Citrus polyphenol hesperidin stimulates production of nitric oxide in endothelial cells while improving endothelial function and reducing inflammatory markers in patients with metabolic syndrome

Abstract

Context: Hesperidin, a citrus flavonoid, and its metabolite hesperetin may have vascular actions relevant to their health benefits. Molecular and physiological mechanisms of hesperetin actions are unknown.

Objective: We tested whether hesperetin stimulates production of nitric oxide (NO) from vascular endothelium and evaluated endothelial function in subjects with metabolic syndrome on oral hesperidin therapy. DESIGN, SETTING, AND INTERVENTIONS: Cellular mechanisms of action of hesperetin were evaluated in bovine aortic endothelial cells (BAEC) in primary culture. A randomized, placebo-controlled, double-blind, crossover trial examined whether oral hesperidin administration (500 mg once daily for 3 wk) improves endothelial function in individuals with metabolic syndrome (n = 24).

Main outcome measure: We measured the difference in brachial artery flow-mediated dilation between placebo and hesperidin treatment periods.

Results: Treatment of BAEC with hesperetin acutely stimulated phosphorylation of Src, Akt, AMP kinase, and endothelial NO synthase to produce NO; this required generation of H(2)O(2). Increased adhesion of monocytes to BAEC and expression of vascular cell adhesion molecule-1 in response to TNF-α treatment was reduced by pretreatment with hesperetin. In the clinical study, when compared with placebo, hesperidin treatment increased flow-mediated dilation (10.26 ± 1.19 vs. 7.78 ± 0.76%; P = 0.02) and reduced concentrations of circulating inflammatory biomarkers (high-sensitivity C-reactive protein, serum amyloid A protein, soluble E-selectin).

Conclusions: Novel mechanisms for hesperetin action in endothelial cells inform effects of oral hesperidin treatment to improve endothelial dysfunction and reduce circulating markers of inflammation in our exploratory clinical trial. Hesperetin has vasculoprotective actions that may explain beneficial cardiovascular effects of citrus consumption.

Trial registration: ClinicalTrials.gov NCT00914251.

Figures

Fig. 1.

Hesperetin acutely stimulates phosphorylation of Akt, AMPK, and eNOS to produce NO in a concentration- and time-dependent manner in vascular endothelial cells. A, BAEC were serum-starved overnight and then treated with hesperetin for 10 min at the indicated concentrations. Cell lysates were immunoblotted with anti-phospho-AMPK (Thr172), anti-phospho-Akt (Ser473), anti-phospho-eNOS (Ser1179), and anti-β actin (loading control) antibodies. Representative blots are shown for experiments that were repeated independently five to six times (top). Scanning densitometry was used to quantify results of multiple independent experiments represented in panel A normalized to loading control (mean ± sem) (bottom). Significant concentration-dependent effects of hesperetin to increase pAMPK, pAkt, and peNOS were observed (P < 0.05; one-way ANOVA); *, P < 0.05 for Dunnett's comparisons to control (0 μm, hesperetin) for pAMPK, pAkt, and peNOS. B, BAEC were serum-starved overnight and then treated with hesperetin (10 μm) for the indicated durations. Cell lysates were immunoblotted as in panel A. Representative blots are shown for experiments that were repeated independently five to six times (top). Scanning densitometry was used to quantify results of multiple independent experiments represented in panel B normalized to loading control (mean ± sem) (bottom). Significant time-dependent effects of hesperetin to increase pAMPK, pAkt, and peNOS were observed (P < 0.05; one-way ANOVA); *, P < 0.05 for Dunnett's comparisons to control (0 min) for pAMPK, pAkt, or peNOS. C, BAEC were loaded with DAF-2-DA as described in Subjects and Methods before treatment with hesperetin for 10 min at the indicated concentrations (top) or with hesperetin (10 μm) for the indicated durations (bottom). Cells were then fixed and viewed as described in Subjects and Methods. Emission of green light (510 nm) from cells excited by light at 480 nm is indicative of NO production. Phase contrast views of cells corresponding to images in the upper panels are also shown. A representative experiment is shown for experiments that were repeated independently three times. D, BAEC prepared as in panel C were treated without or with insulin (100 nm, 5 min), hesperetin (10 μm, 10 min), or LPA (5 μm, 5 min). In some groups of cells, the NO synthase inhibitor L-Nitro-Arginine Methyl Ester (100 μm), PI3K inhibitor wortmannin (100 nm), or AMPK inhibitor compound C was added 30 min before loading cells with DAF-2-DA. A representative set of experiments is shown for experiments that were repeated independently five times.

Fig. 2.

TNF-α-stimulated expression of VCAM-1 and increased adhesion of monocytes to endothelial cells is diminished by hesperetin pretreatment. A, BAEC were serum-starved overnight and then treated without or with TNF-α (10 ng/ml, 5 h). In some groups, cells were pretreated with hesperetin (10 μm, 1 h) before treatment with TNF-α. Cell lysates were immunoblotted using antibodies against VCAM-1 or β-actin (loading control). Representative immunoblots are shown for experiments that were repeated independently four times. B, Scanning densitometry was used to quantify results of multiple independent experiments represented in panel A normalized to loading control (mean ± sem; n = 4). Bars labeled with different letters are significantly different from each other. *, P < 0.001, by one-way ANOVA and Bonferroni's posttest. C, BAEC were treated without or with TNF-α in the absence or presence of pretreatment with hesperetin as in panel A. Then, labeled U937 monocytes were cocultured with BAEC as described in Subjects and Methods. Adherent monocytes were visualized using an epifluorescent microscope (upper panel). Phase contrast view of cells is shown in lower panel. A representative set of experiments is shown for experiments that were repeated independently five times.