Dissociation between metabolic and vascular insulin resistance in aging

Physiological actions of insulin via activation of the phosphatidylinositol 3-kinase/Akt pathway in the endothelium serve to couple regulation of hemodynamic and metabolic homeostasis. Insulin resistance, endothelial dysfunction, and hypertension increase in prevalence with aging. We investigated the metabolic and endothelial actions of insulin in 24- vs. 3-mo Sprague-Dawley rats. With the use of the hyperinsulinemic euglycemic clamp, the rate of glucose infusion necessary to maintain equivalent plasma glucose (5.5 mmol/l) was similar in 24- vs. 3-mo rats, as was fasting glucose (5.2 +/- 0.33 vs. 4.4 +/- 0.37 mmol/l; mean +/- SE) and insulin (0.862 +/- 0.193 vs. 1.307 +/- 0.230 mg/l). Systolic blood pressure was higher in 24-mo rats (133 +/- 5 vs. 110 +/- 4 mmHg; P = 0.005). Endothelial nitric oxide (NO)-dependent relaxation to insulin was impaired in aortas of 24- vs. 3-mo rats (maximal response 8.9 +/- 4.3 vs. 34.9 +/- 3.9%; P = 0.002); N(G)-nitro-l-arginine methyl ester abolished insulin-mediated relaxation in 3- but not 24-mo rats. Endothelium NO-dependent (acetylcholine) and -independent (sodium nitroprusside) relaxation, as well as NADPH oxidase activity, were similar in 3- and 24-mo rats. Insulin increased aortic serine phosphorylation of Akt in 3-mo rats by 120% over 24-mo rats (P < 0.05) and serine phosphorylation of endothelial NO synthase (eNOS) in 3-mo rats by 380% over 24-mo rats (P < 0.05). Aortic expression of phosphorylated c-Jun NH(2)-terminal kinase-1 and serine phosphorylated insulin receptor substrate-1, known mediators of metabolic insulin resistance, was similar in 3- and 24-mo rats. Expression of caveolin-1, a regulator of eNOS activity and insulin signaling, was 55% lower in 24- than 3-mo rats (P = 0.002). In summary, impaired vasorelaxation to insulin in aging was independent of metabolic insulin sensitivity and associated with impaired insulin-mediated activation of the Akt/eNOS pathway, but intact activation of the acetylcholine-mediated Ca(2+)-calmodulin/eNOS pathway. Vascular insulin resistance in aging may add to the increased susceptibility of this population to vascular injury induced by traditional cardiovascular risk factors.     

VEGF increases the proliferative capacity and eNOS/NO levels of endothelial progenitor cells through the calcineurin/NFAT signalling pathway

We have investigated whether VEGF (vascular endothelial growth factor) regulates the proliferative capacity and eNOS (endothelial nitric oxide synthase)/NO (nitric oxide) pathway of EPCs (endothelial progenitor cells) by activating CaN (calcineurin)/NFAT (nuclear factor of activated T-cells) signalling. EPCs were obtained from cultured mononuclear cells isolated from the peripheral blood of healthy adults. Treatment with VEGF (50 ng/ml) potently promoted CaN enzymatic activity, activation of NFAT2, cell proliferation, eNOS protein expression and NO production. Pretreatment with cyclosporin A (10 μg/ml), a pharmacological inhibitor of CaN or 11R-VIVIT, a special inhibitor of NFAT, completely abrogated the aforementioned effects of VEGF treatment and increased apoptosis. The results indicate that VEGF treatment promotes the proliferative capacity of human EPCs by activating CaN/NFAT signalling leading to increased eNOS protein expression and NO production.     

