bims-mascan 2019-07-21 papers

bims-mascan

Biomed News

on Mass spectrometry in cancer research

Issue of 2019–07–21
twenty papers selected by
Giovanny Rodriguez Blanco, The Beatson Institute for Cancer Research

Inhibition of Glucose Transporters and Glutaminase Synergistically Impairs Tumor Cell Growth.
Metabolite profiling of guanfacine in plasma and urine of healthy Japanese subjects after oral administration of guanfacine extended-release tablets.
Impact of 17β-HSD12, the 3-ketoacyl-CoA reductase of long-chain fatty acid synthesis, on breast cancer cell proliferation and migration.
Maximal exercise and plasma cytochrome P450 and lipoxygenase mediators: a lipidomics study.
Exploiting metabolic vulnerabilities for personalized therapy in acute myeloid leukemia.
Fundamental study of ion trapping and multiplexing using drift tube-ion mobility time-of-flight mass spectrometry for non-targeted metabolomics.
Sequential Detection of Lipids, Metabolites and Proteins in One Tissue for Improved Cancer Differentiation Accuracy.
Addressing the challenges of high-throughput cancer tissue proteomics for clinical application: ProCan®.
Role of acyl-CoA synthetase ACSL4 in arachidonic acid metabolism.
GPD1 Specifically Marks Dormant Glioma Stem Cells with a Distinct Metabolic Profile.
Kinome screen of ferroptosis reveals a novel role of ATM in regulating iron metabolism.
Effect of essential amino acid supplementation and aerobic exercise on insulin sensitivity in healthy older adults: A randomized clinical trial.
Mitochondrial function in liver cells is resistant to perturbations in NAD+ salvage capacity.
Metabolomic Analysis Identifies Alterations of Amino Acid Metabolome Signatures in the Postmortem Brain of Alzheimer's Disease.
Small-protein enrichment assay enables the rapid, unbiased analysis of over 100 low abundance factors from human plasma.
Metabolic control of gene transcription in non-alcoholic fatty liver disease: the role of the epigenome.
Proteomic profiling of paired interstitial fluids reveals dysregulated pathways and salivary NID1 as a biomarker of oral cavity squamous cell carcinoma.
Effect of polyunsaturated fatty acids on proliferation and survival of prostate cancer cells.
MolNetEnhancer: Enhanced Molecular Networks by Integrating Metabolome Mining and Annotation Tools.
HILPDA regulates lipid metabolism, lipid droplet abundance and response to microenvironmental stress in solid tumors.

Cell Chem Biol. 2019 Jul 02. pii: S2451-9456(19)30206-5. [Epub ahead of print]

Inhibition of Glucose Transporters and Glutaminase Synergistically Impairs Tumor Cell Growth.

Elena S Reckzeh, George Karageorgis, Melanie Schwalfenberg, Javier Ceballos, Jessica Nowacki, Marcus C M Stroet, Aylin Binici, Lena Knauer, Silke Brand, Axel Choidas, Carsten Strohmann, Slava Ziegler, Herbert Waldmann.

  Cancer cells sustain growth by altering their metabolism to accelerated aerobic glycolysis accompanied by increased glucose demand and employ glutamine as additional nutrient source. This metabolic adaptation induces upregulation of glucose transporters GLUT-1 and -3, and simultaneous targeting of both transporters and of glutamine metabolism may offer a promising approach to inhibit cancer cell growth. We describe the discovery of the very potent glucose uptake inhibitor Glutor, which targets glucose transporters GLUT-1, -2, and -3, attenuates glycolytic flux and potently and selectively suppresses growth of a variety of cancer cell lines. Co-treatment of colon cancer cells with Glutor and glutaminase inhibitor CB-839 very potently and synergistically inhibits cancer cell growth. Such a dual inhibition promises to be particularly effective because it targets the metabolic plasticity as well as metabolic rescue mechanisms in cancer cells.

Keywords:  GLUT-1; GLUT-3; Warburg effect; co-treatment; glutaminase; metabolic plasticity; small molecule

DOI:  https://doi.org/10.1016/j.chembiol.2019.06.005
Biopharm Drug Dispos. 2019 Jul 17.

Metabolite profiling of guanfacine in plasma and urine of healthy Japanese subjects after oral administration of guanfacine extended-release tablets.

Yuji Inoue, Hirotoshi Morita, Kohei Nozawa, Takushi Kanazu.

