Extended Data Figure 8. Effects of aldolase mutants on AMPK activation.

All intact cell experiments in this Figure were performed in the presence of 10% serum. a, ALDOA-D34S mutant increases intracellular FBP levels under glucose starvation. HEK293T cells stably expressing ALDOA or its D34S mutant were incubated in DMEM with or without glucose for 2 h, followed by measuring FBP levels. Results are mean ± SD, n = 3; P value by ANOVA. N.S., not significant. b, D34S mutant of ALDOA that still binds FBP in glucose-free medium exhibits reduced AMPK activation. ALDO-TKD MEFs stably expressing ALDOA-D34S, or wild type ALDOA as control, were incubated in DMEM medium with or without glucose for 2 h, followed by immunoblotting. c, ALDOA-D34S blunts the formation of Ragulator-AXIN/LKB1-AMPK complex. HEK293T cells stably expressing ALDOA and ALDOA-D34S were lysed and the endogenous LAMTOR1 was immunoprecipitated, followed by immunoblotting. d, Adenovirus-mediated expression of the D34S mutant in mouse liver blocks AMPK activation after starvation. Adenovirus expressing ALDOA-D34S, or WT ALDOA as control, was injected into the tail veins of 6-week-old mice. Five days later, mice were fasted for 16 h. Phosphorylation of AMPKα and ACC were then determined by immunoblotting. e, K230A mutant of aldolase promotes the formation of Ragulator-AXIN/LKB1-AMPK complex. Endogenous LAMTOR1 in unstarved or glucose-starved WT MEFs and ALDOA-K230A MEFs were immunoprecipitated, followed by immunoblotting. f, FBP fails to dissociate AXIN/LKB1 from LAMTOR1 on light organelles purified from starved ALDOA-K230A MEFs. Cytosol from unstarved MEFs was mixed with light organelles purified from 2-h-glucose-starved ALDOA-K230A MEFs or WT MEFs as a control, and 200 μM FBP was then added to each mixture. The mixtures were then dissolved and LAMTOR1 was immunoprecipitated, followed by immunoblotting. g, Addition of exogenous FBP fails to block glucose starvation-induced AMPK activation in permeabilized ALDO-K230A expressed, TKD-MEFs (ALDOA-K230A MEFs). FBP (200 μM) was added to the 2-h-glucose-starved ALDOA-K230A MEFs or WT MEFs that had been pre-incubated with SLO for 5 min. After incubating for another 15 min, cells were subjected to immunoblotting. Experiments in c and f were performed 3 times, and that in a, b, d, e and g twice.