Fig. 2: Aldometanib is accumulated in the lysosome.
From: The aldolase inhibitor aldometanib mimics glucose starvation to activate lysosomal AMPK
a–c, Aldometanib activates AMPK through the lysosomal pathway without causing elevation of AMP/ADP, unless high doses are used. In a, results of adenylate ratios are shown as the mean ± s.e.m.; n = 4 dishes of cells for each condition. P values for AMP:ATP (coloured in green, and same hereafter for all comparisons between ratios of AMP:ATP) or ADP:ATP (coloured in red, and same hereafter for ADP:ATP) ratios were determined by one-way ANOVA followed by Dunnet’s (MEFs) or Tukey’s (hepatocytes) test. d,e,k,l, Aldometanib is enriched in the lysosome. MEFs (d, e and k) or HEK293T cells (l; expressing TRPV4-GCaMP6s indicator) were treated with aldometanib, followed by fractionation as in Fig. 1p to analyse aldometanib contents and concentrations (d; see also the lysosomal protein levels of ALDOA, ALDOB and ALDOC in e) and the activity of aldolase (k) in the lysosomal fraction, or determining the activity of TRPV4, which is an outcome of lysosomal aldolase activity in situ15 (l). Data are the mean ± s.e.m. from n = 3 (k) or 4 (d) biological replicates for each treatment; P values were determined by two-sided Student’s t-test (k), or by one-way ANOVA followed by Tukey’s test (d). f–i, Lysosomal pH, but not endocytosis, contributes to the accumulation of aldometanib in the lysosome. MEFs were pretreated with NH4Cl, followed by incubation with aldometanib or starved for glucose (f and g). AMPK activation (f, upper part of h) and lysosomal concentrations of aldometanib (g, lower panel of h) were then determined. Aldometanib does not affect endocytosis, as evidenced by the fluorescence intensity (FI) of FITC in MEFs labelled with FITC-dextran via flow cytometry (i). Data are the mean ± s.e.m., from n = 7 (i) or 6 (h) biological replicates, with P values calculated by two-way ANOVA, followed by Tukey’s test (i), or by one-way ANOVA, followed by Dunn’s test (h). j, Intact lysosomes enhance the potency of aldometanib towards aldolase. Lysosomes from MEFs were purified, followed by incubation with aldometanib. Activities of lysosome-bound aldolase are the mean ± s.e.m. from n = 4 biological replicates for each treatment; P values were determined by one-way ANOVA, followed by Tukey’s test. m–p, Aldometanib activates AMPK in liver and muscle through the lysosomal pathway. Wild-type mice (m and n) or mice with Lamtor1 specifically knocked out in liver (o; LKO) or muscle (p; MKO) were orally gavaged with aldometanib. After 2 h of gavaging, AMPK activation and the AMP:ATP and ADP:ATP ratios were determined. Data are the mean ± s.e.m. from n = 6 (m and n; AMPK activation), 3 (o and p; AMPK activation) or 4 (m and n; adenylate ratios) biological replicates for each treatment; P values were determined by two-sided Student’s t-test (m and n), or by two-way ANOVA followed by Tukey’ test (o and p). Experiments were performed three times.
