Spatial-fluxomics provides a subcellular-compartmentalized view of reductive glutamine metabolism in cancer cells

Abstract

The inability to inspect metabolic activities within 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, rapid subcellular fractionation, LC-MS-based metabolomics, computational deconvolution, and metabolic network modeling. Applied to study reductive glutamine metabolism in cancer cells, shown to mediate fatty acid biosynthesis under hypoxia and defective mitochondria, we find a previously unappreciated role of reductive IDH1 as the sole net contributor of carbons to fatty acid biosynthesis under standard normoxic conditions in HeLa cells. In murine cells with defective SDH, we find that reductive biosynthesis of citrate in mitochondria is followed by a reversed CS 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.

Metabolic and OXPHOS Activities Quantified by Temporal ex vivo Analysis Display Patient-Specific Metabolic Vulnerabilities in Human Breast Cancers

“…For example ( 40 ) used selective permeabilization and isotope tracing on cell monolayer, which, unfortunately, is not applicable on solid tumors. Rapid fractionation combined with isotope tracing, computational deconvolution and network modeling have been applied, but its again only suitable on cell lines ( 14 ). Additionally, respiration has been studied using homogenization of muscle tissue ( 17 ), but this destroys mitochondrial structure, removes regulatory effect of the cytoskeleton ( 22 ) and does not allow measurement of flux across mitochondrial membrane.…”

“…Despite the rapid nature of the metabolic experiments on human cancers, solute transporters and enzymes can have residual activity even at very low temperatures and can severely affect the results ( 13 ). Shortcomings in metabolomic studies have been noted by numerous groups and many of them have developed new methods to differentiate mitochondrial and cytosolic metabolite pools or metabolite fluxes within cells to answer critical questions in understanding cancer metabolism ( 14 – 17 ). Reliable, efficient and widely accepted methods for studying metabolism in solid tumors, however, are still missing.…”

Stable Isotopes for Tracing Mammalian-Cell Metabolism In Vivo

“…Resolving compartmentalized metabolism in vivo thus seems to call for technical approaches allowing physical fractionation of cells into their different organelles. Several methods for rapid isolation of mitochondrial [13,61] and lysosomal [62] fractions from whole cells have been implemented in vitro to probe the metabolism of these organelles. Interestingly, one of these approaches, based on epitope-tagging of mitochondria for further separation via magnetic immunoprecipitation [13], was recently extended to enable its application for specific cell types in vivo [63].…”

Current Status and Future Prospects of Genome-Scale Metabolic Modeling to Optimize the Use of Mesenchymal Stem Cells in Regenerative Medicine

“…Further information may be obtained if the cells are fractionated into different organelles before analysis (Ahn et al, 2017). For example, the subcellular localization of glutamine metabolism in cancer has been elucidated using this technique (Lee et al, 2019).…”