A Single [U-13C6]Glucose Tracer Enables Simultaneous Quantification of Integrated Multi-Tissue Metabolic Fluxes In Vivo

Abstract Precise simultaneous quantification of carbon fluxes in liver and skeletal muscle remains challenging due to overlapping metabolic pathways and incomplete correction of isotopic dilution in conventional stable-isotope tracing. Here we establish an integrated in vivo [13C6]-glucose tracing framework to absolutely quantify core glycolytic, gluconeogenic, lipogenic, TCA cycle and anaplerotic fluxes in mouse liver and skeletal muscle under steady state. Low-dose tracer infusion avoids disrupting endogenous metabolism, and our refined network resolves competing carbon routes with rigorous isotopic dilution correction. Orthogonal 2-deoxyglucose tracing is incorporated to specifically enable accurate quantification of skeletal muscle glucose uptake and support the calculation of carbon metabolic fluxes within skeletal muscle. We applied this platform to characterize tissue-specific metabolic remodeling triggered by fasting, high-fat diet, metformin and Aldometanib. This method detects subtle flux changes undetectable by standard isotopologue analysis. Our unified multi-tissue flux toolkit provides a robust resource to dissect interorgan carbon metabolism and evaluate metabolism-targeting therapeutics.