Angiotensin II promotes NO production, inhibits apoptosis and enhances adhesion potential of bone marrow-derived endothelial progenitor cells

Endothelial progenitor cells (EPCs) participate in the processes of postnatal neovascularization and re-endothelialization in response to tissue ischemia and endothelial injury. The level of EPCs present has been found to be directly associated with the outcome of cardiovascular diseases, and could be regulated by stimulatory or inhibitory factors. Given the close relationship between angiotensin II (AngII) and the cardiovascular system, we investigated the effect of AngII on the activities of bone marrow (BM)-derived EPCs. Cells were isolated from BM of rats by density gradient centrifugation. Administration of AngII significantly promoted nitric oxide (NO) release, inhibited EPC apoptosis and enhanced EPC adhesion potential. All of these AngII-mediated effects on EPCs were attenuated by pretreatment with valsartan or L-NAME. Moreover, both LY294002 and wortmannin abolished the anti-apoptotic effect of AngII. Western blot analyses indicated that endothelial NO synthase (eNOS) protein and phosphorylated Akt increased with the treatment of AngII in EPCs. Thus, AngII improved several activities of EPCs through AngII type 1 receptor (AT1R), which may represent a possible mechanism linking AngII and AT1R with angiogenesis. Additionally, AngII-induced NO synthesis through eNOS in EPCs regulates apoptosis and adhesion, and the PI3-kinase/Akt pathway has an essential role in AngII-induced antiapoptosis signaling.     

N-3 polyunsaturated fatty acids alleviate high glucose-mediated dysfunction of endothelial progenitor cells and prevent ischemic injuries both in vitro and in vivo

Hyperglycemia is associated with a reduced number of endothelial progenitor cells (EPCs) that impairs vascular function. Circulating EPCs play important roles in postnatal neovasculogenesis and the prevention of ischemic injury. Frequent consumption of fish oil (FO) that is abundant with eicosapentaenoic acid (EPA)/docosahexaenoic acid (DHA) is reportedly associated with an alleviation of diabetic complications and a lowered incidence of cardiovascular disease. The aim of this study was to examine whether N-3 polyunsaturated fatty acids such as EPA and DHA would reverse the high glucose-mediated dysfunction of EPCs in vitro and thereby prevent the ischemic injury that occurs under the hyperglycemic conditions in Type 2 diabetes (T2D) db^−/− mice. The results demonstrate that EPA and DHA alleviate high glucose-mediated impairment of tubular formation in EPCs through a rescue of neovasculogenic capability. The molecular mechanisms underlying the effects of EPA and DHA include the activation of the extracellular signal-regulated kinase 1/2, Akt/endothelial nitric oxide synthase (eNOS) and AMP-activated kinase (AMPK) signaling cascades as well as the phosphorylation of the downstream FOXO3a protein in EPCs. Moreover, EPA and DHA up-regulate the expression of c-kit, erythroid 2-related factor and heme oxygenase-1 proteins. Daily consumption of FO at dosages of 4% and 6% (wt/wt) significantly increased the level of bone marrow-derived and circulating EPCs, induced a recovery of blood flow and prevented ischemic injuries in a T2D db^−/− mouse model. The effects of FO consumption were exerted the activation of Akt/eNOS and AMPK signaling cascades without any effect on the plasma VEGF level in vivo.     

Oxidized low density lipoprotein impairs endothelial progenitor cells by regulation of endothelial nitric oxide synthase

Oxidized low density lipoprotein (OxLDL) is one of the most important risk factors of cardiovascular disease. Here, we study the impact of OxLDL on endothelial progenitor cells (EPCs) and determine whether OxLDL affects EPCs by an inhibitory effect on endothelial nitric oxide synthase (eNOS). It was found that OxLDL decreased EPC survival and impaired its adhesive, migratory, and tube-formation capacities in a dose-dependent manner. However, all of the detrimental effects of OxLDL were attenuated by pretreatment of EPCs with lectin-like oxidized low density lipoprotein receptor (LOX-1) monoclonal antibody or l-arginine. Western blot analysis revealed that OxLDL dose-dependently decreased Akt phosphorylation and eNOS protein expression and increased LOX-1 protein expression. Furthermore, OxLDL caused a decrease in eNOS mRNA expression and an increase in LOX-1 mRNA expression. These data indicate that OxLDL inhibits EPC survival and impairs its function, and this action is attributable to an inhibitory effect on eNOS.     