  Guanfacine is used for the treatment of Attention-deficit/hyperactivity disorder (ADHD). Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), metabolite profiling of guanfacine was performed in plasma and urine collected from healthy Japanese adults following repeated oral administration of guanfacine extended-release formulation. Unchanged guanfacine was the most abundant component in both plasma and urine (from the MS signal intensity). In plasma, the M3 metabolite (a sulfate of hydroxy-guanfacine) was the prominent metabolite; the M2 metabolite (a glucuronide of a metabolite formed by monooxidation of guanfacine), 3-hydroxyguanfacine, and several types of glucuronide at different positions on guanfacine were also detected. In urine, the M2 metabolite and 3-hydroxyguanfacine were the principal metabolites. From metabolite analysis, the proposed main metabolic pathway of guanfacine is monooxidation on the dichlorobenzyl moiety, followed by glucuronidation or sulfation. A minor pathway is glucuronidation at different positions on guanfacine. As the prominent metabolites in plasma were glucuronide and sulfate of hydroxyguanfacine, which have no associated toxicity concerns, further toxicity studies of the metabolites, for example in animals, were not deemed necessary.

Keywords:  ADHD; LC-MS/MS; guanfacine extended release; human; metabolic pathway

DOI:  https://doi.org/10.1002/bdd.2201
Cell Mol Life Sci. 2019 Jul 13.

Impact of 17β-HSD12, the 3-ketoacyl-CoA reductase of long-chain fatty acid synthesis, on breast cancer cell proliferation and migration.

Maria Tsachaki, Pirmin Strauss, Anja Dunkel, Hana Navrátilová, Natasa Mladenovic, Alex Odermatt.

  Metabolic reprogramming of tumor cells involves upregulation of fatty acid (FA) synthesis to support high bioenergetic demands and membrane synthesis. This has been shown for cytosolic synthesis of FAs with up to 16 carbon atoms. Synthesis of long-chain fatty acids (LCFAs), including ω-6 and ω-3 polyunsaturated FAs, takes place at the endoplasmic reticulum. Despite increasing evidence for an important role of LCFAs in cancer, the impact of their synthesis in cancer cell growth has scarcely been studied. Here, we demonstrated that silencing of 17β-hydroxysteroid dehydrogenase type 12 (17β-HSD12), essentially catalyzing the 3-ketoacyl-CoA reduction step in LCFA production, modulates proliferation and migration of breast cancer cells in a cell line-dependent manner. Increased proliferation and migration after 17β-HSD12 knockdown were partly mediated by metabolism of arachidonic acid towards COX2 and CYP1B1-derived eicosanoids. Decreased proliferation was rescued by increased glucose concentration and was preceded by reduced ATP production through oxidative phosphorylation and spare respiratory capacity. In addition, 17β-HSD12 silencing was accompanied by alterations in unfolded protein response, including a decrease in CHOP expression and increase in eIF2α activation and the folding chaperone ERp44. Our study highlights the significance of LCFA biosynthesis for tumor cell physiology and unveils unknown aspects of breast cancer cell heterogeneity.

Keywords:  17β-Hydroxysteroid dehydrogenase; Biosynthesis; Cancer; Endoplasmic reticulum; Long-chain fatty acid; Unfolded protein response

DOI:  https://doi.org/10.1007/s00018-019-03227-w
Physiol Rep. 2019 Jul;7(13): e14165

Maximal exercise and plasma cytochrome P450 and lipoxygenase mediators: a lipidomics study.

Benjamin Gollasch, Inci Dogan, Michael Rothe, Maik Gollasch, Friedrich C Luft.

  Epoxides derived from arachidonic acid (AA) are released during exercise and may contribute to vasodilation. However, exercise may also affect circulating levels of other epoxides derived from cytochromes P450 (CYP) monooxygenase and lipoxygenase (LOX) pathways, many of whose exhibit cardiovascular activity in vitro. The effects of exercise on their levels have not been documented. We tested the hypothesis that acute, maximal exercise would influence the plasma concentrations of these vasoactive substances. We measured plasma CYP and LOX mediators derived from both the n - 3 and n - 6 fatty acid (FA) classes in healthy volunteers before, during and after short-term exhaustive exercise. Lipid mediators were profiled by means of LC-MS/MS tandem mass spectrometry. A maximal Bruce treadmill test was performed to voluntary exhaustion. Exhaustive exercise increased the circulating levels of epoxyoctadecenoic (12,13-EpOME), dihydroxyeicosatrienoic (5,6-DHET), dihydroxyeicosatetraenoic acids (5,6-DiHETE, 17,18-DiHETE), but had no effect on the majority of CYP and LOX metabolites. Although our calculations of diol/epoxide ratios revealed preferred hydrolysis of epoxyeicosatrienoic acids (EEQs) into their diols (DiHETEs), this hydrolysis was resistant to maximal exercise. Our study is the first documentation that bioactive endogenous n - 3 and n - 6 CYP lipid mediators are released by short-term exhaustive exercise in humans. In particular, the CYP epoxy-metabolite status, 12,13-EpOME/DiHOME, 5,6-EET/DHET, 5,6-EEQ/DiHETE and 17,18-EEQ/DiHETE may contribute to the cardiovascular response during maximal exercise.

Keywords:  Eicosanoids; exercise; fatty acids; lipidomics

DOI:  https://doi.org/10.14814/phy2.14165
BMC Biol. 2019 Jul 18. 17(1): 57

Exploiting metabolic vulnerabilities for personalized therapy in acute myeloid leukemia.

