Restoration of perivascular adipose tissue function in diet-induced obese mice without changing bodyweight

Abstract

Background and purpose: We have recently shown that a reduced function of endothelial nitric oxide synthase (eNOS) in the perivascular adipose tissue (PVAT) contributes crucially to obesity-induced vascular dysfunction in mice. The current study was conducted to test the hypothesis that vascular dysfunction in obesity can be reversed by in vivo improvement of PVAT eNOS activity.

Experimental approach: Male C57BL/6J mice were fed a high-fat diet (HFD) for 22 weeks to induce obesity. During the last 4 weeks of HFD feeding, the obese mice were treated p.o. with the standardized Crataegus extract WS® 1442, which has been shown previously to improve eNOS activity.

Key results: Diet-induced obesity in mice markedly reduced the vasodilator response of thoracic aorta to acetylcholine in wire myograph experiments. Strikingly, this vascular dysfunction was only evident in PVAT-containing aorta but not in PVAT-free aorta. In vivo treatment of obese mice with WS® 1442 had no effect on body weight or epididymal fat mass, but completely restored the vascular function of PVAT-containing aorta. Feeding a HFD led to a reduced phosphorylation and an enhanced acetylation of PVAT eNOS, both effects were reversed by WS® 1442 treatment.

Conclusion and implications: PVAT plays a key role in vascular dysfunction in diet-induced obese mice. Not obesity itself, but a PVAT dysfunction is responsible for obesity-induced vascular disorders. Improving PVAT function by pharmacological means (e.g. with WS® 1442) can ameliorate vascular function even without reducing body weight or fat mass.

Linked articles: This article is part of a themed section on Molecular Mechanisms Regulating Perivascular Adipose Tissue - Potential Pharmacological Targets? To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.20/issuetoc.

Figure 1

WS® 1442 had no effect on body weight or fat mass. Male C57BL/6J mice were put on a NFD or HFD for 22 weeks starting at the age of 8 weeks. A subgroup of HFD animals were treated with WS® 1442, p.o., during the last 4 weeks (from week 19 to week 22) of HFD feeding. Body weight and epididymal fat were measured at the end of the experiment (week 22). Columns represent mean ± SEM. *P < 0.05, n = 10.

Figure 2

WS® 1442 restores vascular function in HFD mice. Male C57BL/6J mice were put on a NFD or HFD for 22 weeks starting at the age of 8 weeks. A subgroup of HFD animals were treated with WS® 1442, p.o., during the last 4 weeks of HFD feeding. The vasodilator response to acetylcholine was performed in noradrenaline‐precontracted aorta with or without PVAT. Symbols represent mean ± SEM. *P < 0.05, n = 6.

Figure 3

WS® 1442 improves PVAT NO production in HFD mice. (A) eNOS immunohistochemistry staining and western blot analyses were performed using PVAT‐containing aorta samples from C57BL/6J wild‐type mice (WT) or global eNOS knockout mice (KO). E and P indicate endothelium and PVAT respectively. (B) Male C57BL/6J mice were put on a NFD or HFD for 22 weeks starting at the age of 8 weeks. A subgroup of HFD animals were treated with WS® 1442, p.o., during the last 4 weeks of HFD feeding. NO production in PVAT was determined by DAF‐2 DA staining in the absence or presence of the NOS inhibitor L‐NAME. The confocal images shown are representative for five independent experiments with similar results. (C) Shows the quantification fluorescence intensity in PVAT. *P < 0.05, n = 5.

Figure 4

WS® 1442 had no effect on NO synthases or arginases. Male C57BL/6J mice were put on a NFD or HFD or for 22 weeks starting at the age of 8 weeks. A subgroup of HFD animals were treated with WS® 1442, p.o., during the last 4 weeks of HFD feeding. The mRNA expression of NO synthases (nNOS, iNOS and eNOS), arginases (Arg1 and Arg2), the macrophage marker CD68 and phagocyte NADPH oxidase NOX2 was studied with qPCR in aorta (A, C and E) and PVAT (B, D and F). Columns represent mean ± SEM. *P < 0.05, n = 9.

Figure 5

WS® 1442 restores eNOS phosphorylation in PVAT. Male C57BL/6J mice were put on a NFD or HFD for 22 weeks starting at the age of 8 weeks. A subgroup of HFD animals were treated with WS® 1442, p.o., during the last 4 weeks of HFD feeding. The expression and phosphorylation of eNOS at serine 1177 and of upstream kinases for eNOS serine 1177 phosphorylation, Akt and AMPK were analysed by western blotting using samples from aortic PVAT or epididymal visceral adipose tissue (VAT). The blots shown are representative of six experiments with similar results (A). (B) Shows the results of densitometric analyses. Columns represent mean ± SEM. *P < 0.05, n = 6.

Figure 6

WS® 1442 normalizes eNOS acetylation level in PVAT. Male C57BL/6J mice were put on a NFD or HFD for 22 weeks starting at the age of 8 weeks. A subgroup of HFD animals were treated with WS® 1442, p.o., during the last 4 weeks of HFD feeding. Immunoprecipitation (IP) was performed with an anti‐eNOS antibody using PVAT samples followed by immunoblotting (IB) with antibodies against eNOS or acetyl‐lysine respectively. The band detected with the antibody against acetyl‐lysine at the level of eNOS was considered acetyl‐eNOS. In (A) the blots shown are representative of six independent experiments. (B) Results of densitometric analyses for acetyl‐eNOS normalized to total eNOS; n = 6. (C,D and E) Expression of SIRT1 and NAMPT at the protein (C and D) or mRNA (E) levels was analysed by western blotting and qPCR respectively. In (C) the blots shown are representative of six independent experiments. (D and E) Results of densitometric analyses are shown; n = 6. Columns represent mean ± SEM. *P < 0.05.