Figure 4

Argininosuccinate is produced by the reverse activity of the urea cycle enzyme ASL. (A) mRNA expression levels of ASL after acute infection of the indicated cell lines with shRNAs against human (shASL) or mouse (shAsl) ASL. Values are normalized to cells infected with the control plasmid. Argininosuccinate production is decreased in ASL-silenced (B) human or (C) mouse cells. Cells were incubated with U-¹³C-arginine for 24 hours and the isotopologue distribution of argininosuccinate was analyzed by LC-MS. (D) Schematic representation of the fate of arginine in FH-deficient cells. Orange arrows indicate the major metabolic flux from arginine; light yellow arrow indicates low metabolic flux, gray arrows indicate undetectable metabolic flux; and dark gray arrow indicates the crosstalk between the TCA cycle and the urea cycle. All the results were obtained from three independent cultures and expressed as average ± SEM. ASL, argininosuccinate lyase; FH, fumarate hydratase; LC-MS, liquid chromatography-mass spectrometry; NTC, non-targeting control; SEM, standard error of the mean; shRNA, short hairpin RNA; TCA, tricarboxylic acid.