Ascorbic acid prevents high glucose-induced apoptosis in human brain pericytes

High glucose concentrations due to diabetes increase apoptosis of vascular pericytes, impairing vascular regulation and weakening vessels, especially in brain and retina. We sought to determine whether vitamin C, or ascorbic acid, could prevent such high glucose-induced increases in pericyte apoptosis. Culture of human microvascular brain pericytes at 25 mM compared to 5 mM glucose increased apoptosis measured as the appearance of cleaved caspase 3. Loading the cells with ascorbate during culture decreased apoptosis, both at 5 and 25 mM glucose. High glucose-induced apoptosis was due largely to activation of the receptor for advanced glycation end products (RAGE), since it was prevented by specific RAGE inhibition. Culture of pericytes for 24 h with RAGE agonists also increased apoptosis, which was completely prevented by inclusion of 100 μM ascorbate. Ascorbate also prevented RAGE agonist-induced apoptosis measured as annexin V binding in human retinal pericytes, a cell type with relevance to diabetic retinopathy. RAGE agonists decreased intracellular ascorbate and GSH in brain pericytes. Despite this evidence of increased oxidative stress, ascorbate prevention of RAGE-induced apoptosis was not mimicked by several antioxidants. These results show that ascorbate prevents pericyte apoptosis due RAGE activation. Although RAGE activation decreases intracellular ascorbate and GSH, the prevention of apoptosis by ascorbate may involve effects beyond its function as an antioxidant.     

Protective effect of N-acetylcysteine supplementation on mitochondrial oxidative stress and mitochondrial enzymes in cerebral cortex of streptozotocin-treated diabetic rats

Diabetic encephalopathy, characterized by cognitive deficits involves hyperglycemia-induced oxidative stress. Impaired mitochondrial functions might play an important role in accelerated oxidative damage observed in diabetic brain. The aim of the present study was to examine the role of mitochondrial oxidative stress and dysfunctions in the development of diabetic encephalopathy along with the neuroprotective potential of N-acetylcysteine (NAC). Chronic hyperglycemia accentuated mitochondrial oxidative stress in terms of increased ROS production and lipid peroxidation. Significant decrease in Mn-SOD activity along with protein and non-protein thiols was observed in the mitochondria from diabetic brain. The activities of mitochondrial enzymes; NADH dehydrogenase, succinate dehydrogenase and cytochrome oxidase were decreased in the diabetic brain. Increased mitochondrial oxidative stress and dysfunctions were associated with increased cytochrome c and active caspase-3 levels in cytosol. Electron microscopy revealed mitochondrial swelling and chromatin condensation in neurons of diabetic animals. NAC administration, on the other hand was found to significantly improve diabetes-induced biochemical and morphological changes, bringing them closer to the controls. The results from the study provide evidence for the role of mitochondrial oxidative stress and dysfunctions in the development of diabetic encephalopathy and point towards the clinical potential of NAC as an adjuvant therapy to conventional anti-hyperglycemic regimens for the prevention and/or delaying the progression of CNS complications.     

TAT-mediated peroxiredoxin 5 and 6 protein transduction protects against high-glucose-induced cytotoxicity in retinal pericytes

AimsHyperglycemia-induced oxidative stress is implicated in pericyte apoptosis seen in diabetic retinopathy. The six mammalian Peroxiredoxins (PRDXs) comprise a novel family of antioxidative proteins that negatively regulate oxidative stress-induced apoptosis by controlling reactive oxygen species (ROS) levels.Main methodsSprague–Dawley rats were used to detect the retinal expressions of PRDXs1–6. Pig pericytes cultured in high-glucose medium were used to monitor the protective effect of PRDX5 and 6 against high-glucose-associated change. Recombinant PRDX5 and 6 proteins were linked to the Trans-Activating Transduction (TAT) domain from HIV-1 TAT protein for their efficient delivery into cells/tissues.Key findingsWe found higher expression of PRDX5 and 6 mRNAs and PRDX5 and 6 proteins in retina than the other Prdxs (Prdx1–4). Western blotting affirmed the intracellular presence of TAT-linked proteins and revealed the efficient transduction of TAT-HA-PRDX5 and 6 in these cells. Extrinsic supply of TAT-HA-PRDX5 and 6 proteins inhibited the oxidative stress-induced DNA damage after high-glucose exposure in pig pericytes. The cell survival and apoptosis assay revealed that extrinsic supply of TAT-HA-PRDX5 and 6 proteins was responsible for inhibiting hyperglycemia-induced pericyte apoptosis.SignificanceResults suggest that delivery of PRDX5 and 6 might protect hyperglycemia-induced pericyte loss to inhibit oxidative stress.     

