Extended Data Figure 4: Absence of FBP defines a starvation signal for AMPK activation.

All intact cell experiments were performed in the presence of 10% serum. a, FBP dissociates axin–LKB1 from LAMTOR1 in vitro. The same experiment identical to that in Fig. 3b was performed except that the bacterially expressed His-tagged AXIN was used in lieu of AXIN-containing cytosol and incubated with light organelles purified from MEFs glucose-starved for 2 h. b, FBP modulates the interaction between AMPK and LKB1. Cytosol from unstarved MEFs was mixed with light organelles purified from 2-h glucose-starved ALDO-TKD MEFs or wild-type MEFs, followed by addition of 200 μM FBP. The mixtures were then dissolved and LKB1 was immunoprecipitated. c, Concentrations of FBP in mouse liver after starvation for 16 h. FBP levels from mouse livers were determined by CE–MS. The amounts of FBP were estimated according to the standard curve generated by plotting the peak-area ratios of unlabelled to ¹³C-labelled FBP (derived from [U-¹³C]FBP used as internal standard) against the concentrations of unlabelled FBP. The results were obtained by dividing its amount by the estimated volume of liver, and the concentrations of free-state FBP were then estimated as described previously³⁴. Values are presented as mean ± s.d., n = 5 for each condition, P = 9.627 × 10⁻⁵ (Student’s t-test). d, e, Intracellular FBP levels were measured by CE–MS in MEFs after incubation in media containing different concentrations of glucose (d), or in glucose-free DMEM for the indicated time periods (e). Values are presented as mean ± s.d., n = 3 for each treatment; *P = 0.0187, FBP levels in cells incubated in 10 mM glucose (for 1 h) were compared to those in cells incubated with media containing 5 mM glucose (for 1 h); †P = 0.0102, cells incubated with media containing 5 mM to 3 mM glucose (for 1 h); P = 0.831, cells incubated with medium containing 25 mM to 10 mM glucose (for 1 h); P = 0.00577, comparison of incubation in glucose-free medium for 0 min to 20 min (ANOVA). f, Plasma glucose concentrations (left) and intracellular FBP levels (right) in mouse liver. FBP were determined by CE–MS analysis. Results are mean ± s.d., n = 6 for each condition; P value by Student’s t-test. g, h, Linear correlation between extracellular glucose concentrations and intracellular FBP levels. FBP levels in MEFs, measured after 2 h of incubation in media containing various concentrations of glucose as shown in d, and in the starved mouse liver shown in f, were plotted against their corresponding glucose concentrations in the culture medium (g) and plasma (h), respectively. i, FBP levels in SLO-permeabilized MEFs. Levels of FBP in regularly cultured SLO-permeabilized MEFs (left panel), or glucose-starved SLO-permeabilized MEFs treated with exogenous FBP (right panel), were determined by CE–MS analysis. Results are mean ± s.d., n = 3 for each condition. ***P < 0.001, N.S., not significant by Student’s t-test (left panel) and ANOVA (right panel). Experiments shown in left and right panels were performed simultaneously and shared a single control (SLO-permeabilized MEFs incubated in medium containing 25 mM glucose). j–l, Addition of exogenous FBP to permeabilized MEFs blocks glucose starvation-induced AMPK activation. Various glycolytic intermediates (200 μM) as indicated were individually added to the 2-h glucose-starved wild-type (j), PFK1-KD (k) or TPI-KD (l) MEFs pre-incubated with SLO for 5 min. After incubating for another 15 min, cells were lysed and were analysed by immunoblotting. Experiments in this figure were performed twice.

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