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.     

High glucose inhibits insulin-stimulated nitric oxide production without reducing endothelial nitric-oxide synthase Ser1177 phosphorylation in human aortic endothelial cells

Recent studies have indicated that insulin activates endothelial nitric-oxide synthase (eNOS) by protein kinase B (PKB)-mediated phosphorylation at Ser1177 in endothelial cells. Because hyperglycemia contributes to endothelial dysfunction and decreased NO availability in types 1 and 2 diabetes mellitus, we have studied the effects of high glucose (25 mM, 48 h) on insulin signaling pathways that regulate NO production in human aortic endothelial cells. High glucose inhibited insulin-stimulated NO synthesis but was without effect on NO synthesis stimulated by increasing intracellular Ca2+ concentration. This was accompanied by reduced expression of IRS-2 and attenuated insulin-stimulated recruitment of PI3K to IRS-1 and IRS-2, yet insulin-stimulated PKB activity and phosphorylation of eNOS at Ser1177 were unaffected. Inhibition of insulin-stimulated NO synthesis by high glucose was unaffected by an inhibitor of PKC. Furthermore, high glucose down-regulated the expression of CAP and Cbl, and insulin-stimulated Cbl phosphorylation, components of an insulin signaling cascade previously characterized in adipocytes. These data suggest that high glucose specifically inhibits insulin-stimulated NO synthesis and down-regulates some aspects of insulin signaling, including the CAP-Cbl signaling pathway, yet this is not a result of reduced PKB-mediated eNOS phosphorylation at Ser1177. Therefore, we propose that phosphorylation of eNOS at Ser1177 is not sufficient to stimulate NO production in cells cultured at 25 mM glucose.     

Insulin-stimulated activation of eNOS is independent of Ca2+ but requires phosphorylation by Akt at Ser(1179)

Vasodilator actions of insulin are mediated by activation of endothelial nitric-oxide synthase (eNOS) and subsequent production of NO. Phosphatidylinositol 3-kinase and Akt play important roles in insulin-signaling pathways leading to production of NO in vascular endothelium. Here we dissected mechanisms whereby insulin activates eNOS by using the fluorescent dye DAF-2 to directly measure NO production in single cells. Insulin caused a rapid increase in intracellular NO in NIH-3T3(IR) cells transiently transfected with eNOS. The stimulation of NO production by lysophosphatidic acid (LPA) was abrogated by pretreatment of cells with the calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. Remarkably, in the same cells, insulin-stimulated production of NO was unaffected. However, cells expressing the eNOS-S1179A mutant (disrupted Akt phosphorylation site) did not produce detectable NO in response to insulin, whereas the response to LPA was similar to that observed in cells expressing wild-type eNOS. Moreover, production of NO in response to insulin was blocked by coexpression of an inhibitory mutant of Akt, whereas the response to LPA was unaffected. Phosphorylation of eNOS at Ser(1179) was observed only in response to treatment with insulin, but not with LPA. Interestingly, platelet-derived growth factor treatment of cells activated Akt but not eNOS. Results from human vascular endothelial cells were qualitatively similar to those obtained in transfected NIH-3T3(IR) cells, although the magnitude of the responses was smaller. We conclude that insulin regulates eNOS activity using a Ca(2+)-independent mechanism requiring phosphorylation of eNOS by Akt. Importantly, phosphorylation-dependent mechanisms that enhance eNOS activity can operate independently from Ca(2+)-dependent mechanisms.     

