Urotensin Ⅱ-induced insulin resistance is mediated by NADPH oxidase-derived reactive oxygen species in Hep G2 cells

AIM: To investigated the effects of urotensin Ⅱ(UII) on hepatic insulin resistance in Hep G2 cells and the potential mechanisms involved.METHODS: Human hepatoma Hep G2 cells were cultured with or without exogenous UII for 24 h, in the presence or absence of 100 nmol/L insulin for the last 30 min. Glucose levels were detected by the glucoseoxidase method and glycogen synthesis was analyzed by glycogen colorimetric/fluorometric assay. Reactive oxygen species(ROS) levels were detected with a multimode reader using a 2′,7′-dichlorofluorescein diacetate probe. The protein expression and phosphorylation levels of c-Jun N-terminal kinase(JNK), insulin signal essential molecules such as insulin receptor substrate-1(IRS-1), protein kinase B(Akt), glycogen synthase kinase-3β(GSK-3β), and glucose transporter-2(Glut 2), and NADPH oxidase subunits such as gp91 phox, p67 phox, p47 phox, p40 phox, and p22 phox were evaluated by Western blot.RESULTS: Exposure to 100 nmol/L UII reduced the insulin-induced glucose consumption(P 0.05)and glycogen content(P 0.01) in Hep G2 cells compared with cells without UII. UII also abolished insulin-stimulated protein expression(P 0.01) and phosphorylation of IRS-1(P 0.05), associated with down-regulation of Akt(P 0.05) and GSK-3β(P 0.05) phosphorylation levels, and the expression of Glut 2(P 0.001), indicating an insulin-resistance state in Hep G2 cells. Furthermore, UII enhanced the phosphorylation of JNK(P 0.05), while the activity of JNK, insulin signaling, such as total protein of IRS-1(P 0.001), phosphorylation of IRS-1(P 0.001) and GSK-3β(P 0.05), and glycogen synthesis(P 0.001) could be reversed by pretreatment with the JNK inhibitor SP600125. Besides, UII markedly improved ROS generation(P 0.05) and NADPH oxidase subunit expression(P 0.05). However, the antioxidant/NADPH oxidase inhibitor apocynin could decrease UII-induced ROS production(P 0.05), JNK phosphorylation(P 0.05), and insulin resistance(P 0.05) in HepG 2 cells. CONCLUSION: UII induces insulin resistance, and this can be reversed by JNK inhibitor SP600125 and antioxidant/NADPH oxidase inhibitor apocynin targeting the insulin signaling pathway in HepG 2 cells.     

Cocaine, not morphine, causes the generation of reactive oxygen species and activation of NF-κB in transiently cotransfected heart cells

This study was designed to determine levels of NF-κB reporter gene activity and free radical generation in cultured striated myocytes (H9C2 cells) exposed to cocaine or morphine in the presence of free radical scavengers. Cells were transiently transfected with a NF-κB reporter gene and changes in luciferase activity were detected, by bioluminescence. Using confocal microscopy and 2′,7′-dichlorofluorescin diacetate, cocaine-induced or morphine-induced free radicals were quantified in H9C2 cells. Cocaine and morphine (0–1×10⁻² M) were tested separately. Cocaine but not morphine significantly activated Nf-κB reporter gene, activity in H9C2 cells. Overexpression of IκB inhibited NF-κB reporter activity at low (1×10⁻⁴ M) but not high (1×10⁻² M) cocaine concentrations. Free radicals were generated in H9C2 cells stimulated with cocaine but not with morphine. The production of free radicals and NF-κB reporter gene activity could be blocked with N-acetylcysteine, glutathione, and to a lesser extent, lipoic acid. The results suggest that cocaine induces free radical production, which leads to the activation of NF-κB signal transduction and possible inflammatory responses.     

The inhibition in tumor necrosis factor-α-induced attenuation in endothelial thrombomodulin expression by carvedilol is mediated by nuclear factor-κB and reactive oxygen species

Carvedilol, a nonselective β-adrenoceptor antagonist, has been shown to possess antioxidant effects and reduce the risk of hospitalization and death in patients with severe congestive heart failure, which is featured by the activation of pro-inflammatory cytokines, including tumor necrosis factor-α (TNF-α), and leads to thrombotic complications. Thrombomodulin (TM) plays protective roles against thrombosis. Treatment of ECs with TNF-α resulted in a down-regulation in the TM expression in a time-dependent manner. Pre-treatment of ECs with carvedilol (1 and 10 μM) for 1 h significantly up-regulated the TM expression in ECs in response to TNF-α. When ECs were pre-treated with a nuclear factor-κB (NF-κB) inhibitor, i.e., parthenolide, their TNF-α-mediated down-regulation of TM expression was inhibited. Pre-treatment of ECs with carvedilol inhibited the NF-κB-DNA binding activity in ECs induced by TNF-α. Our findings provide insights into the mechanisms by which carvedilol exerts anti-thrombotic effects by inducing TM expression in ECs in response to pro-inflammatory stimulation.     

