Tocomin Restores Endothelium-Dependent Relaxation in the Diabetic Rat Aorta by Increasing NO Bioavailability and Improving the Expression of eNOS

Abstract

We aimed to determine whether tocomin, an extract from palm oil that has a high tocotrienol content, was able to prevent diabetes-induced endothelial dysfunction. To induce type 1 diabetes streptozotocin (50 mg/kg) was injected into the tail vein of Wistar rats. Six weeks later the diabetic rats, and normal rats injected with citrate buffer, commenced treatment with tocomin (40 mg/kg/day sc) or its vehicle (peanut oil) for a further 4 weeks. Aortae isolated from diabetic rats had impaired acetylcholine (ACh)-induced endothelium-dependent relaxation compared to normal rat aortae but there was no change in endothelium-independent relaxation in response to sodium nitroprusside. By contrast, responses to ACh in aortae from diabetic rats treated with tocomin were not different to normal rats. In addition to impaired endothelium-dependent relaxation the diabetic aortae had increased expression of the NADPH oxidase Nox2 subunit, increased generation of superoxide and decreased expression of eNOS and all of these effects were prevented by tocomin treatment. Tocomin did not affect plasma glucose levels. The impaired response to ACh in vitro was maintained in the presence of TRAM-34 and apamin, selective inhibitors of calcium-activated potassium (K _Ca ) channels, indicating diabetes impaired the contribution of NO to endothelium-dependent relaxation. By contrast, neither diabetes nor tocomin treatment influenced EDH-type relaxation as, in the presence of L-NNA, an inhibitor of eNOS, and ODQ, to inhibit soluble guanylate cyclase, responses to ACh were similar in all treatment groups. Thus tocomin treatment improves NO mediated endothelium dependent relaxation in aortae from diabetic rats associated with a decrease in vascular oxidant stress but without affecting hyperglycaemia.

Keywords: diabetes; eNOS; endothelium; nitric oxide; oxidative stress; tocomin.

Figure 1

Superoxide generated in rat aorta in the presence of NADPH from normal, diabetic, and tocomin treated (normal + tocomin/diabetic + tocomin) and in the presence of DPI. Data is expressed as mean ± SEM. ^∗Significantly different to normal. ^#Significantly different to diabetic. Results are shown as mean ± SEM. P < 0.05. Two-way ANOVA, Dunnett’s multiple comparisons test. n = 3–6 experiments.

Figure 2

Cumulative concentration–response curves to ACh and SNP in endothelium-intact aortae isolated from normal, diabetic, and tocomin treated (normal + tocomin/diabetic + tocomin) rats in control (A,B) and in the presence of Tram + apamin (C,D). ^∗R_max Significantly different to normal. ^#R_max Significantly different to diabetic. P < 0.05. Data is expressed as mean ± SEM. Sidak’s multiple comparison test. n = 3–7. See Table 2 for values and statistical comparison.

Figure 3

Cumulative concentration–response curves to ACh and SNP in endothelium-intact aortae isolated from normal, diabetic, and tocomin treated (normal + tocomin/diabetic + tocomin) rats in the presence of L-NNA (A,B) and L-NNA + ODQ (C,D). Data is expressed as mean ± SEM. ^∗p < 0.05. Sidak’s multiple comparison test. n = 4–8. See Table 2 for values and statistical comparison.

Figure 4

Protein expression of NADPH oxidase (Nox2) from isolated aortae from normal, diabetic, and tocomin treated (normal + tocomin/diabetic + tocomin) rats. Representative blots are shown for each corresponding graph. ^∗Significantly different to normal. ^#Significantly different to diabetic. Results are shown as means ± SEM. P < 0.05. Two-way ANOVA Dunnett’s multiple comparison test. n = 3–5 experiments.

Figure 5

Protein expression of total eNOS (A), calmodulin-1 (B), caveolin-1 (C), and pAkt/Akt (D) from isolated aortae from normal, diabetic, and tocomin treated (normal + tocomin/diabetic + tocomin) rats. Representative blots are shown for each corresponding graph. ^∗Significantly different to normal. ^#Significantly different to diabetic. Results are shown as means ± SEM; p < 0.05. Two-way ANOVA Dunnett’s multiple comparisons test. n = 3–5 experiments.