Because systems controlled by basal NAD(P)H oxidase activity appear to contribute to differences in responses of endothelium-removed bovine coronary (BCA) and pulmonary (BPA) arteries to hypoxia, we characterized the Nox oxidases activities present in these vascular segments and how cytosolic NAD(P)H redox systems could be controlling oxidase activity. BPA generated approximately 60-80% more lucigenin (5 microM) chemiluminescence detectable superoxide than BCA. Apocynin (10 microM), a NAD(P)H oxidase inhibitor, and 6-aminonicotinamide (1 mM), a pentose phosphate inhibitor (PPP), both attenuated (approximately by 50-70%) superoxide detected in BPA and BCA. There was no significant difference in the expression of Nox2 or Nox4 mRNA or protein detected by Western blot analysis. NADPH and NADH increased superoxide in homogenates and isolated microsomal membrane fractions in a manner consistent with BPA and BCA having similar levels of oxidase activity. BPA had 4.2-fold higher levels of NADPH than BCA. The activity and protein levels of glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting PPP enzyme generating cytosolic NADPH, were 1.5-fold higher in BPA than BCA. Thus BPA differ from BCA in that they have higher levels of G6PD activity, NADPH, and superoxide. Because both arteries have similar levels of Nox expression and activity, elevated levels of cytosolic NADPH may contribute to increased superoxide in BPA.

Serpillon S, Floyd BC, Gupte RS, George S, Kozicky M, Neito V, Recchia F, Stanley W, Wolin MS, Gupte SA. Superoxide production by NAD(P)H oxidase and mitochondria is increased in genetically obese and hyperglycemic rat heart and aorta before the development of cardiac dysfunction. The role of glucose-6-phosphate dehydrogenase-derived NADPH. Am J Physiol Heart Circ Physiol 297: H153-H162, 2009. First published May 8, 2009 doi:10.1152/ajpheart.01142.2008.-Increased oxidative stress is a known cause of cardiac dysfunction in animals and patients with diabetes, but the sources of reactive oxygen species [e.g., superoxide anion (O 2 Ϫ )] and the mechanisms underlying O 2 Ϫ production in diabetic hearts are not clearly understood. Our aim was to determine whether NADPH oxidase (Nox) is a source of O 2 Ϫ and whether glucose-6-phosphate dehydrogenase (G6PD)-derived NADPH plays a role in augmenting O 2 Ϫ generation in diabetes. We assessed cardiac function, Nox and G6PD activities, NADPH levels, and the activities of antioxidant enzymes in heart homogenates from young (9-11 wk old) Zucker lean and obese (fa/fa) rats. We found that myocardial G6PD activity was significantly higher in fa/fa than in lean rats, whereas superoxide dismutase and glutathione peroxidase activities were decreased (P Ͻ 0.05). O 2 Ϫ levels were elevated (70-90%; P Ͻ 0.05) in the diabetic heart, and this elevation was blocked by the Nox inhibitor gp-91 ds-tat (50 M) or by the mitochondrial respiratory chain inhibitors antimycin (10 M) and rotenone (50 M). Inhibition of G6PD by 6-aminonicotinamide (5 mM) and dihydroepiandrosterone (100 M) also reduced (P Ͻ 0.05) O 2 Ϫ production. Notably, the activities of Nox and G6PD in the fa/fa rat heart were inhibited by chelerythrine, a protein kinase C inhibitor. Although we detected no changes in stroke volume, cardiac output, or ejection fraction, left ventricular diameter was slightly increased during diastole and systole, and left ventricular posterior wall thickness was decreased during systole (P Ͻ 0.05) in Zucker fa/fa rats. Our findings suggest that in a model of severe hyperlipidema and hyperglycemia Nox-derived O 2 Ϫ generation in the myocardium is fueled by elevated levels of G6PD-derived NADPH. Similar mechanisms were found to activate O 2 Ϫ production and induce endothelial dysfunction in aorta. Thus G6PD may be a useful therapeutic target for treating the cardiovascular disease associated with type 2 diabetes, if second-generation drugs specifically reducing the activity of G6PD to near normal levels are developed.pentose phosphate pathway; protein kinase C; adenylate cyclase; cardiac function TYPE 2 DIABETES AFFECTS A number of visceral organs, including the heart. Reduced diastolic function and left ventricular (LV) hypertrophy have been diagnosed in the absence of coronary heart disease in patients with diabetes, and heart failure is a leading cause of death among these patients (2). There is now solid evidence that, despite the elevated plasma glucose levels, the diabetic heart oxidizes less...

