title: Abolition of mitochondrial substrate-level phosphorylation by itaconic acid produced by LPS-induced Irg1 expression in cells of murine macrophage lineage.
abstract: Itaconate is a nonamino organic acid exhibiting antimicrobial effects. It has been recently identified in cells of macrophage lineage as a product of an enzyme encoded by immunoresponsive gene 1 (Irg1), acting on the citric acid cycle intermediate cis-aconitate. In mitochondria, itaconate can be converted by succinate-coenzyme A (CoA) ligase to itaconyl-CoA at the expense of ATP (or GTP), and is also a weak competitive inhibitor of complex II. Here, we investigated specific bioenergetic effects of increased itaconate production mediated by LPS-induced stimulation of Irg1 in murine bone marrow-derived macrophages (BMDM) and RAW-264.7 cells. In rotenone-treated macrophage cells, stimulation by LPS led to impairment in substrate-level phosphorylation (SLP) of in situ mitochondria, deduced by a reversal in the directionality of the adenine nucleotide translocase operation. In RAW-264.7 cells, the LPS-induced impairment in SLP was reversed by short-interfering RNA(siRNA)-but not scrambled siRNA-treatment directed against Irg1. LPS dose-dependently inhibited oxygen consumption rates (61-91%) and elevated glycolysis rates (>21%) in BMDM but not RAW-264.7 cells, studied under various metabolic conditions. In isolated mouse liver mitochondria treated with rotenone, itaconate dose-dependently (0.5-2 mM) reversed the operation of adenine nucleotide translocase, implying impairment in SLP, an effect that was partially mimicked by malonate. However, malonate yielded greater ADP-induced depolarizations (3-19%) than itaconate. We postulate that itaconate abolishes SLP due to 1) a "CoA trap" in the form of itaconyl-CoA that negatively affects the upstream supply of succinyl-CoA from the α-ketoglutarate dehydrogenase complex; 2) depletion of ATP (or GTP), which are required for the thioesterification by succinate-CoA ligase; and 3) inhibition of complex II leading to a buildup of succinate which shifts succinate-CoA ligase equilibrium toward ATP (or GTP) utilization. Our results support the notion that Irg1-expressing cells of macrophage lineage lose the capacity of mitochondrial SLP for producing itaconate during mounting of an immune defense.
authors.affiliation: *Department of Medical Biochemistry and Department of Physiology, Semmelweis University, Budapest, Hungary; and Lendület Neurobiochemistry Research Group, Lendület Inflammation Physiology Research Group, Laboratory for Neurobiochemistry, and Lendület Ion Channel Research Group, Hungarian Academy of Sciences, Budapest, Hungary.
authors.forename: Beáta
authors.initials: B
authors.lastname: Németh
authors.affiliation: *Department of Medical Biochemistry and Department of Physiology, Semmelweis University, Budapest, Hungary; and Lendület Neurobiochemistry Research Group, Lendület Inflammation Physiology Research Group, Laboratory for Neurobiochemistry, and Lendület Ion Channel Research Group, Hungarian Academy of Sciences, Budapest, Hungary.
authors.forename: Judit
authors.initials: J
authors.lastname: Doczi
authors.affiliation: *Department of Medical Biochemistry and Department of Physiology, Semmelweis University, Budapest, Hungary; and Lendület Neurobiochemistry Research Group, Lendület Inflammation Physiology Research Group, Laboratory for Neurobiochemistry, and Lendület Ion Channel Research Group, Hungarian Academy of Sciences, Budapest, Hungary.
authors.forename: Dániel
authors.initials: D
authors.lastname: Csete
authors.affiliation: *Department of Medical Biochemistry and Department of Physiology, Semmelweis University, Budapest, Hungary; and Lendület Neurobiochemistry Research Group, Lendület Inflammation Physiology Research Group, Laboratory for Neurobiochemistry, and Lendület Ion Channel Research Group, Hungarian Academy of Sciences, Budapest, Hungary.
authors.forename: Gergely
authors.initials: G
authors.lastname: Kacso
authors.affiliation: *Department of Medical Biochemistry and Department of Physiology, Semmelweis University, Budapest, Hungary; and Lendület Neurobiochemistry Research Group, Lendület Inflammation Physiology Research Group, Laboratory for Neurobiochemistry, and Lendület Ion Channel Research Group, Hungarian Academy of Sciences, Budapest, Hungary.
authors.forename: Dora
authors.initials: D
authors.lastname: Ravasz
authors.affiliation: *Department of Medical Biochemistry and Department of Physiology, Semmelweis University, Budapest, Hungary; and Lendület Neurobiochemistry Research Group, Lendület Inflammation Physiology Research Group, Laboratory for Neurobiochemistry, and Lendület Ion Channel Research Group, Hungarian Academy of Sciences, Budapest, Hungary.
