RESUMO
Mitochondrial fatty acid oxidation serves as an essential process for cellular survival, differentiation, proliferation, and energy metabolism. Numerous studies have utilized etomoxir (ETO) for the irreversible inhibition of carnitine palmitoylcarnitine transferase 1 (CPT1) which catalyzes the rate-limiting step for mitochondrial long-chain fatty acid ß-oxidation to examine the bioenergetic roles of mitochondrial fatty acid metabolism in many tissues in multiple diverse disease states. Herein, we demonstrate that intact mitochondria robustly metabolize etomoxir to etomoxir-carnitine (ETO-carnitine) prior to nearly complete etomoxir-mediated inhibition of CPT1. The novel pharmaco-metabolite, ETO-carnitine, was conclusively identified by accurate mass, fragmentation patterns, and isotopic fine structure. On the basis of these data, ETO-carnitine was successfully differentiated from isobaric structures (e.g., 3-hydroxy-C18:0 carnitine and 3-hydroxy-C18:1 carnitine). Mechanistically, generation of ETO-carnitine from mitochondria required exogenous Mg2+, ATP or ADP, CoASH, and L-carnitine indicating that thioesterification by long-chain acyl-CoA synthetase to form ETO-CoA precedes its conversion to ETO-carnitine by CPT1. CPT1-dependent generation of ETO-carnitine was substantiated by an orthogonal approach using ST1326 (a CPT1 inhibitor) which effectively inhibits mitochondrial ETO-carnitine production. Surprisingly, purified ETO-carnitine potently inhibited calcium-independent PLA2γ and PLA2ß as well as mitochondrial respiration independent of CPT1. Robust production and release of ETO-carnitine from HepG2 cells incubated in the presence of ETO was also demonstrated. Collectively, this study identifies the chemical mechanism for the biosynthesis of a novel pharmaco-metabolite of etomoxir, ETO-carnitine, that is generated by CPT1 in mitochondria and likely impacts multiple downstream (non-CPT1 related) enzymes and processes in multiple subcellular compartments.
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Recently, eicosanoid-lysophospholipids were identified as novel metabolites generated from the direct cyclooxygenase- or lipoxygenase-catalyzed oxidation of 2-arachidonoyl-lysophospholipids produced from either phospholipase A1-mediated hydrolysis of diacyl arachidonoyl-phospholipids or through the cytochrome c-catalyzed oxidative hydrolysis of the vinyl ether linkage of arachidonoyl-plasmalogens. Although the metabolic pathways generating eicosanoid-lysophospholipids have been increasingly appreciated, the signaling functions of eicosanoid-lysophospholipids remain largely unknown. Herein, we demonstrate that 2-12(S)-HETE-lysophospholipids as well as nonesterified 12(S)-HETE are potent lipid mediators that activate THP-1 human monocytic cells to generate tumor necrosis factor α (TNFα) and interleukin 8 (IL8). Remarkably, low nanomolar concentrations of 12(S)-HETE-lysophospholipids, but not other oxidized signaling lipids examined activated THP-1 cells resulting in the production of large amounts of TNFα. Moreover, TNFα release induced by 12(S)-HETE-lysophospholipids was inhibited by the TNFα converting enzyme inhibitor TAPI-0 indicating normal processing of TNFα in THP-1 cells stimulated with these agonists. Western blotting analyses revealed that 12(S)-HETE-lysophospholipids activated the phosphorylation of NFκB p65, suggesting activation of the canonical NFκB signaling pathway. Importantly, activation of THP-1 cells to release TNFα was stereoselective with 12(S)-HETE favored over 12(R)-HETE. Furthermore, the EC50 of 2-12(S)-HETE-lysophosphatidylcholine in activating THP-1 cells was 2.1 nm, whereas the EC50 of free 12(S)-HETE was 23 nm Additionally, lipid extracts of activated platelets were separated by RP-HPLC demonstrating the coelution of 12(S)-HETE with fractions initiating TNFα release. Collectively, these results demonstrate the potent signaling properties of 2-12(S)-HETE-lysophospholipids and 12(S)-HETE by their ability to release TNFα and activate NFκB signaling thereby revealing a previously unknown role of 2-12(S)-HETE-lysophospholipids in mediating inflammatory responses.
Assuntos
Lisofosfatidilcolinas/metabolismo , Monócitos/metabolismo , Transdução de Sinais , Animais , Ciclo-Oxigenase 1/metabolismo , Humanos , Camundongos , Monócitos/citologia , Células THP-1 , Fator de Transcrição RelA/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The canonical pathway of eicosanoid production in most mammalian cells is initiated by phospholipase A2-mediated release of arachidonic acid, followed by its enzymatic oxidation resulting in a vast array of eicosanoid products. However, recent work has demonstrated that the major phospholipase in mitochondria, iPLA2γ (patatin-like phospholipase domain containing 8 (PNPLA8)), possesses sn-1 specificity, with polyunsaturated fatty acids at the sn-2 position generating polyunsaturated sn-2-acyl lysophospholipids. Through strategic chemical derivatization, chiral chromatographic separation, and multistage tandem MS, here we first demonstrate that human platelet-type 12-lipoxygenase (12-LOX) can directly catalyze the regioselective and stereospecific oxidation of 2-arachidonoyl-lysophosphatidylcholine (2-AA-LPC) and 2-arachidonoyl-lysophosphatidylethanolamine (2-AA-LPE). Next, we identified these two eicosanoid-lysophospholipids in murine myocardium and in isolated platelets. Moreover, we observed robust increases in 2-AA-LPC, 2-AA-LPE, and their downstream 12-LOX oxidation products, 12(S)-HETE-LPC and 12(S)-HETE-LPE, in calcium ionophore (A23187)-stimulated murine platelets. Mechanistically, genetic ablation of iPLA2γ markedly decreased the calcium-stimulated production of 2-AA-LPC, 2-AA-LPE, and 12-HETE-lysophospholipids in mouse platelets. Importantly, a potent and selective 12-LOX inhibitor, ML355, significantly inhibited the production of 12-HETE-LPC and 12-HETE-LPE in activated platelets. Furthermore, we found that aging is accompanied by significant changes in 12-HETE-LPC in murine serum that were also markedly attenuated by iPLA2γ genetic ablation. Collectively, these results identify previously unknown iPLA2γ-initiated signaling pathways mediated by direct 12-LOX oxidation of 2-AA-LPC and 2-AA-LPE. This oxidation generates previously unrecognized eicosanoid-lysophospholipids that may serve as biomarkers for age-related diseases and could potentially be used as targets in therapeutic interventions.
