Cytosolic and mitochondrial NADPH fluxes are independently regulated

Although nicotinamide adenine dinucleotide phosphate (NADPH) is produced and consumed in both the cytosol and mitochondria, the relationship between NADPH fluxes in each compartment has been difficult to assess due to technological limitations. Here we introduce an approach to resolve cytosolic and mitochondrial NADPH fluxes that relies on tracing deuterium from glucose to metabolites of proline biosynthesis localized to either the cytosol or mitochondria. We introduced NADPH challenges in either the cytosol or mitochondria of cells by using isocitrate dehydrogenase mutations, administering chemotherapeutics or with genetically encoded NADPH oxidase. We found that cytosolic challenges influenced NADPH fluxes in the cytosol but not NADPH fluxes in mitochondria, and vice versa. This work highlights the value of using proline labeling as a reporter system to study compartmentalized metabolism and reveals that NADPH homeostasis in the cytosolic and mitochondrial locations of a cell are independently regulated, with no evidence for NADPH shuttle activity.

Automated summary

In this study, an approach to resolve the fluxes of NADPH in the cytosol and mitochondria was introduced using deuterium tracing. It was found that challenges in the cytosol affected NADPH fluxes in the cytosol, while challenges in the mitochondria affected NADPH fluxes in the mitochondria. This work highlights the value of using proline labeling as a reporter system for studying compartmentalized metabolism and reveals independent regulation of NADPH homeostasis in the cytosol and mitochondria, without evidence for NADPH shuttle activity.