Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Quantitative analysis of metabolic fluxes in brown fat and skeletal muscle during thermogenesis

Nature Metabolism volume 5, pages 1204–1220 (2023)Cite this article

Subjects

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.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Arteriovenous metabolomics reveals broadly altered metabolic activities of BAT in cold-adapted mice.
Fig. 2: Acute cold challenge is only partially replicated by pharmacological β-adrenergic receptor activation.
Fig. 3: The landscape of leg metabolic activities under various thermogenic conditions.
Fig. 4: Quantitative analysis of BAT carbon influx and efflux.
Fig. 5: Quantitative analysis of BAT nitrogen influx and efflux.
Fig. 6: Distinct usage of glutamine nitrogen by BAT and liver at different temperatures.
Fig. 7: Cold-induced glutamine synthetase facilitates fuel oxidation in BAT by scavenging ammonia.

Similar content being viewed by others

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.

References

  1. Cannon, B. & Nedergaard, J. Brown adipose tissue: function and physiological significance. Physiol. Rev. 84, 277–359 (2004).

    CAS  Google Scholar 

  2. Rothwell, N. J. & Stock, M. J. A role for brown adipose tissue in diet-induced thermogenesis. Nature 281, 31–35 (1979).

    CAS  Google Scholar 

  3. Feldmann, H. M., Golozoubova, V., Cannon, B. & Nedergaard, J. UCP1 ablation induces obesity and abolishes diet-induced thermogenesis in mice exempt from thermal stress by living at thermoneutrality. Cell Metab. 9, 203–209 (2009).

    CAS  Google Scholar 

  4. von Essen, G., Lindsund, E., Cannon, B. & Nedergaard, J. Adaptive facultative diet-induced thermogenesis in wild-type but not in UCP1-ablated mice. Am. J. Physiol. Endocrinol. Metab. 313, E515–E527 (2017).

    Google Scholar 

  5. Hung, C. M. et al. Rictor/mTORC2 loss in the Myf5 lineage reprograms brown fat metabolism and protects mice against obesity and metabolic disease. Cell Rep. 8, 256–271 (2014).

    CAS  PubMed Central  Google Scholar 

  6. Jung, S. M. et al. Non-canonical mTORC2 signaling regulates brown adipocyte lipid catabolism through SIRT6-FoxO1. Mol. Cell 75, 807–822 e808 (2019).

    CAS  PubMed Central  Google Scholar 

  7. Nedergaard, J. & Cannon, B. Diet-induced thermogenesis: principles and pitfalls. Methods Mol. Biol. 2448, 177–202 (2022).

    Google Scholar 

  8. Betz, M. J. & Enerback, S. Targeting thermogenesis in brown fat and muscle to treat obesity and metabolic disease. Nat. Rev. Endocrinol. 14, 77–87 (2018).

    CAS  Google Scholar 

  9. Cypess, A. M. et al. Identification and importance of brown adipose tissue in adult humans. N. Engl. J. Med. 360, 1509–1517 (2009).

    CAS  PubMed Central  Google Scholar 

  10. Nedergaard, J., Bengtsson, T. & Cannon, B. Unexpected evidence for active brown adipose tissue in adult humans. Am. J. Physiol. Endocrinol. Metab. 293, E444–E452 (2007).

    CAS  PubMed  Google Scholar 

  11. Saito, M. et al. High incidence of metabolically active brown adipose tissue in healthy adult humans: effects of cold exposure and adiposity. Diabetes 58, 1526–1531 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. van Marken Lichtenbelt, W. D. et al. Cold-activated brown adipose tissue in healthy men. N. Engl. J. Med. 360, 1500–1508 (2009).

    PubMed  Google Scholar 

  13. Virtanen, K. A. et al. Functional brown adipose tissue in healthy adults. N. Engl. J. Med. 360, 1518–1525 (2009).

    CAS  PubMed  Google Scholar 

  14. Chen, K. Y. et al. Opportunities and challenges in the therapeutic activation of human energy expenditure and thermogenesis to manage obesity. J. Biol. Chem. 295, 1926–1942 (2020).

