- Email: [email protected]
Epigenetic alterations in skeletal muscle metabolism are associated with weight loss resistance
J.R. Hickok, D. Vasudevan, D.D. Thomas* University of Illinois at Chicago, USA
Cardiovascular, Metabolic & Environmental Disorders I 2:
Introduction: Histone methylation of lysine residues is an important epigenetic regulator of chromatin structure and gene transcription. Lysine methylation status is orchestrated by the activity of both methyltransferases and demethylases. KDM3A is a member of the JMJC domain containing family of Fe(II), a-ketoglutarate dependent histone demethylases. This enzyme demethylates mono- and dimethylated histone lysine residues.
Epigenetic alterations in skeletal muscle metabolism are associated with weight loss resistance
Methods: We examined the ability of nitric oxide (NO) to regulate histone methylation using NO-producing cells and NO-donor compounds. Protein immunoblots measured changes in histone methylation status. Electron paramagnetic imagining was used to determine NO/iron/enzyme interactions. Enzyme demethylase activity was measured by mass spectroscopy. Results: Using MDA-MD-231 breast cancer cells, we uncovered a novel dual regulatory mechanism of NO on global histone methylation. Overall levels of dimethylated Lys9 on histone 3 (H3K9me2) were significantly elevated in a concentration-dependent manner by NO exposure. This increase in H3K9me2 occurred despite a concomitant down regulation of the predominant histone lysine methyltransferase (G9a) and upregulation of KDM3A. These findings were replicated in NO-producing RAW 264.7 macrophages which revealed that endogenous NO production produced patterns of histone methylation consistent with inhibition of demethylase activity. Examination of KDM3A activity upon NO exposure demonstrated inhibition of its demethylase function that correlated to iron nitrosyl formation in the catalytic pocket. Cellular iron supplementation diminished the inhibitory effect of NO on histone demethylation. Discussion: These data are the first to demonstrate both direct and indirect mechanisms of epigenetic regulation by NO through histone methylation. NO directly inhibits KDM3A, alters the expression of both G9a and KDM3A, and sequesters iron via the formation of dinitrosyliron complexes. Take together these results indicate that NO/iron interactions have important epigenetic implications for the etiology of numerous NO-associated diseases; they also provide an explanation for nonclassical NO mediated gene regulation. Keywords: Histone, Demethylation, Methylation, Nitric oxide doi:10.1016/j.freeradbiomed.2012.08.535
B. Beauchamp*1, S. Ghosh2, A. Chu1, A. Blais1, K. Rajamanickam3, E. Tsai3, M.E. Patti4, M.E. Harper1 1 University of Ottawa, Canada, 2North Carolina Central University, USA, 3Ottawa Hospital Research Institute, Canada, 4Harvard Medical School, USA Introduction: Epigenetic mechanisms are hypothesized to contribute to the substantial variation in risk for obesity and type 2 diabetes. We have used an experimental mouse model system of maternal undernutrition during the last term of pregnancy to examine epigenetic effects on skeletal muscle metabolism in the offspring. We hypothesize that predisposition to obesity and type 2 diabetes in the offspring is, in part, due to low oxidative capacity and dysfunctional mitochondrial energetics in skeletal muscle. Methods: Two experimental groups of mice were studied: female offspring from undernourished dams (U) and control offspring from ad libitum fed dams (C). Weight loss of 10 wk old offspring on a 4 week 40% calorie restricted diet was followed. Results: U offspring had increased adiposity and impaired glucose tolerance compared to C offspring, confirming previous studies. Weight gain and food intake post-weaning were similar for both groups. Gene expression analysis demonstrated decreased expression in muscle of genes involved in PPARα /RXRα activation; RAR activation; fatty acid metabolism; and NRF2mediated oxidative stress response (p<0.01) in U. Skeletal muscle mitochondria isolated from U had decreased coupled and uncoupled respiration (states 3 and 4 respiration, respectively) but increased maximal respiration compared to C. Preliminary analyses show a trend for increased superoxide dismutase 2 and decreased uncoupling protein 3 in muscle from U. After calorie restriction, U lost half as much weight as controls (p<0.02), mirroring previously reported weight loss variation in highly compliant obese women on a clinical weight loss program. Calorie restricted U mice had decreased type 1(oxidative) muscle fibers (p<0.01) and decreased skeletal muscle mitochondria content (p<0.04) as compared to calorie restricted C mice. Conclusion: Our data suggest that epigenetic alterations in skeletal muscle metabolism are associated with weight loss resistance. Findings are expected to further our understanding of the influence of epigenetics on obesity and its treatment.
