Ji- Youn

Protein phosphatase 2Cm is a critical regulator of branched-chain amino acid catabolism in mice and cultured cells

The branched-chain amino acids (BCAA) are essential amino acids required for protein homeostasis, energy balance, and nutrient signaling. In individuals with deficiencies in BCAA, these amino acids can be preserved through inhibition of the branched-chain-alpha-ketoacid dehydrogenase (BCKD) complex, the rate-limiting step in their metabolism. BCKD is inhibited by phosphorylation of its E1alpha subunit at Ser293, which is catalyzed by BCKD kinase. During BCAA excess, phosphorylated Ser293 (pSer293) becomes dephosphorylated through the concerted inhibition of BCKD kinase and the activity of an unknown intramitochondrial phosphatase. Using unbiased, proteomic approaches, we have found that a mitochondrial-targeted phosphatase, PP2Cm, specifically binds the BCKD complex and induces dephosphorylation of Ser293 in the presence of BCKD substrates. Loss of PP2Cm completely abolished substrate-induced E1alpha dephosphorylation both in vitro and in vivo. PP2Cm-deficient mice exhibited BCAA catabolic defects and a metabolic phenotype similar to the intermittent or intermediate types of human maple syrup urine disease (MSUD), a hereditary disorder caused by defects in BCKD activity. These results indicate that PP2Cm is the endogenous BCKD phosphatase required for nutrient-mediated regulation of BCKD activity and suggest that defects in PP2Cm may be responsible for a subset of human MSUD.

Oxidative stress in atrial fibrillation: An emerging role of NADPH oxidase

Atrial fibrillation (AF) is the most common cardiac arrhythmia. Patients with AF have up to seven-fold higher risk of suffering from ischemic stroke. Better understanding of etiologies of AF and its thromboembolic complications are required for improved patient care, as current anti-arrhythmic therapies have limited efficacy and off target effects. Accumulating evidence has implicated a potential role of oxidative stress in the pathogenesis of AF. Excessive production of reactive oxygen species (ROS) is likely involved in the structural and electrical remodeling of the heart, contributing to fibrosis and thrombosis. In particular, NADPH oxidase (NOX) has emerged as a potential enzymatic source for ROS production in AF based on growing evidence from clinical and animal studies. Indeed, NOX can be activated by known upstream triggers of AF such as angiotensin II and atrial stretch. In addition, treatments such as statins, antioxidants, ACEI or AT1RB have been shown to prevent post-operative AF; among which ACEI/AT1RB and statins can attenuate NOX activity. On the other hand, detailed molecular mechanisms by which specific NOX isoform(s) are involved in the pathogenesis of AF and the extent to which activation of NOX plays a causal role in AF development remains to be determined. The current review discusses causes and consequences of oxidative stress in AF with a special focus on the emerging role of NOX pathways.

The p47phox- and NADPH oxidase organiser 1 (NOXO1)-dependent activation of NADPH oxidase 1 (NOX1) mediates endothelial nitric oxide synthase (eNOS) uncoupling and endothelial dysfunction in a streptozotocin-induced murine model of diabetes

