Liquid chromatography-high-resolution mass spectrometry (LC-MS)-based metabolomics aims to identify and quantify all metabolites, but most LC-MS peaks remain unidentified. Here we present a global ...network optimization approach, NetID, to annotate untargeted LC-MS metabolomics data. The approach aims to generate, for all experimentally observed ion peaks, annotations that match the measured masses, retention times and (when available) tandem mass spectrometry fragmentation patterns. Peaks are connected based on mass differences reflecting adduction, fragmentation, isotopes, or feasible biochemical transformations. Global optimization generates a single network linking most observed ion peaks, enhances peak assignment accuracy, and produces chemically informative peak-peak relationships, including for peaks lacking tandem mass spectrometry spectra. Applying this approach to yeast and mouse data, we identified five previously unrecognized metabolites (thiamine derivatives and N-glucosyl-taurine). Isotope tracer studies indicate active flux through these metabolites. Thus, NetID applies existing metabolomic knowledge and global optimization to substantially improve annotation coverage and accuracy in untargeted metabolomics datasets, facilitating metabolite discovery.
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
C-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,
C-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.
Liquid chromatography–mass spectrometry (LC-MS) technology allows for rapid quantitation of cellular metabolites, with metabolites identified by mass spectrometry and chromatographic retention time. ...Recently, with the development of rapid scanning high-resolution high accuracy mass spectrometers and the desire for high throughput screening, minimal or no chromatographic separation has become increasingly popular. When analyzing complex cellular extracts, however, the lack of chromatographic separation could potentially result in misannotation of structurally related metabolites. Here, we show that, even using electrospray ionization, a soft ionization method, in-source fragmentation generates unwanted byproducts of identical mass to common metabolites. For example, nucleotide-triphosphates generate nucleotide-diphosphates, and hexose-phosphates generate triose-phosphates. We evaluated yeast intracellular metabolite extracts and found more than 20 cases of in-source fragments that mimic common metabolites. Accordingly, chromatographic separation is required for accurate quantitation of many common cellular metabolites.
Mammalian organs are nourished by nutrients carried by the blood circulation. These nutrients originate from diet and internal stores, and can undergo various interconversions before their eventual ...use as tissue fuel. Here we develop isotope tracing, mass spectrometry, and mathematical analysis methods to determine the direct sources of circulating nutrients, their interconversion rates, and eventual tissue-specific contributions to TCA cycle metabolism. Experiments with fifteen nutrient tracers enabled extensive accounting for both circulatory metabolic cycles and tissue TCA inputs, across fed and fasted mice on either high-carbohydrate or ketogenic diet. We find that a majority of circulating carbon flux is carried by two major cycles: glucose-lactate and triglyceride-glycerol-fatty acid. Futile cycling through these pathways is prominent when dietary content of the associated nutrients is low, rendering internal metabolic activity robust to food choice. The presented in vivo flux quantification methods are broadly applicable to different physiological and disease states.
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•Comprehensive isotope tracer studies reveal TCA substrate usage for 11 major organs•These data also reveal interconversion rates between circulating nutrients•Circulatory fluxes are similar across high-carbohydrate and ketogenic diet•Futile cycling helps render internal metabolic activity robust to food choice
By infusing 15 different 13C-isotope tracers, Hui et al. quantify metabolic fluxes in mice on either high-carbohydrate or ketogenic diet. Results include interconversion fluxes between circulating metabolites and their contributions to the TCA cycle in 11 major organs. Rapid interconversion among circulating carbon carriers renders internal metabolic activity robust to diet.
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.
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.
During insulin-resistant states such as type II diabetes mellitus (T2DM), insulin fails to suppress hepatic glucose production (HGP) yet promotes lipid synthesis. This metabolic state has been termed ...“selective insulin resistance” to indicate a defect in one arm of the insulin-signaling cascade, potentially downstream of Akt. Here we demonstrate that Akt-dependent activation of mTORC1 and inhibition of Foxo1 are required and sufficient for de novo lipogenesis, suggesting that hepatic insulin signaling is likely to be intact in insulin-resistant states. Moreover, cell-nonautonomous suppression of HGP by insulin depends on a reduction of adipocyte lipolysis and serum FFAs but is independent of vagal efferents or glucagon signaling. These data are consistent with a model in which, during T2DM, intact liver insulin signaling drives enhanced lipogenesis while excess circulating FFAs become a dominant inducer of nonsuppressible HGP.
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•Insulin autonomously regulates hepatic lipid synthesis in vivo•Activation of mTORC1 is not sufficient to induce lipogenesis in the absence of Akt•Inhibition of Foxo1 and activation of mTORC1 are sufficient to drive lipogenesis•Foxo1 controls adipocyte lipolysis to nonautonomously regulate glucose production
During diabetes, both gluconeogenesis and lipogenesis occur in the liver. Titchenell et al. (2016) address the issue of selective insulin resistance to show that direct liver insulin signaling is required for lipogenesis, while liver insulin signaling only indirectly suppresses glucose production by reducing serum free fatty acids coming from adipocytes.
