The Sirt1 activator SRT3025 provides atheroprotection in Apoe-/- mice by reducing hepatic Pcsk9 secretion and enhancing Ldlr expression

Abstract

Aims: The deacetylase sirtuin 1 (Sirt1) exerts beneficial effects on lipid metabolism, but its roles in plasma LDL-cholesterol regulation and atherosclerosis are controversial. Thus, we applied the pharmacological Sirt1 activator SRT3025 in a mouse model of atherosclerosis and in hepatocyte culture.

Methods and results: Apolipoprotein E-deficient (Apoe(-/-)) mice were fed a high-cholesterol diet (1.25% w/w) supplemented with SRT3025 (3.18 g kg(-1) diet) for 12 weeks. In vitro, the drug activated wild-type Sirt1 protein, but not the activation-resistant Sirt1 mutant; in vivo, it increased deacetylation of hepatic p65 and skeletal muscle Foxo1. SRT3025 treatment decreased plasma levels of LDL-cholesterol and total cholesterol and reduced atherosclerosis. Drug treatment did not change mRNA expression of hepatic LDL receptor (Ldlr) and proprotein convertase subtilisin/kexin type 9 (Pcsk9), but increased their protein expression indicating post-translational effects. Consistent with hepatocyte Ldlr and Pcsk9 accumulation, we found reduced plasma levels of Pcsk9 after pharmacological Sirt1 activation. In vitro administration of SRT3025 to cultured AML12 hepatocytes attenuated Pcsk9 secretion and its binding to Ldlr, thereby reducing Pcsk9-mediated Ldlr degradation and increasing Ldlr expression and LDL uptake. Co-administration of exogenous Pcsk9 with SRT3025 blunted these effects. Sirt1 activation with SRT3025 in Ldlr(-/-) mice reduced neither plasma Pcsk9, nor LDL-cholesterol levels, nor atherosclerosis.

Conclusion: We identify reduction in Pcsk9 secretion as a novel effect of Sirt1 activity and uncover Ldlr as a prerequisite for Sirt1-mediated atheroprotection in mice. Pharmacological activation of Sirt1 appears promising to be tested in patients for its effects on plasma Pcsk9, LDL-cholesterol, and atherosclerosis.

Keywords: Atherogenesis; LDL receptor; LDL-cholesterol; Pcsk9; Sirt1.

Figure 1

SRT3025 confers atheroprotection, reduces plasma cholesterol and systemic inflammation in Apoe^−/− mice. Eight-week-old Apoe^−/− mice were fed a high-cholesterol diet (1.25% w/w) supplemented with the Sirt1 activator SRT3025 (n = 9) or placebo (n = 9) for 12 weeks. Representative micrographs (left) and quantifications (right) of thoraco-abdominal aortae en face (A) and of aortic root cross sections (B–D) stained with ORO or immunohistochemically for macrophages (Cd68) or Vcam-1; (E) cholesterol distribution in the different lipoprotein subfractions separated by gel filtration chromatography; (F) Plasma total cholesterol, LDL-, and VLDL-cholesterol concentrations; (G) Plasma levels of Mcp-1 and Il-6. Scale bars in photomicrographs: 1 mm (A) and 500 μm (B–D). HCD, high-cholesterol diet; AU, arbitrary units; LDL, low-density lipoprotein; ORO, oil-red O; VLDL, very low-density lipoprotein; Vcam-1, vascular cell adhesion molecule 1; Mcp-1, monocyte chemoattractant protein 1; Il-6, interleukin 6.

Figure 2

SRT3025 mimics Sirt1 activity in vitro and in Apoe^−/− mice. (A) Concentration-response curve of SRT3025 on the activity of wild-type Sirt1 and activation-resistant mutant E230K in vitro. (B, C) Western blots for acetylation status of Sirt1 target proteins (B) p65 and (C) Foxo1 immunoprecipitated from nuclear extracts of liver and skeletal muscle, respectively, from Apoe^−/− mice fed a high-cholesterol diet (1.25% w/w) supplemented with the Sirt1 activator SRT3025 or placebo for 12 weeks. HCD, high-cholesterol diet; Ac-Ly, anti-acetyl-lysine antibody.

