Plasma Extracellular-superoxide Dismutase Levels Research Articles

Effect of endoplasmic reticulum (ER) stress inducer thapsigargin on the expression of extracellular-superoxide dismutase in mouse 3T3-L1 adipocytes

Endoplasmic reticulum stress is related to metabolic disorders, including atherosclerosis and type 2 diabetes. It is known that inflammatory adipocytokines and oxidative stress are increased, while anti-inflammatory adipocytokines such as adiponectin are decreased in adipocytes during above conditions. Extracellular-superoxide dismutase is an anti-inflammatory enzyme that protects cells from oxidative stress. Because plasma extracellular-superoxide dismutase levels in type 2 diabetes patients were inversely related to the body mass index and homeostasis model assessment-insulin resistance index, it is speculated that the regulation of extracellular-superoxide dismutase might lead to the suppression of metabolic disorders. Here, we observed the reduction of extracellular-superoxide dismutase and adiponectin in 3T3-L1 adipocytes treated with thapsigargin, an endoplasmic reticulum stress inducer. Interestingly, tunicamycin, another endoplasmic reticulum stress inducer, did not decrease the expression of extracellular-superoxide dismutase in spite of the induction of glucose regulated protein kinase 78 kDa, an endoplasmic reticulum stress marker. Moreover, eukaryotic translation initiation factor 2α signaling cascade plays a pivotal role in the reduction of extracellular-superoxide dismutase in 3T3-L1 adipocytes during endoplasmic reticulum stress conditions.

Abstract 9760: Plasma Levels of Nitric Oxide Metabolites Are Markedly Reduced In Normotensive Individuals With Electrocardiographically Determined Left Ventricular Hypertrophy

Objective: It has recently been revealed that normotensive subjects with electrocardiographically determined left ventricular hypertrophy (ECG-LVH) are at risk of multiple cardiovascular diseases, but the underlying mechanism remains to be elucidated. To address this point, we examined the hypothesis that normotensive individuals with ECG-LVH show reduced plasma levels of metabolites of nitric oxide (NO), a critical regulator of cardiovascular function. Methods: A total of 579 Japanese male workers (40-65 years of age) were studied. Since hypertension (blood pressure ≥140/90 mmHg) or medications could influence plasma NOx (NO metabolites, nitrite and nitrate), workers with such factors were excluded from the study. ECG-LVH was assessed by the Sokolow-Lyon voltage criteria and the Cornell voltage-duration product. The subjects were categorized by quartiles of plasma NOx level, and a multivariate logistic regression analysis was conducted relating ECG-LVH to NOx quartiles after adjustment for potential confounders. Results: The mean levels of plasma NOx were markedly lower in individuals with ECG-LVH (n=73, 28.9±19.2 μ mol/l) than in those without (n=506, 42.5±31.2 μ mol/l) ( P <0.0001). The multivariate-adjusted odd ratio for the presence of ECG-LVH compared with the forth quartile of NOx (≥51.9 μ mol/l) was 1.38 (95% CI 0.55-3.59) for the third (NOx 33.1-51.8 μ mol/l), 1.83 (0.76-4.64) for the second (NOx 21.3-33.0 μ mol/l) and 4.93 (2.31-11.47) for the first quartile (NOx ≤21.2 μ mol/l). Similar result was obtained when subjects were stratified by waist circumference. The mean levels of plasma extracellular superoxide dismutase (EC-SOD), a major antioxidant in the cardiovascular system, were comparable in individuals with and without ECG-LVH. On the other hand, plasma EC-SOD levels were significantly decreased with lower quartiles of NOx ( P <0.01). Conclusion: This study demonstrated for the first time that plasma NOx levels are markedly reduced in normotensive individuals with ECG-LVH. These results indicate that disrupted NO production through the involvement of down-regulation of the antioxidant enzyme may explain, at least in part, the potential mechanism for increased cardiovascular risk in normotensive individuals with ECG-LVH.

