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Crosstalk between metabolism and circadian rhythms is a fundamental building block of multicellular life, and disruption of this reciprocal communication could be relevant to disease. Here, we investigated whether maintenance of circadian rhythms depends on specific metabolic pathways, particularly in the context of cancer. We found that in adult mouse fibroblasts, ATP levels were a major contributor to signal from a clock gene luciferase reporter, although not necessarily to the strength of circadian cycling. In contrast, we identified significant metabolic control of circadian function across a series of pancreatic adenocarcinoma cell lines. Metabolic profiling of congenic tumor cell clones revealed substantial diversity among these lines that we used to identify clones to generate circadian reporter lines. We observed diverse circadian profiles among these lines that varied with their metabolic phenotype: The most hypometabolic line [exhibiting low levels of oxidative phosphorylation (OxPhos) and glycolysis] had the strongest rhythms, while the most hypermetabolic line had the weakest rhythms. Pharmacological enhancement of OxPhos decreased the amplitude of circadian oscillation in a subset of tumor cell lines. Strikingly, inhibition of OxPhos enhanced circadian rhythms only in the tumor cell line in which glycolysis was also low, thereby establishing a hypometabolic state. We further analyzed metabolic and circadian phenotypes across a panel of human patient-derived melanoma cell lines and observed a significant negative association between metabolic activity and circadian cycling strength. Together, these findings suggest that metabolic heterogeneity in cancer directly contributes to circadian function and that high levels of glycolysis or OxPhos independently disrupt circadian rhythms in these cells. [ABSTRACT FROM AUTHOR]

Polycyclic aromatic hydrocarbons (PAHs) are environmental pollutants generated from the incomplete combustion of organic material. PAHs have been studied as genotoxicants, but some also act via non-genotoxic mechanisms in estrogen-dependent malignancies, such as breast cancer. PAHs require metabolic activation to electrophilic metabolites to exert their genotoxicity but non-genotoxic properties may also contribute to their carcinogenicity. The role of PAHs in endometrial cancer, a cancer associated with unopposed estrogen action is unknown. We assessed the metabolism of the representative PAH, benzo[a]pyrene (B[a]P), to estrogenic compounds in Ishikawa human endometrial cells in the presence and absence of cytochrome P450 induction. Using stable-isotope dilution high-performance liquid chromatography and APCI tandem mass spectrometry in the selected reaction monitoring mode, we analyzed B[a]P metabolism in Ishikawa cells. Estrogenic activity of B[a]P metabolites was determined by the endogenous estrogen inducible alkaline phosphatase reporter gene and an exogenous estrogen response element (ERE) luciferase reporter gene construct. We also assessed whether PAHs can induce a proliferative phenotype via estrogen receptor (ER)- and non-ER-regulated pathways. We demonstrate that B[a]P can be metabolized in human endometrial cells into 3-OH-B[a]P and B[a]P-7,8-dione in sufficient amounts to activate ERs. We also show that only B[a]P-7,8-dione induces endometrial cell proliferation at concentrations lower than required to activate the ER; instead nongenomic signaling by the EGF receptor (EGFR) and activation of the mitogen-activated protein kinase (MAPK) pathway was responsible. This work indicates that human endometrial cells can metabolize PAHs into estrogenic metabolites, which may induce cell proliferation through non-ER-regulated pathways. [ABSTRACT FROM AUTHOR]

Background: Storage of platelets (PLTs) results in a progressive defect termed PLT storage lesion (PSL). The PSL is characterized by poor PLT quality on a variety of assays. Metabolic defects are thought to underlie the PSL; thus this study was designed to quantitatively probe specific metabolic pathways over PLT storage.Study Design and Methods: Relative incorporation of stable isotope-labeled substrates was quantified by isotopologue analysis of key acyl-coenzyme A (CoA) thioester products for fresh, viable (after collection, Days 2-5), and expired PLTs (after Day 5). We examined the incorporation of acetate, glucose, and palmitate into acetyl- and succinyl-CoA via liquid chromatography-tandem mass spectrometry.Results: Storage-related defects in the incorporation of acetyl-CoA derived from acetate and palmitate were observed. Carbon derived from palmitate and acetate in succinyl-CoA was reduced over storage time. Glucose incorporation into succinyl-CoA increased in viable PLTs and then decreased in expired PLTs. Carbon derived from octanoate and pyruvate remained partially able to incorporate into acetyl- and succinyl-CoA in expired PLTs, with high variability in pyruvate incorporation.Conclusion: Isotopologue analysis is useful in probing substrate specific defects in the PSL. [ABSTRACT FROM AUTHOR]

