Oxidative stress and thioredoxin-interacting protein promote intravasation of melanoma cells

Although intravasation may be a critical rate-limiting step in the metastatic cascade, the role of oxidative stress in intravasation is unknown. We tested the hypothesis that reactive oxygen species (ROS), regulated by thioredoxin interacting protein (Txnip) through the action of thioredoxin (Trx), influence human SK-MEL-28 melanoma cell reverse (basolateral-to-apical) transendothelial migration (TEM) in vitro as a model for intravasation. Reverse transendothelial migration was dose-dependently induced by hydrogen peroxide 2.4-fold for 0.1 microM (P < 0.01) and 3.9-fold for 1 microM (P < 0.001) vs. control, and this effect was blocked by the antioxidant N-acetylcysteine. Overexpression of Txnip by infecting melanoma cells with adenovirus increased TEM 3-fold vs. control (P < 0.001), and this increase was blocked by N-acetylcysteine, indicating a redox-sensitive mechanism. Conversely, thioredoxin overexpression blocked hydrogen peroxide-induced TEM. Exposure to ultraviolet-A radiation increased ROS 1.8-fold (P < 0.01), and this was accompanied by a 45% reduction (P < 0.05) in thioredoxin activity and an 11.4-fold (P < 0.001) increase in Txnip gene expression. These data suggest that TEM of melanoma cells during intravasation is in part mediated by ROS-sensitive cellular signaling cascades, may be controlled by Txnip and its interaction with thioredoxin that in turn modulates cellular levels of oxidative stress, and may be initiated by ultraviolet-A induction of this cascade.     

Acute hyperglycaemia enhances oxidative stress and aggravates myocardial ischaemia/reperfusion injury: role of thioredoxin‐interacting protein

Hyperglycaemia during acute myocardial infarction is common and associated with increased mortality. Thioredoxin‐interacting protein (Txnip) is a modulator of cellular redox state and contributes to cell apoptosis. This study aimed to investigate whether or not hyperglycaemia enhances Txnip expression in myocardial ischaemia/reperfusion (MI/R) and consequently exacerbates MI/R injury. Rats were subjected to 30 min. of left coronary artery ligation followed by 4 hrs of reperfusion and treated with saline or high glucose (HG, 500 g/l, 4 ml/kg/h intravenously). In vitro study was performed on cultured rat cardiomyocytes subjected to simulated ischaemia/reperfusion (SI/R) and incubated with HG (25 mM) or normal glucose (5.6 mM) medium. In vivo HG infusion during MI/R significantly impaired cardiac function, aggravated myocardial injury and increased cardiac oxidative stress. Meanwhile, Txnip expression was enhanced whereas thioredoxin activity was inhibited following HG treatment in ischaemia/reperfusion (I/R) hearts. In addition, HG activated p38 MAPK and inhibited Akt in I/R hearts. In cultured cardiomyocytes subjected to SI/R, HG incubation stimulated Txnip expression and reduced thioredoxin activity. Overexpression of Txnip enhanced HG‐induced superoxide generation and aggravated cardiomyocyte apoptosis, whereas Txnip RNAi significantly blunted the deleterious effects of HG. Moreover, inhibition of p38 MAPK or activation of Akt markedly blocked HG‐induced Txnip expression in I/R cardiomyocytes. Most importantly, intramyocardial injection of Txnip siRNA markedly decreased Txnip expression and alleviated MI/R injury in HG‐treated rats. Hyperglycaemia enhances myocardial Txnip expression, possibly through reciprocally modulating p38 MAPK and Akt activation, leading to aggravated oxidative stress and subsequently, amplification of cardiac injury following MI/R.     

