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The fatty acid oxidation enzyme long-chain acyl-CoA dehydrogenase can be a source of mitochondrial hydrogen peroxide.

ABSTRACT: Fatty acid oxidation (FAO)-driven H₂O₂ has been shown to be a major source of oxidative stress in several tissues and disease states. Here, we established that the mitochondrial flavoprotein long-chain acyl-CoA dehydrogenase (LCAD), which catalyzes a key step in mitochondrial FAO, directly produces H₂O₂in vitro by leaking electrons to oxygen. Kinetic analysis of recombinant human LCAD showed that it produces H₂O₂ 15-fold faster than the related mitochondrial enzyme very long-chain acyl-CoA dehydrogenase (VLCAD), but 50-fold slower than a bona fide peroxisomal acyl-CoA oxidase. The rate of H₂O₂ formation by human LCAD is slow compared to its activity as a dehydrogenase (about 1%). However, expression of hLCAD in HepG2 cells is sufficient to significantly increase H₂O₂ in the presence of fatty acids. Liver mitochondria from LCAD-/- mice, but not VLCAD-/- mice, produce significantly less H₂O₂ during incubation with fatty acids. Finally, we observe highest LCAD expression in human liver, followed by kidney, lung, and pancreas. Based on our data, we propose that the presence of LCAD drives H₂O₂ formation in response to fatty acids in these tissues.

SUBMITTER: Zhang Y

PROVIDER: S-EPMC6597861 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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The fatty acid oxidation enzyme long-chain acyl-CoA dehydrogenase can be a source of mitochondrial hydrogen peroxide.

Zhang Yuxun Y Bharathi Sivakama S SS Beck Megan E ME Goetzman Eric S ES

Redox biology 20190615

Fatty acid oxidation (FAO)-driven H2O2 has been shown to be a major source of oxidative stress in several tissues and disease states. Here, we established that the mitochondrial flavoprotein long-chain acyl-CoA dehydrogenase (LCAD), which catalyzes a key step in mitochondrial FAO, directly produces H2O2in vitro by leaking electrons to oxygen. Kinetic analysis of recombinant human LCAD showed that it produces H2O2 15-fold faster th ...[more]

PMID: 31234015

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Project description:Mitochondrial fatty-acid beta-oxidation (mFAO) plays a central role in mammalian energy metabolism. Multiple severe diseases are associated with defects in this pathway. Its kinetic structure is characterized by a complex wiring of which the functional implications have hardly been explored. Repetitive cycles of reversible reactions, each cycle shortening the fatty acid by two carbon atoms, evoke competition between intermediates of different chain lengths for a common set of 'promiscuous' enzymes (enzymes with activity towards multiple substrates). In our validated kinetic model of the pathway, substrate overload causes a steep and detrimental flux decline. Here, we unravel the underlying mechanism and the role of enzyme promiscuity in it. Comparison of alternative model versions elucidated the role of promiscuity of individual enzymes. Promiscuity of the last enzyme of the pathway, medium-chain ketoacyl-CoA thiolase (MCKAT), was both necessary and sufficient to elicit the flux decline. Subsequently, Metabolic Control Analysis revealed that MCKAT had insufficient capacity to cope with high substrate influx. Next, we quantified the internal metabolic regulation, revealing a vicious cycle around MCKAT. Upon substrate overload, MCKAT's ketoacyl-CoA substrates started to accumulate. The unfavourable equilibrium constant of the preceding enzyme, medium/short-chain hydroxyacyl-CoA dehydrogenase, worked as an amplifier, leading to accumulation of upstream CoA esters, including acyl-CoA esters. These acyl-CoA esters are at the same time products of MCKAT and inhibited its already low activity further. Finally, the accumulation of CoA esters led to a sequestration of free CoA. CoA being a cofactor for MCKAT, its sequestration limited the MCKAT activity even further, thus completing the vicious cycle. Since CoA is also a substrate for distant enzymes, it efficiently communicated the 'traffic jam' at MCKAT to the entire pathway. This novel mechanism provides a basis to explore the role of mFAO in disease and elucidate similar principles in other pathways of lipid metabolism.

Project description:Triheptanoin (triheptanoylglycerol) has shown value as anaplerotic therapy for patients with long chain fatty acid oxidation disorders but is contraindicated in medium-chain acyl-CoA dehydrogenase (MCAD) deficiency. In search for anaplerotic therapy for patients with MCAD deficiency, fibroblasts from three patients homozygous for the most common mutation, ACADMG985A/G985A, were treated with fatty acids hypothesized not to require MCAD for their metabolism, including heptanoic (C7; the active component of triheptanoin), 2,6-dimethylheptanoic (dMC7), 6-amino-2,4-dimethylheptanoic (AdMC7), or 4,8-dimethylnonanoic (dMC9) acids. Their effectiveness as anaplerotic fatty acids was assessed in live cells by monitoring changes in cellular oxygen consumption rate (OCR) and mitochondrial protein lysine succinylation, which reflects cellular succinyl-CoA levels, using immunofluorescence (IF) staining. Krebs cycle intermediates were also quantitated in these cells using targeted metabolomics. The four fatty acids induced positive changes in OCR parameters, consistent with their oxidative catalysis and utilization. Increases in cellular IF staining of succinylated lysines were observed, indicating that the fatty acids were effective sources of succinyl-CoA in the absence of media glucose, pyruvate, and lipids. The ability of MCAD deficient cells to metabolize C7 was confirmed by the ability of extracts to enzymatically utilize C7-CoA as substrate but not C8-CoA. To evaluate C7 therapeutic potential in vivo, Acadm-/- mice were treated with triheptanoin for seven days. Dose dependent increase in plasma levels of heptanoyl-, valeryl-, and propionylcarnitine indicated efficient metabolism of the medication. The pattern of the acylcarnitine profile paralleled resolution of liver pathology including reversing hepatic steatosis, increasing hepatic glycogen content, and increasing hepatocyte protein succinylation, all indicating improved energy homeostasis in the treated mice. These results provide the impetus to evaluate triheptanoin and the medium branched chain fatty acids as potential therapeutic agents for patients with MCAD deficiency.

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The fatty acid oxidation enzyme long-chain acyl-CoA dehydrogenase can be a source of mitochondrial hydrogen peroxide.

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The fatty acid oxidation enzyme long-chain acyl-CoA dehydrogenase can be a source of mitochondrial hydrogen peroxide.

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