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.

Nitrogen recycling buffers against ammonia toxicity from skeletal muscle breakdown in hibernating arctic ground squirrels

Abstract

Hibernation is a state of extraordinary metabolic plasticity. The pathways of amino acid metabolism as they relate to nitrogen homeostasis in hibernating mammals in vivo are unknown. Here we show, using pulse isotopic tracing, evidence of increased myofibrillar (skeletal muscle) protein breakdown and suppressed whole-body production of metabolites in vivo throughout deep torpor. As whole-body production of metabolites is suppressed, amino acids with nitrogenous side chains accumulate during torpor, while urea cycle intermediates do not. Using 15N stable isotope methodology in arctic ground squirrels (Urocitellus parryii), we provide evidence that free nitrogen is buffered and recycled into essential amino acids, non-essential amino acids and the gamma-glutamyl system during the inter-bout arousal period of hibernation. In the absence of nutrient intake or physical activity, our data illustrate the orchestration of metabolic pathways that sustain the provision of essential and non-essential amino acids and prevent ammonia toxicity during hibernation.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: Schematic for separate experimental procedures.
Fig. 2: Skeletal muscle breakdown is ongoing and WBP of all metabolites is depressed in deep torpor.
Fig. 3: Circulating nitrogen metabolite pools increase as torpor progresses, while urea cycle intermediates do not increase.
Fig. 4: Free [15N]ammonia is recycled into non-essential amino acids (blue), essential amino acids (red) and 5-oxoproline (turquoise) during arousal from torpor.
Fig. 5: Proposed pathway for [15N]ammonia recycling into amino acids depends on non-essential amino acid incorporation during arousal from torpor.
Fig. 6: Branched-chain keto acid WBP is suppressed in animals in torpor, but ketoacids and ketones increase during arousal from torpor.
Fig. 7: Pathway enrichment analysis shows evidence for amino acid metabolism and BCAA biosynthesis in arousal from torpor and points to the prevalence of transamination reactions involving glutamate, alanine and aspartate during torpor.

Data availability

The datasets generated and/or analysed during the current study are available from the corresponding author on reasonable request.

References

  1. 1.

    Carey, H. V., Andrews, M. T. & Martin, S. L. Mammalian hibernation: cellular and molecular responses to depressed metabolism and low temperature. Physiol. Rev. 83, 1153–1181 (2003).

    CAS  PubMed  Google Scholar 

  2. 2.

    Bogren, L. K. et al. The effects of hibernation and forced disuse (neurectomy) on bone properties in arctic ground squirrels. Physiol. Rep. 4, e12771 (2016).

    PubMed  PubMed Central  Google Scholar 

  3. 3.

    Barnes, B. M. Freeze avoidance in a mammal: body temperatures below 0 degree C in an Arctic hibernator. Science 244, 1593–1595 (1989).

    CAS  PubMed  Google Scholar 

  4. 4.

    Fedorov, V. B. et al. Comparative functional genomics of adaptation to muscular disuse in hibernating mammals. Mol. Ecol. 23, 5524–5537 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. 5.

    Harlow, H. J., Lohuis, T., Beck, T. D. & Iaizzo, P. A. Muscle strength in overwintering bears. Nature 409, 997 (2001).

    CAS  PubMed  Google Scholar 

  6. 6.

    Barboza, P. S., Farley, S. D. & Robbins, C. T. Whole-body urea cycling and protein turnover during hyperphagia and dormancy in growing bears (Ursus americanus and U. arctos). Can. J. Zool. 75, 2129–2136 (1997).

    Google Scholar 

  7. 7.

    Lee, T. N., Buck, C. L., Barnes, B. M. & O’Brien, D. M. A test of alternative models for increased tissue nitrogen isotope ratios during fasting in hibernating arctic ground squirrels. J. Exp. Biol. 215, 3354–3361 (2012).

    CAS  PubMed  Google Scholar 

  8. 8.

