Glycerol-3-phosphate biosynthesis regenerates cytosolic NAD+ to alleviate mitochondrial disease

Electron transport chain (ETC) dysfunction or hypoxia causes toxic NADH accumulation. How cells regenerate NAD+ under such conditions remains elusive. Here, integrating bioinformatic analysis and experimental validation, we identify glycerol-3-phosphate (Gro3P) biosynthesis as an endogenous NAD+-regeneration pathway. Under genetic or pharmacological ETC inhibition, disrupting Gro3P synthesis inhibits yeast proliferation, shortens lifespan of C. elegans, impairs growth of cancer cells in culture and in xenografts, and causes metabolic derangements in mouse liver. Moreover, the Gro3P shuttle selectively regenerates cytosolic NAD+ under mitochondrial complex I inhibition; enhancing Gro3P synthesis promotes shuttle activity to restore proliferation of complex I-impaired cells. Mouse brain has much lower levels of Gro3P synthesis enzymes as compared with other organs. Strikingly, enhancing Gro3P synthesis suppresses neuroinflammation and extends lifespan in the Ndufs4−/− mice. Collectively, our results reveal Gro3P biosynthesis as an evolutionarily conserved coordinator of NADH/NAD+ redox homeostasis and present a therapeutic target for mitochondrial complex I diseases. [Display omitted] •Gro3P biosynthesis regenerates cytosolic NAD+ under ETC dysfunction and hypoxia•Inhibiting Gro3P synthesis is toxic in ETC-defective cells, organs, and organisms•Enhancing Gro3P synthesis rescues complex I deficiency through the Gro3P shuttle•Enhancing Gro3P synthesis extends lifespan of the Ndufs4−/− mice Liu et al. demonstrate that mitochondrial respiratory failure promotes Gro3P biosynthesis to regenerate NAD+ and maintain metabolism. While lower organisms and tumor cells exploit this pathway to antagonize mitochondrial dysfunction, mammalian brain lacks this pathway. Enhancing Gro3P synthesis in the brain rescues a mouse model of mitochondrial complex I disease.