Global 13C tracing and metabolic flux analysis of intact human liver tissue ex vivo

Liver metabolism is central to human physiology and influences the pathogenesis of common metabolic diseases. Yet, our understanding of human liver metabolism remains incomplete, with much of current knowledge based on animal or cell culture models that do not fully recapitulate human physiology. Here, we performed in-depth measurement of metabolism in intact human liver tissue ex vivo using global 13C tracing, non-targeted mass spectrometry and model-based metabolic flux analysis. Cultured liver tissue exhibited normal anatomical structure and retained canonical liver functions such as glucose production, albumin and VLDL synthesis at near-physiological rates. Isotope tracing with a highly 13C-labeled medium generated 13C enrichment in hundreds of compounds, allowing qualitative assessment of a wide range of metabolic pathways within a single experiment. This confirmed well-known features of liver metabolism, but also revealed unexpected metabolic activities such as de novo creatine synthesis and branched-chain amino acid transamination, where human liver appears to differ from rodent models. Metabolic flux analysis identified glycogenolysis as the main source of glucose production, which could be suppressed by pharmacological inhibition of glycogen phosphorylase. Glucose production ex vivo also correlated with donor plasma glucose, suggesting that cultured liver tissue retains individual metabolic phenotypes. Moreover, liver tissue responded to postprandial levels of nutrients and insulin by suppressing glucose production and increasing nutrient uptake. Isotope tracing ex vivo allows measuring human liver metabolism with great depth and resolution in an experimentally tractable system.