Salsalate reduces atherosclerosis through AMPKβ1 in mice

Abstract

Objective: Salsalate is a prodrug of salicylate that lowers blood glucose in people with type 2 diabetes. AMP-activated protein kinase (AMPK) is an αβγ heterotrimer which inhibits macrophage inflammation and the synthesis of fatty acids and cholesterol in the liver through phosphorylation of acetyl-CoA carboxylase (ACC) and HMG-CoA reductase (HMGCR), respectively. Salicylate binds to and activates AMPKβ1-containing heterotrimers that are highly expressed in both macrophages and liver, but the potential importance of AMPK and ability of salsalate to reduce atherosclerosis have not been evaluated.

Methods: ApoE-/- and LDLr-/- mice with or without (-/-) germline or bone marrow AMPKβ1, respectively, were treated with salsalate, and atherosclerotic plaque size was evaluated in serial sections of the aortic root. Studies examining the effects of salicylate on markers of inflammation, fatty acid and cholesterol synthesis and proliferation were conducted in bone marrow-derived macrophages (BMDMs) from wild-type mice or mice lacking AMPKβ1 or the key AMPK-inhibitory phosphorylation sites on ACC (ACC knock-in (KI)-ACC KI) or HMGCR (HMGCR-KI).

Results: Salsalate reduced atherosclerotic plaques in the aortic roots of ApoE-/- mice, but not ApoE-/- AMPKβ1-/- mice. Similarly, salsalate reduced atherosclerosis in LDLr-/- mice receiving wild-type but not AMPKβ1-/- bone marrow. Reductions in atherosclerosis by salsalate were associated with reduced macrophage proliferation, reduced plaque lipid content and reduced serum cholesterol. In BMDMs, this suppression of proliferation by salicylate required phosphorylation of HMGCR and the suppression of cholesterol synthesis.

Conclusions: These data indicate that salsalate suppresses macrophage proliferation and atherosclerosis through an AMPKβ1-dependent pathway, which may involve HMGCR phosphorylation and cholesterol synthesis. Since rapidly-proliferating macrophages are a hallmark of atherosclerosis, these data indicate further evaluation of salsalate as a potential therapeutic agent for treating atherosclerotic cardiovascular disease.

Trial registration: ClinicalTrials.gov NCT00624923.

Keywords: Aspirin; Lipogenesis; Macrophage; Proliferation; Salicylate; Sterol synthesis.

Copyright © 2021 The Author(s). Published by Elsevier GmbH.. All rights reserved.

Figures

Figure 1

Salsalate reduces atherosclerosis through AMPKβ1. A) Serum salicylate levels on western diet +2.5 g/kg salsalate. B) Food intake, C) body mass, D) glucose tolerance and E) GTT Area under the curve of ApoE−/− and ApoE−/−AMPKβ1−/− fed a western diet or western diet containing + salsalate. F) Representative images of H&E–stained aortic roots from control or salsalate-treated ApoE−/− and ApoE−/−AMPKβ1−/− mice. G) Average plaque size, H) plaque necrotic area, I) plaque lipid area, J) plasma triglyceride and K) plasma cholesterol. Data is expressed as mean ± S.E.M. # indicates significant effect of genotype, ∗ indicates p < 0.05 when compared to control. Scale bar is 200 μm.

Figure 2

Salsalate reduces atherosclerosis through hematopoietic AMPKβ1. A) Representative image of genotyping from blood of LDLrWT and LDLr β1KO mice. B) Body mass of LDLrWT and LDLr β1KO mice fed a western diet or western diet + salsalate. C) Representative images of H&E–stained aortic roots from control or salsalate-treated LDLrWT and LDLr β1KO mice. D) Average plaque size, E) plaque necrotic area, F) plaque lipid area, G) serum cholesterol and H) serum triglycerides. Data is expressed as mean ± S.E.M. # indicates significant effect of genotype, & indicates significant effect of salsalate, and ∗ indicates p < 0.05 when compared to control. Scale bar is 200 μm.

Figure 3

Salsalate reduces macrophage proliferation in vivo. A) Representative Mac3- (yellow dashed lines), Ki67- (blue triangles) and BrdU- (green triangles) stained aortic roots from ApoE−/- and ApoE−/−AMPKβ1−/− mice after 6 weeks of western diet or western diet + salsalate. B, C) Ki67-and BrdU-positive cells within the atherosclerotic plaques. Data is expressed as mean ± S.E.M. ∗p < 0.05 for salsalate versus control within genotype. Scale bar is 100 μm.

Figure 4

AMPK phosphorylation of HMGCR is required for the decrease in macrophage proliferation with salicylate. A, B) Representative western blots and quantification of wild-type and AMPKβ1−/− bone marrow–derived macrophages were treated with 1 mM salicylate for 90 min (Statistical test; One-Way ANOVA). C) Proliferation relative to control (MCSF only) of wild-type and AMPKβ1−/−, simultaneously treated with MCSF (2 ng/mL) and salicylate (1 mM) for 72 h (Statistical test non-parametric t-test) D) Wild-type bone marrow–derived macrophages were simultaneously treated with MCSF (2 ng/mL), salicylate (1 mM) ± mevalonate or oleate where indicated for 72 h (Statistical Test Kruskal–Wallis) E, F) Incorporation of radiolabelled acetate into sterol and fatty acid fractions in wild-type, AMPKβ1−/− ACC DKI and HMGCR KI bone marrow–derived macrophages over 4 h. G) Proliferation relative to control (MCSF only) of wild-type, ACC DKI and HMGCR KI bone marrow–derived macrophages simultaneously treated with MCSF (2 ng/mL) and salicylate (1 mM) for 72 h (Statistical Test, Kruskal–Wallis with Dunn's multiple comparison). Data is expressed as mean ± S.E.M. from 3 to 6 individual experiments. ∗p < 0.05 for comparison indicated.