Mitochondrial uncoupling protein-2 (UCP2) mediates free fatty acid (FA)-dependent H+ translocation across the inner mitochondrial membrane (IMM), which leads to acceleration of respiration and suppression of mitochondrial superoxide formation. Redox-activated mitochondrial phospholipase A2 (mt-iPLA2γ) cleaves FAs from the IMM and has been shown to acts in synergy with UCP2. Here, we tested the mechanism of mt-iPLA2γ-dependent UCP2-mediated antioxidant protection using lipopolysaccharide (LPS)-induced pro-inflammatory and pro-oxidative responses and their acute influence on the overall oxidative stress reflected by protein carbonylation in murine lung and spleen mitochondria and tissue homogenates. We provided challenges either by blocking the mt-iPLA 2γ function by the selective inhibitor R-bromoenol lactone (R-BEL) or by removing UCP2 by genetic ablation. We found that the basal levels of protein carbonyls in lung and spleen tissues and isolated mitochondria were higher in UCP2-knockout mice relative to the wild-type (wt) controls. The administration of R-BEL increased protein carbonyl levels in wt but not in UCP2-knockout (UCP2-KO) mice. LPS further increased the protein carbonyl levels in UCP2-KO mice, which correlated with protein carbonyl levels determined in wt mice treated with R-BEL. These results are consistent with the UCP2/mt-iPLA 2γ antioxidant mechanisms in these tissues and support the existence of UCP2-synergic mt-iPLA 2γ-dependent cytoprotective mechanism in vivo.

Department of Mitochondrial Physiology Institute of Physiology Academy of Sciences Prague Czech Republic

Zobrazit více v PubMed

Ježek P, Žáčková M, Růžička M, Škobisová E and Jabůrek M (2004) Mitochondrial uncoupling proteins–facts and fantasies. Physiol Res 53(S1), S199–S211. PubMed

Skulachev VP (1998) Uncoupling: new approaches to an old problem of bioenergetics. Biochim Biophys Acta 1363, 100–124. PubMed

Ježek P, Holendová B, Garlid KD and Jabůrek M (2018) Mitochondrial uncoupling proteins: subtle regulators of cellular redox signaling. Antioxid Redox Signal, in press. https://doi.org/10.1089/ars.2017.7225 PubMed DOI PMC

Plecitá‐Hlavatá L and Ježek P (2016) Integration of superoxide formation and cristae morphology for mitochondrial redox signaling. Int J Biochem Cell Biol 80, 31–50. PubMed

Brand MD (2016) Mitochondrial generation of superoxide and hydrogen peroxide as the source of mitochondrial redox signaling. Free Radic Biol Med 100, 14–31. PubMed

Jabůrek M, Varecha M, Gimeno REE, Dembski M, Jezek P, Zhang MB, Burn P, Tartaglia LA and Garlid KD (1999) Transport function and regulation of mitochondrial uncoupling proteins 2 and 3. J Biol Chem 274, 26003–26007. PubMed

Jabůrek M, Miyamoto S, Di Mascio P, Garlid KD and Ježek P (2004) Hydroperoxy fatty acid cycling mediated by mitochondrial uncoupling protein UCP2. J Biol Chem 279, 53097–53102. PubMed

Negre‐Salvayre A, Hirtz C, Carrera G, Cazenave R, Troly M, Salvayre R, Penicaud L and Casteilla L (1997) A role for uncoupling protein‐2 as a regulator of mitochondrial hydrogen peroxide generation. FASEB J 11, 809–815. PubMed

Arsenijevic D, Onuma H, Pecqueur C, Raimbault S, Manning BS, Miroux B, Couplan E, Alves‐Guerra MC, Goubern M, Surwit R et al (2000) Disruption of the uncoupling protein‐2 gene in mice reveals a role in immunity and reactive oxygen species production. Nat Genet 26, 435–439. PubMed

Jabůrek M, Ježek J, Zelenka J and Ježek P (2013) Antioxidant activity by a synergy of redox‐sensitive mitochondrial phospholipase A2 and uncoupling protein‐2 in lung and spleen. Int J Biochem Cell Biol 45, 816–825. PubMed

Ježek J, Dlasková A, Zelenka J, Jabůrek M and Ježek P (2015) H2O2 ‐activated mitochondrial phospholipase iPLA2γ prevents lipotoxic oxidative stress in synergy with UCP2, amplifies signaling via G‐protein–coupled receptor GPR40, and regulates insulin secretion in pancreatic β‐cells. Antioxid Redox Signal 23, 958–972. PubMed PMC

Murakami M, Taketomi Y, Miki Y, Sato H, Hirabayashi T and Yamamoto K (2011) Recent progress in phospholipase A2 research: from cells to animals to humans. Prog Lipid Res 50, 152–192. PubMed

Ramanadham S, Ali T, Ashley JW, Bone RN, Hancock WD and Lei X (2015) Calcium‐independent phospholipases A2 and their roles in biological processes and diseases. J Lipid Res 56, 1643–1668. PubMed PMC

Rauckhorst AJ, Pfeiffer DR and Broekemeier KM (2015) The iPLA2γ is identified as the membrane potential sensitive phospholipase in liver mitochondria. FEBS Lett 589, 2367–2371. PubMed

