How the Location of Superoxide Generation Influences the β-Cell Response to Nitric Oxide

Cytokines impair the function and decrease the viability of insulin-producing β-cells by a pathway that requires the expression of inducible nitric oxide synthase (iNOS) and generation of high levels of nitric oxide. In addition to nitric oxide, excessive formation of reactive oxygen species, such as superoxide and hydrogen peroxide, has been shown to cause β-cell damage. Although the reaction of nitric oxide with superoxide results in the formation of peroxynitrite, we have shown that β-cells do not have the capacity to produce this powerful oxidant in response to cytokines. When β-cells are forced to generate peroxynitrite using nitric oxide donors and superoxide-generating redox cycling agents, superoxide scavenges nitric oxide and prevents the inhibitory and destructive actions of nitric oxide on mitochondrial oxidative metabolism and β-cell viability. In this study, we show that the β-cell response to nitric oxide is regulated by the location of superoxide generation. Nitric oxide freely diffuses through cell membranes, and it reacts with superoxide produced within cells and in the extracellular space, generating peroxynitrite. However, only when it is produced within cells does superoxide attenuate nitric oxide-induced mitochondrial dysfunction, gene expression, and toxicity. These findings suggest that the location of radical generation and the site of radical reactions are key determinants in the functional response of β-cells to reactive oxygen species and reactive nitrogen species. Although nitric oxide is freely diffusible, its biological function can be controlled by the local generation of superoxide, such that when this reaction occurs within β-cells, superoxide protects β-cells by scavenging nitric oxide.     

Suppressive Role of PPARγ-Regulated Endothelial Nitric Oxide Synthase in Adipocyte Lipolysis

Introduction

Metabolic syndrome causes insulin resistance and is associated with risk factor clustering, thereby increasing the risk of atherosclerosis. Recently, endothelial nitric oxide synthase deficient (eNOS-/-) mice have been reported to show metabolic disorders. Interestingly, eNOS has also been reported to be expressed in non-endothelial cells including adipocytes, but the functions of eNOS in adipocytes remain unclear.

Methods and Results

The eNOS expression was induced with adipocyte differentiation and inhibition of eNOS/NO enhanced lipolysis in vitro and in vivo. Furthermore, the administration of a high fat diet (HFD) was able to induce non-alcoholic steatohepatitis (NASH) in eNOS-/- mice but not in wild type mice. A PPARγ antagonist increased eNOS expression in adipocytes and suppressed HFD-induced fatty liver changes.

Conclusions

eNOS-/- mice induce NASH development, and these findings provide new insights into the therapeutic approach for fatty liver disease and related disorders.     

Exercise Protects against Chronic β-Adrenergic Remodeling of the Heart by Activation of Endothelial Nitric Oxide Synthase

Extensive data have shown that exercise training can provide cardio-protection against pathological cardiac hypertrophy. However, how long the heart can retain cardio-protective phenotype after the cessation of exercise is currently unknown. In this study, we investigated the time course of the loss of cardio-protection after cessation of exercise and the signaling molecules that are responsible for the possible sustained protection. Mice were made to run on a treadmill six times a week for 4 weeks and then rested for a period of 0, 1, 2 and 4 weeks followed by isoproterenol injection for 8 days. Morphological, echocardiographic and hemodynamic changes were measured, gene reactivation was determined by real-time PCR, and the expression and phosphorylation status of several cardio-protective signaling molecules were analyzed by Western-blot. HW/BW, HW/TL and LW/BW decreased significantly in exercise training (ER) mice. The less necrosis and lower fetal gene reactivation induced by isoproterenol injection were also found in ER mice. The echocardiographic and hemodynamic changes induced by β-adrenergic overload were also attenuated in ER mice. The protective effects can be sustained for at least 2 weeks after the cessation of the training. Western-blot analysis showed that the alterations in the phosphorylation status of endothelial nitric oxide synthase (eNOS) (increase in serine 1177 and decrease in threonine 495) continued for 2 weeks after the cessation of the training whereas increases of the phosphorylation of Akt and mTOR disappeared. Further study showed that L-NG-Nitroarginine methyl ester (L-NAME) treatment abolished the cardio-protective effects of ER. Our findings demonstrate that stimulation of eNOS in mice through exercise training provides acute and sustained cardioprotection against cardiac hypertrophy.     

