Apoptosis induced by crocidolite asbestos in human lung epithelial cells involves inactivation of Akt and MAPK pathways

Exposure of human lung epithelial (A549) cells to asbestos fibers causes apoptosis, which is largely attributed to release of iron and generation of reactive oxygen species (ROS) within the cells. To mimic the highly oxidative environment generated by asbestos exposure in the absence of the actual fibers, we used two chemicals; buthione sulfoximine (BSO), an inhibitor of glutathione (GSH) synthesis and ferric ammonium citrate (FAC), a source of iron. Here, we report that exposure of A549 cells to crocidolite asbestos led to a significant time-dependent inactivation of signaling proteins, i.e. Akt and all mitogen-activated protein kinases (MAPKs) (p38, ERK1/2 and SAPK/JNK), and subsequently to apoptosis. Unlike crocidolite treatment, the use of BSO and FAC, independently or combined, did not change the phosphorylation status of proteins, nor did it induce apoptosis. Taken together, our results presented herein point to the possibility that crocidolite-induced apoptosis of human lung epithelial cells is not a mere consequence of generation of oxidants but also requires inactivation of major cell growth and differentiation pathways. A. Baldys, P. Pande contributed equally to this publication.     

Regulation of G6PD acetylation by SIRT2 and KAT9 modulates NADPH homeostasis and cell survival during oxidative stress

Glucose‐6‐phosphate dehydrogenase (G6PD) is a key enzyme in the pentose phosphate pathway (PPP) and plays an essential role in the oxidative stress response by producing NADPH, the main intracellular reductant. G6PD deficiency is the most common human enzyme defect, affecting more than 400 million people worldwide. Here, we show that G6PD is negatively regulated by acetylation on lysine 403 (K403), an evolutionarily conserved residue. The K403 acetylated G6PD is incapable of forming active dimers and displays a complete loss of activity. Knockdown of G6PD sensitizes cells to oxidative stress, and re‐expression of wild‐type G6PD, but not the K403 acetylation mimetic mutant, rescues cells from oxidative injury. Moreover, we show that cells sense extracellular oxidative stimuli to decrease G6PD acetylation in a SIRT2‐dependent manner. The SIRT2‐mediated deacetylation and activation of G6PD stimulates PPP to supply cytosolic NADPH to counteract oxidative damage and protect mouse erythrocytes. We also identified KAT9/ELP3 as a potential acetyltransferase of G6PD. Our study uncovers a previously unknown mechanism by which acetylation negatively regulates G6PD activity to maintain cellular NADPH homeostasis during oxidative stress.     

The toxic effect of fluoride on MG-63 osteoblast cells is also dependent on the production of nitric oxide

Some soda-lime-phospho-silicate glasses, such as Hench's Bioglass(?) 45S5, form bone-like apatite on their surface when bound to living bone. To improve their osteointegration for clinical purposes, the fluoride insertion in their structure has been proposed, but we recently showed that fluoride causes oxidative damage in human MG-63 osteoblasts, via inhibition of pentose phosphate oxidative pathway (PPP) and its key enzyme glucose 6-phosphate dehydrogenase (G6PD). In the same cells we have now investigated the role of nitric oxide (NO) in these effects. Fluoride-containing bioactive glasses and NaF caused, as expected, release of lactate dehydrogenase in the extracellular medium, accumulation of intracellular malonyldialdehyde, inhibition of PPP and G6PD: we have now observed that these effects were significantly reverted not only by superoxide dismutase (SOD) plus catalase (scavengers of reactive oxygen species), but also by N-monomethyl l-arginine (l-NMMA, a NOS inhibitor) and 2-phenyl-4,4,5,5,-tetramethylimidazoline-1oxyl 3-oxide (PTIO, a NO scavenger). Moreover the two highest concentrations of both fluoride-containing bioglasses and NaF caused increase of nitrite (a stable derivative of NO) levels in the culture supernatant, which was inhibited by l-NMMA, erythrocytes, PTIO and SOD/catalase, and increase of intracellular NO synthase (NOS) activity. The incubation with bioglasses or NaF increased also the phosphorylation of Ser(1177) in the endothelial NOS isoform. Furthermore, the NO donor spermine NONOate was able to inhibit G6PD activity in vitro, and this effect was partly reverted by PTIO. Therefore our results suggest that most cytotoxic effects of fluoride are mediated by the production of NO: reactive oxygen species are important, causing NOS phosphorylation. We also observed, for the first time, that Tempol, but not SOD/catalase, besides inhibiting the oxidative stress induced by fluoride, also scavenges fluoride ions. For this reason it is not a selective inhibitor of the oxidative effects of fluoride.     

