Metabolite AutoPlotter - an application to process and visualise metabolite data in the web browser

Background: Metabolomics is gaining popularity as a standard tool for the investigation of biological systems. Yet, parsing metabolomics data in the absence of in-house computational scientists can be overwhelming and time-consuming. As a consequence of manual data processing, the results are often not analysed in full depth, so potential novel findings might get lost. Methods: To tackle this problem, we developed Metabolite AutoPlotter, a tool to process and visualise quantified metabolite data. Other than with bulk data visualisations, such as heat maps, the aim of the tool is to generate single plots for each metabolite. For this purpose, it reads as input pre-processed metabolite-intensity tables and accepts different experimental designs, with respect to the number of metabolites, conditions and replicates. The code was written in the R-scripting language and wrapped into a shiny application that can be run online in a web browser on https://mpietzke.shinyapps.io/autoplotter. Results: We demonstrate the main features and the ease of use with two different metabolite datasets, for quantitative experiments and for stable isotope tracing experiments. We show how the plots generated by the tool can be interactively modified with respect to plot type, colours, text labels and the shown statistics. We also demonstrate the application towards 13C-tracing experiments and the seamless integration of natural abundance correction, which facilitates the better interpretation of stable isotope tracing experiments. The output of the tool is a zip-file containing one single plot for each metabolite as well as restructured tables that can be used for further analysis. Conclusion: With the help of Metabolite AutoPlotter, it is now possible to simplify data processing and visualisation for a wide audience. High-quality plots from complex data can be generated in a short time by pressing a few buttons. This offers dramatic improvements over manual analysis. It is significantly faster and allows researchers to spend more time interpreting the results or to perform follow-up experiments. Further, this eliminates potential copy-and-paste errors or tedious repetitions when things need to be changed. We are sure that this tool will help to improve and speed up scientific discoveries

Formate metabolism in health and disease

Background: Formate is a one-carbon molecule at the crossroad between cellular and whole body metabolism, between host and microbiome metabolism, and between nutrition and toxicology. This centrality confers formate with a key role in human physiology and disease that is currently unappreciated. Scope of review: Here we review the scientific literature on formate metabolism, highlighting cellular pathways, whole body metabolism, and interactions with the diet and the gut microbiome. We will discuss the relevance of formate metabolism in the context of embryonic development, cancer, obesity, immunometabolism, and neurodegeneration. Major conclusions: We will conclude with an outlook of some open questions bringing formate metabolism into the spotlight

Stratification of cancer and diabetes based on circulating levels of formate and glucose

Background: Serum and urine metabolites have been investigated for their use as cancer biomarkers. The specificity of candidate metabolites can be limited by the impact of other disorders on metabolite levels. In particular, the increasing incidence of obesity could become a significant confounding factor. Methods: Here we developed a multinomial classifier for the stratification of cancer, obesity and healthy phenotypes based on circulating glucose and formate levels. We quantified the classifier performance from the retrospective analysis of samples from breast cancer, lung cancer, obese individuals and healthy controls. Results: We discovered that circulating formate levels are significantly lower in breast and lung cancer patients than in healthy controls. However, the performance of a cancer classifier based on formate levels alone is limited because obese patients also have low serum formate levels. By introducing a multinomial classifier based on circulating glucose and formate levels, we were able to improve the classifier performance, reaching a true positive rate of 79% with a false positive rate of 8%. Conclusions: Circulating formate is reduced in HER2+ breast cancer, non-small cell lung cancer and highly obese patients relative to healthy controls. Further studies are required to determine the relevance of these observations in other cancer types and diseases

Amino acid dependent formaldehyde metabolism in mammals

Abstract: Aldehyde dehydrogenase class 3, encoded by ADH5 in humans, catalyzes the glutathione dependent detoxification of formaldehyde. Here we show that ADH5 deficient cells turn over formaldehyde using alternative pathways starting from the reaction of formaldehyde with free amino acids. When mammalian cells are exposed to formaldehyde, the levels of the reaction products of formaldehyde with the amino acids cysteine and histidine - timonacic and spinacine - are increased. These reactions take place spontaneously and the formation of timonacic is reversible. The levels of timonacic are higher in the plasma of Adh5−/− mice relative to controls and they are further increased upon administration of methanol. We conclude that mammals possess pathways of cysteine and histidine dependent formaldehyde metabolism and that timonacic is a formaldehyde reservoir

