Figure 2: Loss of SDH-activity leads to increased pyruvate carboxylation.

(a–c) Both imCC and MAF cell models were incubated for 24 h with ¹³C-[1,2]-glucose, and cell extracts subjected to NMR spectroscopy analysis of resulting incorporation of ¹³C into (a) glutamate, (b) succinate and (c) aspartate (n=3, error bars are s.d.). Reduction in the relative abundance of the ¹³C-[4,5]-glutamate and increase in ¹³C-[2,3]-glutamate isotopomers suggest increase in PC activity and relative decrease in PDH activity in SDH-deficient cells. This is confirmed by change in isotopomer distribution in aspartate towards the ¹³C-[2,3] isotopomer. ¹³C atoms are shown as filled circles, whereas ¹²C atoms are empty circles. (d,e) Diagrams describing the incorporation of carbons from glucose through PDH (red) or PC (blue) activity into (d) wild-type and (e) Sdhb^−/− (KO) cells. Abbreviations in metabolic diagrams: αKG; alpha-ketoglutarate, Ac, acetate; AcCoA, acetyl coenzyme A; Ala, alanine; Asp, aspartate; Cit, citrate; Fum, fumarate; Glc, glucose; Glu, glutamate; Lac, lactate; Mal, malate; OAA, oxaloacetate; PC, pyruvate carboxylase; PDH, pyruvate dehydrogenase; Pyr, pyruvate; Suc, succinate. (f) PC protein expression is increased in Sdhb^−/− (KO) cells compared with wild-type (WT) controls. (g) SDH-mutated PCC also shows significantly upregulated PC expression compared with non-SDH-mutated PCC harbouring a RET mutation. Scale bar, 50 μm. (h) OAA concentrations are decreased in Sdhb^−/− (KO c6 and c8) cells as measured using a citrate synthase-based enzymatic assay (n=3 with 3 replicates/experiment. Error bars are s.e.m.). The glutaminase inhibitor, BPTES, reduces OAA concentrations only in wild-type cells, showing the loss of dependency on glutaminase for OAA synthesis in SDH-deficient cells. MID, mass isotopomer distribution KO, Sdhb knockout.