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Find video protocols related to scientific articles indexed in Pubmed.
The functional characterization of long noncoding RNA SPRY4-IT1 in human melanoma cells.
Oncotarget
PUBLISHED: 10-27-2014
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Expression of the long noncoding RNA (lncRNA) SPRY4-IT1 is low in normal human melanocytes but high in melanoma cells. siRNA knockdown of SPRY4-IT1 blocks melanoma cell invasion and proliferation, and increases apoptosis. To investigate its function further, we affinity purified SPRY4-IT1 from melanoma cells and used mass spectrometry to identify the protein lipin 2, an enzyme that converts phosphatidate to diacylglycerol (DAG), as a major binding partner. SPRY4-IT1 knockdown increases the accumulation of lipin2 protein and upregulate the expression of diacylglycerol O-acyltransferase 2 (DGAT2) an enzyme involved in the conversion of DAG to triacylglycerol (TAG). When SPRY4-IT1 knockdown and control melanoma cells were subjected to shotgun lipidomics, an MS-based assay that permits the quantification of changes in the cellular lipid profile, we found that SPRY4-IT1 knockdown induced significant changes in a number of lipid species, including increased acyl carnitine, fatty acyl chains, and triacylglycerol (TAG). Together, these results suggest the possibility that SPRY4-IT1 knockdown may induce apoptosis via lipin 2-mediated alterations in lipid metabolism leading to cellular lipotoxicity.
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Multidimensional mass spectrometry-based shotgun lipidomics.
Methods Mol. Biol.
PUBLISHED: 10-02-2014
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Multidimensional mass spectrometry-based shotgun lipidomics (MDMS-SL) has become a foundational analytical technology platform among current lipidomics practices due to its high efficiency, sensitivity, and reproducibility, as well as its broad coverage. This platform has been broadly used to determine the altered content and/or composition of lipid classes, subclasses, and individual molecular species induced by diseases, genetic manipulations, drug treatments, and aging, among others. Herein, we briefly discuss the principles underlying this technology and present a protocol for routine analysis of many of the lipid classes and subclasses covered by MDMS-SL directly from lipid extracts of biological samples. In particular, lipid sample preparation from a variety of biological materials, which is one of the key components of MDMS-SL, is described in detail. The protocol for mass spectrometric analysis can readily be expanded for analysis of other lipid classes not mentioned as long as appropriate sample preparation is conducted, and should aid researchers in the field to better understand and manage the technology for analysis of cellular lipidomes.
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Acyl-CoA thioesterase-2 facilitates mitochondrial fatty acid oxidation in the liver.
J. Lipid Res.
PUBLISHED: 08-11-2014
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Acyl-CoA thioesterase-2 (Acot2) localizes to the mitochondrial matrix and hydrolyses long-chain fatty acyl-CoA into free fatty acid (FA) and CoASH. Acot2 is expressed in highly oxidative tissues and is poised to modulate mitochondrial fatty acid oxidation (FAO), yet its biological role is unknown. Using a model of adenoviral Acot2 overexpression in mouse liver (Ad-Acot2), we show that Acot2 increases the utilization of FA substrate during the daytime in ad-libitum fed mice, but the nighttime switch to carbohydrate oxidation is similar to control mice. In further support of elevated FAO in Acot2 liver, daytime serum ketones were higher in Ad-Acot2 mice, and overnight fasting lead to minimal hepatic steatosis as compared to control mice. In liver mitochondria from Ad-Acot2 mice, phosphorylating O2 consumption was higher with lipid but not with non-lipid substrate. This increase depended on whether FA could be activated on the outer mitochondrial membrane, suggesting that the FA released by Acot2 could be effluxed from mitochondria then taken back up again for oxidation. This circuit would prevent the build-up of inhibitory long-chain fatty acyl-CoA esters. Altogether, our findings indicate that Acot2 can enhance FAO, possibly by mitigating the accumulation of FAO intermediates within the mitochondrial matrix.
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Accurate mass searching of individual lipid species candidates from high-resolution mass spectra for shotgun lipidomics.
Rapid Commun. Mass Spectrom.
PUBLISHED: 07-07-2014
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With the increased mass accuracy and resolution in commercialized mass spectrometers, new developments on shotgun lipidomics could be expected with increased speed, dynamic range, and coverage over lipid species and classes. However, we found that the major issue by using high mass accuracy/resolution instruments to search lipid species is the partial overlap between the two-(13) C-atom-containing isotopologue of a species M (i.e., M+2 isotopologue) and the ion of a species with one less double bond than M (assigned here as L). This partial overlap alone could cause a mass shift of the species L to the lower mass end up to 12 ppm around m/z 750 as well as significant peak broadening.
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Synthesis and biological evaluation of antimetastatic agents predicated upon dihydromotuporamine C and its carbocyclic derivatives.
J. Med. Chem.
PUBLISHED: 05-02-2014
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The motuporamines isolated from the sea sponge Xestospongia exigua are of biological interest because of their unique antimigration and antiangiogenic properties. Key bioactive features were found to be a saturated 15-membered heterocycle and a norspermidine motif. This paper describes new analogues that modulate the cytotoxicity of this compound class and have enhanced antimigration properties. By movement of the polyamine chain outside the ring, new carbocycles were discovered that doubled the antimigration potency and reduced compound toxicity by 133-fold. Mice injected with metastatic human L3.6pl pancreatic cancer cells demonstrated significant reduction in liver metastases when treated with N(1)-(3-aminopropyl)-N(3)-(cyclopentadecylmethyl)propane-1,3-diamine compared with dihydromotuporamine C. Significant changes in specific ceramide populations (N16:0 and N22:1) were noted in L3.6pl cells treated with dihydromotuporamine C but not for the cyclopentadecylmethylnorspermidine derivative, which had lower toxicity. Both compounds gave increased levels of specific low molecular weight sphingomyelins, suggesting that they may act upon sphingomyelin processing enzymes.
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Mitochondria-targeted antioxidant prevents cardiac dysfunction induced by tafazzin gene knockdown in cardiac myocytes.
Oxid Med Cell Longev
PUBLISHED: 03-26-2014
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Tafazzin, a mitochondrial acyltransferase, plays an important role in cardiolipin side chain remodeling. Previous studies have shown that dysfunction of tafazzin reduces cardiolipin content, impairs mitochondrial function, and causes dilated cardiomyopathy in Barth syndrome. Reactive oxygen species (ROS) have been implicated in the development of cardiomyopathy and are also the obligated byproducts of mitochondria. We hypothesized that tafazzin knockdown increases ROS production from mitochondria, and a mitochondria-targeted antioxidant prevents tafazzin knockdown induced mitochondrial and cardiac dysfunction. We employed cardiac myocytes transduced with an adenovirus containing tafazzin shRNA as a model to investigate the effects of the mitochondrial antioxidant, mito-Tempo. Knocking down tafazzin decreased steady state levels of cardiolipin and increased mitochondrial ROS. Treatment of cardiac myocytes with mito-Tempo normalized tafazzin knockdown enhanced mitochondrial ROS production and cellular ATP decline. Mito-Tempo also significantly abrogated tafazzin knockdown induced cardiac hypertrophy, contractile dysfunction, and cell death. We conclude that mitochondria-targeted antioxidant prevents cardiac dysfunction induced by tafazzin gene knockdown in cardiac myocytes and suggest mito-Tempo as a potential therapeutic for Barth syndrome and other dilated cardiomyopathies resulting from mitochondrial oxidative stress.
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Circadian clocks and feeding time regulate the oscillations and levels of hepatic triglycerides.
Cell Metab.
PUBLISHED: 02-11-2014
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Circadian clocks play a major role in orchestrating daily physiology, and their disruption can evoke metabolic diseases such as fatty liver and obesity. To study the role of circadian clocks in lipid homeostasis, we performed an extensive lipidomic analysis of liver tissues from wild-type and clock-disrupted mice either fed ad libitum or night fed. To our surprise, a similar fraction of lipids (?17%) oscillated in both mouse strains, most notably triglycerides, but with completely different phases. Moreover, several master lipid regulators (e.g., PPAR?) and enzymes involved in triglyceride metabolism retained their circadian expression in clock-disrupted mice. Nighttime restricted feeding shifted the phase of triglyceride accumulation and resulted in ?50% decrease in hepatic triglyceride levels in wild-type mice. Our findings suggest that circadian clocks and feeding time dictate the phase and levels of hepatic triglyceride accumulation; however, oscillations in triglycerides can persist in the absence of a functional clock.
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Macrophage CGI-58 deficiency activates ROS-inflammasome pathway to promote insulin resistance in mice.
