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. 2020 Feb 4;11:698. doi: 10.1038/s41467-020-14433-7

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© This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 1 — Heat-maps of the log10 ratio from the average peak areas from Gas Chromatography-Mass Spectrometry (GC-MS) analysis of metabolites associated with the arginine metabolism (a), glycolysis (b), and citric acid cycle (c) from bone marrow-derived macrophages (BMDMs) from wild-type (WT) and Nos2^−/− mice activated with LPS for 24 h compared to unstimulated (ctrl). d Schematic illustration of atom transitions in central metabolism using uniformly labeled ¹³C-glucose ([U-¹³C]) (labeled carbons are indicated in blue) as tracer for determination of mass isotopologue distributions (MID) to infer relative intracellular fluxes through oxidation of pyruvate. PDH pyruvate dehydrogenase, ACO2 aconitase 2, IDH isocitrate dehydrogenase, OGDH oxoglutarate dehydrogenase, SDH succinate dehydrogenase, FH fumarate hydratase, MDH malate dehydrogenase. e–g WT and Nos2^−/− BMDMs were activated with LPS + IFNγ and cultured with labeled tracer. Bars show evaluation of the [U-¹³C] glucose-derived carbon incorporation (percentage) into m + 2 isotopologues of the indicated TCA intermediates. e Percentage of ¹³C in m + 2 isotopologues of cis-aconitate (cis-aco) and α-ketoglutarate (α-kG). f Bar graph depicting the ratio between citrate and α-kG for ¹³C fraction of the total metabolite level. g m + 2 isotopologues of glutamate (glu), succinate (succ), fumarate, malate and in m + 1 itaconate (ita) in activated WT and Nos2^−/− BMDMs. Data in a–c (n = 5 biological replicates per group) and e–g (n = 6) were analyzed by two-way ANOVA (interaction < 0.0001) (Sidak’s post-tests). h Expression profile of selected inflammatory genes from microarray analysis (see Table1) shown as heatmap of log2 fold changes of LPS + IFNγ-stimulated Nos2^−/− BMDMs compared to WT (n = 3). I The concentration of IL1β, IL6, IL12p40, TNFα, IL10, MCP1, MIP1α, and KC secreted into the culture media when BMDMs were stimulated for 24 h. Data are pooled from three experiments (n = 7–8) and were analyzed by two-way ANOVA with Sidak’s post-tests. All error bars display mean ± SEM. Source data are provided as a Source Data file.