    CAS  Google Scholar 

  15. Wolfrum, C. & Gerhart-Hines, Z. Fueling the fire of adipose thermogenesis. Science 375, 1229–1231 (2022).

    CAS  Google Scholar 

  16. Seki, T. et al. Brown-fat-mediated tumour suppression by cold-altered global metabolism. Nature 608, 421–428 (2022).

    CAS  PubMed Central  Google Scholar 

  17. Hui, S. et al. Quantitative fluxomics of circulating metabolites. Cell Metab. 32, 676–688 (2020).

    CAS  PubMed Central  Google Scholar 

  18. Villarroya, F., Cereijo, R., Villarroya, J. & Giralt, M. Brown adipose tissue as a secretory organ. Nat. Rev. Endocrinol. 13, 26–35 (2017).

    CAS  Google Scholar 

  19. Cereijo, R. et al. CXCL14, a brown adipokine that mediates brown-fat-to-macrophage communication in thermogenic adaptation. Cell Metab. 28, 750–763 (2018).

    CAS  Google Scholar 

  20. Wang, Z. et al. Chronic cold exposure enhances glucose oxidation in brown adipose tissue. EMBO Rep. 21, e50085 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Trayhurn, P. Fatty acid synthesis in vivo in brown adipose tissue, liver and white adipose tissue of the cold-acclimated rat. FEBS Lett. 104, 13–16 (1979).

    CAS  PubMed  Google Scholar 

  22. Foster, D. O., Frydman, M. L. & Usher, J. R. Nonshivering thermogenesis in the rat. I. The relation between drug-induced changes in thermogenesis and changes in the concentration of plasma cyclic AMP. Can. J. Physiol. Pharmacol. 55, 52–64 (1977).

    CAS  PubMed  Google Scholar 

  23. Foster, D. O. & Frydman, M. L. Nonshivering thermogenesis in the rat. II. Measurements of blood flow with microspheres point to brown adipose tissue as the dominant site of the calorigenesis induced by noradrenaline. Can. J. Physiol. Pharmacol. 56, 110–122 (1978).

    CAS  PubMed  Google Scholar 

  24. Foster, D. O. & Frydman, M. L. Tissue distribution of cold-induced thermogenesis in conscious warm- or cold-acclimated rats reevaluated from changes in tissue blood flow: the dominant role of brown adipose tissue in the replacement of shivering by nonshivering thermogenesis. Can. J. Physiol. Pharmacol. 57, 257–270 (1979).

    CAS  PubMed  Google Scholar 

  25. Lopez-Soriano, F. J. & Alemany, M. Effect of cold-temperature exposure and acclimation on amino acid pool changes and enzyme activities of rat brown adipose tissue. Biochim. Biophys. Acta 925, 265–271 (1987).

    CAS  PubMed  Google Scholar 

  26. Jang, C., Chen, L. & Rabinowitz, J. D. Metabolomics and isotope tracing. Cell 173, 822–837 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Murashige, D. et al. Comprehensive quantification of fuel use by the failing and nonfailing human heart. Science 370, 364–368 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Jang, C. et al. Metabolite exchange between mammalian organs quantified in pigs. Cell Metab. 30, 594–606 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Berg, F., Gustafson, U. & Andersson, L. The uncoupling protein 1 gene (UCP1) is disrupted in the pig lineage: a genetic explanation for poor thermoregulation in piglets. PLoS Genet. 2, e129 (2006).

    PubMed  PubMed Central  Google Scholar 

  30. Hou, L. et al. Pig has no uncoupling protein 1. Biochem. Biophys. Res. Commun. 487, 795–800 (2017).

    CAS  Google Scholar 

  31. Dou, H. et al. Aryl hydrocarbon receptor (AhR) regulates adipocyte differentiation by assembling CRL4B ubiquitin ligase to target PPARγ for proteasomal degradation. J. Biol. Chem. 294, 18504–18515 (2019).

    CAS  PubMed Central  Google Scholar 

  32. Gnad, T. et al. Adenosine activates brown adipose tissue and recruits beige adipocytes via A2A receptors. Nature 516, 395–399 (2014).