Cell Rep
PUBLISHED: 02-04-2014
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Overnutrition activates a proinflammatory program in macrophages to induce insulin resistance (IR), but its molecular mechanisms remain incompletely understood. Here, we show that saturated fatty acid and lipopolysaccharide, two factors implicated in high-fat diet (HFD)-induced IR, suppress macrophage CGI-58 expression. Macrophage-specific CGI-58 knockout (MaKO) in mice aggravates HFD-induced glucose intolerance and IR, which is associated with augmented systemic/tissue inflammation and proinflammatory activation of adipose tissue macrophages. CGI-58-deficient macrophages exhibit mitochondrial dysfunction due to defective peroxisome proliferator-activated receptor (PPAR)? signaling. Consequently, they overproduce reactive oxygen species (ROS) to potentiate secretion of proinflammatory cytokines by activating NLRP3 inflammasome. Anti-ROS treatment or NLRP3 silencing prevents CGI-58-deficient macrophages from oversecreting proinflammatory cytokines and from inducing proinflammatory signaling and IR in the cocultured fat slices. Anti-ROS treatment also prevents exacerbation of inflammation and IR in HFD-fed MaKO mice. Our data thus establish CGI-58 as a suppressor of overnutrition-induced NLRP3 inflammasome activation in macrophages.
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Characterization and quantification of diacylglycerol species in biological extracts after one-step derivatization: a shotgun lipidomics approach.
Anal. Chem.
PUBLISHED: 01-28-2014
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Diacylglycerols (DAGs) are important intermediates of lipid metabolism and cellular signaling. It is well-known that the mass levels of DAG are altered under disease states. Therefore, quantitative analysis of DAGs in biological samples can provide critical information to uncover underlying mechanisms of various cellular functional disorders. Although great efforts on the analysis of individual DAG species have recently been made by utilizing mass spectrometry with or without derivatization, cost-effective and high throughput methodologies for identification and quantification of all DAG species including regioisomers, particularly in an approach of shotgun lipidomics, are still missing. Herein, we described a novel method for directly identifying and quantifying DAG species including regioisomers present in lipid extracts of biological samples after facile one-step derivatization with dimethylglycine based on the principles of multidimensional mass spectrometry-based shotgun lipidomics. The established method provided substantial sensitivity (low limit of quantification at amol/?L), high specificity, and broad linear dynamics range (2500-fold) without matrix effects. By exploiting this novel method, we revealed a 16-fold increase of total DAG mass in the livers of ob/ob mice compared to their wild type controls at 4 months of age (an insulin-resistant state) versus a 5-fold difference between 3 month old mice (with normal insulin). These results demonstrated the importance and power of the method for studying biochemical mechanisms underpinning disease states.
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Alterations in mouse brain lipidome after disruption of CST gene: a lipidomics study.
Mol. Neurobiol.
PUBLISHED: 01-07-2014
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To investigate the effects of a critical enzyme, cerebroside sulfotransferase (CST), involving sulfatide biosynthesis on lipid (particularly sphingolipid) homeostasis, herein, we determined the lipidomes of brain cortex and spinal cord from CST null and heterozygous (CST(-/-) and CST(+/-), respectively) mice in comparison to their wild-type littermates by multi-dimensional mass spectrometry-based shotgun lipidomics. As anticipated, we demonstrated the absence of sulfatide in the tissues from CST(-/-) mice and found that significant reduction of sulfatide mass levels was also present, but in an age-dependent manner, in CST(+/-) mice. Unexpectedly, we revealed that the profiles of sulfatide species in CST(+/-) mice were significantly different from that of littermate controls with an increase in the composition of species containing saturated and hydroxylated fatty acyl chains. Contrary to the changes of sulfatide levels, shotgun lipidomics analysis did not detect significant changes of the mass levels of other lipid classes examined. Taken together, shotgun lipidomics analysis demonstrated anticipated sulfatide mass deficiency in CST defect mouse brain and revealed novel brain lipidome homeostasis in these mice. These results might provide new insights into the role of CST in myelin function.
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A Role for peroxisome proliferator-activated receptor gamma coactivator 1 (PGC-1) in the regulation of cardiac mitochondrial phospholipid biosynthesis.
J. Biol. Chem.
PUBLISHED: 12-11-2013
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The energy demands of the adult mammalian heart are met largely by ATP generated via oxidation of fatty acids (FA) in a high capacity mitochondrial system. Peroxisome proliferator-activated receptor gamma coactivator 1 (PGC-1) ? and ? serve as inducible transcriptional co-regulators of genes involved in mitochondrial biogenesis and metabolism. Whether PGC-1 plays a role in the regulation of mitochondrial structure is unknown. In this study, mice with combined deficiency of PGC-1? and PGC-1? (PGC-1??(-/-)) in adult heart were analyzed. PGC-1??(-/-) hearts exhibited a distinctive mitochondrial cristae-stacking abnormality suggestive of a phospholipid (PL) abnormality as has been described in humans with genetic defects in cardiolipin (CL) synthesis (Barth syndrome). A subset of molecular species, containing n-3 polyunsaturated species in the CL, phosphatidylcholine (PC), and phosphatidylethanolamine (PE) profiles were reduced in PGC-1??-deficient hearts. Gene expression profiling of PGC-1??(-/-) hearts revealed reduced expression of the gene encoding CDP-diacylglycerol synthase 1 (Cds1), an enzyme that catalyzes the proximal step in CL biosynthesis. Cds1 gene promoter-reporter co-transfection experiments and chromatin immunoprecipitation studies demonstrated that PGC-1? co-regulates estrogen-related receptors (ERRs) to activate the transcription of the Cds1 gene. We conclude that the PGC-1/ERR axis coordinately regulates metabolic and membrane structural programs relevant to the maintenance of high capacity mitochondrial function in heart.
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Unremodeled and remodeled cardiolipin are functionally indistinguishable in yeast.
J. Biol. Chem.
PUBLISHED: 11-27-2013
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After biosynthesis, an evolutionarily conserved acyl chain remodeling process generates a final highly homogenous and yet tissue-specific molecular form of the mitochondrial lipid cardiolipin. Hence cardiolipin molecules in different organisms, and even different tissues within the same organism, contain a distinct collection of attached acyl chains. This observation is the basis for the widely accepted paradigm that the acyl chain composition of cardiolipin is matched to the unique mitochondrial demands of a tissue. For this hypothesis to be correct, cardiolipin molecules with different acyl chain compositions should have distinct functional capacities and cardiolipin that has been remodeled should promote cardiolipin-dependent mitochondrial processes better than its unremodeled form. However, functional disparities between different molecular forms of cardiolipin have never been established. Here we interrogate this simple but crucial prediction utilizing the best available model to do so, Saccharomyces cerevisiae. Specifically, we compare the ability of unremodeled and remodeled cardiolipin, which differ markedly in their acyl chain composition, to support mitochondrial activities known to require cardiolipin. Surprisingly, defined changes in the acyl chain composition of cardiolipin do not alter either mitochondrial morphology or oxidative phosphorylation. Importantly, preventing cardiolipin remodeling initiation in yeast lacking TAZ1, an ortholog of the causative gene in Barth syndrome, ameliorates mitochondrial dysfunction. Thus, our data do not support the prevailing hypothesis that unremodeled cardiolipin is functionally distinct from remodeled cardiolipin, at least for the functions examined, suggesting alternative physiological roles for this conserved pathway.
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Quantitative profiling and pattern analysis of triacylglycerol species in Arabidopsis seeds by electrospray ionization mass spectrometry.
Plant J.
PUBLISHED: 09-14-2013
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Plant triacylglycerols (TAGs), or vegetable oils, provide approximately 25% of dietary calories to humans and are becoming an increasingly important source of renewable bioenergy and industrial feedstocks. TAGs are assembled by multiple enzymes in the endoplasmic reticulum from building blocks that include an invariable glycerol backbone and variable fatty acyl chains. It remains a challenge to elucidate the mechanism of synthesis of hundreds of different TAG species in planta. One reason is the lack of an efficient analytical approach quantifying individual molecular species. Here we report a rapid and quantitative TAG profiling approach for Arabidopsis seeds based on electrospray ionization tandem mass spectrometry with direct infusion and multiple neutral loss scans. The levels of 93 TAG molecular species, identified by their acyl components, were determined in Arabidopsis seeds. Quantitative TAG pattern analyses revealed that the TAG assembly machinery preferentially produces TAGs with one elongated fatty acid. The importance of the selectivity in oil synthesis was consistent with an observation that an Arabidopsis mutant overexpressing a patatin-like phospholipase had enhanced seed oil content with elongated fatty acids. This quantitative TAG profiling approach should facilitate investigations aimed at understanding the biochemical mechanisms of TAG metabolism in plants.
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Fatty acidomics: global analysis of lipid species containing a carboxyl group with a charge-remote fragmentation-assisted approach.
Anal. Chem.