Lucille Stuani, Marie Sabatier, Jean-Emmanuel Sarry.

Changes in cell metabolism and metabolic adaptation are hallmark features of many cancers, including leukemia, that support biological processes involved into tumor initiation, growth, and response to therapeutics. The discovery of mutations in key metabolic enzymes has highlighted the importance of metabolism in cancer biology and how these changes might constitute an Achilles heel for cancer treatment. In this Review, we discuss the role of metabolic and mitochondrial pathways dysregulated in acute myeloid leukemia, and the potential of therapeutic intervention targeting these metabolic dependencies on the proliferation, differentiation, stem cell function and cell survival to improve patient stratification and outcomes.

DOI: https://doi.org/10.1186/s12915-019-0670-4
Anal Bioanal Chem. 2019 Jul 13.

Fundamental study of ion trapping and multiplexing using drift tube-ion mobility time-of-flight mass spectrometry for non-targeted metabolomics.

Tim J Causon, Le Si-Hung, Kenneth Newton, Ruwan T Kurulugama, John Fjeldsted, Stephan Hann.

  This study of ion accumulation/release behavior relevant to ion mobility-mass spectrometry (IM-MS) as employed for non-targeted metabolomics involves insight from theoretical studies, and controlled reference experiments involving measurement of low and high molecular mass metabolites in varying concentrations within a complex matrix (yeast extracts). Instrumental settings influencing ion trapping (accumulation time) and release conditions in standard and multiplexed operation have been examined, and translation of these insights to liquid chromatography (LC) in combination with drift tube IM-MS measurements has been made. The focus of the application is non-targeted metabolomics using carefully selected samples to allow quantitative interpretations to be made. Experimental investigation of the IM-MS ion utilization efficiency particularly focusing on the use of the Hadamard transform multiplexing with 4-bit pseudo-random pulsing sequence for assessment of low and high molecular mass metabolites is compared with theoretical modeling of gas-phase behavior of small and large molecules in the IM trapping funnel. Increasing the trapping time for small metabolites with standard IM-MS operation is demonstrated to have a deleterious effect on maintaining a quantitative representation of the metabolite abundance. The application of these insights to real-world non-targeted metabolomics assessment of intracellular extracts from biotechnologically relevant production processes is presented, and the results were compared to LC×IM-MS measurements of the same samples. Spiking of a uniformly 13C-labeled yeast extract (as a standard matrix) with varying amounts of natural metabolites is used to assess the linearity and sensitivity according to the instrument mode of operation (i.e., LC-MS, LC×IM-MS, and LC×[multiplexed]IM-MS). When comparing metabolite quantification using standard and multiplexed operation, sensitivity gain factors of 2-8 were obtained for metabolites with m/z below 250. Taken together, the simulation and experimental results of this study provide insight for optimizing measurement conditions for metabolomics and highlight the need for implementation of multiplexing strategies using short trapping times as relative quantification (e.g., in the context with non-targeted differential analysis) with sufficient sensitivity and working range is a requirement in this field of application.

Keywords:  CCS; Hadamard; Ion mobility; Liquid chromatography; Mass spectrometry; Metabolomics; Multiplexing; Yeast

DOI:  https://doi.org/10.1007/s00216-019-02021-8
Anal Chem. 2019 Jul 16.

Sequential Detection of Lipids, Metabolites and Proteins in One Tissue for Improved Cancer Differentiation Accuracy.

Haiyan Lu, Hua Zhang, Konstantin Chingin, Yiping Wei, Jiaquan Xu, Mufang Ke, Keke Huang, Shouhua Feng, Huanwen Chen.

Traditionally, molecular information on metabolites, lipids and proteins is collected from separate individual tissue samples using different analytical approaches. Herein a novel strategy to minimize the potential material losses and the mismatch between metabolomics, lipidomics and proteomics data has been demonstrated based on internal extractive electrospray ionization mass spectrometry (iEESI-MS). Sequential detection of lipids, metabolites, and proteins from the same tissue sample was achieved without sample re-loading and hardware alteration to MS instrument by sequentially using extraction solutions with different chemical composition. With respect to the individual compound class analysis, the sensitivity, specificity, and accuracy obtained with the integrative information on metabolites, lipids and proteins from 57 samples of 13 patients for lung cancer prediction was substantially improved from 54.0%, 51.0% and 76.0% to 100.0%, respectively. The established method is featured by low sample consumption (ca. 2.0 mg) and easy operation, which is important to minimize systematic errors in precision molecular diagnosis and systems biology studies.

DOI: https://doi.org/10.1021/acs.analchem.9b01507
Proteomics. 2019 Jul 18. e1900109

Addressing the challenges of high-throughput cancer tissue proteomics for clinical application: ProCan®.