    CAS  Google Scholar 

  33. Lahesmaa, M. et al. Regulation of human brown adipose tissue by adenosine and A2A receptors - studies with [(15)O]H2O and [(11)C]TMSX PET/CT. Eur. J. Nucl. Med. Mol. Imaging 46, 743–750 (2019).

    CAS  Google Scholar 

  34. Hui, S. et al. Glucose feeds the TCA cycle via circulating lactate. Nature 551, 115–118 (2017).

    PubMed Central  Google Scholar 

  35. Yoneshiro, T. et al. Metabolic flexibility via mitochondrial BCAA carrier SLC25A44 is required for optimal fever. eLife https://doi.org/10.7554/eLife.66865 (2021).

  36. Yoneshiro, T. et al. BCAA catabolism in brown fat controls energy homeostasis through SLC25A44. Nature 572, 614–619 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Keinan, O. et al. Glycogen metabolism links glucose homeostasis to thermogenesis in adipocytes. Nature 599, 296–301 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Jung, S. M. et al. In vivo isotope tracing reveals the versatility of glucose as a brown adipose tissue substrate. Cell Rep. 36, 109459 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Jabbour, H. N. & Sales, K. J. Prostaglandin receptor signalling and function in human endometrial pathology. Trends Endocrinol. Metab. 15, 398–404 (2004).

    CAS  PubMed  Google Scholar 

  40. Cekic, C. & Linden, J. Purinergic regulation of the immune system. Nat. Rev. Immunol. 16, 177–192 (2016).

    CAS  Google Scholar 

  41. Di Virgilio, F., Sarti, A. C., Falzoni, S., De Marchi, E. & Adinolfi, E. Extracellular ATP and P2 purinergic signalling in the tumour microenvironment. Nat. Rev. Cancer 18, 601–618 (2018).

    Google Scholar 

  42. Boldyrev, A. A., Aldini, G. & Derave, W. Physiology and pathophysiology of carnosine. Physiol. Rev. 93, 1803–1845 (2013).

    CAS  Google Scholar 

  43. Schaalan, M. F., Ramadan, B. K. & Abd Elwahab, A. H. Synergistic effect of carnosine on browning of adipose tissue in exercised obese rats; a focus on circulating irisin levels. J. Cell. Physiol. 233, 5044–5057 (2018).

    CAS  Google Scholar 

  44. Anderson, E. J. et al. A carnosine analog mitigates metabolic disorders of obesity by reducing carbonyl stress. J. Clin. Invest. 128, 5280–5293 (2018).

    PubMed Central  Google Scholar 

  45. Wolfe, R. R. Branched-chain amino acids and muscle protein synthesis in humans: myth or reality? J. Int Soc. Sports Nutr. 14, 30 (2017).

    PubMed Central  Google Scholar 

  46. Laha, A., Singh, M., George, A. K., Homme, R. P. & Tyagi, S. C. Dysregulation of 1-carbon metabolism and muscle atrophy: potential roles of forkhead box O proteins and PPARγ co-activator-1α. Can. J. Physiol. Pharmacol. 97, 1013–1017 (2019).

    CAS  Google Scholar 

  47. Ye, J. et al. Serine catabolism regulates mitochondrial redox control during hypoxia. Cancer Discov. 4, 1406–1417 (2014).

    CAS  PubMed Central  Google Scholar 

  48. Bartelt, A. et al. Brown adipose tissue activity controls triglyceride clearance. Nat. Med. 17, 200–205 (2011).

    CAS  Google Scholar 

  49. Heine, M. et al. Lipolysis triggers a systemic insulin response essential for efficient energy replenishment of activated brown adipose tissue in mice. Cell Metab. 28, 644–655 (2018).

    CAS  Google Scholar 

  50. Fischer, A. W. et al. Lysosomal lipoprotein processing in endothelial cells stimulates adipose tissue thermogenic adaptation. Cell Metab. 33, 547–564 (2021).

    CAS  PubMed  Google Scholar 

  51. Fischer, A. W. et al. Brown adipose tissue lipoprotein and glucose disposal is not determined by thermogenesis in uncoupling protein 1-deficient mice. J. Lipid Res. 61, 1377–1389 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. Berbee, J. F. et al. Brown fat activation reduces hypercholesterolaemia and protects from atherosclerosis development. Nat. Commun. 6, 6356 (2015).

