Insulin-stimulated eNOS Phosphorylation Research Articles

A combination of isolated phytochemicals and botanical extracts lowers diastolic blood pressure in a randomized controlled trial of hypertensive subjects.

Isolated phytochemicals have been shown to reduce blood pressure; however, combinations of phytochemicals have rarely been tested in humans. We hypothesized that a combination of extracts from grape seed and skin (330 mg), green tea (100 mg), resveratrol (60 mg) and a blend of quercetin, ginkgo biloba and bilberry (60 mg) would reduce blood pressure (BP) in hypertensive subjects. Eighteen individuals meeting BP requirements (⩾130 mm Hg systolic or ⩾85 mm Hg diastolic) and criteria for metabolic syndrome were enrolled in a double-blinded, placebo-controlled, crossover trial (ClinicalTrials.gov, NCT01106170). The 28-day placebo and supplement arms were separated by a 2-week washout period, and 14 -h daytime ambulatory BP was assessed at baseline and at the end point of each arm. BP was not altered after placebo. After supplement treatment, diastolic pressure was reduced by 4.4 mm Hg (P=0.024, 95% CI, 0.6-8.1), systolic pressure was unchanged and mean arterial pressure trended (P=0.052) toward reduction. Serum angiotensin-converting enzyme activity was similar between placebo and supplement arms, but urinary nitrate and nitrite concentrations were significantly increased (P=0.022) after supplementation. Human aortic endothelial cells treated with metabolites of the polyphenols used in the human supplement trial had a significant increase (P=0.005) in insulin-stimulated eNOS phosphorylation and greater (P<0.001) accumulation of nitrates/nitrites. Our clinical and in vitro data support the theory that this combination of polyphenols reduced diastolic pressure by potentiating eNOS activation and nitric oxide production. Such supplements may have clinical relevance as stand-alone or adjunct therapy to help reduce BP.

184 Increasing Insulin Sensitivity in the Endothelium Leads to Reduced Nitric Oxide Bioavailability

<h3>Introduction</h3> Insulin resistance is known to precede Type 2 diabetes (T2DM). Insulin mediated release of the endothelial cell (EC) derived anti-atherosclerotic molecule, nitric oxide (NO) is blunted in patients suffering from insulin resistance T2DM. We examined the effects of enhancing EC insulin sensitivity <i>in vivo</i>, by generating a novel transgenic mouse, over-expressing Type A human Insulin Receptor (HIRECO) restricted to EC. <h3>Methods</h3> Western blotting and RT-PCR were carried out on tissues and isolated endothelial cells from lungs to measure protein levels and mRNA expression, respectively. NADPH-dependent lucigenin-enhanced chemiluminescence was used to measure superoxide anion levels. Isolated thoracic aortic rings suspended in an organ bath were used to determine vasomotor functions. eNOS activity was examined by citrulline assay with ¹⁴C-labelled L-arginine. HIRECO were compared to wild type littermates. <h3>Results</h3> Over-expressing human insulin receptors in EC had no significant effect on morphological features, metabolic phenotype or blood pressure of HIRECO. HIRECO demonstrated significant EC dysfunction measured by a blunted endothelium-dependent vasorelaxation to acetylcholine and reduced basal NO release. EC-independent response to sodium nitroprusside remained unchanged. EC dysfunction observed in the organ bath was normalised by a NADPH oxidase-specific inhibitor peptide, gp91ds-tat as well as the superoxide dismutase mimetic, MnTmPyp. HIRECO demonstrated significant increase in superoxide anion release compared to WT littermates. This data was supported by a concomitant increase in NADPH oxidase isoform, NOX2 protein expression. Basal eNOS and Akt phosphorylation levels in isolated EC of HIRECO mice were enhanced compared to WT mice. Interestingly, insulin-stimulated eNOS phosphorylation and activation was decreased, whereas Akt phosphorylation remained unchanged. eNOS tyrosine phosphorylation mediated by proline-rich tyrosine kinase (PYK2) was significantly enhanced in EC from HIRECO mice. In order to investigate, if the perturbations of insulin signalling in the EC have a pathological outcome, HIRECO mice were crossed with ApoE-knockout mice. These mice demonstrated a significant enhancement of plaque formation in aorta, as well as the aortic root. <h3>Conclusions/implications</h3> These data show that increasing EC insulin sensitivity leads to reduced bioavailability of NO. These data demonstrate for the first time that increased insulin signalling in EC increases the generation of superoxide anion via activation of NOX₂ NADPH oxidase and reduced NO production in response to insulin due at least in part to increased EC PYK2 activity leading to a pro-atherosclerotic state.

173 Increasing Insulin Sensitivity in the Endothelium Leads to Reduced Nitric Oxide Bioavailability

IntroductionInsulin resistance is known to precede Type 2 diabetes (T2DM). Insulin mediated release of the endothelial cell (EC) derived anti-atherosclerotic molecule, nitric oxide (NO) is blunted in patients suffering from...

