Hyperammonaemia-induced skeletal muscle mitochondrial dysfunction results in cataplerosis and oxidative stress.
*ammonia; *ATP; *cellular respiration; *cirrhosis; *mitochondria; *Oxidative Stress; *portacaval anastamosis; *reactive oxygen species; *skeletal muscle; Adenosine Triphosphate/metabolism; Aged; Animals; Cell Line; Cell Respiration; Creatine Kinase/metabolism; Female; Humans; Hyperammonemia/*metabolism; Liver Cirrhosis/metabolism; Male; Middle Aged; Mitochondria; Muscle; Muscle/*metabolism; Myosin Heavy Chains/metabolism; NAD/metabolism; Rats; Reactive Oxygen Species/metabolism; Skeletal/*metabolism; Sprague-Dawley; Thiobarbituric Acid Reactive Substances/metabolism
KEY POINTS: Hyperammonaemia occurs in hepatic, cardiac and pulmonary diseases with increased muscle concentration of ammonia. We found that ammonia results in reduced skeletal muscle mitochondrial respiration, electron transport chain complex I dysfunction, as well as lower NAD(+) /NADH ratio and ATP content. During hyperammonaemia, leak of electrons from complex III results in oxidative modification of proteins and lipids. Tricarboxylic acid cycle intermediates are decreased during hyperammonaemia, and providing a cell-permeable ester of alphaKG reversed the lower TCA cycle intermediate concentrations and increased ATP content. Our observations have high clinical relevance given the potential for novel approaches to reverse skeletal muscle ammonia toxicity by targeting the TCA cycle intermediates and mitochondrial ROS. ABSTRACT: Ammonia is a cytotoxic metabolite that is removed primarily by hepatic ureagenesis in humans. Hyperammonaemia occurs in advanced hepatic, cardiac and pulmonary disease, and in urea cycle enzyme deficiencies. Increased skeletal muscle ammonia uptake and metabolism are the major mechanism of non-hepatic ammonia disposal. Non-hepatic ammonia disposal occurs in the mitochondria via glutamate synthesis from alpha-ketoglutarate resulting in cataplerosis. We show skeletal muscle mitochondrial dysfunction during hyperammonaemia in a comprehensive array of human, rodent and cellular models. ATP synthesis, oxygen consumption, generation of reactive oxygen species with oxidative stress, and tricarboxylic acid (TCA) cycle intermediates were quantified. ATP content was lower in the skeletal muscle from cirrhotic patients, hyperammonaemic portacaval anastomosis rat, and C2C12 myotubes compared to appropriate controls. Hyperammonaemia in C2C12 myotubes resulted in impaired intact cell respiration, reduced complex I/NADH oxidase activity and electron leak occurring at complex III of the electron transport chain. Consistently, lower NAD(+) /NADH ratio was observed during hyperammonaemia with reduced TCA cycle intermediates compared to controls. Generation of reactive oxygen species resulted in increased content of skeletal muscle carbonylated proteins and thiobarbituric acid reactive substances during hyperammonaemia. A cell-permeable ester of alpha-ketoglutarate reversed the low TCA cycle intermediates and ATP content in myotubes during hyperammonaemia. However, the mitochondrial antioxidant MitoTEMPO did not reverse the lower ATP content during hyperammonaemia. We provide for the first time evidence that skeletal muscle hyperammonaemia results in mitochondrial dysfunction and oxidative stress. Use of anaplerotic substrates to reverse ammonia-induced mitochondrial dysfunction is a novel therapeutic approach.
Davuluri Gangarao; Allawy Allawy; Thapaliya Samjhana; Rennison Julie H; Singh Dharmvir; Kumar Avinash; Sandlers Yana; Van Wagoner David R; Flask Chris A; Hoppel Charles; Kasumov Takhar; Dasarathy Srinivasan
The Journal of physiology
2016
2016-12
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1113/JP272796" target="_blank" rel="noreferrer noopener">10.1113/JP272796</a>
Stable isotope-based flux studies in nonalcoholic fatty liver disease.
