Division of Molecular Medicine, Department of Medicine, University of Minnesota, 401 East River Parkway, MMC 194, Minneapolis, MN 55455, USA; Center for Metabolic Origins of Disease, Sanford Burnham Prebys Medical Discovery Institute, Orlando, FL 32827, USA
Xiaojing Huang
Center for Metabolic Origins of Disease, Sanford Burnham Prebys Medical Discovery Institute, Orlando, FL 32827, USA; Department of Chemistry, Washington University, St. Louis, MO 63110, USA; Department of Radiation Oncology, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA
Shannon E. Martin
Center for Metabolic Origins of Disease, Sanford Burnham Prebys Medical Discovery Institute, Orlando, FL 32827, USA; Pathobiology Graduate Program, Brown University, Providence, RI 02912, USA
Xianlin Han
Center for Metabolic Origins of Disease, Sanford Burnham Prebys Medical Discovery Institute, Orlando, FL 32827, USA; Barshop Institute for Longevity and Aging Studies, Department of Medicine, Division of Diabetes, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229, USA
Gary J. Patti
Department of Chemistry, Washington University, St. Louis, MO 63110, USA
Peter A. Crawford
Division of Molecular Medicine, Department of Medicine, University of Minnesota, 401 East River Parkway, MMC 194, Minneapolis, MN 55455, USA; Center for Metabolic Origins of Disease, Sanford Burnham Prebys Medical Discovery Institute, Orlando, FL 32827, USA; Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA; Corresponding author
Summary: We apply stable isotope tracing, mass-spectrometry-based untargeted metabolomics, to reveal the biochemical space labeled by 13C-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 13C-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 13C-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 [13C]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 [13C]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. : Components of the Immune System; Immunology; Metabolomics Subject Areas: Components of the Immune System, Immunology, Metabolomics