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Conversion of the redox probe hydroethidine (HE) to 2-chloroethidium (2-Cl-E

AGPATs (1-acylglycerol-3-phosphate O-acyltransferases) catalyze the acylation of lysophosphatidic acid to form phosphatidic acid (PA), a key step in the glycerol-3-phosphate pathway for the synthesis of phospholipids and triacylglycerols. AGPAT2 is the only AGPAT isoform whose loss-of-function mutations cause a severe form of human congenital generalized lipodystrophy. Paradoxically, AGPAT2 deficiency is known to dramatically increase the level of its product, PA. Here, we find that AGPAT2 deficiency impairs the biogenesis and growth of lipid droplets. We show that AGPAT2 deficiency compromises the stability of CDP-diacylglycerol (DAG) synthases (CDSs) and decreases CDS activity in both cell lines and mouse liver. Moreover, AGPAT2 and CDS1/2 can directly interact and form functional complexes, which promote the metabolism of PA along the CDP-DAG pathway of phospholipid synthesis. Our results provide key insights into the regulation of metabolic flux during lipid synthesis and suggest substrate channelling at a major branch point of the glycerol-3-phosphate pathway., AGPATs (1-acylglycerol-3-phosphate O-acyltransferases) catalyze the acylation of lysophosphatidic acid to form phosphatidic acid (PA), a key step in the synthesis of all glycerolipids. Here, the authors show that AGPAT2 and CDP-DAG synthases (CDS1 and CDS2) form functional complexes that promote further conversion of PA along the CDP-DAG pathway of phospholipid synthesis.

Background & aims Plasma concentrations of bilirubin, a product of heme catabolism formed by biliverdin reductase A (BVRA), inversely associate with the risk of metabolic diseases including hepatic steatosis and diabetes mellitus in humans. Bilirubin has antioxidant and anti-inflammatory activities and may also regulate insulin signaling and peroxisome proliferator-activated receptor alpha (PPARα) activity. However, a causal link between bilirubin and metabolic diseases remains to be established. Here, we used the global Bvra gene knockout (Bvra–/–) mouse as a model of deficiency in bilirubin to assess its role in metabolic diseases. Approach & results We fed mice fat-rich diets to induce hepatic steatosis and insulin resistance. Bile pigments were measured by LC-MS/MS, and hepatic lipids by LC-MS/MS (non-targeted lipidomics), HPLC-UV and Oil-Red-O staining. Oxidative stress was evaluated measuring F2-isoprostanes by GC-MS. Glucose metabolism and insulin sensitivity were verified by glucose and insulin tolerance tests, ex vivo and in vivo glucose uptake, and Western blotting for insulin signaling. Compared with wild type littermates, Bvra–/– mice contained negligible bilirubin in plasma and liver, and they had comparable glucose metabolism and insulin sensitivity. However, Bvra–/– mice exhibited an inflamed and fatty liver phenotype, accompanied by hepatic accumulation of oxidized triacylglycerols and F2-isoprostanes, in association with depletion of α-tocopherol. α-Tocopherol supplementation reversed the hepatic phenotype and observed biochemical changes in Bvra–/– mice. Conclusions Our data suggests that BVRA deficiency renders mice susceptible to oxidative stress-induced hepatic steatosis in the absence of insulin resistance., Graphical abstract Image 1, Highlights • Low plasma levels of bilirubin associate with increased metabolic disease risk. • A direct link between bilirubin and metabolic disease remains to be established. • Global BVRA deficiency causes global bilirubin deficiency and a fatty, inflamed liver. • This hepatic phenotype is linked to decreased vitamin E and increased lipid oxidation. • Vitamin E supplements restore normal liver phenotype in BVRA deficiency.

Currently there are no established therapies to treat high-risk patients with unstable atherosclerotic lesions that are prone to rupture and can result in thrombosis, abrupt arterial occlusion, and a precipitous infarction. Rather than being stenotic, rupture-prone non-occlusive plaques are commonly enriched with inflammatory cells and have a thin fibrous cap. We reported previously that inhibition of the pro-inflammatory enzyme myeloperoxidase (MPO) with the suicide inhibitor AZM198 prevents formation of unstable plaque in the Tandem Stenosis (TS) mouse model of plaque instability. However, in our previous study AZM198 was administered to animals before unstable plaque was present and hence it did not test the significant unmet clinical need present in high-risk patients with vulnerable atherosclerosis. In the present study we therefore asked whether pharmacological inhibition of MPO with AZM198 can stabilize pre-existing unstable lesions in an interventional setting using the mouse model of plaque instability. In vivo molecular magnetic resonance imaging of arterial MPO activity using bis-5-hydroxytryptamide-DTPA-Gd and histological analyses revealed that arterial MPO activity was elevated one week after TS surgery, prior to the presence of unstable lesions observed two weeks after TS surgery. Animals with pre-existing unstable plaque were treated with AZM198 for one or five weeks. Both short- and long-term intervention effectively inhibited arterial MPO activity and increased fibrous cap thickness, indicative of a more stable plaque phenotype. Plaque stabilization was observed without AZM198 affecting the arterial content of Ly6B.2

