Etomoxir-carnitine, a novel pharmaco-metabolite of etomoxir, inhibits phospholipases A2 and mitochondrial respiration.

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 ETO to etomoxir-carnitine (ETO-carnitine) prior to nearly complete ETO-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 ETO, ETO-carnitine, that is generated by CPT1 in mitochondria and likely impacts multiple downstream (non-CPT1 related) enzymes and processes in multiple subcellular compartments.

The foundations and development of lipidomics.

For over a century, the importance of lipid metabolism in biology was recognized but difficult to mechanistically understand due to the lack of sensitive and robust technologies for identification and quantification of lipid molecular species. The enabling technological breakthroughs emerged in the 1980s with the development of soft ionization methods (Electrospray Ionization and Matrix Assisted Laser Desorption/Ionization) that could identify and quantify intact individual lipid molecular species. These soft ionization technologies laid the foundations for what was to be later named the field of lipidomics. Further innovative advances in multistage fragmentation, dramatic improvements in resolution and mass accuracy, and multiplexed sample analysis fueled the early growth of lipidomics through the early 1990s. The field exponentially grew through the use of a variety of strategic approaches, which included direct infusion, chromatographic separation, and charge-switch derivatization, which facilitated access to the low abundance species of the lipidome. In this Thematic Review, we provide a broad perspective of the foundations, enabling advances, and predicted future directions of growth of the lipidomics field.

High-fat diet activates liver iPLA2γ generating eicosanoids that mediate metabolic stress.

High-fat (HF) diet-induced obesity precipitates multiple metabolic disorders including insulin resistance, glucose intolerance, oxidative stress, and inflammation, resulting in the initiation of cell death programs. Previously, we demonstrated murine germline knockout of calcium-independent phospholipase A2γ (iPLA2γ) prevented HF diet-induced weight gain, attenuated insulin resistance, and decreased mitochondrial permeability transition pore (mPTP) opening leading to alterations in bioenergetics. To gain insight into the specific roles of hepatic iPLA2γ in mitochondrial function and cell death under metabolic stress, we generated a hepatocyte-specific iPLA2γ-knockout (HEPiPLA2γKO). Using this model, we compared the effects of an HF diet on wild-type versus HEPiPLA2γKO mice in eicosanoid production and mitochondrial bioenergetics. HEPiPLA2γKO mice exhibited higher glucose clearance rates than WT controls. Importantly, HF-diet induced the accumulation of 12-hydroxyeicosatetraenoic acid (12-HETE) in WT liver which was decreased in HEPiPLA2γKO. Furthermore, HF-feeding markedly increased Ca2+ sensitivity and resistance to ADP-mediated inhibition of mPTP opening in WT mice. In contrast, ablation of iPLA2γ prevented the HF-induced hypersensitivity of mPTP opening to calcium and maintained ADP-mediated resistance to mPTP opening. Respirometry revealed that ADP-stimulated mitochondrial respiration was significantly reduced by exogenous 12-HETE. Finally, HEPiPLA2γKO hepatocytes were resistant to calcium ionophore-induced lipoxygenase-mediated lactate dehydrogenase release. Collectively, these results demonstrate that an HF diet increases iPLA2γ-mediated hepatic 12-HETE production leading to mitochondrial dysfunction and hepatic cell death.

A functional role for eicosanoid-lysophospholipids in activating monocyte signaling.

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.

12-LOX catalyzes the oxidation of 2-arachidonoyl-lysolipids in platelets generating eicosanoid-lysolipids that are attenuated by iPLA2γ knockout.

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.

Identifying off-target effects of etomoxir reveals that carnitine palmitoyltransferase I is essential for cancer cell proliferation independent of ß-oxidation.

