The association between eicosanoids and incident atrial fibrillation in the Framingham Heart Study.

Chronic inflammation is a continuous low-grade activation of the systemic immune response. Whereas downstream inflammatory markers are associated with atrial fibrillation (AF), upstream inflammatory effectors including eicosanoids are less studied. To examine the association between eicosanoids and incident AF. We used a liquid chromatography-mass spectrometry for the non-targeted measurement of 161 eicosanoids and eicosanoid-related metabolites in the Framingham Heart Study. The association of each eicosanoid and incident AF was assessed using Cox proportional hazards models and adjusted for AF risk factors, including age, sex, height, weight, systolic/diastolic blood pressure, current smoking, antihypertensive medication, diabetes, history of myocardial infarction and heart failure. False discovery rate (FDR) was used to adjust for multiple testing. Eicosanoids with FDR < 0.05 were considered significant. In total, 2676 AF-free individuals (mean age 66 ± 9 years, 56% females) were followed for mean 10.8 ± 3.4 years; 351 participants developed incident AF. Six eicosanoids were associated with incident AF after adjusting for multiple testing (FDR < 0.05). A joint score was built from the top eicosanoids weighted by their effect sizes, which was associated with incident AF (HR = 2.72, CI = 1.71-4.31, P = 2.1 × 10-5). In conclusion, six eicosanoids were associated with incident AF after adjusting for clinical risk factors for AF.

Association of Physical Activity With Bioactive Lipids and Cardiovascular Events.

BACKGROUND: To clarify the mechanisms underlying physical activity (PA)-related cardioprotection, we examined the association of PA with plasma bioactive lipids (BALs) and cardiovascular disease (CVD) events. We additionally performed genome-wide associations. METHODS: PA-bioactive lipid associations were examined in VITAL (VITamin D and OmegA-3 TriaL)-clinical translational science center (REGISTRATION: URL: https://www. CLINICALTRIALS: gov; Unique identifier: NCT01169259; N=1032) and validated in JUPITER (Justification for the Use of statins in Prevention: an Intervention Trial Evaluating Rosuvastatin)-NC (REGISTRATION: URL: https://www. CLINICALTRIALS: gov; Unique identifier: NCT00239681; N=589), using linear models adjusted for age, sex, race, low-density lipoprotein-cholesterol, total-C, and smoking. Significant BALs were carried over to examine associations with incident CVD in 2 nested CVD case-control studies: VITAL-CVD (741 case-control pairs) and JUPITER-CVD (415 case-control pairs; validation). RESULTS: We detected 145 PA-bioactive lipid validated associations (false discovery rate <0.1). Annotations were found for 6 of these BALs: 12,13-diHOME, 9,10-diHOME, lysoPC(15:0), oxymorphone-3b-D-glucuronide, cortisone, and oleoyl-glycerol. Genetic analysis within JUPITER-NC showed associations of 32 PA-related BALs with 22 single-nucleotide polymorphisms. From PA-related BALs, 12 are associated with CVD. CONCLUSIONS: We identified a PA-related bioactive lipidome profile out of which 12 BALs also had opposite associations with incident CVD events.

Mapping choline metabolites in normal and transformed cells.

INTRODUCTION: Choline is an essential human nutrient that is particular important for proliferating cells, and altered choline metabolism has been associated with cancer transformation. Yet, the various metabolic fates of choline in proliferating cells have not been investigated systematically. OBJECTIVES: This study aims to map the metabolic products of choline in normal and cancerous proliferating cells. METHODS: We performed 13C-choline tracing followed by liquid chromatography-high resolution mass spectrometry (LC-HRMS) analysis of metabolic products in normal and in vitro-transformed (tumor-forming) epithelial cells, and also in tumor-derived cancer cell lines. Selected metabolites were quantified by internal standards. RESULTS: Untargeted analysis revealed 121 LCMS peaks that were 13C-labeled from choline, including various phospholipid species, but also previously unknown products such as monomethyl- and dimethyl-ethanolamines. Interestingly, we observed formation of betaine from choline specifically in tumor-derived cells. Expression of choline dehydrogenase (CHDH), which catalyzes the first step of betaine synthesis, correlated with betaine synthesis across the cell lines studied. RNAi silencing of CHDH did not affect cell proliferation, although we observed an increased fraction of G2M phase cells with some RNAi sequences, suggesting that CHDH and its product betaine may play a role in cell cycle progression. Betaine cell concentration was around 10 µM, arguing against an osmotic function, and was not used as a methyl donor. The function of betaine in these tumor-derived cells is presently unknown. CONCLUSION: This study identifies novel metabolites of choline in cancer and normal cell lines, and reveals altered choline metabolism in cancer cells.

