Fig. 1

G6PD neutralizes stresses by supporting reductive glutamine metabolism and AMPK activation. a A simple schematic for the oxidative PPP and the non-oxidative PPP. G6PD enzymatic activity-deficiency alleles are well tolerated, except that it could bring a risk of acute non-spherocytic hemolytic anemia triggered by exogenous oxidative stressors in red blood cells. Based on the report from the World Health Organization, G6PD deficiency-associated variants are divided into several types, class-I G6PD mutants with an activity often less than 1% of normal are associated with chronic NSHA; class-II have an activity less than 10% of normal; class-III show 10-60% of residual enzyme activity; class-IV are nearly normally active. Intriguingly, up to date, 85 class-I mutants, accounting for about 45% of the total, have been identified, but no null mutation has been reported. These observations suggest that G6PD protein could have other function(s) than the mediation of the robust oxPPP, which is indispensable to embryonic development. b Western blots to validate the knockout of G6PD in HeLa cells, and the re-expression of the empty vector (KO-EV), WT G6PD, or G6PD mutants (indicated as HeLa^KO, HeLa^WT, HeLa^R166H, HeLa^R257G, HeLa^242-243Δ, and HeLa^H263A). IDH1 and ME1 were also detected, and β-actin was used as the loading control. c Survival of HeLa cells with different states of G6PD, as indicated, after treatments either with 100 µM H₂O₂ for 8 h or 1 µM antimycin A for 24 h, or under hypoxia (0.5% O₂) for 24 h, normalized to untreated cells. Here, one class-II variant, R166H, and two class-I mutants, 242-243Δ and R257G, were used. d The relative abundance of cellular 6-phospho-D-glucono-1,5-lactone (6PGL) by LC-MS/MS in HeLa cells with different states of G6PD, as indicated. e Mass isotopomer analysis of NADPH in HeLa cell lines, as indicated, cultured with the medium containing 10 mM of [3-²H] glucose for 8 h. f The NADPH/NADP⁺ ratio in HeLa cells with different states of G6PD, as indicated. g Cellular metabolites were measured by LC-MS/MS with an untargeted metabolomic method. Volcano plot of cellular metabolites in HeLa^KO, relative to HeLa^WT cells. h Glucose uptake (left) and lactate excretion (right) in HeLa cells with different states of G6PD, as indicated, under the normal condition. Data were from triplicate experiments, and all experimental data were verified in at least two independent experiments. i NADH/NAD⁺ ratio in HeLa cells with different states of G6PD, as indicated, under the normal condition. j NADH/NAD⁺ ratio in HeLa^KO and HeLa^WT cells treated with antimycin A (1 µM) and/or α-KB (2 mM) for 4 h. k Survival of HeLa^KO cells treated with antimycin A (1 µM) or H₂O₂ (100 µM), without or with α-KB (1 or 5 mM), Pyruvate (1 or 5 mM), and OAA (1 or 5 mM) for 24 h, normalized to untreated cells. l Confirmation of Dox-induced expression of LbNOX, mitoLbNOX, TPNOX, and mitoTPNOX in HeLa^KO cells. m Survival of cell lines from (G) treated with antimycin A (1 µM) in the presence or absence of Dox (50 nM, 24 h prior to antimycin A treatment) for 24 h, normalized to untreated cells. n Mass isotopomer analysis of isocitrate in HeLa^WT, HeLa^KO, and HeLa^R257G cells cultured with ¹³C₅-glutamine for 4 h in the presence of antimycin A (1 μM). o Western blot analysis of G6PD, pACC1, ACC1, pAMPKα, and AMPKα in HeLa cells with different states of G6PD, as indicated, treated with 1 µM antimycin A for the time as indicated. GAPDH was used as the loading control. p Survival of HeLa^WT and HeLa^KO cells treated without or with 1 µM antimycin A for 24 h, in the presence or absence of 500 µM AICAR (pretreatment for 12 h) and/or 10 µM compound C. q Survival of HeLa/Cas9 and HeLa^IDH-DKO cells treated with 10 µM compound C and/or 1 µM antimycin A for 24 h. r The brief working model for the anti-stress roles of G6PD. Data were from triplicate experiments, and all experimental data were verified in at least two independent experiments. Error bars represent mean ± SD. *p < 0.05; **p < 0.01 (Student’s t-test).