Assuntos
Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/metabolismo , Araquidonato 12-Lipoxigenase/metabolismo , Plaquetas/metabolismo , Fosfolipases A2 do Grupo VI/metabolismo , Lisofosfatidilcolinas/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Ácidos Graxos Insaturados/metabolismo , Fosfolipases A2 do Grupo VI/genética , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Oxirredução , SpodopteraRESUMO
Recently, oxidized phospholipid species have emerged as important signaling lipids in activated immune cells and platelets. The canonical pathway for the synthesis of oxidized phospholipids is through the release of arachidonic acid by cytosolic phospholipase A2α (cPLA2α) followed by its enzymatic oxidation, activation of the carboxylate anion by acyl-CoA synthetase(s), and re-esterification to the sn-2 position by sn-2 acyltransferase activity (i.e. the Lands cycle). However, recent studies have demonstrated the unanticipated significance of sn-1 hydrolysis of arachidonoyl-containing choline and ethanolamine glycerophospholipids by other phospholipases to generate the corresponding 2-arachidonoyl-lysolipids. Herein, we identified a pathway for oxidized phospholipid synthesis comprising sequential sn-1 hydrolysis by a phospholipase A1 (e.g. by patatin-like phospholipase domain-containing 8 (PNPLA8)), direct enzymatic oxidation of the resultant 2-arachidonoyl-lysophospholipids, and the esterification of oxidized 2-arachidonoyl-lysophospholipids by acyl-CoA-dependent sn-1 acyltransferase(s). To circumvent ambiguities associated with acyl migration or hydrolysis, we developed a synthesis for optically active (d- and l-enantiomers) nonhydrolyzable analogs of 2-arachidonoyl-lysophosphatidylcholine (2-AA-LPC). sn-1 acyltransferase activity in murine liver microsomes stereospecifically and preferentially utilized the naturally occurring l-enantiomer of the ether analog of lysophosphatidylcholine. Next, we demonstrated the high selectivity of the sn-1 acyltransferase activity for saturated acyl-CoA species. Importantly, we established that 2-15-hydroxyeicosatetraenoic acid (HETE) ether-LPC sn-1 esterification is markedly activated by thrombin treatment of murine platelets to generate oxidized PC. Collectively, these findings demonstrate the enantiomeric specificity and saturated acyl-CoA selectivity of microsomal sn-1 acyltransferase(s) and reveal its participation in a previously uncharacterized pathway for the synthesis of oxidized phospholipids with cell-signaling properties.
Assuntos
Aciltransferases/metabolismo , Ácidos Hidroxieicosatetraenoicos/metabolismo , Lisofosfolipídeos/metabolismo , Fosfolipases/metabolismo , Fosfolipídeos/metabolismo , Acilação , Aciltransferases/genética , Animais , Plaquetas/metabolismo , Ácidos Hidroxieicosatetraenoicos/química , Lisofosfolipídeos/química , Camundongos , Microssomos Hepáticos/metabolismo , Oxirredução , Fosfolipídeos/química , Especificidade por SubstratoRESUMO
Plasmalogens are phospholipids critical for cell function and signaling that contain a vinyl ether linkage at the sn-1 position and are highly enriched in arachidonic acid (AA) at the sn-2 position. However, the enzyme(s) responsible for the cleavage of the vinyl ether linkage in plasmalogens has remained elusive. Herein, we report that cytochrome c, in the presence of either cardiolipin (CL), O2 and H2O2, or oxidized CL and O2, catalyzes the oxidation of the plasmalogen vinyl ether linkage, promoting its hydrolytic cleavage and resultant production of 2-AA-lysolipids and highly reactive α-hydroxy fatty aldehydes. Using stable isotope labeling in synergy with strategic chemical derivatizations and high-mass-accuracy MS, we deduced the chemical mechanism underlying this long sought-after reaction. Specifically, labeling with either 18O2 or H218O, but not with H218O2, resulted in M + 2 isotopologues of the α-hydroxyaldehyde, whereas reactions with both 18O2 and H218O identified the M + 4 isotopologue. Furthermore, incorporation of 18O from 18O2 was predominantly located at the α-carbon. In contrast, reactions with H218O yielded 18O linked to the aldehyde carbon. Importantly, no significant labeling of 2-AA-lysolipids with 18O2, H218O, or H218O2 was present. Intriguingly, phosphatidylinositol phosphates (PIP2 and PIP3) effectively substituted for cardiolipin. Moreover, cytochrome c released from myocardial mitochondria subjected to oxidative stress cleaved plasmenylcholine in membrane bilayers, and this was blocked with a specific mAb against cytochrome c Collectively, these results identify the first plasmalogenase in biology, reveal the production of previously unanticipated signaling lipids by cytochrome c, and present new perspectives on cellular signaling during oxidative stress.
