Evaluation of Nitric Oxide Levels in Obese Normotensive Children

activation of MMP-9, mitochondria damage, and the development of diabetic retinopathy. Expression of MMP-9 and its translocation to the mitochondria were analyzed in the retina of rats streptozotocin-induced diabetic for 15 days to 12 months. The results were confirmed in retinal endothelial cells incubated in 20mM glucose for 6-96 hours. Although MMP-9 was increased as early as 15 days of diabetes, its mitochondrial accumulation was observed at 2 months, but damage to the mtDNA-encoded gene important in electron transport (ETC) system, ND6, was observed only when the duration of diabetes was extended to 6 months, a duration where apoptosis of retinal capillary cells can be detected. Consistent with the results from the retina, increased MMP-9 activity was observed as early as 6 hours of high glucose insult in retinal endothelial cells, and decrease in ND6 gene was observed only after 48 hours of glucose exposure. The results suggest that in diabetes activated MMP-9 translocates to the mitochondria, and this mitochondrial translocation precedes mtDNA damage. Thus, early modulation of MMP-9 by pharmacological means could provide a potent strategy to prevent the initiation of a vicious cycle of free radicals produced by damaged ETC system, and inhibit the progression of retinopathy in diabetic patients.

doi:10.1016/j.freeradbiomed.2012.10.180

227 Glycated Albumin by Methylglyoxal Promotes Oxidative Stress and Dysregulation of the PI3K/Akt/Bad Survival Signaling Pathway in Insulin-Producing Cells MIN-6 1

Adriano Sartori , Elieti Antônia Gimenes Prates1, and Hugo Pequeno Monteiro1 1 Universidade Federal de São Paulo (UNIFESP), Brazil Tissue damage induced by hyperglycemia in diabetic patients is characterized by increased flux through the polyol pathway, protein kinase C activation, increased hexosamine pathway activity, and intracellular production of advanced glycation end products (AGE) precursors. Hyperglycemic conditions promote oxidative stress and apoptosis in β-cells. Thioredoxin (TRX), a redox active protein, has been shown to protect β-cells against oxidative stress and apoptosis. In addition, TRX activity is regulated by thioredoxin-interacting protein (TXNIP). Recently, it has been demonstrated that an increase on TXNIP protein expression levels promotes inhibition of TRX activity and oxidative stress in a variety of cell types. Furthermore, TXNIP expression levels increase after exposure of ȕ-cells to toxic concentrations of glycated albumin by glucose and ribose. Methylglyoxal (MG) is produced from trioses phosphate, acetone, and threonine catabolism. Glycated albumin by MG yields MGderived AGES which have been associated with microvascular complications in diabetes. In this communication we investigated the effects of MG-derived AGEs on MIN6 mouse β-cells. MIN-6 cells were treated with increasing concentrations of MG-derived AGEs (50-200 ȝg/mL). MG-AGEs inhibited cell proliferation, calcium influx, and NO production. In addition, they stimulate the expression levels of receptor for AGEs, the levels of the apoptosis-related proteins p-BAD/Bcl-xL, and of TRX. Expression levels of iNOs and p-AKT decreased after exposure of MIN6 cells to increasing concentrations of MG-AGEs. MIN6 cells exposed to MG-AGEs showed increases in insulin content and on the ratio GSSG/GSH, suggesting the establishment of oxidative stress conditions for these cells. In conclusion, our data suggest that excess of AGEs produced from MG in diabetic patients may drive oxidative damage and eventually death of pancreatic ȕ-cells. Support: CNPq, CAPES, FAPESP.

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doi:10.1016/j.freeradbiomed.2012.10.181