Mapping metabolic oscillations during cell cycle progression.

Proliferating cells must synthesize a wide variety of macromolecules while progressing through the cell cycle, but the coordination between cell cycle progression and cellular metabolism is still poorly understood. To identify metabolic processes that oscillate over the cell cycle, we performed comprehensive, non-targeted liquid chromatography-high resolution mass spectrometry (LC-HRMS) based metabolomics of HeLa cells isolated in the G1 and SG2M cell cycle phases, capturing thousands of diverse metabolite ions. When accounting for increased total metabolite abundance due to cell growth throughout the cell cycle, 18% of the observed LC-HRMS peaks were at least twofold different between the stages, consistent with broad metabolic remodeling throughout the cell cycle. While most amino acids, phospholipids, and total ribonucleotides were constant across cell cycle phases, consistent with the view that total macromolecule synthesis does not vary across the cell cycle, certain metabolites were oscillating. For example, ribonucleotides were highly phosphorylated in SG2M, indicating an increase in energy charge, and several phosphatidylinositols were more abundant in G1, possibly indicating altered membrane lipid signaling. Within carbohydrate metabolism, pentose phosphates and methylglyoxal metabolites were associated with the cycle. Interestingly, hundreds of yet uncharacterized metabolites similarly oscillated between cell cycle phases, suggesting previously unknown metabolic activities that may be synchronized with cell cycle progression, providing an important resource for future studies.

Effects of Diet versus Gastric Bypass on Metabolic Function in Diabetes.

BACKGROUND: Some studies have suggested that in people with type 2 diabetes, Roux-en-Y gastric bypass has therapeutic effects on metabolic function that are independent of weight loss. METHODS: We evaluated metabolic regulators of glucose homeostasis before and after matched (approximately 18%) weight loss induced by gastric bypass (surgery group) or diet alone (diet group) in 22 patients with obesity and diabetes. The primary outcome was the change in hepatic insulin sensitivity, assessed by infusion of insulin at low rates (stages 1 and 2 of a 3-stage hyperinsulinemic euglycemic pancreatic clamp). Secondary outcomes were changes in muscle insulin sensitivity, beta-cell function, and 24-hour plasma glucose and insulin profiles. RESULTS: Weight loss was associated with increases in mean suppression of glucose production from baseline, by 7.04 µmol per kilogram of fat-free mass per minute (95% confidence interval [CI], 4.74 to 9.33) in the diet group and by 7.02 µmol per kilogram of fat-free mass per minute (95% CI, 3.21 to 10.84) in the surgery group during clamp stage 1, and by 5.39 (95% CI, 2.44 to 8.34) and 5.37 (95% CI, 2.41 to 8.33) µmol per kilogram of fat-free mass per minute in the two groups, respectively, during clamp stage 2; there were no significant differences between the groups. Weight loss was associated with increased insulin-stimulated glucose disposal, from 30.5±15.9 to 61.6±13.0 µmol per kilogram of fat-free mass per minute in the diet group and from 29.4±12.6 to 54.5±10.4 µmol per kilogram of fat-free mass per minute in the surgery group; there was no significant difference between the groups. Weight loss increased beta-cell function (insulin secretion relative to insulin sensitivity) by 1.83 units (95% CI, 1.22 to 2.44) in the diet group and by 1.11 units (95% CI, 0.08 to 2.15) in the surgery group, with no significant difference between the groups, and it decreased the areas under the curve for 24-hour plasma glucose and insulin levels in both groups, with no significant difference between the groups. No major complications occurred in either group. CONCLUSIONS: In this study involving patients with obesity and type 2 diabetes, the metabolic benefits of gastric bypass surgery and diet were similar and were apparently related to weight loss itself, with no evident clinically important effects independent of weight loss. (Funded by the National Institutes of Health and others; ClinicalTrials.gov number, NCT02207777.).