Assuntos
Citocromos c/metabolismo , Hidrolases/metabolismo , Mitocôndrias Cardíacas/metabolismo , Estresse Oxidativo , Plasmalogênios/metabolismo , Compostos de Vinila/química , Animais , Citocromos c/química , Cavalos , Humanos , Hidrólise , Lipídeos/análise , Masculino , Oxirredução , Coelhos , Compostos de Vinila/metabolismoRESUMO
Congestive heart failure typically arises from cardiac myocyte necrosis/apoptosis, associated with the pathological opening of the mitochondrial permeability transition pore (mPTP). mPTP opening decreases the mitochondrial membrane potential leading to the activation of Ca2+-independent phospholipase A2γ (iPLA2γ) and the production of downstream toxic metabolites. However, the array of enzymatic mediators and the exact chemical mechanisms responsible for modulating myocardial mPTP opening remain unclear. Herein, we demonstrate that human heart failure activates specific myocardial mitochondrial phospholipases that increase Ca2+-dependent production of toxic hydroxyeicosatetraenoic acids (HETEs) and attenuate the activity of phospholipases that promote the synthesis of protective epoxyeicosatrienoic acids (EETs). Mechanistically, HETEs activated the Ca2+-induced opening of the mPTP in failing human myocardium, and the highly selective pharmacological blockade of either iPLA2γ or lipoxygenases attenuated mPTP opening in failing hearts. In contrast, pharmacological inhibition of cytochrome P450 epoxygenases opened the myocardial mPTP in human heart mitochondria. Remarkably, the major mitochondrial phospholipase responsible for Ca2+-activated release of arachidonic acid (AA) in mitochondria from non-failing hearts was calcium-dependent phospholipase A2ζ (cPLA2ζ) identified by sequential column chromatographies and activity-based protein profiling. In contrast, iPLA2γ predominated in failing human myocardium. Stable isotope kinetics revealed that in non-failing human hearts, cPLA2ζ metabolically channels arachidonic acid into EETs, whereas in failing hearts, increased iPLA2γ activity channels AA into toxic HETEs. These results mechanistically identify the sequelae of pathological remodeling of human mitochondrial phospholipases in failing myocardium. This remodeling metabolically channels AA into toxic HETEs promoting mPTP opening, which induces necrosis/apoptosis leading to further progression of heart failure.
Assuntos
Fosfolipases A2 do Grupo VI/metabolismo , Insuficiência Cardíaca/metabolismo , Ácidos Hidroxieicosatetraenoicos/biossíntese , Mitocôndrias Cardíacas/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Sequência de Aminoácidos , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Insuficiência Cardíaca/enzimologia , Insuficiência Cardíaca/patologia , Humanos , Ácidos Hidroxieicosatetraenoicos/metabolismo , Potencial da Membrana Mitocondrial , Mitocôndrias Cardíacas/enzimologia , Membranas Mitocondriais/enzimologia , Membranas Mitocondriais/metabolismo , Poro de Transição de Permeabilidade Mitocondrial , Miocárdio/enzimologia , Miocárdio/metabolismo , Miocárdio/patologia , Permeabilidade , Fosfolipases A2/metabolismoRESUMO
Cardiolipin (CL) is a dimeric phospholipid with critical roles in mitochondrial bioenergetics and signaling. Recently, inhibition of the release of oxidized fatty acyl chains from CL by the calcium-independent phospholipase A2γ (iPLA2γ)-selective inhibitor (R)-BEL suggested that iPLA2γ is responsible for the hydrolysis of oxidized CL and subsequent signaling mediated by the released oxidized fatty acids. However, chemical inhibition by BEL is subject to off-target pharmacologic effects. Accordingly, to unambiguously determine the role of iPLA2γ in the hydrolysis of oxidized CL, we compared alterations in oxidized CLs and the release of oxidized aliphatic chains from CL in experiments with purified recombinant iPLA2γ, germ-line iPLA2γ-/- mice, cardiac myocyte-specific iPLA2γ transgenic mice, and wild-type mice. Using charge-switch high mass accuracy LC-MS/MS with selected reaction monitoring and product ion accurate masses, we demonstrated that iPLA2γ is the major enzyme responsible for the release of oxidized aliphatic chains from CL. Our results also indicated that iPLA2γ selectively hydrolyzes 9-hydroxy-octadecenoic acid in comparison to 13-hydroxy-octadecenoic acid from oxidized CLs. Moreover, oxidative stress (ADP, NADPH, and Fe3+) resulted in the robust production of oxidized CLs in intact mitochondria from iPLA2γ-/- mice. In sharp contrast, oxidized CLs were readily hydrolyzed in mitochondria from wild-type mice during oxidative stress. Finally, we demonstrated that CL activates the iPLA2γ-mediated hydrolysis of arachidonic acid from phosphatidylcholine, thereby integrating the production of lipid messengers from different lipid classes in mitochondria. Collectively, these results demonstrate the integrated roles of CL and iPLA2γ in lipid second-messenger production and mitochondrial bioenergetics during oxidative stress.