228 Mitochondrial Fatty Acid Overload and Proximal Tubular Redox Imbalance - A Role for Peroxiredoxin 2? 1

Christine Howard1 and Krisztian Stadler 1 Pennington Biomedical Research Center Obesity has recently been suggested as an independent risk factor for chronic kidney disease (CKD), even in patients lacking diabetes. Obesity is generally associated with oversupply of the nutrient pool including fatty acids. While renal podocytes are insulin sensitive and able to consume various substrates, tubular cells are almost completely lacking glycolysis and preferably oxidize fatty acids. We have hypothesized that a fatty acid overexposure therefore can induce tubular mitochondrial and cellular redox imbalance, ultimately contributing to the activation of redox sensitive fibrotic or apoptotic pathways. To test this, we have exposed NRK-52 proximal tubular epithelial cells to pure albumin, fatty acid contaminated albumin and palmitate in various doses and timepoints. Mitochondria were primary targets in palmitate exposure as indicated by decreased mitochondrial viability (MTT assay), membrane potential and decreased ATP turnover and spare respiratory capacity (Seahorse analyzer). Late apoptosis was evident in cells with longer exposure or higher dose of FA. When we looked at apoptosis related redox sensitive pathways and defense mechanisms, we have found early activation of pJNK at 6 hrs followed by caspase 3 at 24 hrs, and interestingly, this was associated with an almost complete lack of peroxiredoxin 2 expression and an increase of the oxidized form Prx-SO3. This suggests a probably peroxide or H2O2 related redox imbalance where FA exposure targets peroxiredoxin 2, ultimately leading to redox sensitive apoptosis of tubular cells. We are currently investigating this pathway further with mito CAT or Prdx2 transfection and whether increasing catalase in mitochondria or cellular prdx2 has potential beneficial effects on bioenergetics, restoration of redox balance and tubular viability.

doi:10.1016/j.freeradbiomed.2012.10.182

229 Evaluation of Nitric Oxide Levels in Obese Normotensive Children 1

Cleva Villanueva , Lily Rivera-Figueroa2, Irene Mendoza-Lujambio1, Rafael Cobilt-Catana2, Antonio Ruiz-Rivera1, and Robert David Kross3 1 2 Escuela Superior de Medicina del IPN, Mexico Hospital 3 Regional Militar de Irapuato, Mexico Kross Link Laboratories Chronic inflammation and endothelial dysfunction have been related to some features of metabolic syndrome, such as hypertension. Nitric oxide (NO) levels are increased in inflammation and reduced in endothelial dysfunction. The goal of the study was to evaluate NO levels in obese normotensive children, and to look for any relationship with plasma metabolic variables. A case-control study was performed in 28 obese and 21 non-obese children (5-15 years old). Compared to control children, obese patients had significantly higher plasma glucose and triglycerides (TGs), whereas high density lipoproteins (HDLs) were lower in obese children. Total cholesterol and low density lipoproteins were similar in both groups. Plasma reduced nitrates (NOx) were significantly higher in obese children. A positive correlation between NOx and TGs was found in both groups, whereas a negative correlation between NOx and HDLs was

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found only in obese children. Since obese children were normotensive, it is postulated that NO production increased as a result of inflammation and/or adaptation to the vascular changes (e.g. increased shear stress) that precede hypertension.

doi:10.1016/j.freeradbiomed.2012.10.183

230 Carnitine-Palmitoyltransferase-1 Deficiency Impairs Skeletal Muscle Fatty Acid Oxidation, But Preserves Insulin Sensitivity 1

Shawna E Wicks , Bolormaa Vandanmagsar1, Kimberly R Haynie1, Jingying Zhang1, Robert Noland1, and Randall L Mynatt1 1 Pennington Biomedical Research Center The development of insulin resistance has been linked to accumulation of toxic lipid intermediates in skeletal muscle, possibly as a result of decreased fatty acid oxidation (FAO). Alternatively, insulin resistance may arise as a consequence of accelerated, but incomplete, FAO that results in redox imbalance. To differentiate between these hypotheses, we generated mice with muscle-specific deletion of carnitine palmitoyltransferase-I m-/(CPT-1 ), an essential enzyme for mitochondrial fatty acid import for beta-oxidation. Remarkably, despite impaired FAO, m-/CPT-1 mice accumulate less body fat, have preserved insulin sensitivity and improved glucose disposal, as measured by insulin (ITT) and glucose (GTT) tolerance tests. Serum levels of glucose and insulin are lower, indicative of better glycemic control. This occurs despite marked accumulation of lipid droplets in oxidative muscle fibers. Isolated mitochondria respirate normally, indicating that mitochondrial function is not compromised, but citrate synthase activity and mitochondrial DNA content are elevated, suggesting mitochondrial biogenesis. Consistent with increased mitochondrial biogenesis, the mRNA levels of PCG1Į are m-/increased in CPT-1 mice, as are enzymes associated with peroxisomal FAO and the muscle-specific uncoupling protein, UCP3. Electron micrographs show increased number and altered morphology of mitochondria, specifically in oxidative-type fibers. We are utilizing extracellular flux analysis to dissect the contribution of UCP3 and the differences between oxidative and glycolytic muscle fiber mitochondria. We are currently examining the redox implications of these adaptations using both Amplex red and DMPO spin trapping to quantify mitochondrial oxidant emission, analysis of diacylglycerol and ceramide species and measurement of GSH:GSSG ratios.

