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. Author manuscript; available in PMC: 2021 Mar 26.
Published in final edited form as: Nat Metab. 2020 Jul 27;2(9):893–901. doi: 10.1038/s42255-020-0250-5

Figure 3. GAPDH is downstream of the metabolite that signals glucose availability to mTORC1.

Figure 3.

a) Diagram of glycolysis with an emphasis on the role of GAPDH and its inhibitor Koningic Acid (KA). b) Inhibition of GAPDH by KA prevents the suppression of mTORC1 normally caused by glucose starvation, but only if KA is added at the beginning of the starvation period. AMPKα DKO HEK-293T cells were incubated with glucose (Unst) or starved of it for 3 hours. Koningic acid was added to cells either at the beginning of the 3-hour glucose starvation period or for 15 minutes after a 3-hour starvation. Cell lysates were analyzed by immunoblotting for the phosphorylation state or levels of the indicated proteins. c) GAPDH inhibition by 50 μM KA prevents depletion of metabolites upstream of GAPDH upon glucose starvation but only if added at the beginning of the starvation period. Cells were treated as in (B), metabolite extracts were analyzed by LC/MS. Data are shown as mean ± s.e.m. for n = 3 biologically independent replicates. P-values were determined for two-sided Student’s t-test. N.D., peak not detected. d) Overexpression of the KA-resistant version of GAPDH from the fungus T. koningii (TK-GAPDH) eliminated the effects of KA on mTORC1 signaling. Cells stably expressing FLAG-metap2 or FLAG-TK-GAPDH were incubated under the indicated conditions. KA was added at the beginning of the starvation. Cell lysates were analyzed by immunoblotting for the phosphorylation state or levels of the indicated proteins. e) Overexpression of TK-GAPDH prevents the accumulation of metabolites upstream of GAPDH normally caused by KA treatment in glucose-starved cells. Cells were treated as in (d). Metabolite extracts were analyzed by LC/MS. Data are shown as mean ± s.e.m. for n = 3 biologically independent replicates. P-values were determined for two-sided Student’s t-test. f) Loss of GAPDH expression has the same phenotype as inhibition of GAPDH by KA. GAPDH dox-off cells treated with doxycycline maintain mTORC1 activity even after a 3-hour starvation of glucose.