    Steffen, J. M., Rigler, G. L., Moore, A. K. & Riedesel, M. L. Urea recycling in active golden-mantled ground squirrels (Spermophilus lateralis). Am. J. Physiol. 239, R168–R173 (1980).

    CAS  PubMed  Google Scholar 

  9. 9.

    Buck, C. L. & Barnes, B. M. Effects of ambient temperature on metabolic rate, respiratory quotient and torpor in an arctic hibernator. Am. J. Physiol. Regul. Integr. Comp. Physiol. 279, R255–R262 (2000).

    CAS  PubMed  Google Scholar 

  10. 10.

    Karpovich, S. A., Toien, O., Buck, C. L. & Barnes, B. M. Energetics of arousal episodes in hibernating arctic ground squirrels. J. Comp. Physiol. B 179, 691–700 (2009).

    PubMed  Google Scholar 

  11. 11.

    Serkova, N. J., Rose, J. C., Epperson, L. E., Carey, H. V. & Martin, S. L. Quantitative analysis of liver metabolites in three stages of the circannual hibernation cycle in 13-lined ground squirrels by NMR. Physiol. Genomics 31, 15–24 (2007).

    CAS  PubMed  Google Scholar 

  12. 12.

    Emirbekov, E. Z. & Mukailov, M. I. Ammonia, glutamine and urea concentration in the brain tissue of susliks during winter hibernation. Biull. Eksp. Biol. Med. 69, 64–66 (1970).

    CAS  PubMed  Google Scholar 

  13. 13.

    Cooper, A. J. & Freed, B. R. Metabolism of [13N]ammonia in rat lung. Neurochem. Int. 47, 103–118 (2005).

    CAS  PubMed  Google Scholar 

  14. 14.

    Brusilow, S. W. & Cooper, A. J. Encephalopathy in acute liver failure resulting from acetaminophen intoxication: new observations with potential therapy. Crit. Care Med. 39, 2550–2553 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. 15.

    Lockwood, A. H. et al. The dynamics of ammonia metabolism in man. Effects of liver disease and hyperammonemia. J. Clin. Invest. 63, 449–460 (1979).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. 16.

    Wolfe, R. R., Nelson, R. A., Wolfe, M. H. & Rogers, L. Nitrogen cycling in hibernating bears. Proc. Amer. Soc. Mass Spectrometry 30, 426 (1982).

  17. 17.

    Galster, W. & Morrison, P. R. Gluconeogenesis in arctic ground squirrels between periods of hibernation. Am. J. Physiol. 228, 325–330 (1975).

    CAS  PubMed  Google Scholar 

  18. 18.

    Cotton, C. J. & Harlow, H. J. Avoidance of skeletal muscle atrophy in spontaneous and facultative hibernators. Physiol. Biochem. Zool. 83, 551–560 (2010).

    PubMed  Google Scholar 

  19. 19.

    Deutz, N. E. P., Thaden, J. J., ten Have, G. A. M., Walker, D. K. & Engelen, M. Metabolic phenotyping using kinetic measurements in young and older healthy adults. Metabolism 78, 167–178 (2018).

    CAS  PubMed  Google Scholar 

  20. 20.

    Vesali, R. F., Klaude, M., Thunblad, L., Rooyackers, O. E. & Wernerman, J. Contractile protein breakdown in human leg skeletal muscle as estimated by [2H3]-3-methylhistidine: a new method. Metabolism 53, 1076–1080 (2004).

    CAS  PubMed  Google Scholar 

  21. 21.

    Laurent, G. J. Dynamic state of collagen: pathways of collagen degradation in vivo and their possible role in regulation of collagen mass. Am. J. Physiol. 252, C1–C9 (1987).

    CAS  PubMed  Google Scholar 

  22. 22.

