Contribution of insulin and Akt1 signaling to endothelial nitric oxide synthase in the regulation of endothelial function and blood pressure.
ABSTRACT: 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.
Project description:OBJECTIVE:The importance of PI3K/Akt signaling in the vasculature has been demonstrated in several models, as global loss of Akt1 results in impaired postnatal ischemia- and VEGF-induced angiogenesis. The ubiquitous expression of Akt1, however, raises the possibility of cell-type-dependent Akt1-driven actions, thereby necessitating tissue-specific characterization. APPROACH AND RESULTS:Herein, we used an inducible, endothelial-specific Akt1-deleted adult mouse model (Akt1iECKO) to characterize the endothelial cell autonomous functions of Akt1 in the vascular system. Endothelial-targeted ablation of Akt1 reduces eNOS (endothelial nitric oxide synthase) phosphorylation and promotes both increased vascular contractility in isolated vessels and elevated diastolic blood pressures throughout the diurnal cycle in vivo. Furthermore, Akt1iECKO mice subject to the hindlimb ischemia model display impaired blood flow and decreased arteriogenesis. CONCLUSIONS:Endothelial Akt1 signaling is necessary for ischemic resolution post-injury and likely reflects the consequence of NO insufficiency critical for vascular repair.
Project description:Obesity causes inflammation and insulin resistance in the vasculature as well as in tissues involved in glucose metabolism such as liver, muscle, and adipose tissue. To investigate the relative susceptibility of vascular tissue to these effects, we determined the time course over which inflammation and insulin resistance develops in various tissues of mice with diet-induced obesity (DIO) and compared these tissue-based responses to changes in circulating inflammatory markers.Adult male C57BL/6 mice were fed either a control low-fat diet (LF; 10% saturated fat) or a high-fat diet (HF, 60% saturated fat) for durations ranging between 1 to 14 weeks. Cellular inflammation and insulin resistance were assessed by measuring phospho-IkappaBalpha and insulin-induced phosphorylation of Akt, respectively, in extracts of thoracic aorta, liver, skeletal muscle, and visceral fat. As expected, HF feeding induced rapid increases of body weight, fat mass, and fasting insulin levels compared to controls, each of which achieved statistical significance within 4 weeks. Whereas plasma markers of inflammation became elevated relatively late in the course of DIO (eg, serum amyloid A [SAA], by Week 14), levels of phospho-IkappaBalpha in aortic lysates were elevated by 2-fold within the first week. The early onset of vascular inflammation was accompanied by biochemical evidence of both endothelial dysfunction (reduced nitric oxide production; induction of intracellular adhesion molecule-1 and vascular cell adhesion molecule-1) and insulin resistance (impaired insulin-induced phosphorylation of Akt and eNOS). Although inflammation and insulin resistance were also detected in skeletal muscle and liver of HF-fed animals, these responses were observed much later (between 4 and 8 weeks of HF feeding), and they were not detected in visceral adipose tissue until 14 weeks.During obesity induced by HF feeding, inflammation and insulin resistance develop in the vasculature well before these responses are detected in muscle, liver, or adipose tissue. This observation suggests that the vasculature is more susceptible than other tissues to the deleterious effects of nutrient overload.
Project description:20-Hydroxyeicosatetraenoic acid (20-HETE) induces endothelial dysfunction and is correlated with diabetes. This study was designed to investigate the effects of 20-HETE on endothelial insulin signaling.Human umbilical vein endothelial cells (HUVECs) or C57BL/6J mice were treated with 20-HETE in the presence or absence of insulin, and p-ERK1/2, p-JNK, IRS-1/PI3K/AKT/eNOS pathway, were examined in endothelial cells and aortas by immunoblotting. eNOS activity and nitric oxide production were measured. 20-HETE increased ERK1/2 phosphorylation and IRS-1 phosphorylation at Ser616; these effects were reversed by ERK1/2 inhibition. We further observed that 20-HETE treatment resulted in impaired insulin-stimulated IRS-1 phosphorylation at Tyr632 and subsequent PI3-kinase/Akt activation. Furthermore, 20-HETE treatment blocked insulin-stimulated phosphorylation of eNOS at the stimulatory Ser1177 site, eNOS activation and NO production; these effects were reversed by inhibiting ERK1/2. Treatment of C57BL/6J mice with 20-HETE resulted in ERK1/2 activation and impaired insulin-dependent activation of the IRS-1/PI3K/Akt/eNOS pathway in the aorta. Our data suggest that the 20-HETE activation of IRS-1 phosphorylation at Ser616 is dependent on ERK1/2 and leads to impaired insulin-stimulated vasodilator effects that are mediated by the IRS-1/PI3K/AKT/eNOS pathway.
