- Email: [email protected]
Mitochondria-localized lactate dehydrogenase is not a biologically significant contributor to bioenergetic function in striated muscle
A. Newman / Free Radical Biology and Medicine 128 (2018) S79–S97
transformation. These results demonstrate for the first time that MnSOD is a redox sensitive protein that maintains mitochondrial health and prevents UVB-induced mitophagy and cell transformation by regulating the activity of the mTORC2 complex.
https://doi.org/10.1016/j.freeradbiomed.2018.10.186
182
Time-course metabolomic analysis of manganese toxicity reveals biomarkers of oxidative stress and amino acid metabolism as early cellular targets Jolyn Fernandes*, Karan Uppal, Ken H. Liu, Xin Hu, Young-Mi Go, Dean Jones Emory University, GA, USA
Manganese (Mn) is a naturally occurring essential nutrient at low doses that functions as a cofactor in multiple enzymatic reactions, provides structural stability and participates in redox activities. However, high Mn exposure even under federal safety limits for air Mn levels, may cause a dose-dependent progression of Parkinsonism. The molecular mechanisms and critical transitions from normal Mn physiology to low dose toxicity as well as early targets of Mn toxicity nonetheless remains poorly understood. In this temporal study, we used human neuroblastoma SH-SY5Y cells exposed to Mn concentrations varied over a physiologic (5μM MnCl2) and toxicological range (50μM MnCl2) for 0, 0.5, 2, 5 and 10h (n¼ 9 per Mn concentration and time point). Metabolomics analysis was done using liquid chromatography with ultra-high resolution mass spectrometry and cellular Mn accumulation was measured using ICP-MS. Results showed cellular Mn accumulation increased upto 5h in a dose dependent manner, beyond which Mn uptake was stabilized. Pathway enrichment analysis showed that methionine and cysteine metabolism; urea cycle metabolism; glycine, serine, alanine and threonine metabolism; and histidine metabolism were significantly altered within 30min of manganese treatment and persisted upto 10h suggesting these pathways define early targeted mechanisms of Mn toxicity. Discriminatory metabolites involved in methionine and cysteine pathway include methionine, homocysteine, 1,2 dihydroxy-3-keto-5-methylthiopentene and 5’-methylthioadenosine. Discriminatory metabolites involved in glycine, serine, alanine and threonine metabolism include 3-phosphohydroxypyruvate, phosphoserine, serine and creatine. These early responders may reflect rapid transport and redistribution of Mn within the cell. In conclusion, we demonstrate key metabolic biomarkers and pathways that respond dynamically to varying Mn concentration and attempt to provide a chronological fingerprint of Mn dependent toxicity. Time course metabolomics thereby provides a useful tool to predict metabolic status for dynamic systems at the cellular level in response to environmental chemical exposures.
https://doi.org/10.1016/j.freeradbiomed.2018.10.187
183
Mitochondria-localized lactate dehydrogenase is not a biologically significant contributor to bioenergetic function in striated muscle Kyle Fulghum*, Benjamin Rood, Velma Shang, Lindsey McNally, Yu-Ting Zheng, Bradford Hill University of Louisville, USA
S83
Background: Previous studies indicate that mitochondrial lactate dehydrogenase (mLDH) might be a significant contributor to metabolism. The presence of mLDH could provide mitochondria with a higher capacity to generate reducing equivalents for respiration, especially during exercise when circulating lactate levels are high. Furthermore, mitochondrial lactate oxidation could support cardiac output or skeletal muscle function, both of which contribute to exercise capacity. Methods: We measured lactate-, pyruvate-, and glutamate-supported respiration in mitochondria isolated from heart and skeletal muscle of sedentary, acutely exercised, and exercise-adapted mice. We modeled the acute effects of exercise by subjecting male FVB/NJ mice to one, 60 min bout of treadmill running. Exercise adaptation was modeled with progressive treadmill training at 40-60 min/d for 2 weeks, with adaptation determined by measuring exercise capacity and indices of cardiac growth. We performed immunoblotting to assess relative abundance of LDH isoforms in isolated mitochondria. Results: Two weeks of treadmill running increased running distance by 1.3-fold. Compared with sedentary mice, exercise training increased cardiac mass by 15% (n¼ 5/group, po0.01). Cardiac mitochondria energized with 5 mM pyruvateþ2.5 mM malate, and skeletal muscle mitochondria provided with 5 mM glutamateþ2.5 mM malate, showed approximately 10fold higher respiration rates compared with mitochondria provided 5 mM lactateþ2.5 mM malate. Exercise training did not significantly affect respiration on either substrate. We performed similar studies on mitochondria isolated immediately after one intense bout of exercise. In both sedentary and exercised conditions, cardiac and skeletal muscle mitochondria provided with lactate showed 10-fold lower respiration rates than those supplied with pyruvate or glutamate (n¼3/group). Western blotting indicated low levels of LDHB in mitochondrial fractions from heart and low levels of LDHA in mitochondrial fractions from skeletal muscle. Conclusion: Lactate metabolism in striated muscle is primarily a cytosolic phenomenon; mLDH is of minimal biological significance.