Mitochondrial-derived free radicals mediate asbestos-induced alveolar epithelial cell apoptosis

Asbestos causes pulmonary toxicity by mechanisms that in part involve reactive oxygen species (ROS). However, the precise source of ROS is unclear. We showed that asbestos induces alveolar epithelial cell (AEC) apoptosis by a mitochondrial-regulated death pathway. To determine whether mitochondrial-derived ROS are necessary for causing asbestos-induced AEC apoptosis, we utilized A549-rho(omicron) cells that lack mitochondrial DNA and a functional electron transport. As expected, antimycin, which induces an oxidative stress by blocking mitochondrial electron transport at complex III, increased dichlorofluoroscein (DCF) fluorescence in A549 cells but not in A549-rho(omicron) cells. Compared with A549 cells, rho(omicron) cells have less asbestos-induced ROS production, as assessed by DCF fluorescence, and reductions in total glutathione levels as well as less caspase-9 activation and apoptosis, as assessed by TdT-mediated dUTP nick end labeling staining and DNA fragmentation. A mitochondrial anion channel inhibitor that prevents ROS release from the mitochondria to the cytoplasm also blocked asbestos-induced A549 cell caspase-9 activation and apoptosis. Finally, a role for nonmitochondrial-derived ROS with exposure to high levels of asbestos (50 microg/cm(2)) was suggested by our findings that an iron chelator (phytic acid or deferoxamine) or a free radical scavenger (sodium benzoate) provided additional protection against asbestos-induced caspase-9 activation and DNA fragmentation in rho(omicron) cells. We conclude that asbestos fibers affect mitochondrial DNA and functional electron transport, resulting in mitochondrial-derived ROS production that in turn mediates AEC apoptosis. Nonmitochondrial-associated ROS may also contribute to AEC apoptosis, particularly with high levels of asbestos exposure.     

ATM activates the pentose phosphate pathway promoting anti-oxidant defence and DNA repair

Ataxia telangiectasia (A-T) is a human disease caused by ATM deficiency characterized among other symptoms by radiosensitivity, cancer, sterility, immunodeficiency and neurological defects. ATM controls several aspects of cell cycle and promotes repair of double strand breaks (DSBs). This probably accounts for most of A-T clinical manifestations. However, an impaired response to reactive oxygen species (ROS) might also contribute to A-T pathogenesis. Here, we show that ATM promotes an anti-oxidant response by regulating the pentose phosphate pathway (PPP). ATM activation induces glucose-6-phosphate dehydrogenase (G6PD) activity, the limiting enzyme of the PPP responsible for the production of NADPH, an essential anti-oxidant cofactor. ATM promotes Hsp27 phosphorylation and binding to G6PD, stimulating its activity. We also show that ATM-dependent PPP stimulation increases nucleotide production and that G6PD-deficient cells are impaired for DSB repair. These data suggest that ATM protects cells from ROS accumulation by stimulating NADPH production and promoting the synthesis of nucleotides required for the repair of DSBs.     

Neuroprotection by glucose-6-phosphate dehydrogenase and the pentose phosphate pathway

Glucose-6-phosphate dehydrogenase (G6PD), the rate limiting enzyme that channels glucose catabolism from glycolysis into the pentose phosphate pathway (PPP), is vital for the production of reduced nicotinamide adenine dinucleotide phosphate (NADPH) in cells. NADPH is in turn a substrate for glutathione reductase, which reduces oxidized glutathione disulfide to sulfhydryl glutathione. Best known for inherited deficiencies underlying acute hemolytic anemia due to elevated oxidative stress by food or medication, G6PD, and PPP activation have been associated with neuroprotection. Recent works have now provided more definitive evidence for G6PD's protective role in ischemic brain injury and strengthened its links to neurodegeneration. In Drosophila models, improved proteostasis and lifespan extension result from an increased PPP flux due to G6PD induction, which is phenocopied by transgenic overexpression of G6PD in neurons. Moderate transgenic expression of G6PD was also shown to improve healthspan in mouse. Here, the deciphered and implicated roles of G6PD and PPP in protection against brain injury, neurodegenerative diseases, and in healthspan/lifespan extensions are discussed together with an important caveat, namely NADPH oxidase (NOX) activity and the oxidative stress generated by the latter. Activation of G6PD with selective inhibition of NOX activity could be a viable neuroprotective strategy for brain injury, disease, and aging.     