*Citric acid cycle; *Fatty acid oxidation; *Fibrosis; *NAFLD; *Oxidative stress; *Stable isotopes; Animals; Humans; Isotopes/metabolism; Mass Spectrometry/*methods; Non-alcoholic Fatty Liver Disease/*metabolism
Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease and is associated with the worldwide epidemics of obesity, diabetes and cardiovascular diseases. NAFLD ranges from benign fat accumulation in the liver (steatosis) to non-alcoholic steatohepatitis (NASH), and cirrhosis which can progress to hepatocellular carcinoma and liver failure. Mass spectrometry and magnetic resonance spectroscopy-coupled stable isotope-based flux studies provide new insights into the understanding of NAFLD pathogenesis and the disease progression. This review focuses mainly on the utilization of mass spectrometry-based methods for the understanding of metabolic abnormalities in the different stages of NAFLD. For example, stable isotope-based flux studies demonstrated multi-organ insulin resistance, dysregulated glucose, lipids and lipoprotein metabolism in patients with NAFLD. We also review recent developments in the stable isotope-based technologies for the study of mitochondrial dysfunction, oxidative stress and fibrogenesis in NAFLD. We highlight the limitations of current methodologies, discuss the emerging areas of research in this field, and future directions for the applications of stable isotopes to study NAFLD and its complications.
McCullough Arthur; Previs Stephen; Kasumov Takhar
Pharmacology & therapeutics
2018
2018-01
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1016/j.pharmthera.2017.07.008" target="_blank" rel="noreferrer noopener">10.1016/j.pharmthera.2017.07.008</a>
Epigenetic regulation in diabetes-associated oxidative stress and myocardial dysfunction.
*Epigenesis; *Oxidative Stress; Cardiomyopathies; Genetic; Humans
Yin Liya; Chilian William M; Dong Feng
International journal of cardiology
2018
2018-10
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1016/j.ijcard.2018.05.058" target="_blank" rel="noreferrer noopener">10.1016/j.ijcard.2018.05.058</a>
Increased serotransferrin and ceruloplasmin turnover in diet-controlled patients with type 2 diabetes.
*Ceruloplasmin; *Deamidation; *Heavy water metabolic labeling; *High resolution mass spectrometry; *Iron metabolism; *LC-MS/MS; *Non-enzymatic glycation; *Oxidative stress; *Protein Processing; *Proteome dynamics; *Serotransferrin; *Type 2 diabetes mellitus; Adult; Amino Acid Sequence; Case-Control Studies; Ceruloplasmin/genetics/*metabolism; Deuterium/metabolism; Diabetes Mellitus; Diabetic; Diet; Female; Gene Expression Regulation; Glycated Hemoglobin A/genetics/metabolism; Glycosylation; Humans; Iron/*metabolism; Isotope Labeling; Male; Middle Aged; Oxidation-Reduction; Oxidative Stress; Post-Translational; Proteolysis; Transferrin/genetics/*metabolism; Type 2/diet therapy/genetics/*metabolism/pathology
Type 2 diabetes mellitus (T2DM) is associated with oxidative stress and perturbed iron metabolism. Serotransferrin (Trf) and ceruloplasmin (Cp) are two key proteins involved in iron metabolism and anti-oxidant defense. Non-enzymatic glycation and oxidative modification of plasma proteins are known to occur under hyperglycemia and oxidative stress. In this study, shotgun proteomics and (2)H2O-based metabolic labeling were used to characterize post-translational modifications and assess the kinetics of Trf and Cp in T2DM patients and matched controls in vivo. Six early lysine (Amadori) and one advanced arginine glycation were detected in Trf. No glycation, but five asparagine deamidations, were found in Cp. T2DM patients had increased fractional catabolic rates of both Trf and Cp that correlated with HbA1c (p \textless 0.05). The glycated Trf population was subject to an even faster degradation compared to the total Trf pool, suggesting that hyperglycemia contributed to an increased Trf degradation in T2DM patients. Enhanced production of Trf and Cp kept their levels stable. The changes in Trf and Cp turnover were associated with increased systemic oxidative stress without any alteration in iron status in T2DM. These findings can help better understand the potential role of altered Trf and Cp metabolism in the pathogenesis of T2DM and other diseases.