S55
doi:10.1016/j.freeradbiomed.2012.08.536 Cardiovascular, Metabolic & Environmental Disorders I 3: Cardiac mitochondrial bioenergetics in endotoxemia V. Vanasco, N. Magnani, M.C. Cimolai, L.B. Valdez, P. Evelson, S. Alvarez* University of Buenos Aires, Argentina Mitochondrial dysfunction and organ failure are key features in endotoxemia and the associated MOF syndrome including heart failure. Mitochondrial dysfunction in endotoxic shock has been observed including inhibition of electron transfer and ATP synthesis using a series of experimental designs. However, no systematic analysis with a bioenergetic approach has been carried out using a unique experimental model, thus lacking precise information about impaired cellular energy metabolism. Acute endotoxemia (LPS, 10 mg/kg ip, Sprague Dawley rats, 45 days old, 180 g) decreased the O2 consumption of rat heart (1 mm3 tissue cubes) by 33% (from 4.69 to 3.11 µmol O2/min. g tissue). Mitochondrial O2 consumption and complex I activity were also decreased by 27% and 29%, respectively. Impaired respiration was associated to decreased ATP synthesis (from 417 to 168 nmol/min. mg protein) and ATP content (from 5.40 to 4.18 nmol ATP/mg protein), without affecting mitochondrial membrane potential. This scenario is accompanied by an increased production of O2●- and H2O2 due to complex I inhibition. The increased NO production, is expected to fuel an increased ONOO- generation that is considered relevant in terms of the biochemical mechanism. Heart mitochondrial bioenergetic dysfunction with decreased O2 uptake, ATP production and contents may indicate that preservation of mitochondrial function will prevent heart failure in endotoxemia. Overall, the data support the hypothesis that heart complex I activity and mitochondrial O2 consumption impairment in endotoxemia, contribute to a decreased ATP production by FoF1-ATP synthase and to decreased mitochondrial ATP content without affecting inner membrane potential. To our knowledge this is the first study in which "Complex I syndrome" is described in endotoxemia. Keywords: Endotoxemia, Mitochondria, Bioenergetics, ATP doi:10.1016/j.freeradbiomed.2012.08.537
Cardiovascular, Metabolic & Environmental Disorders I 4: Nox4-dependent regulation reticulum stress in cardiac cells
of
C.X. Santos*, A.C. Brewer, M. Zhang, N. Anilkumar, A.M. Ajay King’s College London British Heart Foundation Centre, Cardiovascular Division, UK ROS-dependent signalling is involved in the cardiac response to different stresses, such as Endoplasmic Reticulum Stress (ER-Stress) which activates a signalling cascade called Unfolded Protein Response (UPR) and implicated in numerous heart pathologies. The ROS-generating NADPH oxidase-4 (Nox4) is upregulated during ER stress, but its role in the UPR remains unclear. Here, we investigate the effect of Nox4 on the UPR in cardiomyocytes. Neonatal rat cardiomyocytes (NRC) exposed to an ER stressor tunicamycin (2mg/ml) showed a significant timedependent increase in Nox4 mRNA and protein levels, paralleled by a marked increase in ATF4 transcription factor (~20-fold vs. basal) and increased of ER Stress marker, Grp78/Grp94. This effect was significant reduced by Nox4 siRNA knockdown. Adenoviralmediated overexpression of Nox4 in NRC or H9c2 caused a significant increase in tunicamycin-induced expression of Grp78/Grp94 as compared to Ad.βgal and also increased nuclear protein levels of ATF4 -indicating that Nox4 augments the UPR. The overexpression of Nox4 also resulted in a significant enhancement and prolongation (up to 16 h) of phosphorylation of the translation initiation factor eIF2α as compared to βgalinfected cells. eIF2α phosphorylation is a key consequence of the UPR and is antagonised by the phosphatase PP1c. PP1c protein levels were unaltered after Nox4 overexpression but PP1 activity was significantly inhibited vs.β-gal-expressing cells. This suggests that Nox4 enhances eIF2α phosphorylation by inhibiting PP1 activity, and we found that PP1c was also inactivated in vitro by H2O2. The amplification of the ROS production and UPR by endogenous Nox4 was confirmed using Nox4-deficient mouse embryonic fibroblasts (MEFs) and had a potential protective effect via decrease in the cleavage of ER-associated procaspase-12. This study uncovers mechanisms involved in Nox4-dependent enhancement of the UPR in cardiomyocytes and provides new insights into the redox regulation of PP1c. Keywords: Nox, Endoplasmic reticulum stress, ROS, Signalling doi:10.1016/j.freeradbiomed.2012.08.538
S56
endoplasmic
Cardiovascular, Metabolic & Environmental Disorders I 2:
Introduction: Histone methylation of lysine residues is an important epigenetic regulator of chromatin structure and gene transcription. Lysine methylation status is orchestrated by the activity of both methyltransferases and demethylases. KDM3A is a member of the JMJC domain containing family of Fe(II), a-ketoglutarate dependent histone demethylases. This enzyme demethylates mono- and dimethylated histone lysine residues.
