Fig. 2.

SIRT1 deacetylates eNOS in cells and in vitro, and calorie restriction is associated with deacetylation of eNOS in mice livers. (A) Acetylation of endogenous eNOS on lysine residues, with and without RNAi-mediated knockdown of endogenous SIRT1, and with and without treatment of cells with the SIRT1 inhibitor nicotinamide. Rat aortic endothelial cells were transfected with SIRT1 RNAi or control RNAi, or treated with nicotinamide. Immunoprecipitates of eNOS from whole-cell lysates were immunoblotted with anti-eNOS and anti-acetyllysine antibodies (top two panels). Immunoprecipitates by using nonimmune IgG were used as a control. Whole-cell lysates were immunoblotted with anti-eNOS, anti-SIRT1, and anti-β-actin antibodies (bottom three panels). (B) Acetylation of endogenous eNOS on lysine residues, with and without treatment with the SIRT1 activator resveratrol. Immunoprecipitated eNOS from whole-cell lysates of rat aortic endothelial cells, with and without treatment with resveratrol, was immunoblotted with anti-acetyllysine and anti-eNOS antibodies. The whole-cell lysates were also immunoblotted with anti-eNOS, anti-SIRT1, and anti-β-actin antibodies. (C) Incorporation of [³H]acetate into eNOS expressed in COS7 cells. Cells transfected with eNOS, with and without SIRT1 overexpression, or treated with nicotinamide, were incubated in medium containing sodium [³H]acetate. Immunoprecipitated eNOS from cell lysates was autoradiographed, and immunoblotted with anti-eNOS antibody. Whole-cell lysates were immunoblotted with anti-eNOS and anti-SIRT1 antibodies. (D) Deacetylation of acetylated eNOS by SIRT1 in vitro. Purified full-length GST-eNOS and GST-eNOS acetylated with the p300 acetyltransferase in the presence of the acetyl group donor acetyl-CoA (Ac-CoA) were incubated with active SIRT1 enzyme, in the presence or absence of the SIRT1 cofactor NAD. Reaction mixes were immunoblotted with anti-acetyllysine and anti-eNOS antibodies. (E) Deacetylation of eNOS in mice livers by CR. Male and female mice were fed ad libitum or a calorie-restricted diet (57.4 kcal/week) for 3 weeks. Immunoprecipitates of eNOS from liver homogenates were immunoblotted with anti-acetyllysine and anti-eNOS antibodies (top two panels), and homogenates immunoblotted with anti-SIRT1 and anti-β-actin antibodies (bottom two panels). The bar graph shows the calculated acetylated/total eNOS ratio, expressed relative to the mouse fed ad libitum in each gender group. The weight of the mice at the end of 3 weeks (as a percentage of starting weight) is shown. AL, ad libitum.