doi:10.1016/j.freeradbiomed.2012.10.184

231 Human Serum Albumin Masks Antioxidant Potential of Dietary Polyphenols 2

Hui Cao1, Yixi Xie1, Xiaoqing Chen1, and Jianbo Xiao 1 2 Central South University, China, Normal University, China Polyphenols in plasma are bound to plasma proteins to some degree. The polyphenol-protein interaction (PPI) is reversible in that the polyphenol-protein complex can dissociate and release the free polyphenols. PPI is expected to modulate the bioavailability of polyphenols. Determining the influence of PPI on the antioxidant potential of polyphenols is critical and will directly correlate with the bioavailability of polyphenols. Herein, the -4 antioxidant activities of polyphenols (5.0 × 10 mol/L) in the absence and presence of HSA were measured on the basis of the DPPH free radical, ABTS radical, and superoxide anion

scavenging potential. Polyphenols scavenged free radical depending on their structure. We have found that HSA masked the antioxidant potential (IC50) of polyphenols. For example, DPPH free radical scavenging percentages of kaempferol and kaempeferide were 86.83% and 87.67% in the absence of HSA, respectively; however, free radical scavenging percentages reduced to 59.95% and 62.76% in the presence of HSA. The masking effect of these polyphenols with strong DPPH radical scavenging potential appears higher than those polyphenols with weak DPPH radical scavenging potential. The corresponding consequence of PPI is improving free polyphenols in blood, which causes fewer polyphenols expose to free radicals in blood. Therefore, polyphenols in blood are protected to be oxidized and can be efficiently delivered to other tissues, which enhances the beneficial impact of polyphenols. Acknowledgments: The authors are grateful for financial sponsored by Shanghai Rising-Star Program (11QA1404700), and Shanghai Science and Technology Development Project (11440502300).

doi:10.1016/j.freeradbiomed.2012.10.185

232 Transcription Factor NF-E2-Related Factor 2 (Nrf2)Mediated Antioxidant Defense System in the Development of Diabetic Retinopathy 1

Qing Zhong and Renu Kowluru1 1 Wayne State University Purpose: Increased oxidative stress in diabetes has been associated with the development of diabetic retinopathy. Our previous studies have shown that, in addition to increase in retinal reactive oxygen species in diabetes, the level of the intracellular antioxidant, glutathione (GSH), also becomes subnormal. Furthermore, the catalytic subunit of glutamylcysteine ligase (GCLC), an enzyme important in GSH synthesis, is compromised. GCLC transcript is regulated by transcription factor Nrf2, and Nrf2 is implicated in the regulation of antioxidant genes through binding to the antioxidant-response element (ARE). The aim of this study is to investigate the role of Nrf2-GCLC-GSH pathway in the development of diabetic retinopathy. Methods: Retinal endothelial cells were incubated in 5mM glucose or 20mM glucose media for 4 days with or without pretreatment with an activator of Nrf2, tert-butylhydroquinone (tBHQ; 1.5 μM). The expressions of Nrf2 and GCLC were measured by quantitative PCR and by western blot technique. DNA binding activity of Nrf2 was determined in the nuclear fraction by an ELISA method in which the binding of the activated Nrf2 to the ARE oligonucleotide was quantified using anti-Nrf2 antibody. Nrf2 binding at the GCLC enhancer was detected by chromatin immunoprecipitation technique, and GSH level was quantified by an enzymatic recycling method. Key results were confirmed in the retina from streptozotocin-diabetic rats and also in the retina from human donors with diabetic retinopathy. Results: Exposure of retinal endothelial cells to high glucose increased gene expressions of Nrf2, but the Nrf2 DNA binding activity and the binding of Nrf2 at GCLC enhancer were decreased by 50%. Nrf2 activator effectively prevented glucose-induced inhibition of Nrf2 activity, and restored GCLC gene expression. Consistent with this, although diabetes increased Nrf2 gene expression in rat retina, the activity of Nrf2 and the binding of Nrf2 at the enhancer of GCLC were significantly decreased compared to the values obtained from normal rat retina. Similarly, in the retina from human donors with diabetic retinopathy despite increase in Nrf2 gene expression, GCLC was significantly decreased compared to that from age-matched non-diabetic donors. Conclusions: Although retinal Nrf2 gene expression is

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