Interleukin-6 impairs the insulin signaling pathway, promoting production of nitric oxide in human umbilical vein endothelial cells

Interleukin 6 (IL-6) is an independent predictor of type 2 diabetes and cardiovascular disease and is correlated with insulin resistance. Insulin stimulates nitric oxide (NO) production through the IRS-1/PI3-kinase/Akt/eNOS pathway (where IRS-1 is insulin receptor substrate 1, PI3-kinase is phosphatidylinositol 3-kinase, and eNOS is endothelial NO synthase). We asked if IL-6 affects insulin vasodilator action both in human umbilical vein endothelial cells (HUVEC) and in the aortas of C57BL/6J mice and whether this inhibitory effect was caused by increased Ser phosphorylation of IRS-1. We observed that IL-6 increased IRS-1 phosphorylation at Ser(312) and Ser(616); these effects were paralleled by increased Jun N-terminal protein kinase (JNK) and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation and reversed by JNK and ERK1/2 inhibition. In addition, IL-6 treatment resulted in impaired IRS-1 phosphorylation at Tyr(612), a site essential for engaging PI3-kinase. Furthermore, IL-6 treatment reduced insulin-stimulated phosphorylation of eNOS at the stimulatory Ser(1177) site and impaired insulin-stimulated eNOS dephosphorylation at the inhibitory Thr(495) site. Insulin-stimulated eNOS activation and NO production were also inhibited by IL-6; these effects were reversed by inhibition of JNK and ERK1/2. Treatment of C57BL/6J mice with IL-6 resulted in impaired insulin-dependent activation of the Akt/eNOS pathway in the aorta as a result of JNK and ERK1/2 activation. Our data suggest that IL-6 impairs the vasodilator effects of insulin that are mediated by the IRS-1/PI3-kinase/Akt/eNOS pathway through activation of JNK and ERK1/2.     

Adiponectin stimulates production of nitric oxide in vascular endothelial cells

Adiponectin is secreted by adipose cells and mimics many metabolic actions of insulin. However, mechanisms by which adiponectin acts are poorly understood. The vascular action of insulin to stimulate endothelial production of nitric oxide (NO), leading to vasodilation and increased blood flow is an important component of insulin-stimulated whole body glucose utilization. Therefore, we hypothesized that adiponectin may also stimulate production of NO in endothelium. Bovine aortic endothelial cells in primary culture loaded with the NO-specific fluorescent dye 4,5-diaminofluorescein diacetate (DAF-2 DA) were treated with lysophosphatidic acid (LPA) (a calcium-releasing agonist) or adiponectin (10 microg/ml bacterially produced full-length adiponectin). LPA treatment increased production of NO by approximately 4-fold. Interestingly, adiponectin treatment significantly increased production of NO by approximately 3-fold. Preincubation of cells with wortmannin (phosphatidylinositol 3-kinase inhibitor) blocked only adiponectin- but not LPA-mediated production of NO. Using phospho-specific antibodies, we observed that either adiponectin or insulin treatment (but not LPA treatment) caused phosphorylation of both Akt at Ser473 and endothelial nitric-oxide synthase (eNOS) at Ser1179 that was inhibitable by wortmannin. We next transfected bovine aortic endothelial cells with dominant-inhibitory mutants of Akt (Akt-AAA) or AMP-activated protein kinase (AMPK) (AMPKK45R). Neither mutant affected production of NO in response to LPA treatment. Importantly, only AMPKK45R, but not Akt-AAA, caused a significant partial inhibition of NO production in response to adiponectin. Moreover, AMPK-K45R inhibited phosphorylation of eNOS at Ser1179 in response to adiponectin but not in response to insulin. We conclude that adiponectin has novel vascular actions to directly stimulate production of NO in endothelial cells using phosphatidylinositol 3-kinase-dependent pathways involving phosphorylation of eNOS at Ser1179 by AMPK. Thus, the effects of adiponectin to augment metabolic actions of insulin in vivo may be due, in part, to vasodilator actions of adiponectin.     

