Mapping metabolic dependences and capacities using ATP as a biomarker

Metabolic dependences and capacities highlight a cell's reliance on specific pathways to meet its bioenergetic needs, with these pathways being interrogated using chemical inhibitors to assess their significance. While surrogate markers of bioenergetics (e.g., oxygen consumption, protein translation inhibition) have yielded important insights, we asked whether ATP could be used as a biomarker. To address this gap, we developed Mitochondrial/Energy Flow Cytometry (MitE-Flo), a method that evaluates the contributions of glycolysis, fatty acid/amino acid oxidation (FAO/AAO), and oxidative phosphorylation (OXPHOS) to cellular ATP content. In models of mitochondrial disease due to complex I or complex IV deficiency, we identified impaired OXPHOS with a compensatory shift to glycolysis. To define the utility of ATP monitoring in immune niches, we analyzed lungs from mice infected with influenza. CD4⁺ and CD8⁺ T cells, as well as macrophages, demonstrated a shift from OXPHOS dependence to glycolysis dependence during infection, consistent with activation. Using ATP as a biomarker to define metabolic dependences and capacities provides valuable insights into disease states and mixed cell populations, thereby enhancing the metabolism research toolkit.