Project description:The PI3K/Akt pathway is necessary for several key endothelial cell (EC) functions, including cell growth, migration, survival, and vascular tone. However, existing literature supports the idea that Akt can be either pro- or antiangiogenic, possibly due to compensation by multiple isoforms in the EC when a single isoform is deleted. Thus, biochemical, genetic, and proteomic studies were conducted to examine isoform-substrate specificity for Akt1 vs. Akt2. In vitro, Akt1 preferentially phosphorylates endothelial nitric oxide synthase (eNOS) and promotes NO release, whereas nonphysiological overexpression of Akt2 can bypass the loss of Akt1. Conditional deletion of Akt1 in the EC, in the absence or presence of Akt2, retards retinal angiogenesis, implying that Akt1 exerts a nonredundant function during physiological angiogenesis. Finally, proteomic analysis of Akt substrates isolated from Akt1- or Akt2-deficient ECs documents that phosphorylation of multiple Akt substrates regulating angiogenic signaling is reduced in Akt1-deficient, but not Akt2-deficient, ECs, including eNOS and Forkhead box proteins. Therefore, Akt1 promotes angiogenesis largely due to phosphorylation and regulation of important downstream effectors that promote aspects of angiogenic signaling.
Project description:Obesity is characterized by chronic inflammation of adipose tissue, which contributes to insulin resistance and diabetes. Although nitric oxide (NO) signaling has antiinflammatory effects in the vasculature, whether reduced NO contributes to adipose tissue inflammation is unknown. We sought to determine whether (1) obesity induced by high-fat (HF) diet reduces endothelial nitric oxide signaling in adipose tissue, (2) reduced endothelial nitric oxide synthase (eNOS) signaling is sufficient to induce adipose tissue inflammation independent of diet, and (3) increased cGMP signaling can block adipose tissue inflammation induced by HF feeding.Relative to mice fed a low-fat diet, an HF diet markedly reduced phospho-eNOS and phospho-vasodilator-stimulated phosphoprotein (phospho-VASP), markers of vascular NO signaling. Expression of proinflammatory cytokines was increased in adipose tissue of eNOS-/- mice. Conversely, enhancement of signaling downstream of NO by phosphodiesterase-5 inhibition using sildenafil attenuated HF-induced proinflammatory cytokine expression and the recruitment of macrophages into adipose tissue. Finally, we implicate a role for VASP, a downstream mediator of NO-cGMP signaling in mediating eNOS-induced antiinflammatory effects because VASP-/- mice recapitulated the proinflammatory phenotype displayed by eNOS-/- mice.These results imply a physiological role for endothelial NO to limit obesity-associated inflammation in adipose tissue and hence identify the NO-cGMP-VASP pathway as a potential therapeutic target in the treatment of diabetes.
Project description:Akt1 is critical for many in vivo functions; however, the cell-specific substrates responsible remain to be defined. Here, we examine the importance of endothelial nitric oxide synthase (eNOS) as an Akt1 substrate by generating Akt1-deficient mice (Akt1(-/-) mice) carrying knock-in mutations (serine to aspartate or serine to alanine substitutions) of the critical Akt1 phosphorylation site on eNOS (serine 1176) that render the enzyme "constitutively active" or "less active." The eNOS mutations did not influence several phenotypes in Akt1(-/-) mice; however, the defective postnatal angiogenesis characteristic of Akt1(-/-) mice was rescued by crossing the Akt1(-/-) mice with mice carrying the constitutively active form of eNOS, but not by crossing with mice carrying the less active eNOS mutant. This genetic rescue resulted in the stabilization of hypoxia-inducible factor 1alpha (HIF-1alpha) and increased production of HIF-1alpha-responsive genes in vivo and in vitro. Thus, Akt1 regulates angiogenesis largely through phosphorylation of eNOS and NO-dependent signaling.
Project description:Endothelial nitric oxide (NO) is a critical mediator of vascular function and vascular remodeling. NO is produced by endothelial nitric oxide synthase (eNOS), which is activated by calcium (Ca2+)-dependent and Ca2+-independent pathways. Here, we report that neurogranin (Ng), which regulates Ca2+-calmodulin (CaM) signaling in the brain, is uniquely expressed in endothelial cells (EC) of human and mouse vasculature, and is also required for eNOS regulation. To test the role of Ng in eNOS activation, Ng knockdown in human aortic endothelial cells (HAEC) was performed using Ng SiRNA along with Ng knockout (Ng -/-) in mice. Depletion of Ng expression decreased eNOS activity in HAEC and NO production in mice. We show that Ng expression was decreased by short-term laminar flow and long-them oscillating flow shear stress, and that Ng siRNA with shear stress decreased eNOS expression as well as eNOS phosphorylation at S1177. We further reveled that lack of Ng expression decreases both AKT-dependent eNOS phosphorylation, NF-?B-mediated eNOS expression, and promotes endothelial activation. Our findings also indicate that Ng modulates Ca2+-dependent calcineurin (CaN) activity, which suppresses Ca2+-independent AKT-dependent eNOS signaling. Moreover, deletion of Ng in mice also reduced eNOS activity and caused endothelial dysfunction in flow-mediated dilation experiments. Our results demonstrate that Ng plays a crucial role in Ca2+-CaM-dependent eNOS regulation and contributes to vascular remodeling, which is important for the pathophysiology of cardiovascular disease.