Modulation of the L-arginine/nitric oxide signalling pathway in vascular endothelial cells

Nitric oxide (NO) is synthesized from L-arginine, and in endothelial cells influx of L-arginine is mediated predominantly via Na+-independent cationic amino acid transporters. Constitutive, Ca2+-calmodulin-sensitive eNOS (endothelial nitric oxide synthase) metabolizes L-arginine to NO and L-citrulline. eNOS is present in membrane caveolae and the cytosol and requires tetrahydrobiopterin, NADPH, FAD and FMN as additional cofactors for its activity. Supply of L-arginine for NO synthesis appears to be derived from a membrane-associated compartment distinct from the bulk intracellular amino acid pool, e.g. near invaginations of the plasma membrane referred to as 'lipid rafts' or caveolae. Co-localization of eNOS and the cationic amino acid transport system y+ in caveolae in part explains the 'arginine paradox', related to the phenomenon that in certain disease states eNOS requires an extracellular supply of L-arginine despite having sufficient intracellular L-arginine concentrations. Vasoactive agonists normally elevate [Ca2+]i (intracellular calcium concentration) in endothelial cells, thus stimulating NO production, whereas fluid shear stress, 17beta-oestradiol and insulin cause phosphorylation of the serine/threonine protein kinase Akt/protein kinase B in a phosphoinositide 3-kinase-dependent manner and activation of eNOS at basal [Ca2+]i levels. Adenosine causes an acute activation of p42/p44 mitogen-activated protein kinase and NO release, with membrane hyperpolarization leading to increased system y+ activity in fetal endothelial cells. In addition to acute stimulatory actions of D-glucose and insulin on L-arginine transport and NO synthesis, gestational diabetes, intrauterine growth retardation and pre-eclampsia induce phenotypic changes in the fetal vasculature, resulting in alterations in the L-arginine/NO signalling pathway and regulation of [Ca2+]i. These alterations may have significant implications for long-term programming of the fetal cardiovascular system.     

D-glucose stimulation of L-arginine transport and nitric oxide synthesis results from activation of mitogen-activated protein kinases p42/44 and Smad2 requiring functional type II TGF-beta receptors in human umbilical vein endothelium

Elevated extracellular D-glucose increases transforming growth factor beta1 (TGF-beta1) release from human umbilical vein endothelium (HUVEC). TGF-beta1, via TGF-beta receptors I (TbetaRI) and TbetaRII, activates Smad2 and mitogen-activated protein kinases p44 and p42 (p42/44(mapk)). We studied whether D-glucose-stimulation of L-arginine transport and nitric oxide synthesis involves TGF-beta1 in primary cultures of HUVEC. TGF-beta1 release was higher ( approximately 1.6-fold) in 25 mM (high) compared with 5 mM (normal) D-glucose. TGF-beta1 increases L-arginine transport (half maximal effect approximately 1.6 ng/ml) in normal D-glucose, but did not alter high D-glucose-increased L-arginine transport. TGF-beta1 and high D-glucose increased hCAT-1 mRNA expression ( approximately 8-fold) and maximal transport velocity (V(max)), L-[(3)H]citrulline formation from L-[(3)H]arginine (index of NO synthesis) and endothelial NO synthase (eNOS) protein abundance, but did not alter eNOS phosphorylation. TGF-beta1 and high D-glucose increased p42/44(mapk) and Smad2 phosphorylation, an effect blocked by PD-98059 (MEK1/2 inhibitor). However, TGF-beta1 and high D-glucose were ineffective in cells expressing a truncated, negative dominant TbetaRII. High D-glucose increases L-arginine transport and eNOS expression following TbetaRII activation by TGF-beta1 involving p42/44(mapk) and Smad2 in HUVEC. Thus, TGF-beta1 could play a crucial role under conditions of hyperglycemia, such as gestational diabetes mellitus, which is associated with fetal endothelial dysfunction.     

