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Abstract:
Use of stable isotope labeling in cell culture experiments is a widely applied and powerful technique to study metabolic pathways and fluxes. In particular, the incorporation of 13C-labels at specific carbon positions of a given metabolite can provide important information about cellular metabolism and may even reveal unknown metabolic phenomena. Depending on the cellular model and the
metabolic pathway of interest, one has to take into consideration which labeled substrate will give the most valuable information. Primary hepatocyte cultures are a widely used model for studying liver physiology and liver diseases. We have recently shown that labeled glucose (e.g. [U-13C]glucose) was not feasible for the investigation of cellular and energy metabolism in cultured primary
hepatocytes. But, we were able to demonstrate that [3-13C]pyruvate is readily metabolized by these cells to yield useful information about a number of different metabolic pathways in the liver. However, some difficulties remained in distinguishing the contributions of specific metabolic pathways due to overlap in the signals from different isotopomers after metabolism of [3-13C]pyruvate through pyruvate dehydrogenase (PDH) and pyruvate carboxylase (PC). To overcome these difficulties we applied one- and two-dimensional multinuclear NMR-spectroscopy to characterize the metabolism of labeled [2-13C]pyruvate and to identify its metabolic isotopomers derived through various intermediary pathways in cultured hepatocytes.