Production of reactive oxygen species and expression of inducible nitric oxide synthase in rat isolated Kupffer cells stimulated by Leptospira interrogans and Borrelia burgdorfen

AIM: To evaluate the production of reactive oxygen species (ROS) and the expression of inducible nitric oxide synthase (iNOS) in rat isolated Kupffer cells (KCs) stimulated by Leptospira interrogans and Borrelia burgdorferi. METHODS: Rat Kupffer cells were separated by perfu-sion of the liver with 0.05% collagenase, and purified by Percoll gradients. Purified Kupffer cells were tested in vitro with alive L.interogans and B. burgdorferi preparations. The production of ROS was determined by che-miluminescence, whereas iNOS protein expression was evaluated by Western blot assay using anti-iNOS antibodies. RESULTS: B. burgdorferi and to a less extent L interrogans induced ROS production with a peak 35 min after infection. The chemiluminescence signal progressively diminished and was undetectable by 180 min of incubation. Leptospirae and borreliae induced an increased iNOS expression in Kupffer cells that peaked at 6 hours and was still evident 22 h after infection. CONCLUSION: Both genera of spirochetes induced ROS and iNOS production in rat Kupffer cells. Since the cause of liver damage both in leptospiral as well as in borreliai infections are still unknown, we suggest that leptospira and borrelia damage of the liver can be initially mediated by oxygen radicals, and is then maintained at least in part by nitric oxide.     

Changes of NF—kB,p53,Bcl—2 and caspase in apoptosis induced by JTE—522 in human gastric adenocarcinoma cell line AGS cells：role of reactive oxygen species

AIM:To identify whether JTE-522 can induce apoptosis in AGS cells and ROS also involved in the process,and to investigate the changes in NF-kB,p53,bcl-2 and caspase in the apoptosis process.METHODS:Cell culture,MTT,Electromicroscopy,agarose gel electrophoresis,lucigenin,western blot and electrophoretic mobility shift assay(EMSA)analysis were employed to investigate the effect of JTE-522 on cell proliferation and apoptosis in AGS cells and related molecular mechanisms.RESULTS:JTE-522 inhitied the growth of AGS cells and induced the apoptosis,Lucigenin assay showed thd generation of ROS in cells under incubation with JTE-522,The increased ROS generation might Contribute to the induction of AGS cells to apoptosis.EMSA and Western blot revealed that NF-kB activity was almost completely inhihbited by preventing the degradation of IKBα,Additionally,by using Western blot we confirmed that the level of bcl-2 was decreased,whtereas p53 showed a great inmcrease following JTE-522 treatment.Their changes were in a dose-dependent manner. CONCLUSION：These findings suggest that reactive oxygen species,NF-kB,p53,bcl-2 and caspase-3 may play an important role in the induction of apoptosis,in ags cells after treatment with JTE-522.     

Sca-1 is a marker for cell plasticity in murine pancreatic epithelial cells and induced by IFN-β in vitro

Background & aimsSca-1 is a surface marker for murine hematopoietic stem cells (HSCs) and type-I interferon is a key regulator for Lin^-Sca-1⁺ HSCs expansion through Ifnar/Stat-1/Sca-1-signaling. In this study we aimed to characterize the role and regulation of Sca-1⁺ cells in pancreatic regeneration.MethodsTo characterize Sca-1 in vivo, immunohistochemistry and immunofluorescence staining of Sca-1 was conducted in normal pancreas, in cerulein-mediated acute pancreatitis, and in Kras-triggered cancerous lesions. Ifnar/Stat-1/Sca-1-signaling was studied in type-I IFN-treated epithelial explants of adult wildtype, Ifnar^-/-, and Stat-1^-/- mice. Sca-1 induction was analyzed by gene expression and FACS analysis. After isolation of pancreatic epithelial Lin^-Sca-1⁺cells, pancreatosphere-formation and immunofluorescence-assays were carried out to investigate self-renewal and differentiation capabilities.ResultsSca-1⁺ cells were located in periacinar and periductal spaces and showed an enrichment during cerulein-induced acute pancreatitis (23.2/100 μm² ± 4.9 SEM) and in early inflammation-mediated carcinogenic lesions of the pancreas of Kras^G12D mice (35.8/100 μm² ± SEM 1.9) compared to controls (3.6/100 μm² ± 1.3 SEM). Pancreatic Lin^-Sca-1⁺ cells displayed a small population of 1.46% ± 0.12 SEM in FACS. In IFN-β treated pancreatic epithelial explants, Sca-1 expression was increased, and Lin^-Sca-1⁺ cells were enriched in vitro (from 1.49% ± 0.36 SEM to 3.85% ± 0.78 SEM). Lin^-Sca-1⁺ cells showed a 12 to 51-fold higher capacity for clonal self-renewal compared to Lin^-Sca-1^- cells and generated cells express markers of the acinar and ductal compartment.ConclusionsPancreatic Sca-1⁺ cells enriched during parenchymal damage showed a significant capacity for cell renewal and in vitro plasticity, suggesting that corresponding to the type I interferon-dependent regulation of Lin^-Sca-1⁺ hematopoietic stem cells, pancreatic Sca-1⁺ cells also employ type-I-interferon for regulating progenitor-cell-homeostasis.     

Fatty acids acutely enhance insulin-induced oxidative stress and cause insulin resistance by increasing mitochondrial reactive oxygen species (ROS) generation and nuclear factor-κB inhibitor (IκB)–nuclear factor-κB (NFκB) activation in rat muscle,in the absence of mitochondrial dysfunction

Aims/hypothesis  

Insulin effects reportedly involve reactive oxygen species (ROS) and oxidative stress in vitro, but skeletal muscle oxidative stress is an emerging negative regulator of insulin action following high-fat feeding. NEFA may enhance oxidative stress and insulin resistance. We investigated the acute impact of insulin with or without NEFA elevation on muscle ROS generation and insulin signalling, and the potential association with altered muscle mitochondrial function.