https://doi.org/10.1016/j.freeradbiomed.2018.10.188
184
Dysregulation of MnSOD-K68 acetylation status leads to neurological and tumor-permissive phenotypes Yucheng Gao*, Angela Dean, Yueming Zhu, David Gius Northwestern University, USA
Manganese superoxide dismutase (MnSOD) lysine acetylation is a critical post-translational modification that regulates its detoxification activity to direct cellular ROS levels and metabolic balance. In this regard, our lab has shown the change in structural configuration and gain of peroxidase function upon MnSOD-K68 acetylation. As such, this study aims to investigate the role of MnSOD-K68 acetylation (K68-Ac) status, as well as its potential biological significance both in vitro and in vivo. To experimentally evaluate the effects of MnSOD acetylation, recombinant mutants were constructed where lysine was substituted with glutamine (Q) or arginine (R), an acetylation or deacetylation mimic, respectively. The results showed that enforced expression of MnSODK68Q disrupts mitochondrial metabolism and ultrastructure, and functions as an in vitro oncogene. To examine the physiological significance of MnSOD acetylation, a genetically engineered whole-body MnSODK68Q knockin mouse has been constructed. Biallelic knock-in (MnSODK68Q/K68Q) mice around 3 weeks of age display a significant phenotype that includes (1) small size, (2) lean body mass, (3) significant weight loss, and (4) a fatal neurological disorder involving loss of lower extremity movement, tremors, and dyskinesia. Daily injection of GC4419, a Mn-based chemical drug that replaces MnSOD activity by removing superoxide, extended the lifespan (from 25 days to 45 days), partially rescued motor coordination loss (as tested by rotarod) and delayed the onset of the profound neurological symptoms. In addition, electron microscopy identified disrupted mitochondrial structure in multiple tissues including liver, skeletal muscles, and the substantia nigra. Lastly, wholebody monoallelic female mice (MnSODWT/K68Q) of six months also display
transformation. These results demonstrate for the first time that MnSOD is a redox sensitive protein that maintains mitochondrial health and prevents UVB-induced mitophagy and cell transformation by regulating the activity of the mTORC2 complex.
https://doi.org/10.1016/j.freeradbiomed.2018.10.186
182
Time-course metabolomic analysis of manganese toxicity reveals biomarkers of oxidative stress and amino acid metabolism as early cellular targets Jolyn Fernandes*, Karan Uppal, Ken H. Liu, Xin Hu, Young-Mi Go, Dean Jones Emory University, GA, USA
Manganese (Mn) is a naturally occurring essential nutrient at low doses that functions as a cofactor in multiple enzymatic reactions, provides structural stability and participates in redox activities. However, high Mn exposure even under federal safety limits for air Mn levels, may cause a dose-dependent progression of Parkinsonism. The molecular mechanisms and critical transitions from normal Mn physiology to low dose toxicity as well as early targets of Mn toxicity nonetheless remains poorly understood. In this temporal study, we used human neuroblastoma SH-SY5Y cells exposed to Mn concentrations varied over a physiologic (5μM MnCl2) and toxicological range (50μM MnCl2) for 0, 0.5, 2, 5 and 10h (n¼ 9 per Mn concentration and time point). Metabolomics analysis was done using liquid chromatography with ultra-high resolution mass spectrometry and cellular Mn accumulation was measured using ICP-MS. Results showed cellular Mn accumulation increased upto 5h in a dose dependent manner, beyond which Mn uptake was stabilized. Pathway enrichment analysis showed that methionine and cysteine metabolism; urea cycle metabolism; glycine, serine, alanine and threonine metabolism; and histidine metabolism were significantly altered within 30min of manganese treatment and persisted upto 10h suggesting these pathways define early targeted mechanisms of Mn toxicity. Discriminatory metabolites involved in methionine and cysteine pathway include methionine, homocysteine, 1,2 dihydroxy-3-keto-5-methylthiopentene and 5’-methylthioadenosine. Discriminatory metabolites involved in glycine, serine, alanine and threonine metabolism include 3-phosphohydroxypyruvate, phosphoserine, serine and creatine. These early responders may reflect rapid transport and redistribution of Mn within the cell. In conclusion, we demonstrate key metabolic biomarkers and pathways that respond dynamically to varying Mn concentration and attempt to provide a chronological fingerprint of Mn dependent toxicity. Time course metabolomics thereby provides a useful tool to predict metabolic status for dynamic systems at the cellular level in response to environmental chemical exposures.