Rationale and Objectives: The aim of this study was to assess the feasibility of 18F-fluorodeoxyglucose (FDG)-positron emission tomography/computed tomography (PET/CT) to systematically detect and quantify differential effects of chronic tobacco use in organs of the whole body.Materials and Methods: Twenty healthy male subjects (10 nonsmokers and 10 chronic heavy smokers) were enrolled. Subjects underwent whole-body FDG-PET/CT, diagnostic unenhanced chest CT, mini-mental state examination, urine testing for oxidative stress, and serum testing. The organs of interest (thyroid, skin, skeletal muscle, aorta, heart, lung, adipose tissue, liver, spleen, brain, lumbar spinal bone marrow, and testis) were analyzed on FDG-PET/CT images to determine their metabolic activities using standardized uptake value (SUV) or metabolic volumetric product (MVP). Measurements were compared between subject groups using two-sample t tests or Wilcoxon rank-sum tests as determined by tests for normality. Correlational analyses were also performed.Results: FDG-PET/CT revealed significantly decreased metabolic activity of lumbar spinal bone marrow (MVPmean: 29.8 ± 9.7 cc vs 40.8 ± 11.6 cc, P = 0.03) and liver (SUVmean: 1.8 ± 0.2 vs 2.0 ± 0.2, P = 0.049) and increased metabolic activity of visceral adipose tissue (SUVmean: 0.35 ± 0.10 vs 0.26 ± 0.06, P = 0.02) in chronic smokers compared to nonsmokers. Normalized visceral adipose tissue volume was also significantly decreased (P = 0.04) in chronic smokers. There were no statistically significant differences in the metabolic activity of other assessed organs.Conclusions: Subclinical organ effects of chronic tobacco use are detectable and quantifiable on FDG-PET/CT. FDG-PET/CT may, therefore, play a major role in the study of systemic toxic effects of tobacco use in organs of the whole body for clinical or research purposes. [ABSTRACT FROM AUTHOR]

Release of arachidonic acid (AA) by cytoplasmic phospholipase A2 (cPLA2), followed by metabolism through cyclooxygenase-2 (COX-2) and 15-hydroxyprostaglandin dehydrogenase (15-PGDH), results in the formation of the eicosanoids 11-oxo- and 15-oxo-eicosatetraenoic acid (oxo-ETE). Both 11-oxo- and 15-oxo-ETE have been identified in human biospecimens but their function and further metabolism is poorly described. The oxo-ETEs contain an α,β-unsaturated ketone and a free carboxyclic acid, and thus may form Michael adducts with a nucleophile or a thioester with the free thiol of Coenzyme A (CoA). To examine the potential for eicosanoid-CoA formation, which has not previously been a metabolic route examined for this class of lipids, we applied a semi-targeted neutral loss scanning approach following arachidonic acid treatment in cell culture and detected inducible long-chain acyl-CoAs including a predominant AA-CoA peak. Interestingly, a series of AA-inducible acyl-CoAs at lower abundance but higher mass, likely corresponding to eicosanoid metabolites, was detected. Using a targeted LC–MS/MS approach we detected the formation of CoA thioesters of both 11-oxo- and 15-oxo-ETE and monitored the kinetics of their formation. Subsequently, we demonstrated that these acyl-CoA species undergo up to four double bond reductions. We confirmed the generation of 15-oxo-ETE-CoA in human platelets via LC-high resolution MS. Acyl-CoA thioesters of eicosanoids may provide a route to generate reducing equivalents, substrates for fatty acid oxidation, and substrates for acyl-transferases through cPLA2-dependent eicosanoid metabolism outside of the signaling contexts traditionally ascribed to eicosanoid metabolites. [ABSTRACT FROM AUTHOR]

Abstract Quantification of cellular deoxyribonucleoside mono- (dNMP), di- (dNDP), triphosphates (dNTPs) and related nucleoside metabolites are difficult due to their physiochemical properties and widely varying abundance. Involvement of dNTP metabolism in cellular processes including senescence and pathophysiological processes including cancer and viral infection make dNTP metabolism an important bioanalytical target. We modified a previously developed ion pairing reversed phase chromatography-mass spectrometry method for the simultaneous quantification and 13C isotope tracing of dNTP metabolites. dNMPs, dNDPs, and dNTPs were chromatographically resolved to avoid mis-annotation of in-source fragmentation. We used commercially available 13C15N-stable isotope labeled analogs as internal standards and show that this isotope dilution approach improves analytical figures of merit. At sufficiently high mass resolution achievable on an Orbitrap mass analyzer, stable isotope resolved metabolomics allows simultaneous isotope dilution quantification and 13C isotope tracing from major substrates including 13C-glucose. As a proof of principle, we quantified dNMP, dNDP and dNTP pools from multiple cell lines. We also identified isotopologue enrichment from glucose corresponding to ribose from the pentose-phosphate pathway in dNTP metabolites. Highlights • Deoxyribonucleoside mono-, di-, and tri-phosphates (dNTPs) are important bioanalytical targets. • LC-HRMS provides sufficient sensitivity and specificity for dNTP analysis in cellular metabolism studies. • 13C and 15N labeling can be used orthogonally in dNTPs. • Simultaneous isotope dilution based quantification and isotope tracing with 13C labeled substrates is feasible. [ABSTRACT FROM AUTHOR]