Effects of simulated hyperglycemia, insulin, and glucagon on endothelial nitric oxide synthase expression

Diabetes is associated with endothelial dysfunction and increased risk of hypertension, cardiovascular disease, and renal complications. Earlier studies have revealed that hyperglycemia impairs nitric oxide (NO) production and diabetes causes endothelial dysfunction in humans and experimental animals. This study was designed to test the effects of altered concentrations of glucose, insulin, and glucagon, the principal variables in types I and II diabetes, on NO production and endothelial NO synthase (eNOS) expression in cultured human coronary endothelial cells. Cultured endothelial cells were incubated in the presence of glucose at either normal (5.6 mM) or high (25 mM) concentrations for 7 days. The rates of basal and bradykinin-stimulated NO production (nitrate + nitrite) and eNOS protein expression (Western blot) were then determined at the basal condition and in the presence of insulin (10(-8) and 10(-7) M), glucagon (10(-8) and 10(-7) M), or both. Incubation with a high-glucose concentration for 7 days significantly downregulated, whereas insulin significantly upregulated, basal and bradykinin-stimulated NO production and eNOS expression in cultured endothelial cells. The stimulatory action of insulin was mitigated by high-glucose concentration and abolished by cotreatment of cells with glucagon. Thus hyperglycemia, insulinopenia, and hyperglucagonemia, which frequently coexist in diabetes, can work in concert to suppress NO production by human coronary artery endothelial cells.     

Salvianolic acid inhibits the effects of high glucose on vascular endothelial dysfunction by modulating the Sirt1‐eNOS pathway

Salvianolic acid (SA) is known for improving blood circulation, scavenging hydroxyl radicals, and preventing platelet aggregation. The research explored whether SA can protect against cardiovascular disease induced by high glucose conditions. Our results indicate that SA significantly increases cells viability and nitric oxide levels while decreasing reactive oxygen species generation. SA upregulated the expression levels of Bcl‐2 and decreased the levels of Bax, cleaved caspase‐3, and cleaved caspase‐9. Furthermore, the expression levels of Sirtuin 1 (Sirt1) and p‐endothelial nitric oxide synthase (eNOS) were markedly increased in response to SA treatment. Moreover, exposure of human umbilical vein endothelial cells to Ex527 resulted in reducing expression of p‐eNOS. However, the beneficial effects of SA were abolished partially when Ex527 was added. These findings suggest that SA can be used as a potential therapeutic to protect against high glucose‐induced endothelial injury by modulating Sirt1‐eNOS pathway.     

Fibroblast growth factor 21 reverses high‐fat diet‐induced impairment of vascular function via the anti‐oxidative pathway in ApoE knockout mice

Circulating endothelial progenitor cells (EPCs), which function in vascular repair, are the markers of endothelial dysfunction and vascular health. Fibroblast growth factor 21 (FGF21), a liver‐secreted protein, plays a crucial role in glucose homeostasis and lipid metabolism. FGF21 has been reported to attenuate the progression of atherosclerosis, but its impact on EPCs under high oxidative stress conditions remains unclear. In vitro studies showed that the β‐klotho protein was expressed in cultured EPCs and that its expression was upregulated by FGF21 treatment. Hydrogen peroxide (H₂O₂)‐induced oxidative stress impaired EPC function, including cell viability, migration and tube formation. Pretreatment with FGF21 restored the functions of EPCs after the exposure to H₂O₂. Administration of N(ω)‐nitro‐L‐arginine methyl ester (L‐NAME), an inhibitor of nitric oxide synthase, inhibited the effects of FGF21 in alleviating oxidative injury by suppressing endothelial nitric oxide synthase (eNOS). In an in vivo study, the administration of FGF21 significantly reduced total cholesterol (TC) and blood glucose levels in apolipoprotein E (ApoE)‐deficient mice that were fed a high‐fat diet (HFD). Endothelial function, as reflected by acetylcholine‐stimulated aortic relaxation, was improved after FGF21 treatment in ApoE‐deficient mice. Analysis of mRNA levels in the aorta indicated that FGF21 increased the mRNA expression of eNOS and upregulated the expression of the antioxidant genes superoxide dismutase (SOD)1 and SOD2 in ApoE‐deficient mice. These data suggest that FGF21 improves EPC functions via the Akt/eNOS/nitric oxide (NO) pathway and reverses endothelial dysfunction under oxidative stress. Therefore, administration of FGF21 may ameliorate a HFD‐induced vascular injury in ApoE‐deficient mice.     