Impact of formate supplementation on body weight and plasma amino acids

Current nutritional recommendations are focused on energy, fat, carbohydrate, protein and vitamins. Less attention has been paid to the nutritional demand of one-carbon units for nucleotide and methionine synthesis. Here, we investigated the impact of sodium formate supplementation as a nutritional intervention to increase the dietary intake of one-carbon units. A cohort of six female and six male mice received 125 mM of sodium formate in the drinking water for three months. A control group of another six female and six male mice was also followed up for the same period of time. Tail vein blood samples were collected once a month and profiled with a haematology analyser. At the end of the study, blood and tissues were collected for metabolomics analysis and immune cell profiling. Formate supplementation had no significant physiological effect on male mice, except for a small decrease in body weight. Formate supplementation had no significant effect on the immune cell counts during the intervention or at the end of the study in either gender. In female mice, however, the body weight and spleen wet weight were significantly increased by formate supplementation, while the blood plasma levels of amino acids were decreased. Formate supplementation also increased the frequency of bifidobacteria, a probiotic bacterium, in the stools of female mice. We conclude that formate supplementation induces physiological changes in a gender-specific manner

Amino acid dependent formaldehyde metabolism in mammals.

Aldehyde dehydrogenase class 3, encoded by ADH5 in humans, catalyzes the glutathione dependent detoxification of formaldehyde. Here we show that ADH5 deficient cells turn over formaldehyde using alternative pathways starting from the reaction of formaldehyde with free amino acids. When mammalian cells are exposed to formaldehyde, the levels of the reaction products of formaldehyde with the amino acids cysteine and histidine - timonacic and spinacine - are increased. These reactions take place spontaneously and the formation of timonacic is reversible. The levels of timonacic are higher in the plasma of Adh5-/- mice relative to controls and they are further increased upon administration of methanol. We conclude that mammals possess pathways of cysteine and histidine dependent formaldehyde metabolism and that timonacic is a formaldehyde reservoir

Immune regulated IDO1-dependent tryptophan metabolism is source of one-carbon units for pancreatic cancer and stellate cells

Cancer cells adapt their metabolism to support elevated energetic and anabolic demands of proliferation. Folate-dependent one-carbon metabolism is a critical metabolic process underpinning cellular proliferation supplying carbons for the synthesis of nucleotides incorporated into DNA and RNA. Recent research has focused on the nutrients that supply one-carbons to the folate cycle, particularly serine. Tryptophan is a theoretical source of one-carbon units through metabolism by IDO1, an enzyme intensively investigated in the context of tumor immune evasion. Using in vitro and in vivo pancreatic cancer models, we show that IDO1 expression is highly context dependent, influenced by attachment-independent growth and the canonical activator IFNγ. In IDO1-expressing cancer cells, tryptophan is a bona fide one-carbon donor for purine nucleotide synthesis in vitro and in vivo. Furthermore, we show that cancer cells release tryptophan-derived formate, which can be used by pancreatic stellate cells to support purine nucleotide synthesis

Amino acid dependent formaldehyde metabolism in mammals

Abstract: Aldehyde dehydrogenase class 3, encoded by ADH5 in humans, catalyzes the glutathione dependent detoxification of formaldehyde. Here we show that ADH5 deficient cells turn over formaldehyde using alternative pathways starting from the reaction of formaldehyde with free amino acids. When mammalian cells are exposed to formaldehyde, the levels of the reaction products of formaldehyde with the amino acids cysteine and histidine - timonacic and spinacine - are increased. These reactions take place spontaneously and the formation of timonacic is reversible. The levels of timonacic are higher in the plasma of Adh5−/− mice relative to controls and they are further increased upon administration of methanol. We conclude that mammals possess pathways of cysteine and histidine dependent formaldehyde metabolism and that timonacic is a formaldehyde reservoir