Assuntos
Cardiolipinas/metabolismo , Metabolismo Energético , Fosfolipases A2 do Grupo VI/metabolismo , Mitocôndrias Cardíacas/enzimologia , Estresse Oxidativo , Transdução de Sinais , Animais , Cardiolipinas/genética , Fosfolipases A2 do Grupo VI/genética , Camundongos , Camundongos Knockout , Mitocôndrias Cardíacas/genética , OxirreduçãoRESUMO
Calcium-independent phospholipase A2γ (iPLA2γ) is a mitochondrial enzyme that produces lipid second messengers that facilitate opening of the mitochondrial permeability transition pore (mPTP) and contribute to the production of oxidized fatty acids in myocardium. To specifically identify the roles of iPLA2γ in cardiac myocytes, we generated cardiac myocyte-specific iPLA2γ knock-out (CMiPLA2γKO) mice by removing the exon encoding the active site serine (Ser-477). Hearts of CMiPLA2γKO mice exhibited normal hemodynamic function, glycerophospholipid molecular species composition, and normal rates of mitochondrial respiration and ATP production. In contrast, CMiPLA2γKO mice demonstrated attenuated Ca(2+)-induced mPTP opening that could be rapidly restored by the addition of palmitate and substantially reduced production of oxidized polyunsaturated fatty acids (PUFAs). Furthermore, myocardial ischemia/reperfusion (I/R) in CMiPLA2γKO mice (30 min of ischemia followed by 30 min of reperfusion in vivo) dramatically decreased oxidized fatty acid production in the ischemic border zones. Moreover, CMiPLA2γKO mice subjected to 30 min of ischemia followed by 24 h of reperfusion in vivo developed substantially less cardiac necrosis in the area-at-risk in comparison with their WT littermates. Furthermore, we found that membrane depolarization in murine heart mitochondria was sensitized to Ca(2+) by the presence of oxidized PUFAs. Because mitochondrial membrane depolarization and calcium are known to activate iPLA2γ, these results are consistent with salvage of myocardium after I/R by iPLA2γ loss of function through decreasing mPTP opening, diminishing production of proinflammatory oxidized fatty acids, and attenuating the deleterious effects of abrupt increases in calcium ion on membrane potential during reperfusion.
Assuntos
Ácidos Graxos Insaturados/metabolismo , Fosfolipases A2 do Grupo VI/metabolismo , Potencial da Membrana Mitocondrial , Mitocôndrias Cardíacas/enzimologia , Traumatismo por Reperfusão Miocárdica/enzimologia , Miocárdio/enzimologia , Miócitos Cardíacos/enzimologia , Animais , Cálcio/metabolismo , Fosfolipases A2 do Grupo VI/genética , Camundongos , Camundongos Knockout , Mitocôndrias Cardíacas/genética , Traumatismo por Reperfusão Miocárdica/genética , Especificidade de Órgãos , OxirreduçãoRESUMO
Diglycerides play a central role in lipid metabolism and signaling in mammalian cells. Although diacylglycerol molecular species comprise the majority of cellular diglycerides that are commonly measured using a variety of approaches, identification of extremely low abundance vinyl ether diglycerides has remained challenging. In this work, representative molecular species from the three diglyceride subclasses (diacyl, vinyl ether, and alkyl ether diglycerides; hereafter referred to as diradylglycerols) were interrogated by mass spectrometric analysis. Product ion mass spectra of the synthesized diradylglycerols with varied chain lengths and degrees of unsaturation demonstrated diagnostic fragmentation patterns indicative of each subclass. Multidimensional mass spectrometry-based shotgun lipidomics (MDMS-SL) analysis of mouse brain and heart lipid extracts were performed using the identified informative signature product ions. Through an array of tandem mass spectrometric analyses utilizing the orthogonal characteristics of neutral loss scanning and precursor ion scanning, the differential fragmentation of each subclass was exploited for high-yield structural analyses. Although molecular ion mass spectra readily identified diacylglycerol molecular species directly from the hexane fractions of tissue extracts enriched in nonpolar lipids, molecular ion peaks corresponding to ether-linked diglycerides were not observable. The power of MDMS-SL utilizing the tandem mass spectrometric array analysis was demonstrated by identification and profiling of individual molecular species of vinyl ether diglycerides in mouse brain and heart from their undetectable molecular ion peaks during MS(1) analysis. Collectively, this technology enabled the identification and profiling of previously inaccessible vinyl ether diglyceride molecular species in mammalian tissues directly from extracts of biologic tissues.
Assuntos
Química Encefálica , Diglicerídeos/análise , Miocárdio/química , Compostos de Vinila/análise , Animais , Masculino , Espectrometria de Massas/métodos , Camundongos , Camundongos Endogâmicos C57BLRESUMO
The multifaceted roles of calcium-independent phospholipase A2ß (iPLA2ß) in numerous cellular processes have been extensively examined through utilization of the iPLA2-selective inhibitor (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one (BEL). Herein, we employed accurate mass/high resolution mass spectrometry to demonstrate that the active site serine (S465) and C651 of iPLA2ß are covalently cross-linked during incubations with BEL demonstrating their close spatial proximity. This cross-link results in macroscopic alterations in enzyme molecular geometry evidenced by anomalous migration of the cross-linked enzyme by SDS-PAGE. Molecular models of iPLA2ß constructed from the crystal structure of iPLA2α (patatin) indicate that the distance between S465 and C651 is approximately 10 Å within the active site of iPLA2ß. Kinetic analysis of the formation of the 75 kDa iPLA2ß-BEL species with the (R) and (S) enantiomers of BEL demonstrated that the reaction of (S)-BEL with iPLA2ß was more rapid than for (R)-BEL paralleling the enantioselectivity for the inhibition of catalysis by each inhibitor with iPLA2ß. Moreover, we demonstrate that the previously identified selective acylation of iPLA2ß by oleoyl-CoA occurs at C651 thereby indicating the importance of active site architecture for acylation of this enzyme. Collectively, these results identify C651 as a highly reactive nucleophilic residue within the active site of iPLA2ß which is thioesterified by BEL, acylated by oleoyl-CoA, and located in close spatial proximity to the catalytic serine thereby providing important chemical insights on the mechanisms through which BEL inhibits iPLA2ß and the topology of the active site.