The interaction of thioredoxin with Txnip. Evidence for formation of a mixed disulfide by disulfide exchange

The thioredoxin system plays an important role in maintaining a reducing environment in the cell. Recently, several thioredoxin binding partners have been identified and proposed to mediate aspects of redox signaling, but the significance of these interactions is unclear in part due to incomplete understanding of the mechanism for thioredoxin binding. Thioredoxin-interacting protein (Txnip) is critical for regulation of glucose metabolism, the only currently known function of which is to bind and inhibit thioredoxin. We explored the mechanism of the Txnip-thioredoxin interaction and present evidence that Txnip and thioredoxin form a stable disulfide-linked complex. We identified two Txnip cysteines that are important for thioredoxin binding and showed that this interaction is consistent with a disulfide exchange reaction between oxidized Txnip and reduced thioredoxin. These cysteines are not conserved in the broader family of arrestin domain-containing proteins, and we demonstrate that the thioredoxin-binding property of Txnip is unique. These data suggest that Txnip is a target of reduced thioredoxin and provide insight into the potential role of Txnip as a redox-sensitive signaling protein.     

Thioredoxin-interacting protein (Txnip) is a critical regulator of hepatic glucose production

Thioredoxin-interacting protein (Txnip) has been recently described as a possible link between cellular redox state and metabolism; Txnip binds thioredoxin and inhibits its disulfide reductase activity in vitro, while a naturally occurring strain of Txnip-deficient mice has hyperlipidemia, hypoglycemia, and ketosis exacerbated by fasting. We generated Txnip-null mice to investigate the role of Txnip in glucose homeostasis. Txnip-null mice were hypoglycemic, hypoinsulinemic, and had blunted glucose production following a glucagon challenge, consistent with a central liver glucose-handling defect. Glucose release from isolated Txnip-null hepatocytes was 2-fold lower than wild-type hepatocytes, whereas beta-hydroxybutyrate release was increased 2-fold, supporting an intrinsic defect in hepatocyte glucose metabolism. While hepatocyte-specific gene deletion of Txnip did not alter glucose clearance compared with littermate controls, Txnip expression in the liver was required for maintaining normal fasting glycemia and glucose production. In addition, hepatic overexpression of a Txnip transgene in wild-type mice resulted in elevated serum glucose levels and decreased ketone levels. Liver homogenates from Txnip-null mice had no significant differences in the glutathione oxidation state or in the amount of available thioredoxin. However, overexpression of wild-type Txnip in Txnip-null hepatocytes rescued cellular glucose production, whereas overexpression of a C247S mutant Txnip, which does not bind thioredoxin, had no effect. These data demonstrate that Txnip is required for normal glucose homeostasis in the liver. While available thioredoxin is not changed in Txnip-null mice, the effects of Txnip on glucose homeostasis are abolished by a single cysteine mutation that inhibits binding to thioredoxin.     

Thioredoxin regulates adipogenesis through thioredoxin-interacting protein (Txnip) protein stability

Txnip (thioredoxin-interacting protein) is a critical mediator of metabolism and adipogenesis in vivo. The mechanisms of action of Txnip are believed to operate at least in part by inhibiting the redox signaling functions of thioredoxin. We tested here whether Txnip suppressed adipogenesis by inhibiting thioredoxin and discovered a reversal of roles; Txnip inhibits adipogenesis directly, and thioredoxin binding regulates Txnip by enhancing Txnip protein stability. Unlike Txnip, a Txnip mutant that cannot bind thioredoxin (C247S) did not prevent adipocyte differentiation, but was degraded more quickly by proteasomal targeting. Finding that endogenous Txnip protein is also rapidly degraded at the onset of adipogenesis suggested that Txnip degradation is required for adipocyte differentiation. Thioredoxin overexpression stabilized Txnip protein levels to inhibit adipogenesis, and adipogenic stimulants such as insulin promoted Txnip-thioredoxin dissociation to the more labile free Txnip state. As an α-arrestin protein, Txnip has two C-terminal tail PPXY motifs that mediate E3 ubiquitin ligase binding and Txnip protein stability. Mutating the PPXY motifs prevented Txnip degradation, even when thioredoxin binding was lost, and restored the ability of C247S Txnip to inhibit adipogenesis. These studies present a novel reconsideration of Txnip-thioredoxin signaling by showing that thioredoxin regulates the intrinsic function of Txnip as an inhibitor of adipogenesis through protein stabilization.     