Fatty acid-binding protein 4 impairs the insulin-dependent nitric oxide pathway in vascular endothelial cells

ABSTRACT: BACKGROUND: Recent studies have shown that fatty acid-binding protein 4 (FABP4) plasma levels are associated with impaired endothelial function in type 2 diabetes (T2D). In this work, we analysed the effect of FABP4 on the insulin-mediated nitric oxide (NO) production by endothelial cells in vitro. METHODS: In human umbilical vascular endothelial cells (HUVECs), we measured the effects of FABP4 on the insulin-mediated endothelial nitric oxide synthase (eNOS) expression and activation and on NO production. We also explored the impact of exogenous FABP4 on the insulin-signalling pathway (insulin receptor substrate 1 (IRS1) and Akt). RESULTS: We found that eNOS expression and activation and NO production are significantly inhibited by exogenous FABP4 in HUVECs. FABP4 induced an alteration of the insulin-mediated eNOS pathway by inhibiting IRS1 and Akt activation. These results suggest that FABP4 induces endothelial dysfunction by inhibiting the activation of the insulin-signalling pathway resulting in decreased eNOS activation and NO production. CONCLUSION: These findings provide a mechanistic linkage between FABP4 and impaired endothelial function in diabetes, which leads to an increased cardiovascular risk.     

Beneficial effect of insulin in hyperhomocysteinemia and diabetes mellitus-induced vascular endothelium dysfunction: role of phosphoinositide dependent kinase and protein kinase B

A primary defect in the vascular action of insulin may be the key intermediate mechanism that links endothelial dysfunction with diabetes mellitus and hyperhomocysteinemia. This study investigated the downstream targets of insulin, involved in this process. Hyperhomocysteinemia (serum homocysteine > 10 μm/l) was produced in rats by administering L-methionine (1.7% w/w, p.o.x. 4 weeks) and diabetes mellitus (serum glucose > 140 mg/dl) was induced using streptozotocin (55 mg/kg/day, i.v. once) in another group. Four weeks after L-methionine and streptozotocin administration, vascular endothelium dysfunction was assessed in terms of attenuation of acetylcholine-induced, endothelium-dependent relaxation (isolated aortic ring preparation), decrease in serum nitrate/nitrite level, as well as mRNA expression of eNOS (rtPCR), and disruption of integrity of vascular endothelium. Both hyperhomocysteinemia and diabetes mellitus significantly attenuated acetylcholine-induced endothelial-dependent relaxation, and the increase in serum nitrite/nitrate concentration and the expression of eNOS. Insulin (0.4 and 0.6 IU/kg/day, s.c.) and atorvastatin (30 mg/kg/day, p.o.x. 4 weeks) significantly improved all these parameters. However, this ameliorative effect of insulin was blocked by 7-hydroxystaurosporine (UCN-01) [Inhibitor of phosphoinositide dependent kinase (PDK)], and triciribine (API-2) (protein kinase B/Akt inhibitor). It is suggested that amelioration of vascular endothelium dysfunction by insulin may be due to stimulation of PDK and Akt pathways.     

Up-regulation of the association between heat shock protein 90 and endothelial nitric oxide synthase prevents high glucose-induced apoptosis in human endothelial cells

Hyperglycemia is the hallmark of diabetes mellitus. Poor glycemic control is correlated with increased cardiovascular morbidity and mortality. High glucose can trigger endothelial cell apoptosis by de-activation of endothelial nitric oxide synthase (eNOS). eNOS was recently demonstrated to be extensively regulated by Akt and heat shock protein 90 (HSP90). Yet, little is known about the molecular mechanisms that regulate eNOS activity during high glucose exposure. The present study was designed to determine the involvement of protein interactions between eNOS and HSP90 in high glucose-induced endothelial cell apoptosis. The protein interaction of eNOS/HSP90 and eNOS/Akt were studied in cultured human umbilical vein endothelial cells (HUVECs) exposed to either control-level (5.5 mM) or high-level (33 mM) glucose for different durations (2, 4, 6, and 24 h). The results showed that the protein interactions between eNOS and HSP90 and between eNOS and Akt and the phosphorylation of eNOS were up-regulated by high glucose exposure for 2-4 h. With longer exposures, these effects decreased gradually. During early hours of exposure, the protein interactions of eNOS/HSP90 and eNOS/Akt and the phosphorylation of eNOS were all inhibited by geldanamycin, an HSP90 inhibitor. High glucose-induced endothelial cell apoptosis was also enhanced by geldanamycin and was reversed by NO donors. LY294002, a phosphatidylinositol 3 (PI3) kinase inhibitor, inhibited the association of eNOS/Akt and the phosphorylation of eNOS but had no effect on the interaction between eNOS and HSP90 during early hours of exposure. From our results we propose that, in HUVECs, during early phase of high glucose exposure, apoptosis can be prevented by enhancement of eNOS activity through augmentation of the protein interaction between eNOS and HSP90 and recruitment of the activated Akt. With longer exposure, dysregulation of eNOS activity would result in apoptosis. The present study provides a molecular basis for the effects of eNOS in the prevention of endothelial cells apoptosis during early phase of high glucose exposure. These observations may contribute to the understanding of the pathogenesis of vascular complications in diabetes mellitus.     