It has been suggested that some cancer cells rely upon fatty acid oxidation (FAO) for energy. Here we show that when FAO was reduced approximately 90% by pharmacological inhibition of carnitine palmitoyltransferase I (CPT1) with low concentrations of etomoxir, the proliferation rate of various cancer cells was unaffected. Efforts to pharmacologically inhibit FAO more than 90% revealed that high concentrations of etomoxir (200 µM) have an off-target effect of inhibiting complex I of the electron transport chain. Surprisingly, however, when FAO was reduced further by genetic knockdown of CPT1, the proliferation rate of these same cells decreased nearly 2-fold and could not be restored by acetate or octanoic acid supplementation. Moreover, CPT1 knockdowns had altered mitochondrial morphology and impaired mitochondrial coupling, whereas cells in which CPT1 had been approximately 90% inhibited by etomoxir did not. Lipidomic profiling of mitochondria isolated from CPT1 knockdowns showed depleted concentrations of complex structural and signaling lipids. Additionally, expression of a catalytically dead CPT1 in CPT1 knockdowns did not restore mitochondrial coupling. Taken together, these results suggest that transport of at least some long-chain fatty acids into the mitochondria by CPT1 may be required for anabolic processes that support healthy mitochondrial function and cancer cell proliferation independent of FAO.

Synthesis of oxidized phospholipids by sn-1 acyltransferase using 2-15-HETE lysophospholipids.

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.

Caveolin-1 regulates lipid droplet metabolism in endothelial cells via autocrine prostacyclin-stimulated, cAMP-mediated lipolysis.

Lipid droplets (LD) are dynamic organelles involved in intracellular lipid metabolism in almost all eukaryotic cells, and LD-associated proteins tightly regulate their dynamics. One LD coat protein is caveolin-1 (Cav-1), an essential component for caveola assembly in highly differentiated cells, including adipocytes, smooth muscle cells, and endothelial cells (EC). However, the role of Cav-1 in LD dynamics is unclear. Here we report that EC lacking Cav-1 exhibit impaired LD formation. The decreased LD formation is due to enhanced lipolysis and not caused by reduced triglyceride synthesis or fatty acid uptake. Mechanistically, the absence of Cav-1 increased cAMP/PKA signaling in EC, as indicated by elevated phosphorylation of hormone-sensitive lipase and increased lipolysis. Unexpectedly, we also observed enhanced autocrine production of prostaglandin I2 (PGI2, also called prostacyclin) in Cav-1 KO EC, and this PGI2 increase appeared to stimulate cAMP/PKA pathways, contributing to the enhanced lipolysis in Cav-1 KO cells. Our results reveal an unanticipated role of Cav-1 in regulating lipolysis in non-adipose tissue, indicating that Cav-1 is required for LD metabolism in EC and that it regulates cAMP-dependent lipolysis in part via the autocrine production of PGI2.

Cytochrome c is an oxidative stress-activated plasmalogenase that cleaves plasmenylcholine and plasmenylethanolamine at the sn-1 vinyl ether linkage.

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.

Heart failure-induced activation of phospholipase iPLA2γ generates hydroxyeicosatetraenoic acids opening the mitochondrial permeability transition pore.

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.

The phospholipase iPLA2γ is a major mediator releasing oxidized aliphatic chains from cardiolipin, integrating mitochondrial bioenergetics and signaling.

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.

Lactate metabolism is associated with mammalian mitochondria.

It is well established that lactate secreted by fermenting cells can be oxidized or used as a gluconeogenic substrate by other cells and tissues. It is generally assumed, however, that within the fermenting cell itself, lactate is produced to replenish NAD+ and then is secreted. Here we explore the possibility that cytosolic lactate is metabolized by the mitochondria of fermenting mammalian cells. We found that fermenting HeLa and H460 cells utilize exogenous lactate carbon to synthesize a large percentage of their lipids. Using high-resolution mass spectrometry, we found that both 13C and 2-2H labels from enriched lactate enter the mitochondria. The lactate dehydrogenase (LDH) inhibitor oxamate decreased respiration of isolated mitochondria incubated in lactate, but not of isolated mitochondria incubated in pyruvate. Additionally, transmission electron microscopy (TEM) showed that LDHB localizes to the mitochondria. Taken together, our results demonstrate a link between lactate metabolism and the mitochondria of fermenting mammalian cells.