Moderate dose insulin promotes function of endothelial progenitor cells

EPCs (endothelial progenitor cells) regenerate the vascular endothelial cells and keep the integrity of the vascular endothelium and thus may retard the onset of atherosclerosis. Steady state levels of EPCs in the circulation were found to be correlated with cardiovascular event risks. Given the close relationship between insulin and the cardiovascular system, we tested the long-term effects of moderate-dose insulin treatment on bone marrow-derived EPCs. Rat bone marrow EPCs were exposed to various levels of insulin under normal (5 mmol/l) or high (40 mmol/l) glucose conditions for 7 days. Insulin at levels near the physiological range (0.1, 1 nmol/l) up-regulated EPCs proliferation, stimulated NO (nitric oxide) production and reduced EPC senescence and ROS (reactive oxygen species) generation under both normal- and high-glucose conditions. Glucose exerted deleterious effects on EPCs contrary to insulin. Western blot analysis suggested concomitant decrease of Akt phosphorylation and eNOS (endothelial nitric oxide synthase) expression by high-glucose treatment and increase with insulin administration. Thus, insulin promoted several activities of EPCs, which suggested a potential endothelial protective role of insulin. Akt/eNOS pathway may be involved in the modulation of EPCs function by glucose and insulin.     

AMP-activated protein kinase mediates VEGF-stimulated endothelial NO production

Vascular endothelial growth factor (VEGF) is an important regulator of endothelial cell function. VEGF stimulates NO production, proposed to be a result of phosphorylation and activation of endothelial NO synthase (eNOS) at Ser1177. Phosphorylation of eNOS at this site also occurs after activation of AMP-activated protein kinase (AMPK) in cultured endothelial cells. We therefore determined whether AMPK mediates VEGF-stimulated NO synthesis in endothelial cells. VEGF caused a rapid, dose-dependent stimulation of AMPK activity, with a concomitant increase in phosphorylation of eNOS at Ser1177. Infection of endothelial cells with an adenovirus expressing a dominant negative mutant AMPK partially inhibited both VEGF-stimulated eNOS Ser1177 phosphorylation and NO production. VEGF-stimulated AMPK activity was completely inhibited by the Ca(2+)/calmodulin-dependent protein kinase kinase inhibitor, STO-609. Stimulation of AMPK via Ca(2+)/calmodulin-dependent protein kinase kinase represents a novel signalling mechanism utilised by VEGF in endothelial cells that contributes to eNOS phosphorylation and NO production.     

Mechanisms of shear stress-induced endothelial nitric-oxide synthase phosphorylation and expression in ovine fetoplacental artery endothelial cells

Placental blood flow, nitric-oxide (NO) levels, and endothelial NO synthase (eNOS) expression increase during human and ovine pregnancy. Shear stress stimulates NO production and eNOS expression in ovine fetoplacental artery endothelial (OFPAE) cells. Because eNOS is the rate-limiting enzyme essential for NO synthesis, its activity and expression are both closely regulated. We investigated signaling mechanisms underlying pulsatile shear stress-induced increases in eNOS phosphorylation and protein expression by OFPAE cells. The OFPAE cells were cultured at 3 dynes/cm2 shear stress, then exposed to 15 dynes/cm2 shear stress. Western blot analysis for phosphorylated ERK1/2, Akt, p38 mitogen activated protein kinase (MAPK), and eNOS showed that shear stress rapidly increased phosphorylation of ERK1/2 and Akt but not of p38 MAPK. Phosphorylation of eNOS Ser1177 under shear stress was elevated by 20 min, a response that was blocked by the phosphatidyl inositol-3-kinase (PI-3K)-inhibitors wortmannin and LY294002 but not by the mitogen activated protein kinase kinase (MEK)-inhibitor UO126. Basic fibroblast growth factor (bFGF) enhanced eNOS protein levels in static culture via a MEK-mediated mechanism, but it could not further augment the elevated eNOS protein levels otherwise induced by the 15 dynes/cm2 shear stress. Blockade of either signaling pathway changed the shear stress-induced increase in eNOS protein levels. In conclusion, shear stress induced rapid eNOS phosphorylation on Ser1177 in OFPAE cells through a PI-3K-dependent pathway. The bFGF-induced rise in eNOS protein levels in static culture was much less than those observed under flow and was blocked by inhibition of MEK. Prolonged shear stress-stimulated increases in eNOS protein were not affected by inhibition of MEK- or PI-3K-mediated pathways.     

