Metformin and salicylate synergistically activate liver AMPK, inhibit lipogenesis and improve insulin sensitivity

Abstract

Metformin is the mainstay therapy for type 2 diabetes (T2D) and many patients also take salicylate-based drugs [i.e., aspirin (ASA)] for cardioprotection. Metformin and salicylate both increase AMP-activated protein kinase (AMPK) activity but by distinct mechanisms, with metformin altering cellular adenylate charge (increasing AMP) and salicylate interacting directly at the AMPK β1 drug-binding site. AMPK activation by both drugs results in phosphorylation of ACC (acetyl-CoA carboxylase; P-ACC) and inhibition of acetyl-CoA carboxylase (ACC), the rate limiting enzyme controlling fatty acid synthesis (lipogenesis). We find doses of metformin and salicylate used clinically synergistically activate AMPK in vitro and in vivo, resulting in reduced liver lipogenesis, lower liver lipid levels and improved insulin sensitivity in mice. Synergism occurs in cell-free assays and is specific for the AMPK β1 subunit. These effects are also observed in primary human hepatocytes and patients with dysglycaemia exhibit additional improvements in a marker of insulin resistance (proinsulin) when treated with ASA and metformin compared with either drug alone. These data indicate that metformin-salicylate combination therapy may be efficacious for the treatment of non-alcoholic fatty liver disease (NAFLD) and T2D.

Figure 1. Metformin and salicylate synergistically activate AMPK and inhibit lipogenesis in primary mouse hepatocytes

Dose-dependent inhibition of de novo lipogenesis by (A) Met and (B) salicylate in mouse hepatocytes. (C) Ser^79/212 P-ACC as a downstream target and marker of AMPK activation in primary mouse hepatocytes treated with no drug (Con), Met, salicylate (Sal) or Met + salicylate. (D) Inverse suppression of lipogenesis in mouse hepatocytes using the drug concentrations in (C). (E) CI compared with fractional effect inhibition (F_a) of lipogenesis in mouse hepatocytes treated with Met + salicylate (concentration ratio of 1:10 Met–salicylate) where CI > 1 indicates an antagonistic, CI = 1 additive or CI < 1 synergistic inhibition. Results represent at least two independent experiments performed in triplicate. (F) Fatty acid oxidation in mouse hepatocytes treated with no drug, low dose Met (0.1 mM), salicylate (0.5 mM) or both. Results from two independent experiments performed in triplicate. (G) Activation of AMPK by salicylate + AMP in vitro using purified, dephosphorylated AMPK α1β1γ1. Results were generated from four independent experiments. All densitometry is a ratio of phosphorylated to total protein. Data are means ± S.E.M. except panel (E), which are −means only. *P < 0.05 compared with Con, ‡ P < 0.05 compared with both respective Met-only and Sal-only doses.

Figure 2. Metformin and salicylate synergistically activate AMPK and inhibit lipogenesis in primary human hepatocytes

(A) Dose-dependent inhibition of lipogenesis by Met, salicylate (Sal) or equimolar Met + salicylate (Met + Sal) in human hepatocytes. (B) Ser^79/212 P-ACC as a downstream target and marker of AMPK activation in primary human hepatocytes treated with no drug (Con), 0.1 mM Met, 0.3 mM salicylate or Met + salicylate (0.1 and 0.3 mM respectively). (C) Suppression of lipogenesis in human primary hepatocytes treated with the drug conditions in (B). Results represent experiments from two donors performed in triplicate. All densitometry is a ratio of phosphorylated to total protein. Data are means ± S.E.M. *P < 0.05 compared with Con, ‡P < 0.05 compared with both respective Met only and Sal only doses.

Figure 3. Metformin–salsalate combination treatment synergistically improves metabolic homoeostasis and lowers liver lipids in HFD-fed mice

(A) P-ACC Ser^79/212 (marker of AMPK activation; P-ACC) in clamped liver samples of C57bl/6 mice fed a 60 % HFD for 5 weeks followed by HFD with no drug (Con), 2.5 g/kg Met, 1 g/kg salsalate (SS) or Met and salsalate (Met + SS) for an additional 5 weeks. Densitometry is the ratio of phosphorylated to total protein. n = 5–8 mice per group. (B) Growth curves for mice on the diets indicated. n = 8 mice per group. (C) Percentage body adiposity by CT scan analysis. n = 8 mice per group. (D) Mean RER over a 24-h light–dark cycle. n = 4 mice per group. (E) Representative H & E staining of hepatic sections (scale bars, 100 μm) illustrate liver lipid deposition and (F) total liver TG assayed as total liver glycerol. n = 8–12 mice per group. Legends are conserved across bar graph panels. All data are means ± S.E.M. *P < 0.05 compared with Con, ‡P < 0.01 compared with both respective Met-only and SS-only doses.

Figure 4. Met and salsalate synergistically improve liver insulin sensitivity in mice

Intraperitoneal (A) GTT and (B) ITT for C57bl/6 mice fed a 60 % HFD for 5 weeks followed by HFD with no drug (Con), 2.5 g/kg Met, 1 g/kg salsalate (SS) or Met and salsalate (Met + SS) for an additional 5 weeks. (C) Whole blood glucose and (D) serum insulin concentrations following a 12-h fast. (E) HGP and (F) percentage suppression of hepatic glucose output during hyperinsulinaemic–euglycaemic clamps. Legends are conserved across line or bar graph panels. n = 4–5 mice per group for all measures. All data are means ± S.E.M. *P < 0.05 compared with Con, ‡P < 0.01 compared with both respective Met and SS only doses, †P < 0.05 compared with respective SS only.