硫氧还蛋白系统在COPD抗氧化中的作用研究进展

硫氧还蛋白系统是由硫氧还蛋白（thioredoxin,Trx）,硫氧还蛋白还原酶（thioredoxinreductase,TrxR）和还原型辅酶Ⅱ（NADPH）组成的多功能小分子蛋白系统,广泛表达的硫氧还蛋白作为蛋白质二硫键的还原酶,它参与很多生理过程,并发挥重要生物学功能,包括调节机体的氧化还原反应、抑制细胞凋亡、调节转录因子DNA结合活性以及免疫应答等,其中一重要作用是参与调节细胞氧化还原状态以对抗氧化应激。因此在一些炎症性疾病如慢性阻塞性肺疾病、急性呼吸窘迫综合征、肺间质疾病、哮喘、肺结节病等的发生发展中扮演重要角色,本文对硫氧还蛋白系统在慢性阻塞性肺疾病中的抗氧化作用作一综述。     

Divergent effects of different oxidants on glutathione homeostasis and protein damage in erythrocytes from diabetic patients: effects of high glucose.

Long-term complications of diabetes mellitus have been ascribed to both the effects of prolonged hyperglycemia and to increased oxidative stress. In an attempt to identify the mechanisms underlying the acute effects of hyperglycemia on oxidative stress, we investigated the hypothesis that high glucose might lead to an insufficiency in reducing equivalents (such as NADPH) and thus to a disruption in the glutathione-dependent antioxidant defences and to an incapacity to deal with oxidant attack. For this purpose, erythrocytes from diabetic patients were incubated for 0-90 min in 5.55 or 33.3 mM D-glucose containing tertbutyl hydroperoxide 0.5 and 1 mM, Menadione 100 microM, or glucose oxidase. The time course of the changes in non-protein bound glutathione (reduced and oxidised), lactate and pyruvate, alanine and fluorescent products of oxidative proteolysis, hemolysis and methemoglobin was monitored. The results show that although glucose utilisation was unaffected, all oxidants caused a persistent decrease in total non-protein-bound glutathione suggesting binding to proteins. However, changes in glutathione and redox status differed between the various oxidants and were not directly related to the extent of oxidative cellular damage. In these experimental conditions, with short incubations and using the erythrocyte as the simplest cellular model of glucose metabolism, neither high glucose nor the diabetic condition worsened the susceptibility of erythrocytes to acute in vitro oxidative damage.     

Diabetes,oxidative stress,and antioxidants: a review

Increasing evidence in both experimental and clinical studies suggests that oxidative stress plays a major role in the pathogenesis of both types of diabetes mellitus. Free radicals are formed disproportionately in diabetes by glucose oxidation, nonenzymatic glycation of proteins, and the subsequent oxidative degradation of glycated proteins. Abnormally high levels of free radicals and the simultaneous decline of antioxidant defense mechanisms can lead to damage of cellular organelles and enzymes, increased lipid peroxidation, and development of insulin resistance. These consequences of oxidative stress can promote the development of complications of diabetes mellitus. Changes in oxidative stress biomarkers, including superoxide dismutase, catalase, glutathione reductase, glutathione peroxidase, glutathione levels, vitamins, lipid peroxidation, nitrite concentration, nonenzymatic glycosylated proteins, and hyperglycemia in diabetes, and their consequences, are discussed in this review. In vivo studies of the effects of various conventional and alternative drugs on these biomarkers are surveyed. There is a need to continue to explore the relationship between free radicals, diabetes, and its complications, and to elucidate the mechanisms by which increased oxidative stress accelerates the development of diabetic complications, in an effort to expand treatment options.     

Antioxidative treatment reverses imbalances of nitric oxide synthase isoform expression and attenuates tissue-cGMP activation in diabetic rats

