Fig. 1. Metformin activates AMPK without increasing AMP/ADP levels.
a, Low-dose metformin deacidifies lysosomes in mouse primary hepatocytes (left). Cells were treated with 5 μM metformin (Met) for 2 h, and the relative fluorescence intensities of Lysosensor are shown (right). b, Metformin does not increase AMP/ADP levels in mouse primary hepatocytes. Cells were treated with 5 μM metformin for the indicated time periods followed by analysis of phosophrylated (p)-AMPKα and p-ACC by immunoblotting (IB; left), AMP/ATP and ADP/ATP ratios, and the absolute concentrations of AMP, ADP and ATP by mass spectrometry (bottom right). After washing three times with PBS, the intracellular metformin concentrations (conc.) were measured by mass spectrometry (top right). For gel source data, see Supplementary Fig. 1. Data are the mean ± s.e.m., n values are labelled on each panel. P values were calculated using two-sided Mann–Whitney test (a) or one-way analysis of variance (ANOVA) followed by Tukey’s (b, bottom right) or Sidak’s test (b, top right). Experiments in a were performed three times and experiments in b were performed five times.