PUBLISHED: 09-09-2013
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Charge-remote fragmentation has been well recognized as an effective approach for dissociation of long aliphatic chains. Herein, we exploited this approach for structural identification of all fatty acids including saturated, unsaturated, and modified ones by using electrospray ionization tandem mass spectrometry after one-step derivatization of a charge-carried reagent through an amidation reaction. We tested the approach with different charge-carried reagents with respect to the hydrophobicity, charge strength, and distance from the charge to the carboxyl group. We found that all of the derivatives with these reagents could yield informative charge-remote fragmentation patterns regardless of the different chemical and physical properties of the reagents. These informative fragmentation patterns all could be effectively used for structural elucidation of lipid species containing a carboxyl group. We further found that the distinguished charge-remote fragmentations of fatty acid isomers enabled us to determine the composition of these isomers without any chromatographic separation. Finally, the abundant fragments yielded from an individual derivatized moiety enabled us to sensitively quantify the individual species containing a carboxyl group. The described approach was a great extension to the multidimensional mass-spectrometry-based shotgun lipidomics for global analysis of fatty acids including isomers and modifications. We believe that this approach could greatly facilitate identification of the biochemical mechanisms underlying numerous pathological conditions.
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Tafazzin knockdown interrupts cell cycle progression in cultured neonatal ventricular fibroblasts.
Am. J. Physiol. Heart Circ. Physiol.
PUBLISHED: 08-30-2013
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Mutation of the mitochondrial protein tafazzin causes dilated cardiomyopathy in Barth syndrome. Previous studies have shown that tafazzin knockdown promotes hypertrophy of neonatal cardiac myocytes. The current investigation was designed to show whether tafazzin knockdown affects cardiac fibroblast proliferation and collagen secretion, which contribute to fibrosis in dilated cardiomyopathy. In primary cultures of neonatal ventricular fibroblasts (NVFs) transduced with a tafazzin short hairpin RNA adenovirus, tafazzin knockdown increased production of reactive oxygen species and activation of mitogen-activated protein kinases and induced protein and DNA synthesis via cell cycle regulators. It also reduced intracellular ATP, activated AMPK, and caused multinucleation, hypertrophy, and enhanced collagen secretion. We concluded that tafazzin knockdown interrupts the NVF cell cycle and this in turn may contribute to fibrosis and dilated cardiomyopathy in Barth syndrome.
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Cardiolipin remodeling in diabetic heart.
Chem. Phys. Lipids
PUBLISHED: 08-20-2013
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Cardiolipin, a signature phospholipid of mitochondria, is predominantly present in the mitochondrial inner membrane and plays an important role in keeping optimal mitochondrial function. In addition to the cardiolipin content, the composition of four fatty acid chain is thought determine cardiolipin biological function. These acyl chains of cardiolipin are dynamically remodeled via tafazzin, monolysocardiolipin acyltransferase, and acyl-CoA lysocardiolipin acyltransferase especially in the heart under pathological conditions. The major species of cardiolipin in the normal heart, tetralinoleoyl cardiolipin, is dramatically decreased in the diabetic heart, but other species, typically those containing long fatty acyl chains, are increased. This remodeling of cardiolipin has detrimental effects on mitochondrial function and thereafter cardiac function. Approaches for manipulating cardiolipin acyl chains have been examined including via molecular biology and through supplementation of linoleic acid. The efficiency of cardiolipin remodeling and functional improvement is still under investigation.
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Rotenone induces reductive stress and triacylglycerol deposition in C2C12 cells.
Int. J. Biochem. Cell Biol.
PUBLISHED: 06-07-2013
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Environmental rotenone is associated with Parkinsons disease due to its inhibitory property to the complex I of mitochondrial respiration chain. Although environmental pollution has been postulated as a causal factor for the increasing prevalence of obesity, the role of rotenone in the pathogenesis of obesity has not been studied. We employed muscle-derived cell C2C12 as a model and shotgun lipidomics as a tool for lipid analysis and found that treatment with rotenone led to the profound deposition of intracellular triacylglycerol (TAG) in a time- and dose-dependent fashion. The TAG deposition resulted from complex I inhibition. Further studies revealed that rotenone induced mitochondrial stress as shown by decreased mitochondrial oxygen consumption rate, increased NADH/NAD+ ratio (i.e., reductive stress) and mitochondrial metabolites. We demonstrated that rotenone activated fatty acid de novo synthesis and TAG synthesis and ultimately resulted in intracellular TAG deposition. These studies suggested that increased mitochondrial stresses might be an underlying mechanism responsible for TAG accumulation manifest in obesity.
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Specific changes of sulfatide levels in individuals with pre-clinical Alzheimers disease: an early event in disease pathogenesis.
J. Neurochem.
PUBLISHED: 05-06-2013
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To explore the hypothesis that alterations in cellular membrane lipids are present at the stage of pre-clinical Alzheimers disease (AD) (i.e., cognitively normal at death, but with AD neuropathology), we performed targeted shotgun lipidomics of lipid extracts from post-mortem brains of subjects with pre-clinical AD. We found sulfatide levels were significantly lower in subjects with pre-clinical AD compared to those without AD neuropathology. We also found that the level of ethanolamine glycerophospholipid was marginally lower at this stage of AD, whereas changes of the ceramide levels were undetectable with the available samples. These results indicate that cellular membrane defects are present at the earliest stages of AD pathogenesis and also suggest that sulfatide loss is among the earliest events of AD development, while alterations in the levels of ethanolamine glycerophospholipid and ceramide occur relatively later in disease.
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Dysfunctional cardiac mitochondrial bioenergetic, lipidomic, and signaling in a murine model of Barth syndrome.
J. Lipid Res.
PUBLISHED: 02-14-2013
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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 iPLA2? 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.
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Simulation of triacylglycerol ion profiles: bioinformatics for interpretation of triacylglycerol biosynthesis.
J. Lipid Res.
PUBLISHED: 01-30-2013
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Although the synthesis pathways of intracellular triacylglycerol (TAG) species have been well elucidated, assessment of the contribution of an individual pathway to TAG pools in different mammalian organs, particularly under pathophysiological conditions, is difficult, although not impossible. Herein, we developed and validated a novel bioinformatic approach to assess the differential contributions of the known pathways to TAG pools through simulation of TAG ion profiles determined by shotgun lipidomics. This powerful approach was applied to determine such contributions in mouse heart, liver, and skeletal muscle and to examine the changes of these pathways in mouse liver induced after treatment with a high-fat diet. It was clearly demonstrated that assessment of the altered TAG biosynthesis pathways under pathophysiological conditions can be readily achieved through simulation of lipidomics data. Collectively, this new development should greatly facilitate our understanding of the biochemical mechanisms underpinning TAG accumulation at the states of obesity and lipotoxicity.
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Chronic caloric restriction attenuates a loss of sulfatide content in PGC-1?-/- mouse cortex: a potential lipidomic role of PGC-1? in neurodegeneration.
J. Lipid Res.
PUBLISHED: 11-23-2011
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Peroxisome proliferator-activated receptor gamma coactivator-1? (PGC-1?), a key regulator of energy metabolism and lipid homeostasis in multiple highly oxidative tissues, has been implicated in the metabolic derangements of diabetes and obesity. However, relatively less is known regarding its role in neurological functions. Using shotgun lipidomics, we investigated the lipidome of mouse cerebral cortex with generalized deficiency of PGC-1? (PGC-1?(-/-)) versus wild-type (WT) mice under standard diet and chronically calorically restricted conditions. Specific deficiency in sulfatide, a myelin-specific lipid class critically involved in maintaining neurological function, was uncovered in the cortex of PGC-1?(-/-) mice compared with WT mice at all ages examined. Chronic caloric restriction (CR) for 22 months essentially restored the sulfatide reduction in PGC-1?(-/-) mice compared with WT, but sulfatide reduction was not restored in PGC-1?(-/-) with CR for a short term (i.e., 3 months). Mechanistic studies uncovered and differentiated the biochemical mechanisms underpinning the two conditions of altered sulfatide homeostasis. The former is modulated through PGC-1?-MAL pathway, whereas the latter is under the control of LXR/RXR-apoE metabolism pathway. These results suggest a novel mechanistic role of PGC-1? in sulfatide homeostasis, provide new insights into the importance of PGC-1? in neurological functions, and indicate a potential therapeutic approach for treatment of deficient PGC-1?-induced alterations in sulfatide homeostasis.
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Accurate quantification of lipid species by electrospray ionization mass spectrometry - Meet a key challenge in lipidomics.
Metabolites
PUBLISHED: 11-11-2011
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Electrospray ionization mass spectrometry (ESI-MS) has become one of the most popular and powerful technologies to identify and quantify individual lipid species in lipidomics. Meanwhile, quantitative analysis of lipid species by ESI-MS has also become a major obstacle to meet the challenges of lipidomics. Herein, we discuss the principles, advantages, and possible limitations of different mass spectrometry-based methodologies for lipid quantification, as well as a few practical issues important for accurate quantification of individual lipid species. Accordingly, accurate quantification of individual lipid species, one of the key challenges in lipidomics, can be practically met.
