Wenyun Lu

Analytical strategies for LC-MS-based targeted metabolomics.

Recent advances in mass spectrometry are enabling improved analysis of endogenous metabolites. Here we discuss several issues relevant to developing liquid chromatography-electrospray ionization-mass spectrometry methods for targeted metabolomics (i.e., quantitative analysis of dozens to hundreds of specific metabolites). Sample preparation and liquid chromatography approaches are discussed, with an eye towards the challenge of dealing with a diversity of metabolite classes in parallel. Evidence is presented that heated electrospray ionization (ESI) generally gives improved signal compared to the more traditional unheated ESI. Applicability to targeted metabolomics of triple quadrupole mass spectrometry operating in multiple reaction monitoring (MRM) mode and high mass resolution full scan mass spectrometry (e.g., time-of-flight, Orbitrap) are described. We suggest that both are viable solutions, with MRM preferred when targeting a more limited number of analytes, and full scan preferred for its potential ability to bridge targeted and untargeted metabolomics.

Autophagy suppresses progression of K-ras-induced lung tumors to oncocytomas and maintains lipid homeostasis

Macroautophagy (autophagy hereafter) degrades and recycles proteins and organelles to support metabolism and survival in starvation. Oncogenic Ras up-regulates autophagy, and Ras-transformed cell lines require autophagy for mitochondrial function, stress survival, and engrafted tumor growth. Here, the essential autophagy gene autophagy-related-7 (atg7) was deleted concurrently with K-ras(G12D) activation in mouse models for non-small-cell lung cancer (NSCLC). atg7-deficient tumors accumulated dysfunctional mitochondria and prematurely induced p53 and proliferative arrest, which reduced tumor burden that was partly relieved by p53 deletion. atg7 loss altered tumor fate from adenomas and carcinomas to oncocytomas-rare, predominantly benign tumors characterized by the accumulation of defective mitochondria. Surprisingly, lipid accumulation occurred in atg7-deficient tumors only when p53 was deleted. atg7- and p53-deficient tumor-derived cell lines (TDCLs) had compromised starvation survival and formed lipidic cysts instead of tumors, suggesting defective utilization of lipid stores. atg7 deficiency reduced fatty acid oxidation (FAO) and increased sensitivity to FAO inhibition, indicating that with p53 loss, Ras-driven tumors require autophagy for mitochondrial function and lipid catabolism. Thus, autophagy is required for carcinoma fate, and autophagy defects may be a molecular basis for the occurrence of oncocytomas. Moreover, cancers require autophagy for distinct roles in metabolism that are oncogene- and tumor suppressor gene-specific.

Conservation of the metabolomic response to starvation across two divergent microbes

We followed 68 cellular metabolites after carbon or nitrogen starvation of Escherichia coli and Saccharomyces cerevisiae, using a filter-culture methodology that allows exponential growth, nondisruptive nutrient removal, and fast quenching of metabolism. Dynamic concentration changes were measured by liquid chromatography–tandem mass spectrometry and viewed in clustered heat-map format. The major metabolic responses anticipated from metabolite-specific experiments in the literature were observed as well as a number of novel responses. When the data were analyzed by singular value decomposition, two dominant characteristic vectors were found, one corresponding to a generic starvation response and another to a nutrient-specific starvation response that is similar in both organisms. Together these captured a remarkable 72% of the metabolite concentration changes in the full data set. The responses described by the generic starvation response vector (42%) included, for example, depletion of most biosynthetic intermediates. The nutrient-specific vector (30%) included key responses such as increased phosphoenolpyruvate signaling glucose deprivation and increased α-ketoglutarate signaling ammonia deprivation. Metabolic similarity across organisms extends from the covalent reaction network of metabolism to include many elements of metabolome response to nutrient deprivation as well.

