Fig. 5. ASNS and GABA shunt are alternative pathways to metabolize glutamine.

A Heatmap representation of metabolite levels, as determined by LC–MS analysis, in amino acid-starved U2OS cells incubated with glutamine and/or leucine. The heatmap was created with MetaboAnalyst3.0 with the total pools of the detected metabolites. B ¹³C-labeled metabolite levels, as determined by LC–MS analysis, in U2OS cells incubated with or without all amino acids or leucine alone as indicated, in the presence of (U)-¹³C-glutamine during 72 h. Total ion counts of glutamate m + 5, asparagine m + 4, aspartate m + 4, GABA m + 4, citrate m + 4, and malate m + 4 are graphed. The percentage of labeling with respect to total metabolite levels is shown in parenthesis for each metabolite. C ¹⁵N-labeled metabolite levels, as determined by LC–MS analysis, in U2OS cells incubated with or without all amino acids or leucine alone as indicated, in the presence of ¹⁵N2-glutamine during 72 h. Ion counts of ¹⁵N -glutamate, ¹⁵N -asparagine, ¹⁵N aspartate, and ¹⁵N2-asparagine are plotted. The percentage of labeling relative to the total metabolite is shown in parenthesis for each metabolite. D ASNS expression was knocked down using small interfering RNA (siRNA) in U2OS cells during 48 h. Cells were then treated with glutamine and BPTES as indicated for 72 h and the ATP/ADP ratio was measured. Scramble non-targeting siRNA was used as a control. Immunoblot of ASNS levels is presented as a control of the knockdown. E Immunoblot analysis of AMPK phosphorylation in U2OS cells treated as in (D). F Relative mRNA expression levels of ASNS as determined by qPCR in amino acid-starved U2OS cells incubated as indicated during 72 h. G ATP/ADP ratio of amino acid-starved U2OS cells incubated with glutamine and/or DMKG during 72 h. H Immunoblot analysis of mTORC1 downstream target (S6K, S6, and 4EBP1 phosphorylation) and AMPK phosphorylation in U2OS cells incubated during 24 h in the presence of all amino acids with dual inhibition of GLS and/or ASNS. ASNS expression was knocked down using small interfering RNA (siRNA) in U2OS cells during 48 h. Scramble non-targeting siRNA was used as a control. Cells were then incubated in the presence of absence of all amino acids and/or BPTES as indicated during 24 h. I Schematic representation of the two branches model connecting glutamine metabolism and mTORC1 signaling. Graphs show mean values ± SEM (n = 3 biologically independent experiments). *p < 0.05 (ANOVA analysis followed by a post hoc Bonferroni test). Source data are provided as a Source Data file.