Project description:Vascular endothelial growth factor (VEGF) stimulated fetoplacental artery endothelial (oFPAE) cell migration and activated multiple signaling pathways including ERK2/1, p38MAPK, Jun N-terminal kinase (JNK1/2), v-Akt murine thymoma viral oncogene homolog 1 (Akt1), and c-Src in oFPAE cells. VEGF-induced cell migration was blocked by specific kinase inhibitors of JNK1/2 (SP600125), c-Src (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d] pyrimidine), and phosphatidylinositol 3-kinase/Akt (wortmannin) but not ERK2/1 (U0126) and p38MAPK (SB203580). VEGF-induced cell migration was associated with dynamic actin reorganization and focal adhesion as evidenced by increased stress fiber formation and phosphorylation of cofilin-1 and focal adhesion kinase (FAK) and paxillin. Inhibition of JNK1/2, c-Src, and phosphatidylinositol 3-kinase/Akt suppressed VEGF-induced stress fiber formation and cofilin-1 phosphorylation. c-Src inhibition suppressed VEGF-induced phosphorylation of focal adhesion kinase, paxillin, and focal adhesion. VEGF-induced cell migration requires endogenous nitric oxide (NO) as: 1) VEGF-stimulated phosphorylation of endothelial NO synthase (eNOS) via activation of Akt, JNK1/2, and Src; 2) a NO donor diethylenetriamine-NO-stimulated cell migration; and 3) NO synthase inhibition blocked VEGF-induced cell migration. Targeted down-regulation and overexpression of caveolin-1 both inhibited VEGF-induced cell migration. Caveolin-1 down-regulation suppressed VEGF-stimulated phosphorylation of Akt, JNK, eNOS, c-Src, and FAK; however, basal activities of c-Src and FAK were elevated in parallel with increased stress fiber formation and focal adhesion. Caveolin-1 overexpression also inhibited VEGF-induced phosphorylation of Akt, JNK, c-Src, FAK, and eNOS. Thus, VEGF-induced placental endothelial cell migration requires activation of complex pathways that are paradoxically regulated by caveolin-1.
Project description:In addition to an extensively characterized role of high-density lipoprotein (HDL) in reverse cholesterol transport, bioactive lipids bound to HDL can also exert diverse vascular effects. Despite this, integration of HDL action in the vasculature with pathways that metabolize HDL and release bioactive lipids has been much less explored. The effects of HDL on endothelial cells are mediated in part by HDL-associated sphingosine 1-phosphate (S1P), which binds to S1P1 receptors and promotes activation of endothelial NO synthase (eNOS) and the kinase Akt. In these studies, we characterized the role of endothelial lipase (EL) in the control of endothelial signaling and biology, including those mediated by HDL-associated S1P.HDL-induced angiogenesis in aortic rings from EL-deficient (EL(-/-)) mice was markedly decreased compared with wild-type controls. In cultured endothelial cells, small interfering RNA-mediated knockdown of EL abrogated HDL-promoted endothelial cell migration and tube formation. Small interfering RNA-mediated EL knockdown also attenuated HDL-induced phosphorylation of eNOS(1179) and Akt(473). S1P stimulation restored HDL-induced endothelial migration and Akt/eNOS phosphorylation that had been blocked by small interfering RNA-mediated EL knockdown. HDL-induced endothelial cell migration and Akt/eNOS phosphorylation were completely inhibited by the S1P1 antagonist W146 but not by the S1P3 antagonist CAY10444.EL is a critical determinant of the effects of HDL on S1P-mediated vascular responses and acts on HDL to promote activation of S1P1, leading to Akt/eNOS phosphorylation and subsequent endothelial migration and angiogenesis. The role of EL in HDL-associated S1P effects provides new insights into EL action, the responses seen through EL and HDL interaction, and S1P signaling.
Project description:Prior studies have implicated accumulation of ceramide in blood vessels as a basis for vascular dysfunction in diet-induced obesity via a mechanism involving type 2 protein phosphatase (PP2A) dephosphorylation of endothelial nitric oxide synthase (eNOS). The current study sought to elucidate the mechanisms linking ceramide accumulation with PP2A activation and determine whether pharmacological inhibition of PP2A in vivo normalizes obesity-associated vascular dysfunction and limits the severity of hypertension. We show in endothelial cells that ceramide associates with the inhibitor 2 of PP2A (I2PP2A) in the cytosol, which disrupts the association of I2PP2A with PP2A leading to its translocation to the plasma membrane. The increased association between PP2A and eNOS at the plasma membrane promotes dissociation of an Akt-Hsp90-eNOS complex that is required for eNOS phosphorylation and activation. A novel small-molecule inhibitor of PP2A attenuated PP2A activation, prevented disruption of the Akt-Hsp90-eNOS complex in the vasculature, preserved arterial function, and maintained normal blood pressure in obese mice. These findings reveal a novel mechanism whereby ceramide initiates PP2A colocalization with eNOS and demonstrate that PP2A activation precipitates vascular dysfunction in diet-induced obesity. Therapeutic strategies targeted to reducing PP2A activation might be beneficial in attenuating vascular complications that exist in the context of type 2 diabetes, obesity, and conditions associated with insulin resistance.