Macrophage AMPK β1 activation by PF-06409577 reduces the inflammatory response, cholesterol synthesis, and atherosclerosis in mice

Abstract

Atherosclerotic cardiovascular disease is characterized by both chronic low-grade inflammation and dyslipidemia. The AMP-activated protein kinase (AMPK) inhibits cholesterol synthesis and dampens inflammation but whether pharmacological activation reduces atherosclerosis is equivocal. In the current study, we found that the orally bioavailable and highly selective activator of AMPKβ1 complexes, PF-06409577, reduced atherosclerosis in two mouse models in a myeloid-derived AMPKβ1 dependent manner, suggesting a critical role for macrophages. In bone marrow-derived macrophages (BMDMs), PF-06409577 dose dependently activated AMPK as indicated by increased phosphorylation of downstream substrates ULK1 and acetyl-CoA carboxylase (ACC), which are important for autophagy and fatty acid oxidation/de novo lipogenesis, respectively. Treatment of BMDMs with PF-06409577 suppressed fatty acid and cholesterol synthesis and transcripts related to the inflammatory response while increasing transcripts important for autophagy through AMPKβ1. These data indicate that pharmacologically targeting macrophage AMPKβ1 may be a promising strategy for reducing atherosclerosis.

Keywords: Immunology.

Graphical abstract

Figure 1

PF-064095777 reduces Atherosclerosis via AMPK β1 in ApoE^-/- mice independently of reductions in circulating triglycerides or cholesterol ApoE^−/− and ApoE^−/− AMPK β1^−/− mice were fed a Western diet and treated with vehicle or 100 mg/kg PF-06409577 by oral gavage. (A) Representative images of plaques in the aortic root, with plaques outlined in red, scale bar represents 100 μM, (B) plaque quantification, (C) necrotic area, (D) serum cholesterol, and (E) triglycerides. Data are presented as mean ± SEM, ∗ indicates p < 0.05 by two-way ANOVA with Fisher’s LSD post-hoc testing.

Figure 2

PF-06409577 reduces atherosclerosis via macrophage AMPK activation (A–D) Peritoneal macrophages were isolated from AMPK β1^fl/fl and AMPK β1^LysM mice. (A) AMPK levels and phosphorylation of ACC following treatment with PF-06409577 (10 μM, 90 min) were detected by western blot and (B–D) quantified using ImageJ data are presented as mean ± SEM, ∗ indicates p < 0.05 and ∗∗∗ indicates <0.005 by unpaired t test. AMPK β1^fl/fl and AMPK β1 ^LysM mice were injected with PCSK9 AAV, fed a Western diet and treated with Vehicle or 100 mg/kg PF-06409577 by oral gavage for 6 weeks. (E and F) Representative plaque images, scale bar represents 100 μM and quantification. Data are presented as mean ± SEM, # indicates p < 0.05 in overall group effect my two-way ANOVA, ∗ indicates p < 0.05 by two-way ANOVA with Fisher’s LSD post-hoc testing.

Figure 3

PF-06409577 activates AMPK in macrophages Bone marrow derived macrophages were isolated from wildtype and AMPK β1^−/− mice. (A–C) cells were treated with indicated concentrations of PF-06409577(PF) or A-769662(A-76) (100 μM) for 90 min in serum free media. Total and phosphorylation ACC and ULK were measured by immunoblotting. Data are mean ± SEM, ∗ indicates p < 0.05 by two-way ANOVA with Dunnet's post-hoc testing, n = 3.

Figure 4

PF-06409577 reduces inflammatory pathways and lipid synthesis in macrophages Wild type and AMPK β1^−/− BMDMs were treated with 10 μM PF-06409577 for 6 h and analyzed by RNA-Seq. (A) PCA plots, (B and C) differentially regulated genes in wild type and AMPK β1^−/−, respectively, (D) heatmap showing log-fold change of the top 50 differentially regulated genes, (E and F) AMPK β1 specific downregulated and upregulated biological process with PF-06409577 treatment. Wild type and AMPK β1^−/− BMDMs were treated with 10 μM PF-06409577(PF) for 4 h. (G) Fatty acid and (H) cholesterol synthesis. Data are presented as mean ± SEM, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 by two-way ANOVA with Dunnet's post-hoc testing.