Formate induces a metabolic switch in nucleotide and energy metabolism

Formate is a precursor for the de novo synthesis of purine and deoxythymidine nucleotides. Formate also interacts with energy metabolism by promoting the synthesis of adenine nucleotides. Here we use theoretical modelling together with metabolomics analysis to investigate the link between formate, nucleotide and energy metabolism. We uncover that endogenous or exogenous formate induces a metabolic switch from low to high adenine nucleotide levels, increasing the rate of glycolysis and repressing the AMPK activity. Formate also induces an increase in the pyrimidine precursor orotate and the urea cycle intermediate argininosuccinate, in agreement with the ATP-dependent activities of carbamoyl-phosphate and argininosuccinate synthetase. In vivo data for mouse and human cancers confirms the association between increased formate production, nucleotide and energy metabolism. Finally, the in vitro observations are recapitulated in mice following and intraperitoneal injection of formate. We conclude that formate is a potent regulator of purine, pyrimidine and energy metabolism

Analyse der metabolischen Kontrolle des Zellwachstums mit Hilfe von Metabolitmessungen und stabiler Isotopenmarkierung

1\. INTRODUCTION 1.1. Metabolomics and the central carbon metabolism 1.2. The use of stable isotopes for flux measurements 1.3. The role of the cell cycle 1.4. The metabolic difference of cancer cells 1.5. Glycolytic inhibition as a potential therapeutic target against cancer cells 1.5.1. Inhibition by 2-deoxyglucose 1.5.2. Inhibition by glyceraldehyde 1.5.3. Inhibition by 3-bromopyruvate 1.6. Aim of this study 2\. CHEMICALS AND MATERIAL 2.1. Lab Equipment 2.2. Materials 2.2.1. Cell lines: 2.2.2. Chemicals for cell culture 2.2.3. Chemicals for metabolite harvest and measurement 3\. METHODS 3.1. Growth media composition for cell culture 3.2. Standard cell culture 3.3. Sample preparation for metabolomics 3.3.1. Standard metabolomic harvest for adherent cells 3.3.2. Standard metabolomic harvest for weakly adherent cells 3.3.3. Performing labeling experiments 3.3.4. Preparation of the Ident-mix 3.3.5. Preparation of the Quant-mix 3.3.6. Derivatization 3.3.7. GC-MS measurement 3.3.8. GC-MS data analysis 3.4. Flow cytometry for detecting cell cycle phases 3.5. Proteomics Preparation 3.5.1. Solutions 3.5.2. Harvest and extraction 3.6. Additional Data analysis 3.7. Experimental description 3.7.1. Experimental verification of correcting strategies by mixing 13C with 12C- glucose 3.7.2. Reproducibility of harvest procedure and label incorporation 3.7.3. Cell Cycle experiment 3.7.4. Growth inhibition under influence of glycolytic inhibitors 3.7.5. Incorporation of 13C-Glyceraldehyde 3.7.6. Effect of glycolytic inhibition to metabolism 3.7.7. In vivo study in mice to monitor gluconeogenesis from pyruvate 3.7.8. In vivo study to elucidate differential use of substrates in HCC tumor models 4\. RESULTS 4.1. Section 1 - Method development of pulsed stable isotope resolved metabolomics (pSIRM) of cell culture samples 4.1.1. A high percentage of the central carbon metabolism can be measured by GC-MS 4.1.2. The Ident-mixture enables a more reliable and semiautomated identification in complex biological samples 4.1.3. Multiple metabolites can be simultaneously quantified with external calibration curves 4.1.4. The sample preparation and the quantification of compounds in cell culture samples is reproducible 4.1.5. The natural 13C abundance must be subtracted 4.1.6. Correction of natural abundance and calculation of label incorporation can be done in a single step in an untargeted way 4.1.7. Mass isotopomer distributions are concentration dependent 4.1.8. Many metabolites of the CCM in mammalian cell lines can be found labeled and have a turnover on the minute time scale 4.1.9. Label incorporation and quantification of metabolites can be obtained from a single sample with high precision 4.2. Section 2 – Cell cycle Experiment 