Positive and negative regulation of insulin action by genistein in the endothelium

Genistein is an isoflavone phytoestrogen with biological activities in management of metabolic disorders. This study aims to evaluate the regulation of insulin action by genistein in the endothelium. Genistein inhibited insulin-stimulated tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and attenuated downstream Akt and endothelial nitric oxide synthase (eNOS) phosphorylation, leading to a decreased nitric oxide (NO) production in endothelial cells. These results demonstrated its negative regulation of insulin action in the endothelium. Palmitate (PA) stimulation evoked inflammation and induced insulin resistance in endothelial cells. Genistein inhibited IKKβ and nuclear factor-кB (NF-кB) activation with down-regulation of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) production and expression. Genistein inhibited inflammation-stimulated IRS-1 serine phosphorylation and restored insulin-mediated tyrosine phosphorylation. Genistein restored insulin-mediated Akt and eNOS phosphorylation, and then led to an increased NO production from endothelial cells, well demonstrating its positive regulation of insulin action under insulin-resistant conditions. Meanwhile, genistein effectively inhibited inflammation-enhanced mitogenic actions of insulin by down-regulation of endothelin-1 and vascular cell adhesion protein-1 overexpression. PA stimulation impaired insulin-mediated vessel dilation in rat aorta, while genistein effectively restored the lost vasodilation in a concentration-dependent manner (0.1, 1 and 10 μM). These results suggested that genistein inhibited inflammation and ameliorated endothelial dysfunction implicated in insulin resistance. Better understanding of genistein action in regulation of insulin sensitivity in the endothelium could be beneficial for its possible applications in controlling endothelial dysfunction associated with diabetes and insulin resistance.     

Nutrient-dependent and insulin-stimulated phosphorylation of insulin receptor substrate-1 on serine 302 correlates with increased insulin signaling

Ser/Thr phosphorylation of insulin receptor substrate IRS-1 regulates insulin signaling, but the relevant phosphorylated residues and their potential functions during insulin-stimulated signal transduction are difficult to resolve. We used a sequence-specific polyclonal antibody directed against phosphorylated Ser(302) to study IRS-1-mediated signaling during insulin and insulin-like growth factor IGF-I stimulation. Insulin or IGF-I stimulated phosphorylation of Ser(302) in various cell backgrounds and in murine muscle. Wortmannin or rapamycin inhibited Ser(302) phosphorylation, and amino acids or glucose stimulated Ser(302) phosphorylation, suggesting a role for the mTOR cascade. The Ser(302) kinase associates with IRS-1 during immunoprecipitation, but its identity is unknown. The NH(2)-terminal c-Jun kinase did not phosphorylate Ser(302). Replacing Ser(302) with alanine significantly reduced insulin-stimulated tyrosine phosphorylation of IRS-1 and p85 binding and reduced insulin-stimulated phosphorylation of p70(S6K), ribosomal S6 protein, and 4E-BP1; however, this mutation had no effect on insulin-stimulated Akt or glycogen synthase kinase 3beta phosphorylation. Replacing Ser(302) with alanine reduced insulin/IGF-I-stimulated DNA synthesis. We conclude that Ser(302) phosphorylation integrates nutrient availability with insulin/IGF-I signaling to promote mitogenesis and cell growth.     

Muscle glycogen inharmoniously regulates glycogen synthase activity, glucose uptake, and proximal insulin signaling