Genetic Ablation of Calcium-independent Phospholipase A2γ Induces Glomerular Injury in Mice.

Glomerular visceral epithelial cells (podocytes) play a critical role in the maintenance of glomerular permselectivity. Podocyte injury, manifesting as proteinuria, is the cause of many glomerular diseases. We reported previously that calcium-independent phospholipase A2γ (iPLA2γ) is cytoprotective against complement-mediated glomerular epithelial cell injury. Studies in iPLA2γ KO mice have demonstrated an important role for iPLA2γ in mitochondrial lipid turnover, membrane structure, and metabolism. The aim of the present study was to employ iPLA2γ KO mice to better understand the role of iPLA2γ in normal glomerular and podocyte function as well as in glomerular injury. We show that deletion of iPLA2γ did not cause detectable albuminuria; however, it resulted in mitochondrial structural abnormalities and enhanced autophagy in podocytes as well as loss of podocytes in aging KO mice. Moreover, after induction of anti-glomerular basement membrane nephritis in young mice, iPLA2γ KO mice exhibited significantly increased levels of albuminuria, podocyte injury, and loss of podocytes compared with wild type. Thus, iPLA2γ has a protective functional role in the normal glomerulus and in glomerulonephritis. Understanding the role of iPLA2γ in glomerular pathophysiology provides opportunities for the development of novel therapeutic approaches to glomerular injury and proteinuria.

Cardiac Myocyte-specific Knock-out of Calcium-independent Phospholipase A2γ (iPLA2γ) Decreases Oxidized Fatty Acids during Ischemia/Reperfusion and Reduces Infarct Size.

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.

The evolution of lipidomics through space and time.

Although the foundations of mass spectrometry-based lipidomics have been practiced for over 30 years, recent technological advances in ionization modalities in conjunction with robust increases in mass accuracy and resolution have greatly accelerated the emergence, growth and importance of the field of lipidomics. Moreover, advances in the separation sciences, bioinformatic strategies and the availability of robust databases have been synergistically integrated into modern lipidomic technologies leading to unprecedented improvements in the depth, penetrance and precision of lipidomic analyses and identification of their biological and mechanistic significance. The purpose of this "opinion" article is to briefly review the evolution of lipidomics, critique the platforms that have evolved and identify areas that are likely to emerge in the years to come. Through seamlessly integrating a rich repertoire of mass spectrometric, chemical and bioinformatic strategies, the chemical identities and quantities of tens of thousands to hundreds of thousands of different lipid molecular species and their metabolic alterations during physiologic or pathophysiologic perturbations can be obtained. Thus, the field of lipidomics which already has a distinguished history of exciting new discoveries in many disease states holds unparalleled potential to identify the pleiotropic roles of lipids in health and disease at the chemical level. This article is part of a Special Issue entitled: BBALIP_Lipidomics Opinion Articles edited by Sepp Kohlwein.

Shotgun Lipidomics Approach to Stabilize the Regiospecificity of Monoglycerides Using a Facile Low-Temperature Derivatization Enabling Their Definitive Identification and Quantitation.

Monoglycerides play a central role in lipid metabolism and are important signaling metabolites. Quantitative analysis of monoglyceride molecular species has remained challenging due to rapid isomerization via α-hydroxy acyl migration. Herein, we describe a shotgun lipidomics approach that utilizes a single-phase methyl tert-butyl ether extraction to minimize acyl migration, a facile low temperature diacetyl derivatization to stabilize regiospecificity, and tandem mass spectrometric analysis to identify and quantify regioisomers of monoglycerides in biological samples. The rapid and robust diacetyl derivatization at low temperatures (e.g., -20 °C, 30 min) prevents postextraction acyl migration and preserves regiospecificity of monoglyceride structural isomers. Furthermore, ionization of ammonium adducts of diacetyl monoglyceride derivatives in positive-ion mode markedly increases analytic sensitivity (low fmol/µL). Critically, diacetyl derivatization enables the differentiation of discrete monoglyceride regioisomers without chromatography through their distinct signature fragmentation patterns during collision induced dissociation. The application of this approach in the analysis of monoglycerides in multiple biologic tissues demonstrated diverse profiles of molecular species. Remarkably, the regiospecificity of individual monoglyceride molecular species is also diverse from tissue to tissue. Collectively, this developed approach enables the profiling, identification and quantitation of monoglyceride regioisomers directly from tissue extracts.

