Isotope Tracing Untargeted Metabolomics Reveals Macrophage Polarization-State-Specific Metabolic Coordination across Intracellular Compartments

Patrycja Puchalska; Xiaojing Huang; Shannon E. Martin; Xianlin Han; Gary J. Patti; Peter A. Crawford

doi:10.1016/j.isci.2018.10.029

Isotope Tracing Untargeted Metabolomics Reveals Macrophage Polarization-State-Specific Metabolic Coordination across Intracellular Compartments

Patrycja Puchalska, Xiaojing Huang, Shannon E. Martin, Xianlin Han, Gary J. Patti, Peter A. Crawford

Research output: Contribution to journal › Article › peer-review

50 Scopus citations

Abstract

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.

Original language	English (US)
Pages (from-to)	298-313
Number of pages	16
Journal	iScience
Volume	9
DOIs	https://doi.org/10.1016/j.isci.2018.10.029
State	Published - Nov 30 2018

Bibliographical note

Funding Information:
The authors thank Alisa Nelson for helpful discussions, J. Matthew Gandy and Matthew Longo for assistance with mouse husbandry, Laura Kyro for graphics expertise, and Peter Phelan for guidance on primary macrophage isolation and culture. This work was supported in part by grants from the NIH (DK091538, CA235482, ES028365, and OD024624). Conceptualization, P.P. X. Huang, and P.A.C.; Methodology, P.P. X. Huang, S.M. G.J.P. and P.A.C.; Investigation, P.P. X. Huang, S.M.; Resources, P.A.C.; Writing – Original Draft, P.P. and P.A.C.; Writing – Review & Editing, all authors; Visualization, P.P. X. Huang, and P.A.C.; Supervision, X. Han, G.J.P. and P.A.C.; Funding Acquisition, P.A.C. The authors declare no conflict of interest.

Funding Information:
The authors thank Alisa Nelson for helpful discussions, J. Matthew Gandy and Matthew Longo for assistance with mouse husbandry, Laura Kyro for graphics expertise, and Peter Phelan for guidance on primary macrophage isolation and culture. This work was supported in part by grants from the NIH ( DK091538 , CA235482 , ES028365 , and OD024624 ).

Publisher Copyright:
© 2018 The Authors

Keywords

Components of the Immune System
Immunology
Metabolomics

Access

10.1016/j.isci.2018.10.029

OpenUrl availability

Full text

Cite this

@article{2f3757eca92044b9a4584c57e4f6fe61,

title = "Isotope Tracing Untargeted Metabolomics Reveals Macrophage Polarization-State-Specific Metabolic Coordination across Intracellular Compartments",

abstract = "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.",

keywords = "Components of the Immune System, Immunology, Metabolomics",

author = "Patrycja Puchalska and Xiaojing Huang and Martin, {Shannon E.} and Xianlin Han and Patti, {Gary J.} and Crawford, {Peter A.}",

note = "Funding Information: The authors thank Alisa Nelson for helpful discussions, J. Matthew Gandy and Matthew Longo for assistance with mouse husbandry, Laura Kyro for graphics expertise, and Peter Phelan for guidance on primary macrophage isolation and culture. This work was supported in part by grants from the NIH (DK091538, CA235482, ES028365, and OD024624). Conceptualization, P.P. X. Huang, and P.A.C.; Methodology, P.P. X. Huang, S.M. G.J.P. and P.A.C.; Investigation, P.P. X. Huang, S.M.; Resources, P.A.C.; Writing – Original Draft, P.P. and P.A.C.; Writing – Review & Editing, all authors; Visualization, P.P. X. Huang, and P.A.C.; Supervision, X. Han, G.J.P. and P.A.C.; Funding Acquisition, P.A.C. The authors declare no conflict of interest. Funding Information: The authors thank Alisa Nelson for helpful discussions, J. Matthew Gandy and Matthew Longo for assistance with mouse husbandry, Laura Kyro for graphics expertise, and Peter Phelan for guidance on primary macrophage isolation and culture. This work was supported in part by grants from the NIH ( DK091538 , CA235482 , ES028365 , and OD024624 ). Publisher Copyright: {\textcopyright} 2018 The Authors",

year = "2018",

month = nov,

day = "30",

doi = "10.1016/j.isci.2018.10.029",

language = "English (US)",

volume = "9",

pages = "298--313",

journal = "iScience",

issn = "2589-0042",

publisher = "Elsevier Inc.",

}

TY - JOUR

T1 - Isotope Tracing Untargeted Metabolomics Reveals Macrophage Polarization-State-Specific Metabolic Coordination across Intracellular Compartments

AU - Puchalska, Patrycja

AU - Huang, Xiaojing

AU - Martin, Shannon E.

AU - Han, Xianlin

AU - Patti, Gary J.

AU - Crawford, Peter A.

N1 - Funding Information: The authors thank Alisa Nelson for helpful discussions, J. Matthew Gandy and Matthew Longo for assistance with mouse husbandry, Laura Kyro for graphics expertise, and Peter Phelan for guidance on primary macrophage isolation and culture. This work was supported in part by grants from the NIH (DK091538, CA235482, ES028365, and OD024624). Conceptualization, P.P. X. Huang, and P.A.C.; Methodology, P.P. X. Huang, S.M. G.J.P. and P.A.C.; Investigation, P.P. X. Huang, S.M.; Resources, P.A.C.; Writing – Original Draft, P.P. and P.A.C.; Writing – Review & Editing, all authors; Visualization, P.P. X. Huang, and P.A.C.; Supervision, X. Han, G.J.P. and P.A.C.; Funding Acquisition, P.A.C. The authors declare no conflict of interest. Funding Information: The authors thank Alisa Nelson for helpful discussions, J. Matthew Gandy and Matthew Longo for assistance with mouse husbandry, Laura Kyro for graphics expertise, and Peter Phelan for guidance on primary macrophage isolation and culture. This work was supported in part by grants from the NIH ( DK091538 , CA235482 , ES028365 , and OD024624 ). Publisher Copyright: © 2018 The Authors

PY - 2018/11/30

Y1 - 2018/11/30

N2 - 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.

AB - 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.

KW - Components of the Immune System

KW - Immunology

KW - Metabolomics

UR - http://www.scopus.com/inward/record.url?scp=85060711802&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85060711802&partnerID=8YFLogxK

U2 - 10.1016/j.isci.2018.10.029

DO - 10.1016/j.isci.2018.10.029

M3 - Article

C2 - 30448730

AN - SCOPUS:85060711802

SN - 2589-0042

VL - 9

SP - 298

EP - 313

JO - iScience

JF - iScience

ER -

Isotope Tracing Untargeted Metabolomics Reveals Macrophage Polarization-State-Specific Metabolic Coordination across Intracellular Compartments

Abstract

Bibliographical note

Keywords

Access

OpenUrl availability

Other files and links

Fingerprint

Cite this