Polyphenol fraction of extra virgin olive oil protects against endothelial dysfunction induced by high glucose and free fatty acids through modulation of nitric oxide and endothelin-1

Abstract

Epidemiological and clinical studies have reported that olive oil reduces the incidence of cardiovascular disease. However, the mechanisms involved in this beneficial effect have not been delineated. The endothelium plays an important role in blood pressure regulation through the release of potent vasodilator and vasoconstrictor agents such as nitric oxide (NO) and endothelin-1 (ET-1), respectively, events that are disrupted in type 2 diabetes. Extra virgin olive oil contains polyphenols, compounds that exert a biological action on endothelial function. This study analyzes the effects of olive oil polyphenols on endothelial dysfunction using an in vitro model that simulates the conditions of type 2 diabetes. Our findings show that high glucose and linoleic and oleic acids decrease endothelial NO synthase phosphorylation, and consequently intracellular NO levels, and increase ET-1 synthesis by ECV304 cells. These effects may be related to the stimulation of reactive oxygen species production in these experimental conditions. Hydroxytyrosol and the polyphenol extract from extra virgin olive oil partially reversed the above events. Moreover, we observed that high glucose and free fatty acids reduced NO and increased ET-1 levels induced by acetylcholine through the modulation of intracellular calcium concentrations and endothelial NO synthase phosphorylation, events also reverted by hydroxytyrosol and polyphenol extract. Thus, our results suggest a protective effect of olive oil polyphenols on endothelial dysfunction induced by hyperglycemia and free fatty acids.

Keywords: Cell oxidative stress; ECV304 cells; Hyperglycemia; Linoleic acid; Oleic acid; Reactive oxygen species; Type 2 diabetes.

Graphical abstract

Fig. 1

Effect of oleic and linoleic acids on nitric oxide (NO) and reactive oxygen species (ROS) produced by ECV304 cells. Cells were cultured in 199 medium and 10% FBS for 48 h with oleic (triangle) or linoleic (romb) acid (0–120 µM). NO (A) and ROS (B) levels were measured using DAF-DA or CM-H₂DCF-DA, respectively. Results are means±SEM of at least three experiments performed in triplicate. *p<0.05 vs control cells. The changes in endothelial NO synthase (eNOS) and phosphorylated eNOS (p-eNOS) in the presence of oleic or linoleic acid (60 µM) were measured by Western blot. A representative blot (C) and the means of three blots (D) are shown.

Fig. 2

Effect of hydroxytyrosol (HT) and polyphenol extract from extra virgin olive oil (PEEVOO) on the changes in nitric oxide (NO), reactive oxygen species (ROS) and endothelin-1 (ET-1) induced by the presence of high glucose (HG) and/or linoleic and oleic acids in ECV304 cells. Cells were cultured in 199 medium and 10% FBS for 48 h in normal glucose (NG, 5 mM), HG (30 mM) or HG together with linoleic or oleic acid (60 µM) in the presence or absence of HT (10 µM) or PEEVOO (10 µM gallic acid equivalents). NO (A), ROS (B) and ET-1 (C) concentrations in the culture supernatants were determined. Results are means±SEM of at least three experiments performed in triplicate. *p<0.05 vs control cells; ^‡p<0.05 vs HG condition; ^†p<0.05 vs HG plus linoleic acid; ^#p<0.05 vs HG plus oleic acid.

Fig. 3

Effect of hydroxytyrosol (HT) and polyphenol extract from extra virgin olive oil (PEEVOO) on endothelial nitric oxide synthase (eNOS) phosphorylation induced by high glucose (HG) and oleic acid. ECV304 cells were cultured in 199 medium and 10% FBS for 48 h in normal glucose (NG, 5 mM), HG (30 mM) or HG together with oleic acid (60 µM) in the presence or absence of HT (10 µM) or PEEVOO (10 µM gallic acid equivalents). Cells were scraped off and eNOS and p-eNOS were determined by Western blot. A representative blot (A) and means from three westerns blots (B) are shown. Bars are means±SEM. *p<0.05 vs control cells; ^‡p<0.05 vs HG plus oleic acid.

Fig. 4

Effect of hydroxytyrosol (HT) and polyphenol extract from extra virgin olive oil (PEEVOO) on the changes in nitric oxide (NO) and intracellular calcium concentration ([Ca²⁺]_i) induced by acetylcholine (Ach) in the presence of high glucose (HG) and/or oleic and linoleic acids in ECV304 cells. Cells were cultured in 199 medium and 10% FBS for 48 h in normal glucose (NG, 5 mM), HG (30 mM) or HG together with linoleic or oleic acid (60 µM) in the presence or absence of HT (10 µM) or PEEVOO (10 µM gallic acid equivalents). Ach (0.1 µM) was added and NO concentration (A) and [Ca²⁺]_i (B) were determined. Results are means±SEM of at least three experiments performed in triplicate. *p<0.05 vs control cells; ^‡p<0.05 vs HG condition; ^†p<0.05 vs HG/linoleic acid; ^#p<0.05 vs HG/oleic acid.