Glycolysis plays a central role in organismal metabolism, but its quantitative inputs across mammalian tissues remain unclear. Here we use 13C-tracing in mice to quantify glycolytic intermediate ...sources: circulating glucose, intra-tissue glycogen, and circulating gluconeogenic precursors. Circulating glucose is the main source of circulating lactate, the primary end product of tissue glycolysis. Yet circulating glucose highly labels glycolytic intermediates in only a few tissues: blood, spleen, diaphragm, and soleus muscle. Most glycolytic intermediates in the bulk of body tissue, including liver and quadriceps muscle, come instead from glycogen. Gluconeogenesis contributes less but also broadly to glycolytic intermediates, and its flux persists with physiologic feeding (but not hyperinsulinemic clamp). Instead of suppressing gluconeogenesis, feeding activates oxidation of circulating glucose and lactate to maintain glucose homeostasis. Thus, the bulk of the body slowly breaks down internally stored glycogen while select tissues rapidly catabolize circulating glucose to lactate for oxidation throughout the body.
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•Most circulating lactate comes from circulating glucose•But most tissue glycolytic intermediates come from glycogen•This discrepancy reflects compartmentalization of glycolysis•Glycolytic flux is concentrated in red muscle and other specialized cell populations
Glycolysis, glycogen metabolism, and gluconeogenesis produce glycolytic intermediates. Here, TeSlaa et al. quantify the sources of glycolytic intermediates in mammalian tissues, finding that the bulk of the body makes glycolytic intermediates mainly using glycogen, but most glycolytic flux comes from circulating glucose and localizes to selected cell types including red muscle.
Scope
The tea polyphenol (‐)‐epigallocatechin‐3‐gallate (EGCG) has been shown to ameliorate metabolic abnormalities and fatty liver. The present study investigates the mechanisms of actions of EGCG ...on bile acid homeostasis and lipid metabolism.
Methods
Male C57BL/6J mice are fed a low‐fat diet, a high‐fat western‐style diet, or a high‐fat western‐style diet containing 0.32% EGCG. The effects of the treatments on biochemical parameters, gene expression, and lipidomics are analyzed.
Results
EGCG treatment significantly reduces body weight gain, mesenteric fat mass, fasting blood glucose, insulin resistance, serum cholesterol, and severity of fatty liver after treatment for 17 weeks, but most of these effects were less apparent at week 33. At week 17, EGCG treatment significantly elevates the mRNA levels of cholesterol 7α‐hydroxylase, HMG‐CoA reductase, low‐density lipoprotein receptor, and scavenger receptor B1, and partially normalizes the high‐fat diet induced lipidomic profile. The intestinal bile acid content is significantly decreased by EGCG, while fecal excretion of bile acids, cholesterol, and total lipids are increased.
Conclusion
EGCG decreases bile acid reabsorption, results in lower intestinal bile acid levels, which further decreases the absorption of lipids. These actions contribute to the alleviation of metabolic abnormalities and fatty liver disease caused by the high‐fat diet.
The green tea polyphenol EGCG (0.32% in the diet) was found to reduce body weight gain, mesenteric fat mass, fasting blood glucose, insulin resistance, serum cholesterol, and severity of fatty liver in mice fed a high‐fat Western style diet. A proposed mechanism is that EGCG inhibits bile acid reabsorption and lipid absorption. EGCG also altered the gene expression of some key hepatic enzymes, resulting in partial alleviation of abnormal metabolism induced by the high‐fat diet.
We present a liquid chromatography−mass spectrometry (LC−MS) method that capitalizes on the mass-resolving power of the orbitrap to enable sensitive and specific measurement of known and ...unanticipated metabolites in parallel, with a focus on water-soluble species involved in core metabolism. The reversed phase LC method, with a cycle time 25 min, involves a water−methanol gradient on a C18 column with tributylamine as the ion pairing agent. The MS portion involves full scans from 85 to 1000 m/z at 1 Hz and 100 000 resolution in negative ion mode on a stand alone orbitrap (“Exactive”). The median limit of detection, across 80 metabolite standards, was 5 ng/mL with the linear range typically ≥100-fold. For both standards and a cellular extract from Saccharomyces cerevisiae (Baker’s yeast), the median inter-run relative standard deviation in peak intensity was 8%. In yeast exact, we detected 137 known compounds, whose 13C-labeling patterns could also be tracked to probe metabolic flux. In yeast engineered to lack a gene of unknown function (YKL215C), we observed accumulation of an ion of m/z 128.0351, which we subsequently confirmed to be oxoproline, resulting in annotation of YKL215C as an oxoprolinase. These examples demonstrate the suitability of the present method for quantitative metabolomics, fluxomics, and discovery metabolite profiling.
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