Figure 3

SRT3025 increases hepatic Ldlr protein expression while decreasing plasma Pcsk9 in Apoe^−/− mice. Eight week-old Apoe^−/− mice were fed a high-cholesterol diet (1.25% w/w) supplemented with the Sirt1 activator SRT3025 (n = 9) or placebo (n = 9) for 12 weeks. (A) Relative mRNA expression levels of hepatic genes involved in cholesterol regulation. (B) Western blots of liver lysates for Ldlr, Pcsk9, and β-actin. (C) Plasma levels of Pcsk9. HCD, high-cholesterol diet; Pcsk9, proprotein convertase subtilisin/kexin type 9.

Figure 4

SRT3025 increases Ldlr expression in AML12 hepatocytes and decreases Pcsk9 in the supernatant. Western blots of Ldlr, Pcsk9, and β-actin in cultured AML12 cells (A) treated with SRT3025 at indicated concentrations for 24 h and (B) incubated with 10 μM SRT3025 for the times indicated. (C) Relative mRNA expression levels of Ldlr and Pcsk9 in AML12 cells after incubation with 10 μM SRT3025 for 24 h. (D) Pcsk9 protein levels in the supernatant of AML12 cells incubated with 10 μM SRT3025 for the times indicated. (E) Pcsk9 immunoprecipiated from AML12 cells incubated with vehicle (Veh, DMSO) or 10 μM SRT3025, and blotted for Ldlr and Pcsk9. Pcsk9, proprotein convertase subtilisin/kexin type 9.

Figure 5

Exogenous Pcsk9 prevents SRT3025-induced increase in Ldlr expression and activity in AML12 hepatocytes. (A) Western blot and corresponding quantifications of AML12 cells treated with vehicle (Veh, DMSO) or 10 μM SRT3025 and incubated with or without Pcsk9 active protein (3 ng ml⁻¹) for 1 h. (B) BODIPY-labelled LDL uptake in AML12 cells incubated for 24 h with 10 μM SRT3025 or Veh and incubated with or without Pcsk9 active protein (3 ng ml⁻¹) for 1 h. Fluorescence intensity and western blot quantifications are given as percentage of Veh control. BODIPY, 4,4-difluoro-3a,4a-diaza-s-indacene; AU, arbitrary units; Pcsk9, proprotein convertase subtilisin/kexin type 9.

Figure 6

Sirt1 knockdown reduces and Sirt1 overexpression enhances SRT3025-induced increase in Ldlr expression in AML12 hepatocytes. (A) Western blots of Ldlr, Sirt1, and β-actin with corresponding quantifications of AML12 cell lysates following transfection with Sirt1 siRNA or scrambled siRNA for 24 h and incubated with 10 μM SRT3025 or vehicle (Veh, DMSO) for additional 24 h. (B) Western blots of Ldlr, Sirt1, and β-actin with corresponding quantifications of AML12 cell lysates following Sirt1 overexpression plasmid or scramble control for 24 h and incubated with 10 μM SRT3025 or Veh for additional 24 h. Western blot quantifications are given as a percentage of Veh control. Scr, scramble plasmid; Sirt1 OE, Sirt1 overexpression plasmid.

Figure 7

Genetic deletion of Ldlr abolishes anti-atherosclerotic effects of SRT3025 in vivo. Eight-week-old Ldlr^−/− mice were fed a high-cholesterol diet (1.25% w/w) supplemented with the Sirt1 activator SRT3025 (n = 9) or placebo (n = 9) for 12 weeks and fasted for 12 h before blood was drawn and aortae were explanted. Representative micrographs (left) and quantifications (right) of thoraco-abdominal aortae en face (A) and of aortic root cross sections (B–D) stained with oil-red O (ORO) or immunohistochemically for macrophages (CD68) or Vcam-1; (E) cholesterol distribution in the different lipoprotein subfractions separated by gel filtration chromatography; (F) plasma total cholesterol, LDL-, and VLDL-cholesterol concentrations; (G) plasma levels of Mcp-1, Il-6, and Pcsk9. Scale bars in photomicrographs = 1 mm (A) and 500 μm (B–D). HCD, high-cholesterol diet; AU, arbitrary units; LDL, low-density lipoprotein; ORO, oil-red O; VLDL, very low-density lipoprotein; Vcam-1, vascular cell adhesion molecule 1; Mcp-1, monocyte chemoattractant protein 1; Il-6, interleukin 6; Pcsk9, proprotein convertase subtilisin/kexin type 9.