Insulin-stimulated glucose uptake and incorporation of glucose into skeletal muscle glycogen contribute to physiological regulation of blood glucose concentration. In the present study, glucose handling and insulin signaling in isolated rat muscles with low glycogen (LG, 24-h fasting) and high glycogen (HG, refed for 24 h) content were compared with muscles with normal glycogen (NG, rats kept on their normal diet). In LG, basal and insulin-stimulated glycogen synthesis and glycogen synthase activation were higher and glycogen synthase phosphorylation (Ser(645), Ser(649), Ser(653), Ser(657)) lower than in NG. GLUT4 expression, insulin-stimulated glucose uptake, and PKB phosphorylation were higher in LG than in NG, whereas insulin receptor tyrosyl phosphorylation, insulin receptor substrate-1-associated phosphatidylinositol 3-kinase activity, and GSK-3 phosphorylation were unchanged. Muscles with HG showed lower insulin-stimulated glycogen synthesis and glycogen synthase activation than NG despite similar dephosphorylation. Insulin signaling, glucose uptake, and GLUT4 expression were similar in HG and NG. This discordant regulation of glucose uptake and glycogen synthesis in HG resulted in higher insulin-stimulated glucose 6-phosphate concentration, higher glycolytic flux, and intracellular accumulation of nonphosphorylated 2-deoxyglucose. In conclusion, elevated glycogen synthase activation, glucose uptake, and GLUT4 expression enhance glycogen resynthesis in muscles with low glycogen. High glycogen concentration per se does not impair proximal insulin signaling or glucose uptake. "Insulin resistance" is observed at the level of glycogen synthase, and the reduced glycogen synthesis leads to increased levels of glucose 6-phosphate, glycolytic flux, and accumulation of nonphosphorylated 2-deoxyglucose.     

Protein kinase Cdelta regulates endothelial nitric oxide synthase expression via Akt activation and nitric oxide generation

In this study, we explore the roles of the delta isoform of PKC (PKCdelta) in the regulation of endothelial nitric oxide synthase (eNOS) activity in pulmonary arterial endothelial cells isolated from fetal lambs (FPAECs). Pharmacological inhibition of PKCdelta with either rottlerin or with the peptide, deltaV1-1, acutely attenuated NO production, and this was associated with a decrease in phosphorylation of eNOS at Ser1177 (S1177). The chronic effects of PKCdelta inhibition using either rottlerin or the overexpression of a dominant negative PKCdelta mutant included the downregulation of eNOS gene expression that was manifested by a decrease in both eNOS promoter activity and protein expression after 24 h of treatment. We also found that PKCdelta inhibition blunted Akt activation as observed by a reduction in phosphorylated Akt at position Ser473. Thus, we conclude that PKCdelta is actively involved in the activation of Akt. To determine the effect of Akt on eNOS signaling, we overexpressed a dominant negative mutant of Akt and determined its effect of NO generation, eNOS expression, and phosphorylation of eNOS at S1177. Our results demonstrated that Akt inhibition was associated with decreased NO production that correlated with reduced phosphorylation of eNOS at S1177, and decreased eNOS promoter activity. We next evaluated the effect of endogenously produced NO on eNOS expression by incubating FPAECs with the eNOS inhibitor 2-ethyl-2-thiopseudourea (ETU). ETU significantly inhibited NO production, eNOS promoter activity, and eNOS protein levels. Together, our data indicate involvement of PKCdelta-mediated Akt activation and NO generation in maintaining eNOS expression.     

Involvement of androgen receptor in nitric oxide production induced by icariin in human umbilical vein endothelial cells

Icariin, a flavonoid isolated from Epimedii herba, stimulated phosphorylation of endothelial nitric oxide synthase (eNOS) at Ser1177, Akt (Ser473) and ERK1/2 (Thr202/Tyr204). The icariin-induced eNOS phosphorylation was abolished by an androgen receptor (AR) antagonist, nilutamide in human umbilical vein endothelial cells (HUVECs). Furthermore, it was also reduced in the cells transfected with small interfering RNA in which the expression of AR was broken down. The icariin-induced eNOS phosphorylation was inhibited by wortmannin, a phosphatidylinositol 3-kinase (PI3K) inhibitor and partially attenuated by PD98059, an upstream inhibitor for ERK1/2. These data suggest that icariin stimulates release of NO by AR-dependent activation of eNOS in HUVECs. PI3K/Akt and MAPK-ERK kinase (MEK)/ERK1/2 pathways were involved in the phosphorylation of eNOS by icariin.     

