Spatial-Fluxomics Provides a Subcellular-Compartmentalized View of Reductive Glutamine Metabolism in Cancer Cells

The inability to directly inspect metabolic activities within distinct subcellular compartments has been a major barrier to our understanding of eukaryotic cell metabolism. Here, we describe a spatial-fluxomics approach for inferring metabolic fluxes in mitochondria and cytosol under physiological conditions, combining isotope tracing in intact cells followed by a rapid subcellular fractionation, LC-MS based metabolomics, computational deconvolution, and metabolic network modelling. We apply this approach to study reductive glutamine metabolism in mitochondria and cytosol, shown to mediate fatty acid biosynthesis under hypoxia and defective mitochondria. We find a previously unappreciated role of reductive isocitrate dehydrogenase (IDH1) as the sole net contributor of carbons to fatty acid biosynthesis under standard normoxic conditions in HeLa cells. Surprisingly, while the relative contribution of reductive glutamine metabolism to fatty acid biosynthesis (versus that of glucose oxidation) increases in hypoxia, the total reductive flux drops due to a decrease in mitochondrial IDH2 flux. In cells with defective mitochondrial succinate dehydrogenase, we find that reductive biosynthesis of citrate in mitochondria is followed by a reversed citrate synthase activity, suggesting a new route for supporting pyrimidine biosynthesis. We expect this spatial-fluxomics approach to be a highly useful tool for elucidating the role of metabolic dysfunction in human disease.