The Malate Aspartate Shuttle is important for de novo serine biosynthesis. Broeks et al.

We hypothesized that all MAS defects lead to a secondary serine biosynthesis defect. To study whether MAS defects lead to diminished serine and glycine biosynthesis, we performed isotopic tracing experiments in 33 HEK293 cell lines with a genetic disruption of the MAS enzymes and transporters (GOT1, MDH1, SLC25A11, MDH2, GOT2, SLC25A12/13) and corresponding WT lines, including a double knockout of the AGC transporter. Cells were incubated with [U-13C]-glucose, as serine and glycine are synthesized de novo from the glycolytic intermediate 3-phosphoglycerate (3-PG). After 8 hours of incubation with [U-13C]-glucose, 13C3-serine and 13C2-glycine concentrations were most strongly decreased in MDH1 KO cells, followed by AGC, GOT1, GOT2, OGC and MDH2 KO cells when compared to control cells. The fraction of 13C3-serine and 13C2-glycine over time confirmed diminished de novo serine and glycine biosynthesis in all MAS KO cells, serine and glycine biosynthesis being only partially hampered in OGC and MDH2 KO cells. Overall, these findings demonstrate that all MAS defects lead to decreased de novo serine and glycine biosynthesis on a cellular level. [Figures 1 & S2-3]

To further investigate the underlying causes of diminished de novo serine biosynthesis in MAS KO cells, we analyzed the intracellular metabolomes of the cells incubated with [U-13C]-glucose for 8 hours using direct infusion high resolution mass spectrometry (DI-HRMS). Our data demonstrates that the abundant availability of intermediates in the first steps of glycolysis leads to an increased flux to glycerol biosynthesis in MAS KO cells, while flux from glucose to pyruvate was decreased in all KO cells. The lactate (m+3)/pyruvate (m+3) ratio was increased in all KO cells and strongly correlated with serine and glycerol 3-P synthesis from glucose. Analysis of isotope enrichment in the TCA cycle and MAS demonstrated impaired flux through TCA cycle in most MAS defects. In addition, our findings indicated that a defect in the MAS cycle from GOT2 (-AGC-GOT1-) to MDH1 results in diminished metabolite flux through the NAD+ regenerating enzyme MDH1. [Figure 2, 3, S4 & S5]