Mitochondrial energy production and function rely on optimal concentrations of the essential redox-active lipid, coenzyme Q (CoQ). CoQ deficiency results in mitochondrial dysfunction associated with increased mitochondrial oxidative stress and a range of pathologies. What drives CoQ deficiency in many of these pathologies is unknown, just as there currently is no effective therapeutic strategy to overcome CoQ deficiency in humans. To date, large-scale studies aimed at systematically interrogating endogenous systems that control CoQ biosynthesis and their potential utility to treat disease have not been carried out. Therefore, we developed a quantitative high-throughput method to determine CoQ concentrations in yeast cells. Applying this method to the Yeast Deletion Collection as a genome-wide screen, 30 genes not known previously to regulate cellular concentrations of CoQ were discovered. In combination with untargeted lipidomics and metabolomics, phosphatidylethanolamine N-methyltransferase (PEMT) deficiency was confirmed as a positive regulator of CoQ synthesis, the first identified to date. Mechanistically, PEMT deficiency alters mitochondrial concentrations of one-carbon metabolites, characterized by an increase in the S-adenosylmethionine to S-adenosylhomocysteine (SAM-to-SAH) ratio that reflects mitochondrial methylation capacity, drives CoQ synthesis, and is associated with a decrease in mitochondrial oxidative stress. The newly described regulatory pathway appears evolutionary conserved, as ablation of PEMT using antisense oligonucleotides increases mitochondrial CoQ in mouse-derived adipocytes that translates to improved glucose utilization by these cells, and protection of mice from high-fat diet-induced insulin resistance. Our studies reveal a previously unrecognized relationship between two spatially distinct lipid pathways with potential implications for the treatment of CoQ deficiencies, mitochondrial oxidative stress/dysfunction, and associated diseases., Graphical abstract Image 1, Highlights • Mitochondrial CoQ deficiency results in oxidative stress and a range of pathologies • The drivers of mitochondrial CoQ deficiency remain largely unknown • PEMT deficiency is the first identified positive regulator of mitochondrial CoQ • PEMT deficiency increases CoQ by increasing the mitochondrial SAM-to-SAH ratio • PEMT deficiency prevents insulin resistance by increasing mitochondrial CoQ

Succinate dehydrogenase (SDH) is a hetero-tetrameric nuclear-encoded complex responsible for the oxidation of succinate to fumarate in the tricarboxylic acid (TCA) cycle. Loss-of-function mutations in any of the SDH genes are associated with cancer formation. However, the impact of SDH loss on cell metabolism and the mechanisms enabling growth of SDH-defective cells are largely unknown. Here, we generated Sdhb-ablated kidney mouse cells and employed comparative metabolomics and stable isotope-labelling approaches to identify nutritional requirements and metabolic adaptations to SDH loss. We found that lack of SDH activity commits cells to consume extracellular pyruvate, which sustains Warburg-like bioenergetic features. We further demonstrated that pyruvate carboxylation diverts glucose-derived carbons into aspartate biosynthesis, thus sustaining cell growth. By identifying pyruvate carboxylase as an essential gene for the proliferation and tumorigenic capacity of SDH-deficient cells, this study revealed a metabolic vulnerability for potential future treatment of SDH-associated malignancies.

Cultures of dissociated hippocampal neurons are often used to study neuronal cell biology. We report that the development of these neurons is strongly affected by chemicals leaching from commonly used disposable medical-grade syringes and syringe filters. Contamination of culture medium by bioactive substance(s) from syringes and filters occurred with multiple manufacturing lots and filter types under normal use conditions and resulted in changes to neurite growth, axon formation and the neuronal microtubule cytoskeleton. The effects on neuronal morphology were concentration dependent and significant effects were detected even after substantial dilution of the contaminated medium. Gas chromatography-mass spectrometry analyses revealed many chemicals eluting from the syringes and filters. Three of these chemicals (stearic acid, palmitic acid and 1,2-ethanediol monoacetate) were tested but showed no effects on neurite growth. Similar changes in neuronal morphology were seen with high concentrations of bisphenol A and dibutyl phthalate, two hormonally active plasticisers. Although no such compounds were detected by gas chromatography–mass spectrometry, unknown plasticisers in leachates may affect neurites. This is the first study to show that leachates from laboratory consumables can alter the growth of cultured hippocampal neurons. We highlight important considerations to ensure leachate contamination does not compromise cell biology experiments.