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Cellular mechanism of insulin resistance in nonalcoholic fatty liver disease.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 09-19-2011
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Insulin resistance is associated with nonalcoholic fatty liver disease (NAFLD) and is a major factor in the pathogenesis of type 2 diabetes. The development of hepatic insulin resistance has been ascribed to multiple causes, including inflammation, endoplasmic reticulum (ER) stress, and accumulation of hepatocellular lipids in animal models of NAFLD. However, it is unknown whether these same cellular mechanisms link insulin resistance to hepatic steatosis in humans. To examine the cellular mechanisms that link hepatic steatosis to insulin resistance, we comprehensively assessed each of these pathways by using flash-frozen liver biopsies obtained from 37 obese, nondiabetic individuals and correlating key hepatic and plasma markers of inflammation, ER stress, and lipids with the homeostatic model assessment of insulin resistance index. We found that hepatic diacylglycerol (DAG) content in cytoplasmic lipid droplets was the best predictor of insulin resistance (R = 0.80, P < 0.001), and it was responsible for 64% of the variability in insulin sensitivity. Hepatic DAG content was also strongly correlated with activation of hepatic PKC? (R = 0.67, P < 0.001), which impairs insulin signaling. In contrast, there was no significant association between insulin resistance and other putative lipid metabolites or plasma or hepatic markers of inflammation. ER stress markers were only partly correlated with insulin resistance. In conclusion, these data show that hepatic DAG content in lipid droplets is the best predictor of insulin resistance in humans, and they support the hypothesis that NAFLD-associated hepatic insulin resistance is caused by an increase in hepatic DAG content, which results in activation of PKC?.
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A mathematical model for the determination of steady-state cardiolipin remodeling mechanisms using lipidomic data.
PLoS ONE
PUBLISHED: 05-23-2011
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Technical advances in lipidomic analysis have generated tremendous amounts of quantitative lipid molecular species data, whose value has not been fully explored. We describe a novel computational method to infer mechanisms of de novo lipid synthesis and remodeling from lipidomic data. We focus on the mitochondrial-specific lipid cardiolipin (CL), a polyglycerol phospholipid with four acyl chains. The lengths and degree of unsaturation of these acyl chains vary across CL molecules, and regulation of these differences is important for mitochondrial energy metabolism. We developed a novel mathematical approach to determine mechanisms controlling the steady-state distribution of acyl chain combinations in CL . We analyzed mitochondrial lipids from 18 types of steady-state samples, each with at least 3 replicates, from mouse brain, heart, lung, liver, tumor cells, and tumors grown in vitro. Using a mathematical model for the CL remodeling mechanisms and a maximum likelihood approach to infer parameters, we found that for most samples the four chain positions have an independent and identical distribution, indicating they are remodeled by the same processes. Furthermore, for most brain samples and liver, the distribution of acyl chains is well-fit by a simple linear combination of the pools of acyl chains in phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylglycerol (PG). This suggests that headgroup chemistry is the key determinant of acyl donation into CL, with chain length/saturation less important. This canonical remodeling behavior appears damaged in some tumor samples, which display a consistent excess of CL molecules having particular masses. For heart and lung, the "proportional incorporation" assumption is not adequate to explain the CL distribution, suggesting additional acyl CoA-dependent remodeling that is chain-type specific. Our findings indicate that CL remodeling processes can be described by a small set of quantitative relationships, and that bioinformatic approaches can help determine these processes from high-throughput lipidomic data.
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Identification and quantitation of unsaturated fatty acid isomers by electrospray ionization tandem mass spectrometry: a shotgun lipidomics approach.
Anal. Chem.
PUBLISHED: 05-02-2011
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Identification and quantification of unsaturated fatty acid (FA) isomers in a biological system are significant in the study of lipid metabolism and catabolism, membrane biophysics, and pathogenesis of diseases but are challenging in lipidomics. We developed a novel approach for identification and quantitation of unsaturated FA isomers by exploiting two facts: (1) unsaturated FA anions yield fragment ion(s) from loss of CO(2) or H(2)O from the anions upon collision-induced dissociation; and (2) the fragment ions yielded from discrete FA isomers have distinct profiles of the fragment ion intensity vs. collision conditions. These distinct profiles likely result from the differential interactions of the negative charge of the fragment ion with the electron clouds of the double bonds due to their different distances in discrete FA isomers. The novel approach was also extended to analyze the double bond isomers of FA chains present in phospholipids by multistage tandem mass spectrometry. Collectively, we developed a new approach for identification and quantification of the double bond isomers of endogenous FA species or FA chains present in intact phospholipid species. We believe that this approach should further advance the lipidomic power for identification of the biochemical mechanisms underlying metabolic diseases.
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A practical approach for determination of mass spectral baselines.
J. Am. Soc. Mass Spectrom.
PUBLISHED: 04-14-2011
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Precise determination of the baseline levels of mass spectra is critical for identification and quantification of analytes. Herein, we present a practical approach for determination of the baselines of mass spectra acquired under differential conditions. The baseline determined by this approach was the sum of baseline drift and noise level. The baseline drift was determined by averaging a number of lowest ion intensities. The noise level was determined based on the fact that an accelerated intensity change exists from noise to signal. This change was best revealed by the established accumulative layer thickness curve that was derived from the thicknesses of individual deducted layers. Deductions were performed sequentially layer by layer, each of which has a thickness of averaged lowest ion intensities from existing spectral data. The layer where the accelerated intensity change occurred was defined as a transition layer, which was determined from the polynomial regression in the sixth order of the accumulative layer thickness curve followed by resolving the roots of its fourth derivative. We validated the presence of this transition layer through determination of its convergence from various accumulative layer thickness curves generated by varying either the ending or the fineness of the sequential layer deductions. This simple, practical, program-based baseline determination approach should greatly increase the accuracy and consistency of identification and quantification by mass spectrometry, and facilitate the automation of data processing, thereby increasing the power of any high throughput methodology in general and of shotgun lipidomics in particular.
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Multi-dimensional mass spectrometry-based shotgun lipidomics and novel strategies for lipidomic analyses.
Mass Spectrom Rev
PUBLISHED: 03-31-2011
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Since our last comprehensive review on multi-dimensional mass spectrometry-based shotgun lipidomics (Mass Spectrom. Rev. 24 (2005), 367), many new developments in the field of lipidomics have occurred. These developments include new strategies and refinements for shotgun lipidomic approaches that use direct infusion, including novel fragmentation strategies, identification of multiple new informative dimensions for mass spectrometric interrogation, and the development of new bioinformatic approaches for enhanced identification and quantitation of the individual molecular constituents that comprise each cells lipidome. Concurrently, advances in liquid chromatography-based platforms and novel strategies for quantitative matrix-assisted laser desorption/ionization mass spectrometry for lipidomic analyses have been developed. Through the synergistic use of this repertoire of new mass spectrometric approaches, the power and scope of lipidomics has been greatly expanded to accelerate progress toward the comprehensive understanding of the pleiotropic roles of lipids in biological systems.
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Metabolomics in early Alzheimers disease: identification of altered plasma sphingolipidome using shotgun lipidomics.
PLoS ONE
PUBLISHED: 03-29-2011
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The development of plasma biomarkers could facilitate early detection, risk assessment and therapeutic monitoring in Alzheimers disease (AD). Alterations in ceramides and sphingomyelins have been postulated to play a role in amyloidogensis and inflammatory stress related neuronal apoptosis; however few studies have conducted a comprehensive analysis of the sphingolipidome in AD plasma using analytical platforms with accuracy, sensitivity and reproducibility.
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Central nervous system dysfunction in a mouse model of FA2H deficiency.
Glia
PUBLISHED: 03-03-2011
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Fatty acid 2-hydroxylase (FA2H) is responsible for the synthesis of myelin galactolipids containing hydroxy fatty acid (hFA) as the N-acyl chain. Mutations in the FA2H gene cause leukodystrophy, spastic paraplegia, and neurodegeneration with brain iron accumulation. Using the Cre-lox system, we developed two types of mouse mutants, Fa2h(-/-) mice (Fa2h deleted in all cells by germline deletion) and Fa2h(flox/flox) Cnp1-Cre mice (Fa2h deleted only in oligodendrocytes and Schwann cells). We found significant demyelination, profound axonal loss, and abnormally enlarged axons in the CNS of Fa2h(-/-) mice at 12 months of age, while structure and function of peripheral nerves were largely unaffected. Fa2h(-/-) mice also exhibited histological and functional disruption in the cerebellum at 12 months of age. In a time course study, significant deterioration of cerebellar function was first detected at 7 months of age. Further behavioral assessments in water T-maze and Morris water maze tasks revealed significant deficits in spatial learning and memory at 4 months of age. These data suggest that various regions of the CNS are functionally compromised in young adult Fa2h(-/-) mice. The cerebellar deficits in 12-month-old Fa2h(flox/flox) Cnp1-Cre mice were indistinguishable from Fa2h(-/-) mice, indicating that these phenotypes likely stem from the lack of myelin hFA-galactolipids. In contrast, Fa2h(flox/flox) Cnp1-Cre mice did not show reduced performance in water maze tasks, indicating that oligodendrocytes are not involved in the learning and memory deficits found in Fa2h(-/-) mice. These findings provide the first evidence that FA2H has an important function outside of oligodendrocytes in the CNS.