Assuntos
Cisteína/química , Fosfolipases A2 Independentes de Cálcio/antagonistas & inibidores , Animais , Catálise , Domínio Catalítico , Cristalografia por Raios X , Desenho de Fármacos , Hidrólise , Insetos , Lipídeos/química , Espectrometria de Massas , Modelos Químicos , Fosfolipases A2 Independentes de Cálcio/química , Fosfolipases A2 Independentes de Cálcio/genética , Serina/química , EspectrofotometriaRESUMO
Calcium-independent phospholipase A(2)γ (iPLA(2)γ) (PNPLA8) is the predominant phospholipase activity in mammalian mitochondria. However, the chemical mechanisms that regulate its activity are unknown. Here, we utilize iPLA(2)γ gain of function and loss of function genetic models to demonstrate the robust activation of iPLA(2)γ in murine myocardial mitochondria by Ca(2+) or Mg(2+) ions. Calcium ion stimulated the production of 2-arachidonoyl-lysophosphatidylcholine (2-AA-LPC) from 1-palmitoyl-2-[(14)C]arachidonoyl-sn-glycero-3-phosphocholine during incubations with wild-type heart mitochondrial homogenates. Furthermore, incubation of mitochondrial homogenates from transgenic myocardium expressing iPLA(2)γ resulted in 13- and 25-fold increases in the initial rate of radiolabeled 2-AA-LPC and arachidonic acid (AA) production, respectively, in the presence of calcium ion. Mass spectrometric analysis of the products of calcium-activated hydrolysis of endogenous mitochondrial phospholipids in transgenic iPLA(2)γ mitochondria revealed the robust production of AA, 2-AA-LPC, and 2-docosahexaenoyl-LPC that was over 10-fold greater than wild-type mitochondria. The mechanism-based inhibitor (R)-(E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one (BEL) (iPLA(2)γ selective), but not its enantiomer, (S)-BEL (iPLA(2)ß selective) or pyrrolidine (cytosolic PLA(2)α selective), markedly attenuated Ca(2+)-dependent fatty acid release and polyunsaturated LPC production. Moreover, Ca(2+)-induced iPLA(2)γ activation was accompanied by the production of downstream eicosanoid metabolites that were nearly completely ablated by (R)-BEL or by genetic ablation of iPLA(2)γ. Intriguingly, Ca(2+)-induced iPLA(2)γ activation was completely inhibited by long-chain acyl-CoA (IC(50) â¼20 µm) as well as by a nonhydrolyzable acyl-CoA thioether analog. Collectively, these results demonstrate that mitochondrial iPLA(2)γ is activated by divalent cations and inhibited by acyl-CoA modulating the generation of biologically active metabolites that regulate mitochondrial bioenergetic and signaling functions.
Assuntos
Ácido Araquidônico/metabolismo , Cálcio/metabolismo , Fosfolipases A2 do Grupo VI/metabolismo , Magnésio/metabolismo , Mitocôndrias Cardíacas/enzimologia , Animais , Ácido Araquidônico/genética , Cátions Bivalentes/metabolismo , Ativação Enzimática/efeitos dos fármacos , Fosfolipases A2 do Grupo VI/antagonistas & inibidores , Fosfolipases A2 do Grupo VI/genética , Lisofosfatidilcolinas/genética , Lisofosfatidilcolinas/metabolismo , Camundongos , Camundongos Knockout , Mitocôndrias Cardíacas/genética , Modelos Genéticos , Naftalenos/farmacologia , Inibidores de Fosfodiesterase/farmacologia , Pironas/farmacologiaRESUMO
Herein, we demonstrate that calcium-independent phospholipase A(2)γ (iPLA(2)γ) is a critical mechanistic participant in the calcium-induced opening of the mitochondrial permeability transition pore (mPTP). Liver mitochondria from iPLA(2)γ(-/-) mice were markedly resistant to calcium-induced swelling in the presence or absence of phosphate in comparison with wild-type littermates. Furthermore, the iPLA(2)γ enantioselective inhibitor (R)-(E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one ((R)-BEL) was markedly more potent than (S)-BEL in inhibiting mPTP opening in mitochondria from wild-type liver in comparison with hepatic mitochondria from iPLA(2)γ(-/-) mice. Intriguingly, low micromolar concentrations of long chain fatty acyl-CoAs and the non-hydrolyzable thioether analog of palmitoyl-CoA markedly accelerated Ca(2+)-induced mPTP opening in liver mitochondria from wild-type mice. The addition of l-carnitine enabled the metabolic channeling of acyl-CoA through carnitine palmitoyltransferases (CPT-1/2) and attenuated the palmitoyl-CoA-mediated amplification of calcium-induced mPTP opening. In contrast, mitochondria from iPLA(2)γ(-/-) mice were insensitive to fatty acyl-CoA-mediated augmentation of calcium-induced mPTP opening. Moreover, mitochondria from iPLA(2)γ(-/-) mouse liver were resistant to Ca(2+)/t-butyl hydroperoxide-induced mPTP opening in comparison with wild-type littermates. In support of these findings, cytochrome c release from iPLA(2)γ(-/-) mitochondria was dramatically decreased in response to calcium in the presence or absence of either t-butyl hydroperoxide or phenylarsine oxide in comparison with wild-type littermates. Collectively, these results identify iPLA(2)γ as an important mechanistic component of the mPTP, define its downstream products as potent regulators of mPTP opening, and demonstrate the integrated roles of mitochondrial bioenergetics and lipidomic flux in modulating mPTP opening promoting the activation of necrotic and necroapoptotic pathways of cell death.