β-Amyloid-Induced Endothelial Necrosis and Inhibition of Nitric Oxide Production

Deposits of amyloid β-peptide (Aβ) in senile plaques and cerebral blood vessels is the prominent feature of Alzheimer's disease (AD), regardless of genetic predisposition. The cellular origin of cerebral deposits of Aβ or its precise role in the neurodegenerative process has not been established. Recently we demonstrated a novel action of β-amyloid on blood vessels—vasoactivity and endothelial damage through superoxide radicals. Since endothelial dysfunction is associated with vascular degenerative diseases, we examined the direct action of Aβ on endothelial cells in culture. Cells treated with Aβ displayed characteristics of necrotic cell death which was prevented by the free radical scavenging enzyme superoxide dismutase. Stimulation of endothelial nitric oxide (NO) production by the calcium ionophore, A23187, or bradykinin was inhibited by β-amyloid. We conclude that an imbalance of NO and oxygen radicals may mediate the Aβ-induced endothelial damage on endothelial cells in culture and may also contribute to a variety of pathophysiological conditions associated with aging: hypertension, cerebral ischemia, vasospasm, or stroke.     

Insulin Reverses D-Glucose–Increased Nitric Oxide and Reactive Oxygen Species Generation in Human Umbilical Vein Endothelial Cells

Vascular tone is controlled by the L-arginine/nitric oxide (NO) pathway, and NO bioavailability is strongly affected by hyperglycaemia-induced oxidative stress. Insulin leads to high expression and activity of human cationic amino acid transporter 1 (hCAT-1), NO synthesis and vasodilation; thus, a protective role of insulin on high D-glucose–alterations in endothelial function is likely. Vascular reactivity to U46619 (thromboxane A₂ mimetic) and calcitonin gene related peptide (CGRP) was measured in KCl preconstricted human umbilical vein rings (wire myography) incubated in normal (5 mmol/L) or high (25 mmol/L) D-glucose. hCAT-1, endothelial NO synthase (eNOS), 42 and 44 kDa mitogen-activated protein kinases (p42/44^mapk), protein kinase B/Akt (Akt) expression and activity were determined by western blotting and qRT-PCR, tetrahydrobiopterin (BH₄) level was determined by HPLC, and L-arginine transport (0–1000 μmol/L) was measured in response to 5–25 mmol/L D-glucose (0–36 hours) in passage 2 human umbilical vein endothelial cells (HUVECs). Assays were in the absence or presence of insulin and/or apocynin (nicotinamide adenine dinucleotide phosphate-oxidase [NADPH oxidase] inhibitor), tempol or Mn(III)TMPyP (SOD mimetics). High D-glucose increased hCAT-1 expression and activity, which was biphasic (peaks: 6 and 24 hours of incubation). High D-glucose–increased maximal transport velocity was blocked by insulin and correlated with lower hCAT-1 expression and SLC7A1 gene promoter activity. High D-glucose–increased transport parallels higher reactive oxygen species (ROS) and superoxide anion (O₂^•–) generation, and increased U46619-contraction and reduced CGRP-dilation of vein rings. Insulin and apocynin attenuate ROS and O₂^•– generation, and restored vascular reactivity to U46619 and CGRP. Insulin, but not apocynin or tempol reversed high D-glucose–increased NO synthesis; however, tempol and Mn(III)TMPyP reversed the high D-glucose–reduced BH₄ level. Insulin and tempol blocked the high D-glucose–increased p42/44^mapk phosphorylation. Vascular dysfunction caused by high D-glucose is likely attenuated by insulin through the L-arginine/NO and O₂^•–/NADPH oxidase pathways. These findings are of interest for better understanding vascular dysfunction in states of foetal insulin resistance and hyperglycaemia.     

Nitric Oxide Synthase 2 Improves Proliferation and Glycolysis of Peripheral γδ T Cells

γδ T cells play critical roles in host defense against infections and cancer. Although advances have been made in identifying γδ TCR ligands, it remains essential to understand molecular mechanisms responsible for in vivo expansion of γδ T cells in periphery. Recent findings identified the expression of the inducible NO synthase (NOS2) in lymphoid cells and highlighted novel immunoregulatory functions of NOS2 in αβ T cell differentiation and B cell survival. In this context, we wondered whether NOS2 exerts an impact on γδ T cell properties. Here, we show that γδ T cells express NOS2 not only in vitro after TCR triggering, but also directly ex vivo. Nos2 deficient mice have fewer γδ T cells in peripheral lymph nodes (pLNs) than their wild-type counterparts, and these cells exhibit a reduced ability to produce IL-2. Using chemical NOS inhibitors and Nos2 deficient γδ T cells, we further evidence that the inactivation of endogenous NOS2 significantly reduced γδ T cell proliferation and glycolysis metabolism that can be restored in presence of exogenous IL-2. Collectively, we demonstrate the crucial role of endogenous NOS2 in promoting optimal IL-2 production, proliferation and glycolysis of γδ T cells that may contribute to their regulation at steady state.     

