p38 mitogen-activated protein kinase is involved in arginase-II-mediated eNOS-Uncoupling in Obesity

Abstract

BackgroundEndothelial 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.MethodsObesity 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.ResultsHFD 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.ConclusionsTaking together, we demonstrate here for the first time that Arg-II causes eNOS-uncoupling through activation of p38 mapk in HFD-induced obesity.

A Novel Arginase Inhibitor Derived from Scutellavia indica Restored Endothelial Function in ApoE-Null Mice Fed a High-Cholesterol Diet

“…Therefore, arginase inhibition may have an alternative mechanism for eNOS coupling associated with phosphorylation that accompanies the increase in L-arginine concentration. Indeed, increased arginase activity with a high-fat diet (HFD) has been associated with p38 MAPK activation and subsequent eNOS uncoupling, whereas arginase II-null mice fed a HFD have reduced activation of p38 MAPK in the aorta, which protects them from eNOS uncoupling and endothelial dysfunction (Yu et al, 2014).…”

<em>En Face</em> Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

“…eNOS-uncoupling has been shown to be an important mechanism of endothelial dysfunction under numerous physiological and pathological conditions including aging 22 , atherosclerosis, and obesity 14 . Therefore, here we compare the lean and obese mice to show the representative result of NO and O 2 .− levels in the aortas.…”

“…Many research groups including ours have in recent years used the fluorescence dye method to detect intracellular production of NO [14][15][16][17][18][19] . In this method the cell permeable fluorescence indicator diaminofluorescein-2 diacetate (DAF-2DA) was used to measure free NO and NOS function in living cells and tissues in vitro or ex vivo.…”

“…The principle is that in the living cells, DAF-2DA is deacetylated by intracellular esterase to non-fluorescent 4,5-diaminofluorescein (DAF-2) which was then converted to fluorescent DAF-2 triazole (DAF-2T) by reacting with NO. The fluorescence from DAF-2T can be observed under a fluorescence microscope or a fluorescence confocal microscope 14 . The intracellular fluorescence intensity therefore reflects the intracellular NO production in the cells or the endothelium of an in intact blood vessel.…”

“…The intracellular fluorescence intensity therefore reflects the intracellular NO production in the cells or the endothelium of an in intact blood vessel. Combined with a specific fluorescence dye such as dihydroethidium (DHE), one can simultaneously assess intracellular NO and O 2 .− generation in the cells or in blood vessels 14 . Similarly, DHE is also a cell-permeable compound that is oxidized by O 2 .− inside the cells, and the oxidative product then intercalates with nucleic acids to emit a bright red color detectable quantitatively by fluorescent microscope or fluorescence confocal microscope.…”