Abstract 340: Psychological Stress Impairs Both Endothelial Function and Insulin Sensitivity via Activation of Inflammation and ER Stress Pathways Mediated by Corticotropin-Releasing Hormone

Background: Psychological stress may contribute to atherogenesis, although the underlying mechanism is poorly understood yet. We studied the role of corticotropin-releasing hormone (CRH) in the pathogenesis of stress-induced endothelial dysfunction and insulin resistance with specific focus on inflammation and ER stress pathways. Methods and Results: Immobilization stress (IS) was applied (2 hr/d) for 14 d to male 8-wk OLETF rats, whereas control groups comprised of rats without IS (n≥7 for each). 14d-IS decreased acetylcholine (Ach)-induced NO production, measured by direct NO electrochemical microsensor, from the aorta along with decreased expression of eNOS mRNA compared with control group. 14d-IS did not significantly change plasma glucose, but increased plasma insulin (342.5±6.3 vs. 496.0±51.6 pg/ml, p&lt;0.05) and HOMA-IR (3.1±0.2 vs.4.7±0.5 mmol/LxμIU/mL, p&lt;0.05) compared with control group. IS increased phosphorylation of both IRS1 serine and ERK, and increased expression of ER stress marker BiP. IS also increased translocation of NF-kB subunit p65 into nucleal fraction, and increased mRNA expression of both TNF-α and MCP-1 in the liver. Real-time RT-qPCR study showed that 1d IS increased expression of CRH mRNA and urocortin 2 mRNA in the hypothalamus by 2.5 folds and expression of CRH receptor type 2 mRNA in the aorta by 7.8 folds compared with control group. In cultured monocyte U937 cells, CRH induced translocation of NF-kB subunit p65 into nuclear fraction, and increased expression of BiP, phosphorylated inositol requiring enzyme 1, and MCP-1 mRNA. In cultured HUVECs, CRH pretreatment inhibited insulin-stimulated eNOS phosphorylation at Ser 1177 . Conclusions: Psychological stress impairs both endothelial function and insulin sensitivity probably via activation of NF-kB-mediated inflammation and ER stress pathways by CRH.

Intrauterine growth restriction decreases pulmonary alveolar and vessel growth and causes pulmonary artery endothelial cell dysfunction in vitro in fetal sheep.

Intrauterine growth restriction (IUGR) increases the risk for bronchopulmonary dysplasia (BPD). Abnormal lung structure has been noted in animal models of IUGR, but whether IUGR adversely impacts fetal pulmonary vascular development and pulmonary artery endothelial cell (PAEC) function is unknown. We hypothesized that IUGR would decrease fetal pulmonary alveolarization, vascular growth, and in vitro PAEC function. Studies were performed in an established model of severe placental insufficiency and IUGR induced by exposing pregnant sheep to elevated temperatures. Alveolarization, quantified by radial alveolar counts, was decreased 20% (P < 0.005) in IUGR fetuses. Pulmonary vessel density was decreased 44% (P < 0.01) in IUGR fetuses. In vitro, insulin increased control PAEC migration, tube formation, and nitric oxide (NO) production. This response was absent in IUGR PAECs. VEGFA stimulated tube formation, and NO production also was absent. In control PAECs, insulin increased cell growth by 68% (P < 0.0001). Cell growth was reduced in IUGR PAECs by 29% at baseline (P < 0.01), and the response to insulin was attenuated (P < 0.005). Despite increased basal and insulin-stimulated Akt phosphorylation in IUGR PAECs, endothelial NO synthase (eNOS) protein expression as well as basal and insulin-stimulated eNOS phosphorylation were decreased in IUGR PAECs. Both VEGFA and VEGFR2 also were decreased in IUGR PAECs. We conclude that fetuses with IUGR are characterized by decreased alveolar and vascular growth and PAEC dysfunction in vitro. This may contribute to the increased risk for adverse respiratory outcomes and BPD in infants with IUGR.

Insulin rapidly stimulates l-arginine transport in human aortic endothelial cells via Akt