    CAS  PubMed  Google Scholar 

  53. Wade, G., McGahee, A., Ntambi, J. M. & Simcox, J. Lipid transport in brown adipocyte thermogenesis. Front. Physiol. 12, 787535 (2021).

    PubMed  PubMed Central  Google Scholar 

  54. Simcox, J. et al. Global analysis of plasma lipids identifies liver-derived acylcarnitines as a fuel source for brown fat thermogenesis. Cell Metab. 26, 509–522 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  55. M, U. D. et al. Postprandial oxidative metabolism of human brown fat indicates thermogenesis. Cell Metab. 28, 207–216 (2018).

    Google Scholar 

  56. Adlanmerini, M. et al. Circadian lipid synthesis in brown fat maintains murine body temperature during chronic cold. Proc. Natl Acad. Sci. USA 116, 18691–18699 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  57. Fedorenko, A., Lishko, P. V. & Kirichok, Y. Mechanism of fatty-acid-dependent UCP1 uncoupling in brown fat mitochondria. Cell 151, 400–413 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  58. Mottillo, E. P. et al. Coupling of lipolysis and de novo lipogenesis in brown, beige, and white adipose tissues during chronic β3-adrenergic receptor activation. J. Lipid Res. 55, 2276–2286 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  59. Sanchez-Gurmaches, J. et al. Brown fat AKT2 is a cold-induced kinase that stimulates ChREBP-mediated de novo lipogenesis to optimize fuel storage and thermogenesis. Cell Metab. 27, 195–209 (2018).

    CAS  Google Scholar 

  60. Veliova, M. et al. Blocking mitochondrial pyruvate import in brown adipocytes induces energy wasting via lipid cycling. EMBO Rep. 21, e49634 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  61. Mills, E. L. et al. Accumulation of succinate controls activation of adipose tissue thermogenesis. Nature 560, 102–106 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  62. Bisbach, C. M. et al. Succinate can shuttle reducing power from the hypoxic retina to the O(2)-rich pigment epithelium. Cell Rep. 31, 107606 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  63. Heim, T. & Hull, D. The blood flow and oxygen consumption of brown adipose tissue in the new-born rabbit. J. Physiol. 186, 42–55 (1966).

    CAS  PubMed  PubMed Central  Google Scholar 

  64. Foster, D. O. & Frydman, M. L. Brown adipose tissue: the dominant site of nonshivering thermogenesis in the rat. Exp. Suppl. 32, 147–151 (1978).

    CAS  Google Scholar 

  65. Foster, D. O., Depocas, F. & Frydman, M. L. Noradrenaline-induced calorigenesis in warm- and cold-acclimated rats: relations between concentration of noradrenaline in arterial plasma, blood flow to differently located masses of brown adipose tissue, and calorigenic response. Can. J. Physiol. Pharmacol. 58, 915–924 (1980).

    CAS  PubMed  Google Scholar 

  66. Bean, C. et al. The mitochondrial protein Opa1 promotes adipocyte browning that is dependent on urea cycle metabolites. Nat. Metab. 3, 1633–1647 (2021).

    CAS  PubMed  Google Scholar 

  67. Ramirez, A. K. et al. Integrating extracellular flux measurements and genome-scale modeling reveals differences between brown and white adipocytes. Cell Rep. 21, 3040–3048 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  68. Wang, C. H. et al. CRISPR-engineered human brown-like adipocytes prevent diet-induced obesity and ameliorate metabolic syndrome in mice. Sci. Transl. Med. https://doi.org/10.1126/scitranslmed.aaz8664 (2020).

  69. Labbe, S. M. et al. mTORC1 is required for brown adipose tissue recruitment and metabolic adaptation to cold. Sci. Rep. 6, 37223 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  70. Villarroya, J. et al. New insights into the secretory functions of brown adipose tissue. J. Endocrinol. 243, R19–R27 (2019).

    CAS  Google Scholar 

  71. Scheele, C. & Wolfrum, C. Brown adipose crosstalk in tissue plasticity and human metabolism. Endocr. Rev. https://doi.org/10.1210/endrev/bnz007 (2020).