Brett Tully, Rosemary L Balleine, Peter G Hains, Qing Zhong, Roger R Reddel, Phillip J Robinson.

  The cancer tissue proteome has enormous potential as a source of novel predictive biomarkers in oncology. Progress in development of mass spectrometry (MS)-based tissue proteomics now presents an opportunity to exploit this by applying the strategies of comprehensive molecular profiling and big-data analytics that have been refined in other fields of 'omics research. ProCan® is a program aiming to generate high-quality tissue proteomic data across a broad spectrum of cancer types. It is based on data-independent acquisition (DIA)-MS proteomic analysis of annotated tissue samples sourced through collaboration with expert clinical and cancer research groups. The practical requirements of a high-throughput translational research program have shaped the approach that ProCan is taking to address challenges in study design, sample preparation, raw data acquisition and data analysis. The ultimate goal is to establish a large proteomics knowledge-base that, in combination with other cancer 'omics data, will accelerate cancer research. This article is protected by copyright. All rights reserved.

Keywords:  SWATH-MS; cancer; data analysis; data-independent acquisition; proteomics

DOI:  https://doi.org/10.1002/pmic.201900109
Prostaglandins Other Lipid Mediat. 2019 Jul 12. pii: S1098-8823(19)30054-1. [Epub ahead of print] 106363

Role of acyl-CoA synthetase ACSL4 in arachidonic acid metabolism.

Hiroshi Kuwata, Shuntaro Hara.

  The activation of long-chain free fatty acids is the first step reaction of their usage in the cells and tissues, which are catalyzed by a family of enzymes called acyl-coenzyme A synthetases long-chain isoform (ACSL). The five ACSL enzymes identified in mammals are thought to have specific and differing functions. Among them, ACSL4 is a unique isozyme that preferentially catalyzes several polyunsaturated fatty acids (PUFAs) such as arachidonic acid (AA), and ACSL4 is thought to be an important isozyme for PUFA metabolism. Recent studies revealed that ACSL4 is involved in biological responses including inflammation, steroidogenesis, cell death, female fertility, and cancer. ACSL4 and its substrate PUFAs are thus likely to contribute to these responses. However, the roles of ACSL4 in PUFA metabolism are not fully understood. In this review, we describe the recent progress in ACSL4 research including the involvement of this enzyme in AA metabolism.

Keywords:  ACSL4; acyl-CoA synthetase; arachidonic acid; eicosanoid; polyunsaturated fatty acid

DOI:  https://doi.org/10.1016/j.prostaglandins.2019.106363
Cell Stem Cell. 2019 Jul 03. pii: S1934-5909(19)30268-1. [Epub ahead of print]

GPD1 Specifically Marks Dormant Glioma Stem Cells with a Distinct Metabolic Profile.

Patricia Rusu, Chunxuan Shao, Anna Neuerburg, Azer Aylin Acikgöz, Yonghe Wu, Peng Zou, Prasad Phapale, Tchirupura S Shankar, Kristina Döring, Steffen Dettling, Huiqin Körkel-Qu, Gözde Bekki, Barbara Costa, Te Guo, Olga Friesen, Magdalena Schlotter, Mathias Heikenwalder, Darjus F Tschaharganeh, Bernd Bukau, Günter Kramer, Peter Angel, Christel Herold-Mende, Bernhard Radlwimmer, Hai-Kun Liu.

  Brain tumor stem cells (BTSCs) are a chemoresistant population that can drive tumor growth and relapse, but the lack of BTSC-specific markers prevents selective targeting that spares resident stem cells. Through a ribosome-profiling analysis of mouse neural stem cells (NSCs) and BTSCs, we find glycerol-3-phosphate dehydrogenase 1 (GPD1) expression specifically in BTSCs and not in NSCs. GPD1 expression is present in the dormant BTSC population, which is enriched at tumor borders and drives tumor relapse after chemotherapy. GPD1 inhibition prolongs survival in mouse models of glioblastoma in part through altering cellular metabolism and protein translation, compromising BTSC maintenance. Metabolomic and lipidomic analyses confirm that GPD1+ BTSCs have a profile distinct from that of NSCs, which is dependent on GPD1 expression. Similar GPD1 expression patterns and prognostic associations are observed in human gliomas. This study provides an attractive therapeutic target for treating brain tumors and new insights into mechanisms regulating BTSC dormancy.

Keywords:  cancer stem cell; dormancy; glioblastoma; glycerol-3-phosphate dehydrogenase 1; mouse model; ribosome profiling

DOI:  https://doi.org/10.1016/j.stem.2019.06.004
Cell Death Differ. 2019 Jul 18.

Kinome screen of ferroptosis reveals a novel role of ATM in regulating iron metabolism.

Po-Han Chen, Jianli Wu, Chien-Kuang Cornelia Ding, Chao-Chieh Lin, Samuel Pan, Nathan Bossa, Yitong Xu, Wen-Hsuan Yang, Bernard Mathey-Prevot, Jen-Tsan Chi.