Signaling pathway of nitric oxide production induced by ginsenoside Rb1 in human aortic endothelial cells: a possible involvement of androgen receptor

Ginsenosides have been shown to stimulate nitric oxide (NO) production in aortic endothelial cells. However, the signaling pathways involved have not been well studied in human aortic endothelial cells. The present study was designed to examine whether purified ginsenoside Rb1, a major active component of ginseng could actually induce NO production and to clarify the signaling pathway in human aortic endothelial cells. NO production was rapidly increased by Rb1. The rapid increase in NO production was abrogated by treatment with nitric oxide synthetase inhibitor, L-NAME. Rb1 stimulated rapid phosphorylation of Akt (Ser473), ERK1/2 (Thr202/Thr204) and eNOS (Ser1177). Rapid phosphorylation of eNOS (Ser1177) was prevented by SH-5, an Akt inhibitor or wortmannin, PI3-kinase inhibitor and partially attenuated by PD98059, an upstream inhibitor for ERK1/2. Interestingly, NO production and eNOS phosphorylation at Ser1177 by Rb1 were abolished by androgen receptor antagonist, nilutamide. The results suggest that PI3kinase/Akt and MEK/ERK pathways and androgen receptor are involved in the regulation of acute eNOS activation by Rb1 in human aortic endothelial cells.     

HSP90 and Akt modulate Ang-1-induced angiogenesis via NO in coronary artery endothelium.

This study examines the notion that heat shock protein (HSP) 90 binding to nitric oxide (NO), endothelial NO synthase (eNOS), and PI3K-Akt regulate angiopoietin (Ang)-1-induced angiogenesis in porcine coronary artery endothelial cells (PCAEC). Exposure to Ang-1 (250 ng/ml) for periods up to 2 h resulted in a time-dependent increase in eNOS phosphorylation at Ser 1177 that occurred by 5 min and peaked at 60 min. This was accompanied by a gradual increase in NO release. Ang-1 also led to stimulation of HSP90 binding to eNOS and a significant increase in Akt phosphorylation. Thirty minutes of pretreatment of cells with either 1 microg/ml geldanamycin (a specific inhibitor of HSP90) or 500 nM wortmannin [a specific phosphatidylinositol 3 (PI3)-kinase (PI3K) inhibitor] significantly attenuated Ang-1-stimulated eNOS phosphorylation and NO production. Exposure to Ang-1 caused an increase in endothelial cell migration, tube formation, and sprouting from PCAEC spheroids, and pharmacological blockage of HSP90 function or inhibition of PI3K-Akt pathway completely abolished these effects. Inhibition of nitric oxide synthase by NG-nitro-l-arginine methyl ester (2.5 mM) also resulted in a significant decrease in Ang-1-induced angiogenesis. We conclude that stimulated HSP90 binding to eNOS and activation of the PI3-Akt pathway contribute to Ang-1-induced eNOS phosphorylation, NO production, and angiogenesis in PCAEC.     

Rho-kinase phosphorylates eNOS at threonine 495 in endothelial cells

Endothelial nitric oxide synthase (eNOS) produces nitric oxide (NO), which is involved in various physiological functions of the cardiovascular system. eNOS is activated by dephosphorylation at Thr495 and phosphorylation at Ser1177. Inhibition of Rho-kinase, an effector of the small GTPase RhoA, leads to activation of Akt/PKB, which phosphorylates eNOS at Ser1177 and thereby promotes NO production. However, little is known about the effects of Rho-kinase on phosphorylation of Thr495. We here found that the constitutively active form of Rho-kinase phosphorylated eNOS at Thr495 in vitro. Expression of the constitutively active form of RhoA or Rho-kinase increased this phosphorylation in COS-7 cells. Addition of thrombin to cultured human umbilical vein endothelial cells induced phosphorylation of eNOS at Thr495. Treatment with Y27632, a Rho-kinase inhibitor, suppressed thrombin-induced phosphorylation at Thr495. These results indicate that Rho-kinase can directly phosphorylate eNOS at Thr495 to suppress NO production in endothelium.     