Modulation of genotoxic effects in asbestos-exposed primary human mesothelial cells by radical scavengers, metal chelators and a glutathione precursor

The genotoxicity of asbestos fibers is generally mediated by reactive oxygen species (ROS) and by insufficient antioxidant protection. To further elucidate which radicals are involved in asbestos-mediated genotoxicity and to which extent, we have carried out experiments with the metal chelators deferoxamine (DEF) and phytic acid (PA), and with the radical scavengers superoxide dismutase (SOD), dimethylthiourea (DMTU) and the glutathione precursor Nacystelyn trade mark (NAL). We investigated the influence of these compounds on the potency of crocidolite, an amphibole asbestos fiber with a high iron content (27%), and chrysotile, a serpentine asbestos fiber with a low iron content (2%), to induce micronuclei (MN) in human mesothelial cells (HMC) after an exposure time of 24-72 h. Our results show that the number of crocidolite-induced MN is significantly reduced after pretreatment of fibers with PA and DEF. This effect was not observed with chrysotile. In contrast, simultaneous treatment of cells with asbestos and the OH*scavenging DMTU or the O2- -scavenging SOD significantly decreased the number of MN induced by chrysotile and crocidolite. In particular, DMTU almost completely suppressed micronucleus induction by both fiber types. A similar effect was observed in the presence of the H(2)O(2)-scavenging NAL after chrysotile treatment of HMC. By means of kinetochore analysis, it could be shown that the number of clastogenic events is decreased after PA and DEF pretreatment of fibers as well as after application of the above-mentioned scavengers. Our results show that chrysotile asbestos induces an increased release of H(2)O(2) in contrast to crocidolite. Also, the iron content of the fiber plays an important role in radical formation, but nevertheless, chrysotile produces oxy radicals to a similar extent as crocidolite, probably by phagocytosis-mediated oxidative bursting.     

Glycolytic cancer cells lacking 6-phosphogluconate dehydrogenase metabolize glucose to induce senescence

We show that knockdown of 6-phosphogluconate dehydrogenase (6PGD) of the pentose phosphate pathway (PPP) inhibits growth of lung cancer cells by senescence induction. This inhibition is not due to a defect in the oxidative PPP per se. NADPH and ribose phosphate production are normal in 6PGD knockdown cells and shutdown of PPP by knockdown of glucose-6-phosphate dehydrogenase (G6PD) has little effect on cell growth. Moreover, 6PGD knockdown cells can proliferate when the PPP is bypassed by using fructose instead of glucose in medium. Significantly, G6PD knockdown rescues proliferation of cells lacking 6PGD, suggesting an accumulation of growth inhibitory glucose metabolics in cells lacking 6PGD. Therefore, 6PGD inhibition may provide a novel strategy to treat glycolyic tumors such as lung cancer.     

SIRT5 promotes IDH2 desuccinylation and G6PD deglutarylation to enhance cellular antioxidant defense

Excess in mitochondrial reactive oxygen species (ROS) is considered as a major cause of cellular oxidative stress. NADPH, the main intracellular reductant, has a key role in keeping glutathione in its reduced form GSH, which scavenges ROS and thus protects the cell from oxidative damage. Here, we report that SIRT5 desuccinylates and deglutarylates isocitrate dehydrogenase 2 (IDH2) and glucose‐6‐phosphate dehydrogenase (G6PD), respectively, and thus activates both NADPH‐producing enzymes. Moreover, we show that knockdown or knockout of SIRT5 leads to high levels of cellular ROS. SIRT5 inactivation leads to the inhibition of IDH2 and G6PD, thereby decreasing NADPH production, lowering GSH, impairing the ability to scavenge ROS, and increasing cellular susceptibility to oxidative stress. Our study uncovers a SIRT5‐dependent mechanism that regulates cellular NADPH homeostasis and redox potential by promoting IDH2 desuccinylation and G6PD deglutarylation.     