Upstream reactive oxidative species (ROS) signals in exogenous oxidative stress-induced mitochondrial dysfunction

Exogenous oxidative stress induces cell death, but the upstream molecular mechanisms involved of the process remain relatively unknown. We determined the instant dynamic reactions of intracellular reactive oxygen species (ROS, including hydrogen peroxide (H₂O₂), superoxide radical (O₂⁻), and nitric oxide (NO)) in cells exposed to exogenous oxidative stress by using a confocal laser scanning microscope. Stimulation with extracellular H₂O₂ significantly increased the production of intracellular H₂O₂, O₂⁻, and NO (P < 0.01) through certain mechanisms. Increased levels of intracellular ROS resulted in mitochondrial dysfunction, involving the impairment of mitochondrial activity and the depolarization of mitochondrial membrane potential. Mitochondrial dysfunction significantly inhibited the proliferation of human hepatoblastoma G2 (HepG2) cells and resulted in mitochondrial cytochrome c (cyt c) release. The results indicate that upstream ROS signals play a potential role in exogenous oxidative stress-induced cell death through mitochondrial dysfunction and cyt c release.     

The role of oxidative stress in diabetic complications

The morbidity and mortality associated with diabetes is the result of the myriad complications related to the disease. One of the most explored hypotheses to explain the onset of complications is a hyperglycemia-induced increase in oxidative stress. Reactive oxygen species (ROS) are produced by oxidative phosphorylation, nicotinamide adenine dinucleotide phosphate oxidase (NADPH), xanthine oxidase, the uncoupling of lipoxygenases, cytochrome P450 monooxygenases, and glucose autoxidation. Once formed, ROS deplete antioxidant defenses, rendering the affected cells and tissues more susceptible to oxidative damage. Lipid, DNA, and protein are the cellular targets for oxidation, leading to changes in cellular structure and function. Recent evidence suggests ROS are also important as second messengers in the regulation of intracellular signaling pathways and, ultimately, gene expression. This review explores the production of ROS and the propagation and consequences of oxidative stress in diabetes.     

4-Hydroxy-2(E)-nonenal inhibits CNS mitochondrial respiration at multiple sites

A destructive cycle of oxidative stress and mitochondrial dysfunction is proposed in neurodegenerative disease. Lipid peroxidation, one outcome of oxidative challenge, can lead to the formation of 4-hydroxy-2(E)-nonenal (HNE), a lipophilic alkenal that forms stable adducts on mitochondrial proteins. In this study, we characterized the effects of HNE on brain mitochondrial respiration. We used whole rat brain mitochondria and concentrations of HNE comparable to those measured in patients with Alzheimer's disease. Our results showed that HNE inhibited respiration at multiple sites. Complex I-linked and complex II-linked state 3 respirations were inhibited by HNE with IC50 values of approximately 200 microM HNE. Respiration was apparently diminished owing to the inhibition of complex III activity. In addition, complex II activity was reduced slightly. The lipophilicity and adduction characteristics of HNE were responsible for the effects of HNE on respiration. The inhibition of respiration was not prevented by N-acetylcysteine or aminoguanidine. Studies using mitochondria isolated from porcine cerebral cortex also demonstrated an inhibition of complex I- and complex II-linked respiration. Thus, in neurodegenerative disease, oxidative stress may impair mitochondrial respiration through the production of HNE.     