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DGAT enzymes are required for triacylglycerol synthesis and lipid droplets in adipocytes.
J. Lipid Res.
PUBLISHED: 02-11-2011
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The total contribution of the acyl CoA:diacylglycerol acyltransferase (DGAT) enzymes, DGAT1 and DGAT2, to mammalian triacylglycerol (TG) synthesis has not been determined. Similarly, whether DGAT enzymes are required for lipid droplet (LD) formation is unknown. In this study, we examined the requirement for DGAT enzymes in TG synthesis and LDs in differentiated adipocytes with genetic deletions of DGAT1 and DGAT2. Adipocytes with a single deletion of either enzyme were capable of TG synthesis and LD formation. In contrast, adipocytes with deletions of both DGATs were severely lacking in TG and did not have LDs, indicating that DGAT1 and DGAT2 account for nearly all TG synthesis in adipocytes and appear to be required for LD formation during adipogenesis. DGAT enzymes were not absolutely required for LD formation in mammalian cells, however; macrophages deficient in both DGAT enzymes were able to form LDs when incubated with cholesterol-rich lipoproteins. Although adipocytes lacking both DGATs had no TG or LDs, they were fully differentiated by multiple criteria. Our findings show that DGAT1 and DGAT2 account for the vast majority of TG synthesis in mice, and DGAT function is required for LDs in adipocytes, but not in all cell types.
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Barth syndrome mutations that cause tafazzin complex lability.
J. Cell Biol.
PUBLISHED: 02-09-2011
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Deficits in mitochondrial function result in many human diseases. The X-linked disease Barth syndrome (BTHS) is caused by mutations in the tafazzin gene TAZ1. Its product, Taz1p, participates in the metabolism of cardiolipin, the signature phospholipid of mitochondria. In this paper, a yeast BTHS mutant tafazzin panel is established, and 18 of the 21 tested BTHS missense mutations cannot functionally replace endogenous tafazzin. Four BTHS mutant tafazzins expressed at low levels are degraded by the intermembrane space AAA (i-AAA) protease, suggesting misfolding of the mutant polypeptides. Paradoxically, each of these mutant tafazzins assembles in normal protein complexes. Furthermore, in the absence of the i-AAA protease, increased expression and assembly of two of the BTHS mutants improve their function. However, the BTHS mutant complexes are extremely unstable and accumulate as insoluble aggregates when disassembled in the absence of the i-AAA protease. Thus, the loss of function for these BTHS mutants results from the inherent instability of the mutant tafazzin complexes.
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Lipidomics at the interface of structure and function in systems biology.
Chem. Biol.
PUBLISHED: 01-03-2011
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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.
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Neuronal LRP1 knockout in adult mice leads to impaired brain lipid metabolism and progressive, age-dependent synapse loss and neurodegeneration.
J. Neurosci.
PUBLISHED: 12-17-2010
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The vast majority of Alzheimers disease (AD) cases are late onset with progressive synapse loss and neurodegeneration. Although the amyloid hypothesis has generated great insights into the disease mechanism, several lines of evidence indicate that other risk factors might precondition the brain to amyloid toxicity. Here, we show that the deletion of a major lipoprotein receptor, low-density lipoprotein receptor-related protein 1 (LRP1), in forebrain neurons in mice leads to a global defect in brain lipid metabolism characterized by decreased brain levels of cholesterol, sulfatide, galactosylceramide, and triglyceride. These lipid deficits correlate with progressive, age-dependent dendritic spine degeneration, synapse loss, neuroinflammation, memory loss, and eventual neurodegeneration. We further show that the levels of glutamate receptor subunits NMDA receptor 1 and Glu receptor 1 are selectively reduced in LRP1 forebrain knock-out mice and in LRP1 knockdown neurons, which is partially rescued by restoring neuronal cholesterol. Together, these studies support a critical role for LRP1 in maintaining brain lipid homeostasis and associated synaptic and neuronal integrity, and provide important insights into the pathophysiological mechanisms in AD.
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[Preliminary research of endothelial growth stimulation of 125I-vascular endothelial growth factor-coated artificial vascular patch].
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi
PUBLISHED: 10-14-2010
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To explore whether 125I-vascular endothelial growth factor (VEGF)-coated artificial vascular patch accelerate the vessel endothelialization and inhibit thrombosis.
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Genetic ablation of calcium-independent phospholipase A2gamma prevents obesity and insulin resistance during high fat feeding by mitochondrial uncoupling and increased adipocyte fatty acid oxidation.
J. Biol. Chem.
PUBLISHED: 09-03-2010
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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)? (iPLA(2)?(-/-)) are completely resistant to high fat diet-induced weight gain, adipocyte hypertrophy, hyperinsulinemia, and insulin resistance, which occur in iPLA(2)?(+/+) mice after high fat feeding. Notably, iPLA(2)?(-/-) 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)?(-/-) 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)?(-/-) mice fed either a standard diet or a high fat diet. Respirometry of skeletal muscle mitochondria from iPLA(2)?(-/-) 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)?(-/-) mouse. Collectively, these results identify iPLA(2)? 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.
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CGI-58 knockdown in mice causes hepatic steatosis but prevents diet-induced obesity and glucose intolerance.
J. Lipid Res.
PUBLISHED: 08-27-2010
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Mutations of Comparative Gene Identification-58 (CGI-58) in humans cause triglyceride (TG) accumulation in multiple tissues. Mice genetically lacking CGI-58 die shortly after birth due to a skin barrier defect. To study the role of CGI-58 in integrated lipid and energy metabolism, we utilized antisense oligonucleotides (ASOs) to inhibit CGI-58 expression in adult mice. Treatment with two distinct CGI-58-targeting ASOs resulted in ?80-95% knockdown of CGI-58 protein expression in both liver and white adipose tissue. In chow-fed mice, ASO-mediated depletion of CGI-58 did not alter weight gain, plasma TG, or plasma glucose, yet raised hepatic TG levels ?4-fold. When challenged with a high-fat diet (HFD), CGI-58 ASO-treated mice were protected against diet-induced obesity, but their hepatic contents of TG, diacylglycerols, and ceramides were all elevated, and intriguingly, their hepatic phosphatidylglycerol content was increased by 10-fold. These hepatic lipid alterations were associated with significant decreases in hepatic TG hydrolase activity, hepatic lipoprotein-TG secretion, and plasma concentrations of ketones, nonesterified fatty acids, and insulin. Additionally, HFD-fed CGI-58 ASO-treated mice were more glucose tolerant and insulin sensitive. Collectively, this work demonstrates that CGI-58 plays a critical role in limiting hepatic steatosis and maintaining hepatic glycerophospholipid homeostasis and has unmasked an unexpected role for CGI-58 in promoting HFD-induced obesity and insulin resistance.
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Phospholipid transfer protein in human plasma associates with proteins linked to immunity and inflammation.
Biochemistry
PUBLISHED: 07-30-2010
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Phospholipid transfer protein (PLTP), which associates with apolipoprotein A-I (the major HDL protein), plays a key role in lipoprotein remodeling. Because its level in plasma increases during acute inflammation, it may also play previously unsuspected roles in the innate immune system. To gain further insight into its potential physiological functions, we isolated complexes containing PLTP from plasma by immunoaffinity chromatography and determined their composition. Shotgun proteomics revealed that only 6 of the 24 proteins detected in the complexes were apolipoproteins. The most abundant proteins were clusterin (apoJ), PLTP itself, coagulation factors, complement factors, and apoA-I. Remarkably, 20 of the 24 proteins had known protein-protein interactions. Biochemical studies confirmed two previously established interactions and identified five new ones between PLTP and proteins. Moreover, clusterin, apoA-I, and apoE preserved the lipid-transfer activity of recombinant PLTP in the absence of lipid, indicating that these interactions may have functional significance. Unexpectedly, lipids accounted for only 3% of the mass of the PLTP complexes. Collectively, our observations indicate that PLTP in human plasma resides on lipid-poor complexes dominated by clusterin and proteins implicated in host defense and inflammation. They further suggest that protein-protein interactions drive the formation of PLTP complexes in plasma.
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Lipidomics analysis reveals efficient storage of hepatic triacylglycerides enriched in unsaturated fatty acids after one bout of exercise in mice.