Fructose stimulated de novo lipogenesis is promoted by inflammation.

Benign hepatosteatosis, affected by lipid uptake, de novo lipogenesis and fatty acid (FA) oxidation, progresses to non-alcoholic steatohepatitis (NASH) on stress and inflammation. A key macronutrient proposed to increase hepatosteatosis and NASH risk is fructose. Excessive intake of fructose causes intestinal-barrier deterioration and endotoxaemia. However, how fructose triggers these alterations and their roles in hepatosteatosis and NASH pathogenesis remain unknown. Here we show, using mice, that microbiota-derived Toll-like receptor (TLR) agonists promote hepatosteatosis without affecting fructose-1-phosphate (F1P) and cytosolic acetyl-CoA. Activation of mucosal-regenerative gp130 signalling, administration of the YAP-induced matricellular protein CCN1 or expression of the antimicrobial peptide Reg3b (beta) peptide counteract fructose-induced barrier deterioration, which depends on endoplasmic-reticulum stress and subsequent endotoxaemia. Endotoxin engages TLR4 to trigger TNF production by liver macrophages, thereby inducing lipogenic enzymes that convert F1P and acetyl-CoA to FA in both mouse and human hepatocytes.

Cellular sensing of extracellular purine nucleosides triggers an innate IFN-ß response.

Mechanisms linking immune sensing of DNA danger signals in the extracellular environment to innate pathways in the cytosol are poorly understood. Here, we identify a previously unidentified immune-metabolic axis by which cells respond to purine nucleosides and trigger a type I interferon-ß (IFN-ß) response. We find that depletion of ADA2, an ectoenzyme that catabolizes extracellular dAdo to dIno, or supplementation of dAdo or dIno stimulates IFN-ß. Under conditions of reduced ADA2 enzyme activity, dAdo is transported into cells and undergoes catabolysis by the cytosolic isoenzyme ADA1, driving intracellular accumulation of dIno. dIno is a functional immunometabolite that interferes with the cellular methionine cycle by inhibiting SAM synthetase activity. Inhibition of SAM-dependent transmethylation drives epigenomic hypomethylation and overexpression of immune-stimulatory endogenous retroviral elements that engage cytosolic dsRNA sensors and induce IFN-ß. We uncovered a previously unknown cellular signaling pathway that responds to extracellular DNA-derived metabolites, coupling nucleoside catabolism by adenosine deaminases to cellular IFN-ß production.

HORMA Domain Proteins and a Trip13-like ATPase Regulate Bacterial cGAS-like Enzymes to Mediate Bacteriophage Immunity.

Bacteria are continually challenged by foreign invaders, including bacteriophages, and have evolved a variety of defenses against these invaders. Here, we describe the structural and biochemical mechanisms of a bacteriophage immunity pathway found in a broad array of bacteria, including E. coli and Pseudomonas aeruginosa. This pathway uses eukaryotic-like HORMA domain proteins that recognize specific peptides, then bind and activate a cGAS/DncV-like nucleotidyltransferase (CD-NTase) to generate a cyclic triadenylate (cAAA) second messenger; cAAA in turn activates an endonuclease effector, NucC. Signaling is attenuated by a homolog of the AAA+ ATPase Pch2/TRIP13, which binds and disassembles the active HORMA-CD-NTase complex. When expressed in non-pathogenic E. coli, this pathway confers immunity against bacteriophage λ through an abortive infection mechanism. Our findings reveal the molecular mechanisms of a bacterial defense pathway integrating a cGAS-like nucleotidyltransferase with HORMA domain proteins for threat sensing through protein detection and negative regulation by a Trip13 ATPase.

Biliopancreatic Diversion Induces Greater Metabolic Improvement Than Roux-en-Y Gastric Bypass.