Ferroptosis is a specialized iron-dependent cell death that is associated with lethal lipid peroxidation. Modulation of ferroptosis may have therapeutic potential since it has been implicated in various human diseases as well as potential antitumor activities. However, much remains unknown about the underlying mechanisms and genetic determinants of ferroptosis. Given the critical role of kinases in most biological processes and the availability of various kinase inhibitors, we sought to systemically identify kinases essential for ferroptosis. We performed a forward genetic-based kinome screen against ferroptosis in MDA-MB-231 cells triggered by cystine deprivation. This screen identified 34 essential kinases involved in TNFα and NF-kB signaling. Unexpectedly, the DNA damage response serine/threonine kinase ATM (mutated in Ataxia-Telangiectasia) was found to be essential for ferroptosis. The pharmacological or genetic inhibition of ATM consistently rescued multiple cancer cells from ferroptosis triggered by cystine deprivation or erastin. Instead of the canonical DNA damage pathways, ATM inhibition rescued ferroptosis by increasing the expression of iron regulators involved in iron storage (ferritin heavy and light chain, FTH1 and FTL) and export (ferroportin, FPN1). The coordinated changes of these iron regulators during ATM inhibition resulted in a lowering of labile iron and prevented the iron-dependent ferroptosis. Furthermore, we found that ATM inhibition enhanced the nuclear translocation of metal-regulatory transcription factor 1 (MTF1), responsible for regulating expression of Ferritin/FPN1 and ferroptosis protection. Genetic depletion of MTF-1 abolished the regulation of iron-regulatory elements by ATM and resensitized the cells to ferroptosis. Together, we have identified an unexpected ATM-MTF1-Ferritin/FPN1 regulatory axis as novel determinants of ferroptosis through regulating labile iron levels.

DOI: https://doi.org/10.1038/s41418-019-0393-7
Clin Nutr. 2019 Jun 28. pii: S0261-5614(19)30273-0. [Epub ahead of print]

Effect of essential amino acid supplementation and aerobic exercise on insulin sensitivity in healthy older adults: A randomized clinical trial.

Amanda C Randolph, Melissa M Markofski, Blake B Rasmussen, Elena Volpi.

   BACKGROUND & AIMS: The combination of prolonged essential amino acid (EAA) supplementation and aerobic exercise training (Ex) improves muscle protein metabolism, strength and function in healthy older adults. However, excess EAA intake may worsen insulin sensitivity. Here we report the effects of EAA supplementation (EAA, n = 11), placebo (PLA, n = 10), aerobic exercise with placebo (Ex + PLA, n = 11) or Ex with EAA supplementation (Ex + EAA, n = 10) for 22 weeks on insulin sensitivity in non-diabetic older adults.
METHODS: A 2 × 2 design with block randomization and double blinding for supplement or placebo was used. Subjects ingested EAA (15 g) or placebo daily. Exercising subjects participated in supervised progressive vigorous treadmill walking 3 times weekly. Measures of insulin sensitivity by oral glucose tolerance testing were collected at baseline and 22 weeks. Dietary intakes of protein and specific amino acids were determined in a subset of subjects.
RESULTS: Overall, exercise improved insulin sensitivity, while EAA supplementation had no effect. In the dietary subset, post-intervention insulin sensitivity did not correlate significantly with the total intake of EAA, anti-angiogenic amino acids (cysteine, methionine), or branched-chain amino acids (isoleucine, leucine, valine).
CONCLUSIONS: Overall, we conclude that in healthy older adults with moderate protein intake, EAA supplementation is metabolically safe as it does not decrease insulin sensitivity regardless of its combination with aerobic exercise. Thus, daily protein intake should be controlled for when modeling insulin sensitivity. Future studies should explore the role of increased blood flow as a potential explanatory factor for the observed interaction between aerobic exercise and supplementation.
CLINICAL TRIAL REGISTRATION NUMBER: NCT00872911.

Keywords:  Glucose metabolism; Matsuda index; Sarcopenia

DOI:  https://doi.org/10.1016/j.clnu.2019.06.017
J Biol Chem. 2019 Jul 18. pii: jbc.RA118.006756. [Epub ahead of print]

Mitochondrial function in liver cells is resistant to perturbations in NAD+ salvage capacity.

Morten Dall, Samuel A J Trammell, Magnus Asping, Anna Skab Hassing, Marianne Agerholm, Sara G Vienberg, Matthew P Gillum, Steen Larsen, Jonas T Treebak.