Bao S, Song H, Tan M, Wohltmann M, Ladenson JH and Turk J (2012) Group VIB phospholipase A2 promotes proliferation of INS‐1 insulinoma cells and attenuates lipid peroxidation and apoptosis induced by inflammatory cytokines and oxidant agents. Oxid Med Cell Longev 2012, 1–16. PubMed PMC

Elimam H, Papillon J, Takano T and Cybulsky AV (2013) Complement‐mediated activation of calcium‐independent phospholipase A2γ: role of protein kinases and phosphorylation. J Biol Chem 288, 3871–3885. PubMed PMC

Moon SH, Jenkins CM, Kiebish MA, Sims HF, Mancuso DJ and Gross RW (2012) Genetic ablation of calcium‐independent phospholipase A2γ (iPLA2γ) attenuates calcium‐induced opening of the mitochondrial permeability transition pore and resultant cytochrome c release. J Biol Chem 287, 29837–29850. PubMed PMC

Elimam H, Papillon J, Kaufman DR, Guillemette J, Aoudjit L, Gross RW, Takano T and Cybulsky AV (2016) Genetic ablation of calcium‐independent phospholipase A2γ induces glomerular injury in mice. J Biol Chem 291, 14468–14482. PubMed PMC

Liu G‐YY, Moon SH, Jenkins CM, Li M, Sims HF, Guan S, Gross RW, Ho Moon S, Jenkins CM, Li M et al (2017) The phospholipase iPLA2 is a major mediator releasing oxidized aliphatic chains from cardiolipin, integrating mitochondrial bioenergetics and signaling. J Biol Chem 292, 10672–10684. PubMed PMC

Ježek J, Jabůrek M, Zelenka J and Ježek P (2010) Mitochondrial phospholipase A2 activated by reactive oxygen species in heart mitochondria induces mild uncoupling. Physiol Res 59, 737–747. PubMed

Augustyniak E, Adam A, Wojdyla K, Rogowska‐Wrzesinska A, Willetts R, Korkmaz A, Atalay M, Weber D, Grune T, Borsa C et al (2015) Validation of protein carbonyl measurement: a multi‐centre study. Redox Biol 4, 149–157. PubMed PMC

Weber D, Davies MJ and Grune T (2015) Determination of protein carbonyls in plasma, cell extracts, tissue homogenates, isolated proteins: focus on sample preparation and derivatization conditions. Redox Biol 5, 367–380. PubMed PMC

Buss H, Chan TP, Sluis KB, Domigan NM and Winterbourn CC (1997) Protein carbonyl measurement by a sensitive ELISA method. Free Radic Biol Med 23, 361–366. PubMed

Kizaki T, Suzuki K, Hitomi Y, Taniguchi N, Saitoh D, Watanabe K, Onoé K, Day NK, Good RA, Ohno H et al (2002) Uncoupling protein 2 plays an important role in nitric oxide production of lipopolysaccharide‐stimulated macrophages. Proc Natl Acad Sci USA 99, 9392–9397. PubMed PMC

Emre Y, Hurtaud C, Nübel T, Criscuolo F, Ricquier D and Cassard‐Doulcier AM (2007) Mitochondria contribute to LPS‐induced MAPK activation via uncoupling protein UCP2 in macrophages. Biochem J 402, 271–278. PubMed PMC

Copeland S, Warren HS, Lowry SF, Calvano SE and Remick D (2005) Acute inflammatory response to endotoxin in mice and humans. Clin Diagn Lab Immunol 12, 60–67. PubMed PMC

Jenkins CM, Han X, Mancuso DJ and Gross RW (2002) Identification of calcium‐independent phospholipase A2 (iPLA2) β, and not iPLA2γ, as the mediator of arginine vasopressin‐induced arachidonic acid release in A‐10 smooth muscle cells. Enantioselective mechanism‐based discrimination of mammalian iPLA2s. J Biol Chem 277, 32807–32814. PubMed

Daniels SB, Cooney E, Sofia MJ, Chakravarty PK and Katzenellenbogen JA (1983) Haloenol lactones. Potent enzyme‐activated irreversible inhibitors for alpha‐chymotrypsin. J Biol Chem 258, 15046–15053. PubMed

Balsinde J and Dennis EA (1996) Bromoenol lactone inhibits magnesium‐dependent phosphatidate phosphohydrolase and blocks triacylglycerol biosynthesis in mouse P388D1 macrophages. J Biol Chem 271, 31937–31941. PubMed

Piantadosi CA and Suliman HB (2017) Mitochondrial dysfunction in lung pathogenesis. Annu Rev Physiol 79, 495–515. PubMed

Varga I, Babala J and Kachlik D (2018) Anatomic variations of the spleen: current state of terminology, classification, and embryological background. Surg Radiol Anat 40, 21–29. PubMed

Ježek P and Hlavatá L (2005) Mitochondria in homeostasis of reactive oxygen species in cell, tissues, and organism. Int J Biochem Cell Biol 37, 2478–2503. PubMed

Antioxidant Role and Cardiolipin Remodeling by Redox-Activated Mitochondrial Ca2+-Independent Phospholipase A2γ in the Brain

Antioxidant Synergy of Mitochondrial Phospholipase PNPLA8/iPLA2γ with Fatty Acid-Conducting SLC25 Gene Family Transporters