Extended Data Figure 1: Glucose deprivation activates AMPK via an AMP/ADP-independent mechanism.
From: Fructose-1,6-bisphosphate and aldolase mediate glucose sensing by AMPK
a, Phosphorylation of AMPK (pT172) and ACC (p-ACC) analysed by immunoblotting from the experiment shown in Fig. 1a; the upper panel shows quantification of the pT172:AMPKα ratios (mean ± s.d., n = 2). b, c, Glucose deprivation rapidly activates AMPK without altering cellular AMP/ATP or ADP/ATP ratios in HEK293T cells. Cells were grown in full medium (25 mM glucose, 4 mM alanyl-glutamine, 1 mM pyruvate, 10% serum) and then switched to medium without glucose, with other components unchanged, for 15 min or 2 h. AMPK activation was monitored by immunoblotting for p-AMPKα and p-ACC (b), and intracellular AMP/ATP and ADP/ATP ratios determined by CE–MS (c, results are mean ± s.d., n = 3, N.S., not significant by ANOVA). d, Adenine nucleotide ratios were unaltered in mouse liver after starvation. Mice were fed ad libitum or starved for 16 h, freeze-clamped liver samples prepared, and AMP/ATP and ADP/ATP ratios measured by CE–MS. Results are mean ± s.d., n = 6; ns, not significant by Student’s t-test. e, Decrease in plasma glucose concentrations induced by starving mice for 16 h. Mice from the same experiment shown in Extended Data Fig. 1d were either fed ad libitum or were deprived of food for 16 h and plasma glucose determined. Circles and squares are individual data points and horizontal lines are means ± s.d., n = 6; P value computed by Student’s t-test. f, Extracts were prepared from livers of mice treated as in d (6 mice per group) and analysed by western blotting using the indicated antibodies. g, Western blots from the experiment shown in Fig. 1c. Although shown as separate panels, the blots with each antibody were from the same gel scanned at the same time, and are therefore directly comparable. h, Quantification of blots in g. Results are mean ± s.d., n = 2. i, ADP/ATP ratios determined by LC–MS, from the same experiments shown in Fig. 1c, d. j, Time course of effects of starving MEFs for glucose only, or glucose and glutamine, in the presence of serum. The experiment was as in Fig. 1c except that the medium contained 10% fetal calf serum throughout (n = 2). k, l, ADP/ATP ratios determined by LC–MS, from the experiments shown in Figs 1e–h (n = 3). All experiments were performed at least twice.
