Circulating Sirt1 Protects Against Atherosclerosis Through Its Modulation of Pcsk9 Activity

Introduction: Sirtuin 1 (SIRT1), an NAD + - dependent deacetylase enzyme is an important regulator of nutrient metabolism. Hepatic LDL receptor (LDL-R) expression confers atheroprotection by clearing cholesterol from blood. Previously, we have shown that SIRT1 activation protects against atherosclerosis through its effect on PCSK9 expression. However, the role of secreted SIRT1 in atherosclerosis is yet unknown. Hypothesis: Circulating SIRT1 protects against atherosclerosis through an increase in hepatic LDL-R expression and a decrease in plasma PCSK9 activity. Methods: Male ApoE -/- mice (n=6) on a high-cholesterol diet (1.25% w/w) were treated with recombinant SIRT1 (rSIRT1) or PBS (vehicle) for 10 weeks and were assessed for severity of aortic atherosclerosis, plasma lipids and hepatic protein expression. PCSK9 and SIRT1 expression was measured in healthy subjects of different age groups (n=80) and in hypercholesterolaemic patients (n=70) by ELISA. In vitro experiments were performed in Huh7 hepatoma cells. Results: Genetic overexpression of GFP-tagged SIRT1 confirms that SIRT1 is indeed secreted in Huh7 cells. Plasma SIRT1 levels decrease in an age-dependent manner in healthy subjects and inversely correlates with plasma LDL-C and PCSK9 in healthy subjects and hypercholestrolaemic patients. ApoE -/- mice treated with rSIRT1 showed less atherosclerosis and had lower levels of plasma cholesterol (LDL- and total cholesterol) compared vehicle-treated controls. Hepatic LDL-R protein expression increased with no change in LDL-R or PCSK9 mRNA levels. SIRT1 was found bound to PCSK9 immunoprecipitated from plasma of healthy subjects. Correspondingly, mass spectrometry revealed PCSK9 to have four acetylation sites which are deacetylated by SIRT1. In vitro, rSIRT1 increased LDL-R expression in a time-dependent manner in Huh7 cells. LDL-R-PCSK9 binding assay showed that deacetylated PCSK9 binds to a lower extent to LDL-R EGF-A domain compared to control PCSK9. Conclusion: SIRT1 secretion reduces with age. Pharmacological treatment with rSIRT1 protects against atherosclerosis through a decrease in LDL-C and an increase in LDL-R. Our findings demonstrate a link into why LDL-C increases as we age, causing an increase risk to atherosclerosis.