Tracing Compartmentalized NADPH Metabolism in the Cytosol and Mitochondria of Mammalian Cells

Abstract

Summary Eukaryotic cells compartmentalize biochemical processes in different organelles, often relying on metabolic cycles to shuttle reducing equivalents across intracellular membranes. NADPH serves as the electron carrier for the maintenance of redox homeostasis and reductive biosynthesis, with separate cytosolic and mitochondrial pools providing reducing power in each respective location. This cellular organization is critical for numerous functions but complicates analysis of metabolic pathways using available methods. Here we develop an approach to resolve NADP(H)-dependent pathways present within both the cytosol and the mitochondria. By tracing hydrogen in compartmentalized reactions that use NADPH as a cofactor, including the production of 2-hydroxyglutarate by mutant isocitrate dehydrogenase enzymes, we can observe metabolic pathway activity in these distinct cellular compartments. Using this system we determine the direction of serine/glycine interconversion within the mitochondria and cytosol, highlighting the ability of this approach to resolve compartmentalized reactions in intact cells.

Targeting MTHFD2 in acute myeloid leukemia

“…possibly due in part to the fact that the whole cell NAD + / NADH ratio is dominated by cofactor levels in the cytosol, and MTH FD2 knockdown is expected to affect the mitochondrial NAD + /NADH ratio. Tracing compartmentalized NAD PH metabolism (Lewis et al, 2014) was also not possible because MTH FD2 suppression resulted in a profound decrease in cellular α-ketoglutarate levels. In fact, levels of all TCA cycle metabolites were suppressed, potentially because loss of MTH FD2 alters mitochondrial redox metabolism.…”

Non-targeted Tracer Fate Detection

“…Genome-scale metabolic networks [40] are used for the stoichiometric modeling of metabolism to infer intracellular metabolic fluxes [41]. These metabolic networks are generated from transcriptomics or proteomics data.…”

“…Lewis et al [41] and Fan et al [43] used deuterated glucose tracers to selectively label the cellular NADH or NADPH pool. [3-2 H]glucose specifically labels NADPH that is generated in the pentose phosphate pathway.…”

“…These products can then be detected by NTFD in a nontargeted manner. Analogously, [4-2 H]glucose can be used to label the cellular NADH pool and to trace NADH-dependent reductions [41].…”

Coping with the metabolic stress of leaving home

“…Given the importance of these processes in cancer cells, there has been considerable interest in understanding how cancer cells generate NADPH. Studies addressing this question have identifi ed key roles for multiple metabolic pathways that contribute to cellular NADPH pools, including the pentose phosphate pathway (PPP) and the folate pathway [11,12]. Key to these studies has been the use of deuterium ( 2 H) tracers that allow quantitative analysis of NADPH biosynthesis.…”