Modulation of Nrf2 expression alters high glucose-induced oxidative stress and antioxidant gene expression in mouse mesangial cells

Reactive oxygen species (ROS) play an important role in the pathogenesis of diabetic nephropathy. Nuclear factor erythroid 2-related factor 2 (Nrf2) can up-regulate the expression of antioxidant genes and protect cells from oxidative damage. The current study is aimed at examining the effect of modulation of Nrf2 expression on high glucose-induced oxidative stress and Nrf2-targeting antioxidant expression in mouse mesangial cells. In this study, mouse mesangial cells were transiently transfected with Nrf2-plasmid or the Nrf2-specific siRNA. The high glucose-induced intracellular ROS, malondialdehyde, cell proliferation, and TGF-β1 secretion were measured. The levels of Nrf2, heme oxygenase-1 (HO-1), γ-glutamylcysteine synthethase (γ-GCS) expression, and nuclear expression of Nrf2 in mouse mesangial cells were determined. We found that high glucose induced ROS and malondialdehyde generation in mouse mesangial cells. Induction of Nrf2 over-expression reduced the high glucose-induced ROS and malondialdehyde production, inhibited cell proliferation and TGF-β1 secretion, accompanied by up-regulating the expressions of HO-1 and γ-GCS in mouse mesangial cells. However, knockdown of Nrf2 expression displayed reverse effects in mouse mesangial cells. All these results indicated that Nrf2 and its downstream antioxidants, HO-1 and γ-GCS, are negative regulators of high glucose-induced ROS-related mouse mesangial cell dysfunction.     

Reactive oxygen species generation is modulated by mitochondrial kinases: correlation with mitochondrial antioxidant peroxidases in rat tissues

Mitochondrial hexokinase (mt-HK) and creatine kinase (mt-CK) activities have been recently proposed to reduce the rate of mitochondrial ROS generation through an ADP re-cycling mechanism. Here, we determined the role of mt-HK and mt-CK activities in regulate mitochondrial ROS generation in rat brain, kidney, heart and liver, relating them to the levels of classical antioxidant enzymes. The activities of both kinases were significantly higher in the brain than in other tissues, whereas the activities of catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GR) were higher in both liver and kidney mitochondria. In contrast, manganese superoxide dismutase (Mn-SOD) activity was not significantly different among these tissues. Activation of mitochondrial kinases by addition of their substrates increased the ADP re-cycling and thus the respiration by enhancing the oxidative phosphorylation. Succinate induced hydrogen peroxide (H(2)O(2)) generation was higher in brain than in kidney and heart mitochondria, and the lowest in liver mitochondria. Mitochondrial membrane potential (DeltaPsi(m)) and H(2)O(2) production, decreased with additions of 2-DOG or Cr to respiring brain and kidney mitochondria but not to liver. The inhibition of H(2)O(2) production by 2-DOG and Cr correspond to almost 100% in rat brain and about 70% in kidney mitochondria. Together our data suggest that mitochondrial kinases activities are potent preventive antioxidant mechanism in mitochondria with low peroxidase activities, complementing the classical antioxidant enzymes against oxidative stress.     

Reduced TOR signaling extends chronological life span via increased respiration and upregulation of mitochondrial gene expression

The relationships between mitochondrial respiration, reactive oxygen species (ROS), and life span are complex and remain controversial. Inhibition of the target of rapamycin (TOR) signaling pathway extends life span in several model organisms. We show here that deletion of the TOR1 gene extends chronological life span in Saccharomyces cerevisiae, primarily by increasing mitochondrial respiration via enhanced translation of mtDNA-encoded oxidative phosphorylation complex subunits. Unlike previously reported pathways regulating chronological life span, we demonstrate that deletion of TOR1 delays aging independently of the antioxidant gene SOD2. Furthermore, wild-type and tor1 null strains differ in life span only when respiration competent and grown in normoxia in the presence of glucose. We propose that inhibition of TOR signaling causes derepression of respiration during growth in glucose and that the subsequent increase in mitochondrial oxygen consumption limits intracellular oxygen and ROS-mediated damage during glycolytic growth, leading to lower cellular ROS and extension of chronological life span.     