    Rennie, M. J. & Millward, D. J. 3-methylhistidine excretion and the urinary 3-methylhistidine/creatinine ratio are poor indicators of skeletal muscle protein breakdown. Clin. Sci. 65, 217–225 (1983).

    CAS  Google Scholar 

  23. 23.

    Epperson, L. E., Karimpour-Fard, A., Hunter, L. E. & Martin, S. L. Metabolic cycles in a circannual hibernator. Physiol. Genomics 43, 799–807 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. 24.

    Gehrke, S. et al. Red blood cell metabolic responses to torpor and arousal in the hibernator arctic ground squirrel. J. Proteome Res. 18, 1827–1841 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. 25.

    D’Alessandro, A., Nemkov, T., Bogren, L. K., Martin, S. L. & Hansen, K. C. Comfortably numb and back: plasma metabolomics reveals biochemical adaptations in the hibernating 13-lined ground squirrel. J. Proteome Res. 16, 958–969 (2017).

    PubMed  Google Scholar 

  26. 26.

    Moy, R. M. Renal function in the hibernating ground squirrel Spermophilus columbianus. Am. J. Physiol. 220, 747–753 (1971).

    CAS  PubMed  Google Scholar 

  27. 27.

    Cynober, L. A. Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition 2nd edn (CRC Press, 2003).

  28. 28.

    Deutz, N. E. The 2007 ESPEN Sir David Cuthbertson Lecture: amino acids between and within organs. The glutamate-glutamine-citrulline-arginine pathway. Clin. Nutr. 27, 321–327 (2008).

    CAS  PubMed  Google Scholar 

  29. 29.

    Lesser, R. W., Moy, R., Passmore, J. C. & Pfeiffer, E. W. Renal regulation of urea excretion in arousing and homeothermic ground squirrels (Citellus columbianus). Comp. Biochem Physiol. 36, 291–296 (1970).

    CAS  PubMed  Google Scholar 

  30. 30.

    Bell, R. A. & Storey, K. B. Regulation of liver glutamate dehydrogenase by reversible phosphorylation in a hibernating mammal. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 157, 310–316 (2010).

    PubMed  Google Scholar 

  31. 31.

    Thatcher, B. J. & Storey, K. B. Glutamate dehydrogenase from liver of euthermic and hibernating Richardson’s ground squirrels: evidence for two distinct enzyme forms. Biochem. Cell Biol. 79, 11–19 (2001).

    CAS  PubMed  Google Scholar 

  32. 32.

    Spanaki, C. & Plaitakis, A. The role of glutamate dehydrogenase in mammalian ammonia metabolism. Neurotox. Res 21, 117–127 (2012).

    CAS  PubMed  Google Scholar 

  33. 33.

    Wu, G. et al. Arginine metabolism and nutrition in growth, health and disease. Amino Acids 37, 153–168 (2009).

    CAS  PubMed  Google Scholar 

  34. 34.

    Mugahid, D. A. et al. Proteomic and transcriptomic changes in hibernating grizzly bears reveal metabolic and signaling pathways that protect against muscle atrophy. Sci. Rep. 9, 19976 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. 35.

    Lohuis, T. D., Beck, T. D. & Harlow, H. J. Hibernating black bears have blood chemistry and plasma amino acid profiles that are indicative of long-term adaptive fasting. Can. J. Zool. 9, 1257–1263 (2005).

    Google Scholar 

  36. 36.

    Hissa, R., Puukka, M., Hohtola, E., Sassi, M. & Risteli, J. Seasonal changes in plasma nitrogenous compounds of the european brown bear (Ursus arctos arctos). Ann. Zool. Fennici 35, 205–213 (1998).

    Google Scholar 

  37. 37.

    Harper, A. E., Miller, R. H. & Block, K. P. Branched-chain amino acid metabolism. Annu Rev. Nutr. 4, 409–454 (1984).

    CAS  PubMed  Google Scholar 

  38. 38.

