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Obesity Related Markers in Catalase Transgenic Mice
TG
141 Glutathionylation of Hepatic and Visceral Adipose Proteins Decreases in Obese-Prone, GlucoseIntolerant Rats Matthew J Picklo1, Matthew I Jackson1, and Joseph Idso1 1 USDA/ARS Grand Forks Human Nutrition Research Center Obesity and insulin resistance are associated with increases in oxidative stress and lipid peroxidation. On the other hand, adipocytes from obese animals have elevated GSH content, and insulin resistance can be reversed by GSH depletion. Oxidation of active site cysteines of protein tyrosine phosphatases (PTP) improves insulin sensitivity indicating that limited levels of protein thiol oxidation has beneficial physiological properties. We tested the hypothesis that the oxidized thiol content of proteins from tissues important to glucose uptake is decreased in obesity. This study utilized obese-prone Sprague-Dawley (OPSD) rats that developed obesity and glucose intolerance when fed a high-fat (45% fat calories) diet versus OPSD rats fed a control, normal fat (10% fat calories) diet. Liver, visceral (perirenal) adipose and skeletal (gastrocnemius) muscle were studied. Proteins from these tissues were analyzed for content of sulfenic acids (PSOH), protein glutathione disulfides (PSSG), and PTPbased sulfonic acids (oxPTP) by dot blot and immunoblot methods. Hepatic proteins from obese animals demonstrated a decrease in PSSG (45%) and oxPTP (40%) content versus the control animals while there was no change in PSOH. There was no obvious change in the masses of modified proteins as assessed by western blot. Like liver, adipose proteins from obese rats showed a decrease in PSSG (26%) content but not PSOH or oxPTP. There were no changes in the PSOH, PSSG, or oxPTP in skeletal muscle. PSSG is reduced by glutaredoxin (GRX) activity. However there was no difference in hepatic and muscular GRX activity or GRX-1 protein content between obese and control animals. While GRX activity was not detectable in adipose tissue, GRX-1 content was increased 72% in visceral adipose from obese animals. Our data indicate that glutathionylation of proteins is decreased in select tissues in the obese state in some cases by elevated GRX1. These data suggest that obesity alters protein redox state in a tissue-specific manner during obesity and that reduced protein thiol oxidation may influence obesity-induced pathology.
doi:10.1016/j.freeradbiomed.2011.10.168
142 Nitric Oxide Prevents Diet-Induced Obesity by Regulating Adipose Tissue Phenotype Brian E Sansbury1, Jason Hellman1, Yunan Tang1, Matthew Spite1, Aruni Bhatnagar1, and Bradford G Hill1 1 University of Louisville Obesity and diabetes are the most pressing health issues of our time. Although a decrease in nitric oxide (NO) bioavailability has been linked to insulin resistance, it is not known how increasing NO affects diet-induced obesity and diabetes. We hypothesize that overexpression of endothelial nitric oxide synthase (eNOS) prevents diet-induced obesity and insulin resistance by regulating adipose tissue metabolism. To test this hypothesis, mice TG overexpressing eNOS (eNOS ) and wild-type C57BL/6 mice (WT) were placed on either 60% high fat or 10% low fat diet for TG six weeks. the eNOS mice showed a striking resistance to dietinduced obesity compared with WT mice as evidenced by decreased weight gain (4.29±1.04g vs. 9.00±1.52g; n=13, P<0.05) despite nearly identical increases in plasma cholesterol and similar glucose and insulin tolerance. the mean adipocyte
area of the high fat-fed eNOS mice was decreased compared 2 2 with WT mice (1949±461µm vs. 4022±249µm n=7, P<0.05). Interestingly, plasma triglycerides and free fatty acids were TG decreased twofold in eNOS mice (P<0.001) with no change in glycerol levels indicating that fat utilization, and not adipose tissue TG lipolysis, is likely responsible for lower adiposity in eNOS mice. Consistent with this, mitochondrial content was increased in TG adipose tissue from eNOS mice compared with WT mice; protein levels of the mitochondrial proteins cytochrome c oxidase subunit 1, Sirt3, and aldehyde dehydrogenase 2, as well as PGC1α, were increased by two fold or greater (P<0.05). However, no change in UCP-1 was detected. Extracellular flux analysis of differentiated 3T3-L1 adipocytes treated chronically with DETANONOate showed increases in oxidative phosphorylation, glycolysis, and fatty acid oxidation compared with untreated adipocytes (P<0.05). the triglyceride content of the 3T3 adipocytes was decreased by nearly half after incubation with NO in culture. These data suggest that overexpression of eNOS prevents diet-induced obesity and promotes transdifferentiation of adipocytes to a previously un-described phenotype characterized by increased mitochondria and high fat oxidation capacity.