  Supplementation with nicotinamide adenine dinucleotide (NAD+) precursors such as nicotinamide riboside (NR) has been shown to enhance mitochondrial function in the liver and prevent hepatic lipid accumulation in high-fat diet (HFD)-fed rodents. Hepatocyte-specific knockout of the NAD+-synthesizing enzyme nicotinamide phosphoribosyltransferase (NAMPT) reduces liver NAD+ levels, but the metabolic phenotype of Nampt-deficient hepatocytes in mice is unknown. Here, we assessed Nampt's role in maintaining mitochondrial and metabolic functions in the mouse liver. Using the Cre-LoxP system, we generated hepatocyte-specific Nampt knockout (HNKO) mice, having a 50% reduction of liver NAD+ levels. We screened the HNKO mice for signs of metabolic dysfunction following 60% HFD feeding for 20 weeks +/- NR supplementation and found that NR increases hepatic NAD+ levels without affecting fat mass or glucose tolerance in HNKO or WT animals. High-resolution respirometry revealed that NR supplementation of the HNKO mice did not increase state III respiration, which was observed in WT mice following NR supplementation. Mitochondrial oxygen consumption and fatty-acid oxidation was unaltered in primary HNKO hepatocytes. Mitochondria isolated from whole HNKO livers had only a 20% reduction in NAD+, suggesting that the mitochondrial NAD+ pool is less affected by HNKO than the whole-tissue pool. When stimulated with tryptophan in the presence of 15N-glutamine, HNKO hepatocytes had a higher 15N-NAD+ enrichment than WT hepatocytes, indicating that HNKO mice compensate through de novo NAD+ synthesis. We conclude that NAMPT-deficient hepatocytes can maintain substantial NAD+ levels and that the Nampt knockout has only minor consequences for mitochondrial function in the mouse liver.

Keywords:  NAD biosynthesis; NAMPT; fatty acid metabolism; hepatocyte; mitochondrial metabolism; nicotinamide adenine dinucleotide (NAD); respiration; tryptophan metabolism

DOI:  https://doi.org/10.1074/jbc.RA118.006756
Exp Neurobiol. 2019 Jun;28(3): 376-389

Metabolomic Analysis Identifies Alterations of Amino Acid Metabolome Signatures in the Postmortem Brain of Alzheimer's Disease.

Yoon Hwan Kim, Hyun Soo Shim, Kyoung Heon Kim, Junghee Lee, Bong Chul Chung, Neil W Kowall, Hoon Ryu, Jeongae Lee.

  Despite significant advances in neuroscience research over the past several decades, the exact cause of AD has not yet fully understood. The metabolic hypothesis as well as the amyloid and tau hypotheses have been proposed to be associated with AD pathogenesis. In order to identify metabolome signatures from the postmortem brains of sporadic AD patients and control subjects, we performed ultra performance liquid chromatography coupled with linear ion trap-Orbitrap mass spectrometer (UPLC-LTQ-Orbitrap-MS). Not only our study identified new metabolome signatures but also verified previously known metabolome profiles in the brain. Statistical modeling of the analytical data and validation of the structural assignments discovered metabolic biomarkers associated with the AD pathogenesis. Interestingly, hypotaurin, myo-inositol and oxo-proline levels were markedly elevated in AD while lutamate and N-acetyl-aspartate were decreased in the postmortem brain tissue of AD patients. In addition, neurosteroid level such as cortisol was significantly increased in AD. Together, our data indicate that impaired amino acid metabolism is associated with AD pathogenesis and the altered amino acid signatures can be useful diagnostic biomarkers of AD. Thus, modulation of amino acid metabolism may be a possible therapeutic approach to treat AD.

Keywords:  Alzheimer's disease; Biomarkers; Liquid chromatography mass spectrometry; Metabolomics; amino acid metabolism

DOI:  https://doi.org/10.5607/en.2019.28.3.376
Mol Cell Proteomics. 2019 Jul 15. pii: mcp.TIR119.001562. [Epub ahead of print]

Small-protein enrichment assay enables the rapid, unbiased analysis of over 100 low abundance factors from human plasma.

Dylan Harney, Amy T Hutchison, Zhiduan Su, Luke Hatchwell, Leonie K Heilbronn, Samantha Hocking, David E James, Mark Larance.

  Unbiased and sensitive quantification of low abundance small proteins in human plasma (e.g. hormones, immune factors, metabolic regulators) remains an unmet need. These small protein factors are typically analysed individually and using antibodies that can lack specificity. Mass spectrometry (MS)-based proteomics has the potential to address these problems, however the analysis of plasma by MS is plagued by the extremely large dynamic range of this body fluid, with protein abundances spanning at least 13 orders of magnitude. Here we describe an enrichment assay (SPEA), that greatly simplifies the plasma dynamic range problem by enriching small-proteins of 2-10 kDa, enabling the rapid, specific and sensitive quantification of >100 small-protein factors in a single untargeted LC-MS/MS acquisition. Applying this method to perform deep-proteome profiling of human plasma we identify C5ORF46 as a previously uncharacterized human plasma protein. We further demonstrate the reproducibility of our workflow for low abundance protein analysis using a stable-isotope labelled protein standard of insulin spiked into human plasma. SPEA provides the ability to study numerous important hormones in a single rapid assay, which we applied to study the intermittent fasting response and observed several unexpected changes including decreased plasma abundance of the iron homeostasis regulator hepcidin.