In the failing heart, NADPH oxidase and uncoupled NO synthase utilize cytosolic NADPH to form superoxide. NADPH is supplied principally by the pentose phosphate pathway, whose rate-limiting enzyme is glucose 6-phosphate dehydrogenase (G6PD). Therefore, we hypothesized that cardiac G6PD activation drives part of the excessive superoxide production implicated in the pathogenesis of heart failure. Pacing-induced heart failure was performed in eight chronically instrumented dogs. Seven normal dogs served as control. End-stage failure occurred after 28 +/- 1 days of pacing, when left ventricular end-diastolic pressure reached 25 mm Hg. In left ventricular tissue homogenates, spontaneous superoxide generation measured by lucigenin (5 microM) chemiluminescence was markedly increased in heart failure (1338 +/- 419 vs. 419 +/- 102 AU/mg protein, P < 0.05), as were NADPH levels (15.4 +/- 1.5 vs. 7.5 +/- 1.5 micromol/gww, P < 0.05). Superoxide production was further stimulated by the addition of NADPH. The NADPH oxidase inhibitor gp91(ds-tat) (50 microM) and the NO synthase inhibitor L-NAME (1 mM) both significantly lowered superoxide generation in failing heart homogenates by 80% and 76%, respectively. G6PD was upregulated and its activity higher in heart failure compared to control (0.61 +/- 0.10 vs. 0.24 +/- 0.03 nmol/min/mg protein, P < 0.05), while superoxide production decreased to normal levels in the presence of the G6PD inhibitor 6-aminonicotinamide. We conclude that the activation of myocardial G6PD is a novel mechanism that enhances NADPH availability and fuels superoxide-generating enzymes in heart failure.

Glucose metabolism through glycolysis and hexosamine pathway has been shown to be altered in type 2 diabetes. However, its fate through the pentose phosphate pathway (PPP) is currently unclear. In this study, we determined whether the activity of glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme in the PPP, is modulated in the liver of Zucker obese fa/fa rats (9–11 weeks old). We found that G6PD expression and activity, NADPH levels and 6-phosphogluconate generation was significantly increased in liver of fa/fa rats. Inhibition of PI3 kinase and Src kinases decreased (P<0.05) G6PD activity in the fa/fa but not in the lean rat liver, suggesting that G6PD activity is regulated by PI3/Src kinase signaling pathways. G6PD-derived NADPH increased (P<0.05) superoxide anion levels by 70–90% in the fa/fa vs the lean rat liver, which was inhibited by NADPH oxidase inhibitor, gp91ds-tat (50 μM), and G6PD inhibitors, 6-aminonicotinamide (1 mM) and dihydroepiandrosterone (100 μM); therefore, indicating that elevated G6PD activity may be responsible for mediating superoxide generation. Interestingly, we also found a positive correlation between liver hypertrophy/increased G6PD activity (r2=0.77; P=0.0009) and liver hypertrophy/superoxide production (r2=0.51; P=0.0091) in fa/fa rats. Increased G6PD and NADPH oxidase expression and activity, in young hyper-glycemic and -insulinemic rats prior to the development of diabetes, appear to be contributing factors for the induction of oxidative stress. Since inhibition of G6PD activity decreases oxidative stress, we conclude that it behaves as a pro-oxidant in the fa/fa rat liver, in type 2 diabetes.

No abstract