The effect of hypoxia mimetic cobalt chloride on the expression of EC-SOD in 3T3-L1 adipocytes

It is well known that adipose tissue is not only a passive reservoir for energy storage but also produces and secretes a variety of bioactive molecules called adipocytokines, including adiponectin and tumor necrosis factor-α (TNF-α). Recently, it has been reported that adipose tissue can suffer a chronic hypoxic condition during hypertrophy of adipocytes, and this condition leads to the dysregulation of adipocytokines. Further, hypoxic adipocytes are in an increased oxidative stress. Extracellular-superoxide dismutase (EC-SOD) is an anti-inflammatory enzyme that protects cells from reactive oxygen species (ROS) by scavenging superoxide anion. Previous reports showed that plasma EC-SOD levels in type 2 diabetes patients were significantly and inversely related to the body mass index, homeostasis model assessment-insulin resistance index; however, the mechanisms of EC-SOD and adiponectin reductions during hypoxia remain poorly understood. Here, we demonstrate that cobalt chloride (CoCl2), a hypoxia mimetic, decreases EC-SOD and adiponectin in 3T3-L1 adipocytes by intracellular ROS-independent, but TNF-α and c-jun N-terminal kinase (JNK)-dependent mechanisms. From these results, it is possible that TNF-α is a key regulator of the reduction of EC-SOD and adiponectin in CoCl2-treated 3T3-L1 adipocytes, and we speculated that the reduction of EC-SOD and adiponectin would lead to and/or promote metabolic disorders.

Expression of extracellular superoxide dismutase during adipose differentiation in 3T3-L1 cells

Obesity is known to be the primary causal component in metabolic syndrome. Adipocytes in obese patients exhibit increased oxidative stress via the activation of reactive oxygen species (ROS)-producing systems and inactivation of antioxidant enzymes. Extracellular superoxide dismutase (EC-SOD) is an anti-inflammatory enzyme that protects cells from the damaging effects of ROS. An earlier report showed that plasma EC-SOD levels in type 2 diabetic patients were significantly and inversely related to body mass index and homeostasis model assessment-insulin resistance index. Moreover, the administration of pioglitazone, an antidiabetic agent, significantly increased the plasma level of EC-SOD. In this report, the expression of EC-SOD was compared to other adipocytokines in mice 3T3-L1 pre-adipocytes. EC-SOD expression levels were increased after the induction of differentiation and then declined, which was similar to adiponectin and transcription factors such as peroxisome proliferator-activated receptor-γ (PPAR-γ) and CCAAT/enhancer-binding protein-α (C/EBP-α). On the other hand, the expression levels of pro-inflammatory adipocytokines, such as tumor necrosis factor-α (TNF-α) and monocyte chemo-attractant protein-1 (MCP-1), increased markedly in the development stage of cells. It was observed that the expression of EC-SOD in differentiated 3T3-L1 cells co-cultured with LPS-stimulated J774 macrophages was up-regulated, while the addition of TNF-α down-regulated EC-SOD and adiponectin expression in adipocytes. It is known that infiltrated and activated macrophages produce extracellular ROS at high levels in adipose tissue. It is possible that the expression of EC-SOD in adipocytes was stimulated to protect them from oxidative stress in the co-culture system.

Infliximab Neutralizes the Suppressive Effect of TNF-α on Expression of Extracellular-Superoxide Dismutase <i>in Vitro</i>

Extracellular-superoxide dismutase (EC-SOD) is the major SOD isozyme in blood vessel walls, normal cartilage and synovial fluid and may be important for the antioxidant capability of these tissues. We have reported that EC-SOD gene transferred mice exhibited significant suppression of clinical symptoms of type II collagen induced arthritis [Iyama, et al., Arthritis Rheum., 44, 2160-2167 (2001)] and plasma EC-SOD levels in type 2 diabetic patients were significantly negatively related to indices of insulin resistance [Adachi, et al., J. Endocrinol., 181, 413-417 (2004)]. Tumor necrosis factor-alpha (TNF-alpha) has been implicated in the pathological conditions of the above diseases and is a major therapeutic target, based on clinical studies with anti-TNF-alpha monoclonal antibodies such as infliximab. In this report, we investigated the effect of TNF-alpha on the expression of EC-SOD in cultured cells and the cooperating effect of infliximab. In the in vitro assays examined, expression of EC-SOD, but not other SOD isozymes, in smooth muscle and fibroblast cells were suppressed by the addition of TNF-alpha. Simultaneous addition of infliximab dose-dependently and significantly prevented the suppressive effects of TNF-alpha. p38 mitogen-activated protein kinase (MAPK) inhibitor, SB203580, prevented significantly the suppressive effect of TNF-alpha suggesting that p38 MAPK is an important signaling molecule downstream of TNF-alpha to inhibit the EC-SOD expression. From the results, it is speculated that the decline in TNF-alpha activity by the administration of infliximab results in the liberation of EC-SOD from the suppressed state of gene expression. This reveals a potential usefulness of infliximab on TNF-alpha related pathological conditions such as arthritis and insulin resistance.