Human Pancreatic Cancer Tumors Are Nutrient Poor and Tumor Cells Actively Scavenge Extracellular Protein

Glucose and amino acids are key nutrients supporting cell growth. Amino acids are imported as monomers, but an alternative route induced by oncogenic KRAS involves uptake of extracellular proteins via macropinocytosis and subsequent lysosomal degradation of these proteins as a source of amino acids. In this study, we examined the metabolism of pancreatic ductal adenocarcinoma (PDAC), a poorly vascularized lethal KRAS-driven malignancy. Metabolomic comparisons of human PDAC and benign adjacent tissue revealed that tumor tissue was low in glucose, upper glycolytic intermediates, creatine phosphate, and the amino acids glutamine and serine, two major metabolic substrates. Surprisingly, PDAC accumulated essential amino acids. Such accumulation could arise from extracellular proteins being degraded through macropinocytosis in quantities necessary to meet glutamine requirements, which in turn produces excess of most other amino acids. Consistent with this hypothesis, active macropinocytosis is observed in primary human PDAC specimens. Moreover, in the presence of physiologic albumin, we found that cultured murine PDAC cells grow indefinitely in media lacking single essential amino acids and replicate once in the absence of free amino acids. Growth under these conditions was characterized by simultaneous glutamine depletion and essential amino acid accumulation. Overall, our findings argue that the scavenging of extracellular proteins is an important mode of nutrient uptake in PDAC.

A high-performance liquid chromatography-tandem mass spectrometry method for quantitation of nitrogen-containing intracellular metabolites

A comprehensive method of quantifying intracellular metabolite concentrations would be a valuable addition to the arsenal of tools for holistic biochemical studies. Here, we describe a step toward the development of such method: a quantitative assay for 90 nitrogen-containing cellular metabolites. The assay involves reverse-phase high-performance liquid chromatography separation followed by electrospray ionization and detection of the resulting ions using triple-quadrupole mass spectrometry in selected reaction monitoring mode. For 79 of the 90 metabolites, the assay is linear with a limit of detection of 10 ng/mL or less. Using this method, 36 metabolites can be reliably detected in extracts of the bacterium Salmonella enterica, with the identity of each metabolite confirmed by the presence, on growing of the bacteria in 13C-glucose, of a peak corresponding to the isotope-labeled form of the compound. Quantitation in biological samples is performed by mixing unlabeled test cell extract with 13C-labeled standard extract, and determining the 12C/13C-ratio for each metabolite. Using this approach, the metabolomes of growing (exponential phase) and carbon-starved (stationary phase) bacteria were compared, revealing 16 metabolites that are significantly down-regulated and five metabolites that are significantly up-regulated, in stationary phase.

Ammonium toxicity and potassium limitation in yeast.

DNA microarray analysis of gene expression in steady-state chemostat cultures limited for potassium revealed a surprising connection between potassium and ammonium: potassium limits growth only when ammonium is the nitrogen source. Under potassium limitation, ammonium appears to be toxic for Saccharomyces cerevisiae. This ammonium toxicity, which appears to occur by leakage of ammonium through potassium channels, is recapitulated under high-potassium conditions by over-expression of ammonium transporters. Although ammonium toxicity is well established in metazoans, it has never been reported for yeast. To characterize the response to ammonium toxicity, we examined the filtrates of these cultures for compounds whose excretion might serve to detoxify the ammonium (such as urea in mammals). Using liquid chromatography–tandem mass spectrometry to assay for a wide array of metabolites, we detected excreted amino acids. The amounts of amino acids excreted increased in relation to the severity of growth impairment by ammonium, suggesting that amino acid excretion is used by yeast for ammonium detoxification.

Cyclic-di-GMP-mediated repression of swarming motility by Pseudomonas aeruginosa: the pilY1 gene and its impact on surface-associated behaviors.

The intracellular signaling molecule cyclic-di-GMP (c-di-GMP) has been shown to influence surface-associated behaviors of Pseudomonas aeruginosa, including biofilm formation and swarming motility. Previously, we reported a role for the bifA gene in the inverse regulation of biofilm formation and swarming motility. The bifA gene encodes a c-di-GMP-degrading phosphodiesterase (PDE), and the Delta bifA mutant exhibits increased cellular pools of c-di-GMP, forms hyperbiofilms, and is unable to swarm. In this study, we isolated suppressors of the Delta bifA swarming defect. Strains with mutations in the pilY1 gene, but not in the pilin subunit pilA gene, show robust suppression of the swarming defect of the Delta bifA mutant, as well as its hyperbiofilm phenotype. Despite the ability of the pilY1 mutation to suppress all the c-di-GMP-related phenotypes, the global pools of c-di-GMP are not detectably altered in the Delta bifA Delta pilY1 mutant relative to the Delta bifA single mutant. We also show that enhanced expression of the pilY1 gene inhibits swarming motility, and we identify residues in the putative VWA domain of PilY1 that are important for this phenotype. Furthermore, swarming repression by PilY1 specifically requires the diguanylate cyclase (DGC) SadC, and epistasis analysis indicates that PilY1 functions upstream of SadC. Our data indicate that PilY1 participates in multiple surface behaviors of P. aeruginosa, and we propose that PilY1 may act via regulation of SadC DGC activity but independently of altering global c-di-GMP levels.