  72. Niemann, B. et al. Apoptotic brown adipocytes enhance energy expenditure via extracellular inosine. Nature https://doi.org/10.1038/s41586-022-05041-0 (2022).

    Article  PubMed Central  Google Scholar 

  73. Gnad, T. et al. Adenosine/A2B receptor signaling ameliorates the effects of aging and counteracts obesity. Cell Metab. 34, 649 (2022).

    CAS  Google Scholar 

  74. Yoo, H., Antoniewicz, M. R., Stephanopoulos, G. & Kelleher, J. K. Quantifying reductive carboxylation flux of glutamine to lipid in a brown adipocyte cell line. J. Biol. Chem. 283, 20621–20627 (2008).

    CAS  PubMed Central  Google Scholar 

  75. Mullen, A. R. et al. Reductive carboxylation supports growth in tumour cells with defective mitochondria. Nature 481, 385–388 (2011).

    PubMed Central  Google Scholar 

  76. Metallo, C. M. et al. Reductive glutamine metabolism by IDH1 mediates lipogenesis under hypoxia. Nature 481, 380–384 (2011).

    PubMed  PubMed Central  Google Scholar 

  77. McCormack, J. G. & Denton, R. M. Evidence that fatty acid synthesis in the interscapular brown adipose tissue of cold-adapted rats is increased in vivo by insulin by mechanisms involving parallel activation of pyruvate dehydrogenase and acetyl-coenzyme A carboxylase. Biochem. J. 166, 627–630 (1977).

    CAS  PubMed Central  Google Scholar 

  78. Shimazu, T. & Takahashi, A. Stimulation of hypothalamic nuclei has differential effects on lipid synthesis in brown and white adipose tissue. Nature 284, 62–63 (1980).

    CAS  Google Scholar 

  79. Yu, X. X., Lewin, D. A., Forrest, W. & Adams, S. H. Cold elicits the simultaneous induction of fatty acid synthesis and β-oxidation in murine brown adipose tissue: prediction from differential gene expression and confirmation in vivo. FASEB J. 16, 155–168 (2002).

    PubMed  Google Scholar 

  80. Weir, G. et al. Substantial metabolic activity of human brown adipose tissue during warm conditions and cold-induced lipolysis of local triglycerides. Cell Metab. 27, 1348–1355 (2018).

    CAS  PubMed Central  Google Scholar 

  81. Imai, K., Tsujisaki, M. & Yachi, A. [Application of monoclonal antibodies to cancer therapy: idiotype mapping of monoclonal antibodies to tumor-associated antigens]. Gan To Kagaku Ryoho 315, 1051–1059 (1988).

    Google Scholar 

  82. Spinelli, J. B. et al. Metabolic recycling of ammonia via glutamate dehydrogenase supports breast cancer biomass. Science 358, 941–946 (2017).

    CAS  PubMed Central  Google Scholar 

  83. Isler, D., Hill, H. P. & Meier, M. K. Glucose metabolism in isolated brown adipocytes under β-adrenergic stimulation. Quantitative contribution of glucose to total thermogenesis. Biochem. J. 245, 789–793 (1987).

    CAS  PubMed Central  Google Scholar 

  84. Ma, S. W. & Foster, D. O. Uptake of glucose and release of fatty acids and glycerol by rat brown adipose tissue in vivo. Can. J. Physiol. Pharmacol. 64, 609–614 (1986).

    CAS  Google Scholar 

  85. Saggerson, E. D., McAllister, T. W. & Baht, H. S. Lipogenesis in rat brown adipocytes. Effects of insulin and noradrenaline, contributions from glucose and lactate as precursors and comparisons with white adipocytes. Biochem. J. 251, 701–709 (1988).

    CAS  PubMed Central  Google Scholar 

  86. Lopez-Soriano, F. J. & Alemany, M. Activities of enzymes of amino acid metabolism in rat brown adipose tissue. Biochem. Int. 12, 471–478 (1986).

    CAS  Google Scholar 

  87. Lopez-Soriano, F. J. et al. Amino acid and glucose uptake by rat brown adipose tissue. Effect of cold-exposure and acclimation. Biochem. J. 252, 843–849 (1988).