Effects of PPARγ ligands and C/EBPβ enhancer on expression of extracellular-superoxide dismutase

Extracellular-superoxide dismutase (EC-SOD) is the major SOD isozyme in the blood vessel walls and may be important for antioxidant capability of the vascular walls. Expression of EC-SOD is known to be regulated by numerous substances such as cytokines and vasoactive factors. Recently, we found that the plasma EC-SOD levels in type 2 diabetic patients were significantly and inversely related to indices of insulin resistance, whereas they were strongly and positively related to adiponectin. Administration of pioglitazone significantly increased the plasma level of EC-SOD and adiponectin. Transcription factors such as CCAAT/enhancer-binding proteins (C/EBPs) and peroxisome proliferator-activated receptors (PPARs) are known to regulate genes associated with insulin resistance. The aim of this study was to assess the contribution of these transcription factors on the EC-SOD level. We found that a C/EBPβ enhancer, prolactin, significantly induced the EC-SOD mRNA and protein levels in cultured fibroblast cell lines, but PPARγ ligands, pioglitazone and other thiazolidinedione agents did not. Deletion analysis of the EC-SOD promoter-luciferase construct showed that an important element responsible for prolactin is located between –242 and –178 in the promoter region of the EC-SOD gene in which a known C/EBPβ-binding site is located. Increasing the EC-SOD expression by treatment with ligands of transcription factors might be one approach to ameliorate the pathological conditions of insulin resistance.

Plasma levels of extracellular superoxide dismutase in an Australian population: genetic contribution to normal variation and correlations with plasma nitric oxide and apolipoprotein A-I levels.

Extracellular superoxide dismutase (EC-SOD) is a major superoxide scavenger and may be important to normal vascular function and cardiovascular health. We analyzed family data from 610 healthy Australians to detect and quantify the effects of genes on normal variation in plasma levels of EC-SOD and to test for pleiotropy with plasma nitric oxide (NO) and apolipoprotein A-I (apoA-I). Using maximum-likelihood-based variance decomposition methods, we determined that sex, age, and plasma levels of HDL cholesterol, apoA-I, and creatinine accounted for 38.6% of the variance in plasma EC-SOD levels and that additive genes accounted for 35% (P<0.00002). Multivariate analyses of plasma levels of EC-SOD, NO(x) (a measure of basal NO production), and apoA-I detected significant genetic correlations, indicating pleiotropy between EC-SOD and apoA-I (genetic correlation [rho(G)]=-0.45) and between NO(x) and apoA-I (rho(G)=0.58) but not between EC-SOD and NO(x). Genes shared by EC-SOD and apoA-I account for 20% of the genetic variance and, respectively, 7% and 9% of the phenotypic variance in both traits. Shared genes also account for >33% of the genetic variance and 5% and 15% of the respective phenotypic variance in NO(x) and apoA-I. In healthy individuals, over a third of the variance in EC-SOD plasma levels is due to the additive effects of genes. Some genes influence EC-SOD and apoA-I levels. The same is true of NO(x) and apoA-I but not of EC-SOD and NO(x). These patterns of pleiotropy can guide subsequent attempts to identify the genes and physiological mechanisms underlying them.