Epigenetic alterations in skeletal muscle metabolism are associated with weight loss resistance
Methods: We examined the ability of nitric oxide (NO) to regulate histone methylation using NO-producing cells and NO-donor compounds. Protein immunoblots measured changes in histone methylation status. Electron paramagnetic imagining was used to determine NO/iron/enzyme interactions. Enzyme demethylase activity was measured by mass spectroscopy. Results: Using MDA-MD-231 breast cancer cells, we uncovered a novel dual regulatory mechanism of NO on global histone methylation. Overall levels of dimethylated Lys9 on histone 3 (H3K9me2) were significantly elevated in a concentration-dependent manner by NO exposure. This increase in H3K9me2 occurred despite a concomitant down regulation of the predominant histone lysine methyltransferase (G9a) and upregulation of KDM3A. These findings were replicated in NO-producing RAW 264.7 macrophages which revealed that endogenous NO production produced patterns of histone methylation consistent with inhibition of demethylase activity. Examination of KDM3A activity upon NO exposure demonstrated inhibition of its demethylase function that correlated to iron nitrosyl formation in the catalytic pocket. Cellular iron supplementation diminished the inhibitory effect of NO on histone demethylation. Discussion: These data are the first to demonstrate both direct and indirect mechanisms of epigenetic regulation by NO through histone methylation. NO directly inhibits KDM3A, alters the expression of both G9a and KDM3A, and sequesters iron via the formation of dinitrosyliron complexes. Take together these results indicate that NO/iron interactions have important epigenetic implications for the etiology of numerous NO-associated diseases; they also provide an explanation for nonclassical NO mediated gene regulation. Keywords: Histone, Demethylation, Methylation, Nitric oxide doi:10.1016/j.freeradbiomed.2012.08.535
B. Beauchamp*1, S. Ghosh2, A. Chu1, A. Blais1, K. Rajamanickam3, E. Tsai3, M.E. Patti4, M.E. Harper1 1 University of Ottawa, Canada, 2North Carolina Central University, USA, 3Ottawa Hospital Research Institute, Canada, 4Harvard Medical School, USA Introduction: Epigenetic mechanisms are hypothesized to contribute to the substantial variation in risk for obesity and type 2 diabetes. We have used an experimental mouse model system of maternal undernutrition during the last term of pregnancy to examine epigenetic effects on skeletal muscle metabolism in the offspring. We hypothesize that predisposition to obesity and type 2 diabetes in the offspring is, in part, due to low oxidative capacity and dysfunctional mitochondrial energetics in skeletal muscle. Methods: Two experimental groups of mice were studied: female offspring from undernourished dams (U) and control offspring from ad libitum fed dams (C). Weight loss of 10 wk old offspring on a 4 week 40% calorie restricted diet was followed. Results: U offspring had increased adiposity and impaired glucose tolerance compared to C offspring, confirming previous studies. Weight gain and food intake post-weaning were similar for both groups. Gene expression analysis demonstrated decreased expression in muscle of genes involved in PPARα /RXRα activation; RAR activation; fatty acid metabolism; and NRF2mediated oxidative stress response (p<0.01) in U. Skeletal muscle mitochondria isolated from U had decreased coupled and uncoupled respiration (states 3 and 4 respiration, respectively) but increased maximal respiration compared to C. Preliminary analyses show a trend for increased superoxide dismutase 2 and decreased uncoupling protein 3 in muscle from U. After calorie restriction, U lost half as much weight as controls (p<0.02), mirroring previously reported weight loss variation in highly compliant obese women on a clinical weight loss program. Calorie restricted U mice had decreased type 1(oxidative) muscle fibers (p<0.01) and decreased skeletal muscle mitochondria content (p<0.04) as compared to calorie restricted C mice. Conclusion: Our data suggest that epigenetic alterations in skeletal muscle metabolism are associated with weight loss resistance. Findings are expected to further our understanding of the influence of epigenetics on obesity and its treatment.