    CAS  PubMed Central  Google Scholar 

  88. Kaikaew, K., Grefhorst, A. & Visser, J. A. Sex differences in brown adipose tissue function: sex hormones, glucocorticoids, and their crosstalk. Front. Endocrinol. 12, 652444 (2021).

    Google Scholar 

  89. Keuper, M. & Jastroch, M. The good and the BAT of metabolic sex differences in thermogenic human adipose tissue. Mol. Cell. Endocrinol. 533, 111337 (2021).

    CAS  Google Scholar 

  90. Ouellet, V. et al. Brown adipose tissue oxidative metabolism contributes to energy expenditure during acute cold exposure in humans. J. Clin. Invest. 122, 545–552 (2012).

    PubMed  PubMed Central  Google Scholar 

  91. Carneheim, C., Nedergaard, J. & Cannon, B. β-adrenergic stimulation of lipoprotein lipase in rat brown adipose tissue during acclimation to cold. Am. J. Physiol. 246, E327–E333 (1984).

    CAS  PubMed  Google Scholar 

  92. Schreiber, R. et al. Cold-induced thermogenesis depends on ATGL-mediated lipolysis in cardiac muscle, but not brown adipose tissue. Cell Metab. 26, 753–763 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  93. Wollenberger, A., Ristau, O. & Schoffa, G. [A simple technic for extremely rapid freezing of large pieces of tissue]. Pflug. Arch. Gesamt. Physiol. Menschen Tiere 270, 399–412 (1960).

    CAS  Google Scholar 

  94. Heinrich, P. et al. Correcting for natural isotope abundance and tracer impurity in MS-, MS/MS- and high-resolution-multiple-tracer-data from stable isotope labeling experiments with IsoCorrectoR. Sci. Rep. 8, 17910 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  95. Respress, J. L. & Wehrens, X. H. Transthoracic echocardiography in mice. J. Vis. Exp. https://doi.org/10.3791/1738 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  96. Scherrer-Crosbie, M. & Thibault, H. B. Echocardiography in translational research: of mice and men. J. Am. Soc. Echocardiogr. 21, 1083–1092 (2008).

    PubMed  PubMed Central  Google Scholar 

  97. Fasshauer, M. et al. Essential role of insulin receptor substrate 1 in differentiation of brown adipocytes. Mol. Cell. Biol. 21, 319–329 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  98. Isidor, M. S. et al. An siRNA-based method for efficient silencing of gene expression in mature brown adipocytes. Adipocyte 5, 175–185 (2016).

    CAS  PubMed  Google Scholar 

  99. Spinelli, J. B., Kelley, L. P. & Haigis, M. C. An LC–MS approach to quantitative measurement of ammonia isotopologues. Sci. Rep. 7, 10304 (2017).

    PubMed  PubMed Central  Google Scholar 

Download references

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.

Author information

Author notes

  1. These authors contributed equally: Grace Park, John A. Haley, Johnny Le, Su Myung Jung.

Authors and Affiliations

  1. Department of Biological Chemistry, University of California Irvine, Irvine, CA, USA

    Grace Park, Johnny Le & Cholsoon Jang

  2. Program in Molecular Medicine, UMass Chan Medical School, Worcester, MA, USA

    John A. Haley, Ekaterina D. Korobkina, Huawei Li, Shelagh M. Fluharty, Qingbo Chen, Jessica B. Spinelli & David A. Guertin

  3. Department of Biological Sciences, Sungkyunkwan University, Suwon, South Korea

    Su Myung Jung

  4. Division of Cardiovascular Medicine, Department of Medicine, UMass Chan Medical School, Worcester, MA, USA

    Timothy P. Fitzgibbons & Chinmay M. Trivedi

  5. Department of Molecular, Cell and Cancer Biology, UMass Chan Medical School, Worcester, MA, USA

    Chinmay M. Trivedi

  6. Li-Weibo Institute for Rare Diseases Research, UMass Chan Medical School, Worcester, MA, USA

    Chinmay M. Trivedi

Authors
  1. Grace Park
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. John A. Haley
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Johnny Le
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Su Myung Jung
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Timothy P. Fitzgibbons
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Ekaterina D. Korobkina
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Huawei Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Shelagh M. Fluharty
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Qingbo Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Jessica B. Spinelli
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Chinmay M. Trivedi
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Cholsoon Jang
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. David A. Guertin
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

D.A.G. and C.J. conceived the project and supervised the study. G.P. performed sample processing and LC–MS 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 LC–MS 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.

