Mitochondrial Ca2+-independent phospholipase A2γ (iPLA2γ/PNPLA8) was previously shown to be directly activated by H2O2 and release free fatty acids (FAs) for FA-dependent H+ transport mediated by the adenine nucleotide translocase (ANT) or uncoupling protein 2 (UCP2). The resulting mild mitochondrial uncoupling and consequent partial attenuation of mitochondrial superoxide production lead to an antioxidant effect. However, the antioxidant role of iPLA2γ in the brain is not completely understood. Here, using wild-type and iPLA2γ-KO mice, we demonstrate the ability of tert-butylhydroperoxide (TBHP) to activate iPLA2γ in isolated brain mitochondria, with consequent liberation of FAs and lysophospholipids. The liberated FA caused an increase in respiratory rate, which was fully inhibited by carboxyatractyloside (CATR), a specific inhibitor of ANT. Employing detailed lipidomic analysis, we also demonstrate a typical cleavage pattern for TBHP-activated iPLA2γ, reflecting cleavage of glycerophospholipids from both sn-1 and sn-2 positions releasing saturated FAs, monoenoic FAs, and predominant polyunsaturated FAs. The acute antioxidant role of iPLA2γ-released FAs is supported by monitoring both intramitochondrial superoxide and extramitochondrial H2O2 release. We also show that iPLA2γ-KO mice were more sensitive to stimulation by pro-inflammatory lipopolysaccharide, as reflected by the concomitant increase in protein carbonyls in the brain and pro-inflammatory IL-6 release in the serum. These data support the antioxidant and anti-inflammatory role of iPLA2γ in vivo. Our data also reveal a substantial decrease of several high molecular weight cardiolipin (CL) species and accumulation of low molecular weight CL species in brain mitochondria of iPLA2γ-KO mice. Collectively, our results support a key role of iPLA2γ in the remodeling of lower molecular weight immature cardiolipins with predominantly saturated acyl chains to high molecular weight mature cardiolipins with highly unsaturated PUFA acyl chains, typical for the brain.

• Klíčová slova
• adenine nucleotide translocase, cardiolipin remodeling, mitochondria, phospholipase iPLA2γ/PNPLA8, redox homeostasis,
• Publikační typ
• časopisecké články MeSH

Patatin-like phospholipase domain-containing protein PNPLA8, also termed Ca2+-independent phospholipase A2γ (iPLA2γ), is addressed to the mitochondrial matrix (or peroxisomes), where it may manifest its unique activity to cleave phospholipid side-chains from both sn-1 and sn-2 positions, consequently releasing either saturated or unsaturated fatty acids (FAs), including oxidized FAs. Moreover, iPLA2γ is directly stimulated by H2O2 and, hence, is activated by redox signaling or oxidative stress. This redox activation permits the antioxidant synergy with mitochondrial uncoupling proteins (UCPs) or other SLC25 mitochondrial carrier family members by FA-mediated protonophoretic activity, termed mild uncoupling, that leads to diminishing of mitochondrial superoxide formation. This mechanism allows for the maintenance of the steady-state redox status of the cell. Besides the antioxidant role, we review the relations of iPLA2γ to lipid peroxidation since iPLA2γ is alternatively activated by cardiolipin hydroperoxides and hypothetically by structural alterations of lipid bilayer due to lipid peroxidation. Other iPLA2γ roles include the remodeling of mitochondrial (or peroxisomal) membranes and the generation of specific lipid second messengers. Thus, for example, during FA β-oxidation in pancreatic β-cells, H2O2-activated iPLA2γ supplies the GPR40 metabotropic FA receptor to amplify FA-stimulated insulin secretion. Cytoprotective roles of iPLA2γ in the heart and brain are also discussed.

• Klíčová slova
• SLC25 gene family, adenine nucleotide translocase, antioxidant synergy, cytoprotection, lipid peroxidation, mitochondrial carriers, mitochondrial phospholipase A2γ, mitochondrial uncoupling proteins,
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

SIGNIFICANCE: Mitochondria are the energetic, metabolic, redox, and information signaling centers of the cell. Substrate pressure, mitochondrial network dynamics, and cristae morphology state are integrated by the protonmotive force Δp or its potential component, ΔΨ, which are attenuated by proton backflux into the matrix, termed uncoupling. The mitochondrial uncoupling proteins (UCP1-5) play an eminent role in the regulation of each of the mentioned aspects, being involved in numerous physiological events including redox signaling. Recent Advances: UCP2 structure, including purine nucleotide and fatty acid (FA) binding sites, strongly support the FA cycling mechanism: UCP2 expels FA anions, whereas uncoupling is achieved by the membrane backflux of protonated FA. Nascent FAs, cleaved by phospholipases, are preferential. The resulting Δp dissipation decreases superoxide formation dependent on Δp. UCP-mediated antioxidant protection and its impairment are expected to play a major role in cell physiology and pathology. Moreover, UCP2-mediated aspartate, oxaloacetate, and malate antiport with phosphate is expected to alter metabolism of cancer cells. CRITICAL ISSUES: A wide range of UCP antioxidant effects and participations in redox signaling have been reported; however, mechanisms of UCP activation are still debated. Switching off/on the UCP2 protonophoretic function might serve as redox signaling either by employing/releasing the extra capacity of cell antioxidant systems or by directly increasing/decreasing mitochondrial superoxide sources. Rapid UCP2 degradation, FA levels, elevation of purine nucleotides, decreased Mg2+, or increased pyruvate accumulation may initiate UCP-mediated redox signaling. FUTURE DIRECTIONS: Issues such as UCP2 participation in glucose sensing, neuronal (synaptic) function, and immune cell activation should be elucidated. Antioxid. Redox Signal. 29, 667-714.