Golizeh Makan; Lee Kwangwon; Ilchenko Serguei; Osme Abdullah; Bena James; Sadygov Rovshan G; Kashyap Sangeeta R; Kasumov Takhar
Free radical biology & medicine
2017
2017-12
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1016/j.freeradbiomed.2017.10.373" target="_blank" rel="noreferrer noopener">10.1016/j.freeradbiomed.2017.10.373</a>
A Western diet induced NAFLD in LDLR(-/)(-) mice is associated with reduced hepatic glutathione synthesis.
*Flux; *Glutathione; *Heavy water; *Mass spectrometer; *NAFLD; *NASH; *Oxidative stress; *Western Diet; Animal; Animals; Antioxidants/metabolism; Diet; Disease Models; Glutathione/blood/*metabolism; Humans; LDL/blood/*genetics/metabolism; Liver/*metabolism/pathology; Mice; Non-alcoholic Fatty Liver Disease/blood/*genetics/pathology; Oxidative Stress/genetics; Receptors; Western/adverse effects
Oxidative stress plays a key role in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). Glutathione is the major anti-oxidant involved in cellular oxidative defense, however there are currently no simple non-invasive methods for assessing hepatic glutathione metabolism in patients with NAFLD. As a primary source of plasma glutathione, liver plays an important role in interorgan glutathione homeostasis. In this study, we have tested the hypothesis that measurements of plasma glutathione turnover could be used to assess the hepatic glutathione metabolism in LDLR(-/)(-) mice, a mouse model of diet-induced NAFLD. Mice were fed a standard low fat diet (LFD) or a high fat diet containing cholesterol (a Western type diet (WD)). The kinetics of hepatic and plasma glutathione were quantified using the (2)H2O metabolic labeling approach. Our results show that a WD leads to reduced fractional synthesis rates (FSR) of hepatic (25%/h in LFD vs. 18%/h in WD, P\textless0.05) and plasma glutathione (43%/h in LFD vs. 21%/h in WD, P\textless0.05), without any significant effect on their absolute production rates (PRs). WD-induced concordant changes in both hepatic and plasma glutathione turnover suggest that the plasma glutathione turnover measurements could be used to assess hepatic glutathione metabolism. The safety, simplicity, and low cost of the (2)H2O-based glutathione turnover approach suggest that this method has the potential for non-invasive probing of hepatic glutathione metabolism in patients with NAFLD and other diseases.
Li Ling; Zhang Guo-Fang; Lee Kwangwon; Lopez Rocio; Previs Stephen F; Willard Belinda; McCullough Arthur; Kasumov Takhar
Free radical biology & medicine
2016
2016-07
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1016/j.freeradbiomed.2016.03.032" target="_blank" rel="noreferrer noopener">10.1016/j.freeradbiomed.2016.03.032</a>
Protein thiyl radical mediates S-glutathionylation of complex I.
*Oxidative Stress; Amino Acid Motifs; Amino Acid Sequence; Animals; Binding Sites; Cattle; Cell Line; Cyclic N-Oxides/chemistry/pharmacology; Cysteine/chemistry/*metabolism; Electron Transport Complex I/chemistry/*metabolism; Free Radical Scavengers/chemistry/pharmacology; Free Radicals/chemistry/*metabolism; Glutathione/chemistry/*metabolism; Heart/enzymology/metabolism; Mice; Mitochondria; Models; Molecular; Molecular Sequence Data; Muscle Cells/drug effects/metabolism; Onium Compounds/pharmacology; Peptide Fragments/chemistry; Peptide Mapping; Protein; Rats; Rotenone/pharmacology; Structural Homology; Superoxides/metabolism
Complex I is a critical site of O(2)(*-) production and the major host of reactive protein thiols in mitochondria. In response to oxidative stress, complex I protein thiols at the 51- and 75-kDa subunits are reversibly
Kang Patrick T; Zhang Liwen; Chen Chwen-Lih; Chen Jingfeng; Green Kari B; Chen Yeong-Renn
Free radical biology & medicine
2012
2012-08
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1016/j.freeradbiomed.2012.05.025" target="_blank" rel="noreferrer noopener">10.1016/j.freeradbiomed.2012.05.025</a>
Impairment of pH gradient and membrane potential mediates redox dysfunction in the mitochondria of the post-ischemic heart.