Loss of function variants in human PNPLA8 encoding calcium-independent phospholipase A2 γ recapitulate the mitochondriopathy of the homologous null mouse.

Mitochondriopathies are a group of clinically heterogeneous genetic diseases caused by defects in mitochondrial metabolism, bioenergetic efficiency, and/or signaling functions. The large majority of proteins involved in mitochondrial function are encoded by nuclear genes, with many yet to be associated with human disease. We performed exome sequencing on a young girl with a suspected mitochondrial myopathy that manifested as progressive muscle weakness, hypotonia, seizures, poor weight gain, and lactic acidosis. She was compound heterozygous for two frameshift mutations, p.Asn112HisfsX29 and p.Leu659AlafsX4, in the PNPLA8 gene, which encodes mitochondrial calcium-independent phospholipase A2 γ (iPLA2 γ). Western blot analysis of affected muscle displayed the absence of PNPLA8 protein. iPLA2 s are critical mediators of a variety of cellular processes including growth, metabolism, and lipid second messenger generation, exerting their functions through catalyzing the cleavage of the acyl groups in glycerophospholipids. The clinical presentation, muscle histology and the mitochondrial ultrastructural abnormalities of this proband are highly reminiscent of Pnpla8 null mice. Although other iPLA2 -related diseases have been identified, namely, infantile neuroaxonal dystrophy and neutral lipid storage disease with myopathy, this is the first report of PNPLA8-related disease in a human. We suggest PNPLA8 join the increasing list of human genes involved in lipid metabolism associated with neuromuscular diseases due to mitochondrial dysfunction.

Liver-specific loss of lipin-1-mediated phosphatidic acid phosphatase activity does not mitigate intrahepatic TG accumulation in mice.

Lipin proteins (lipin 1, 2, and 3) regulate glycerolipid homeostasis by acting as phosphatidic acid phosphohydrolase (PAP) enzymes in the TG synthesis pathway and by regulating DNA-bound transcription factors to control gene transcription. Hepatic PAP activity could contribute to hepatic fat accumulation in response to physiological and pathophysiological stimuli. To examine the role of lipin 1 in regulating hepatic lipid metabolism, we generated mice that are deficient in lipin-1-encoded PAP activity in a liver-specific manner (Alb-Lpin1(-/-) mice). This allele of lipin 1 was still able to transcriptionally regulate the expression of its target genes encoding fatty acid oxidation enzymes, and the expression of these genes was not affected in Alb-Lpin1(-/-) mouse liver. Hepatic PAP activity was significantly reduced in mice with liver-specific lipin 1 deficiency. However, hepatocytes from Alb-Lpin1(-/-) mice had normal rates of TG synthesis, and steady-state hepatic TG levels were unaffected under fed and fasted conditions. Furthermore, Alb-Lpin1(-/-) mice were not protected from intrahepatic accumulation of diacylglycerol and TG after chronic feeding of a diet rich in fat and fructose. Collectively, these data demonstrate that marked deficits in hepatic PAP activity do not impair TG synthesis and accumulation under acute or chronic conditions of lipid overload.

Multidimensional mass spectrometry-based shotgun lipidomics analysis of vinyl ether diglycerides.

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.

Mechanism-based inhibition of iPLA2ß demonstrates a highly reactive cysteine residue (C651) that interacts with the active site: mass spectrometric elucidation of the mechanisms underlying inhibition.

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.