Keywords:  Chromatography; Hormones*; Insulin resistance; Plasma or serum analysis; Serum/Plasma*; chemokine; hepcidin; insulin; intermittent fasting

DOI:  https://doi.org/10.1074/mcp.TIR119.001562
Clin Epigenetics. 2019 Jul 18. 11(1): 104

Metabolic control of gene transcription in non-alcoholic fatty liver disease: the role of the epigenome.

Matthew C Sinton, David C Hay, Amanda J Drake.

  Non-alcoholic fatty liver disease (NAFLD) is estimated to affect 24% of the global adult population. NAFLD is a major risk factor for the development of cirrhosis and hepatocellular carcinoma, as well as being strongly associated with type 2 diabetes and cardiovascular disease. It has been proposed that up to 88% of obese adults have NAFLD, and with global obesity rates increasing, this disease is set to become even more prevalent. Despite intense research in this field, the molecular processes underlying the pathology of NAFLD remain poorly understood. Hepatic intracellular lipid accumulation may lead to dysregulated tricarboxylic acid (TCA) cycle activity and associated alterations in metabolite levels. The TCA cycle metabolites alpha-ketoglutarate, succinate and fumarate are allosteric regulators of the alpha-ketoglutarate-dependent dioxygenase family of enzymes. The enzymes within this family have multiple targets, including DNA and chromatin, and thus may be capable of modulating gene transcription in response to intracellular lipid accumulation through alteration of the epigenome. In this review, we discuss what is currently understood in the field and suggest areas for future research which may lead to the development of novel preventative or therapeutic interventions for NAFLD.

Keywords:  Alpha-ketoglutarate-dependent dioxygenases; Metabolite; Mitochondria; Non-alcoholic fatty liver disease; TCA cycle

DOI:  https://doi.org/10.1186/s13148-019-0702-5
Mol Cell Proteomics. 2019 Jul 17. pii: mcp.RA119.001654. [Epub ahead of print]

Proteomic profiling of paired interstitial fluids reveals dysregulated pathways and salivary NID1 as a biomarker of oral cavity squamous cell carcinoma.

Chia-Wei Hsu, Kai-Ping Chang, Yenlin Huang, Hao-Ping Liu, Pei-Chun Hsueh, Po-Wen Gu, Wei-Chen Yen, Chih-Ching Wu.

  Patients with oral cavity squamous cell carcinoma (OSCC) are frequently first diagnosed at an advanced stage, leading to poor prognosis and high mortality rates. Early detection of OSCC using body fluid-accessible biomarkers may improve the prognosis and survival rate of OSCC patients. As tumor interstitial fluid is a proximal fluid enriched with cancer-related proteins, it is a useful reservoir suitable for the discovery of cancer biomarkers and dysregulated biological pathways in tumor microenvironments. Thus, paired interstitial fluids of tumor (TIF) and adjacent noncancerous (NIF) tissues from 10 OSCC patients were harvested and analyzed using one-dimensional gel electrophoresis coupled with liquid chromatography-tandem mass spectrometry (GeLC-MS/MS). Using label-free spectral counting-based quantification, 113 proteins were found to be upregulated in the TIFs compared to the NIFs. The gene set enrichment analysis (GSEA) revealed that the differentially expressed TIF proteins were highly associated with aminoacyl tRNA biosynthesis pathway. The elevated levels of 4 proteins (IARS, KARS, WARS, and YARS) involved in the aminoacyl tRNA biosynthesis were verified in the OSCC tissues with immunohistochemistry (IHC). In addition, nidogen-1 (NID1) was selected for verification as an OSCC biomarker. Salivary level of NID1 in OSCC patients (n = 48) was significantly higher than that in the healthy individuals (n = 51) and subjects with oral potentially malignant disorder (OPMD; n = 53). IHC analysis showed that NID1 level in OSCC tissues was increased compared to adjacent noncancerous epithelium (n =222). Importantly, the elevated NID1 level was correlated with the advanced stages of OSCC, as well as the poor survival of OSCC patients. Collectively, the results suggested that TIF analysis facilitates understanding of the OSCC microenvironment and that salivary NID1 may be a useful biomarker for OSCC.

Keywords:  Biofluids*; Biomarker: Diagnostic; Biomarker: Prognostic; Head and neck cancer; Label-free quantification; interstitial fluids; oral cancer

DOI:  https://doi.org/10.1074/mcp.RA119.001654
PLoS One. 2019 ;14(7): e0219822

Effect of polyunsaturated fatty acids on proliferation and survival of prostate cancer cells.

Brenden A Bratton, Ivan V Maly, Wilma A Hofmann.