Corresponding authors

Correspondence to Cholsoon Jang or David A. Guertin.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature Metabolism thanks Brandon Faubert and the other, anonymous, reviewers for their contribution to the peer review of this work. Primary Handling Editor: Alfredo Giménez-Cassina, in collaboration with the Nature Metabolism team

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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 Sulzer’s 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 (22 °C) or severe cold (6 °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. ****p = 3×10−12 by unpaired two-tailed Student’s t-test. b, Final body weight of TN, CC, AC, CL-treated mice. Data are mean ± s.e. **p = 0.001 and p = 0.009 by one-way ANOVA with Tukey’s 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.

Source data

Extended Data Fig. 3 AV concentration gradients of the 35 primary fuel metabolites.

Median values from mice in Figs. 1–3 are shown.

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 ‘LIPID’ 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 5 min after tracer administration in BAT for TN, MC and CC. Data are mean ± s.e. N = 6 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. N = 6 mice for TN, N = 6 mice for MC, N = 7 mice for CC at 2.5 min, N = 6 mice for all temperature conditions at 5 min, N = 6 mice for TN, N = 6 mice for MC, N = 5 mice for CC at 15 min for BAT and N = 6 mice for all temperature conditions at 15 min for liver and serum. c, Total normalized labeling fraction of carbon atoms in representative TCA intermediates in BAT. Data are mean ± s.e. ****p < 0.0001 by two-way ANOVA with post-hoc Tukey HSD Test. Malate MC vs TN **p = 0.0021, Succinate CC vs TN ***p = 0.0002 and MC vs TN **p = 0.0027. N = 6 mice for TN, N = 6 mice for MC, N = 7 mice for CC at 2.5 min, N = 6 mice for all temperature conditions at 5 min, N = 6 mice for TN, N = 6 mice for MC, N = 5 mice for CC at 15 min. d, Normalized carbon labeling fraction of representative TCA intermediates at 5 min after tracer administration. Data are mean ± s.e. N = 6 mice per temperature condition. e, Schematic of TCA cycle labeling from glutamine. Conventional TCA cycle predicts M + 4 labeling of succinate, malate and citrate from glutamine tracer, whereas reversed TCA cycle (that is, reductive carboxylation) predicts M + 5 labeling of citrate. PC flux can also generate M + 1 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. N = 6 mice for TN, N = 6 mice for MC, N = 7 mice for CC at 2.5 min, N = 6 mice for all temperature conditions at 5 min, N = 6 mice for TN, N = 6 mice for MC, N = 5 mice for CC at 15 min. 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, qRT–PCR comparing Glul gene expression in BAT for TN and CC. N = 8 mice per condition. Data are mean ± s.e. ***p = 0.0003 by unpaired two-tailed Student’s t-test. b, Western blot of GS in brown adipocytes during adipogenesis. c, Western blot of GS in different tissues from mice at 22 °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. N = 5 mice were used for each time point except TN 15-minute and CC 5-minute N = 4 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. N = 15 biological replicates. Data are mean ± s.e.

Source data

Extended Data Table 1 List of metabolites significantly taken up or released by BAT and leg across TN, CC, AC and CL
Full size table
Extended Data Table 2 Carbon flux values in BAT across TN, CC, AC and CL
Full size table
Extended Data Table 3 Nitrogen flux values in BAT across TN, CC, AC and CL
Full size table

Supplementary information

Source data

Source Data Fig. 6

Statistical Source Data.

Source Data Fig. 7

Unprocessed western blots.

Source Data Extended Data Fig. 2

Unprocessed western blots.

Source Data Extended Data Fig. 7

Unprocessed western blots.

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

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, 1204–1220 (2023). https://doi.org/10.1038/s42255-023-00825-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s42255-023-00825-8

This article is cited by

Search

Advanced search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research