authors.forename: Daniel
authors.initials: D
authors.lastname: Adams
authors.affiliation: *Department of Medical Biochemistry and Department of Physiology, Semmelweis University, Budapest, Hungary; and Lendület Neurobiochemistry Research Group, Lendület Inflammation Physiology Research Group, Laboratory for Neurobiochemistry, and Lendület Ion Channel Research Group, Hungarian Academy of Sciences, Budapest, Hungary.
authors.forename: Gergely
authors.initials: G
authors.lastname: Kiss
authors.affiliation: *Department of Medical Biochemistry and Department of Physiology, Semmelweis University, Budapest, Hungary; and Lendület Neurobiochemistry Research Group, Lendület Inflammation Physiology Research Group, Laboratory for Neurobiochemistry, and Lendület Ion Channel Research Group, Hungarian Academy of Sciences, Budapest, Hungary.
authors.forename: Adam M
authors.initials: AM
authors.lastname: Nagy
authors.affiliation: *Department of Medical Biochemistry and Department of Physiology, Semmelweis University, Budapest, Hungary; and Lendület Neurobiochemistry Research Group, Lendület Inflammation Physiology Research Group, Laboratory for Neurobiochemistry, and Lendület Ion Channel Research Group, Hungarian Academy of Sciences, Budapest, Hungary.
authors.forename: Gergo
authors.initials: G
authors.lastname: Horvath
authors.affiliation: *Department of Medical Biochemistry and Department of Physiology, Semmelweis University, Budapest, Hungary; and Lendület Neurobiochemistry Research Group, Lendület Inflammation Physiology Research Group, Laboratory for Neurobiochemistry, and Lendület Ion Channel Research Group, Hungarian Academy of Sciences, Budapest, Hungary.
authors.forename: Laszlo
authors.initials: L
authors.lastname: Tretter
authors.affiliation: *Department of Medical Biochemistry and Department of Physiology, Semmelweis University, Budapest, Hungary; and Lendület Neurobiochemistry Research Group, Lendület Inflammation Physiology Research Group, Laboratory for Neurobiochemistry, and Lendület Ion Channel Research Group, Hungarian Academy of Sciences, Budapest, Hungary.
authors.forename: Attila
authors.initials: A
authors.lastname: Mócsai
authors.affiliation: *Department of Medical Biochemistry and Department of Physiology, Semmelweis University, Budapest, Hungary; and Lendület Neurobiochemistry Research Group, Lendület Inflammation Physiology Research Group, Laboratory for Neurobiochemistry, and Lendület Ion Channel Research Group, Hungarian Academy of Sciences, Budapest, Hungary.
authors.forename: Roland
authors.initials: R
authors.lastname: Csépányi-Kömi
authors.affiliation: *Department of Medical Biochemistry and Department of Physiology, Semmelweis University, Budapest, Hungary; and Lendület Neurobiochemistry Research Group, Lendület Inflammation Physiology Research Group, Laboratory for Neurobiochemistry, and Lendület Ion Channel Research Group, Hungarian Academy of Sciences, Budapest, Hungary.
authors.forename: Iordan
authors.initials: I
authors.lastname: Iordanov
authors.affiliation: *Department of Medical Biochemistry and Department of Physiology, Semmelweis University, Budapest, Hungary; and Lendület Neurobiochemistry Research Group, Lendület Inflammation Physiology Research Group, Laboratory for Neurobiochemistry, and Lendület Ion Channel Research Group, Hungarian Academy of Sciences, Budapest, Hungary.
authors.forename: Vera
authors.initials: V
authors.lastname: Adam-Vizi
authors.affiliation: *Department of Medical Biochemistry and Department of Physiology, Semmelweis University, Budapest, Hungary; and Lendület Neurobiochemistry Research Group, Lendület Inflammation Physiology Research Group, Laboratory for Neurobiochemistry, and Lendület Ion Channel Research Group, Hungarian Academy of Sciences, Budapest, Hungary chinopoulos.christos@eok.sote.hu.
authors.forename: Christos
authors.initials: C
authors.lastname: Chinopoulos
mesh_terms: D000818:Animals; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D005260:Female; D006019:Glycolysis; D006836:Hydro-Lyases / Q000235:genetics / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D008314:Malonates / Q000494:pharmacology; D053078:Membrane Potential, Mitochondrial; D051379:Mice; D008810:Mice, Inbred C57BL; D008930:Mitochondria, Liver / Q000187:drug effects / Q000378:metabolism*; D000226:Mitochondrial ADP, ATP Translocases / Q000378:metabolism; D010085:Oxidative Phosphorylation; D012402:Rotenone / Q000494:pharmacology; D013389:Succinate-CoA Ligases / Q000378:metabolism; D013386:Succinates / Q000494:pharmacology*
chemical_list: D008070:Lipopolysaccharides; D008314:Malonates; D013386:Succinates; D012402:Rotenone; D000226:Mitochondrial ADP, ATP Translocases; C030290:malonic acid; D006836:Hydro-Lyases; C487648:Acod1 protein, mouse; D013389:Succinate-CoA Ligases; C005229:itaconic acid
keywords: cadA; lipopolysaccharide; succinate-CoA ligase