Assuntos
Cálcio/metabolismo , Fosfolipases A2 do Grupo VI/metabolismo , Mitocôndrias Hepáticas/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Animais , Carnitina/genética , Carnitina/metabolismo , Carnitina O-Palmitoiltransferase/genética , Carnitina O-Palmitoiltransferase/metabolismo , Morte Celular , Citocromos c/genética , Citocromos c/metabolismo , Metabolismo Energético/efeitos dos fármacos , Metabolismo Energético/genética , Inibidores Enzimáticos/farmacologia , Fosfolipases A2 do Grupo VI/antagonistas & inibidores , Fosfolipases A2 do Grupo VI/genética , Metabolismo dos Lipídeos/efeitos dos fármacos , Metabolismo dos Lipídeos/genética , Camundongos , Camundongos Knockout , Mitocôndrias Hepáticas/genética , Proteínas de Transporte da Membrana Mitocondrial/genética , Poro de Transição de Permeabilidade Mitocondrial , Palmitoil Coenzima A/genética , Palmitoil Coenzima A/metabolismo , CoelhosRESUMO
Lipidomic regulation of mitochondrial cardiolipin content and molecular species composition is a prominent regulator of bioenergetic efficiency. However, the mechanisms controlling cardiolipin metabolism during health or disease progression have remained elusive. Herein, we demonstrate that cardiac myocyte-specific transgenic expression of cardiolipin synthase results in accelerated cardiolipin lipidomic flux that impacts multiple aspects of mitochondrial bioenergetics and signaling. During the postnatal period, cardiolipin synthase transgene expression results in marked changes in the temporal maturation of cardiolipin molecular species during development. In adult myocardium, cardiolipin synthase transgene expression leads to a marked increase in symmetric tetra-18:2 molecular species without a change in total cardiolipin content. Mechanistic analysis demonstrated that these alterations result from increased cardiolipin remodeling by sequential phospholipase and transacylase/acyltransferase activities in conjunction with a decrease in phosphatidylglycerol content. Moreover, cardiolipin synthase transgene expression results in alterations in signaling metabolites, including a marked increase in the cardioprotective eicosanoid 14,15-epoxyeicosatrienoic acid. Examination of mitochondrial bioenergetic function by high resolution respirometry demonstrated that cardiolipin synthase transgene expression resulted in improved mitochondrial bioenergetic efficiency as evidenced by enhanced electron transport chain coupling using multiple substrates as well as by salutary changes in Complex III and IV activities. Furthermore, transgenic expression of cardiolipin synthase attenuated maladaptive cardiolipin remodeling and bioenergetic inefficiency in myocardium rendered diabetic by streptozotocin treatment. Collectively, these results demonstrate the unanticipated role of cardiolipin synthase in maintaining physiologic membrane structure and function even under metabolic stress, thereby identifying cardiolipin synthase as a novel therapeutic target to attenuate mitochondrial dysfunction in diabetic myocardium.
Assuntos
Diabetes Mellitus Experimental/metabolismo , Metabolismo Energético , Proteínas de Membrana/metabolismo , Miocárdio/enzimologia , Miócitos Cardíacos/enzimologia , Fosfatidilgliceróis/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo , Animais , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/patologia , Complexo III da Cadeia de Transporte de Elétrons/genética , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Humanos , Proteínas de Membrana/genética , Camundongos , Camundongos Transgênicos , Mitocôndrias Cardíacas/enzimologia , Mitocôndrias Cardíacas/genética , Mitocôndrias Cardíacas/patologia , Miocárdio/patologia , Miócitos Cardíacos/metabolismo , Fosfatidilgliceróis/genética , Transferases (Outros Grupos de Fosfato Substituídos)/genéticaRESUMO
A highly sensitive, specific, and robust method for the analysis of oxidized metabolites of linoleic acid (LA), arachidonic acid (AA), and docosahexaenoic acid (DHA) was developed using charge-switch derivatization, liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI MS/MS) with selected reaction monitoring (SRM) and quantitation by high mass accuracy analysis of product ions, thereby minimizing interferences from contaminating ions. Charge-switch derivatization of LA, AA, and DHA metabolites with N-(4-aminomethylphenyl)-pyridinium resulted in a 10- to 30-fold increase in ionization efficiency. Improved quantitation was accompanied by decreased false positive interferences through accurate mass measurements of diagnostic product ions during SRM transitions by ratiometric comparisons with stable isotope internal standards. The limits of quantitation were between 0.05 and 6.0pg, with a dynamic range of 3 to 4 orders of magnitude (correlation coefficient r(2)>0.99). This approach was used to quantitate the levels of representative fatty acid metabolites from wild-type (WT) and iPLA2γ(-/-) mouse liver identifying the role of iPLA2γ in hepatic lipid second messenger production. Collectively, these results demonstrate the utility of high mass accuracy product ion analysis in conjunction with charge-switch derivatization for the highly specific quantitation of diminutive amounts of LA, AA, and DHA metabolites in biologic systems.
Assuntos
Ácido Araquidônico/análise , Ácido Araquidônico/química , Ácidos Docosa-Hexaenoicos/análise , Ácidos Docosa-Hexaenoicos/química , Ácido Linoleico/análise , Ácido Linoleico/química , Animais , Ácido Araquidônico/metabolismo , Cromatografia Líquida , Ácidos Docosa-Hexaenoicos/metabolismo , Ácido Linoleico/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Oxirredução , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em TandemRESUMO
Skeletal muscle is the major site of glucose utilization in mammals integrating serum glucose clearance with mitochondrial respiration. To mechanistically elucidate the roles of iPLA2γ in skeletal muscle mitochondria, we generated a skeletal muscle-specific calcium-independent phospholipase A2γ knockout (SKMiPLA2γKO) mouse. Genetic ablation of skeletal muscle iPLA2γ resulted in pronounced muscle weakness, muscle atrophy, and increased blood lactate resulting from defects in mitochondrial function impairing metabolic processing of pyruvate and resultant bioenergetic inefficiency. Mitochondria from SKMiPLA2γKO mice were dysmorphic displaying marked changes in size, shape, and interfibrillar juxtaposition. Mitochondrial respirometry demonstrated a marked impairment in respiratory efficiency with decreases in the mass and function of oxidative phosphorylation complexes and cytochrome c. Further, a pronounced decrease in mitochondrial membrane potential and remodeling of cardiolipin molecular species were prominent. Collectively, these alterations prevented body weight gain during high-fat feeding through enhanced glucose disposal without efficient capture of chemical energy thereby altering whole-body bioenergetics.
