The nitroxide radical TEMPOL prevents obesity, hyperlipidaemia, elevation of inflammatory cytokines, and modulates atherosclerotic plaque composition in apoE-/- mice

Abstract

Objective: The nitroxide compound TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl radical) has been shown to prevent obesity-induced changes in adipokines in cell and animal systems. In this study we investigated whether supplementation with TEMPOL inhibits inflammation and atherosclerosis in apoE-/- mice fed a high fat diet (HFD).

Methods: ApoE-/- mice were fed for 12 weeks on standard chow diet or a high-fat diet. Half the mice were supplemented with 10 mg/g TEMPOL in their food. Plasma samples were analysed for triglycerides, cholesterol, low- and high-density lipoprotein cholesterol, inflammatory cytokines and markers (interleukin-6, IL-6; monocyte-chemotactic protein, MCP-1; myeloperoxidase, MPO; serum amyloid A, SAA; adiponectin; leptin). Plaques in the aortic sinus were analysed for area, and content of collagen, lipid, macrophages and smooth muscle cells.

Results: High fat feeding resulted in marked increases in body mass and plasma lipid levels. Dietary TEMPOL decreased both parameters. In the high-fat-fed mice significant elevations in plasma lipid levels and the inflammatory markers IL-6, MCP-1, MPO, SAA were detected, along with an increase in leptin and a decrease in adiponectin. TEMPOL supplementation reversed these effects. When compared to HFD-fed mice, TEMPOL supplementation increased plaque collagen content, decreased lipid content and increased macrophage numbers.

Conclusions: These data indicate that in a well-established model of obesity-associated hyperlipidaemia and atherosclerosis, TEMPOL had a significant impact on body mass, atherosclerosis, hyperlipidaemia and inflammation. TEMPOL may therefore be of value in suppressing obesity, metabolic disorders and increasing atherosclerotic plaque stability.

Keywords: Adipokines; Atherosclerosis; Cardiovascular disease; Inflammatory cytokines; Lipid; Nitroxide; TEMPOL.

Fig. 1.

Body mass and plasma lipid parameters for apoE^−/− mice that were fed either a chow or HFD, with or without TEMPOL supplementation. (A) Mean body mass (g) of mice over the time course of the study: ◻, apoE^−/− mice fed HFD; ∎, apoE^−/− mice fed HFD plus added TEMPOL; ◯, apoE^−/− mice fed standard chow; ●, apoE^−/− mice fed standard chow plus added TEMPOL. Data were compared using the non-parametric Mann–Whitney U-test of significance using WinSTAT® for Excel, with comparisons made between HFD vs. HFD plus TEMPOL (*p < 0.001) and standard chow vs. standard chow plus TEMPOL (†p < 0.001). Error bars (±SD) are only shown for the mice fed HFD, and HFD plus added TEMPOL; in some cases these are smaller than the symbols. Panel (B), plasma triglycerides; (C) total cholesterol; (D) HDL-cholesterol; (E) LDL-cholesterol. Data in (C)–(E) are given as mmol/L. For panels (B)–(E) values are expressed as mean ± SEM and * indicates statistical difference against the chow-fed group, + versus the chow-fed group administered TEMPOL, and # versus the HFD-fed mice using two-way ANOVA analysis followed by Bonferroni’s post-hoc test.

Fig. 2.

Plasma cytokines and inflammatory agents in apoE^−/− mice that were fed either a chow or HFD, with or without TEMPOL supplementation. Plasma cytokines; MCP-1 (Panel A) and IL-6 (B) and inflammatory agents; SAA (C) and MPO (D) were measured in mice that were fed normal chow or a HFD, with or without TEMPOL supplementation. Values are expressed as mean ± SEM. * Indicates statistical difference against the chow-fed group, + versus the chow-fed group administered TEMPOL, and # versus the HFD-fed mice using two-way ANOVA analysis followed by Bonferroni’s post-hoc test.

Fig. 3.

Plasma levels of (A) adiponectin, (B) leptin, and (C) resistin, in apoE^−/− mice fed either a chow or HFD, with or without TEMPOL supplementation. Values are expressed as mean ± SEM. * Indicates statistical difference against the chow-fed group, and # versus the HFD-fed mice using two-way ANOVA analysis followed by Bonferroni’s post-hoc test.

Fig. 4.

(A) Plaque area, (B,C) collagen content, and (D) content of α-actin positive cells (each expressed as a percentage over the total area) in the aortic sinus in apoE^−/− mice that were fed either a chow or HFD, with or without TEMPOL supplementation. Representative images of atherosclerotic plaques stained for collagen with picrosirius red in sinus crosssections are shown in panel (C) and quantified in panel (B) from animals fed control chow (CTL), control chow plus TEMPOL (CTL + T), high-fat diet (HFD) or HDF plus TEMPOL (HFD + T). Values are expressed as mean ± SEM. * Indicates statistical difference against the chow-fed group, + versus the chow-fed group administered TEMPOL, and # versus the HFD-fed mice using two-way ANOVA analysis followed by Bonferroni’s post-hoc test. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 5.

(A,B) Plaque macrophage (F4/80 staining) and (C) lipid content in the aortic sinus in apoE^−/− mice that were fed either a chow or HFD, with or without TEMPOL supplementation. Panel (A) represents representative images of atherosclerotic plaques stained with antibody F4/80 to detect macrophages (pink staining); legend as Fig. 4. Quantification of macrophage staining is expressed relative to plaque area in panel (B). Lipid levels were assessed as white areas (arising from sample delipidation) in images stained with picrosirius red (cf. Fig. 4) and are quantified relative to plaque area in panel (C). Values are expressed as mean ± SEM. * Indicates statistical difference against the chow-fed group, + versus the chow-fed group administered TEMPOL, and # versus the HFD-fed mice using two-way ANOVA analysis followed by Bonferroni’s post-hoc test. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)