Melanoma cells steer out of tumours using self-generated lysophosphatidic acid (LPA) gradients. The cells break down LPA, which is present at high levels around the tumours, creating a dynamic gradient that is low in the tumour and high outside. They then migrate up this gradient, creating a complex and evolving outward chemotactic stimulus. Here, we introduce a new assay for self-generated chemotaxis, and show that raising LPA levels causes a delay in migration rather than loss of chemotactic efficiency. Knockdown of the lipid phosphatase LPP3 – but not of its homologues LPP1 or LPP2 – diminishes the cell's ability to break down LPA. This is specific for chemotactically active LPAs, such as the 18:1 and 20:4 species. Inhibition of autotaxin-mediated LPA production does not diminish outward chemotaxis, but loss of LPP3-mediated LPA breakdown blocks it. Similarly, in both 2D and 3D invasion assays, knockdown of LPP3 diminishes the ability of melanoma cells to invade. Our results demonstrate that LPP3 is the key enzyme in the breakdown of LPA by melanoma cells, and confirm the importance of attractant breakdown in LPA-mediated cell steering. This article has an associated First Person interview with the first author of the paper., Highlighted Article: Melanoma cells can create and follow their own gradients of attractant, via a new mechanism by which tumour cells may undergo metastasis.

Serine catabolism results in formate efflux that exceeds anabolic demands for purine synthesis., Serine catabolism to glycine and a one-carbon unit has been linked to the anabolic requirements of proliferating mammalian cells. However, genome-scale modeling predicts a catabolic role with one-carbon release as formate. We experimentally prove that in cultured cancer cells and nontransformed fibroblasts, most of the serine-derived one-carbon units are released from cells as formate, and that formate release is dependent on mitochondrial reverse 10-CHO-THF synthetase activity. We also show that in cancer cells, formate release is coupled to mitochondrial complex I activity, whereas in nontransformed fibroblasts, it is partially insensitive to inhibition of complex I activity. We demonstrate that in mice, about 50% of plasma formate is derived from serine and that serine starvation or complex I inhibition reduces formate synthesis in vivo. These observations transform our understanding of one-carbon metabolism and have implications for the treatment of diabetes and cancer with complex I inhibitors.

Technological advances in the area of analytical chemistry and the development of state-of-the-art analytical instrumentation have allowed for a shift in the focus from a previously strict targeted approach towards the approach adopted in metabolomics, the essence of which is non-targeted providing an unbiased analysis of metabolites in biological samples. Metabolite profiling methods have served their purpose in providing descriptive information about biological systems through qualitative and relative semi-quantitative data. However, quantitative characterization of a system cannot be fully accomplished without using absolute metabolite concentrations, an area which is lacking in most current metabolomics platforms. The objective of this work was to develop a calibration-curve free method for quantitation of amino and non-amino organic acids in biological samples. We developed a novel calibration curve-free GC–MS method based on isotope-coded derivatization for absolute non-targeted quantification of polar metabolites. A new R-based package MetabQ was created for automated data processing of GC–MS data files performing data extraction and calculation of absolute metabolite values. The new method requires metabolite response factors which should be calculated only once for each equipment, and was validated for metabolite quantification of different biological matrices. The method showed high reproducibility and accuracy, and does not require the use of calibration curves using standards to be analyzed in parallel with every sample batch. However, there is a small group of metabolites where their quantification required additional steps of correction due to their chemical instability. The introduced R package significantly increased the throughput in the data analysis process, extensively reducing the time required to perform the task manually. Our novel approach gives the potential to identify and quantify hundreds of metabolites, far exceeding the capabilities of any absolute quantitative targeted metabolite analysis, limited only by the size of the mass spectral library.

Cells can synthesize fatty acids by ligating multiple acetyl units from acetyl-CoA. This is followed by desaturation and elongation reactions to produce a variety of fatty acids required for proper cellular functioning. Alternatively, exogenous lipid sources can contribute to cellular fatty acid pools. Here, we present a method based on incorporation of (13)C-carbon from labeled substrates into fatty acids and subsequent mass spectrometry analysis. The resulting labeling patterns can be used to determine (1) (13)C-enrichment of lipogenic acetyl-CoA, (2) the relative contributions of synthesis and uptake, and (3) absolute fatty acid fluxes. We begin by providing a background and general principles regarding the use of stable isotopes to study fatty acid metabolism. We then proceed with detailing procedures for sample preparation and both GC-MS and LC-MS analysis of isotope incorporation. Finally, we discuss the interpretation of the resulting fatty acid-labeling patterns.

