In Vivo Estimates of Liver Metabolic Flux Assessed by C-Propionate and C-Lactate Are Impacted by Tracer Recycling and Equilibrium Assumptions

Isotope-based assessment of metabolic flux is achieved through a judicious balance of measurements and assumptions. Recent publications debate the validity of key assumptions used to model stable isotope labeling of liver metabolism in vivo. Here, we examine the controversy surrounding estimates of liver citric acid cycle and gluconeogenesis fluxes using a flexible modeling platform that enables rigorous testing of standard assumptions. Fasted C57BL/6J mice are infused with [C]lactate or [C]propionate isotopes, and hepatic fluxes are regressed using models with gradually increasing complexity and relaxed assumptions. We confirm that liver pyruvate cycling fluxes are incongruent between different C tracers in models with conventional assumptions. When models are expanded to include more labeling measurements and fewer constraining assumptions, however, liver pyruvate cycling is significant, and inconsistencies in hepatic flux estimates using [C]lactate and [C]propionate isotopes emanate, in part, from peripheral tracer recycling and incomplete isotope equilibration within the citric acid cycle.