High glucose promotes retinal endothelial cell migration through activation of Src, PI3K/Akt1/eNOS, and ERKs

Hyperglycemia impacts retinal vascular function and promotes the development and progression of diabetic retinopathy, which ultimately results in growth of new blood vessels and loss of vision. How high glucose affects retinal endothelial cell (EC) properties requires further investigation. Here we determined the impact of high glucose on mouse retinal EC function in vitro. High glucose significantly enhanced the migration of retinal EC without impacting their proliferation, apoptosis, adhesion, and capillary morphogenesis. The enhanced migration of retinal EC under high glucose was reversed in the presence of the antioxidant N-acetylcysteine, suggesting increased oxidative stress under high-glucose conditions. Retinal EC under high-glucose conditions also expressed increased levels of fibronectin, osteopontin, and alpha(v)beta(3)-integrin, and reduced levels of thrombospondin-1. These changes were concomitant with sustained activation of the downstream prosurvival and promigratory signaling pathways, including Src kinase, phosphatidylinositol 3-kinase/Akt1/endothelial nitric oxide synthase, and ERKs. The sustained activation of these signaling pathways was essential for enhanced migration of retinal EC under high-glucose conditions. Together, our results indicate the exposure of retinal EC to high glucose promotes a promigratory phenotype. Thus alterations in the proangiogenic properties of retinal EC during diabetes may contribute to the development and pathogenesis of diabetic retinopathy.     

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.     

Ghrelin has novel vascular actions that mimic PI 3-kinase-dependent actions of insulin to stimulate production of NO from endothelial cells

Ghrelin is an orexigenic peptide hormone secreted by the stomach. In patients with metabolic syndrome and low ghrelin levels, intra-arterial ghrelin administration acutely improves their endothelial dysfunction. Therefore, we hypothesized that ghrelin activates endothelial nitric oxide synthase (eNOS) in vascular endothelium, resulting in increased production of nitric oxide (NO) using signaling pathways shared in common with the insulin receptor. Similar to insulin, ghrelin acutely stimulated increased production of NO in bovine aortic endothelial cells (BAEC) in primary culture (assessed using NO-specific fluorescent dye 4,5-diaminofluorescein) in a time- and dose-dependent manner. Production of NO in response to ghrelin (100 nM, 10 min) in human aortic endothelial cells was blocked by pretreatment of cells with NG-nitro-L-arginine methyl ester (nitric oxide synthase inhibitor), wortmannin [phosphatidylinositol (PI) 3-kinase inhibitor], or (D-Lys3)-GHRP-6 (selective antagonist of ghrelin receptor GHSR-1a), as well as by knockdown of GHSR-1a using small-interfering (si) RNA (but not by mitogen/extracellular signal-regulated kinase inhibitor PD-98059). Moreover, ghrelin stimulated increased phosphorylation of Akt (Ser473) and eNOS (Akt phosphorylation site Ser1179) that was inhibitable by knockdown of GHSR-1a using siRNA or by pretreatment of cells with wortmannin but not with PD-98059. Ghrelin also stimulated phosphorylation of mitogen-activated protein (MAP) kinase in BAEC. However, unlike insulin, ghrelin did not stimulate MAP kinase-dependent secretion of the vasoconstrictor endothelin-1 from BAEC. We conclude that ghrelin has novel vascular actions to acutely stimulate production of NO in endothelium using a signaling pathway that involves GHSR-1a, PI 3-kinase, Akt, and eNOS. Our findings may be relevant to developing novel therapeutic strategies to treat diabetes and related diseases characterized by reciprocal relationships between endothelial dysfunction and insulin resistance.     