    Abumrad, N. N., Wise, K. L., Williams, P. E., Abumrad, N. A. & Lacy, W. W. Disposal of alpha-ketoisocaproate: roles of liver, gut and kidneys. Am. J. Physiol. 243, E123–E131 (1982).

    CAS  PubMed  Google Scholar 

  39. 39.

    Sandovici, M. et al. Differential regulation of glomerular and interstitial endothelial nitric oxide synthase expression in the kidney of hibernating ground squirrel. Nitric Oxide 11, 194–200 (2004).

    CAS  PubMed  Google Scholar 

  40. 40.

    Jani, A. et al. Kidney proteome changes provide evidence for a dynamic metabolism and regional redistribution of plasma proteins during torpor-arousal cycles of hibernation. Physiol. Genomics 44, 717–727 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. 41.

    Nelson, R. A. & Jones, J. D. Leucine metabolism in the black bear. In International Conference on Bear Research and Management 7, 329–331 (1986).

  42. 42.

    Metges, C. C. et al. Incorporation of urea and ammonia nitrogen into ileal and fecal microbial proteins and plasma free amino acids in normal men and ileostomates. Am. J. Clin. Nutr. 70, 1046–1058 (1999).

    CAS  PubMed  Google Scholar 

  43. 43.

    Nelson, D. L. & Cox, M. M. Lehninger Principles of Biochemistry 5th edn (W.H. Freeman, 2008).

  44. 44.

    Millward, D. J. et al. The transfer of 15N from urea to lysine in the human infant. Br. J. Nutr. 83, 505–512 (2000).

    CAS  PubMed  Google Scholar 

  45. 45.

    Drew, K. L. et al. Ascorbate and glutathione regulation in hibernating ground squirrels. Brain Res. 851, 1–8 (1999).

    CAS  PubMed  Google Scholar 

  46. 46.

    Fenves, A. Z., Kirkpatrick, H. M. 3rd, Patel, V. V., Sweetman, L. & Emmett, M. Increased anion gap metabolic acidosis as a result of 5-oxoproline (pyroglutamic acid): a role for acetaminophen. Clin. J. Am. Soc. Nephrol. 1, 441–447 (2006).

    CAS  PubMed  Google Scholar 

  47. 47.

    Yu, Y. M. et al. Plasma l-5-oxoproline kinetics and whole-blood glutathione synthesis rates in severely burned adult humans. Am. J. Physiol. Endocrinol. Metab. 282, E247–E258 (2002).

    CAS  PubMed  Google Scholar 

  48. 48.

    D’Alessandro, A. et al. Trauma/hemorrhagic shock instigates aberrant metabolic flux through glycolytic pathways, as revealed by preliminary [13C]glucose labeling metabolomics. J. Transl. Med 13, 253 (2015).

    PubMed  PubMed Central  Google Scholar 

  49. 49.

    Koga, M. et al. Glutathione is a physiologic reservoir of neuronal glutamate. Biochem. Biophys. Res. Commun. 409, 596–602 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. 50.

    Kumar, A. & Bachhawat, A. K. Pyroglutamic acid: throwing light on a lightly studied metabolite. Curr. Sci. 102, 288–297 (2012).

    CAS  Google Scholar 

  51. 51.

    Griffith, O. W., Bridges, R. J. & Meister, A. Transport of gamma-glutamyl amino acids: role of glutathione and gamma-glutamyl transpeptidase. Proc. Natl Acad. Sci. USA 76, 6319–6322 (1979).

    CAS  PubMed  Google Scholar 

  52. 52.

    Stevenson, T. J., Duddleston, K. N. & Buck, C. L. Effects of season and host physiological state on the diversity, density, and activity of the arctic ground squirrel cecal microbiota. Appl. Environ. Microbiol. 80, 5611–5622 (2014).

    PubMed  PubMed Central  Google Scholar 

  53. 53.

