Richard W. Gross

Matrix-assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometric Analysis of Cellular Glycerophospholipids Enabled by Multiplexed Solvent Dependent Analyte-Matrix Interactions

A matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) based approach was developed for the rapid analyses of cellular glycerophospholipids. Through multiplexed solvent-enabled optimization of analyte-matrix interactions during the crystallization process, over a 30-fold increase in S/N was achieved using 9-aminoacridine as the matrix. The linearity of response (r(2) = 0.99) and dynamic range of this method (over 2 orders of magnitude) were excellent. Moreover, through multiplexing ionization conditions by generating suites of different analyte-matrix interactions in the absence or presence of different alkali metal cations in the matrix, discrete lipid classes were highly and selectively ionized under different conditions resulting in the de facto resolution of lipid classes without chromatography. The resultant decreases in spectral complexity facilitated tandem mass spectrometric analysis through high energy fragmentation of lithiated molecular ions that typically resulted in informative fragment ions. Anionic phospholipids were also detected as singly negatively charged species that could be fragmented using MALDI tandem mass spectrometry leading to structural assignments. Collectively, these results identify a rapid, sensitive, and highly informative MALDI-TOF MS approach for analysis of cellular glycerophospholipids directly from extracts of mammalian tissues without the need for prior chromatographic separation.

Genetic Ablation of Calcium-independent Phospholipase A2γ Leads to Alterations in Mitochondrial Lipid Metabolism and Function Resulting in a Deficient Mitochondrial Bioenergetic Phenotype

Previously, we identified a novel calcium-independent phospholipase, designated calcium-independent phospholipase A2 γ (iPLA2γ), which possesses dual mitochondrial and peroxisomal subcellular localization signals. To identify the roles of iPLA2γ in cellular bioenergetics, we generated mice null for the iPLA2γ gene by eliminating the active site of the enzyme through homologous recombination. Mice null for iPLA2γ display multiple bioenergetic dysfunctional phenotypes, including 1) growth retardation, 2) cold intolerance, 3) reduced exercise endurance, 4) greatly increased mortality from cardiac stress after transverse aortic constriction, 5) abnormal mitochondrial function with a 65% decrease in ascorbate-induced Complex IV-mediated oxygen consumption, and 6) a reduction in myocardial cardiolipin content accompanied by an altered cardiolipin molecular species composition. We conclude that iPLA2γ is essential for maintaining efficient bioenergetic mitochondrial function through tailoring mitochondrial membrane lipid metabolism and composition.

Lipidomics at the Interface of Structure and Function in Systems Biology

Cells, tissues, and biological fluids contain a diverse repertoire of many tens of thousands of structurally distinct lipids that play multiple roles in cellular signaling, bioenergetics, and membrane structure and function. In an era where lipid-related disease states predominate, lipidomics has assumed a prominent role in systems biology through its unique ability to directly identify functional alterations in multiple lipid metabolic and signaling networks. The development of shotgun lipidomics has led to the facile accrual of high density information on alterations in the lipidome mediating physiologic cellular adaptation during health and pathologic alterations during disease. Through both targeted and nontargeted investigations, lipidomics has already revealed the chemical mechanisms underlying many lipid-related disease states.

Genetic Ablation of Calcium-independent Phospholipase A2γ Prevents Obesity and Insulin Resistance during High Fat Feeding by Mitochondrial Uncoupling and Increased Adipocyte Fatty Acid Oxidation

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 A2γ (iPLA2γ−/−) are completely resistant to high fat diet-induced weight gain, adipocyte hypertrophy, hyperinsulinemia, and insulin resistance, which occur in iPLA2γ+/+ mice after high fat feeding. Notably, iPLA2γ−/− 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 iPLA2γ−/− 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 iPLA2γ−/− mice fed either a standard diet or a high fat diet. Respirometry of skeletal muscle mitochondria from iPLA2γ−/− 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 iPLA2γ−/− mouse. Collectively, these results identify iPLA2γ 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.

Dysfunctional cardiac mitochondrial bioenergetic, lipidomic, and signaling in a murine model of Barth syndrome

Barth syndrome is a complex metabolic disorder caused by mutations in the mitochondrial transacylase tafazzin. Recently, an inducible tafazzin shRNA knockdown mouse model was generated to deconvolute the complex bioenergetic phenotype of this disease. To investigate the underlying cause of hemodynamic dysfunction in Barth syndrome, we interrogated the cardiac structural and signaling lipidome of this mouse model as well as its myocardial bioenergetic phenotype. A decrease in the distribution of cardiolipin molecular species and robust increases in monolysocardiolipin and dilysocardiolipin were demonstrated. Additionally, the contents of choline and ethanolamine glycerophospholipid molecular species containing precursors for lipid signaling at the sn-2 position were altered. Lipidomic analyses revealed specific dysregulation of HETEs and prostanoids, as well as oxidized linoleic and docosahexaenoic metabolites. Bioenergetic interrogation uncovered differential substrate utilization as well as decreases in Complex III and V activities. Transgenic expression of cardiolipin synthase or iPLA2γ ablation in tafazzin-deficient mice did not rescue the observed phenotype. These results underscore the complex nature of alterations in cardiolipin metabolism mediated by tafazzin loss of function. Collectively, we identified specific lipidomic, bioenergetic, and signaling alterations in a murine model that parallel those of Barth syndrome thereby providing novel insights into the pathophysiology of this debilitating disease.