Glucose-6-Phosphate Dehydrogenase (G6PD)-Deficient Epithelial Cells Are Less Tolerant to Infection by Staphylococcus aureus

Glucose-6-phosphate dehydrogenase (G6PD) is a key enzyme in the pentose phosphate pathway and provides reducing energy to all cells by maintaining redox balance. The most common clinical manifestations in patients with G6PD deficiency are neonatal jaundice and acute hemolytic anemia. The effects of microbial infection in patients with G6PD deficiency primarily relate to the hemolytic anemia caused by Plasmodium or viral infections and the subsequent medication that is required. We are interested in studying the impact of bacterial infection in G6PD-deficient cells. G6PD knock down A549 lung carcinoma cells, together with the common pathogen Staphylococcus aureus, were employed in our cell infection model. Here, we demonstrate that a lower cell viability was observed among G6PD-deficient cells when compared to scramble controls upon bacterial infection using the MTT assay. A significant increase in the intracellular ROS was detected among S. aureus-infected G6PD-deficient cells by observing dichlorofluorescein (DCF) intensity within cells under a fluorescence microscope and quantifying this signal using flow cytometry. The impairment of ROS removal is predicted to enhance apoptotic activity in G6PD-deficient cells, and this enhanced apoptosis was observed by annexin V/PI staining under a confocal fluorescence microscope and quantified by flow cytometry. A higher expression level of the intrinsic apoptotic initiator caspase-9, as well as the downstream effector caspase-3, was detected by Western blotting analysis of G6PD-deficient cells following bacterial infection. In conclusion, we propose that bacterial infection, perhaps the secreted S. aureus α-hemolysin in this case, promotes the accumulation of intracellular ROS in G6PD-deficient cells. This would trigger a stronger apoptotic activity through the intrinsic pathway thereby reducing cell viability when compared to wild type cells.     

SIRT2 controls the pentose phosphate switch

The most common enzyme defect in humans is glucose‐6‐phosphate dehydrogenase (G6PD) deficiency, which affects more than 400 million people. G6PD shunts glucose into the pentose phosphate pathway (PPP) to generate nucleotides and reducing potential in the form of NADPH. In this issue, Wang et al ( 2014 ) show that G6PD activity is post‐translationally regulated by SIRT2, a cytoplasmic NAD⁺‐dependent deacetylase, thereby linking NAD⁺ levels to DNA repair and oxidative defences, and identifying potential new approaches to treating this common genetic disease.     

Glucose-6-phosphate metabolism in Plasmodium falciparum

Malaria is still one of the most threatening diseases worldwide. The high drug resistance rates of malarial parasites make its eradication difficult and furthermore necessitate the development of new antimalarial drugs. Plasmodium falciparum is responsible for severe malaria and therefore of special interest with regard to drug development. Plasmodium parasites are highly dependent on glucose and very sensitive to oxidative stress; two observations that drew interest to the pentose phosphate pathway (PPP) with its key enzyme glucose-6-phosphate dehydrogenase (G6PD). A central position of the PPP for malaria parasites is supported by the fact that human G6PD deficiency protects to a certain degree from malaria infections. Plasmodium parasites and the human host possess a complete PPP, both of which seem to be important for the parasites. Interestingly, there are major differences between parasite and human G6PD, making the enzyme of Plasmodium a promising target for antimalarial drug design. This review gives an overview of the current state of research on glucose-6-phosphate metabolism in P. falciparum and its impact on malaria infections. Moreover, the unique characteristics of the enzyme G6PD in P. falciparum are discussed, upon which its current status as promising target for drug development is based.     

Peroxynitrite protects neurons against nitric oxide-mediated apoptosis. A key role for glucose-6-phosphate dehydrogenase activity in neuroprotection

Peroxynitrite is thought to be a nitric oxide-derived neurotoxic effector molecule involved in the disruption of key energy-related metabolic targets. To assess the consequences of such interference in cellular glucose metabolism and viability, we studied the possible modulatory role played by peroxynitrite in glucose oxidation in neurons and astrocytes in primary culture. Here, we report that peroxynitrite triggered rapid stimulation of pentose phosphate pathway (PPP) activity and the accumulation of NADPH, an essential cofactor for glutathione regeneration. In contrast to peroxynitrite, nitric oxide elicited NADPH depletion, glutathione oxidation, and apoptotic cell death in neurons, but not in astrocytes. These events were noticeably counteracted by pretreatment of neurons with peroxynitrite. In an attempt to elucidate the mechanism responsible for this PPP stimulation and neuroprotection, we found evidence consistent with both exogenous and endogenous peroxynitrite-mediated activation of glucose-6-phosphate dehydrogenase (G6PD), an enzyme that catalyzes the first rate-limiting step in the PPP. Moreover, functional overexpression of the G6PD gene in stably transformed PC12 cells induced NADPH accumulation and offered remarkable resistance against nitric oxide-mediated apoptosis, whereas G6PD gene-targeted antisense inhibition depleted NADPH levels and exacerbated cellular vulnerability. In light of these results, we suggest that G6PD activation represents a novel role for peroxynitrite in neuroprotection against nitric oxide-mediated apoptosis.     

