Insulin Resistance, Anti-inflammatory, and Antioxidant In vitro Activity of Heterologously Expressed Arenin from Dryophytes arenicolor

Abstracts

Green tea consumption is associated with reduced cardiovascular mortality in some epidemiological studies. Epigallocatechin gallate (EGCG), a bioactive polyphenol in green tea, mimics metabolic actions of insulin to inhibit gluconeogenesis in hepatocytes. Because signaling pathways regulating metabolic and vasodilator actions of insulin are shared in common, we hypothesized that EGCG may also have vasodilator actions to stimulate production of nitric oxide (NO) from endothelial cells. Acute intra-arterial administration of EGCG to mesenteric vascular beds isolated ex vivo from WKY rats caused dose-dependent vasorelaxation. This was inhibitable by L-NAME (NO synthase inhibitor), wortmannin (phosphatidylinositol 3-kinase inhibitor), or PP2 (Src family kinase inhibitor). Treatment of bovine aortic endothelial cells (BAEC) with EGCG (50 microm) acutely stimulated production of NO (assessed with NO-specific fluorescent dye DAF-2) that was inhibitable by l-NAME, wortmannin, or PP2. Stimulation of BAEC with EGCG also resulted in dose- and time-dependent phosphorylation of eNOS that was inhibitable by wortmannin or PP2 (but not by MEK inhibitor PD98059). Specific knockdown of Fyn (but not Src) with small interfering RNA inhibited both EGCG-stimulated phosphorylation of Akt and eNOS as well as production of NO in BAEC. Treatment of BAEC with EGCG generated intracellular H(2)O(2) (assessed with H(2)O(2)-specific fluorescent dye CM-H(2)DCF-DA), whereas treatment with N-acetylcysteine inhibited EGCG-stimulated phosphorylation of Fyn, Akt, and eNOS. We conclude that EGCG has endothelial-dependent vasodilator actions mediated by intracellular signaling pathways requiring reactive oxygen species and Fyn that lead to activation of phosphatidylinositol 3-kinase, Akt, and eNOS. This mechanism may explain, in part, beneficial vascular and metabolic health effects of green tea consumption.

Skin, our first interface to the external environment, is subjected to oxidative stress caused by a variety of factors such as solar ultraviolet, infrared and visible light, environmental pollution, including ozone and particulate matters, and psychological stress. Excessive reactive species, including reactive oxygen species and reactive nitrogen species, exacerbate skin pigmentation and aging, which further lead to skin tone unevenness, pigmentary disorder, skin roughness and wrinkles. Besides these, skin microbiota are also a very important factor ensuring the proper functions of skin. While environmental factors such as UV and pollutants impact skin microbiota compositions, skin dysbiosis results in various skin conditions. In this review, we summarize the generation of oxidative stress from exogenous and endogenous sources. We further introduce current knowledge on the possible roles of oxidative stress in skin pigmentation and aging, specifically with emphasis on oxidative stress and skin pigmentation. Meanwhile, we summarize the science and rationale of using three well-known antioxidants, namely vitamin C, resveratrol and ferulic acid, in the treatment of hyperpigmentation. Finally, we discuss the strategy for preventing oxidative stress-induced skin pigmentation and aging.

Unravelling the reactive oxygen and reactive nitrogen signalling networks in plants.

Nitric Oxide (NO) is a second messenger related to development and (a)biotic stress responses in plants. We have studied the role of NO in signaling during plant defense responses upon xylanase elicitation. Treatment of tomato cell cultures with the fungal elicitor xylanase resulted in a rapid and dose-dependent NO accumulation. We have demonstrated that NO is required for the production of the lipid second messenger phosphatidic acid (PA) via the activation of the phospholipase C (PLC) and diacylglycerol kinase (DGK) pathway. Defense-related responses downstream of PA were studied. PA and, correspondingly, xylanase were shown to induce reactive oxygen species production. Scavenging of NO or inhibition of either the PLC or the DGK enzyme diminished xylanase-induced reactive oxygen species production. Xylanase-induced PLDbeta1 and PR1 mRNA levels decreased when NO or PA production were compromised. Finally, we have shown that NO and PA are involved in the induction of cell death by xylanase. Treatment with NO scavenger cPTIO, PLC inhibitor U73122, or DGK inhibitor R59022 diminished xylanase-induced cell death. On the basis of biochemical and pharmacological experimental results, we have shown that PLC/DGK-derived PA represents a novel downstream component of NO signaling cascade during plant defense.