Reversed-phase high-performance liquid chromatographic separation of molecular species of alkyl ether, vinyl ether, and monoacyl lysophospholipids

An isocratic reversed-phase high-performance liquid chromatographic method was developed which resolved individual molecular species of choline and ethanolamine lysophospholipids utilizing a C13 bonded porous silica stationary phase with a mobile phase comprised of methanol--water--acetonitrile (57:23:20) containing 20 mM choline chloride. Solute retention was primarily determined by hydrophobic interactions with the stationary phase permitting separation of individual molecular species of lysophospholipids according to the composition of the aliphatic chain and the nature of its covalent attachment to the sn-1 hydroxyl group. The elution profile of unsaturated monoacyllysophospholipid or lysoplasmalogen molecular species was readily obtained by measuring UV absorbance at 203 nm. Identification of column eluates containing saturated monoacyl and alkyl ether lysophospholipids was possible utilizing relative retention factors that were obtained for the majority of molecular species present in animal tissues.

Identification of hepatic peroxisomal phospholipase A2 and characterization of arachidonic acid-containing choline glycerophospholipids in hepatic peroxisomes

Recently, a sequence encoding a novel mammalian calcium‐independent phospholipase A2 (iPLA2γ) was identified in the human genome and subsequently cloned and expressed in Sf9 insect cells. Unexpectedly, expression studies in recombinant systems demonstrated the usage of multiple translation initiation codons resulting in different polypeptides. Herein, we demonstrate that hepatic iPLA2γ is localized to rat liver peroxisomes, possesses a molecular mass of 63 kDa and that peroxisomal membranes are highly enriched in arachidonic acid‐containing phospholipids. Collectively, these results provide the first demonstration of iPLA2γ in mammalian tissue and suggest the possibility that iPLA2γ can contribute to lipid second messenger generation by hydrolysis of peroxisomal arachidonic acid‐containing phospholipids.

Reversible High Affinity Inhibition of Phosphofructokinase-1 by Acyl-CoA A MECHANISM INTEGRATING GLYCOLYTIC FLUX WITH LIPID METABOLISM

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 Ki ∼1 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 [1-14C]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.

Identification and quantitation of fatty acid double bond positional isomers: a shotgun lipidomics approach using charge-switch derivatization.

The specific locations of double bonds in mammalian lipids have profound effects on biological membrane structure, dynamics and lipid second messenger production. Herein, we describe a shotgun lipidomics approach that exploits charge-switch derivatization with N-(4-aminomethylphenyl) pyridinium (AMPP) and tandem mass spectrometry for identification and quantification of fatty acid double bond positional isomers. Through charge-switch derivatization of fatty acids followed by positive-ion mode ionization and fragmentation analysis, a marked increase in analytic sensitivity (low fmol/μL) and the identification of double bond positional isomers can be obtained. Specifically, the locations of proximal double bonds in AMPP-derivatized fatty acids are identified by diagnostic fragment ions resulting from the markedly reduced 1,4-hydrogen elimination from the proximal olefinic carbons. Additional fragmentation patterns resulting from allylic cleavages further substantiated the double bond position assignments. Moreover, quantification of fatty acid double bond positional isomers is achieved by the linear relationship of the normalized intensities of characteristic fragment ions vs the isomeric compositions of discrete fatty acid positional isomers. The application of this approach for the analysis of fatty acids in human serum demonstrated the existence of two double bond isomers of linolenic acid (i.e., Δ(6,9,12) 18:3, γ-linolenic acid (GLA), and Δ(9,12,15) 18:3, α-linolenic acid (ALA)). Remarkably, the isomeric ratio of GLA vs ALA esterified in neutral lipids was 3-fold higher than the ratio of their nonesterified moieties. Through this developed method, previously underestimated or unidentified alterations in fatty acid structural isomers can be determined facilitating the identification of novel biomarkers and maladaptive alterations in lipid metabolism during disease.

Myocardial regulation of lipidomic flux by cardiolipin synthase: Setting the beat for bioenergetic efficiency

Background: Maintenance of cardiolipin molecular speciation by remodeling directly regulates mitochondrial bioenergetic efficiency. Results: Transgenic expression of cardiolipin synthase accelerates cardiolipin remodeling, improves mitochondrial function, modulates mitochondrial signaling, and attenuates mitochondrial dysfunction during diabetes. Conclusion: Cardiolipin synthase integrates multiple aspects of mitochondrial bioenergetic and signaling functions. Significance: Cardiolipin synthase expression attenuates mitochondrial dysfunction in diabetic myocardium. 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.