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<p>Amino acid and polyamine metabolism underpins many cellular processes, such as cell growth, stress adaptation, and signaling. However, the usage of specific metabolic pathways is highly context-dependent, and there are many compensatory mechanisms in place for the biosynthesis of amino acids. Here, we establish low-dose heavy water (D₂O) labeling as a tracer to monitor amino acid and polyamine metabolism in mammalian systems. Using targeted HPLC–MS of primary amines, we quantified deuterium incorporation in mouse embryonic fibroblasts, pancreatic β-cell–derived MIN6 cells, and mouse tissues, which we then benchmarked with orthogonal tracers (<sup>13</sup>C-glucose and <sup>15</sup>NH₄⁺). We demonstrated D₂O labels nonessential amino acids and polyamines. We validated specificity, as inhibition of key metabolic steps altered deuterium incorporation into Ala/Ser/Gly and polyamines and revealed differential engagement of branched-chain amino acid metabolism. We found that glutamine starvation induces integrated stress response-linked remodeling, increasing deuterium incorporation into Glu and glycolytic amino acids while identifying changes in amino acids efflux. Finally, in vivo short-term D₂O exposure distinguishes tissue-specific biosynthetic capacities. Collectively, these data challenge the assumption of uniform alanine labeling by D<sub>2</sub>O and demonstrate that D₂O provides a sensitive readout of metabolic flexibility, transport crosstalk, and pathway regulation across cell types and tissues.</p>

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Heavy water labeling reveals metabolic flexibility of amino acid and polyamine pathways in mammalian cells

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年5月29日

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  • Alan Gonzalez-Ibarra,
  • Missael Arroyo-Negrete,
  • Wioleta Banaszuk-Krupa,
  • Katarzyna Socała,
  • Nikola Gapińska,
  • Piotr Wlaź,
  • Julio Cesar Torres-Elguera,
  • Tomasz Sawoszczuk,
  • Marek Tchórzewski,
  • Maria Hatzoglou,
  • Dawid Krokowski

摘要

Amino acid and polyamine metabolism underpins many cellular processes, such as cell growth, stress adaptation, and signaling. However, the usage of specific metabolic pathways is highly context-dependent, and there are many compensatory mechanisms in place for the biosynthesis of amino acids. Here, we establish low-dose heavy water (D₂O) labeling as a tracer to monitor amino acid and polyamine metabolism in mammalian systems. Using targeted HPLC–MS of primary amines, we quantified deuterium incorporation in mouse embryonic fibroblasts, pancreatic β-cell–derived MIN6 cells, and mouse tissues, which we then benchmarked with orthogonal tracers (13C-glucose and 15NH₄⁺). We demonstrated D₂O labels nonessential amino acids and polyamines. We validated specificity, as inhibition of key metabolic steps altered deuterium incorporation into Ala/Ser/Gly and polyamines and revealed differential engagement of branched-chain amino acid metabolism. We found that glutamine starvation induces integrated stress response-linked remodeling, increasing deuterium incorporation into Glu and glycolytic amino acids while identifying changes in amino acids efflux. Finally, in vivo short-term D₂O exposure distinguishes tissue-specific biosynthetic capacities. Collectively, these data challenge the assumption of uniform alanine labeling by D2O and demonstrate that D₂O provides a sensitive readout of metabolic flexibility, transport crosstalk, and pathway regulation across cell types and tissues.