Unique responses of stem cell-derived vascular endothelial and mesenchymal cells to high levels of glucose

Diabetes leads to complications in selected organ systems, and vascular endothelial cell (EC) dysfunction and loss is the key initiating and perpetuating step in the development of these complications. Experimental and clinical studies have shown that hyperglycemia leads to EC dysfunction in diabetes. Vascular stem cells that give rise to endothelial progenitor cells (EPCs) and mesenchymal progenitor cells (MPCs) represent an attractive target for cell therapy for diabetic patients. Whether these vascular stem/progenitor cells succumb to the adverse effects of high glucose remains unknown. We sought to determine whether adult vascular stem/progenitor cells display cellular activation and dysfunction upon exposure to high levels of glucose as seen in diabetic complications. Mononuclear cell fraction was prepared from adult blood and bone marrow. EPCs and MPCs were derived, characterized, and exposed to either normal glucose (5 mmol/L) or high glucose levels (25 mmol/L). We then assayed for cell activity and molecular changes following both acute and chronic exposure to high glucose. Our results show that high levels of glucose do not alter the derivation of either EPCs or MPCs. The adult blood-derived EPCs were also resistant to the effects of glucose in terms of growth. Acute exposure to high glucose levels increased caspase-3 activity in EPCs (1.4x increase) and mature ECs (2.3x increase). Interestingly, MPCs showed a transient reduction in growth upon glucose challenge. Our results also show that glucose skews the differentiation of MPCs towards the adipocyte lineage while suppressing other mesenchymal lineages. In summary, our studies show that EPCs are resistant to the effects of high levels of glucose, even following chronic exposure. The findings further show that hyperglycemia may have detrimental effects on the MPCs, causing reduced growth and altering the differentiation potential.     

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.     

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.     

Synergistic activation of endothelial nitric-oxide synthase (eNOS) by HSP90 and Akt: calcium-independent eNOS activation involves formation of an HSP90-Akt-CaM-bound eNOS complex

Endothelial nitric-oxide synthase (eNOS), which generates the endogenous vasodilator, nitric oxide (NO), is highly regulated by post-translational modifications and protein interactions. We recently used purified proteins to characterize the mechanisms by which heat shock protein 90 (HSP90) increases eNOS activity at low and high Ca2+ levels (Takahashi, S. and Mendelsohn, M. E. (2003) J. Biol. Chem. 278, 9339-9344). Here we extend these studies to explore interactions between HSP90, Akt, and eNOS. In studies with purified proteins, HSP90 increased the initial rate and maximal extent of Akt-mediated eNOS phosphorylation and activation at low Ca2+ levels. Akt was not observed in the eNOS complex in the absence of HSP90, but both active and inactive Akt associated with eNOS in the presence of HSP90. Direct binding of Akt to HSP90 was observed even in the absence of eNOS. HSP90 also facilitated CaM binding to eNOS irrespective of Akt presence. Geldanamycin (GA) disrupted HSP90-eNOS binding, reduced HSP90-stimulated CaM binding, and blocked both recruitment of Akt to the eNOS complex and phosphorylation of eNOS at Ser-1179. Akt phosphorylated only CaM-bound eNOS, in an HSP90-independent manner. HSP90 and active Akt together increased eNOS activity synergistically, which was reversed by GA. In bovine aortic endothelial cells (BAECs), the effects of vascular endothelial growth factor (VEGF) and insulin on eNOS-HSP90-Akt complex formation and eNOS activation were compared. BAPTA-AM inhibited VEGF- but not insulin-induced eNOS-HSP90-Akt complex formation and eNOS phosphorylation. Insulin caused rapid, transient increase in eNOS activity correlated temporally with the formation of eNOS-HSP90-Akt complex. GA prevented insulin-induced association of HSP90, Akt and CaM with eNOS and inhibited eNOS activation in BAECs. Both platelet-derived growth factor (PDGF) and insulin induced activation of Akt in BAECs, but only insulin caused HSP90-Akt-eNOS association and eNOS phosphorylation. These results demonstrate that HSP90 and Akt synergistically activate eNOS and suggest that this synergy contributes to Ca2+-independent eNOS activation in response to insulin.     