PLoS ONE
PUBLISHED: 07-14-2010
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Endurance exercise induces lipolysis, increases circulating concentrations of free fatty acids (FFA) and the uptake and oxidation of fatty acids in the working muscle. Less is known about the regulation of lipid metabolism in the liver during and post-exercise.
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Cardiolipin remodeling by ALCAT1 links oxidative stress and mitochondrial dysfunction to obesity.
Cell Metab.
PUBLISHED: 05-24-2010
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Oxidative stress causes mitochondrial dysfunction and metabolic complications through unknown mechanisms. Cardiolipin (CL) is a key mitochondrial phospholipid required for oxidative phosphorylation. Oxidative damage to CL from pathological remodeling is implicated in the etiology of mitochondrial dysfunction commonly associated with diabetes, obesity, and other metabolic diseases. Here, we show that ALCAT1, a lyso-CL acyltransferase upregulated by oxidative stress and diet-induced obesity (DIO), catalyzes the synthesis of CL species that are highly sensitive to oxidative damage, leading to mitochondrial dysfunction, ROS production, and insulin resistance. These metabolic disorders were reminiscent of those observed in type 2 diabetes and were reversed by rosiglitazone treatment. Consequently, ALCAT1 deficiency prevented the onset of DIO and significantly improved mitochondrial complex I activity, lipid oxidation, and insulin signaling in ALCAT1(-/-) mice. Collectively, these findings identify a key role of ALCAT1 in regulating CL remodeling, mitochondrial dysfunction, and susceptibility to DIO.
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Dynamic simulation of cardiolipin remodeling: greasing the wheels for an interpretative approach to lipidomics.
J. Lipid Res.
PUBLISHED: 04-21-2010
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Cardiolipin is a class of mitochondrial specific phospholipid, which is intricately involved in mitochondrial functionality. Differences in cardiolipin species exist in a variety of tissues and diseases. It has been demonstrated that the cardiolipin profile is a key modulator of the functions of many mitochondrial proteins. However, the chemical mechanism(s) leading to normal and/or pathological distribution of cardiolipin species remain elusive. Herein, we describe a novel approach for investigating the molecular mechanism of cardiolipin remodeling through a dynamic simulation. This approach applied data from shotgun lipidomic analyses of the heart, liver, brain, and lung mitochondrial lipidomes to model cardiolipin remodeling, including relative content, regiospecificity, and isomeric composition of cardiolipin species. Generated cardiolipin profiles were nearly identical to those determined by shotgun lipidomics. Importantly, the simulated isomeric compositions of cardiolipin species were further substantiated through product ion analysis. Finally, unique enzymatic activities involved in cardiolipin remodeling were assessed from the parameters used in the dynamic simulation of cardiolipin profiles. Collectively, we described, verified, and demonstrated a novel approach by integrating both lipidomic analysis and dynamic simulation to study cardiolipin biology. We believe this study provides a foundation to investigate cardiolipin metabolism and bioenergetic homeostasis in normal and disease states.
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Selective desorption/ionization of sulfatides by MALDI-MS facilitated using 9-aminoacridine as matrix.
J. Lipid Res.
PUBLISHED: 02-01-2010
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Recently, we used the favorable properties of 9-aminoacridine (9-AA) as matrix for the quantitative analysis of acidic metabolites and glycerophospholipids from extracts of biological materials [Sun, G., Yang, K., Zhao, Z., Guan, S., Han, X., and Gross, R.W. (2007) A shotgun metabolomics approach for rapid analysis of negatively-charged water-soluble cellular metabolites from mouse heart tissue. Anal. Chem. 79: 6629-6640; Sun, G., Yang, K., Zhao, Z., Guan, S., Han, X., and Gross, R.W. (2008) Matrix-assisted laser desorption/ionization-time of flight mass spectrometric analysis of cellular glycerophospholipids enabled by multiplexed solvent dependent analyte-matrix interactions. Anal. Chem. 80: 7576-7585.] by MALDI-MS. Herein, we extend this discovery and identified the selective desorption/ionization of sulfatides over other examined anionic lipids present in lipid extracts of biological samples by MALDI-MS using 9-AA as matrix. Through this approach, a high throughput method for the quantitative analysis of low to very low abundance sulfatide molecular species directly from crude lipid extracts has been developed. This method possessed a linear dynamic range of over 1,000-fold, a detection limit at the high attomole level, and a reproducibility of approximately 10% deviation. Many potential factors that might affect the quantitation of sulfatide species employing the method were examined and their effects were found to be negligible within experimental error. Collectively, these results demonstrate a powerful high throughput method for the measurement of sulfatides directly from extracts of biological samples, facilitating the study of sulfatide metabolism, trafficking, and homeostasis in health and disease.
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Multi-dimensional mass spectrometry-based shotgun lipidomics and the altered lipids at the mild cognitive impairment stage of Alzheimers disease.
Biochim. Biophys. Acta
PUBLISHED: 01-13-2010
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Multi-dimensional mass spectrometry-based shotgun lipidomics (MDMS-SL) is a well-developed technology for global lipid analysis, which identifies and quantifies individual lipid molecular species directly from lipid extracts of biological samples. By using this technology, we have revealed three marked changes of lipids in brain samples of subjects with mild cognitive impairment of Alzheimers disease including sulfatides, ceramides, and plasmalogens. Further studies using MDMS-SL lead us to the identification of the potential biochemical mechanisms responsible for the altered lipids at the disease state, which are thoroughly discussed in this minireview. Specifically, in studies to identify the causes responsible for sulfatide depletion at the mild cognitive impairment stage of Alzheimers disease, we have found that apolipoprotein E is associated with sulfatide transport and mediates sulfatide homeostasis in the nervous system through lipoprotein metabolism pathways and that alterations in apolipoprotein E-mediated sulfatide trafficking can lead to sulfatide depletion in the brain. Collectively, the results obtained from lipidomic analyses of brain samples provide important insights into the biochemical mechanisms underlying the pathogenesis of Alzheimers disease.
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Qualitative and quantitative comparison of brand name and generic protein pharmaceuticals using isotope tags for relative and absolute quantification and matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry.
Anal. Biochem.
PUBLISHED: 01-07-2010
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The capability of iTRAQ (isotope tags for relative and absolute quantification) reagents coupled with matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF-MS) as a qualitative and quantitative technique for the analysis of complicated protein pharmaceutical mixtures was evaluated. Mixtures of Somavert and Miacalcin with a small amount of bovine serum albumin (BSA) as an impurity were analyzed. Both Somavert and Miacalcin were qualitatively identified, and BSA was detected at levels as low as 0.8mol%. Genotropin and Somavert were compared in a single experiment, and all of the distinct amino acid residues from the two proteins were readily identified. Four somatropin drug products (Genotropin, Norditropin, Jintropin, and Omnitrope) were compared using the iTRAQ/MALDI-MS method to determine the similarity between their primary structures and quantify the amount of protein in each product. All four product samples were well labeled and successfully compared when a filtration cleanup step preceded iTRAQ labeling. The quantitative accuracy of the iTRAQ method was evaluated. In all cases, the accuracy of experimentally determined protein ratios was higher than 90%, and the relative standard deviation (RSD) was less than 10%. The iTRAQ and global internal standard technology (GIST) methods were compared, and the iTRAQ method provided both higher sequence coverage and enhanced signal intensity.
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The pathogenic implication of abnormal interaction between apolipoprotein E isoforms, amyloid-beta peptides, and sulfatides in Alzheimers disease.
Mol. Neurobiol.
PUBLISHED: 01-07-2010
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Alzheimers disease (AD) is the most common cause of dementia in the aging population. Prior work has shown that the epsilon4 allele of apolipoprotein E (apoE4) is a major risk factor for "sporadic" AD, which accounts for >99% of AD cases without a defined underlying mechanism. Recently, we have demonstrated that sulfatides are substantially and specifically depleted at the very early stage of AD. To identify the mechanism(s) of sulfatide loss concurrent with AD onset, we have found that: (1) sulfatides are specifically associated with apoE-associated particles in cerebrospinal fluid (CSF); (2) apoE modulates cellular sulfatide levels; and (3) the modulation of sulfatide content is apoE isoform dependent. These findings not only lead to identification of the potential mechanisms underlying sulfatide depletion at the earliest stages of AD but also serve as mechanistic links to explain the genetic association of apoE4 with AD. Moreover, our recent studies further demonstrated that (1) apoE mediates sulfatide depletion in amyloid-beta precursor protein transgenic mice; (2) sulfatides enhance amyloid beta (Abeta) peptides binding to apoE-associated particles; (3) Abeta42 content notably correlates with sulfatide content in CSF; (4) sulfatides markedly enhance the uptake of Abeta peptides; and (5) abnormal sulfatide-facilitated Abeta uptake results in the accumulation of Abeta in lysosomes. Collectively, our studies clearly provide a link between apoE, Abeta, and sulfatides in AD and establish a foundation for the development of effective therapeutic interventions for AD.