RESUMO
The enzyme phosphofructokinase-1 (PFK-1) catalyzes the first committed step of glycolysis and is regulated by a complex array of allosteric effectors that integrate glycolytic flux with cellular bioenergetics. Here, we demonstrate the direct, potent, and reversible inhibition of purified rabbit muscle PFK-1 by low micromolar concentrations of long chain fatty acyl-CoAs (apparent Kiâ¼1 µM). In sharp contrast, short chain acyl-CoAs, palmitoylcarnitine, and palmitic acid in the presence of CoASH were without effect. Remarkably, MgAMP and MgADP but not MgATP protected PFK-1 against inhibition by palmitoyl-CoA indicating that acyl-CoAs regulate PFK-1 activity in concert with cellular high energy phosphate status. Furthermore, incubation of PFK-1 with [1-(14)C]palmitoyl-CoA resulted in robust acylation of the enzyme that was reversible by incubation with acyl-protein thioesterase-1 (APT1). Importantly, APT1 reversed palmitoyl-CoA-mediated inhibition of PFK-1 activity. Mass spectrometric analyses of palmitoylated PFK-1 revealed four sites of acylation, including Cys-114, Cys-170, Cys-351, and Cys-577. PFK-1 in both skeletal muscle extracts and in purified form was inhibited by S-hexadecyl-CoA, a nonhydrolyzable palmitoyl-CoA analog, demonstrating that covalent acylation of PFK-1 was not required for inhibition. Tryptic footprinting suggested that S-hexadecyl-CoA induced a conformational change in PFK-1. Both palmitoyl-CoA and S-hexadecyl-CoA increased the association of PFK-1 with Ca2+/calmodulin, which attenuated the binding of palmitoylated PFK-1 to membrane vesicles. Collectively, these results demonstrate that fatty acyl-CoA modulates phosphofructokinase activity through both covalent and noncovalent interactions to regulate glycolytic flux and enzyme membrane localization via the branch point metabolic node that mediates lipid flux through anabolic and catabolic pathways.
Assuntos
Acil Coenzima A/metabolismo , Fosfofrutoquinase-1/metabolismo , Tioléster Hidrolases/metabolismo , Animais , Cálcio/metabolismo , Calmodulina/metabolismo , Eletroforese em Gel de Poliacrilamida , Glicólise , Humanos , Metabolismo dos Lipídeos , Espectrometria de Massas , Músculo Esquelético/metabolismo , Palmitoil Coenzima A/metabolismo , Ligação Proteica , Coelhos , Lipossomas Unilamelares/metabolismoRESUMO
The scavenger receptor FAT/CD36 contributes to the inflammation associated with diabetes, atherosclerosis, thrombosis, and Alzheimer disease. Underlying mechanisms include CD36 promotion of oxidative stress and its signaling to stress kinases. Here we document an additional mechanism for the role of CD36 in inflammation. CD36 regulates membrane calcium influx in response to endoplasmic reticulum (ER) stress, release of arachidonic acid (AA) from cellular membranes by cytoplasmic phospholipase A(2)α (cPLA(2)α) and contributes to the generation of proinflammatory eicosanoids. CHO cells stably expressing human CD36 released severalfold more AA and prostaglandin E(2) (PGE(2)), a major product of AA metabolism by cyclooxygenases, in response to thapsigargin-induced ER stress as compared with control cells. Calcium influx after ER calcium release resulted in phosphorylation of cPLA(2) and its translocation to membranes in a CD36-dependent manner. Peritoneal macrophages from CD36(-/-) mice exhibited diminished calcium transients and reduced AA release after thapsigargin or UTP treatment with decreased ERK1/2 and cPLA(2) phosphorylation. However, PGE(2) production was unexpectedly enhanced in CD36(-/-) macrophages, which probably resulted from a large induction of cyclooxygenase 2 mRNA and protein. The data demonstrate participation of CD36 in membrane calcium influx in response to ER stress or purinergic receptor stimulation resulting in AA liberation for PGE(2) formation. Collectively, these results identify a mechanism contributing to the pleiotropic proinflammatory effects of CD36 and suggest that its targeted inhibition may reduce the acute inflammatory response.