    Kondo, H., Miura, M. & Itokawa, Y. Antioxidant enzyme systems in skeletal muscle atrophied by immobilization. Pflugers Arch. 422, 404–406 (1993).

    CAS  PubMed  Google Scholar 

  54. 54.

    Wickler, S. J., Hoyt, D. F. & van Breukelen, F. Disuse atrophy in the hibernating golden-mantled ground squirrel, Spermophilus lateralis. Am. J. Physiol. 261, R1214–R1217 (1991).

    CAS  PubMed  Google Scholar 

  55. 55.

    Bodine, S. C. Hibernation: the search for treatments to prevent disuse-induced skeletal muscle atrophy. Exp. Neurol. 248, 129–135 (2013).

    PubMed  Google Scholar 

  56. 56.

    James, R. S., Staples, J. F., Brown, J. C., Tessier, S. N. & Storey, K. B. The effects of hibernation on the contractile and biochemical properties of skeletal muscles in the thirteen-lined ground squirrel, Ictidomys tridecemlineatus. J. Exp. Biol. 216, 2587–2594 (2013).

    PubMed  Google Scholar 

  57. 57.

    Lee, K. et al. Overcoming muscle atrophy in a hibernating mammal despite prolonged disuse in dormancy: proteomic and molecular assessment. J. Cell. Biochem. 104, 642–656 (2008).

    CAS  PubMed  Google Scholar 

  58. 58.

    Lohuis, T. D., Harlow, H. J. & Beck, T. D. Hibernating black bears (Ursus americanus) experience skeletal muscle protein balance during winter anorexia. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 147, 20–28 (2007).

    CAS  PubMed  Google Scholar 

  59. 59.

    Goropashnaya, A. V., Barnes, B. M. & Fedorov, V. B. Transcriptional changes in muscle of hibernating arctic ground squirrels (Urocitellus parryii): implications for attenuation of disuse muscle atrophy. Sci. Rep. 10, 9010 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  60. 60.

    Hindle, A. G. et al. Prioritization of skeletal muscle growth for emergence from hibernation. J. Exp. Biol. 218, 276–284 (2015).

    PubMed  PubMed Central  Google Scholar 

  61. 61.

    Regan, M. D. et al. Shifts in metabolic fuel use coincide with maximal rates of ventilation and body surface rewarming in an arousing hibernator. Am. J. Physiol. Regul. Integr. Comp. Physiol. 316, R764–R775 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  62. 62.

    Perez-Pinzon, M. A., Gidday, J. M. & Zhang, J. H. Innate Tolerance in the CNS: Translational Neuroprotection by Pre- and Post-Conditioning. (Springer Science + Business Media, New York, 2013).

    Google Scholar 

  63. 63.

    van Breukelen, F. & Martin, S. L. Translational initiation is uncoupled from elongation at 18 degrees C during mammalian hibernation. Am. J. Physiol. Regul. Integr. Comp. Physiol. 281, R1374–R1379 (2001).

    PubMed  Google Scholar 

  64. 64.

    Laurent, M. R. et al. Muscle–bone interactions: from experimental models to the clinic? A critical update. Mol. Cell. Endocrinol. 432, 14–36 (2016).

    CAS  PubMed  Google Scholar 

  65. 65.

    Kornfeld, S. F., Biggar, K. K. & Storey, K. B. Differential expression of mature microRNAs involved in muscle maintenance of hibernating little brown bats, Myotis lucifugus: a model of muscle atrophy resistance. Genomics Proteomics Bioinformatics 10, 295–301 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  66. 66.

    Secombe, P., Harley, S., Chapman, M. & Aromataris, E. Feeding the critically ill obese patient: a systematic review protocol. JBI Database System Rev. Implement Rep. 13, 95–109 (2015).

    PubMed  Google Scholar 

  67. 67.

    Wolfe, R. R. & Chinkes, D. L. Isotope Tracers in Metabolic Research: Principles and Practice of Kinetic Analysis 2nd edn (Wiley, 2005).

