Long term treatment with quercetin in contrast to the sulfate and glucuronide conjugates affects HIF1α stability and Nrf2 signaling in endothelial cells and leads to changes in glucose metabolism

Highlights

• Circulating polyphenol metabolites can affect endothelial physiology and consequently cardiovascular health.

• Polyphenol quercetin affected glucose uptake and gene expression in a manner distinct from its conjugates.

• Quercetin increased HIF1α stability indicating a potential to regulate endothelial glucose metabolism.

Abstract

Endothelial functionality profoundly contributes to cardiovascular health. The effects of flavonoids shown to improve endothelial performance include regulating blood pressure by modulating endothelial nitric oxide synthase and NADPH oxidases, but their impact on glucose uptake and metabolism has not been explored. We treated human umbilical vein endothelial cells (HUVEC) with the flavonoid quercetin and its circulating metabolites acutely and chronically, then assessed glucose uptake, glucose metabolism, gene transcription and protein expression. Acute treatment had no effect on glucose uptake, ruling out any direct interaction with sugar transporters. Long term treatment with quercetin, but not quercetin 3-O-glucuronide or 3′-O-sulfate, significantly increased glucose uptake. Heme oxygenase-1 (HO-1) was induced by quercetin but not its conjugates, but was not implicated in the glucose uptake stimulation since hemin, a classical inducer of HO-1, did not affect glucose metabolism. Quercetin increased stability of the transcription factor hypoxia induced factor 1α (HIF1α), a powerful stimulant of glucose metabolism, which was also paralleled by treatment with a prolyl-4-hydroxylase inhibitor dimethyloxalylglycine (DMOG). 6-Phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), which regulates the rate of glycolysis, was upregulated by both quercetin and DMOG. Pyruvate dehydrogenase kinase (PDK) isoforms regulate pyruvate dehydrogenase; PDK2 and PDK4 were down-regulated by both effectors, but only DMOG also upregulated PDK1 and PDK3. Quercetin, but not DMOG, increased glucose-6-phosphate dehydrogenase. Chronic quercetin treatment also stimulated glucose transport across the HUVEC monolyer in a 3D culture model. Gene expression of several flavonoid transporters was repressed by quercetin, but this was either abolished (Organic anion transporter polypeptide 4C1) or reversed (Multidrug resistance gene 1) by both conjugates. We conclude that quercetin and its circulating metabolites differentially modulate glucose uptake/metabolism in endothelial cells, through effects on HIF1α and transcriptional regulation of energy metabolism.

Introduction

Endothelial cells in blood vessels form a boundary between the blood and underlying tissues. The endothelial lining provides a semi-permeable barrier to regulate exchange of nutrients and signaling factors, and participates in the control of blood pressure, coagulation and inflammatory pathways [[1], [2], [3], [4]]. The impairment of endothelial function, generally referred to as endothelial dysfunction, precedes development of cardiovascular diseases, hypertension and diabetes [5,6]. There has been substantial effort to identify pharmacological or dietary factors that would help to prevent disease development by preserving endothelial functionality [7,8]. Various epidemiological studies and human trials (reviewed in [[9], [10], [11], [12]]) show association between consumption of polyphenol-rich foods and protective effects on endothelial function or improved cardiometabolic biomarkers. In vitro and animal studies suggest a range of pathways affected by the polyphenolic compounds, including nitric oxide (NO) production [13,14], oxidative stress responses [15] and inflammation [16]. The NO generated by endothelial nitric oxide synthase (eNOS) induces vasorelaxation via signaling to smooth muscle cells and flavonoids such as quercetin, resveratrol, epicatechin or epigallocatechin gallate have been shown to increase eNOS activity via phoshoinositol 3-kinase/protein kinase B (Akt) [16] or forkhead box family (FOXO)/sirtuin 1 (SIRT1) [17] signaling, via reactive oxygen species (ROS) and tyrosine protein kinase Fyn [18], or by inhibiting enzymes which adversely affect the eNOS function, such as NADPH oxidases [19,20] and arginase [21]. Flavonoids have also been shown to counteract hypertension in human studies [22]. Molecules such as quercetin, resveratrol or epigallocatechin gallate can alleviate detrimental effects of hyperglycemia or hyperlipidemia and reduce inflammation in various settings [[23], [24], [25]]. In addition, the endothelial function could be affected indirectly by lowered blood glucose levels, resulting from activities of polyphenols that impact postprandial glycemic reponse, such as the ability to inhibit sugar uptake and transport in the gut [[26], [27], [28], [29]], regulate glucose homestasis in liver [30] or stimulate glucose uptake and metabolism in adipocytes and muscle cells [31,32].

Since the endothelium is one of the primary targets of exposure to polyphenols and their conjugates, we examined whether polyphenols affect endothelial metabolism directly. We used human umbilical vein endothelial cells (HUVEC) to study the acute and long-term effects of quercetin and its most abundant conjugates, quercetin-3′-O-sulfate (Q3′S) and quercetin-3-O-glucuronide (Q3G) [33]. Since glucose metabolism is a prominent feature of endothelial physiology and polyphenols are known to regulate glucose uptake in other cells, we have evaluated the impact of physiologically relevant quercetin conjugates as well as the parental aglycone on glucose uptake, transport and metabolism in HUVEC. At the same time we examined their impact on the expression of metabolism-related genes and genes implicated in polyphenol conjugate transport and assessed their role in the observed effects.