Involvement of the pentose phosphate pathway and redox regulation in fertilization in the mouse

Glucose metabolism is necessary for successful fertilization in the mouse. Both spermatozoa and oocytes metabolize glucose through the pentose phosphate pathway (PPP), and NADPH appears required for gamete fusion. The aims of this study were to further characterize the utilization of glucose by the fertilizing spermatozoon and the fertilized oocyte, to demonstrate the importance of the PPP in different steps of fertilization, and to examine whether the beneficial effect of glucose could be mediated by a NADPH-dependent enzyme involved in redox regulation. By using a fluorescent analog of 2-deoxyglucose, glucose uptake was evidenced in both the head and flagellum of motile spermatozoa. After sperm-oocyte fusion, an increase in glucose uptake by the fertilized oocyte was observed but not before the formation of the male and female pronuclei. By using a microphotometric technique, activity of glucose 6-phosphate dehydrogenase (G6PDH), the key enzyme of the PPP, was localized to the sperm head and midpiece. When epididymal spermatozoa were released into a glucose-containing medium, the NADPH/NADP ratio increased with capacitation. Sperm-oocyte fusion and meiosis reinitiation of the fertilized oocyte was inhibited by the PPP inhibitor 6-aminonicotinamide (6-AN); inhibition of sperm-oocyte fusion was relieved by NADPH. Sperm-oocyte fusion and meiosis reinitiation were also inhibited by diphenylamine iodonium, which is a flavoenzyme inhibitor reported to prevent reactive oxygen species (ROS) generation in mouse spermatozoa and embryos. These findings indicate that the PPP is involved in different steps of fertilization. Subsequent regulation of a NADPH-dependent flavoenzyme responsible of ROS production is envisaged.     

Redistribution of pulmonary EC-SOD after exposure to asbestos.

Inhalation of asbestos fibers leads to interstitial lung disease (asbestosis) characterized by inflammation and fibrosis. The pathogenesis of asbestosis is not fully understood, but reactive oxygen species are thought to play a central role. Extracellular superoxide dismutase (EC-SOD) is an antioxidant enzyme that protects the lung in a bleomycin-induced pulmonary fibrosis model, but its role has not been studied in asbestos-mediated disease. EC-SOD is found in high levels in the extracellular matrix of lung alveoli because of its positively charged heparin-binding domain. Proteolytic removal of this domain results in clearance of EC-SOD from the matrix of tissues. We treated wild-type C57BL/6 mice with 0.1 mg of crocidolite asbestos by intratracheal instillation and euthanized them 24 h later. Compared with saline- or titanium dioxide-treated control mice, bronchoalveolar lavage fluid (BALF) from asbestos-treated mice contained significantly higher total protein levels and increased numbers of inflammatory cells, predominantly neutrophils, indicating acute lung injury in response to asbestos. Decreased EC-SOD protein and activity were found in the lungs of asbestos-treated mice, whereas more EC-SOD was found in the BALF of these mice. The EC-SOD in the BALF was predominantly in the proteolyzed form, which lacks the heparin-binding domain. This redistribution of EC-SOD correlated with development of fibrosis 14 days after asbestos exposure. These data suggest that asbestos injury leads to enhanced proteolysis and clearance of EC-SOD from lung parenchyma into the air spaces. The depletion of EC-SOD from the extracellular matrix may increase susceptibility of the lung to oxidative stress during asbestos-mediated lung injury.     

