Isotopomer analysis of cellular metabolism in tissue culture: A comparative study between the pathway and network-based methods

Understanding the metabolism of a cell requires knowledge about the intracellular biochemical structure as well as cellular responses to extracellular nutrients. Towards this goal, genomics and proteomics seek a complete description of the cell's metabolic network, while the field of metabolomics aims to identify new metabolites and profile their distribution in such a network. Here we employed tracer-based metabolomics to characterize HepG2 metabolic responses to the nutritional environments of two DMEM media containing [1,2 ¹³C₂] glucose. A computational model describing 254 reactions of the HepG2 metabolic network was developed to systemically analyze the intracellular flux distribution based on tracer data. This is the largest and most comprehensive model used for isotopomer analysis to date. Estimated reaction fluxes from the model were benchmarked with those obtained from the traditional pathway-based method. Results from this study were as follows: (1) HepG2 cells grow equally well in two test media, including one where asparagine is substituted for the commonly used amino acid glutamine; (2) intracellular flux distributions, particularly in the TCA cycle, are markedly different between cells grown in the two cultures; and (3) compared to the pathway-based method, the network-based approach provides a more complete and detailed picture of substrate utilization as well as informs ways to improve the current media. In short, this network-based, systems biology-driven modeling approach to isotopomer analysis has proven to be a valuable tool for metabolic phenotyping and elucidating the nutrient-gene interactions.