4.2.1. After release from mimosine block cells immediately start entering S-phase 4.2.2. Proteins showed a continuous increase in their heavy to light ratios 4.2.3. Glycolysis and glutaminolysis are differentially regulated 4.3. Section 3 – Measuring the effect of glycolytic inhibitors on the central carbon metabolism and growth 4.3.1. All compounds inhibit cell growth and induced a stress phenotype in a dose dependent manner 4.3.2. Further estimation of effective concentrations 4.3.3. L-Glyceraldehyde inhibits glycolysis downstream of the hexokinase reaction 4.3.4. Bromopyruvate inhibits glycolysis primarily at GAPDH-reaction 4.3.5. 2-Deoxyglucose is rapidly phosphorylated but only acts indirectly on glycolysis 4.3.6. The fate of glyceraldehyde can be monitored 4.3.7. Sorbose itself has no effect to growth or viability 4.3.8. Glyceraldehyde further induces a growth crises which depends on the availability of glucose 4.4. Section 4 - in vivo applications of pSIRM 4.4.1. In vivo monitoring of gluconeogenesis from 13C-pyruvate 4.4.2. In vivo usage of glucose and glutamine in HCC model-mouse 5\. DISCUSSION 5.1. Labeling – a shorter timescale reveals new insights 5.2. Calculation of label incorporation – sometimes the wheel needs to be reinvented 5.3. Label incorporation can be summarized into a single number 5.4. Interpretation of labeling data under changing conditions improves with inclusion of quantities 5.5. Analysis of metabolites and proteins during cell cycle progression 5.6. pSIRM as an strategy to understand short termed processes like the inhibition of glycolysis 5.7. The L-isomer of glyceraldehyde is an more effective inhibitor of glycolysis than the D-isomer 5.8. BrPyr is very effective inhibitor of glycolysis in the tested concentration 5.9. The failure of 2DG to inhibit glycolysis 5.10. Comparison of the tested compounds and characterization as possible therapeutics 5.11. GA and further effects to glucose sensing 5.12. In vivo application of isotopic labeled compounds in mice 5.13. Conclusion and outlook 6\. BIBLIOGRAPHY 7\. PUBLICATIONS 8\. APPENDIXCancer cells typically collected a number of mutations resulting in modified metabolism optimized to fulfill their needs to a maximize growth. In the recent years -with development of reliable high-throughput techniques- there was a shift from measuring single actors to an integrated view of the complex regulatory network in a biological system. This thesis aimed at elucidation and under-standing of the metabolic difference between cancer and healthy cells in order to target these differences therapeutically. Firstly, cancer and healthy cells differ metabolically in many aspects. It is generally accepted that most cancer cells have a higher rate of glycolysis that results in elevated level of lactate production, even in the presence of oxygen (known as Warburg effect). Further the function and activity of the TCA cycle and the glutaminolysis in cancer cells is heavily debated. Current, static metabolomics protocols deliver only a limited amount of information and need careful interpretation. With the help of heavy-labeled isotopes the metabolism can be monitored in a more dynamic way. In frame of this thesis a method was established that allows for monitoring changes in the carbon routing of the highly connected central carbon metabolism in human tumor model cell lines. The usage of substrates such as glucose can be compared under different conditions. With this method the high activity of glycolysis and a tight connection to lactate production as well as a truncated TCA cycle could be shown. Secondly, cancer cells can be characterized by their permanent and unlimited replication. During different phases of the cell cycle, cells are expected to have different needs, with respect to precursors for synthesis or energy production. The difference in metabolism during various phases of the cell cycle was monitored with the established approach in synchronized cells. Further the protein-turnover was measured in a