Bleaching (dinoflagellate symbiont loss) is one of the greatest threats facing coral reefs. The functional cnidarian-dinoflagellate symbiosis, which forms coral reefs, is based on the bi-directional exchange of nutrients. During thermal stress this exchange breaks down, however major gaps remain in our understanding of the roles of free metabolite pools in symbiosis and homeostasis. In this study we applied gas chromatography-mass spectrometry (GC-MS) to explore thermally induced changes in intracellular pools of amino and non-amino organic acids in each partner of the model sea anemone Aiptasia sp. and its dinoflagellate symbiont. Elevated temperatures (32°C for 6 d) resulted in symbiont photoinhibition and bleaching. Thermal stress induced distinct changes in the metabolite profiles of both partners, associated with alterations to central metabolism, oxidative state, cell structure, biosynthesis and signalling. Principally, we detected elevated pools of polyunsaturated fatty acids (PUFAs) in the symbiont, indicative of modifications to lipogenesis/lysis, membrane structure and nitrogen assimilation. In contrast, reductions of multiple PUFAs were detected in host pools, indicative of increased metabolism, peroxidation and/or reduced translocation of these groups. Accumulations of glycolysis intermediates were also observed in both partners, associated with photoinhibition and downstream reductions in carbohydrate metabolism. Correspondingly, we detected accumulations of amino acids and intermediate groups in both partners, with roles in gluconeogenesis and acclimation responses to oxidative stress. These data further our understanding of cellular responses to thermal stress in the symbiosis and generates hypotheses relating to the secondary roles of a number of compounds in homeostasis and heat stress resistance.

Oxygen and oxidative stress have become relevant components in clarifying the mechanism that weakens bacterial cells in parallel to the mode of action of bactericidal antibiotics. Given the importance of oxidative stress in the overall defense mechanism of bacteria and their apparent role in the antimicrobial mode of action, it is important to understand how bacteria respond to this stress at a metabolic level. The aim of this study was to determine the impact of oxygen on the metabolism of the facultative anaerobe Enterococcus faecalis using continuous culture, metabolomics and 13C-enrichment of metabolic intermediates. When E. faecalis was rapidly transitioned from anaerobic to aerobic growth, cellular metabolism was directed towards intracellular glutathione production and glycolysis was upregulated two-fold, which increased the supply of critical metabolite precursors (e.g. glycine and glutamate) for sulfur metabolism and glutathione biosynthesis as well as reducing power for cellular respiration in the presence of haemin. The ultimate metabolic response of E. faecalis to an aerobic environment was the upregulation of fatty acid metabolism and benzoate degradation, which was linked to important changes in the bacterial membrane composition as evidenced by changes in membrane fatty acid composition and the reduction of membrane-associated demethylmenaquinone. These key metabolic pathways associated with the response of E. faecalis to oxygen may represent potential new targets to increase the susceptibility of this bacterium to bactericidal drugs., This work was funded by the HRC (Health and Research Council of New Zealand) and the FCT (Portuguese Foundation for Science and Technology), with grant reference SFRH/BD/47016/2008.

Phenylethyl alcohol was one of the first quorum sensing molecules (QSMs) identified in C. albicans. This extracellular signalling molecule inhibits the hyphal formation of C. albicans at high cell density. Little is known, however, about the underlying mechanisms by which this QSM regulates the morphological switches of C. albicans. Therefore, we have applied metabolomics and isotope labelling experiments to investigate the metabolic changes that occur in C. albicans in response to phenylethyl alcohol under defined hyphae-inducing conditions. Our results showed a global upregulation of central carbon metabolism when hyphal development was suppressed by phenylethyl alcohol. By comparing the metabolic changes in response to phenylethyl alcohol to our previous metabolomic studies, we were able to short-list 7 metabolic pathways from central carbon metabolism that appear to be associated with C. albicans morphogenesis. Furthermore, isotope-labelling data showed that phenylethyl alcohol is indeed taken up and catabolised by yeast cells. Isotope-labelled carbon atoms were found in the majority of amino acids as well as in lactate and glyoxylate. However, isotope-labelled carbon atoms from phenylethyl alcohol accumulated mainly in the pyridine ring of NAD(+)/NADH and NADP(-/)NADPH molecules, showing that these nucleotides were the main products of phenylethyl alcohol catabolism. Interestingly, two metabolic pathways where these nucleotides play an important role, nitrogen metabolism and nicotinate/nicotinamide metabolism, were also short-listed through our previous metabolomics works as metabolic pathways likely to be closely associated with C. albicans morphogenesis.