Insulin stimulates endothelial NO synthesis, at least in part mediated by phosphorylation and activation of endothelial NO synthase at Ser1177 and Ser615 by Akt. We have previously demonstrated that insulin-stimulated NO synthesis is inhibited under high culture glucose conditions, without altering Ca2+-stimulated NO synthesis or insulin-stimulated phosphorylation of eNOS. This indicates that stimulation of endothelial NO synthase phosphorylation may be required, yet not sufficient, for insulin-stimulated nitric oxide synthesis. In the current study we investigated the role of supply of the eNOS substrate, l-arginine as a candidate parallel mechanism underlying insulin-stimulated NO synthesis in cultured human aortic endothelial cells. Insulin rapidly stimulated l-arginine transport, an effect abrogated by incubation with inhibitors of phosphatidylinositol-3′-kinase or infection with adenoviruses expressing a dominant negative mutant Akt. Furthermore, supplementation of endothelial cells with extracellular l-arginine enhanced insulin-stimulated NO synthesis, an effect reversed by co-incubation with the l-arginine transport inhibitor, l-lysine. Basal l-arginine transport was significantly increased under high glucose culture conditions, yet insulin-stimulated l-arginine transport remained unaltered. The increase in l-arginine transport elicited by high glucose was independent of the expression of the cationic amino acid transporters, hCAT1 and hCAT2 and not associated with any changes in the activity of ERK1/2, Akt or protein kinase C (PKC). We propose that rapid stimulation of L-arginine transport contributes to insulin-stimulated NO synthesis in human endothelial cells, yet attenuation of this is unlikely to underlie the inhibition of insulin-stimulated NO synthesis under high glucose conditions.

Contribution of Insulin and Akt1 Signaling to Endothelial Nitric Oxide Synthase in the Regulation of Endothelial Function and Blood Pressure

Impaired insulin signaling via phosphatidylinositol 3-kinase/Akt to endothelial nitric oxide synthase (eNOS) in the vasculature has been postulated to lead to arterial dysfunction and hypertension in obesity and other insulin resistant states. To investigate this, we compared insulin signaling in the vasculature, endothelial function, and systemic blood pressure in mice fed a high-fat (HF) diet to mice with genetic ablation of insulin receptors in all vascular tissues (TTr-IR(-/-)) or mice with genetic ablation of Akt1 (Akt1-/-). HF mice developed obesity, impaired glucose tolerance, and elevated free fatty acids that was associated with endothelial dysfunction and hypertension. Basal and insulin-mediated phosphorylation of extracellular signal-regulated kinase 1/2 and Akt in the vasculature was preserved, but basal and insulin-stimulated eNOS phosphorylation was abolished in vessels from HF versus lean mice. In contrast, basal vascular eNOS phosphorylation, endothelial function, and blood pressure were normal despite absent insulin-mediated eNOS phosphorylation in TTr-IR(-/-) mice and absent insulin-mediated eNOS phosphorylation via Akt1 in Akt1-/- mice. In cultured endothelial cells, 6 hours of incubation with palmitate attenuated basal and insulin-stimulated eNOS phosphorylation and NO production despite normal activation of extracellular signal-regulated kinase 1/2 and Akt. Moreover, incubation of isolated arteries with palmitate impaired endothelium-dependent but not vascular smooth muscle function. Collectively, these results indicate that lower arterial eNOS phosphorylation, hypertension, and vascular dysfunction following HF feeding do not result from defective upstream signaling via Akt, but from free fatty acid-mediated impairment of eNOS phosphorylation.

Epigallocatechin Gallate, a Green Tea Polyphenol, Mediates NO-dependent Vasodilation Using Signaling Pathways in Vascular Endothelium Requiring Reactive Oxygen Species and Fyn

Green tea consumption is associated with reduced cardiovascular mortality in some epidemiological studies. Epigallocatechin gallate (EGCG), a bioactive polyphenol in green tea, mimics metabolic actions of insulin to inhibit gluconeogenesis in hepatocytes. Because signaling pathways regulating metabolic and vasodilator actions of insulin are shared in common, we hypothesized that EGCG may also have vasodilator actions to stimulate production of nitric oxide (NO) from endothelial cells. Acute intra-arterial administration of EGCG to mesenteric vascular beds isolated ex vivo from WKY rats caused dose-dependent vasorelaxation. This was inhibitable by L-NAME (NO synthase inhibitor), wortmannin (phosphatidylinositol 3-kinase inhibitor), or PP2 (Src family kinase inhibitor). Treatment of bovine aortic endothelial cells (BAEC) with EGCG (50 microm) acutely stimulated production of NO (assessed with NO-specific fluorescent dye DAF-2) that was inhibitable by l-NAME, wortmannin, or PP2. Stimulation of BAEC with EGCG also resulted in dose- and time-dependent phosphorylation of eNOS that was inhibitable by wortmannin or PP2 (but not by MEK inhibitor PD98059). Specific knockdown of Fyn (but not Src) with small interfering RNA inhibited both EGCG-stimulated phosphorylation of Akt and eNOS as well as production of NO in BAEC. Treatment of BAEC with EGCG generated intracellular H(2)O(2) (assessed with H(2)O(2)-specific fluorescent dye CM-H(2)DCF-DA), whereas treatment with N-acetylcysteine inhibited EGCG-stimulated phosphorylation of Fyn, Akt, and eNOS. We conclude that EGCG has endothelial-dependent vasodilator actions mediated by intracellular signaling pathways requiring reactive oxygen species and Fyn that lead to activation of phosphatidylinositol 3-kinase, Akt, and eNOS. This mechanism may explain, in part, beneficial vascular and metabolic health effects of green tea consumption.