The Involvement of Sirtuin 1 Dysfunction in High-Fat Diet-Induced Vascular Dysfunction in Mice

Figures

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  Figure 1. The role of perivascular adipose tissue (PVAT) in obesity-induced vascular dysfunction. Male C57BL/6J mice were treated with normal control diet (NCD) or high-fat diet (HFD) for 20 weeks. Aortic rings without (A) or with PVAT (B) were used for vascular function assessment with a wire myograph system. The rings were precontracted with noradrenaline, followed by relaxation in response to increasing concentrations of acetylcholine. Symbols represent mean ± SEM. ns, not significant; * p < 0.05; n = 8.

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  Figure 2. Reduced SIRT1 activity despite enhanced SIRT1 expression in PVAT of diet-induced obese mice. Male C57BL/6J mice were treated with normal control diet (NCD) or high-fat diet (HFD) for 20 weeks. The mRNA expression of SIRT1 (A) was studied with qPCR. The protein expression of SIRT1 (B) was studied with Western blot. The blots shown are representative for 3 independent experiments with similar results. * p < 0.05; ns, not significant; n = 10. SIRT1 activity (C) in mouse PVAT lysate was measured with SIRT1 deacetylase fluorometric assay kit. Columns represent mean ± SEM. ** p < 0.01; n = 10.

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  Figure 3. Reduced NAD+ level, NAD+/NADH ratio and NMAPT expression in PVAT of diet-induced obese mice. Male C57BL/6J mice were treated with NCD or HFD for 20 weeks. NAD+ level and NADH in mouse PVAT lysate were measured with NAD+/NADH Colorimetric Assay kit (A). Columns represent mean ± SEM. * p < 0.05, ** p < 0.01, n = 6. The mRNA expression of NAMPT (B) was studied with qPCR. Columns represent mean ± SEM. ns, not significant; ** p < 0.01, n = 6. The protein expression of NAMPT (C) was studied with Western blot. The blots shown are representative for 3 independent experiments with similar results. ns, not significant; * p < 0.05; n = 10. Parp1 and Parp2 expression (D) was studied with (qPCR). n = 10. ns, not significant.

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  Figure 4. Enhanced eNOS acetylation in PVAT of diet-induced obese mice. Male C57BL/6J mice were treated with NCD or HFD for 20 weeks. eNOS immunoprecipitation (IP) was performed with the PVAT samples using an eNOS antibody. Immunoblotting (IB) was performed with the eNOS IP product using an antibody against eNOS and an antibody against acetyl-lysine, respectively. The band detected with the antibody against acetyl-lysine at the level of eNOS was considered acetyl-eNOS. The blots shown (A) are representative of 3 independent experiments with similar results. Results of densitometric analyses for the acetyl-eNOS normalized to a total eNOS are shown in (B). Columns represent mean ± SEM. *** p < 0.001, n = 6. In separate experiments, Western blot analyses (WB) were performed with an antibody against acetyl-lysine using the PVAT samples without IP. The blots shown (C) are representative of 3 independent experiments with similar results. The results of densitometric analyses for acetylated proteins normalized to GAPDH are shown in (D). Columns represent mean ± SEM. ns, not significant; * p < 0.05, ** p < 0.01, n = 10.

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  Figure 5. Improvement of the PVAT function by exogenous NAD+ in diet-induced obese mice. Male C57BL/6J mice were treated with (A) NCD or (B) HFD for 20 weeks. The PVAT-containing aortas were isolated and incubated in the myograph system with 350 μmol/L NAD+ for 6 h. Then, the vessels were contracted with noradrenaline before acetylcholine was added to induce vasodilation. Symbols represent mean ± SEM. * p < 0.05, n = 6. ns, not significant.

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  Figure 6. PVAT eNOS dysregulation in diet-induced obese mice. Diet-induced obesity leads to inhibition of Akt phosphorylation. The downregulation of NAMPT reduces cellular NAD+ levels and SIRT1 activity. The resulting reduction in eNOS phosphorylation and enhancement of eNOS acetylation both decrease eNOS activity. In addition, HFD feeding causes eNOS uncoupling due to arginine deficiency because of arginase induction. The illustrated mechanisms are a summary of results from the present study and two previous publications [7,11].