Novel Stable Isotope Method to Determine Liver Metabolic Fluxes In Vivo

Mouse models designed to examine hepatic metabolism are critical to diabetes and obesity research. Thus, a microscale method to quantitatively assess hepatic glucose and intermediary metabolism in conscious, unrestrained mice was developed. [¹³C₃]propionate, [²H₂]water, and [6,6-²H₂]glucose isotopes were delivered intravenously in short- (9 h) and long-term-fasted (19 h) C57BL/6J mice. GC-MS and mass isotopomer distribution (MID) analysis were performed on three 40-microliter arterial plasma glucose samples obtained during the euglycemic isotopic steady state. Model-based regression of hepatic glucose and citric acid cycle (CAC)-related fluxes was performed using a comprehensive isotopomer model to track carbon and hydrogen atom transitions through the network and thereby simulate the MIDs of measured fragment ions. Glucose-6-phosphate production from glycogen diminished, and endogenous glucose production was exclusively gluconeogenic with prolonged fasting. Gluconeogenic flux from phosphoenolpyruvate (PEP) remained stable, whereas that from glycerol modestly increased from short- to long-term fasting. CAC flux was reduced with long-term fasting. Interestingly, anaplerosis and cataplerosis increased with fast duration. We demonstrate for the first time that the infusion of [¹³C₃]propionate does not significantly perturb gluconeogic fluxes. We also found evidence of significant ¹³CO₂ recycling in liver, which may bias flux calculations using prior methods that do not take ¹³CO₂ recycling into account. This new approach utilizes state-of-the-art in vivo methodology and comprehensive isotopomer modeling to quantify hepatic glucose and intermediary fluxes during physiological stress in mice. The small plasma requirements permit serial sampling without stress and the affirmation of steady-state glucose kinetics. Furthermore, the approach can accommodate a broad range of modeling assumptions, isotope tracers, and measurement inputs without the need to introduce ad hoc mathematical approximations. I will discuss the development and validation of this approach, as well as our initial forays into applying this approch to assess the liver response to genetic knockouts, exercise, and diet manipulation in mice.