Aims/hypothesisWe have previously shown that NADPH oxidase (NOX) lies upstream of uncoupled endothelial nitric oxide synthase (eNOS), which is known to occur in diabetic endothelium. However, it remains unclear which specific NOX isoform(s) is responsible for eNOS uncoupling and endothelial dysfunction in diabetic mouse models. The aim of the present study was to test the hypothesis that one or more NOX isoform(s) mediate(s) diabetic uncoupling of eNOS, which has been shown to occur in patients with diabetes to contribute to endothelial dysfunction.MethodsDiabetes was induced by streptozotocin administration. The Nω-nitro-l-arginine methyl ester (L-NAME)-sensitive superoxide production of aortic segments, reflective of eNOS uncoupling activity, was determined by electron spin resonance.ResultsThe L-NAME-sensitive superoxide production was more than doubled in wild-type diabetic mice, implicating uncoupling of eNOS. This was abolished in diabetic p47phox−/− (also known as Ncf1−/−) mice, but preserved in Nox2−/y (also known as Cybb−/−) mice made diabetic. The eNOS uncoupling activity was markedly attenuated in diabetic mice transfected with Nox1 or Nox1 organiser 1 (Noxo1) short interfering RNA (siRNA), and abolished in Nox1−/y diabetic mice. Diabetes-induced impairment in endothelium-dependent vasorelaxation was also significantly attenuated in the Nox1−/y mice made diabetic. By contrast, Nox4 siRNA, or inhibition of mitochondrial complex I or III with rotenone or siRNA, respectively, had no effect on diabetic uncoupling of eNOS. Overexpression of Dhfr, or oral administration of folic acid to improve dihydrofolate reductase (DHFR) function, recoupled eNOS in diabetes to improve endothelial function.Conclusions/interpretationOur data demonstrate for the first time that the p47phox and NOXO1-dependent activation of NOX1, but not that of NOX2, NOX4 or mitochondrion, mediates diabetic uncoupling of eNOS. NOX1-null mice are protected from diabetic endothelial dysfunction. Novel approaches to inhibit NOX1 and/or improve DHFR function, may prove to have therapeutic potential for diabetic endothelial dysfunction.

Role of Vascular Oxidative Stress in Obesity and Metabolic Syndrome

Obesity is associated with vascular diseases that are often attributed to vascular oxidative stress. We tested the hypothesis that vascular oxidative stress could induce obesity. We previously developed mice that overexpress p22phox in vascular smooth muscle, tgsm/p22phox, which have increased vascular ROS production. At baseline, tgsm/p22phox mice have a modest increase in body weight. With high-fat feeding, tgsm/p22phox mice developed exaggerated obesity and increased fat mass. Body weight increased from 32.16 ± 2.34 g to 43.03 ± 1.44 g in tgsm/p22phox mice (vs. 30.81 ± 0.71 g to 37.89 ± 1.16 g in the WT mice). This was associated with development of glucose intolerance, reduced HDL cholesterol, and increased levels of leptin and MCP-1. Tgsm/p22phox mice displayed impaired spontaneous activity and increased mitochondrial ROS production and mitochondrial dysfunction in skeletal muscle. In mice with vascular smooth muscle–targeted deletion of p22phox (p22phoxloxp/loxp/tgsmmhc/cre mice), high-fat feeding did not induce weight gain or leptin resistance. These mice also had reduced T-cell infiltration of perivascular fat. In conclusion, these data indicate that vascular oxidative stress induces obesity and metabolic syndrome, accompanied by and likely due to exercise intolerance, vascular inflammation, and augmented adipogenesis. These data indicate that vascular ROS may play a causal role in the development of obesity and metabolic syndrome.

Intralipid, a Clinically Safe Compound, Protects the Heart Against Ischemia-Reperfusion Injury More Efficiently Than Cyclosporine-A