https://doi.org/10.1016/j.freeradbiomed.2018.10.187
183
Mitochondria-localized lactate dehydrogenase is not a biologically significant contributor to bioenergetic function in striated muscle Kyle Fulghum*, Benjamin Rood, Velma Shang, Lindsey McNally, Yu-Ting Zheng, Bradford Hill University of Louisville, USA
S83
Background: Previous studies indicate that mitochondrial lactate dehydrogenase (mLDH) might be a significant contributor to metabolism. The presence of mLDH could provide mitochondria with a higher capacity to generate reducing equivalents for respiration, especially during exercise when circulating lactate levels are high. Furthermore, mitochondrial lactate oxidation could support cardiac output or skeletal muscle function, both of which contribute to exercise capacity. Methods: We measured lactate-, pyruvate-, and glutamate-supported respiration in mitochondria isolated from heart and skeletal muscle of sedentary, acutely exercised, and exercise-adapted mice. We modeled the acute effects of exercise by subjecting male FVB/NJ mice to one, 60 min bout of treadmill running. Exercise adaptation was modeled with progressive treadmill training at 40-60 min/d for 2 weeks, with adaptation determined by measuring exercise capacity and indices of cardiac growth. We performed immunoblotting to assess relative abundance of LDH isoforms in isolated mitochondria. Results: Two weeks of treadmill running increased running distance by 1.3-fold. Compared with sedentary mice, exercise training increased cardiac mass by 15% (n¼ 5/group, po0.01). Cardiac mitochondria energized with 5 mM pyruvateþ2.5 mM malate, and skeletal muscle mitochondria provided with 5 mM glutamateþ2.5 mM malate, showed approximately 10fold higher respiration rates compared with mitochondria provided 5 mM lactateþ2.5 mM malate. Exercise training did not significantly affect respiration on either substrate. We performed similar studies on mitochondria isolated immediately after one intense bout of exercise. In both sedentary and exercised conditions, cardiac and skeletal muscle mitochondria provided with lactate showed 10-fold lower respiration rates than those supplied with pyruvate or glutamate (n¼3/group). Western blotting indicated low levels of LDHB in mitochondrial fractions from heart and low levels of LDHA in mitochondrial fractions from skeletal muscle. Conclusion: Lactate metabolism in striated muscle is primarily a cytosolic phenomenon; mLDH is of minimal biological significance.
https://doi.org/10.1016/j.freeradbiomed.2018.10.188
184
Dysregulation of MnSOD-K68 acetylation status leads to neurological and tumor-permissive phenotypes Yucheng Gao*, Angela Dean, Yueming Zhu, David Gius Northwestern University, USA
Manganese superoxide dismutase (MnSOD) lysine acetylation is a critical post-translational modification that regulates its detoxification activity to direct cellular ROS levels and metabolic balance. In this regard, our lab has shown the change in structural configuration and gain of peroxidase function upon MnSOD-K68 acetylation. As such, this study aims to investigate the role of MnSOD-K68 acetylation (K68-Ac) status, as well as its potential biological significance both in vitro and in vivo. To experimentally evaluate the effects of MnSOD acetylation, recombinant mutants were constructed where lysine was substituted with glutamine (Q) or arginine (R), an acetylation or deacetylation mimic, respectively. The results showed that enforced expression of MnSODK68Q disrupts mitochondrial metabolism and ultrastructure, and functions as an in vitro oncogene. To examine the physiological significance of MnSOD acetylation, a genetically engineered whole-body MnSODK68Q knockin mouse has been constructed. Biallelic knock-in (MnSODK68Q/K68Q) mice around 3 weeks of age display a significant phenotype that includes (1) small size, (2) lean body mass, (3) significant weight loss, and (4) a fatal neurological disorder involving loss of lower extremity movement, tremors, and dyskinesia. Daily injection of GC4419, a Mn-based chemical drug that replaces MnSOD activity by removing superoxide, extended the lifespan (from 25 days to 45 days), partially rescued motor coordination loss (as tested by rotarod) and delayed the onset of the profound neurological symptoms. In addition, electron microscopy identified disrupted mitochondrial structure in multiple tissues including liver, skeletal muscles, and the substantia nigra. Lastly, wholebody monoallelic female mice (MnSODWT/K68Q) of six months also display