doi:10.1016/j.freeradbiomed.2011.10.169
143 Obesity Related Markers in Catalase Transgenic Mice Courtney Crain1, Preeya Shah2, Carla Cook1, Holly King1, and Nalini Santanam1 1 Joan C Edwards School of Medicine, Marshall University, 2 Oberlin College, OH Major attributes of obesity are related to increased abdominal fat mass leading to insulin resistance and ultimately to Type 2 diabetes and cardiovascular disease. An increase in adipose mass is also accompanied by an increase in oxidative and inflammatory stress. Though increasing antioxidant defense might prevent these stresses, reports indicate that low-grade oxidative stress might actually be helpful for cellular function. the goal of this study was to investigate if high tissue levels of the antioxidant enzyme, catalase would alter adipose mass and obesity related markers in a high fat fed catalase transgenic mice. Catalase transgenic mice that express high levels of the human Catalase gene, currently being bred in our laboratory, were used for this study. Female catalase transgenic mice (CAT) and wild type (C57Bl/6J) mice (WT) were fed either a high fat diet (HF) or a normal chow diet (NC) for 6 weeks (n=5/group). Lean and obese (Lepob/J) mice were used as controls. Since the CAT mice were heavier to begin with, these mice appeared to have gained more body weight on high fat diet compared to wild type mice. At the end of feeding, obesity related markers (lipid levels and adipose derived factors) in the mice plasma and tissues were measured using cholestech kits, adipokine arrays or qPCR. High fat feeding showed significant changes in both circulating and tissue levels of adipose derived markers such as leptin, CRP and circulating ketones and a decrease in adiponectin in CAT vs. WT groups. Mechanisms related to this increased body weight and leptin/CRP levels in CAT mice compared to wild type mice are currently being investigated.
doi:10.1016/j.freeradbiomed.2011.10.170
SFRBM 2011
S65
141 Glutathionylation of Hepatic and Visceral Adipose Proteins Decreases in Obese-Prone, GlucoseIntolerant Rats Matthew J Picklo1, Matthew I Jackson1, and Joseph Idso1 1 USDA/ARS Grand Forks Human Nutrition Research Center Obesity and insulin resistance are associated with increases in oxidative stress and lipid peroxidation. On the other hand, adipocytes from obese animals have elevated GSH content, and insulin resistance can be reversed by GSH depletion. Oxidation of active site cysteines of protein tyrosine phosphatases (PTP) improves insulin sensitivity indicating that limited levels of protein thiol oxidation has beneficial physiological properties. We tested the hypothesis that the oxidized thiol content of proteins from tissues important to glucose uptake is decreased in obesity. This study utilized obese-prone Sprague-Dawley (OPSD) rats that developed obesity and glucose intolerance when fed a high-fat (45% fat calories) diet versus OPSD rats fed a control, normal fat (10% fat calories) diet. Liver, visceral (perirenal) adipose and skeletal (gastrocnemius) muscle were studied. Proteins from these tissues were analyzed for content of sulfenic acids (PSOH), protein glutathione disulfides (PSSG), and PTPbased sulfonic acids (oxPTP) by dot blot and immunoblot methods. Hepatic proteins from obese animals demonstrated a decrease in PSSG (45%) and oxPTP (40%) content versus the control animals while there was no change in PSOH. There was no obvious change in the masses of modified proteins as assessed by western blot. Like liver, adipose proteins from obese rats showed a decrease in PSSG (26%) content but not PSOH or oxPTP. There were no changes in the PSOH, PSSG, or oxPTP in skeletal muscle. PSSG is reduced by glutaredoxin (GRX) activity. However there was no difference in hepatic and muscular GRX activity or GRX-1 protein content between obese and control animals. While GRX activity was not detectable in adipose tissue, GRX-1 content was increased 72% in visceral adipose from obese animals. Our data indicate that glutathionylation of proteins is decreased in select tissues in the obese state in some cases by elevated GRX1. These data suggest that obesity alters protein redox state in a tissue-specific manner during obesity and that reduced protein thiol oxidation may influence obesity-induced pathology.
