Upregulation of ERK1/2-eNOS via AT2 Receptors Decreases the Contractile Response to Angiotensin II in Resistance Mesenteric Arteries from Obese Rats

It has been clearly established that mitogen-activated protein kinases (MAPKS) are important mediators of angiotensin II (Ang II) signaling via AT1 receptors in the vasculature. However, evidence for a role of these kinases in changes of Ang II-induced vasoconstriction in obesity is still lacking. Here we sought to determine whether vascular MAPKs are differentially activated by Ang II in obese animals. the role of AT2 receptors was also evaluated. Male monosodium glutamate-induced obese (obese) and non-obese Wistar rats (control) were used. the circulating concentrations of Ang I and Ang II, determined by HPLC, were increased in obese rats. Ang II-induced isometric contraction was decreased in endothelium-intact resistance mesenteric arteries from obese compared with control rats and exhibited a retarded AT1 receptor antagonist response. Blocking of AT2 receptors and inhibition of either endothelial nitric oxide synthase (eNOS) or extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) restored Ang II-induced contraction in obese rats. Western blot analysis revealed increased protein expression of AT2 receptors in arteries from obese rats. Basal and Ang II-induced ERK1/2 phosphorylation was also increased in obese rats. Blockade of either AT1 or AT2 receptors corrected the increased ERK1/2 phosphorylation in arteries from obese rats to levels observed in control preparations. Phosphorylation of eNOS was increased in obese rats. Incubation with the ERK1/2 inhibitor before Ang II stimulation did not affect eNOS phosphorylation in control rats; however, it corrected the increased phosphorylation of eNOS in obese rats. These results clearly demonstrate that enhanced AT2 receptor and ERK1/2-induced, NO-mediated vasodilation reduces Ang II-induced contraction in an endothelium-dependent manner in obese rats.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Univ São Paulo, Inst Biomed Sci, Dept Pharmacol, São Paulo, BrazilUniv Fed Goias, Div Cardiovasc Physiol, Dept Biol Sci, Jatai, BrazilUniversidade Federal de São Paulo, Div Nephrol, Dept Med, Escola Paulista Med, São Paulo, BrazilUniversidade Federal de São Paulo, Div Nephrol, Dept Med, Escola Paulista Med, São Paulo, BrazilFAPESP: 2007/58311-0FAPESP: 2008/51622-3FAPESP: 2010/03642-5Web of Scienc

Oxidative stress and inflammatory mediators contribute to endothelial dysfunction in high-fat diet-induced obesity in mice

Objective We investigated the effects of high-fat diet-induced obesity on vascular proinflammatory factors and oxidative stress on endothelium-dependent relaxation of the aorta. Methods Female Swiss mice were submitted to a high-fat diet for 16 weeks. At the end of the experimental period, we evaluated blood pressure, relaxation in response to acetylcholine in aortic rings in the absence and the presence of the superoxide anion scavenger, superoxide dismutase (SOD, 150 U/ml), and the nuclear factor (NF)-kappa B inhibitor, sodium salicylate (5 mmol/l). Aortic protein expression of endothelial nitric oxide synthase, Cu/Zn-SOD, NF-kappa B, I kappa B-alpha, and proinflammatory cytokines were also evaluated. Results Obese mice presented higher systolic and diastolic blood pressure than control mice (P<0.05). The relaxation of aortas to acetylcholine, but not to sodium nitroprusside, was significantly decreased in obese mice and was corrected by both SOD and sodium salicylate (P<0.05). The protein expression of endothelial nitric oxide synthase and Cu/Zn-SOD was significantly decreased in aorta from obese mice (P<0.05). Total p65 NF-kappa B subunit protein expression was not affected by obesity, but the protein expression of NF-kappa B inhibitor I kappa B-alpha was lower in aorta from obese mice (P<0.05). There were no significant differences in the interleukin (IL)-1 beta and IL-6 protein expression between groups. In contrast, the expression of TNF-alpha was significantly increased in aortas from obese mice. Conclusion Our resultssuggest that the reducedantioxidant defense and the local NF-kappa B pathway play an important role in the impairment of endothelium-dependent relaxation in aorta from obese mice. J Hypertens 28: 2111-2119 (C) 2010 Wolters Kluwer Health vertical bar Lippincott Williams & Wilkins.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq), BrazilConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Obesity induced by high-fat diet promotes insulin resistance in the ovary

Besides the effects on peripheral energy homeostasis, insulin also has an important role in ovarian function. Obesity has a negative effect on fertility, and may play a role in the development of the polycystic ovary syndrome in susceptible women. Since insulin resistance in the ovary could contribute to the impairment of reproductive function in obese women, we evaluated insulin signaling in the ovary of high-fat diet-induced obese rats. Female Wistar rats were submitted to a high-fat diet for 120 or 180 days, and the insulin signaling pathway in the ovary was evaluated by immunoprecipitation and immunoblotting. At the end of the diet period, we observed insulin resistance, hyperinsulinemia, an increase in progesterone serum levels, an extended estrus cycle, and altered ovarian morphology in obese female rats. Moreover, in female obese rats treated for 120 days with the high-fat diet, the increase in progesterone levels occurred together with enhancement of LH levels. The ovary from high-fat-fed female rats showed a reduction in the insulin receptor substrate/phosphatidylinositol 3-kinase/AKT intracellular pathway, associated with an increase in FOXO3a, IL1B, and TNF alpha protein expression. These changes in the insulin signaling pathway may have a role in the infertile state associated with obesity. Journal of Endocrinology (2010) 206, 65-74Fundacao de Amparo a Pesquisa do Estado de Sao Paulo-FAPESP[2006/52163-7]Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Fundacao de Amparo a Pesquisa do Estado de Sao Paulo-FAPESP[2006/60215-7]Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Conselho Nacional de Desenvolvimento Cientifico e Tecnologico-CNPq[477906/2006-0]Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)FAPESPEHAEH

