Isotope Tracing Untargeted Metabolomics Reveals Macrophage Polarization-State-Specific Metabolic Coordination across Intracellular Compartments
Open accessAbstract
We apply stable isotope tracing, mass-spectrometry-based untargeted metabolomics, to reveal the biochemical space labeled by 13 C-substrates in bone-marrow-derived macrophages. At the pathway level, classically (lipopolysaccharide [LPS]-polarized, M1) and alternatively (interleukin [IL]-4-polarized, M2) polarized macrophages were 13 C-labeled with surprising concordance. Total pools of uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), an intermediate in the hexosamine biosynthetic pathway, were equally abundant in LPS- and IL-4-polarized macrophages. Informatic scrutiny of 13 C-isotopologues revealed that LPS-polarized macrophages leverage the pentose phosphate pathway to generate UDP-GlcNAc, whereas IL-4-polarized macrophages rely on intact glucose and mitochondrial metabolism of glucose carbon. Labeling from [ 13 C]glucose is competed by unlabeled fatty acids and acetoacetate, underscoring the broad roles for substrate metabolism beyond energy conversion. Finally, the LPS-polarized macrophage metabolite itaconate is imported into IL-4-polarized macrophages, in which it reprograms [ 13 C]glucose metabolism. Thus, use of fully unsupervised isotope tracing metabolomics in macrophages reveals polarization-state-specific metabolic pathway connectivity, substrate competition, and metabolite allocation among cellular compartments.
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Metabolite extraction and LC/MS analysis was performed as described previously.
“Metabolite extraction and LC/MS analysis was performed as described previously [55][56][57] .”
From APOE4-drives brain bioenergetic shifts independent of Alzheimer’s disease pathology (2026) · cites this paper as [55]
The Introduction describes LC–high-resolution MS-based untargeted metabolomics, supporting the citation for LC/MS analysis. The Methods section directs readers to a supplemental file, but our copy does not describe metabolite extraction or establish that the citing paper followed the same procedures. (LLM, conf=0.78)
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- Authors
- Puchalska, P., Huang, X., Martin, S. E., Han, X., Patti, G. J., & Crawford, P. A.
- Affiliations
- University of Minnesota (US)
- Memorial Sloan Kettering Cancer Center (US)
- Sanford Burnham Prebys Medical Discovery Institute (US)
- The University of Texas at San Antonio Health Science Center (US)
- Washington University in St. Louis (US)
- Corresponding author
- Crawford, Peter A.
- Venue
- iScience
- Publisher
- Elsevier BV
- Published
- 2018-11
- Volume
- 9
- Pages
- 298-313
- ISSN
- 2589-0042 (ISSN-L)
- PMC
- PMC6240706
- OpenAlex
- W2899243179
- Cited by
- 79
- References
- 7
- Citations by year
- 2026: 7 · 2025: 8 · 2024: 13 · 2023: 12 · 2022: 9 · 2021: 10 · 2020: 10 · 2019: 9 · 2018: 1
- FWCI
- 2.62
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- Locations
- iScience (publishedVersion, cc-by-nc-nd)
- Europe PMC (PubMed Central) (publishedVersion, cc-by-nc-nd)
- PubMed Central (publishedVersion, cc-by-nc-nd)
- PubMed (publishedVersion)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- Funders
- NIH (DK091538, CA235482, ES028365, OD024624)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Metabolomics and Mass Spectrometry Studies
- Life Sciences › Immunology and Microbiology › Immunology › Immune cells in cancer
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Microbial Metabolic Engineering and Bioproduction
- Keywords
- macrophage polarization, M1/M2 macrophages, 13C isotope tracing, untargeted metabolomics, bone marrow-derived macrophages, UDP-GlcNAc, hexosamine biosynthetic pathway, pentose phosphate pathway, glucose metabolism, substrate competition, itaconate, mitochondrial metabolism, fatty acids, acetoacetate
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