Abstract
Adaptive thermogenesis by brown adipose tissue (BAT) dissipates calories as heat, making it an attractive anti-obesity target. Yet how BAT contributes to circulating metabolite exchange remains unclear. Here, we quantified metabolite exchange in BAT and skeletal muscle by arteriovenous metabolomics during cold exposure in fed male mice. This identified unexpected metabolites consumed, released and shared between organs. Quantitative analysis of tissue fluxes showed that glucose and lactate provide ~85% of carbon for adaptive thermogenesis and that cold and CL316,243 trigger markedly divergent fuel utilization profiles. In cold adaptation, BAT also dramatically increases nitrogen uptake by net consuming amino acids, except glutamine. Isotope tracing and functional studies suggest glutamine catabolism concurrent with synthesis via glutamine synthetase, which avoids ammonia buildup and boosts fuel oxidation. These data underscore the ability of BAT to function as a glucose and amino acid sink and provide a quantitative and comprehensive landscape of BAT fuel utilization to guide translational studies.
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Data availability
All data needed to evaluate the conclusions in the paper are present in the paper and/or the extended tables. R scripts to conduct bootstrapping to calculate the confidence interval of the difference between tissue and serum area under the curve are available on GitHub at https://github.com/johnnl15/Bootstrapping_AUC_NitrogenFL_BATLiverSerum.Source data are provided with this paper.
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Acknowledgements
We thank all members of the Jang and Guertin laboratories for the discussion. We thank J. Park for help with bootstrapping and statistical methods and F. Roberts and T. Cashman for their technical assistance with blood flow measurements. This work was funded by R01DK116005, R01DK127175 and R01DK094004 to D.A.G.; the AASLD Foundation Pinnacle Research Award in Liver Disease, The Edward Mallinckrodt, Jr. Foundation Award, R01DK127175 and R01 AA029124 to C.J.; F31DK129018 to J.A.H.; T32GM008620 and F31DK134173 to J.L.; Basic Science Research Program of the Ministry of Education (South Korea) NRF-2019R1A6A1A10073079 to S.M.J.; and R01HL118100 and R01HL141377 to C.M.T.
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D.A.G. and C.J. conceived the project and supervised the study. G.P. performed sample processing and LCâMS analysis for the AV experiments. J.A.H. performed most of the animal experiments, sample preparation and the experiments and analysis related to GS activity, including ammonia-tracing studies. J.L. performed sample processing and LCâMS analysis for glucose and glutamine-tracing experiments. S.M.J. helped develop the AV collection strategy and assisted in early animal experiments and performed animal experiments for glutamine tracing. T.P.F. performed Doppler imaging and analysis. E.D.K. assisted with animal dissections for AV experiments and ammonia tracing and provided protein samples for the tissue panel and adipogenesis panel. H.L. assisted with the glutamine- and ammonia-tracing experiments and animal colony management. S.M.F. and Q.C. assisted with AV experiments and ammonia tracing and animal dissections for blood flow, respectively. J.B.S. assisted with ammonia-tracing experiments. C.M.T. assisted with blood flow experiments. G.P., J.A.H., J.L., S.M.J., C.J. and D.A.G. wrote the manuscript.
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Extended data
Extended Data Fig. 1 Establishment of AV metabolomics for BAT and hind limb in mice.
a, Schematic of blood vessels used for AV sampling. The Sulzerâs vein (SV) and femoral vein (FV) were used to characterize BAT and hind limb activity. Systemic arterial blood was collected from the left ventricle (LV). Made with BioRender.com. b, Different biological scenarios reflected by AV gradients across BAT. Positive and negative values indicate net release and absorption, while net zero values indicate metabolite bypass (neither uptake nor release). intracellular futile cycling (release equal to uptake) or intercellular cross-exchange between adipocytes and non-adipocytes. c, Heat map shows different metabolite abundances between LV, SV and FV blood collected from mice adapted to mild (22â°C) or severe cold (6â°C). Box 1 highlights metabolites more abundant in BAT-draining blood (SV) than blood from other sites, regardless of temperature, boxes 2-3 highlight metabolites more abundant in BAT-draining blood (SV) or systemic arterial blood (LV) and temperature-sensitive and box 4 highlights metabolites sensitive to temperature across organs. Each column shows an individual mouse.
Extended Data Fig. 2 Characterization of BAT in TN, CC, AC and CL.
a, Daily food intake of TN and CC-adapted mice. Data are mean ± s.e. ****pâ=â3Ã10â12 by unpaired two-tailed Studentâs t-test. b, Final body weight of TN, CC, AC, CL-treated mice. Data are mean ± s.e. **pâ=â0.001 and pâ=â0.009 by one-way ANOVA with Tukeyâs multiple comparisons test. c, Western blot of key markers in BAT from mice in TN, CC, AC and CL. S.E., short exposure; L.E., long exposure. d, H&E images of BAT from mice in TN, CC, AC and CL. Scale bar = 50 μm.