Mitochondrial reactive oxygen species reduce insulin secretion by pancreatic beta-cells

Pancreatic beta-cells exposed to hyperglycemia produce reactive oxygen species (ROS). Because beta-cells are sensitive to oxidative stress, excessive ROS may cause dysfunction of beta-cells. Here we demonstrate that mitochondrial ROS suppress glucose-induced insulin secretion (GIIS) from beta-cells. Intracellular ROS increased 15min after exposure to high glucose and this effect was blunted by inhibitors of the mitochondrial function. GIIS was also suppressed by H(2)O(2), a chemical substitute for ROS. Interestingly, the first-phase of GIIS could be suppressed by 50 microM H(2)O(2). H(2)O(2) or high glucose suppressed the activity of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), a glycolytic enzyme, and inhibitors of the mitochondrial function abolished the latter effects. Our data suggested that high glucose induced mitochondrial ROS, which suppressed first-phase of GIIS, at least in part, through the suppression of GAPDH activity. We propose that mitochondrial overwork is a potential mechanism causing impaired first-phase of GIIS in the early stages of diabetes mellitus.     

Involvement of mitogen-activated protein kinases and reactive oxygen species in the inotropic action of ouabain on cardiac myocytes. A potential role for mitochondrial KATP channels

Binding of ouabain to Na⁺/K⁺-ATPase activated multiple signal transduction pathways including stimulation of Src, Ras, p42/44 MAPKs and production of reactive oxygen species (ROS) in rat cardiac myocytes. Inhibition of either Src or Ras ablated ouabain-induced increase in both [Ca²⁺]_i and contractility. While PD98059 abolished the effects of ouabain on [Ca²⁺]_i, it only caused a partial inhibition of ouabain-induced increases in contractility. On the other hand, pre-incubation of myocytes with N-acetyl cysteine (NAC) reduced the effects of ouabain on contractility, but not [Ca²⁺]_i. Furthermore, 5-hydroxydecanoate (5-HD) blocked ouabain-induced ROS production and partially inhibited ouabain-induced increases in contractility in cardiac myocytes. Pre-incubation of myocytes with both 5-HD and PD98059 completely blocked ouabain's effect on contractility. Finally, we found that opening of mitochondrial K_ATP channel by diazoxide increased intracellular ROS and significantly raised contractility in cardiac myocytes. These new findings indicate that ouabain regulates cardiac contractility via both [Ca²⁺]_i and ROS. While activation of MAPKs leads to increases in [Ca²⁺]_i, opening of mitochondrial K_ATP channel relays the ouabain signal to increased ROS production in cardiac myocytes.     

Inhibition of xanthine oxidase reduces hyperglycemia-induced oxidative stress and improves mitochondrial alterations in skeletal muscle of diabetic mice

Reactive oxygen species (ROS) have been widely implicated in the pathogenesis of diabetes and more recently in mitochondrial alterations in skeletal muscle of diabetic mice. However, so far the exact sources of ROS in skeletal muscle have remained elusive. Aiming at better understanding the causes of mitochondrial alterations in diabetic muscle, we designed this study to characterize the sites of ROS production in skeletal muscle of streptozotocin (STZ)-induced diabetic mice. Hyperglycemic STZ mice showed increased markers of systemic and muscular oxidative stress, as evidenced by increased circulating H(2)O(2) and muscle carbonylated protein levels. Interestingly, insulin treatment reduced hyperglycemia and improved systemic and muscular oxidative stress in STZ mice. We demonstrated that increased oxidative stress in muscle of STZ mice is associated with an increase of xanthine oxidase (XO) expression and activity and is mediated by an induction of H(2)O(2) production by both mitochondria and XO. Finally, treatment of STZ mice, as well as high-fat and high-sucrose diet-fed mice, with oxypurinol reduced markers of systemic and muscular oxidative stress and prevented structural and functional mitochondrial alterations, confirming the in vivo relevance of XO in ROS production in diabetic mice. These data indicate that mitochondria and XO are the major sources of hyperglycemia-induced ROS production in skeletal muscle and that the inhibition of XO reduces oxidative stress and improves mitochondrial alterations in diabetic muscle.     