• Klíčová slova
• UCP2, anion transport, attenuation of superoxide formation, fatty acid cycling, mitochondrial uncoupling proteins, redox signaling,
• MeSH
• antioxidancia metabolismus   MeSH
• lidé MeSH
• mitochondriální odpřahující proteiny metabolismus   MeSH
• oxidace-redukce MeSH
• signální transdukce * MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• antioxidancia MeSH
• mitochondriální odpřahující proteiny MeSH

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.

• Klíčová slova
• antioxidative synergy, cytoprotection, mitochondrial phospholipase iPLA2γ, mitochondrial uncoupling protein UCP2, protein carbonylation,
• Publikační typ
• časopisecké články MeSH

AIMS: Pancreatic β-cell chronic lipotoxicity evolves from acute free fatty acid (FA)-mediated oxidative stress, unprotected by antioxidant mechanisms. Since mitochondrial uncoupling protein-2 (UCP2) plays antioxidant and insulin-regulating roles in pancreatic β-cells, we tested our hypothesis, that UCP2-mediated uncoupling attenuating mitochondrial superoxide production is initiated by FA release due to a direct H2O2-induced activation of mitochondrial phospholipase iPLA2γ. RESULTS: Pro-oxidant tert-butylhydroperoxide increased respiration, decreased membrane potential and mitochondrial matrix superoxide release rates of control but not UCP2- or iPLA2γ-silenced INS-1E cells. iPLA2γ/UCP2-mediated uncoupling was alternatively activated by an H2O2 burst, resulting from palmitic acid (PA) β-oxidation, and it was prevented by antioxidants or catalase overexpression. Exclusively, nascent FAs that cleaved off phospholipids by iPLA2γ were capable of activating UCP2, indicating that the previously reported direct redox UCP2 activation is actually indirect. Glucose-stimulated insulin release was not affected by UCP2 or iPLA2γ silencing, unless pro-oxidant activation had taken place. PA augmented insulin secretion via G-protein-coupled receptor 40 (GPR40), stimulated by iPLA2γ-cleaved FAs (absent after GPR40 silencing). INNOVATION AND CONCLUSION: The iPLA2γ/UCP2 synergy provides a feedback antioxidant mechanism preventing oxidative stress by physiological FA intake in pancreatic β-cells, regulating glucose-, FA-, and redox-stimulated insulin secretion. iPLA2γ is regulated by exogenous FA via β-oxidation causing H2O2 signaling, while FAs are cleaved off phospholipids, subsequently acting as amplifying messengers for GPR40. Hence, iPLA2γ acts in eminent physiological redox signaling, the impairment of which results in the lack of antilipotoxic defense and contributes to chronic lipotoxicity.

• MeSH
• antioxidancia farmakologie   MeSH
• beta-buňky účinky léků   MeSH
• fosfolipasy A2, skupina II metabolismus   MeSH
• inzulin metabolismus   MeSH
• iontové kanály metabolismus   MeSH
• krysa rodu Rattus MeSH
• lipidy toxicita   MeSH
• membránový potenciál mitochondrií účinky léků   MeSH
• mitochondriální proteiny metabolismus   MeSH
• mitochondrie účinky léků   MeSH
• nádorové buněčné linie MeSH
• oxidační stres účinky léků   MeSH
• peroxid vodíku metabolismus   MeSH
• receptory spřažené s G-proteiny metabolismus   MeSH
• sekrece inzulinu MeSH
• signální transdukce účinky léků   MeSH
• superoxidy metabolismus   MeSH
• terc-butylhydroperoxid farmakologie   MeSH
• uncoupling protein 2 MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antioxidancia MeSH
• fosfolipasy A2, skupina II MeSH
• G-protein-coupled receptor 40, rat MeSH Prohlížeč
• inzulin MeSH
• iontové kanály MeSH
• lipidy MeSH
• mitochondriální proteiny MeSH
• peroxid vodíku MeSH
• receptory spřažené s G-proteiny MeSH
• superoxidy MeSH
• terc-butylhydroperoxid MeSH
• Ucp2 protein, rat MeSH Prohlížeč
• uncoupling protein 2 MeSH