Oxidative stress and impaired bioactivity of vascular nitric oxide (NO) play an important role in the pathogenesis of macro- as well as microangiopathic complications in diabetes mellitus. To determine the cause of this impaired bioactivity, we tested the effect of long-term hyperglycemia and antioxidative treatment on tissue-specific endothelial (e)NOS- and inducible (i)NOS-expression and the main target of NO action, cGMP, in diabetic rats. After 4 weeks of hyperglycemia, eNOS-mRNA expression was significantly down-regulated in all tissues tested. In contrast, iNOS-mRNA was significantly up-regulated and tissue generation of cGMP significantly increased. Treatment with alpha-lipoicacid reversed changes of NOS-isoform expression as well as cGMP-concentration without changing blood glucose levels. In addition, oxidative stress significantly decreased in diabetic rats treated with alpha-lipoicacid. Together, diabetes regulates NOS-isoforms differentially by down-regulating eNOS and up-regulating iNOS. In addition, our data suggest that the cause of impaired endothelial vasodilatation in experimental diabetes is not degradation or inactivation of NO. On the contrary, these results support the concept of decreased reactivity of the vascular smooth muscle to NO or increased NO activity as a possible vascular damaging agent, e.g., by inducing apoptosis in vascular cells. Furthermore, our data show that antioxidative treatment is capable of reversing changes in the NO-cGMP system and may therefore be an important therapeutic option for preventing vascular damage in diabetes mellitus.     

High glucose inhibits glucose-6-phosphate dehydrogenase via cAMP in aortic endothelial cells

Recent studies have shown that hyperglycemia is a principal cause of cellular damage in patients with diabetes mellitus. A major consequence of hyperglycemia is increased oxidative stress. Glucose-6-phosphate dehydrogenase (G6PD) plays an essential role in the regulation of oxidative stress by primarily regulating NADPH, the main intracellular reductant. In this paper we show that increased glucose (10-25 mm) caused inhibition of G6PD resulting in decreased NADPH levels in bovine aortic endothelial cells (BAEC). Inhibition was seen within 15 min. High glucose-induced inhibition of G6PD predisposed cells to cell death. High glucose via increased activity of adenylate cyclase also stimulated an increase in cAMP levels in BAEC. Agents that increased cAMP caused a decrease in G6PD activity. Inhibition of cAMP-dependent protein kinase A ameliorated the high glucose-induced inhibition of G6PD. Finally, high glucose stimulated phosphorylation of G6PD. These results suggest that, in BAEC, high glucose stimulated increased cAMP, which led to increased protein kinase A activity, phosphorylation of G6PD, and inhibition of G6PD activity. We conclude that these changes in G6PD activity play an important role in high glucose-induced cell damage/death.     

Aminoguanidine prevented impairment of blood antioxidant system in insulin-dependent diabetic rats

Non-enzymatic glycation is implicated in the development of various diseases such as Alzheimer's and diabetes mellitus. However, it is also observed during the physiologic process of aging. There is considerable interest in the contribution of oxidative stress to diabetes mellitus. An increase in the generation of reactive oxygen species can occur by non-enzymatic glycation and glucose autoxidation. Both of these processes lead to the formation of AGEs (Advanced glycation end-products) that contribute to the irreversible modification of enzymes, proteins, lipids and DNA. In this study, the effect of chronic hyperglycemia on the antioxidant system of diabetic rats was evaluated. The working hypothesis is that the loss of glucose homeostasis reduces the capacity to respond to oxidative damage. The enzymatic activities of CAT (catalase), GPx (gluthatione peroxidase), GR (gluthatione reductase) and GSH (reduced gluthatione) were increased in the blood of healthy rats subjected to endurance training, whereas, in diabetic rats the activities of CAT, GPx and GR were unaltered by similar training. SOD showed low activity in endurance-trained rats. The administration of aminoguanidine (an inhibitor of glycation reactions) in the drinking water increased the activities of CAT, GPx and GR, suggesting that glycation may be responsible for the partial inactivation of these enzymes. These results indicate that the association of hyperglycemia with strenuous physical exercise may induce cellular damage by impairing the antioxidant defense system.     

Oxidative stress, glutathione status, sirtuin and cellular stress response in type 2 diabetes