Neuroprotective Effects of Ginsenoside Rb1 on High Glucose-Induced Neurotoxicity in Primary Cultured Rat Hippocampal Neurons

Ginsenoside Rb1 is one of the main active principles in traditional herb ginseng and has been reported to have a wide variety of neuroprotective effects. Endoplasmic reticulum (ER) stress has been implicated in neurodegenerative diseases, so the present study aimed to observe the effects of ginsenoside Rb1 on ER stress signaling pathways in high glucose-treated hippocampal neurons. The results from MTT, TUNEL labeling and Annexin V-FITC/PI/Hoechst assays showed that incubating neurons with 50 mM high glucose for 72h decreased cell viability and increased the number of apoptotic cells whereas treating neurons with 1 μM Rb1 for 72h protected the neurons against high glucose-induced cell damage. Further molecular mechanism study demonstrated that Rb1 suppressed the activation of ER stress-associated proteins including protein kinase RNA (PKR)-like ER kinase (PERK) and C/EBP homology protein (CHOP) and downregulation of Bcl-2 induced by high glucose. Moreover, Rb1 inhibited both the elevation of intracellular reactive oxygen species (ROS) and the disruption of mitochondrial membrane potential induced by high glucose. In addition, the high glucose-induced cell apoptosis, activation of ER stress, ROS accumulation and mitochondrial dysfunction can also be attenuated by the inhibitor of ER stress 4-phenylbutyric acid (4-PBA) and anti-oxidant N-acetylcysteine(NAC). In conclusion, these results suggest that Rb1 may protect neurons against high glucose-induced cell injury through inhibiting CHOP signaling pathway as well as oxidative stress and mitochondrial dysfunction.     

Induction of mitochondrial oxidative stress in astrocytes by nitric oxide precedes disruption of energy metabolism

Inhibition of the mitochondrial electron transport chain (ETC) ultimately limits ATP production and depletes cellular ATP. However, the individual complexes of the ETC in brain mitochondria need to be inhibited by approximately 50% before causing significant depression of ATP synthesis. Moreover, the ETC is the key site for the production of intracellular reactive oxygen species (ROS) and inhibition of one or more of the complexes of the ETC may increase the rate of mitochondrial ROS generation. We asked whether partial inhibition of the ETC, to a degree insufficient to perturb oxidative phosphorylation, might nonetheless induce ROS production. Chronic increase in mitochondrial ROS might then cause oxidative damage to the ETC sufficient to produce prolonged changes in ETC function and so compound the defect. We show that the exposure of astrocytes in culture to low concentrations of nitric oxide (NO) induces an increased rate of O2*- generation that outlasts the presence of NO. No effect was seen on oxygen consumption, lactate or ATP content over the 4-6 h that the cells were exposed to NO. These data suggest that partial ETC inhibition by NO may initially cause oxidative stress rather than ATP depletion, and this may subsequently induce irreversible changes in ETC function providing the basis for a cycle of damage.     

Oxidative stress by <Emphasis Type="Italic">Helicobacter pylori</Emphasis> causes apoptosis through mitochondrial pathway in gastric epithelial cells

Helicobacter pylori is a gram negative bacterium that infects the human stomach of approximately half of the world’s population. It produces oxidative stress, and mitochondria are one of the possible targets and the major intracellular source of free radicals. The present study was aimed at determining mitochondrial alterations in H. pylori-infected gastric epithelial cells and its relationship with oxidative stress, one of the recognized causes of apoptotic processes. Cells were treated with a strain of H. pylori for 24 h. Cellular oxidative burst, antioxidant defense analysis, mitochondrial alterations and apoptosis-related processes were measured. Our data provide evidence on how superoxide acts on mitochondria to initiate apoptotic pathways, with these changes occurring in the presence of mitochondrial depolarization and other morphological and functional changes. Treatment of infected cells with Vitamin E prevented increases in intracellular ROS and mitochondrial damage consistent with H. pylori inducing a mitochondrial ROS mediated programmed cell death pathway.     

