The ancient drug salicylate directly activates AMP-activated protein kinase

Abstract

Salicylate, a plant product, has been in medicinal use since ancient times. More recently, it has been replaced by synthetic derivatives such as aspirin and salsalate, both of which are rapidly broken down to salicylate in vivo. At concentrations reached in plasma after administration of salsalate or of aspirin at high doses, salicylate activates adenosine monophosphate-activated protein kinase (AMPK), a central regulator of cell growth and metabolism. Salicylate binds at the same site as the synthetic activator A-769662 to cause allosteric activation and inhibition of dephosphorylation of the activating phosphorylation site, threonine-172. In AMPK knockout mice, effects of salicylate to increase fat utilization and to lower plasma fatty acids in vivo were lost. Our results suggest that AMPK activation could explain some beneficial effects of salsalate and aspirin in humans.

Figure 1

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).

Figure 2

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.

Figure 3

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.

Figure 4

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).