Summary Non-enzymatic protein modification driven by thioester reactivity is thought to play a major role in the establishment of cellular lysine acylation. However, the specific protein targets of this process are largely unknown. Here we report an experimental strategy to investigate non-enzymatic acylation in cells. Specifically, we develop a chemoproteomic method that separates thioester reactivity from enzymatic utilization, allowing selective enrichment of non-enzymatic acylation targets. Applying this method to cancer cell lines identifies numerous candidate targets of non-enzymatic acylation, including several enzymes in lower glycolysis. Functional studies highlight malonyl-CoA as a reactive thioester metabolite that can modify and inhibit glycolytic enzyme activity. Finally, we show that synthetic thioesters can be used as novel reagents to probe non-enzymatic acylation in living cells. Our studies provide new insights into the targets and drivers of non-enzymatic acylation, and demonstrate the utility of reactivity-based methods to experimentally investigate this phenomenon in biology and disease. [ABSTRACT FROM AUTHOR]

Background: The failing human heart is characterized by metabolic abnormalities, but these defects remains incompletely understood. In animal models of heart failure there is a switch from a predominance of fatty acid utilization to the more oxygen-sparing carbohydrate metabolism. Recent studies have reported decreases in myocardial lipid content, but the inclusion of diabetic and nondiabetic patients obscures the distinction of adaptations to metabolic derangements from adaptations to heart failure per se. Methods and Results: We performed both unbiased and targeted myocardial lipid surveys using liquid chromatography-mass spectroscopy in nondiabetic, lean, predominantly nonischemic, advanced heart failure patients at the time of heart transplantation or left ventricular assist device implantation. We identified significantly decreased concentrations of the majority of myocardial lipid intermediates, including long-chain acylcarnitines, the primary subset of energetic lipid substrate for mitochondrial fatty acid oxidation. We report for the first time significantly reduced levels of intermediate and anaplerotic acyl-coenzyme A (CoA) species incorporated into the Krebs cycle, whereas the myocardial concentration of acetyl-CoA was significantly increased in end-stage heart failure. In contrast, we observed an increased abundance of ketogenic β-hydroxybutyryl-CoA, in association with increased myocardial utilization of β-hydroxybutyrate. We observed a significant increase in the expression of the gene encoding succinyl-CoA:3-oxoacid-CoA transferase, the rate-limiting enzyme for myocardial oxidation of β-hydroxybutyrate and acetoacetate. Conclusions: These findings indicate increased ketone utilization in the severely failing human heart independent of diabetes mellitus, and they support the role of ketone bodies as an alternative fuel and myocardial ketone oxidation as a key metabolic adaptation in the failing human heart. [ABSTRACT FROM AUTHOR]

Quantitative subcellular metabolomic measurements can explain the roles of metabolites in cellular processes but are subject to multiple confounding factors. We developed stable isotope labeling of essential nutrients in cell culture-subcellular fractionation (SILEC-SF), which uses isotope-labeled internal standard controls that are present throughout fractionation and processing to quantify acyl-coenzyme A (acyl-CoA) thioesters in subcellular compartments by liquid chromatography-mass spectrometry. We tested SILEC-SF in a range of sample types and examined the compartmentalized responses to oxygen tension, cellular differentiation, and nutrient availability. Application of SILEC-SF to the challenging analysis of the nuclear compartment revealed a nuclear acyl-CoA profile distinct from that of the cytosol, with notable nuclear enrichment of propionyl-CoA. Using isotope tracing, we identified the branched chain amino acid isoleucine as a major metabolic source of nuclear propionyl-CoA and histone propionylation, thus revealing a new mechanism of crosstalk between metabolism and the epigenome. [Display omitted] • SILEC-SF allows metabolite quantitation in the subcellular compartments by LC-MS • Acyl-coenzyme A thioesters from mitochondria, cytosol, and nucleus were measured • Nuclear profile was distinct from other compartments and enriched in propionyl-CoA • Isoleucine is a major source of nuclear propionyl-CoA and histone propionylation Trefely et al., developed and applied a technique termed SILEC-SF to measure acyl-coenzyme A thioesters in subcellular compartments by liquid chromatography-mass spectrometry. SILEC-SF was applied to different cell and tissue types to examine the compartmentalized responses across the cytosol, mitochondria, and nucleus to oxygen tension, cellular differentiation, and nutrient availability. [ABSTRACT FROM AUTHOR]