pulsed-SILAC approach. Third, both permanent growth and elevated glycolytic activity are potential targets for therapy, aiming at cancer cells but leaving healthy cells unharmed. The established method is perfectly suitable to detect rearrangements of metabolism on the minute time scale, therefore differentiating between direct inhibition of enzymes targeted by inhibitors and long-termed rearrangements of metabolism. The strategy was tested with three different, putative inhibitors of glycolysis: 3-bromopyruvate, glyceraldehyde and 2-deoxyglucose. The monitored effects partially resembled effects previously described in the literature and further evidenced criticism of 2-deoxyglucose as selective inhibitor of glycolysis. Finally, the methods and strategies developed in cancer-model cell lines were and will be further translated to in vivo mouse models. Pulsed labeling with stable isotope enriched substrates such as glucose or glutamine showed differences in the substrate utilization between female and male HCC tumor model mice.Krebszellen sammeln in ihrer Entwicklung zahlreiche verschiedene Mutationen, die sich häufig in einem veränderten Stoffwechsel manifestieren, einem Stoffwechsel optimiert auf maximales Wachstum. Die Entwicklung von zuverlässigen Hochdurchsatz-Methoden ermöglichte einen Wechsel von der Betrachtung einzelner Aspekte zu einer integrierten Betrachtung des gesamten, komplexen Systems. Diese Arbeit zielt auf ein tieferes Verständnis der Unterschiede zwischen Krebs und gesunden Zellen mit dem Ziel diese Unterschiede therapeutisch auszunutzen. Krebs- und gesunde Zellen unterscheiden sich in ihrem Metabolismus. Die meisten Krebszellen zeigen eine höhere Glykolyse, die sich in einer starken Laktatausscheidung auch in Gegenwart von Sauerstoff zeigt, der wohlbekannte „Warburg-Effekt“. Darüber hinaus werden die Rolle des Citratzyklus und die Rolle des Glutaminstoffwechsels intensiv diskutiert. Klassische Metabolitmessungen liefern nur eingeschränkte Ergebnisse, die vorsichtig interpretiert werden müssen. Das ändert sich durch die Verwendung stabiler, schwerer Isotope. In dieser Arbeit wurde eine Methode etabliert um Veränderungen im Kohlenstoff- Stoffwechsel in menschlichen Tumormodell-Zelllinien mit Hilfe von stabilen Isotopen zu messen. Die Verwendung verschiedener Susbtrate, wie Glukose oder Glutamin, kann somit unter verschiedenen Konditionen verglichen werden. Mit dieser Methode konnte eine starke glykolytische Aktivität, eine enge Kopplung von Pyruvat und Laktat und ein unvollständiger Citratzyklus gezeigt werden. Krebszellen sind weiterhin charakterisiert durch ein unbegrenztes Wachstum. Während verschiedener Phasen des Zell-Zyklus können unterschiedliche metabolische Profile erwartet werden. Das wurde überprüft durch Anwendung der etablierten Methode in verschiedenen Phasen des Zellzyklus von synchronisierten Zellen. Zusätzlich wurde die Protein-Neusynthese mit Hilfe eines „pulsed SILAC“ Ansatzes bestimmt. Beide Faktoren, erhöhte Glykolyse und permanentes Wachstum sind potentielle Ziele für Krebstherapien. Die etablierte Methode ist perfekt geeignet um kurzfristige Veränderungen des Stoffwechsels zu beobachten. Damit können direkte Wirkungen auf die Enzyme von langfristigen metabolischen Umstrukturierungen unterschieden werden. Mit diesem Ansatz wurden drei verschiedene mögliche Inhibitoren der Glykolyse getestet und verglichen, 3-Brompyruvate, Glyceraldehyd und 2-Deoxyglukose. Die beobachteten Effekte spiegeln zum Teil bekannte Effekte wider, liefern aber auch neue Hinweise und stärken die Kritik an 2-Deoxyglukose als selektiven Inhibitor der Glykolyse. Die gleichen Methoden und Strategien die in Zellkulturproben entwickelt wurden können schließlich auch in in vivo Mäuse Modellen benutzt werden. Der kurzzeitige Einbau von stabilen Isotopen in Intermediate des Stoffwechsels lieferte Hinweise über die notwendigen Zeiträume und über die verschiedenartige Nutzung von Glukose und Glutamin in Mäusen die als Modelle für menschlichen HCC-Tumor dienen