Echinocystic acid,isolated from Gleditsia sinensis fruit,protects endothelial progenitor cells from damage caused by oxLDL via the Akt/eNOS pathway

Aims

Our previous studies revealed that echinocystic acid (EA) showed obvious attenuation of atherosclerosis in rabbits fed a high-fat diet. However, the underlying mechanisms remain to be elucidated. Considering the importance of endothelial progenitor cells (EPCs) in atherosclerosis, we hypothesise that EPCs may be one of the targets for the anti-atherosclerotic potential of EA.

Main methods

After in vitro cultivation, EPCs were exposed to 100 μg/mL of oxidised low-density lipoprotein (oxLDL) and incubated with or without EA (5 and 20 μM) for 48 h. An additional two groups of EPCs (oxLDL + 20 μM EA) were pre-treated with either wortmannin, an inhibitor of the phosphoinositide 3-kinase (PI3K) pathway, or nitro-l-arginine methyl ester (l-NAME), an endothelial nitric oxide synthase (eNOS)-specific inhibitor. Assessment of EPC apoptosis, adhesion, migration, and nitric oxide (NO) release was performed using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) staining, cell counting, caspase-3 activity assay, transwell chamber assay, and Griess reagent, respectively. The protein expression of protein kinase B (Akt) and eNOS was detected using Western blot.

Key findings

Treatment of EPCs with oxLDL induced significant apoptosis and impaired adhesion, migration, and NO production. The deleterious effects of oxLDL on EPCs were attenuated by EA. However, when EPCs were pre-treated with wortmannin or l-NAME, the effects of EA were abrogated. Additionally, oxLDL significantly down-regulated eNOS protein expression as well as repression of eNOS and Akt phosphorylation.

Significance

The inhibitory effect of oxLDL on Akt/eNOS phosphorylation was attenuated by EA. Taken together, the results indicate that EA protects EPCs from damage caused by oxLDL via the Akt/eNOS pathway.     

Overexpression of long non‐coding RNA ANRIL promotes post‐ischaemic angiogenesis and improves cardiac functions by targeting Akt

Angiogenesis is critical for re‐establishing the blood supply to the surviving myocardium after myocardial infarction (MI). Long non‐coding RNA ANRIL (lncRNA‐ANRIL) has been reported to regulate endothelial functions in cardiovascular diseases. This study was to determine the role of lncRNA‐ANRIL in Akt regulation and cardiac functions after MI. Human umbilical vein endothelial cells (HUVECs) were exposed to oxygen‐glucose deprivation (OGD) to mimic in vivo ischaemia. The MI model in mice was induced by ligating left anterior descending coronary artery. OGD remarkably decreased lncRNA‐ANRIL expression level, reduced the phosphorylated levels of Akt and eNOS proteins, and inhibited NO release and cell viability, which were duplicated by shRNA‐mediated gene knockdown of lncRNA‐ANRIL. Conversely, all these effects induced by OGD were abolished by adenovirus‐mediated overexpression of lncRNA‐ANRIL in HUVECs. Further, OGD impaired cell migrations and tube formations in HUVECs, which were reversed by lncRNA‐ANRIL overexpression or Akt up‐regulation. RNA immunoprecipitation analysis indicated that the affinity of lncRNA‐ANRIL to Akt protein was increased in OGD‐treated cells. In animal studies, adenovirus‐mediated lncRNA‐ANRIL overexpression increased the phosphorylated levels of Akt and eNOS, promoted post‐ischaemic angiogenesis and improved heart functions in mice with MI surgery. LncRNA‐ANRIL regulates Akt phosphorylation to improve endothelial functions, which promotes angiogenesis and improves cardiac functions in mice following MI. In this perspective, targeting lncRNA‐ANRIL/Akt may be considered to develop a drug to treat angiogenesis‐related diseases.