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Genetic ablation of calcium-independent phospholipase A2{gamma} leads to alterations in hippocampal cardiolipin content and molecular species distribution, mitochondrial degeneration, autophagy, and cognitive dysfunction.
J. Biol. Chem.
PUBLISHED: 10-19-2009
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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.
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Examination of the brain mitochondrial lipidome using shotgun lipidomics.
Methods Mol. Biol.
PUBLISHED: 09-19-2009
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Contamination from subcellular organelles and myelin has hindered attempts to characterize the lipidome of brain mitochondria. A high degree of mitochondrial purity is required for accurate measurements of the content and molecular species composition of mitochondrial lipids. We devised a discontinuous Ficoll and sucrose gradient procedure for the isolation and purification of brain mitochondria free from any detectable contamination. Shotgun lipidomics was used to analyze the lipid composition of the brain mitochondria. These procedures can be used to determine whether intrinsic lipid abnormalities underlie mitochondrial dysfunction associated with neurological and neurodegenerative diseases.
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A review of lipidomic technologies applicable to sphingolipidomics and their relevant applications.
Eur J Lipid Sci Technol
PUBLISHED: 08-20-2009
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Sphingolipidomics, a branch of lipidomics, focuses on the large-scale study of the cellular sphingolipidomes. In the current review, two main approaches for the analysis of cellular sphingolipidomes (i.e. LC-MS- or LC-MS/MS-based approach and shotgun lipidomics-based approach) are briefly discussed. Their advantages, some considerations of these methods, and recent applications of these approaches are summarized. It is the authors sincere hope that this review article will add to the readers understanding of the advantages and limitations of each developed method for the analysis of a cellular sphingolipidome.
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In vitro growth environment produces lipidomic and electron transport chain abnormalities in mitochondria from non-tumorigenic astrocytes and brain tumours.
ASN Neuro
PUBLISHED: 07-03-2009
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The mitochondrial lipidome influences ETC (electron transport chain) and cellular bioenergetic efficiency. Brain tumours are largely dependent on glycolysis for energy due to defects in mitochondria and oxidative phosphorylation. In the present study, we used shotgun lipidomics to compare the lipidome in highly purified mitochondria isolated from normal brain, from brain tumour tissue, from cultured tumour cells and from non-tumorigenic astrocytes. The tumours included the CT-2A astrocytoma and an EPEN (ependymoblastoma), both syngeneic with the C57BL/6J (B6) mouse strain. The mitochondrial lipidome in cultured CT-2A and EPEN tumour cells were compared with those in cultured astrocytes and in solid tumours grown in vivo. Major differences were found between normal tissue and tumour tissue and between in vivo and in vitro growth environments for the content or composition of ethanolamine glycerophospholipids, phosphatidylglycerol and cardiolipin. The mitochondrial lipid abnormalities in solid tumours and in cultured cells were associated with reductions in multiple ETC activities, especially Complex I. The in vitro growth environment produced lipid and ETC abnormalities in cultured non-tumorigenic astrocytes that were similar to those associated with tumorigenicity. It appears that the culture environment obscures the boundaries of the Crabtree and the Warburg effects. These results indicate that in vitro growth environments can produce abnormalities in mitochondrial lipids and ETC activities, thus contributing to a dependency on glycolysis for ATP production.
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Automated lipid identification and quantification by multidimensional mass spectrometry-based shotgun lipidomics.
Anal. Chem.
PUBLISHED: 05-05-2009
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This article presents the strategies underlying the automated identification and quantification of individual lipid molecular species through array analysis of multidimensional mass spectrometry-based shotgun lipidomics (MDMS-SL) data, which are acquired directly from lipid extracts after direct infusion and intrasource separation. The automated analyses of individual lipid molecular species in the program employ a strategy in which MDMS-SL data from building block analyses using precursor ion scans, neutral loss scans, or both are used to identify individual molecular species, followed by quantitation. Through this strategy, the program screens and identifies species in a high-throughput fashion from a built-in database of over 36,000 potential lipid molecular species constructed employing known building blocks. The program then uses a two-step procedure for quantitation of the identified species possessing a linear dynamic range over 3 orders of magnitude and reverifies the results when necessary through redundant quantification of multidimensional mass spectra. This program is designed to be easily adaptable for other shotgun lipidomics approaches that are currently used for mass spectrometric analysis of lipids. Accordingly, the development of this program should greatly accelerate high-throughput analysis of lipids using MDMS-based shotgun lipidomics.
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Direct quantitation of psychosine from alkaline-treated lipid extracts with a semi-synthetic internal standard.
J. Lipid Res.
PUBLISHED: 04-03-2009
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Psychosine is an important bioactive sphingolipid metabolite and plays an essential role in the pathogenesis of Krabbes disease. Herein, we extended shotgun lipidomics for the characterization and quantitation of psychosine in alkaline-treated crude lipid extracts by using neutral loss scan of 180 amicro (i.e., galactose) in the positive-ion mode. Specifically, we semi-synthesized N,N-dimethyl psychosine and used it as an internal standard for quantitation of psychosine. After characterization of the fragmentation patterns of psychosine and the selected internal standard and optimization of the experimental conditions, we demonstrated that a broad linear dynamic range for the quantitation of psychosine and a limit of detection at a concentration of low fmol/microl were achieved using this approach. The developed method is generally simpler and more efficient than other previously reported methods. Multiple factors influencing quantitation of psychosine were extensively examined and/or discussed. The levels of psychosine in diabetic mouse nerve tissue samples were determined by the developed methodology. Collectively, the developed approach, as a new addition to the shotgun lipidomics technology, will be extremely useful for understanding the pathways/networks of sphingolipid metabolism and for exploring the important roles of psychosine in a variety of physiological and pathological conditions.
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Lack of cytochrome c in mouse fibroblasts disrupts assembly/stability of respiratory complexes I and IV.
J. Biol. Chem.
PUBLISHED: 04-01-2009
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Cytochrome c (cyt c) is a heme-containing protein that participates in electron transport in the respiratory chain and as a signaling molecule in the apoptotic cascade. Here we addressed the effect of removing mammalian cyt c on the integrity of the respiratory complexes in mammalian cells. Mitochondria from cyt c knockout mouse cells lacked fully assembled complexes I and IV and had reduced levels of complex III. A redox-deficient mutant of cyt c was unable to rescue the levels of complexes I and IV. We found that cyt c is associated with both complex IV and respiratory supercomplexes, providing a potential mechanism for the requirement for cyt c in the assembly/stability of complex IV.
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Side chain oxygenated cholesterol regulates cellular cholesterol homeostasis through direct sterol-membrane interactions.
J. Biol. Chem.
PUBLISHED: 03-13-2009
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Side chain oxysterols exert cholesterol homeostatic effects by suppression of sterol regulatory element-binding protein maturation and promoting degradation of hydroxymethylglutaryl-CoA reductase. To examine whether oxysterol-membrane interactions contribute to the regulation of cellular cholesterol homeostasis, we synthesized the enantiomer of 25-hydroxycholesterol. Using this unique oxysterol probe, we provide evidence that oxysterol regulation of cholesterol homeostatic responses is not mediated by enantiospecific oxysterol-protein interactions. We show that side chain oxysterols, but not steroid ring-modified oxysterols, exhibit membrane expansion behavior in phospholipid monolayers and bilayers in vitro. This behavior is non-enantiospecific and is abrogated by increasing the saturation of phospholipid acyl chain constituents. Moreover, we extend these findings into cultured cells by showing that exposure to saturated fatty acids at concentrations that lead to endoplasmic reticulum membrane phospholipid remodeling inhibits oxysterol activity. These studies implicate oxysterol-membrane interactions in acute regulation of sterol homeostatic responses and provide new insights into the mechanism through which oxysterols regulate cellular cholesterol balance.
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Systematic analysis of choline-containing phospholipids using multi-dimensional mass spectrometry-based shotgun lipidomics.
J. Chromatogr. B Analyt. Technol. Biomed. Life Sci.
PUBLISHED: 01-15-2009
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Herein, a systematic study on the identification and quantitation of choline-containing phospholipid molecular species, including choline glycerophospholipid (PC), lysoPC, and sphingomyelin (SM), is described using multi-dimensional mass spectrometry-based shotgun lipidomics after intrasource separation (MDMS-SL). Current methods for analysis of choline-containing lipids were improved through multiple modifications leading to: (1) identification of constituents present in the PC and SM classes, subclasses of PC, and individual molecular species using MDMS-SL analysis in the positive-ion mode; (2) identification of the fatty acyl constituents and their regiospecificity in diacyl PC molecular species through the neutral loss of trimethylamine plus fatty acids; (3) direct identification of the alkenyl chains of plasmenylcholine species using precursor ion scans of the fragment ions carrying the alkenyl chains; (4) elimination of the effects of polyunsaturation on the quantitation of PC species by multiple ratiometric comparisons; (5) accurate identification and quantitation of lysoPC molecular species including regioisomers by diagnostic fragment ions; and (6) accurate identification and quantitation of SM molecular species by neutral loss scans of phosphocholine plus methyl aldehyde which is specific to SM molecular species. With these enhancements, the application of MDMS-SL for the analyses of choline-containing phospholipid molecular species in biomedical research has been extended to a much larger number of molecular species with greater quantitative accuracy and an increased depth of structural information.
