Assessment and Expansion of Models for ¹³C-Metabolic Flux Analysis (MFA) In Vivo

Isotope-based MFA is a valuable tool to determine rates of biochemical reactions in vivo, which cannot be assessed by other approaches. Its application in biomedicine may help to uncover metabolic disease determinants in diabetes and obesity. However, various simplifying assumptions have been applied to constrain models used for in vivo MFA studies. These constraints are approximate, and their validity may change with varying experimental conditions. Here we used our flexible flux modeling platform (INCA) to test and eliminate several assumptions imposed on prior estimates of liver gluconeogenesis, anaplerosis, and citric acid cycle (CAC) fluxes in overnight fasted, conscious mice. Male C57Bl/6J mice were given a primed, continuous infusion (160µmol/kg and 40µmol/kg/minutes) of [¹³C₃]lactate intravenously for ∼2 hour. ¹³C-enrichment of plasma and liver metabolites obtained at the end of the study were measured by GC-MS and used to regress liver metabolic fluxes using several different models of in vivo metabolism. In particular, we developed models to assess (1) the extent of reversibility of 4C reactions of the CAC, (2) the impact of pyruvate-decarboxylation, and (3) the influence of secondary isotope recycling on liver flux estimates. Confidence intervals were computed a posteriori to assess uncertainties of individual flux estimates. Glucose production originated largely from PEP. Cataplerosis (PEPCK flux) was ∼3.9-4.5 fold higher than citrate synthase, with ∼56-67% recycling back to the CAC through pyruvate. The modeling of pyruvate decarboxylation increased the relative ratio of gluconeogenesis to citrate synthase flux by ∼18-32% but also reduced the precision of flux estimates. Interestingly, Cori cycling reduced pyruvate kinase flux ∼16% relative to cataplerosis. Thus, interorgan crosstalk with the liver can be estimated with fewer assumptions in vivo, providing a potential path for understanding metabolic disease with greater consideration of whole-body physiology.