<i>Circulation Research</i> Thematic Synopsis Diabetes and Obesity

Hyperinsulinemia is a critical risk factor for the pathogenesis of insulin resistance (IR) in metabolic tissues, including the liver. Ethanolamine phosphate phospholyase (ETNPPL), a newly discovered metabolic enzyme that converts phosphoethanolamine (PEA) to ammonia, inorganic phosphate, and acetaldehyde, is abundantly expressed in liver tissue. Whether it plays a role in the regulation of hyperinsulinemia-induced IR in hepatocytes remains elusive. Here, we established an in vitro hyperinsulinemia-induced IR model in the HepG2 human liver cancer cell line and primary mouse hepatocyte via a high dose of insulin treatment. Next, we overexpressed ETNPPL by using lentivirus-mediated ectopic to investigate the effects of ETNPPL per se on IR without insulin stimulation. To explore the underlying mechanism of ETNPPL mediating hyperinsulinemia-induced IR in HepG2, we performed genome-wide transcriptional analysis using RNA sequencing (RNA-seq) to identify the downstream target gene of ETNPPL. The results showed that ETNPPL expression levels in both mRNA and protein were significantly upregulated in hyperinsulinemia-induced IR in HepG2 and primary mouse hepatocytes. Upon silencing ETNPPL, hyperinsulinemia-induced IR was ameliorated. Under normal conditions without IR in hepatocytes, overexpressing ETNPPL promotes IR, reactive oxygen species (ROS) generation, and AKT inactivation. Transcriptome analysis revealed that salt-inducible kinase 1 (SIK1) is markedly downregulated in the ETNPPL knockdown HepG2 cells. Moreover, disrupting SIK1 prevents ETNPPL-induced ROS accumulation, damage to the PI3K/AKT pathway and IR. Our study reveals that ETNPPL mediates hyperinsulinemia-induced IR through the SIK1/ROS-mediated inactivation of the PI3K/AKT signaling pathway in hepatocyte cells. Targeting ETNPPL may present a potential strategy for hyperinsulinemia-associated metabolic disorders such as type 2 diabetes.

Kallistatin is an endogenous protein that exerts pleiotropic effects, including vasodilation and inhibition of angiogenesis, inflammation, oxidative stress, apoptosis, fibrosis, and tumor progression. Through its two functional domains – an active site and a heparin-binding site – kallistatin regulates differential signaling pathways and a wide spectrum of biological functions. Kallistatin's active site is key for inhibiting tissue kallikrein activity, and stimulating the expression of endothelial nitric oxide synthase (eNOS), sirtuin 1 (SIRT1) and suppressor of cytokine signaling 3 (SOCS3). Kallistatin via its heparin-binding site blocks signaling pathways mediated by growth factors and cytokines, such as vascular endothelial growth factor (VEGF), tumor necrosis factor-α (TNF-α), high mobility group box-1 (HMGB1), Wnt, transforming growth factor-β (TGF-β), and epidermal growth factor (EGF). Kallistatin gene or protein delivery protects against the pathogenesis of hypertension, heart and kidney damage, arthritis, sepsis, influenza virus infection, tumor growth and metastasis in animal models. Conversely, depletion of endogenous kallistatin by neutralizing antibody injection exacerbates cardiovascular and renal injury in hypertensive rats. Kallistatin levels are markedly reduced in rodents with hypertension, sepsis, streptozotocin-induced diabetes, and cardiac and renal injury. Kallistatin levels are also diminished in patients with liver disease, septic syndrome, diabetic retinopathy, severe pneumonia, inflammatory bowel disease, and obesity, prostate and colon cancer. Therefore, circulating kallistatin levels may serve as a new biomarker for human diseases. This review summarizes kallistatin's protective roles and mechanisms in vascular and organ injury, and highlights the therapeutic potential of kallistatin for multiple disease states.

