Clin Nutr 1999;70:13

:10.1016/j.freeradbiomed.2011.10.160 134 Morin Modulates High Glucose Induced Apoptosis in Vitro and in Vivo Radhika Kapoor1, and Poonam Kakkar1 1Indian Institute of Toxicology and Research Excessive ROS generation has been implicated in high glucose toxicity in diabetics. We examined the oxidative stress generated due to high glucose in primary hepatocytes and its amelioration by a flavanoid, naringenin. Hepatocytes treated with 40mM glucose for 1.5 h exhibited a significant decrease in cell viability assessed by MTT assay, Alamar Blue and LDH release. Apoptosis indicators such as nuclear AIF, Endo-G, cytosolic Cyt-c, DNA fragmentation, chromatin condensation, mitochondrial membrane potential and externalization of phosphatidyl-serine were significantly altered in glucose treated hepatocytes, suggesting an apoptotic mode of cell death. Glucose induced apoptosis in hepatocytes was a consequence of increased oxidative stress as evidenced by the increased reactive oxygen species (ROS) level assessed by fluoroprobe DCFH-DA. the intracellular antioxidant glutathione was found to be decreased in hepatocytes treated with glucose, while activity of antioxidant enzyme SOD was enhanced in treated hepatocytes to combat oxidative stress due to high glucose levels. Morin, a flavanoid from Dsidium guava was effective in reverting the morphological changes, increasing the cell viability, decreasing the ROS level, ameliorating the levels of apoptotic proteins and DNA damage. Hyperglycemia was induced by giving 65 mg/kg b.w. streptozotozin morin modulated changes observed in fasting glucose, liver biomarker enzymes SGOT,SGPT, carbohydrate metabolizing enzymes like glucokinase, glucose -6 –phosphate dehydrogenase release of apoptotic and antiapoptotic proteins, modulation in MMP and ROS generation was also observed in morin treatment .Similar significant changes was observed in histopathological findings. This study indicates that morin may play an important role in regulating the apoptosis induced by high glucose presumably through intervention of the mitochondria mediated pathway involving oxidative stress.

doi:10.1016/j.freeradbiomed.2011.10.161

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