Background:We have recently shown that postischemic administration of intralipid protects the heart against ischemia-reperfusion injury. Here we compared the cardioprotective effects of intralipid with cyclosporine-A, a potent inhibitor of the mitochondrial permeability transition pore opening. Methods:In vivo rat hearts or isolated Langendorff-perfused mouse hearts were subjected to ischemia followed by reperfusion with intralipid (0.5%, 1% and 2% ex-vivo, and 20% in vivo), cyclosporine-A (0.2 &mgr;M, 0.8 &mgr;M, and 1.5 &mgr;M ex- vivo and 10 mg/kg in vivo), or vehicle. The hemodynamic function, infarct size, calcium retention capacity, mitochodrial superoxide production, and phosphorylation levels of protein kinase B (Akt)/glycogen synthase kinase-3&bgr; (GSK-3&bgr;) were measured. The values are mean ± SEM. Results:Administration of intralipid at reperfusion significantly reduced myocardial infarct size compared with cyclosporine-A in vivo (infarct size/area at risk)%: 22.9 ± 2.5% vs. 35.2 ± 3.5%; P = 0.030, n = 7/group). Postischemic administration of intralipid at its optimal dose (1%) was more effective than cyclosporine-A (0.8 &mgr;M) in protecting the ex vivo heart against ischemia-reperfusion injury, as the rate pressure product at the end of reperfusion was significantly higher (mmHg · beats/min: 12,740 ± 675 [n = 7] vs. 9,203 ± 10,781 [n = 5], P = 0.024), and the infarct size was markedly smaller (17.3 ± 2.9 [n = 7] vs. 29.2 ± 2.7 [n = 5], P = 0.014). Intralipid was as efficient as cyclosporine-A in inhibiting the mitochondrial permeability transition pore opening (calcium retention capacity = 280 ± 8.2 vs. 260.3 ± 2.9 nmol/mg mitochondria protein in cyclosporine-A, P = 0.454, n = 6) and in reducing cardiac mitochondrial superoxide production. Unlike intralipid, which increased phosphorlyation of Akt (6-fold) and GSK-3&bgr; (5-fold), cyclosporine-A had no effect on the activation of these prosurvival kinases. Conclusions:Although intralipid inhibits the opening of the mitochondrial permeability transition pore as efficiently as cyclosporine-A, intralipid is more effective in reducing the infarct size and improving the cardiac functional recovery.

Mechanisms and consequences of endothelial nitric oxide synthase dysfunction in hypertension.

Reduced nitric oxide bioavailability contributes to endothelial dysfunction and hypertension. The endothelial isoform of nitric oxide synthase (eNOS) is responsible for the production of nitric oxide within the endothelium. Loss of eNOS cofactor tetrahydrobiopterin to initial increase in oxidative stress leads to uncoupling of eNOS, in which the enzyme produces superoxide anion rather than nitric oxide, further substantiating oxidative stress to induce vascular pathogenesis. The current review focuses on recent advances on the molecular mechanisms and consequences of eNOS dysfunction in hypertension, and potential novel therapeutic strategies restoring eNOS function to treat hypertension.

Endothelium-specific sepiapterin reductase deficiency in DOCA-salt hypertension

The endothelial nitric oxide synthase (eNOS) requires tetrahydrobiopterin (H(4)B) as a cofactor and, in its absence, produces superoxide (O(2)(·-)) rather than nitric oxide (NO(·)), a condition referred to as eNOS uncoupling. DOCA-salt-induced hypertension is associated with H(4)B oxidation and uncoupling of eNOS. The present study investigated whether administration of sepiapterin or H(4)B recouples eNOS in DOCA-salt hypertension. Bioavailable NO(·) detected by electron spin resonance was markedly reduced in aortas of DOCA-salt hypertensive mice. Preincubation with sepiapterin (10 μmol/l for 30 min) failed to improve NO(·) bioavailability in hypertensive aortas while it augmented NO(·) production from control vessels, implicating a hypertension-associated deficiency in sepiapterin reductase (SPR), the rate-limiting enzyme for sepiapterin conversion to H(4)B. Indeed, a decreased SPR expression was observed in aortic endothelial cells, but not in endothelium-denuded aortic remains, implicating an endothelium-specific SPR deficiency. Administration of hypertensive aortas with H(4)B (10 μmol/l, 30 min) partially restored vascular NO(·) production. Combined administration of H(4)B and the NADPH oxidase inhibitor apocynin (100 μmol/l, 30 min) fully restored NO(·) bioavailability while reducing O(2)(·-) production. In angiotensin II-induced hypertension, however, aortic endothelial SPR expression was not affected. In summary, administration of sepiapterin is not effective in recoupling eNOS in DOCA-salt hypertension, due to an endothelium-specific loss in SPR, whereas coadministration of H(4)B and apocynin is highly efficient in recoupling eNOS. This is consistent with our previous observations that in angiotensin II hypertension, endothelial deficiency in dihydrofolate reductase is alternatively responsible for uncoupling of eNOS. Taken together, these data indicate that strategies specifically targeting at different H(4)B metabolic enzymes might be necessary in restoring eNOS function in different types of hypertension.