Glucose feeds the TCA cycle via circulating lactate

Mammalian tissues are fuelled by circulating nutrients, including glucose, amino acids, and various intermediary metabolites. Under aerobic conditions, glucose is generally assumed to be burned fully by tissues via the tricarboxylic acid cycle (TCA cycle) to carbon dioxide. Alternatively, glucose can be catabolized anaerobically via glycolysis to lactate, which is itself also a potential nutrient for tissues and tumours. The quantitative relevance of circulating lactate or other metabolic intermediates as fuels remains unclear. Here we systematically examine the fluxes of circulating metabolites in mice, and find that lactate can be a primary source of carbon for the TCA cycle and thus of energy. Intravenous infusions of 13C-labelled nutrients reveal that, on a molar basis, the circulatory turnover flux of lactate is the highest of all metabolites and exceeds that of glucose by 1.1-fold in fed mice and 2.5-fold in fasting mice; lactate is made primarily from glucose but also from other sources. In both fed and fasted mice, 13C-lactate extensively labels TCA cycle intermediates in all tissues. Quantitative analysis reveals that during the fasted state, the contribution of glucose to tissue TCA metabolism is primarily indirect (via circulating lactate) in all tissues except the brain. In genetically engineered lung and pancreatic cancer tumours in fasted mice, the contribution of circulating lactate to TCA cycle intermediates exceeds that of glucose, with glutamine making a larger contribution than lactate in pancreatic cancer. Thus, glycolysis and the TCA cycle are uncoupled at the level of lactate, which is a primary circulating TCA substrate in most tissues and tumours.

The Vibrio cholerae quorum-sensing autoinducer CAI-1: analysis of the biosynthetic enzyme CqsA

Vibrio cholerae, the bacterium that causes the disease cholera, controls virulence factor production and biofilm development in response to two extracellular quorum-sensing molecules, called autoinducers. The strongest autoinducer, called CAI-1 (for cholera autoinducer-1), was previously identified as (S)-3-hydroxytridecan-4-one. Biosynthesis of CAI-1 requires the enzyme CqsA. Here, we determine the CqsA reaction mechanism, identify the CqsA substrates as (S)-2-aminobutyrate and decanoyl coenzyme A, and demonstrate that the product of the reaction is 3-aminotridecan-4-one, dubbed amino-CAI-1. CqsA produces amino-CAI-1 by a pyridoxal phosphate (PLP)-dependent acyl-CoA transferase reaction. Amino-CAI-1 is converted to CAI-1 in a subsequent step via a CqsA-independent mechanism. Consistent with this, we find cells release ≥100 times more CAI-1 than amino-CAI-1. Nonetheless, V. cholerae responds to amino-CAI-1 as well as CAI-1, whereas other CAI-1 variants do not elicit a quorum-sensing response. Thus, both CAI-1 and amino-CAI-1 have potential as lead molecules in the development of an anti-cholera treatment.

Itaconic Acid Is a Mammalian Metabolite Induced during Macrophage Activation

Itaconic acid (ITA), or methylenesuccinic acid, is not generally classified as a mammalian metabolite. Using NMR-based metabolomics and (13)C-labeling, we have detected ITA in both macrophage-like VM-M3 and RAW 264.7 tumor cell lines as well as stimulated and unstimulated primary murine macrophages. Macrophage activation by addition of lipopolysaccharide and IFN-γ markedly increased ITA production and secretion. Crude cell extracts synthesize ITA via decarboxylation of cis-aconitate, indicative of a novel mammalian cis-aconitic decarboxylase activity. Our results highlight a previously unidentified biosynthetic pathway related to TCA cycle metabolism in mammalian cells and a novel metabolite that likely plays a role in macrophage-based immune response.