Extended Data Fig. 1: LCA activates AMPK through the lysosomal AMPK pathway.
From: Lithocholic acid binds TULP3 to activate sirtuins and AMPK to slow down ageing
a, LCA triggers lysosomal translocation of AXIN. MEFs were incubated with 1 μM LCA for 4 h, followed by immunostaining to determine the lysosomal localization of AXIN (accessed by the co-localization, i.e., the Mander’s overlap coefficients, with the lysosomal marker LAMP2). Results are shown as mean ± s.e.m.; n = 29 (DMSO) or 20 (LCA) cells, and P value by two-sided Student’s t-test with Welch’s correction. b, LCA triggers lysosomal translocation of LKB1. Wildtype and AXIN knockout (AXIN−/−) MEFs were incubated with 1 μM LCA for 4 h, followed by immunostaining to determine the lysosomal translocation of LKB1. Mander’s overlap coefficients are shown as mean ± s.e.m.; n = 26 (DMSO, WT) 32 (LCA, WT), 24 (DMSO, AXIN−/−) or 28 (LCA, AXIN−/−) cells, and P value by two-way ANOVA, followed by Tukey. c, LCA induces the formation of the lysosomal AMPK-activating complex. MEFs were treated with 1 μM LCA for 4 h, and the cell lysates were immunoprecipitated (IP) with an antibody against AXIN. Immunoblotting (IB) reveals that AXIN co-immunoprecipitates with v-ATPase, Ragulator, AXIN and LKB1. TCL, total cell lysate. d, e, LCA inhibits v-ATPase. MEFs were incubated with 1 μM LCA for 4 h, followed by determination of the activity of v-ATPase (d, accessed by the decreased intensities of the Lysosensor that monitors acidification of lysosomes; representative images are shown on the left panel, and the statistical analysis data on the right as mean ± s.e.m., normalized to the DMSO group; n = 21 (DMSO) or 23 (LCA) cells, and P value by two-sided Mann-Whitney test) and the proton transport rates of v-ATPase in vitro (e, data are shown as mean ± s.e.m., normalized to the DMSO group; n = 3 replicates, and P value by two-sided Student’s t-test). f-n, The lysosomal AMPK pathway is required for the LCA-induced AMPK activation. HEK293T cells with ATP6v0c (v0c) knockdown (shATP6v0c; f), MEFs with AXIN knockout (g), LAMTOR1 knockout (LAMTOR1−/−; h-j), mice with liver- or muscle-specific LAMTOR1 knockout (LAMTOR1-LKO, in k; LAMTOR1-MKO, in l), or mice with liver- or muscle-specific AXIN knockout (AXIN1-LKO, in m; or AXIN1/2-MKO, in n), were treated with LCA at 1 μM for 4 h (f-j; for cells) or at 1 g/l with LCA coated with (2-hydroxypropyl)-β-cyclodextrin in drinking water for 1 month (k-n; for mice), followed by determination for AMPK activation (f-h, k-n; the levels of p-AMPKα and p-ACC) by immunoblotting, the v-ATPase inhibition (i, see representative images on the left panel, and the statistical analysis data on the right panel), and the lysosomal translocation of AXIN evidenced by co-localization with LAMP2 (j, see representative images on the left panel, and the statistical analysis data on the right panel). Results in i, j are shown as mean ± s.e.m. (in i, data are normalized to the DMSO group); n = 22 (DMSO group of i), 25 (LCA group of i), 29 (DMSO group of j) or 23 (LCA group of j) cells, and P value by two-sided Student’s t-test. Experiments in this figure were performed three times, except a and d four times.