Aminoguanidine inhibits aortic hydrogen peroxide production, VSMC NOX activity and hypercontractility in diabetic mice

BackgroundDysfunctionally uncoupled endothelial nitric oxide synthase (eNOS) is involved in producing reactive oxygen species (ROS) in the diabetic endothelium. The present study investigated whether anti-diabetes drug Aminoguanidine (AG) has any effect on eNOS function and vascular oxidant stress.Methods and ResultsBlood glucose levels were increased to 452.0 ± 15.1 mg/dl in STZ-treated male C57BL/6J mice (148.4 ± 3.2 mg/dl in untreated controls). Aortic productions of NO• and O2•- were measured specifically and sensitively using electron spin resonance. Diabetic mice had a marked increase in aortic O2•- production. Aortic hydrogen peroxide (H2O2) production was also increased in diabetic aortas and significantly attenuated by AG. AG however had only a marginal effect in reducing aortic O2•- production, which corresponded to a minimal effect in improving aortic nitric oxide (NO•) bioavailability. The endothelium-dependent vasodilatation however was modestly but significantly improved by AG, likely consequent to AG-induced reduction in hyper-contractility. N AD(P)H ox idase (NOX)-dependent O2•- production was completely attenuated by AG in endothelium-denuded diabetic aortas.ConclusionIn summary, despite that AG is not an effective eNOS recoupling agent presumably consequent to its ineffectiveness in preventing endothelial NOX activation, it is inhibitory of aortic H2O2 production, VSMC NOX activity, and hypercontractility in diabetes.

Bone Morphogenic Protein 4 Mediates NOX1-Dependent eNOS Uncoupling, Endothelial Dysfunction, and COX2 Induction in Type 2 Diabetes Mellitus

We have recently shown that angiotensin II-mediated uncoupling of endothelial nitric oxide synthase (eNOS) contributes to endothelial dysfunction in streptozotocin-induced type 1 diabetes mellitus. However, it has remained unclear whether and how eNOS uncoupling occurs in type 2 diabetes mellitus (T2DM) and the consequences of such in regulating vascular function. Here we investigated a role of bone morphogenic protein (BMP)-4 in mediating eNOS uncoupling, endothelial dysfunction, and inflammation in db/db mice. Circulating levels of BMP4 were markedly elevated in db/db mice but not in mice with type 1 diabetes mellitus, in which angiotensin II levels were significantly increased. Infusion of BMP4 antagonist noggin into db/db mice (15 μg/kg/day, 4 weeks) abolished eNOS uncoupling activity while restoring tetrahydrobiopterin (H(4)B) bioavailability. The impaired endothelium-dependent vasorelaxation in db/db aortas was significantly improved by noggin infusion. Exposure of aortic endothelial cells to BMP4 (50 ng/mL, 24 hours) resulted in eNOS uncoupling, which was attenuated by H(4)B precursor sepiapterin or small interfering RNA silencing nicotinamide adenine dinucleotide phosphate oxidase isoform 1 (NOX1). Interestingly, BMP4-dependent NOX1 up-regulation was abrogated by sepiapterin, implicating a NOX1-uncoupled eNOS-NOX1 feed-forward loop. BMP4 induction of cyclooxygenase 2 (COX2) expression and vascular cell adhesion protein 1 was found in db/db mice. Consistently, COX2 was up-regulated by BMP4 in endothelial cells, which was attenuated by sepiapterin, implicating an upstream role of eNOS uncoupling in COX2-mediated inflammatory activation. Taken together, our data for the first time reveal a novel role of BMP4 in inducing NOX1-dependent eNOS uncoupling in T2DM, which may promote development of novel therapeutics restoring endothelial function in T2DM.

Reply: To PMID 25882860.

We highly appreciate the quick responce from Mr. Karamat [1]. His letter provides a novel research area in regard to the relationship between creatine kinase (CK) and nitric oxide (NO), and his group have given some evidences that CK system is associated with blood pressure [2, 3]. However, our review mainly focused on the role of H4B deficiency in eNOS uncoupling in hypertension.