p38 mitogen-activated protein kinase is involved in arginase-II-mediated eNOS-uncoupling in obesity - PubMed (original) (raw)
p38 mitogen-activated protein kinase is involved in arginase-II-mediated eNOS-uncoupling in obesity
Yi Yu et al. Cardiovasc Diabetol. 2014.
Abstract
Background: Endothelial nitric oxide synthase (eNOS)-uncoupling links obesity-associated insulin resistance and type-II diabetes to the increased incidence of cardiovascular disease. Studies have indicated that increased arginase is involved in eNOS-uncoupling through competing with the substrate L-arginine. Given that arginase-II (Arg-II) exerts some of its biological functions through crosstalk with signal transduction pathways, and that p38 mitogen-activated protein kinase (p38mapk) is involved in eNOS-uncoupling, we investigated here whether p38mapk is involved in Arg-II-mediated eNOS-uncoupling in a high fat diet (HFD)-induced obesity mouse model.
Methods: Obesity was induced in wild type (WT) and Arg-II-deficient (Arg-II(-/-)) mice on C57BL/6 J background by high-fat diet (HFD, 55% fat) for 14 weeks starting from age of 7 weeks. The entire aortas were isolated and subjected to 1) immunoblotting analysis of the protein level of eNOS, Arg-II and p38mapk activation; 2) arginase activity assay; 3) endothelium-dependent and independent vasomotor responses; 4) en face staining of superoxide anion and NO production with Dihydroethidium and 4,5-Diaminofluorescein Diacetate, respectively, to assess eNOS-uncoupling. To evaluate the role of p38mapk, isolated aortas were treated with p38mapk inhibitor SB203580 (10 μmol/L, 1 h) prior to the analysis. In addition, the role of p38mapk in Arg-II-induced eNOS-uncoupling was investigated in cultured human endothelial cells overexpressing Arg-II in the absence or presence of shRNA against p38mapk.
Results: HFD enhanced Arg-II expression/activity and p38mapk activity, which was associated with eNOS-uncoupling as revealed by decreased NO and enhanced L-NAME-inhibitable superoxide in aortas of WT obese mice. In accordance, WT obese mice revealed decreased endothelium-dependent relaxations to acetylcholine despite of higher eNOS protein level, whereas Arg-II(-/-) obese mice were protected from HFD-induced eNOS-uncoupling and endothelial dysfunction, which was associated with reduced p38mapk activation in aortas of the Arg-II(-/-) obese mice. Moreover, overexpression of Arg-II in human endothelial cells caused eNOS-uncoupling and augmented p38mapk activation. The Arg-II-induced eNOS-uncoupling was prevented by silencing p38mapk. Furthermore, pharmacological inhibition of p38mapk recouples eNOS in isolated aortas from WT obese mice.
Conclusions: Taking together, we demonstrate here for the first time that Arg-II causes eNOS-uncoupling through activation of p38 mapk in HFD-induced obesity.
Figures
Figure 1
HFD feeding enhances eNOS, Arg-II expression/activity, and p38mapk activation in mouse aortas. (A) Immunoblotting analysis of eNOS total protein and eNOS-S1177 (p-eNOS) levels (n = 5). (B) Immunoblotting analysis of Arg-II (Arg-I not detectable, tubulin served as loading control), p38mapk activation, i.e., Thr180/Tyr182-phosphorylated p38mapk (p-p38mapk) and total p38mapk in the aortas of WT mice fed NC or HFD (n = 5), and arginase activity assay (n = 7-9). *p < 0.05 vs NC. In case of analyzing total protein levels such as Arg-II and eNOS, the ratio of Arg-II/tubulin or eNOS/tubulin in NC group serves as reference. The levels of eNOS and Arg-II expression in HFD group are calculated as fold changes to those of the NC group.
Figure 2
Arg-II gene deficiency prevents HFD-induced impairment of endothelium-dependent relaxation. (A) Endothelium-dependent relaxations in response to acetylcholine (ACh) were significantly impaired by HFD feeding in WT mice () and preserved in Arg-II-/- mice (). Arg-II deficiency had no significant effects in mice fed NC. (B) Arg-II deficiency does not affect endothelium-independent relaxations in response to the NO donor sodium nitroprusside (SNP) in animals fed HFD. n = 6, **p < 0.01 between WT HFD and Arg-II-/- HFD groups.
Figure 3
eNOS-uncoupling in obesity. Confocal microscopic en face detection of NO and O2- by DAF-2DA and DHE staining, respectively, followed by counterstaining with DAPI of aortas. Aortas of WT mice fed NC or HFD were treated with or without the eNOS inhibitor L-NAME (1 mmol/L) for 1 hour followed by acetylcholine (ACh) stimulation (1 μmol/L, 10 minutes) and then DAF-2DA and DHE staining. n = 5; ***p < 0.005 vs NC; †††p < 0.005 vs HFD group. Scale bar = 0.1 mm.
Figure 4
Decreased p38mapk activation in aortas of obese Arg-II -/- mouse. Immunoblotting analysis of the Thr180/Tyr182 phosphorylated p38mapk (p-p38mapk) and total p38mapk in aortas of WT and Arg-II-/- mice fed HFD. n = 11, ***p < 0.005 vs WT.
Figure 5
Arg-II gene deficiency and inhibition of p38mapk prevent eNOS-uncoupling in obesity. Confocal microscopic en face detection of NO with DAF-2DA and superoxide with DHE followed by counterstaining with DAPI in intact mouse aortas. Aortas from WT and Arg-II-/- mice fed HFD were cleaned of perivascular tissues, cut into two parts and subjected to treatment with either DMSO as control (Con) or SB203580 (SB, 10 μmol/L) for 1 hour followed by treatment with acetylcholine (ACh, 1 μmol/L) for 10 minutes before DAF-2DA and DHE staining. n = 5, **p < 0.01 and ***p < 0.005 vs WT/Con group. Scale bar = 0.1 mm.
Figure 6
p38mapk is involved in Arg-II-induced eNOS-uncoupling. (A) In cultured HUVECs, overexpressing Arg-II gene led to elevated p38mapk activation as measured by the Thr180/Tyr182 phosphorylated p38mapk (p-p38mapk). n = 6, **p < 0.01 vs Con group. (B) HUVECs were transduced first with rAd/U6-LacZshRNA as control or rAd/U6-p38shRNA. Twelve hours after the 1st transduction with the rAd/U6-shRNAs, the cells were then transduced either with rAd/CMV as control (Con) or with rAd/CMV-Arg-II to overexpress Arg-II (Arg-II). Experiments were performed 60 hours post the 2nd transduction (48 hours in 5% FCS-RPMI-1640 medium plus overnight serum-starvation in 0.2% FCS-RPMI-1640). Shown are representative images of four independent experiments. ***p < 0.005 vs Con/LacZ group, †††p < 0.005 vs Arg-II/LacZ group. Scale bar = 0.2 mm.
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