Increase of urinary extracellular-superoxide dismutase level correlated with cyclic adenosine monophosphate

Extracellular superoxide dismutase (EC-SOD) is a secretory protein that is the major SOD isozyme in extracellular fluids. Plasma EC-SOD levels are distributed in two discrete groups with the rare group having an enzyme with glycine instead of arginine-213, which causes a 10-fold higher serum level. Within the common phenotype group, the urinary EC-SOD level was significantly correlated with the urinary excretion of N-acetyl-β- D-glucosaminidase (NAG), but not with serum EC-SOD. EC-SOD appears not to be leaked from the plasma by glomerular filtration, but rather to be secreted from the renal tubule or its surrounding tissues. The urinary EC-SOD level was also significantly correlated with the urinary cyclic adenosine monophosphate (cAMP) level. cAMP analogues and adenylate cyclase modulators significantly stimulated the expression of EC-SOD but not other SOD isozymes in cultured fibroblast cell lines. Moreover, injection of parathyroid hormone, in Ellsworth-Howard tests, increased urinary EC-SOD accompanied with the elevations of urinary cAMP and NAG. Together these observations suggest that factor(s) that stimulate the adenylate cyclase-cAMP system regulate the urinary EC-SOD level.

Plasma Extracellular Superoxide Dismutase Levels in an Australian Population With Coronary Artery Disease

In vitro experiments suggest that free radicals may contribute importantly to atherogenesis. Superoxide dismutase (SOD), particularly extracellular SOD (EC-SOD), which accounts for the majority of SOD biological activity, is a major superoxide scavenger. We explored factors that may affect plasma EC-SOD levels measured by ELISA and assessed the association between plasma EC-SOD and coronary artery disease documented angiographically in 590 white Australian patients </=65 years old. Mean+/-SEM plasma EC-SOD in female patients (113.6+/-13.2 ng/mL) was significantly higher than in male patients (86.6+/-5.1 ng/mL, P<0.0001), and all 19 patients with levels >400 ng/mL were heterozygous for the Arg213-->Gly mutation at the EC-SOD gene; there was also a positive correlation with age (r=0.131, P=0.0016). Plasma EC-SOD in current smokers (75. 0+/-9.3 ng/mL) was much lower than in nonsmokers (111.7+/-8.2 ng/mL, P<0.01), and ex-smokers had intermediate levels (84.3+/-7.1 ng/mL). Levels were significantly lower in patients with than in those without a history of acute myocardial infarction (MI) (76.1+/-7.5 versus 110.1+/-6.0 ng/mL, P<0.05), and low plasma EC-SOD was independently associated with an increased likelihood of a history of MI (OR, 2.04; 95% CI, 1.10 to 3.82); higher EC-SOD levels also tended to be associated with delayed onset of MI. In conclusion, our study establishes that in patients assessed by coronary angiography, circulating EC-SOD is lower in men than in women and in smokers of each sex and that low levels are independently associated with a history of MI. These findings are consistent with EC-SOD's being protective and contributing to reduced coronary risk.

Two variants of extracellular-superoxide dismutase: relationship to cardiovascular risk factors in an unselected middle-aged population.

Description of the distribution of plasma levels of two variants of extracellular-superoxide dismutase (EC-SOD) and their relationship to cardiovascular risk factors in the general population. A cross-sectional study. Norrbotten and Västerbotten counties in northern Sweden. Four thousand, nine hundred 25-74-year-old randomly selected subjects. Plasma levels of EC-SOD. Genotyping by an allele-specific PCR method. Plasma EC-SOD levels showed a distinct bimodal distribution with the smaller group (3.8%) having a variant of the enzyme with about eight-fold-higher plasma levels. Genotyping was performed in 65 individuals with the high-level variant. All but one were found to carry the same mutation, Arg213Gly, affecting the heparin-binding domain of EC-SOD. Subjects with the high-level variant of EC-SOD had modestly higher body mass index and higher levels of serum cholesterol, serum triglycerides and plasma fibrinogen than those with the common EC-SOD phenotype. Within the population with common EC-SOD, the plasma levels were lower in men than in women and increased with age. Low levels of common phenotype EC-SOD were associated with smoking, high plasma levels of fibrinogen and low activity of tissue plasminogen activator in both univariate and multivariate analyses. Obesity and total serum cholesterol were associated with high common phenotype EC-SOD levels. A high-level variant of EC-SOD caused by one and the same mutation and with low tissue binding and high plasma levels is present in approximately four per cent of an unselected middle-aged population in northern Sweden. Plasma levels of EC-SOD may be modulated by lifestyle factors such as smoking and show a complex covariation with many of the conventional cardiovascular risk factors.