Diabetes remission is greater after biliopancreatic diversion (BPD) than Roux-en-Y gastric bypass (RYGB) surgery. We used a mixed-meal test with ingested and infused glucose tracers and the hyperinsulinemic-euglycemic clamp procedure with glucose tracer infusion to assess the effect of 20% weight loss induced by either RYGB or BPD on glucoregulation in people with obesity (ClinicalTrials.gov number: NCT03111953). The rate of appearance of ingested glucose into the circulation was much slower, and the postprandial increases in plasma glucose and insulin concentrations were markedly blunted after BPD compared to after RYGB. Insulin sensitivity, assessed as glucose disposal rate during insulin infusion, was ∼45% greater after BPD than RYGB, whereas ß cell function was not different between groups. These results demonstrate that compared with matched-percentage weight loss induced by RYGB, BPD has unique beneficial effects on glycemic control, manifested by slower postprandial glucose absorption, blunted postprandial plasma glucose and insulin excursions, and greater improvement in insulin sensitivity.

Mapping Metabolic Events in the Cancer Cell Cycle Reveals Arginine Catabolism in the Committed SG2M Phase.

Alterations in cell-cycle regulation and cellular metabolism are associated with cancer transformation, and enzymes active in the committed cell-cycle phase may represent vulnerabilities of cancer cells. Here, we map metabolic events in the G1 and SG2M phases by combining cell sorting with mass spectrometry-based isotope tracing, revealing hundreds of cell-cycle-associated metabolites. In particular, arginine uptake and ornithine synthesis are active during SG2M in transformed but not in normal cells, with the mitochondrial arginase 2 (ARG2) enzyme as a potential mechanism. While cancer cells exclusively use ARG2, normal epithelial cells synthesize ornithine via ornithine aminotransferase (OAT). Knockdown of ARG2 markedly reduces cancer cell growth and causes G2M arrest, while not inducing compensation via OAT. In human tumors, ARG2 is highly expressed in specific tumor types, including basal-like breast tumors. This study sheds light on the interplay between metabolism and cell cycle and identifies ARG2 as a potential metabolic target.

Profiling the Metabolism of Human Cells by Deep 13C Labeling.

Studying metabolic activities in living cells is crucial for understanding human metabolism, but facile methods for profiling metabolic activities in an unbiased, hypothesis-free manner are still lacking. To address this need, we here introduce the deep-labeling method, which combines a custom 13C medium with high-resolution mass spectrometry. A proof-of-principle study on human cancer cells demonstrates that deep labeling can identify hundreds of endogenous metabolites as well as active and inactive pathways. For example, protein and nucleic acids were almost exclusively de novo synthesized, while lipids were partly derived from serum; synthesis of cysteine, carnitine, and creatine was absent, suggesting metabolic dependencies; and branched-chain keto acids (BCKAs) were formed and metabolized to short-chain acylcarnitines, but did not enter the tricarboxylic acid cycle. Remarkably, BCKAs could substitute for essential amino acids to support growth. The deep-labeling method may prove useful to map metabolic phenotypes across a range of cell types and conditions.

The glutamate/cystine xCT antiporter antagonizes glutamine metabolism and reduces nutrient flexibility.

As noted by Warburg, many cancer cells depend on the consumption of glucose. We performed a genetic screen to identify factors responsible for glucose addiction and recovered the two subunits of the xCT antiporter (system xc-), which plays an antioxidant role by exporting glutamate for cystine. Disruption of the xCT antiporter greatly improves cell viability after glucose withdrawal, because conservation of glutamate enables cells to maintain mitochondrial respiration. In some breast cancer cells, xCT antiporter expression is upregulated through the antioxidant transcription factor Nrf2 and contributes to their requirement for glucose as a carbon source. In cells carrying patient-derived mitochondrial DNA mutations, the xCT antiporter is upregulated and its inhibition improves mitochondrial function and cell viability. Therefore, although upregulation of the xCT antiporter promotes antioxidant defence, it antagonizes glutamine metabolism and restricts nutrient flexibility. In cells with mitochondrial dysfunction, the potential utility of xCT antiporter inhibition should be further tested.

Thiopeptide antibiotics stimulate biofilm formation in Bacillus subtilis.