the discovery of nitric oxide (NO) production from l-arginine by NO synthases has revolutionized our understanding of many aspects of human physiology. Since the seminal studies of Furchgott and Zawadzki ([7][1]), tremendous progress has been made in understanding the regulation of vascular tone by

Hyperhomocysteinemia is believed to induce endothelial dysfunction and promote atherosclerosis; however, the pathogenic mechanism has not been clearly elucidated. In this study, we examined the molecular mechanism by which homocysteine (HCy) causes endothelial cell apoptosis and by which nitric oxide (NO) affects HCy-induced apoptosis. Our data demonstrated that HCy caused caspase-dependent apoptosis in cultured human umbilical vein endothelial cells, as determined by cell viability, nuclear condensation, and caspase-3 activation and activity. These apoptotic characteristics were correlated with reactive oxygen species (ROS) production, lipid peroxidation, p53 and Noxa expression, and mitochondrial cytochrome c release following HCy treatment. HCy also induced p53 and Noxa expression and apoptosis in endothelial cells from wild type mice but not in the p53-deficient cells. The NO donor S-nitroso-N-acetylpenicillamine, adenoviral transfer of inducible NO synthase gene, and antioxidants (alpha-tocopherol and superoxide dismutase plus catalase) but not oxidized SNAP, 8-Br-cGMP, nitrite, and nitrate, suppressed ROS production, p53-dependent Noxa expression, and apoptosis induced by HCy. The cytotoxic effect of HCy was decreased by small interfering RNA-mediated suppression of Noxa expression, indicating that Noxa up-regulation plays an important role in HCy-induced endothelial cell apoptosis. Overexpression of inducible NO synthase increased the formation of S-nitroso-HCy, which was inhibited by the NO synthase inhibitor N-monomethyl-l-arginine. Moreover, S-nitroso-HCy did not increase ROS generation, p53-dependent Noxa expression, and apoptosis. These results suggest that up-regulation of p53-dependent Noxa expression may play an important role in the pathogenesis of atherosclerosis induced by HCy and that an increase in vascular NO production may prevent HCy-induced endothelial dysfunction by S-nitrosylation.

Gupta, K. J., Hinch, D. K., Mur, L. A. (2011). NO way to treat a cold. New Phytologist, 189, (2), 360-363. IMPF: 06.64

Laminar shear stress (LSS) is known to increase endothelial nitric oxide (NO) production, which is essential for vascular health, through expression and activation of nitric oxide synthase 3 (NOS3). Recent studies demonstrated that LSS also increases the expression of argininosuccinate synthetase 1 (ASS1) that regulates the provision of L-arginine, the substrate of NOS3. It was thus hypothesized that ASS1 might contribute to vascular health by enhancing NO production in response to LSS. This hypothesis was pursued in the present study by modulating NOS3 and ASS1 levels in cultured endothelial cells. Exogenous expression of either NOS3 or ASS1 in human umbilical vein endothelial cells increased NO production and decreased monocyte adhesion stimulated by tumor necrosis factor-α (TNF-α). The latter effect of overexpressed ASS1 was reduced when human umbilical vein endothelial cells were co-treated with small interfering RNAs (siRNAs) for ASS1 or NOS3. SiRNAs of NOS3 and ASS1 attenuated the increase of NO production in human aortic endothelial cells stimulated by LSS (12 dynes·cm(-2)) for 24 h. LSS inhibited monocyte adhesion to human aortic endothelial cells stimulated by TNF-α, but this effect of LSS was abrogated by siRNAs of NOS3 and ASS1 that recovered the expression of vascular cell adhesion molecule-1. The current study suggests that the expression of ASS1 harmonized with that of NOS3 may be important for the optimized endothelial NO production and the prevention of the inflammatory monocyte adhesion to endothelial cells.

