Tocotrienol-Rich Tocomin Attenuates Oxidative Stress and Improves Endothelium-Dependent Relaxation in Aortae from Rats Fed a High-Fat Western Diet

Abstract

We have previously reported that tocomin, a mixture high in tocotrienol content and also containing tocopherol, acutely preserves endothelial function in the presence of oxidative stress. In this study, we investigated whether tocomin treatment would preserve endothelial function in aortae isolated from rats fed a high-fat diet known to cause oxidative stress. Wistar hooded rats were fed a western diet (WD, 21% fat) or control rat chow (standard diet, 6% fat) for 12 weeks. Tocomin (40 mg/kg/day sc) or its vehicle (peanut oil) was administered for the last 4 weeks of the feeding regime. Aortae from WD rats showed an impairment of endothelium-dependent relaxation that was associated with an increased expression of the NADPH oxidase Nox2 subunit and an increase in the vascular generation of superoxide measured using L-012 chemiluminescence. The increase in vascular oxidative stress was accompanied by a decrease in basal NO release and impairment of the contribution of NO to ACh-induced relaxation. The impaired relaxation is likely contributed to by a decreased expression of eNOS, calmodulin, and phosphorylated Akt and an increase in caveolin. Tocotrienol rich tocomin, which prevented the diet-induced changes in vascular function, reduced vascular superoxide production and abolished the diet-induced changes in eNOS and other protein expression. Using selective inhibitors of nitric oxide synthase (NOS), soluble guanylate cyclase (sGC) and calcium-activated potassium (K_Ca) channels we demonstrated that tocomin increased NO-mediated relaxation, without affecting the contribution of endothelium-dependent hyperpolarization type relaxation to the endothelium-dependent relaxation. The beneficial actions of tocomin in this diet-induced model of obesity suggest that it may have potential to be used as a therapeutic agent to prevent vascular disease in obesity.

Keywords: endothelium; high-fat diet; oxidative stress; tocotrienol; vascular relaxation.

Figure 1

Superoxide generated in rat aorta in the presence of NADPH from standard diet (SD), western diet (WD), and tocomin treated (SD + tocomin/WD + tocomin) groups in the absence and presence of apocynin (300 μM). Data are expressed as mean ± SEM. *Significantly different to WD p ≤ 0.05, two-way ANOVA, Dunnett’s multiple comparisons test. n = 3–6.

Figure 2

Response to l-NAME in the presence of KPSS (SD), western diet (WD), and tocomin-treated (SD + tocomin/WD + tocomin) groups. Data are expressed as mean ± SEM. *Significantly different to WD. p ≤ 0.05. n = 10, Dunnett’s multiple comparisons test. n = 3–6.

Figure 3

Cumulative concentration–response curves to ACh and SNP in the absence or presence of tocomin in endothelium-intact aortae isolated from standard diet (SD), western diet (WD), and tocomin treated (SD + tocomin/WD + tocomin) rats in the presence of vehicle (A,B), TRAM + apamin (C,D), and l-NAME (E,F). *pEC₅₀ significantly different to SD. ^#pEC₅₀ significantly different to WD p ≤ 0.05. n = 4–10. Two-way ANOVA Sidak’s post test. Results are shown as mean ± SEM. See Table 2 for values and statistical comparison.

Figure 4

Cumulative concentration–response curves to ACh and SNP in the absence or presence of tocomin in endothelium-intact aortae isolated from standard diet (SD), western diet (WD), and tocomin-treated (SD + tocomin/WD + tocomin) rats in the presence of l-NAME + ODQ (A,B) and l-NAME + ODQ + TRAM + apamin (C,D). *R_max significantly different to SD n = 4–8. Two-way ANOVA Sidak’s post test. Results are shown as mean ± SEM. See Table 2 for values and statistical comparison.

Figure 5

Protein expression of NADPH oxidase (Nox-2) from isolated aortae from standard diet (SD), western diet (WD), and tocomin treated (SD + tocomin/WD + tocomin) rats. Representative blots are shown for each corresponding graph. *Significantly different to SD. ^#Significantly different to WD. Results are shown as means ± SEM; n = 6 experiments. p ≤ 0.05. Two-way ANOVA Dunnett’s post test.

Figure 6

Protein expression of total eNOS (A), calmodulin-1 (B), caveolin-1 (C), and pAkt/Akt (D) from isolated aortae from standard diet (SD), western diet (WD), and tocomin treated (SD + tocomin/WD + tocomin) rats. Representative blots are shown for each corresponding graph. *Significantly different to SD. ^#Significantly different to WD. Results are shown as means ± SEM; n = 6 experiments. p ≤ 0.05. Two-way ANOVA Dunnett’s post test.