*Energy Metabolism; *Membrane potential; *Membrane Potential; *Mitochondria; *Myocardial ischemia and reperfusion; *Oxidative Stress; *pH gradient; *Redox dysfunction; Aconitate Hydratase/metabolism; Animal; Animals; Cell Line; Disease Models; Electron Transport Chain Complex Proteins/metabolism; Heart/*metabolism/pathology; Hydrogen Peroxide/metabolism; Hydrogen-Ion Concentration; Ionophores/pharmacology; Male; Mitochondria; Mitochondrial; Myocardial Infarction/*metabolism/pathology; Myocardium/*metabolism/pathology; Oxidation-Reduction; Potassium/metabolism; Rats; Sprague-Dawley; Superoxides/metabolism
The mitochondrial electrochemical gradient (Deltap), which comprises the pH gradient (DeltapH) and the membrane potential (DeltaPsi), is crucial in controlling energy transduction. During myocardial ischemia and reperfusion (IR), mitochondrial dysfunction mediates superoxide ((.)O2(-)) and H2O2 overproduction leading to oxidative injury. However, the role of DeltapH and DeltaPsi in post-ischemic injury is not fully established. Here we studied mitochondria from the risk region of rat hearts subjected to 30 min of coronary ligation and 24 h of reperfusion in vivo. In the presence of glutamate, malate and ADP, normal mitochondria (mitochondria of non-ischemic region, NR) exhibited a heightened state 3 oxygen consumption rate (OCR) and reduced (.)O2(-) and H2O2 production when compared to state 2 conditions. Oligomycin (increases DeltapH by inhibiting ATP synthase) increased (.)O2(-) and H2O2 production in normal mitochondria, but not significantly in the mitochondria of the risk region (IR mitochondria or post-ischemic mitochondria), indicating that normal mitochondrial (.)O2(-) and H2O2 generation is dependent on DeltapH and that IR impaired the DeltapH of normal mitochondria. Conversely, nigericin (dissipates DeltapH) dramatically reduced (.)O2(-) and H2O2 generation by normal mitochondria under state 4 conditions, and this nigericin quenching effect was less pronounced in IR mitochondria. Nigericin also increased mitochondrial OCR, and predisposed normal mitochondria to a more oxidized redox status assessed by increased oxidation of cyclic hydroxylamine, CM-H. IR mitochondria, although more oxidized than normal mitochondria, were not responsive to nigericin-induced CM-H oxidation, which is consistent with the result that IR induced DeltapH impairment in normal mitochondria. Valinomycin, a K(+) ionophore used to dissipate DeltaPsi, drastically diminished (.)O2(-) and H2O2 generation by normal mitochondria, but less pronounced effect on IR mitochondria under state 4 conditions, indicating that DeltaPsi also contributed to (.)O2(-) generation by normal mitochondria and that IR mediated DeltaPsi impairment. However, there was no significant difference in valinomycin-induced CM-H oxidation between normal and IR mitochondria. In conclusion, under normal conditions the proton backpressure imposed by DeltapH restricts electron flow, controls a limited amount of (.)O2(-) generation, and results in a more reduced myocardium; however, IR causes DeltapH impairment and prompts a more oxidized myocardium.
Kang Patrick T; Chen Chwen-Lih; Lin Paul; Chilian William M; Chen Yeong-Renn
Basic research in cardiology
2017
2017-07
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1007/s00395-017-0626-1" target="_blank" rel="noreferrer noopener">10.1007/s00395-017-0626-1</a>