Progression of prostate cancer to lethal forms is marked by emergence of hormone-independent proliferation of the cancer cells. Nutritional and epidemiological studies have indicated that prostate cancer progression is correlated with the consumption of polyunsaturated fatty acids (PUFA). To shed additional light on the cell-level mechanisms of the observed correlation, we compared the sensitivity of hormone-dependent and hormone-independent prostate cancer cells to growth medium supplementation with free PUFAs in a cell proliferation and viability assay. Our data show that the hormone-dependent cells are comparatively insensitive to various PUFAs, at the same time as the growth and viability of hormone-independent cells lines are strongly inhibited by most of the tested PUFAs, whether n-3 or n-6. We speculate that this difference may be at least partially responsible for the observed effects of specific dietary lipids in prostate cancer. The new data strengthen the case for dietary intervention as part of potential new therapeutic strategies seeking to impede prostate cancer progression.

DOI: https://doi.org/10.1371/journal.pone.0219822
Metabolites. 2019 Jul 16. pii: E144. [Epub ahead of print]9(7):

MolNetEnhancer: Enhanced Molecular Networks by Integrating Metabolome Mining and Annotation Tools.

Madeleine Ernst, Kyo Bin Kang, Andrés Mauricio Caraballo-Rodríguez, Louis-Felix Nothias, Joe Wandy, Christopher Chen, Mingxun Wang, Simon Rogers, Marnix H Medema, Pieter C Dorrestein, Justin J J van der Hooft.

  Metabolomics has started to embrace computational approaches for chemical interpretation of large data sets. Yet, metabolite annotation remains a key challenge. Recently, molecular networking and MS2LDA emerged as molecular mining tools that find molecular families and substructures in mass spectrometry fragmentation data. Moreover, in silico annotation tools obtain and rank candidate molecules for fragmentation spectra. Ideally, all structural information obtained and inferred from these computational tools could be combined to increase the resulting chemical insight one can obtain from a data set. However, integration is currently hampered as each tool has its own output format and efficient matching of data across these tools is lacking. Here, we introduce MolNetEnhancer, a workflow that combines the outputs from molecular networking, MS2LDA, in silico annotation tools (such as Network Annotation Propagation or DEREPLICATOR), and the automated chemical classification through ClassyFire to provide a more comprehensive chemical overview of metabolomics data whilst at the same time illuminating structural details for each fragmentation spectrum. We present examples from four plant and bacterial case studies and show how MolNetEnhancer enables the chemical annotation, visualization, and discovery of the subtle substructural diversity within molecular families. We conclude that MolNetEnhancer is a useful tool that greatly assists the metabolomics researcher in deciphering the metabolome through combination of multiple independent in silico pipelines.

Keywords:  chemical classification; in silico workflows; metabolite annotation; metabolite identification; metabolome mining; molecular families; networking; substructures

DOI:  https://doi.org/10.3390/metabo9070144
Mol Cancer Res. 2019 Jul 15. pii: molcanres.1343.2018. [Epub ahead of print]

HILPDA regulates lipid metabolism, lipid droplet abundance and response to microenvironmental stress in solid tumors.

Matthew J VandeKopple, Jinghai Wu, Erich N Auer, Amato J Giaccia, Nicholas C Denko, Ioanna Papandreou.

Accumulation of lipid droplets has been observed in an increasing range of tumors. However, the molecular determinants of this phenotype and the impact of the tumor microenvironment on lipid droplet dynamics are not well defined. The hypoxia-inducible lipid droplet associated protein HILPDA is known to regulate lipid storage and physiological responses to feeding conditions in mice, and was recently shown to promote hypoxic lipid droplet formation through inhibition of the rate-limiting adipose triglyceride lipase (ATGL). Here, we identify fatty acid loading and nutrient deprivation-induced autophagy as stimuli of HILPDA-dependent lipid droplet growth. Using mouse embryonic fibroblasts and human tumor cells, we found that genetic ablation of HILPDA compromised hypoxia- fatty acid- and starvation-induced lipid droplet formation and triglyceride storage. Nutrient deprivation upregulated HILPDA protein post-transcriptionally by a mechanism requiring autophagic flux and lipid droplet turnover, independent of HIF1 transactivation. Mechanistically, loss of HILPDA led to elevated lipolysis which could be corrected by inhibition of ATGL. Lipidomic analysis revealed not only quantitative but also qualitative differences in the glycerolipid and phospholipid profile of HILPDA WT and KO cells, indicating additional HILPDA functions impacting lipid metabolism. Deletion studies of HILPDA mutants identified the N-terminal hydrophobic domain as sufficient for targeting to lipid droplets and restoration of triglyceride storage. In vivo, HILPDA ablated cells showed decreased intratumoral triglyceride levels and impaired xenograft tumor growth associated with elevated levels of apoptosis. Implications: Tumor microenvironmental stresses induce changes in lipid droplet dynamics via HILPDA. Regulation of triglyceride hydrolysis is crucial for cell homeostasis and tumor growth.

DOI: https://doi.org/10.1158/1541-7786.MCR-18-1343