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

Nature Metabolism volume 2, pages 1459–1471 (2020)Cite this article

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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.

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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.

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Data availability

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

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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.

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Authors and Affiliations

  1. Department of Chemistry and Biochemistry, University of Alaska Fairbanks, Fairbanks, AK, USA

    Sarah A. Rice, Carla Frare & Kelly L. Drew

  2. Institute of Arctic Biology, Center for Transformative Research in Metabolism, University of Alaska Fairbanks, Fairbanks, AK, USA

    Sarah A. Rice, Carla Frare, Zeinab Barati, Robert H. Coker & Kelly L. Drew

  3. Center for Translational Research in Aging and Longevity, Department of Health and Kinesiology, Texas A&M University, College Station, TX, USA

    Gabriella A. M. Ten Have & Nicolaas E. P. Deutz

  4. Department of Biochemistry and Molecular Genetics, University of Colorado Anschutz Medical Campus, Aurora, CO, USA

    Julie A. Reisz, Sarah Gehrke, Davide Stefanoni & Angelo D’Alessandro

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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.

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Correspondence to Kelly L. Drew.

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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.

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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.

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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.

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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

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