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Lipin 2 is a liver-enriched phosphatidate phosphohydrolase enzyme that is dynamically regulated by fasting and obesity in mice.
J. Biol. Chem.
PUBLISHED: 01-10-2009
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Lipin 1 is a bifunctional intracellular protein that regulates fatty acid metabolism in the nucleus via interactions with DNA-bound transcription factors and at the endoplasmic reticulum as a phosphatidic acid phosphohydrolase enzyme (PAP-1) to catalyze the penultimate step in triglyceride synthesis. However, livers of 8-day-old mice lacking lipin 1 (fld mice) exhibited normal PAP-1 activity and a 20-fold increase in triglyceride levels. We sought to further analyze the hepatic lipid profile of these mice by electrospray ionization mass spectrometry. Surprisingly, hepatic content of phosphatidate, the substrate of PAP-1 enzymes, was markedly diminished in fld mice. Similarly, other phospholipids derived from phosphatidate, phosphatidylglycerol and cardiolipin, were also depleted. Another member of the lipin family (lipin 2) is enriched in liver, and hepatic lipin 2 protein content was markedly increased by lipin 1 deficiency, food deprivation, and obesity, often independent of changes in steady-state mRNA levels. Importantly, RNAi against lipin 2 markedly reduced PAP-1 activity in hepatocytes from both wild type and fld mice and suppressed triglyceride synthesis under conditions of high fatty acid availability. Collectively, these data suggest that lipin 2 plays an important role as a hepatic PAP-1 enzyme.
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Shotgun lipidomics of neutral lipids as an enabling technology for elucidation of lipid-related diseases.
Am. J. Physiol. Endocrinol. Metab.
PUBLISHED: 01-06-2009
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Neutral lipids fulfill multiple specialized roles in cellular function. These roles include energy storage and utilization, the synthesis of complex lipids in cellular membranes, lipid second messengers for cellular signaling, and the modulation of membrane molecular dynamics. We have developed a novel mass spectrometric technology, now termed shotgun lipidomics, that can identify the types and amounts of thousands of lipids directly from extracts of biological samples. Shotgun lipidomics is well suited for the identification and measurement of the types and amounts of neutral lipid classes and individual molecular species through the use of multidimensional mass spectrometry. This review summarizes the basic principles underlying the use of shotgun lipidomics for the direct measurement of neutral lipids from extracts of biological tissues or fluids. Through exploiting the high information content inherent in shotgun lipidomics, this technology promises to greatly facilitate advances in our understanding of alterations in neutral lipid metabolism in health and disease.
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Diabetes diminishes phosphatidic acid in the retina: a putative mediator for reduced mTOR signaling and increased neuronal cell death.
Invest. Ophthalmol. Vis. Sci.
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We demonstrated previously that pro-survival insulin receptor, PI3K-Akt, and p70 S6K signaling is diminished in models of diabetic retinopathy. As mammalian target of rapamycin (mTOR), an upstream activator of p70 S6Kinase is, in part, regulated by lipid-derived second messengers, such as phosphatidic acid (PA), we sought to determine if diminished mTOR/p70 S6Kinase signaling in diabetic retinas may reflect diminished PA levels.
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Lipidomic profiling in Crohns disease: abnormalities in phosphatidylinositols, with preservation of ceramide, phosphatidylcholine and phosphatidylserine composition.
Int. J. Biochem. Cell Biol.
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Crohns disease is a chronic inflammatory condition largely affecting the terminal ileum and large bowel. A contributing cause is the failure of an adequate acute inflammatory response as a result of impaired secretion of pro-inflammatory cytokines by macrophages. This defective secretion arises from aberrant vesicle trafficking, misdirecting the cytokines to lysosomal degradation. Aberrant intestinal permeability is also well-established in Crohns disease. Both the disordered vesicle trafficking and increased bowel permeability could result from abnormal lipid composition. We thus measured the sphingo- and phospholipid composition of macrophages, using mass spectrometry and stable isotope labelling approaches. Stimulation of macrophages with heat-killed Escherichia coli resulted in three main changes; a significant reduction in the amount of individual ceramide species, an altered composition of phosphatidylcholine, and an increased rate of phosphatidylcholine synthesis in macrophages. These changes were observed in macrophages from both healthy control individuals and patients with Crohns disease. The only difference detected between control and Crohns disease macrophages was a reduced proportion of newly-synthesised phosphatidylinositol 16:0/18:1 over a defined time period. Shotgun lipidomics analysis of macroscopically non-inflamed ileal biopsies showed a significant decrease in this same lipid species with overall preservation of sphingolipid, phospholipid and cholesterol composition.
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Identification of naturally occurring fatty acids of the myelin sheath that resolve neuroinflammation.
Sci Transl Med
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Lipids constitute 70% of the myelin sheath, and autoantibodies against lipids may contribute to the demyelination that characterizes multiple sclerosis (MS). We used lipid antigen microarrays and lipid mass spectrometry to identify bona fide lipid targets of the autoimmune response in MS brain, and an animal model of MS to explore the role of the identified lipids in autoimmune demyelination. We found that autoantibodies in MS target a phosphate group in phosphatidylserine and oxidized phosphatidylcholine derivatives. Administration of these lipids ameliorated experimental autoimmune encephalomyelitis by suppressing activation and inducing apoptosis of autoreactive T cells, effects mediated by the lipids saturated fatty acid side chains. Thus, phospholipids represent a natural anti-inflammatory class of compounds that have potential as therapeutics for MS.
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Statistical analysis of the processes controlling choline and ethanolamine glycerophospholipid molecular species composition.
PLoS ONE
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The regulation and maintenance of the cellular lipidome through biosynthetic, remodeling, and catabolic mechanisms are critical for biological homeostasis during development, health and disease. These complex mechanisms control the architectures of lipid molecular species, which have diverse yet highly regulated fatty acid chains at both the sn1 and sn2 positions. Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) serve as the predominant biophysical scaffolds in membranes, acting as reservoirs for potent lipid signals and regulating numerous enzymatic processes. Here we report the first rigorous computational dissection of the mechanisms influencing PC and PE molecular architectures from high-throughput shotgun lipidomic data. Using novel statistical approaches, we have analyzed multidimensional mass spectrometry-based shotgun lipidomic data from developmental mouse heart and mature mouse heart, lung, brain, and liver tissues. We show that in PC and PE, sn1 and sn2 positions are largely independent, though for low abundance species regulatory processes may interact with both the sn1 and sn2 chain simultaneously, leading to cooperative effects. Chains with similar biochemical properties appear to be remodeled similarly. We also see that sn2 positions are more regulated than sn1, and that PC exhibits stronger cooperative effects than PE. A key aspect of our work is a novel statistically rigorous approach to determine cooperativity based on a modified Fishers exact test using Markov Chain Monte Carlo sampling. This computational approach provides a novel tool for developing mechanistic insight into lipidomic regulation.
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Myocardial regulation of lipidomic flux by cardiolipin synthase: setting the beat for bioenergetic efficiency.
J. Biol. Chem.
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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.
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Shotgun lipidomics analysis of 4-hydroxyalkenal species directly from lipid extracts after one-step in situ derivatization.
Anal. Chem.
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4-Hydroxyalkenal species are a class of peroxidative products of polyunsaturated fatty acids, which serve as "toxic second messengers" in cellular systems. Investigation of their cellular role is hindered due to the lack of sensitive, reliable, robust method for identification and quantification of these metastable metabolites. Herein, we explored the facile Michael adduct of carnosine with 4-hydroxyalkenal species and developed a sensitive, facile, shotgun lipidomics-based method for quantification of these compounds directly from organic solvent lipid extracts of biological samples. In the study, we extensively examined the factors that may affect the accurate quantification of 4-hydroxyalkenal species and found that this method possessed high reproducibility (<8%) and nearly 3 orders of linear dynamic range with a limit of quantification at lower than 0.56 fmol/?L. Mass levels of 4-hydroxyalkenal species in various biological samples, including mouse heart, kidney, liver, and skeletal muscle, were determined by this developing method. In addition, the effects of sample collection methods and sample storage time on 4-hydroxyalkenal mass levels were also determined. We believe that development of this novel methodology should provide a powerful tool for us to better understand the role of 4-hydroxyalkenal species in biological processes.
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