Assuntos
Antígenos CD36/metabolismo , Cálcio/metabolismo , Dinoprostona/biossíntese , Fosfolipases A2 do Grupo IV/metabolismo , Animais , Antígenos CD36/genética , Células CHO , Cricetinae , Cricetulus , Ciclo-Oxigenase 2/biossíntese , Ciclo-Oxigenase 2/genética , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/genética , Inibidores Enzimáticos/farmacologia , Feminino , Fosfolipases A2 do Grupo IV/genética , Humanos , Inflamação/genética , Inflamação/metabolismo , Camundongos , Camundongos Knockout , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Tapsigargina/farmacologia , Uridina Trifosfato/farmacologiaRESUMO
Phospholipases are critical enzyme mediators participating in many aspects of cellular function through modulating the generation of lipid 2nd messengers, membrane physical properties, and cellular bioenergetics. Here, we demonstrate that mice null for calcium-independent phospholipase A(2)γ (iPLA(2)γ(-/-)) are completely resistant to high fat diet-induced weight gain, adipocyte hypertrophy, hyperinsulinemia, and insulin resistance, which occur in iPLA(2)γ(+/+) mice after high fat feeding. Notably, iPLA(2)γ(-/-) mice were lean, demonstrated abdominal lipodystrophy, and remained insulin-sensitive despite having a marked impairment in glucose-stimulated insulin secretion after high fat feeding. Respirometry of adipocyte explants from iPLA(2)γ(-/-) mice identified increased rates of oxidation of multiple different substrates in comparison with adipocyte explants from wild-type littermates. Shotgun lipidomics of adipose tissue from wild-type mice demonstrated the anticipated 2-fold increase in triglyceride content after high fat feeding. In sharp contrast, the adipocyte triglyceride content was identical in iPLA(2)γ(-/-) mice fed either a standard diet or a high fat diet. Respirometry of skeletal muscle mitochondria from iPLA(2)γ(-/-) mice demonstrated marked decreases in state 3 respiration using multiple substrates whose metabolism was uncoupled from ATP production. Shotgun lipidomics of skeletal muscle revealed a decreased content of cardiolipin with an altered molecular species composition thereby identifying the mechanism underlying mitochondrial uncoupling in the iPLA(2)γ(-/-) mouse. Collectively, these results identify iPLA(2)γ as an obligatory upstream enzyme that is necessary for efficient electron transport chain coupling and energy production through its participation in the alterations of cellular bioenergetics that promote the development of the metabolic syndrome.
Assuntos
Adipócitos/metabolismo , Cálcio/metabolismo , Gorduras na Dieta/administração & dosagem , Ácidos Graxos/química , Fosfolipases A2 do Grupo IV/fisiologia , Resistência à Insulina , Mitocôndrias Musculares/fisiologia , Obesidade/prevenção & controle , Animais , Western Blotting , Composição Corporal , Metabolismo Energético , Ácidos Graxos/metabolismo , Insulina/metabolismo , Metabolismo dos Lipídeos , Masculino , Camundongos , Camundongos Knockout , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Oxirredução , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Triglicerídeos/metabolismoRESUMO
Genetic ablation of calcium-independent phospholipase A(2)gamma (iPLA(2)gamma) results in profound alterations in hippocampal phospholipid metabolism and mitochondrial phospholipid homeostasis resulting in enlarged and degenerating mitochondria leading to autophagy and cognitive dysfunction. Shotgun lipidomics demonstrated multiple alterations in hippocampal lipid metabolism in iPLA(2)gamma(-/-) mice including: 1) a markedly elevated hippocampal cardiolipin content with an altered molecular species composition characterized by a shift to shorter chain length molecular species; 2) alterations in both choline and ethanolamine glycerophospholipids, including a decreased plasmenylethanolamine content; 3) increased oxidized phosphatidylethanolamine molecular species; and 4) an increased content of ceramides. Electron microscopic examination demonstrated the presence of enlarged heteromorphic lamellar structures undergoing degeneration accompanied by the presence of ubiquitin positive spheroid inclusion bodies. Purification of these enlarged heteromorphic lamellar structures by buoyant density centrifugation and subsequent SDS-PAGE and proteomics identified them as degenerating mitochondria. Collectively, these results identify the obligatory role of iPLA(2)gamma in neuronal mitochondrial lipid metabolism and membrane structure demonstrating that iPLA(2)gamma loss of function results in a mitochondrial neurodegenerative disorder characterized by degenerating mitochondria, autophagy, and cognitive dysfunction.
Assuntos
Autofagia , Cardiolipinas/metabolismo , Transtornos Cognitivos/enzimologia , Hipocampo/enzimologia , Metabolismo dos Lipídeos , Mitocôndrias/enzimologia , Fosfolipases A2 Independentes de Cálcio/metabolismo , Animais , Cardiolipinas/genética , Transtornos Cognitivos/genética , Transtornos Cognitivos/patologia , Hipocampo/ultraestrutura , Camundongos , Camundongos Knockout , Mitocôndrias/ultraestrutura , Membranas Mitocondriais/enzimologia , Membranas Mitocondriais/ultraestrutura , Fosfolipases A2 Independentes de Cálcio/genéticaRESUMO
Herein, we employ the unique chemical properties of the quaternary amine present in thiocholine (2-mercapto-N,N,N-trimethyl-ethanaminium) in conjunction with alkaline beta-elimination and Michael addition (BEMA) reactions for the specific detection, identification, and quantitation of phosphorylated serine/threonine containing peptides. Through replacement of the phosphate with thiocholine, the negative charge on the phosphopeptide is switched to a quaternary amine containing a permanent positive charge. This strategy resulted in a 100-fold increase in ionization sensitivity during ESI (sub-500 amol/microL detection limit) accompanied by a markedly enhanced production of informative peptidic fragment ions during CID that dramatically increase sequence coverage. Moreover, the definitive localization of phosphorylated residues is greatly facilitated through the generation of diagnostic triads of fragmentation ions resulting from peptide bond cleavage and further neutral loss of either trimethylamine (-59 Da) or thiocholine thiolate (-119 Da) during collision induced dissociation (CID) in tandem mass spectrometry (MS(2) and MS(3)). Synthesis of stable isotope labeled thiocholine enabled the quantitation of protein phosphorylation with high precision by ratiometric comparisons using heavy and light thiocholine. Collectively, this study demonstrates a sensitive and efficient strategy for mapping of phosphopeptides by BEMA using thiocholine through the production of a diagnostic repertoire of unique fragment ions during liquid chromatography-tandem mass spectrometry (LC-MS(2)/MS(3)) analyses, facilitating phosphosite identification and quantitative phosphoproteomics.