Inhibition of aldehyde dehydrogenase 2 by oxidative stress is associated with cardiac dysfunction in diabetic rats

Left ventricular (LV) dysfunction is a common comorbidity in diabetic patients, although the molecular mechanisms underlying this cardiomyopathic feature are not completely understood. Aldehyde dehydrogenase 2 (ALDH2) has been considered a key cardioprotective enzyme susceptible to oxidative inactivation. We hypothesized that hyperglycemia-induced oxidative stress would influence ALDH2 activity, and ALDH2 inhibition would lead to cardiac functional alterations in diabetic rats. Diabetes was induced by intraperitoneal (i.p.) injection of 60 mg/kg streptozotocin. Rats were divided randomly into four groups: control, untreated diabetic, diabetic treated with N-acetylcysteine (NAC) and diabetic treated with α-lipoic acid (α-LA). Cardiac contractile function, oxidative stress markers and reactive oxygen species (ROS) levels were assessed. ALDH2 activity and expression also were determined. The role of ALDH2 activity in change in hyperglycemia-induced mitochondrial membrane potential (Δψ) was tested in cultured neonatal cardiomyocytes. Myocardial MDA content and ROS were significantly higher in diabetic rats than in controls, whereas GSH content and Mn-SOD activity were decreased in diabetic rats. Compared with controls, diabetic rats exhibited significant reduction in LV ejection fraction and fractional shortening, accompanied by decreases in ALDH2 activity and expression. NAC and α-LA attenuated these changes. Mitochondrial Δψ was decreased greatly with hyperglycemia treatment, and high glucose combined with ALDH2 inhibition with daidzin further decreased Δψ. The ALDH2 activity can be regulated by oxidative stress in the diabetic rat heart. ALDH2 inhibition may be associated with LV reduced contractility, and mitochondrial impairment aggravated by ALDH2 inhibition might reflect an underlying mechanism which causes cardiac dysfunction in diabetic rats.     

Decreased mitochondrial OGG1 expression is linked to mitochondrial defects and delayed hepatoma cell growth

Many solid tumor cells exhibit mitochondrial respiratory impairment; however, the mechanisms of such impairment in cancer development remain unclear. Here, we demonstrate that SNU human hepatoma cells with declined mitochondrial respiratory activity showed decreased expression of mitochondrial 8-oxoguanine DNA glycosylase/lyase (mtOGG1), a mitochondrial DNA repair enzyme; similar results were obtained with human hepatocellular carcinoma tissues. Among several OGG1-2 variants with a mitochondrial-targeting sequence (OGG1-2a, -2b, -2c, -2d, and -2e), OGG1-2a was the major mitochondrial isoform in all examined hepatoma cells. Interestingly, hepatoma cells with low mtOGG1 levels showed delayed cell growth and increased intracellular reactive oxygen species (ROS) levels. Knockdown of OGG1-2 isoforms in Chang-L cells, which have active mitochondrial respiration with high mtOGG1 levels, significantly decreased cellular respiration and cell growth, and increased intracellular ROS. Overexpression of OGG1-2a in SNU423 cells, which have low mtOGG1 levels, effectively recovered cellular respiration and cell growth activities, and decreased intracellular ROS. Taken together, our results suggest that mtOGG1 plays an important role in maintaining mitochondrial respiration, thereby contributing to cell growth of hepatoma cells.     

Dieckol Isolated from Ecklonia cava Protects against High-Glucose Induced Damage to Rat Insulinoma Cells by Reducing Oxidative Stress and Apoptosis

Pancreatic β cells are very sensitive to oxidative stress and this might play an important role in β cell death with diabetes. The protective effect of dieckol, one of the phlorotannin polyphenol compounds purified from Ecklonia cava (E. cava), against high glucose-induced oxidative stress was investigated by using rat insulinoma cells. A high-glucose (30 mM) treatment induced the death of rat insulinoma cells, but dieckol, at a concentration 17.5 or 70 μM, significantly inhibited the high-glucose induced glucotoxicity. Treatment with dieckol also dose-dependently reduced thiobarbituric acid reactive substances (TBARS), the generation of intracellular reactive oxygen species (ROS), and the nitric oxide level increased by a high glucose concentration. In addition, the dieckol treatment increased the activities of antioxidative enzymes including catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GSH-px) in high glucose-pretreated rat insulinoma cells. Dieckol protected rat insulinoma cells damage under high glucose conditions. These effects were mediated by suppressing apoptosis and were associated with increased anti-apoptotic Bcl-2 expression, and reduced pro-apoptotic cleaved caspase-3 expression. These findings indicate that dieckol might be useful as a potential pharmaceutical agent to protect against the glucotoxicity caused by hyperglycemia-induced oxidative stress associated with diabetes.