We reviewed mechanisms that determine reactive oxygen species (redox) homeostasis, redox information signaling and metabolic/regulatory function of autocrine insulin signaling in pancreatic β cells, and consequences of oxidative stress and dysregulation of redox/information signaling for their dysfunction. We emphasize the role of mitochondrion in β cell molecular physiology and pathology, including the antioxidant role of mitochondrial uncoupling protein UCP2. Since in pancreatic β cells pyruvate cannot be easily diverted towards lactate dehydrogenase for lactate formation, the respiration and oxidative phosphorylation intensity are governed by the availability of glucose, leading to a certain ATP/ADP ratio, whereas in other cell types, cell demand dictates respiration/metabolism rates. Moreover, we examine the possibility that type 2 diabetes mellitus might be considered as an inevitable result of progressive self-accelerating oxidative stress and concomitantly dysregulated information signaling in peripheral tissues as well as in pancreatic β cells. It is because the redox signaling is inherent to the insulin receptor signaling mechanism and its impairment leads to the oxidative and nitrosative stress. Also emerging concepts, admiting participation of redox signaling even in glucose sensing and insulin release in pancreatic β cells, fit in this view. For example, NADPH has been firmly established to be a modulator of glucose-stimulated insulin release.

• MeSH
• beta-buňky metabolismus   patologie   MeSH
• homeostáza * MeSH
• inzulin metabolismus   MeSH
• lidé MeSH
• mitochondrie metabolismus   MeSH
• oxidace-redukce MeSH
• oxidační stres MeSH
• sekrece inzulinu MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• inzulin MeSH

Mitochondrial uncoupling proteins (UCPs) are pure anion uniporters, which mediate fatty acid (FA) uniport leading to FA cycling. Protonated FAs then flip-flop back across the lipid bilayer. An existence of pure proton channel in UCPs is excluded by the equivalent flux-voltage dependencies for uniport of FAs and halide anions, which are best described by the Eyring barrier variant with a single energy well in the middle of two peaks. Experiments with FAs unable to flip and alkylsulfonates also support this view. Phylogenetically, UCPs took advantage of the common FA-uncoupling function of SLC25 family carriers and dropped their solute transport function.

• MeSH
• biologické modely MeSH
• elektroforéza MeSH
• iontové kanály metabolismus   MeSH
• lidé MeSH
• mitochondriální proteiny metabolismus   MeSH
• protony MeSH
• uncoupling protein 1 MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• iontové kanály MeSH
• mitochondriální proteiny MeSH
• protony MeSH
• uncoupling protein 1 MeSH

Existing controversies led us to analyze absolute mRNA levels of mitochondrial uncoupling proteins (UCP1-UCP5). Individual UCP isoform mRNA levels varied by up to four orders of magnitude in rat and mouse tissues. UCP2 mRNA content was relatively high (0.4 to 0.8 pg per 10 ng of total mRNA) in rat spleen, rat and mouse lung, and rat heart. Levels of the same order of magnitude were found for UCP3 mRNA in rat and mouse skeletal muscle, for UCP4 and UCP5 mRNA in mouse brain, and for UCP2 and UCP5 mRNA in mouse white adipose tissue. Significant differences in pattern were found for rat vs. mouse tissues, such as the dominance of UCP3/UCP5 vs. UCP2 transcript in mouse heart and vice versa in rat heart; or UCP2 (UCP5) dominance in rat brain contrary to 10-fold higher UCP4 and UCP5 dominance in mouse brain. We predict high antioxidant/antiapoptotic UCP function in tissues with higher UCP mRNA content.

• MeSH
• DNA primery genetika   MeSH
• druhová specificita MeSH
• iontové kanály metabolismus   MeSH
• krysa rodu Rattus MeSH
• membránové transportní proteiny metabolismus   MeSH
• messenger RNA metabolismus   MeSH
• mitochondriální odpřahující proteiny MeSH
• mitochondriální proteiny metabolismus   MeSH
• mozek metabolismus   MeSH
• myokard metabolismus   MeSH
• myši MeSH
• plíce metabolismus   MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• proteiny nervové tkáně metabolismus   MeSH
• slezina metabolismus   MeSH
• transportní proteiny mitochondriální membrány MeSH
• uncoupling protein 2 MeSH
• uncoupling protein 3 MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• DNA primery MeSH
• iontové kanály MeSH
• membránové transportní proteiny MeSH
• messenger RNA MeSH
• mitochondriální odpřahující proteiny MeSH
• mitochondriální proteiny MeSH
• proteiny nervové tkáně MeSH
• Slc25a14 protein, rat MeSH Prohlížeč
• Slc25a27 protein, rat MeSH Prohlížeč
• transportní proteiny mitochondriální membrány MeSH
• Ucp2 protein, mouse MeSH Prohlížeč
• Ucp2 protein, rat MeSH Prohlížeč
• Ucp3 protein, mouse MeSH Prohlížeč
• Ucp3 protein, rat MeSH Prohlížeč
• uncoupling protein 2 MeSH
• uncoupling protein 3 MeSH