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

Improvement of metabolic parameters and vascular function by metformin in obese non-diabetic rats

Aims: Metformin is an insulin sensitizing agent with beneficial effects in diabetic patients on glycemic levels and in the cardiovascular system. We examined whether the metabolic changes and the vascular dysfunction in monosodium glutamate-induced obese non-diabetic (MSG) rats might be improved by metformin. Main methods: 16 week-old MSG rats were treated with metformin for 15 days and compared with age-matched untreated MSG and non-obese non-diabetic rats (control). Blood pressure, insulin sensitivity, vascular reactivity and prostanoid release in the perfused mesenteric arteriolar bed as well as nitric oxide production and reactive oxygen species generation in isolated mesenteric arteries were analyzed. Key findings: 18-week-old MSG rats displayed higher Lee index, fat accumulation, dyslipidemia, insulin resistance and hyperinsulinemia. Metformin treatment improved these alterations. The norepinephrine-induced response, increased in the mesenteric arteriolar bed from MSG rats, was corrected by metformin. Indomethacin corrected the enhanced contractile response in MSG rats but did not affect metformin effects. The sensitivity to acetylcholine, reduced in MSG rats, was also corrected by metformin. Indomethacin corrected the reduced sensitivity to acetylcholine in MSG rats but did not affect metformin effects. The sensitivity to sodium nitroprusside was increased in preparations from metformin-treated rats. Metformin treatment restored both the reduced PGI2/TXA2 ratio and the increased reactive oxygen species generation in preparations from MSG rats. Significance: Metformin improved the vascular function in MSG rats through reduction in reactive oxygen species generation, modulation of membrane hyperpolarization. correction of the unbalanced prostanoids release and increase in the sensitivity of the smooth muscle to nitric oxide. (c) 2011 Elsevier Inc. All rights reserved.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Fundacao de Amparo a Pesquisa doEstado de Sao Paulo (FAPESP)Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq)Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq)INCT Obesity and Diabetes/CNPq, BrazilINCT Obesity and Diabetes/CNPq, Brazi

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 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 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

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 angiotensin II receptors activation to the vascular effects of angiotensin.

<p>Mesenteric arteries with intact endothelium from control and monosodium glutamate-induced obese rats were pretreated with the AT1 receptor antagonist losartan (0.3 and 10 µM) (<b>A</b>) or the AT2 receptor antagonist PD 123319 (1 µM) (<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. ^†, P<0.05 vs. Control + losartan. N = 5–6/group.</p

Effects of angiotensin II on ERK 1/2 phosphorylation in mesenteric arteries.

<p>Panels show densitometric analysis of the Western blots for ERK1/2 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 AT1 receptor antagonist losartan (0.3 µM, 30 min), the AT2 receptor antagonist PD 123319 (1 µM, 30 min) or the ERK1/2 inhibitor PD98059 (1 µM, 30 min) and the phosphorylation of ERK 1/2 was examined. Total protein levels are shown as loading controls. Data are expressed as mean ± SEM. *, P<0.05 vs. Control. N = 5–6/group.</p