Identification of Macrolepiota procera extract as a novel G6PD inhibitor for the treatment of lung cancer

Tumor metabolism, an emerging hallmark of cancer, is characterized by aberrant expression of enzymes from various metabolic pathways including glycolysis and PPP (pentose phosphate pathway). Glucose 6 phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD), oxidative carboxylases of PPP, have been reported to accomplish different biosynthetic and energy requirements of cancer cells. G6PD and 6PGD have been proposed as potential therapeutic targets for cancer therapy during recent years due to their overexpression in various cancers. Here, we have employed enzymatic assay based screening using in-house G6PD and 6PGD assay protocols for the identification of mushroom extracts which could inhibit G6PD or 6PGD enzymatic activity for implications in cancer therapy. For the fulfillment of the objectives of present study, nine edible mushrooms were subjected to green extraction for preparation of ethanolic extracts. 6xhis-G6PD and pET-28a-h6PGD plasmids were expressed in BL21-DE3 E. coli cells for the expression and purification of protein of interests. Using purified proteins, in house enzymatic assay protocols were established. The preliminary screening identified two extracts (Macrolepiota procera and Terfezia boudieri) as potent and selective G6PD inhibitors, while no extract was found highly active against 6PGD. Further, evaluation of anticancer potential of mushroom extracts against lung cancer cells revealed Macrolepiota procera as potential inhibitor of cancer cell proliferation with IC₅₀ value of 6.18 μg/ml. Finally, screening of M. procera-derived compounds against G6PD via molecular docking has identified paraben, quercetin and syringic acid as virtual hit compounds possessing good binding affinity with G6PD. The result of present study provides novel findings for possible mechanism of action of M. procera extract against A549 via G6PD inhibition suggesting that M. procera might be of therapeutic interest for lung cancer treatment.     

Asbestos-induced alveolar epithelial cell apoptosis: role of mitochondrial dysfunction caused by iron-derived free radicals

Asbestos causes asbestosis and malignancies by mechanisms that are not fully understood. Alveolar epithelial cell (AEC) injury by iron-derived reactive oxygen species (ROS) is one important mechanism implicated. We previously showed that iron-catalyzed ROS in part mediate asbestos-inducedAEC DNA damage and apoptosis. Mitochondria have a critical role in regulating apoptosis after exposure to agents causing DNA damage but their role in regulating asbestos-induced apoptosis is unknown. To determine whether asbestos causes AEC mitochondrial dysfunction, we exposed A549 cells to amosite asbestos and assessed mitochondrial membrane potential changes (delta(psi)m) using a fluorometric technique involving tetremethylrhodamine ethyl ester (TMRE) and mitotracker green. We show that amosite asbestos, but not an inert particulate, titanium dioxide, reduces delta(psi)m after a 4 h exposure period. Further, the delta(psi)m after 4 h was inversely proportional to the levels of apoptosis noted at 24 h as assessed by nuclear morphology as well as by DNA nucleosome formation. A role for iron-derived ROS was suggested by the finding that phytic acid, an iron chelator, blocked asbestos-induced reductions in A549 cell delta(psi)m and attenuated apoptosis. Finally, overexpression of Bcl-xl, an anti-apoptotic protein that localizes to the mitochondria, prevented asbestos-induced decreases in A549 cell delta(psi)m after 4 h and diminished apoptosis. We conclude that asbestos alters AEC mitochondrial function in part by generating iron-derived ROS, which in turn can result in apoptosis. This suggests that the mitochondrial death pathway is important in regulating pulmonary toxicity from asbestos.     

Increasing Glucose 6-Phosphate Dehydrogenase Activity Restores Redox Balance in Vascular Endothelial Cells Exposed to High Glucose

Previous studies have shown that high glucose increases reactive oxygen species (ROS) in endothelial cells that contributes to vascular dysfunction and atherosclerosis. Accumulation of ROS is due to dysregulated redox balance between ROS-producing systems and antioxidant systems. Previous research from our laboratory has shown that high glucose decreases the principal cellular reductant, NADPH by impairing the activity of glucose 6-phosphate dehydrogenase (G6PD). We and others also have shown that the high glucose-induced decrease in G6PD activity is mediated, at least in part, by cAMP-dependent protein kinase A (PKA). As both the major antioxidant enzymes and NADPH oxidase, a major source of ROS, use NADPH as substrate, we explored whether G6PD activity was a critical mediator of redox balance. We found that overexpression of G6PD by pAD-G6PD infection restored redox balance. Moreover inhibition of PKA decreased ROS accumulation and increased redox enzymes, while not altering the protein expression level of redox enzymes. Interestingly, high glucose stimulated an increase in NADPH oxidase (NOX) and colocalization of G6PD with NOX, which was inhibited by the PKA inhibitor. Lastly, inhibition of PKA ameliorated high glucose mediated increase in cell death and inhibition of cell growth. These studies illustrate that increasing G6PD activity restores redox balance in endothelial cells exposed to high glucose, which is a potentially important therapeutic target to protect ECs from the deleterious effects of high glucose.