Enhancement of Nitric Oxide and Superoxide Generations by α-Tocopheryl Succinate and Its Apoptotic and Anticancer Effects

Tocopheryl succinate (TS), a succinyl ester of alpha-tocopherol (alpha-T), has been reported to have various biological activities. In this communication, we review the current findings about TS including our recent studies of its effects on nitric oxide (NO) and superoxide (O2-) generations implicated in cancer and atherosclerosis. First, we investigated the effect of TS on NO production in vascular smooth muscle cells (VSMC) under atherosclerosis-like conditions using lipopolysaccharide (LPS) and interferon-gamma (IFN). TS enhanced LPS/IFN-dependent NO production, but alpha-T itself did not. The enhancement by TS of NO production was inhibited by alpha-T but not by antioxidants such as ascorbic acid and 2[3]-t-butyl-4-hydroxyanisole (BHA). TS enhanced the amount of protein kinase Calpha (PKCalpha) in VSMC, and PKC inhibitors inhibited TS-enhanced NO production, suggesting that the enhancing effect of TS on NO production is caused by up-regulation of PKC. Second, we found that TS induced apoptosis in VSMC associated with increase in O2- generation via NADPH-dependent oxidase. We further observed that a mouse breast cancer cell line C127I was more susceptible for TS-induced apoptosis than a mouse breast normal cell line NmuMG, and that superoxide dismutase, alpha-T, and BHA inhibited TS-caused morphological cell damage in C127I. From these results, O2- itself and/or other reactive oxygen species are assumed to associate with TS-induced cell toxicity, and antioxidative defense systems are supposed to be lowered in cancer cells. Finally, we found that intravenous injection of TS vesicles completely inhibited the growth of melanoma cells B16-F1 inoculated on the back of hairless mice and enhanced their survival time.     

Nitric Oxide from IFNγ-Primed Macrophages Modulates the Antimicrobial Activity of β-Lactams against the Intracellular Pathogens Burkholderia pseudomallei and Nontyphoidal Salmonella

Our investigations show that nonlethal concentrations of nitric oxide (NO) abrogate the antibiotic activity of β-lactam antibiotics against Burkholderia pseudomallei, Escherichia coli and nontyphoidal Salmonella enterica serovar Typhimurium. NO protects B. pseudomallei already exposed to β-lactams, suggesting that this diatomic radical tolerizes bacteria against the antimicrobial activity of this important class of antibiotics. The concentrations of NO that elicit antibiotic tolerance repress consumption of oxygen (O₂), while stimulating hydrogen peroxide (H₂O₂) synthesis. Transposon insertions in genes encoding cytochrome c oxidase-related functions and molybdenum assimilation confer B. pseudomallei a selective advantage against the antimicrobial activity of the β-lactam antibiotic imipenem. Cumulatively, these data support a model by which NO induces antibiotic tolerance through the inhibition of the electron transport chain, rather than by potentiating antioxidant defenses as previously proposed. Accordingly, pharmacological inhibition of terminal oxidases and nitrate reductases tolerizes aerobic and anaerobic bacteria to β-lactams. The degree of NO-induced β-lactam antibiotic tolerance seems to be inversely proportional to the proton motive force (PMF), and thus the dissipation of ΔH⁺ and ΔΨ electrochemical gradients of the PMF prevents β-lactam-mediated killing. According to this model, NO generated by IFNγ-primed macrophages protects intracellular Salmonella against imipenem. On the other hand, sublethal concentrations of imipenem potentiate the killing of B. pseudomallei by NO generated enzymatically from IFNγ-primed macrophages. Our investigations indicate that NO modulates the antimicrobial activity of β-lactam antibiotics.     

Elevated Body Fat in Rats by the Dietary Nitric Oxide Synthase Inhibitor,L-Nω Nitroarginine

The influence of the dietary nitric oxide (NO) synthase inhibitor, L-N^ω nitroarginine (L-NNA) on body fat was examined in rats. In experiment 1, all rats were fed with the same amount of diet with or without 0.02% L-NNA for 8 wk. L-NNA intake caused elevations in serum triglyceride and body fat, and reduction in serum nitrate (a metabolite of nitric oxide). The activity of hepatic carnitine palmitoyltransferase was reduced by L-NNA. In experiment 2, rats were fed for 8 wk with the same amount of diets with or without 0.02% L-NNA supplemented or not with 4% L-arginine. The elevation in body fat, and the reductions in serum nitrate and in the activity of hepatic carnitine palmitoyltransfererase by L-NNA were all suppressed by supplemental L-arginine. The results suggest that lower NO generation elevated not only serum triglyceride, but also body fat by reduced fatty acid oxidation.