Sleep-disordered breathing (SDB), which includes obstructive sleep apnea (OSA) as its most extreme variant, is characterized by intermittent episodes of partial or complete obstruction of the upper airway during sleep that disrupts normal ventilation and sleep architecture and is typically associated with snoring and daytime sleepiness. SDB is common, with an incidence in middle-aged men and women of 4% and 2%, respectively.1 Major risk factors for SDB include obesity, male gender, increasing age, and abnormalities of craniofacial morphology.2 There is an increasing perception that SDB/OSA via various mechanisms increases cardiovascular morbidity and mortality (Figure 1). However, many risk factors for SDB/OSA, such as obesity and male gender, are the same as for hypertension and cardiovascular disease.3 Thus, only recently has there been converging evidence that SDB is a risk factor for their development.4–6 Moreover, there is increasing information to indicate that SDB/OSA is linked to metabolic, vascular, hematologic, and genetic markers associated with increased cardiovascular disease risk. In addition, central sleep apnea (CSA), another form of SDB, appears to be an important factor that influences morbidity and mortality among those with heart failure (HF). Nevertheless, responsible mechanisms, the role of SDB as a risk factor “independent” of associated comorbidities, and whether treatment of SDB will mitigate this risk are unknown and remain to be determined. Figure 1. This illustrates the range of cardiovascular diseases and potential mechanisms that may be associated with sleep-disordered breathing. As illustrated in this figure, there is likely an interaction between multiple potential mechanisms, particularly in individuals who may have a genetic susceptibility for sleep-disordered breathing. This report summarizes the proceedings of a workshop sponsored by the National Center on Sleep Disorders Research and the National Heart, Lung, and Blood Institute on September 12 to 13, 2002, to assess a broad array …

Although infants with persistent pulmonary hypertension of the newborn (PPHN) experience some relief and therapeutic benefit from current therapies, over 50% have a limited or transient response and significant morbidity. There is no consistency in the best first line treatment throughout hospitals in the United States. Ventilation with high levels of oxygen or inhaled nitric oxide (NO) are typical strategies for improving the extracorporeal membrane oxygen, although they remain unproven to increase survival rates. While oxygen may stimulate endothelial nitric oxide synthase (eNOS) and NO production dilating the pulmonary vasculature, it also fuels the production of reactive oxygen species (ROS). ROS is likely to have counterproductive effects; in addition to stimulating vascular smooth muscle cell proliferation and increasing vascular tone, ROS may directly regulate eNOS and NO. The recent article by Farrow and colleagues (3) in AJP-Lung investigates the role of ROS on eNOS. By using recombinant human superoxide dismutase (rhSOD), they observed 1) increased eNOS activity and expression, 2) increased tetrahydrobiopterin (BH4), a cofactor critical to the function of eNOS, and 3 )a decrease in oxidative stress, in addition to the stimulation of NO production and ultimately pulmonary vasodilatation. The observations they made may be paramount to increasing the survival of infants with PPHN and may lead to an adapted treatment regimen that addresses the pitfalls of current therapeutic approaches. PPHN When the pulmonary circulation fails to respond to natural stimuli, including increased oxygen tension, ventilation, and shear stress, it does not undergo the shift from the high resistance state in utero to a postnatal low resistance system, enabling efficient pulmonary gas exchange and oxygenation. Impaired NO-cGMP signaling has been shown to be critical to the regulation of pulmonary circulation in the newborn, and clinical strategies have involved administration of inhaled NO since the early 1990s (6, 10). While effective in immediate relief due to vasodilatation, the infants can enter an inhaled NO dependency state, and thus inhaled NO proffers poor long-term relief. The necessity for extensive research into the regulation of perinatal circulation and the changes that occur upon ventilation have led to improved and more specific therapeutic approaches for infants with PPHN over the past 30 years. Despite this, PPHN is still associated with significant shortterm and long-term morbidity. Farrow et al. (3) strive to dissect the signaling pathways, determining the impact of ROS and elucidating the potential of decreasing oxidative stress as a therapeutic approach in PPHN. This study is published in a milieu of recent publications exploring the functional abnor

Periodontal disease in polycystic ovary syndrome