Oxidative stress has been suggested to play a main role in the pathogenesis of type 2 diabetes mellitus and its complications. As a consequence of this increased oxidative status a cellular adaptive response occurs requiring functional chaperones, antioxidant production and protein degradation. This study was designed to evaluate systemic oxidative stress and cellular stress response in patients suffering from type 2 diabetes and in age-matched healthy subjects. Systemic oxidative stress has been evaluated by measuring plasma reduced and oxidized glutathione, as well as pentosidine, protein carbonyls lipid oxidation products 4-hydroxy-2-nonenal and F2-isoprostanes in plasma, and lymphocytes, whereas the lymphocyte levels of the heat shock proteins (HSP) HO-1, Hsp72, Sirtuin-1, Sirtuin-2 and thioredoxin reductase-1 (TrxR-1) have been measured to evaluate the systemic cellular stress response. Plasma GSH/GSSG showed a significant decrease in type 2 diabetes as compared to control group, associated with increased pentosidine, F2-isoprostanes, carbonyls and HNE levels. In addition, lymphocyte levels of HO-1, Hsp70, Trx and TrxR-1 (P<0.05 and P<0.01) in diabetic patients were higher than in normal subjects, while sirtuin-1 and sirtuin-2 protein was significantly decreased (p<0.05). In conclusion, patients affected by type 2 diabetes are under condition of systemic oxidative stress and, although the relevance of downregulation in sirtuin signal has to be fully understood, however induction of HSPs and thioredoxin protein system represent a maintained response in counteracting systemic pro-oxidant status. This article is part of a Special Issue entitled: Antioxidants and Antioxidant Treatment in Disease.     

Impaired redox signaling and mitochondrial uncoupling contributes vascular inflammation and cardiac dysfunction in type 1 diabetes: Protective role of arjunolic acid

Vascular inflammation and cardiac dysfunction are the leading causes of mortality and morbidity among the diabetic patients. Type 1 diabetic mellitus (T1DM) is associated with increased cardiovascular complications at an early stage of the disease. The purpose of the present study was to explore whether arjunolic acid (AA) plays any protective role against cardiovascular complications in T1DM and if so, what molecular pathways it utilizes for the mechanism of its protective action. Streptozotocin (STZ) was used to induce T1DM in experimental rats. Alteration in plasma lipid profile and release of membrane bound enzymes like LDH (lactate dehydrogenase) and CK (creatine kinase) established the association of hyperlipidemia and cell membrane disintegration with hyperglycemia. Hyperglycemia altered the levels of oxidative stress related biomarkers, decreased the intracellular NAD and ATP concentrations. Hyperglycemia-induced enhanced levels of VEGF, ICAM-1, MCP-1 and IL-6 in the plasma of STZ treated animals indicate vascular inflammation in T1DM. Histological studies and FACS analysis revealed that hyperglycemia caused cell death mostly via the apoptotic pathway. Investigating molecular mechanism, we observed NF-κB and MAPKs (p38 and ERK1/2) activations, mitochondrial membrane depolarization, cytochrome C release, caspase 3 activation and PARP cleavage in apoptotic cell death in the diabetic cardiac tissue. Treatment with AA (20 mg/kg body weight) reduced hyperglycemia, membrane disintegration, oxidative stress, vascular inflammation and prevented the activation of oxidative stress induced signaling cascades leading to cell death. Results suggest that AA possesses the potential to be a beneficial therapeutic agent in diabetes and its associated cardiac complications.     

Antioxidant and vascular effects of gliclazide in type 2 diabetic rats fed high-fat diet

Diabetes mellitus is characterized by oxidative stress, which in turn determines endothelial dysfunction. Gliclazide is a sulphonylurea antidiabetic drug with antioxidant effects due to its azabicyclo-octyl ring. It has been reported to potentially protect the vasculature through improvements in plasma lipid levels and platelet function. We hypothesized that gliclazide has a beneficial effect on endothelial function in Goto-Kakizaki rats (GK), an animal model of type 2 diabetes fed an atherogenic diet for 4 months. We evaluated the influence of gliclazide on both metabolic and oxidative status and NO-mediated vasodilation. GKAD rats showed increased oxidative stress and impaired endothelium-dependent vasodilation. GKAD rats treated with gliclazide showed increased sensitivity to NO-mediated vasodilation, a significant decrease in fasting glycemia and insulinemia, and a significant decrease in systemic oxidative stress. In conclusion, our results suggest that gliclazide treatment improves NO-mediated vasodilation in diabetic GK rats with dyslipidemia probably due to its antioxidant effects, although we cannot rule out substantial benefits due to a reduction in fasting blood glucose. The availability of a compound that simultaneously decreases hyperglycemia, hyperinsulinemia, and inhibits oxidative stress is a promising therapeutic candidate for the prevention of vascular complications of diabetes.