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Carnitine-Palmitoyltransferase-1 Deficiency Impairs Skeletal Muscle Fatty Acid Oxidation, But Preserves Insulin Sensitivity
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
SFRBM 2012
S99
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
SFRBM 2012
S99