S55
doi:10.1016/j.freeradbiomed.2012.08.536 Cardiovascular, Metabolic & Environmental Disorders I 3: Cardiac mitochondrial bioenergetics in endotoxemia V. Vanasco, N. Magnani, M.C. Cimolai, L.B. Valdez, P. Evelson, S. Alvarez* University of Buenos Aires, Argentina Mitochondrial dysfunction and organ failure are key features in endotoxemia and the associated MOF syndrome including heart failure. Mitochondrial dysfunction in endotoxic shock has been observed including inhibition of electron transfer and ATP synthesis using a series of experimental designs. However, no systematic analysis with a bioenergetic approach has been carried out using a unique experimental model, thus lacking precise information about impaired cellular energy metabolism. Acute endotoxemia (LPS, 10 mg/kg ip, Sprague Dawley rats, 45 days old, 180 g) decreased the O2 consumption of rat heart (1 mm3 tissue cubes) by 33% (from 4.69 to 3.11 µmol O2/min. g tissue). Mitochondrial O2 consumption and complex I activity were also decreased by 27% and 29%, respectively. Impaired respiration was associated to decreased ATP synthesis (from 417 to 168 nmol/min. mg protein) and ATP content (from 5.40 to 4.18 nmol ATP/mg protein), without affecting mitochondrial membrane potential. This scenario is accompanied by an increased production of O2●- and H2O2 due to complex I inhibition. The increased NO production, is expected to fuel an increased ONOO- generation that is considered relevant in terms of the biochemical mechanism. Heart mitochondrial bioenergetic dysfunction with decreased O2 uptake, ATP production and contents may indicate that preservation of mitochondrial function will prevent heart failure in endotoxemia. Overall, the data support the hypothesis that heart complex I activity and mitochondrial O2 consumption impairment in endotoxemia, contribute to a decreased ATP production by FoF1-ATP synthase and to decreased mitochondrial ATP content without affecting inner membrane potential. To our knowledge this is the first study in which "Complex I syndrome" is described in endotoxemia. Keywords: Endotoxemia, Mitochondria, Bioenergetics, ATP doi:10.1016/j.freeradbiomed.2012.08.537
Cardiovascular, Metabolic & Environmental Disorders I 4: Nox4-dependent regulation reticulum stress in cardiac cells
of
C.X. Santos*, A.C. Brewer, M. Zhang, N. Anilkumar, A.M. Ajay King’s College London British Heart Foundation Centre, Cardiovascular Division, UK ROS-dependent signalling is involved in the cardiac response to different stresses, such as Endoplasmic Reticulum Stress (ER-Stress) which activates a signalling cascade called Unfolded Protein Response (UPR) and implicated in numerous heart pathologies. The ROS-generating NADPH oxidase-4 (Nox4) is upregulated during ER stress, but its role in the UPR remains unclear. Here, we investigate the effect of Nox4 on the UPR in cardiomyocytes. Neonatal rat cardiomyocytes (NRC) exposed to an ER stressor tunicamycin (2mg/ml) showed a significant timedependent increase in Nox4 mRNA and protein levels, paralleled by a marked increase in ATF4 transcription factor (~20-fold vs. basal) and increased of ER Stress marker, Grp78/Grp94. This effect was significant reduced by Nox4 siRNA knockdown. Adenoviralmediated overexpression of Nox4 in NRC or H9c2 caused a significant increase in tunicamycin-induced expression of Grp78/Grp94 as compared to Ad.βgal and also increased nuclear protein levels of ATF4 -indicating that Nox4 augments the UPR. The overexpression of Nox4 also resulted in a significant enhancement and prolongation (up to 16 h) of phosphorylation of the translation initiation factor eIF2α as compared to βgalinfected cells. eIF2α phosphorylation is a key consequence of the UPR and is antagonised by the phosphatase PP1c. PP1c protein levels were unaltered after Nox4 overexpression but PP1 activity was significantly inhibited vs.β-gal-expressing cells. This suggests that Nox4 enhances eIF2α phosphorylation by inhibiting PP1 activity, and we found that PP1c was also inactivated in vitro by H2O2. The amplification of the ROS production and UPR by endogenous Nox4 was confirmed using Nox4-deficient mouse embryonic fibroblasts (MEFs) and had a potential protective effect via decrease in the cleavage of ER-associated procaspase-12. This study uncovers mechanisms involved in Nox4-dependent enhancement of the UPR in cardiomyocytes and provides new insights into the redox regulation of PP1c. Keywords: Nox, Endoplasmic reticulum stress, ROS, Signalling doi:10.1016/j.freeradbiomed.2012.08.538
S56
endoplasmic