Effects of salicylate in HEK-293 cells. (A) Effects of salicylate or aspirin on AMPK activity (mean ± SD, n = 4) and phosphorylation of AMPK (Thr172) and ACC (Ser79) (n = 2). (B) Effects of salicylate on AMPK activity (mean ± SD, n = 6) and phosphorylation (n = 2) in HEK-293 cells stably expressing wild type (WT) γ2 or an R531G substitution (RG). In Figs. 1A and 1B, the activity is plotted on a logarithmic scale as % of control without drug, and effects significantly different from control without drug (2-way ANOVA, with Bonferroni’s test comparing each drug concentration to control without drug) are shown (*p<0.05, ***p<0.001). (C) Effect of salicylate on oxygen uptake in WT and RG cells (mean ± SD, n = 7 to 13; significant differences by 2-way ANOVA, using Bonferroni’s test to compare with basal values without salicylate or DNP, are shown (*p<0.05, **p<0.01, ***p<0.001)). (D) Effects of salicylate or H₂O₂ (1 mM) on ADP:ATP ratios (means of duplicate cell incubations).

Effects of salicylate and A-769662 in intact cells. (A) Expression of β subunits assessed using pan-β antibody in parental cells or cells stably expressing β1 WT, β1-S108A, or β2 WT, and of endogenous α1, α2 and γ1 in the same cells. (B-D) Activity and phosphorylation of AMPK after treatment with various activators in cells expressing: (B) β1 WT; (C) β1-S108A; (D) β2 WT. Kinase assays [mean ± SEM, n = 6 except for 100 μM A-769662 (n = 4) and quercetin (n = 2); significantly different from control without drug, by 1-way ANOVA with Dunnett’s multiple comparison test, ***p<0.001] and Western blots (n = 2) were of immunoprecipitates made using anti-FLAG antibody. (E) Palmitate oxidation in hepatocytes isolated from β1-KO mice and WT controls (mean ± SEM, n = 6 to 14, significantly different from control without drug by 2-way ANOVA with Bonferroni’s test, ***p<0.001; †††significantly different from WT, p<0.001). (F) Phosphorylation of AMPK and ACC in hepatocytes isolated from β1-KO or WT mice.

Effect of salicylate on AMPK in cell-free assays. (A-E) Effects of salicylate on activity of purified rat liver AMPK; (A) effect of salicylate; (B) effect of salicylate ± 200 μM AMP; (C) effect of AMP ± 10 mM salicylate; (D) effect of salicylate ± 30 and 100 nM A-769662; (E) effect of A-769662 ± 10 mM salicylate. Data points in (A-E) are means of duplicate assays; lines were generated by fitting data to the equation: Y = basal + (basal*(activation - basal)*X/(A_0.5 + X)); values obtained for A_0.5 and activation are quoted in the text. (F-H) Effects of AMP, A-769662 and salicylate on dephosphorylation of bacterially expressed human AMPK complexes by PP2Cα (all incubations contained PP2Cα, but control lacked Mg²⁺); bar graphs show AMPK activity (% of control without Mg²⁺, mean ± SD, n = 6; pictures show Thr172 phosphorylation (n = 2). (F) WT α1β1γ1 complex; (G) α1β1γ1 complex with β1 S108A substitution; (H) WT α1β2γ1 complex. Significant differences from the control plus Mg²⁺, using 1-way ANOVA with Dunnett’s multiple comparison test, are shown: **p<0.01, ***p<0.001. Also shown are significant differences in the size of the effect of A-76962 or salicylate between the α1β1γ1 and α1β2γ1 complexes (2F and 2H): †††p<0.001. For the latter comparisons, the difference between the “+Mg²⁺+A-76922” and “+Mg²⁺ only” columns was first expressed as a fraction of the difference between the “+Mg²⁺+AMP” and “+Mg²⁺ only” columns.

Effects of salicylate and A-769662 treatment in β1-KO and WT mice in vivo. (A) Phosphorylation/expression of ACC, AMPK and GAPDH in liver of mice treated with salicylate or A-769662 (doublet in ACC blots represents ACC1/ACC2). (B) Quantification of phosphorylation of ACC (pACC:total ACC, mean ± SEM; n = 6 or 7 for WT, n = 3 for β1-KO; statistical significance by 1-way ANOVA with Bonferroni’s test compared with vehicle only are shown (*p<0.05, ***p<0.001). (C)-(F) Respiratory exchange ratio measured in β1-KO and WT mice after injection of vehicle, salicylate (250 mg/kg) or A-769662 (30 mg/kg) at the start of a period of fasting. Results are mean ± SEM (n = 6 to 13). By 2-way ANOVA, effects of salicylate (p<0.05) or A-769662 (p<0.001) were only significant in WT mice; significant differences by Bonferroni’s test at individual time points are shown (*p<0.05, **p<0.01). (G) Fatty acid utilization calculated from data in (C)-(F). Results are mean ± SEM (n = 7 or 8). Significant differences by 2-way ANOVA with Bonferroni’s test are shown (*p<0.05, **p<0.01). (H) Plasma non-esterified fatty acids in mice treated with salicylate or A-769662 for 90 min. Results are mean ± SEM (n = 7 or 8). Significant differences by 2-way ANOVA with Bonferroni’s test are shown (*p<0.05, **p<0.01).