  68. 68.

    Mason, A., Engelen, M., Ivanov, I., Toffolo, G. M. & Deutz, N. E. P. A four-compartment compartmental model to assess net whole-body protein breakdown using a pulse of phenylalanine and tyrosine stable isotopes in humans. Am. J. Physiol. Endocrinol. Metab. 313, E63–E74 (2017).

    PubMed  PubMed Central  Google Scholar 

  69. 69.

    Moore, H. B. et al. The metabolic time line of pancreatic cancer: opportunities to improve early detection of adenocarcinoma. Am. J. Surg. 218, 1206–1212 (2019).

    PubMed  Google Scholar 

  70. 70.

    Nemkov, T., Reisz, J. A., Gehrke, S., Hansen, K. C. & D’Alessandro, A. High-throughput metabolomics: isocratic and gradient mass spectrometry-based methods. Methods Mol. Biol. 1978, 13–26 (2019).

    CAS  PubMed  Google Scholar 

  71. 71.

    Kanehisa, M. The KEGG database. Novartis Found. Symp. 247, 91–101 (2002).

    CAS  PubMed  Google Scholar 

  72. 72.

    Kanehisa, M. & Goto, S. KEGG: kyoto encyclopedia of genes and genomes. Nucleic Acids Res. 28, 27–30 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  73. 73.

    Nemkov, T., Hansen, K. C. & D’Alessandro, A. A three-minute method for high-throughput quantitative metabolomics and quantitative tracing experiments of central carbon and nitrogen pathways. Rapid Commun. Mass Spectrom. 31, 663–673 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  74. 74.

    Williams, C. T. et al. Hibernating above the permafrost: effects of ambient temperature and season on expression of metabolic genes in liver and brown adipose tissue of arctic ground squirrels. J. Exp. Biol. 214, 1300–1306 (2011).

    PubMed  Google Scholar 

Download references

Acknowledgements

We thank C. Terzi and C. Willetto for their veterinary assistance and H. Sugiura, M. Mikes and M. Roed for assistance with animal husbandry. We also thank J. Reakoff for generous use of his cabin while trapping and J. Moore for thoughtful and wise advice. Funding: research reported in this publication was supported by grants from the Division of Integrative Organismal Systems at the National Science Foundation (1258179) and the Institutional Development Award programme from the National Institute of General Medical Sciences of the National Institutes of Health (2P20GM103395 and P20GM130443). A.D. was supported by funding from the Boettcher Foundation Webb-Waring Early Career Award 2017, the National Institute of General Medical Sciences (RM1GM131968) and the National Heart, Lung and Blood Institute (R01HL146442 and R01HL148151). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Author information

Affiliations

Authors

Contributions

S.A.R., K.L.D., G.A.M.H., A.D., R.H.C. and N.E.P.D. conceived the project and designed the research. S.A.R. and C.F. performed the experiments. G.A.M.H., N.E.P.D., J.A.R., S.G., S.A.R., D.S. and Z.B. analysed the data and samples. S.A.R. wrote the manuscript with intellectual input from all authors.

Corresponding author

Correspondence to Kelly L. Drew.

Ethics declarations

Competing interests

S.A.R., A.D., J.A.R., S.G., D.S., C.F., G.A.M.H. and N.E.P.D. declare no competing interests. K.L.D. has a financial interest in Be Cool Pharmaceutics. R.H.C. has a financial interest in Essential Blends. Z.B. has a financial interest in Barati Medical.

Additional information

Peer review information Primary Handling Editors: Pooja Jha; George Caputa. Nature Metabolism thanks C. Loren Buck and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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

Extended data

Extended Data Fig. 1 Tracer to tracee ratio (TTR) decay curves show slow decay in torpor (red, n=9) compared to summer euthermic AGS (blue, n=5).