Extended Data Fig. 4 Quantitative analysis of BAT total carbon and nitrogen influx and efflux in TN and CL.
Colors indicate different metabolite categories. Metabolites are ordered based on their relative contributions from greatest to least. Fatty acids from lipoprotein particles are indicated as âLIPIDâ after each fatty acid species (for example, C16:0 LIPID).
Extended Data Fig. 5 Temperature-dependent glutamine carbon usage by BAT and liver.
a, Glutamine fractional labeling (both carbon and nitrogen) at 5âmin after tracer administration in BAT for TN, MC and CC. Data are mean ± s.e. Nâ=â6 mice per temperature condition. b, Heat map shows median of the total 13C-labeled carbons in metabolites in BAT, liver and serum for TN, MC and CC, scaled for each metabolite and organ. Nâ=â6 mice for TN, Nâ=â6 mice for MC, Nâ=â7 mice for CC at 2.5âmin, Nâ=â6 mice for all temperature conditions at 5âmin, Nâ=â6 mice for TN, Nâ=â6 mice for MC, Nâ=â5 mice for CC at 15âmin for BAT and Nâ=â6 mice for all temperature conditions at 15âmin for liver and serum. c, Total normalized labeling fraction of carbon atoms in representative TCA intermediates in BAT. Data are mean ± s.e. ****pâ<â0.0001 by two-way ANOVA with post-hoc Tukey HSD Test. Malate MC vs TN **pâ=â0.0021, Succinate CC vs TN ***pâ=â0.0002 and MC vs TN **pâ=â0.0027. Nâ=â6 mice for TN, Nâ=â6 mice for MC, Nâ=â7 mice for CC at 2.5âmin, Nâ=â6 mice for all temperature conditions at 5âmin, Nâ=â6 mice for TN, Nâ=â6 mice for MC, Nâ=â5 mice for CC at 15âmin. d, Normalized carbon labeling fraction of representative TCA intermediates at 5âmin after tracer administration. Data are mean ± s.e. Nâ=â6 mice per temperature condition. e, Schematic of TCA cycle labeling from glutamine. Conventional TCA cycle predicts Mâ+â4 labeling of succinate, malate and citrate from glutamine tracer, whereas reversed TCA cycle (that is, reductive carboxylation) predicts Mâ+â5 labeling of citrate. PC flux can also generate Mâ+â1 citrate with labeled CO2 incorporation. Citrate can be used for de novo lipogenesis. Made with BioRender.com.
Extended Data Fig. 6 Temperature-dependent glutamine nitrogen usage by BAT.
a, Heat map shows median of total 15N-labeled nitrogen in BAT metabolites for TN, MC and CC. Nâ=â6 mice for TN, Nâ=â6 mice for MC, Nâ=â7 mice for CC at 2.5âmin, Nâ=â6 mice for all temperature conditions at 5âmin, Nâ=â6 mice for TN, Nâ=â6 mice for MC, Nâ=â5 mice for CC at 15âmin. b, Schematic of nitrogen exchange reactions between glutamine, glutamate and keto acids. Made with BioRender.com.
Extended Data Fig. 7 Cold-induced glutamine synthetase in BAT.
a, qRTâPCR comparing Glul gene expression in BAT for TN and CC. Nâ=â8 mice per condition. Data are mean ± s.e. ***pâ=â0.0003 by unpaired two-tailed Studentâs t-test. b, Western blot of GS in brown adipocytes during adipogenesis. c, Western blot of GS in different tissues from mice at 22â°C. iB, interscapular BAT; sB, subcutaneous BAT; iW, inguinal white fat; pW, perigonadal white fat; LV, liver; Q, quadricep; S, spleen; H, heart; Lu, lung; B, brain; K, kidney. d-g,15N1-labeled glutamine and glutamate abundances in liver (d,e) or serum (f,g) after 15N-ammonia tracer administration. Nâ=â5 mice were used for each time point except TN 15-minute and CC 5-minute Nâ=â4 mice were used. Data are mean ± s.e. h, Full graph of oxygen consumption rate in mature brown adipocytes transfected with control or Glul targeting siRNAs with or without norepinephrine (NE) stimulation. Nâ=â15 biological replicates. Data are mean ± s.e.
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Statistical Source Data.
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Unprocessed western blots.
Source Data Extended Data Fig. 7
Unprocessed western blots.
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Park, G., Haley, J.A., Le, J. et al. Quantitative analysis of metabolic fluxes in brown fat and skeletal muscle during thermogenesis. Nat Metab 5, 1204â1220 (2023). https://doi.org/10.1038/s42255-023-00825-8
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DOI: https://doi.org/10.1038/s42255-023-00825-8
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