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

Figures

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  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.

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  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.

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  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.

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  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.

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  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.

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  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.