Tracer to tracee ratio (TTR) decay curves show slow decay in torpor (red, n=9) compared to summer euthermic AGS (blue, n=5). Decay of TTR amino acid isotopes were fitted to the equation y=a*exp(-k1*x)+b*exp(-k2*x), and area under the curve (AUC) was calculated from the integral of the two exponential curves. Rate of appearance (Ra) for each amino acid is calculated by: dose of metabolite in the pulse infusion/AUC. Whole body rate of appearance (Ra) of amino acids is a proxy for whole body production (WBP).

Extended Data Fig. 2 Linear regression analysis indicates 15N incorporation into leucine/isoleucine is correlated to core body temperature during arousal from torpor.

Linear regression analysis indicates 15N incorporation into leucine/isoleucine is correlated to core body temperature during arousal from torpor (72 mg/kg 15N ammonium acetate pulse infusion n=6, 360 mg/kg 15N ammonium acetate pulse infusion n=5). In tissues where nitrogen incorporation was not observed in more than one animal, regression analysis was not preformed.

Extended Data Fig. 3 Linear regression analysis indicates 15N incorporation glutamate in skeletal muscle is correlated to core body temperature during arousal from torpor.

Linear regression analysis indicates 15N incorporation glutamate in skeletal muscle is correlated to core body temperature during arousal from torpor (72 mg/kg 15N ammonium acetate pulse infusion n=6, 360 mg/kg 15N ammonium acetate pulse infusion n=5). In tissues where nitrogen incorporation was not observed in more than one animal, regression analysis was not preformed.

Extended Data Fig. 4 Linear regression analysis indicates 15N incorporation into glutamine is correlated to core body temperature during arousal from torpor in kidney, plasma and liver.

Linear regression analysis indicates 15N incorporation into glutamine is correlated to core body temperature during arousal from torpor in kidney, plasma and liver (72 mg/kg 15N ammonium acetate pulse infusion n=6, 360 mg/kg 15N ammonium acetate pulse infusion n=5). In tissues where nitrogen incorporation was not observed in more than one animal, regression analysis was not preformed.

Extended Data Fig. 5 Linear regression analysis indicates 15N incorporation into Glutamine M+2 is correlated to core body temperature in kidney and liver during arousal from torpor.

Linear regression analysis indicates 15N incorporation into Glutamine M+2 is correlated to core body temperature in kidney and liver during arousal from torpor (72 mg/kg 15N ammonium acetate pulse infusion n=6, 360 mg/kg 15N ammonium acetate pulse infusion n=5). In tissues where nitrogen incorporation was not observed in more than one animal, regression analysis was not preformed.

Extended Data Fig. 6 Linear regression in analysis indicates 15N incorporation into valine cannot be verified to rely on core body temperature during arousal from torpor.

Linear regression in analysis indicates 15N incorporation into valine cannot be verified to rely on core body temperature during arousal from torpor. In tissues where nitrogen incorporation was not observed in more than one animal, regression analysis was not preformed.

Extended Data Fig. 7 Free 15N ammonia is recycled into nonessential amino acids (blue), essential amino acids (red) and 5-oxoproline (turquoise) during arousal from torpor (360 mg/kg 15N ammonium acetate pulse infusion, n=5, mean ±SEM).

Free 15N ammonia is recycled into nonessential amino acids (blue), essential amino acids (red) and 5-oxoproline (turquoise) during arousal from torpor (360 mg/kg 15N ammonium acetate pulse infusion, n=5, mean ±SEM). Percent 15N incorporation calculated as: (15N metabolite peak area/(15N metabolite peak area + 14N metabolite peak area))*100 following natural abundance correction.

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Rice, S.A., Ten Have, G.A.M., Reisz, J.A. et al. Nitrogen recycling buffers against ammonia toxicity from skeletal muscle breakdown in hibernating arctic ground squirrels. Nat Metab 2, 1459–1471 (2020). https://doi.org/10.1038/s42255-020-00312-4

Download citation

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing