The malate-aspartate shuttle is important for de novo serine biosynthesis.

The malate-aspartate shuttle (MAS) is a redox shuttle that transports reducing equivalents across the inner mitochondrial membrane while recycling cytosolic NADH to NAD⁺. We genetically disrupted each MAS component to generate a panel of MAS-deficient HEK293 cell lines in which we performed [U-¹³C]-glucose tracing. MAS-deficient cells have reduced serine biosynthesis, which strongly correlates with the lactate M+3/pyruvate M+3 ratio (reflective of the cytosolic NAD⁺/NADH ratio), consistent with the NAD⁺ dependency of phosphoglycerate dehydrogenase in the serine synthesis pathway. Among the MAS-deficient cells, those lacking malate dehydrogenase 1 (MDH1) show the most severe metabolic disruptions, whereas oxoglutarate-malate carrier (OGC)- and MDH2-deficient cells are less affected. Increasing the NAD⁺-regenerating capacity using pyruvate supplementation resolves most of the metabolic disturbances. Overall, we show that the MAS is important for de novo serine biosynthesis, implying that serine supplementation could be used as a therapeutic strategy for MAS defects and possibly other redox disorders. [Display omitted] • Loss of MAS components reduces de novo serine biosynthesis • Loss of MAS components lowers the NAD⁺/NADH ratio and constrains glycolysis • Losses of MDH1, GOT1, AGC, and GOT2 show the most severe metabolic disruptions • Pyruvate restores NAD⁺/NADH, glycolysis, and serine biosynthesis in MDH1 KO cells Broeks et al. find that disruption of the malate-aspartate shuttle reduces de novo serine biosynthesis. Using ¹³C 6 -glucose tracer analysis in a comprehensive panel of knockout cell lines, they show that MAS-deficient cells have a low NAD⁺/NADH ratio, which affects metabolite fluxes of glycolysis and the TCA cycle. [ABSTRACT FROM AUTHOR]