Detrimental effects of testosterone addition to estrogen therapy involve cytochrome P-450-induced 20-HETE synthesis in aorta of ovariectomized spontaneously hypertensive rat (SHR), a model of postmenopausal hypertension

Postmenopausal period has been associated to different symptoms such as hot flashes, vulvovaginal atrophy, hypoactive sexual desire disorder (HSDD) and others. Clinical studies have described postmenopausal women presenting HSDD can benefit from the association of testosterone to conventional hormonal therapy. Testosterone has been linked to development of cardiovascular diseases including hypertension and it also increases cytochrome P-450-induced 20-HETE synthesis which in turn results in vascular dysfunction. However, the effect of testosterone plus estrogen in the cardiovascular system is still very poorly studied. The aim of the present study is to evaluate the role of cytochrome P-450 pathway in a postmenopausal hypertensive female treated with testosterone plus estrogen. For that, hypertensive ovariectomized rats (OVX-SHR) were used as a model of postmenopausal hypertension and four groups were created: SHAM-operated (SHAM), ovariectomized SHR (OVX), OVX treated for 15 days with conjugated equine estrogens [(CEE) 9.6 μg/Kg/day/po] or CEE associated to testosterone [(CEE+T) 2.85 mg/kg/weekly/im]. Phenylephrine-induced contraction and generation of reactive oxygen species (ROS) were markedly increased in aortic rings from OVX-SHR compared to SHAM rats which were restored by CEE treatment. On the other hand, CEE+T abolished vascular effects by CEE and augmented both systolic and diastolic blood pressure of SHR. Treatment of aortic rings with the CYP/20-HETE synthesis inhibitor HET0016 (1 μM) reduced phenylephrine hyperreactivity and the augmented ROS generation in the CEE+T group. These results are paralleled by the increased CYP4F3 protein expression and activity in aortas of CEE+T. In conclusion, we showed that association of testosterone to estrogen therapy produces detrimental effects in cardiovascular system of ovariectomized hypertensive females via CYP4F3/20-HETE pathway. Therefore, our findings support the standpoint that the CYP/20-HETE pathway is an important therapeutic target for the prevention of cardiovascular disease in menopausal women in the presence of high levels of testosterone

Effect of obesity on contraction of mesenteric arteries to angiotensin II and norepinephrine.

<p><b>A-</b> Non-cumulative concentration–response curves to angiotensin II (ANG II) obtained in different segments of endothelium intact and endothelium denuded mesenteric arteries from control and monosodium glutamate (MSG)-induced obese rats. The curves were performed on a top of a submaximal tone (30 to 40% of the maximum response) induced by norepinephrine (NE). <b>B</b>- Cumulative concentration–response curves to NE in endothelium intact and endothelium denuded mesenteric arteries from control and monosodium glutamate-induced obese rats. Each point represents the mean ± SEM. *, P<0.05 vs. Control; ^#, P<0.05 vs. respective group in the absence of endothelium. N = 5–6/group.</p

Effect of obesity on angiotensin II receptors protein expression in mesenteric arteries.

<p>Panels show densitometric analysis of the Western blots for AT1 and AT2 protein expression in endothelium intact mesenteric arteries from control and monosodium glutamate-induced obese rats. In <b>A</b> and <b>C</b>, Western blots for AT1 and AT2 receptors, respectively. Results were normalized to α-actin expression and expressed as units of change from the control. Data are expressed as mean ± SEM. *, P<0.05 vs. Control. N =  5/group.</p

General characteristics of sixteen-week-old control and obese rats.

<p>WAT, white adipose tissue; HDL, high density lipoprotein; LDL, low density lipoprotein; VLDL, very low density lipoprotein; HOMA-IR, homeostasis model assessment-insulin resistance; Values are mean ± SEM. *<i>P</i><0.05 vs. control. N = 7–10/group.</p><p>General characteristics of sixteen-week-old control and obese rats.</p

Contribution of nitric oxide and NADPH oxidase to the vascular effects of angiotensin.

<p>Mesenteric arteries with intact endothelium from control and monosodium glutamate-induced obese rats were pretreated with Nω-nitro-L-arginine methyl ester (L-NAME, 100 µM), a nitric oxide synthase inhibitor (<b>A</b>) or apocynin (100 µM), a NADPH inhibitor (<b>B</b>) for 30 min and non-cumulative concentration–response curves to angiotensin II (ANG II) were obtained. Each point represents the mean ± SEM. *, P<0.05 vs. Control. ^#, P<0.05 vs. respective group in the absence of blockade. N =  6/group.</p

Effect of angiotensin II on eNOS phosphorylation in mesenteric arteries.

<p>Panels show densitometric analysis of the Western blots for eNOS protein expression in endothelium intact mesenteric arteries from control and monosodium glutamate-induced obese rats. Vessels from both groups were stimulated with ANG II (0.1 µmol/L) or vehicle for 10 min in the absence or in the presence of the ERK1/2 inhibitor PD98059 (1 µM, 30 min) and the phosphorylation of eNOS was examined. Total protein levels are shown as loading controls. Data are expressed as mean ± SEM. *, P<0.05 vs. Control, ^#, P<0.05 vs. respective group in the presence of ANG II. N = 5–6/group.</p