doi:10.1016/j.freeradbiomed.2011.10.168
142 Nitric Oxide Prevents Diet-Induced Obesity by Regulating Adipose Tissue Phenotype Brian E Sansbury1, Jason Hellman1, Yunan Tang1, Matthew Spite1, Aruni Bhatnagar1, and Bradford G Hill1 1 University of Louisville Obesity and diabetes are the most pressing health issues of our time. Although a decrease in nitric oxide (NO) bioavailability has been linked to insulin resistance, it is not known how increasing NO affects diet-induced obesity and diabetes. We hypothesize that overexpression of endothelial nitric oxide synthase (eNOS) prevents diet-induced obesity and insulin resistance by regulating adipose tissue metabolism. To test this hypothesis, mice TG overexpressing eNOS (eNOS ) and wild-type C57BL/6 mice (WT) were placed on either 60% high fat or 10% low fat diet for TG six weeks. the eNOS mice showed a striking resistance to dietinduced obesity compared with WT mice as evidenced by decreased weight gain (4.29±1.04g vs. 9.00±1.52g; n=13, P<0.05) despite nearly identical increases in plasma cholesterol and similar glucose and insulin tolerance. the mean adipocyte
area of the high fat-fed eNOS mice was decreased compared 2 2 with WT mice (1949±461µm vs. 4022±249µm n=7, P<0.05). Interestingly, plasma triglycerides and free fatty acids were TG decreased twofold in eNOS mice (P<0.001) with no change in glycerol levels indicating that fat utilization, and not adipose tissue TG lipolysis, is likely responsible for lower adiposity in eNOS mice. Consistent with this, mitochondrial content was increased in TG adipose tissue from eNOS mice compared with WT mice; protein levels of the mitochondrial proteins cytochrome c oxidase subunit 1, Sirt3, and aldehyde dehydrogenase 2, as well as PGC1α, were increased by two fold or greater (P<0.05). However, no change in UCP-1 was detected. Extracellular flux analysis of differentiated 3T3-L1 adipocytes treated chronically with DETANONOate showed increases in oxidative phosphorylation, glycolysis, and fatty acid oxidation compared with untreated adipocytes (P<0.05). the triglyceride content of the 3T3 adipocytes was decreased by nearly half after incubation with NO in culture. These data suggest that overexpression of eNOS prevents diet-induced obesity and promotes transdifferentiation of adipocytes to a previously un-described phenotype characterized by increased mitochondria and high fat oxidation capacity.
doi:10.1016/j.freeradbiomed.2011.10.169
143 Obesity Related Markers in Catalase Transgenic Mice Courtney Crain1, Preeya Shah2, Carla Cook1, Holly King1, and Nalini Santanam1 1 Joan C Edwards School of Medicine, Marshall University, 2 Oberlin College, OH Major attributes of obesity are related to increased abdominal fat mass leading to insulin resistance and ultimately to Type 2 diabetes and cardiovascular disease. An increase in adipose mass is also accompanied by an increase in oxidative and inflammatory stress. Though increasing antioxidant defense might prevent these stresses, reports indicate that low-grade oxidative stress might actually be helpful for cellular function. the goal of this study was to investigate if high tissue levels of the antioxidant enzyme, catalase would alter adipose mass and obesity related markers in a high fat fed catalase transgenic mice. Catalase transgenic mice that express high levels of the human Catalase gene, currently being bred in our laboratory, were used for this study. Female catalase transgenic mice (CAT) and wild type (C57Bl/6J) mice (WT) were fed either a high fat diet (HF) or a normal chow diet (NC) for 6 weeks (n=5/group). Lean and obese (Lepob/J) mice were used as controls. Since the CAT mice were heavier to begin with, these mice appeared to have gained more body weight on high fat diet compared to wild type mice. At the end of feeding, obesity related markers (lipid levels and adipose derived factors) in the mice plasma and tissues were measured using cholestech kits, adipokine arrays or qPCR. High fat feeding showed significant changes in both circulating and tissue levels of adipose derived markers such as leptin, CRP and circulating ketones and a decrease in adiponectin in CAT vs. WT groups. Mechanisms related to this increased body weight and leptin/CRP levels in CAT mice compared to wild type mice are currently being investigated.
doi:10.1016/j.freeradbiomed.2011.10.170
SFRBM 2011
S65