Losartan improves aortic endothelium-dependent relaxation via proline-rich tyrosine kinase 2/Src/Akt pathway in type 2 diabetic Goto-Kakizaki rats

In diabetic states, endothelial dysfunction is related to vascular complications. We hypothesized that insulin-induced relaxation and the associated proline-rich tyrosine kinase 2 (Pyk2)/Src/Akt pathway would be abnormal in aortas from the Goto-Kakizaki (GK) type 2 diabetic rat, which exhibits hyperglycemia/insulin resistance, and that losartan treatment of such rats (25 mg·kg(-1)·day(-1) for 2 wk) would correct these abnormalities. Endothelium-dependent relaxation was by measuring isometric force in helical strips of aortas from four groups, each of 30 rats: normal Wistar (control), GK (diabetic), losartan-treated normal, and losartan-treated GK. Pyk2, Src, and Akt/endothelial nitric oxide synthase (eNOS) signaling-pathway protein levels and activities were assayed mainly by Western blotting and partly by immunohistochemistry. In GK (vs. age-matched control) aortas, various insulin-stimulated levels [nitric oxide production and the phosphorylations of eNOS at Ser(1177), of Akt at Thr(308), of phosphoinositide-dependent kinase-1 (PDK1) at Ser(241), of Src at Tyr(416), and of Pyk2 at Tyr(579)] were all significantly decreased and unaffected by either Src inhibitor (PP2) or Pyk2 inhibitor (AG17), while the insulin-stimulated levels of insulin receptor substrate (IRS)-1 phosphorylation at Ser(307), total-eNOS, and total-Akt were significantly increased. Losartan treatment normalized these altered levels. The insulin-stimulated phosphorylation levels of Src/PDK1/Akt/eNOS, but not of Pyk2, were decreased by PP2 in control and losartan-treated GK, but not in GK, aortas. These results suggest that in the GK diabetic aorta increased phospho-IRS-1 (at Ser(307)) and decreased Pyk2/Src activity inhibit insulin-induced stimulation of the PDK/Akt/eNOS pathway. The observed increase in phospho-IRS-1 (at Ser(307)) may result from increased angiotensin II activity.     

Role of Akt2 in contraction-stimulated cell signaling and glucose uptake in skeletal muscle

The serine/threonine kinase Akt/PKB plays diverse roles in cells, and genetic studies have indicated distinct roles for the three Akt isoforms expressed in mammalian cells and tissues. Akt2 is a key signaling intermediate for insulin-stimulated glucose uptake and glycogen synthesis in skeletal muscle. Akt2 has also been shown to be activated by exercise and muscle contraction in both rodents and humans. In this study, we used Akt2 knockout mice to explore the role of Akt2 in exercise-stimulated glucose uptake and glycogen synthesis as well as intracellular signaling pathways that regulate glycogen metabolism in skeletal muscle. We found that Akt2 deficiency does not affect basal or exercise-stimulated glucose uptake or intracellular glycogen content in the soleus muscle. In addition, lack of Akt2 did not result in alterations in basal Akt Thr(308) or basal and contraction-stimulated glycogen synthase kinase-3beta (GSK-3beta) Ser(9) phosphorylation, glycogen synthase phosphorylation, or glycogen synthase activity. In contrast, in situ contraction failed to elicit normal increases in Akt T-loop Thr(308) phosphorylation and GSK-3alpha Ser(21) phosphorylation in tibialis anterior muscles from Akt2-deficient animals. Our data establish a key role for Akt2 in the regulation of GSK-3alpha Ser(21) phosphorylation with contraction and add genetic evidence to support the separation of the intracellular pathways regulated by insulin and exercise that converge on glucose uptake and glycogen synthesis in skeletal muscle.