Bacteria have evolved the ability to produce a wide range of structurally complex natural products historically called "secondary" metabolites. Although some of these compounds have been identified as bacterial communication cues, more frequently natural products are scrutinized for antibiotic activities that are relevant to human health. However, there has been little regard for how these compounds might otherwise impact the physiology of neighboring microbes present in complex communities. Bacillus cereus secretes molecules that activate expression of biofilm genes in Bacillus subtilis. Here, we use imaging mass spectrometry to identify the thiocillins, a group of thiazolyl peptide antibiotics, as biofilm matrix-inducing compounds produced by B. cereus. We found that thiocillin increased the population of matrix-producing B. subtilis cells and that this activity could be abolished by multiple structural alterations. Importantly, a mutation that eliminated thiocillin's antibiotic activity did not affect its ability to induce biofilm gene expression in B. subtilis. We go on to show that biofilm induction appears to be a general phenomenon of multiple structurally diverse thiazolyl peptides and use this activity to confirm the presence of thiazolyl peptide gene clusters in other bacterial species. Our results indicate that the roles of secondary metabolites initially identified as antibiotics may have more complex effects--acting not only as killing agents, but also as specific modulators of microbial cellular phenotypes.

Interspecies interactions stimulate diversification of the Streptomyces coelicolor secreted metabolome.

UNLABELLED: Soils host diverse microbial communities that include filamentous actinobacteria (actinomycetes). These bacteria have been a rich source of useful metabolites, including antimicrobials, antifungals, anticancer agents, siderophores, and immunosuppressants. While humans have long exploited these compounds for therapeutic purposes, the role these natural products may play in mediating interactions between actinomycetes has been difficult to ascertain. As an initial step toward understanding these chemical interactions at a systems level, we employed the emerging techniques of nanospray desorption electrospray ionization (NanoDESI) and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) imaging mass spectrometry to gain a global chemical view of the model bacterium Streptomyces coelicolor interacting with five other actinomycetes. In each interaction, the majority of secreted compounds associated with S. coelicolor colonies were unique, suggesting an idiosyncratic response from S. coelicolor. Spectral networking revealed a family of unknown compounds produced by S. coelicolor during several interactions. These compounds constitute an extended suite of at least 12 different desferrioxamines with acyl side chains of various lengths; their production was triggered by siderophores made by neighboring strains. Taken together, these results illustrate that chemical interactions between actinomycete bacteria exhibit high complexity and specificity and can drive differential secondary metabolite production. IMPORTANCE: Actinomycetes, filamentous actinobacteria from the soil, are the deepest natural source of useful medicinal compounds, including antibiotics, antifungals, and anticancer agents. There is great interest in developing new strategies that increase the diversity of metabolites secreted by actinomycetes in the laboratory. Here we used several metabolomic approaches to examine the chemicals made by these bacteria when grown in pairwise coculture. We found that these interspecies interactions stimulated production of numerous chemical compounds that were not made when they grew alone. Among these compounds were at least 12 different versions of a molecule called desferrioxamine, a siderophore used by the bacteria to gather iron. Many other compounds of unknown identity were also observed, and the pattern of compound production varied greatly among the interaction sets. These findings suggest that chemical interactions between actinomycetes are surprisingly complex and that coculture may be a promising strategy for finding new molecules from actinomycetes.

MS/MS networking guided analysis of molecule and gene cluster families.

The ability to correlate the production of specialized metabolites to the genetic capacity of the organism that produces such molecules has become an invaluable tool in aiding the discovery of biotechnologically applicable molecules. Here, we accomplish this task by matching molecular families with gene cluster families, making these correlations to 60 microbes at one time instead of connecting one molecule to one organism at a time, such as how it is traditionally done. We can correlate these families through the use of nanospray desorption electrospray ionization MS/MS, an ambient pressure MS technique, in conjunction with MS/MS networking and peptidogenomics. We matched the molecular families of peptide natural products produced by 42 bacilli and 18 pseudomonads through the generation of amino acid sequence tags from MS/MS data of specific clusters found in the MS/MS network. These sequence tags were then linked to biosynthetic gene clusters in publicly accessible genomes, providing us with the ability to link particular molecules with the genes that produced them. As an example of its use, this approach was applied to two unsequenced Pseudoalteromonas species, leading to the discovery of the gene cluster for a molecular family, the bromoalterochromides, in the previously sequenced strain P. piscicida JCM 20779(T). The approach itself is not limited to 60 related strains, because spectral networking can be readily adopted to look at molecular family-gene cluster families of hundreds or more diverse organisms in one single MS/MS network.