3-nitrotyrosine and Diabetes-Mellitus

The review deals with impairment of Ca(2+)-ATPases by high glucose or its derivatives in vitro, as well as in human diabetes and experimental animal models. Acute increases in glucose level strongly correlate with oxidative stress. Dysfunction of Ca(2+)-ATPases in diabetic and in some cases even in nondiabetic conditions may result in nitration of and in irreversible modification of cysteine-674. Nonenyzmatic protein glycation might lead to alteration of Ca(2+)-ATPase structure and function contributing to Ca(2+) imbalance and thus may be involved in development of chronic complications of diabetes. The susceptibility to glycation is probably due to the relatively high percentage of lysine and arginine residues at the ATP binding and phosphorylation domains. Reversible glycation may develop into irreversible modifications (advanced glycation end products, AGEs). Sites of SERCA AGEs are depicted in this review. Finally, several mechanisms of prevention of Ca(2+)-pump glycation, and their advantages and disadvantages are discussed.

Topics: Calcium; Calcium-Transporting ATPases; Diabetes Mellitus; Glucose; Glycation End Products, Advanced; Humans; Lysine; Oxidative Stress; Polymers; Tyrosine

Oxidative stress is a key driver of vascular dysfunction in diabetes mellitus. Ebselen is a glutathione peroxidase mimetic. A single-site, randomized, double-masked, placebo-controlled, crossover trial was carried out in 26 patients with type 1 or type 2 diabetes to evaluate effects of high-dose ebselen (150 mg po twice daily) administration on oxidative stress and endothelium-dependent vasodilation. Treatment periods were in random order of 4 wk duration, with a 4-wk washout between treatments. Measures of oxidative stress included nitrotyrosine, plasma 8-isoprostanes, and the ratio of reduced to oxidized glutathione. Vascular ultrasound of the brachial artery and plethysmographic measurement of blood flow were used to assess flow-mediated and methacholine-induced endothelium-dependent vasodilation of conduit and resistance vessels, respectively. Ebselen administration did not affect parameters of oxidative stress or conduit artery or forearm arteriolar vascular function compared with placebo treatment. There was no difference in outcome by diabetes type. Ebselen, at the dose and duration evaluated, does not improve the oxidative stress profile, nor does it affect endothelium-dependent vasodilation in patients with diabetes mellitus.

Topics: Adult; Antioxidants; Azoles; Brachial Artery; Case-Control Studies; Cross-Over Studies; Diabetes Mellitus; Dinoprost; Double-Blind Method; Endothelium, Vascular; Female; Forearm; Glutathione; Humans; Isoindoles; Male; Methacholine Chloride; Middle Aged; Organoselenium Compounds; Oxidative Stress; Parasympathomimetics; Plethysmography; Tyrosine; Ultrasonography; Vasodilation

Glucagon-like peptide 1 (GLP-1) stimulates insulin secretion. However, GLP-1 also improves endothelial function in diabetes.. Sixteen type 2 diabetic patients and 12 control subjects received a meal, an oral glucose tolerance test (OGTT), and two hyperglycemic clamps, with or without GLP-1. The clamps were repeated in diabetic patients after 2 months of strict glycemic control.. During the meal, glycemia, nitrotyrosine, and plasma 8-iso prostaglandin F2α (8-iso-PGF2a) remained unchanged in the control subjects, whereas they increased in diabetic patients. Flow-mediated vasodilation (FMD) decreased in diabetes, whereas GLP-1 increased in both groups. During the OGTT, an increase in glycemia, nitrotyrosine, and 8-iso-PGF2a and a decrease in FMD were observed at 1 h in the control subjects and at 1 and 2 h in the diabetic patients. In the same way, GLP-1 increased in both groups at the same levels of the meal. During the clamps, in both the control subjects and the diabetic patients, a significant increase in nitrotyrosine and 8-iso-PGF2a and a decrease in FMD were observed, effects that were significantly reduced by GLP-1. After improved glycemic control, hyperglycemia during the clamps was less effective in producing oxidative stress and endothelial dysfunction and the GLP-1 administration was most effective in reducing these effects.. Our data suggest that during the meal GLP-1 can simultaneously exert an incretin effect on insulin secretion and a protective effect on endothelial function, reasonably controlling oxidative stress generation. The ability of GLP-1 in protecting endothelial function seems to depend on the level of glycemia, a phenomenon already described for insulin secretion.

Topics: Diabetes Mellitus; Endothelium, Vascular; Female; Glucagon-Like Peptide 1; Glucose Tolerance Test; Humans; Male; Middle Aged; Tyrosine; Vasodilation

Adhesion molecules, particularly intracellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, and E-selectin, have been associated with cardiovascular disease. Elevated levels of these molecules have been reported in diabetic patients. Postprandial hypertriglyceridemia and hyperglycemia are considered risk factors for cardiovascular disease, and evidence suggests that postprandial hypertriglyceridemia and hyperglycemia may induce an increase in circulating adhesion molecules. However, the distinct role of these two factors is a matter of debate. Thirty type 2 diabetic patients and 20 normal subjects ate three different meals: a high-fat meal, 75 g of glucose alone, and a high-fat meal plus glucose. Glycemia, triglyceridemia, plasma nitrotyrosine, ICAM-1, VCAM-1, and E-selectin were assayed during the tests. Subsequently, diabetic subjects took simvastatin 40 mg/day or placebo for 12 weeks. The three tests were performed again at baseline, between 3 and 6 days after starting the study, and at the end of each study. High-fat load and glucose alone produced an increase of nitrotyrosine, ICAM-1, VCAM-1, and E-selectin plasma levels in normal and diabetic subjects. These effects were more pronounced when high fat and glucose were combined. Short-term simvastatin treatment had no effect on lipid parameters, but reduced the effect on adhesion molecules and nitrotyrosine, which was observed during every different test. Long-term simvastatin treatment was accompanied by a lower increase in postprandial triglycerides, which was followed by smaller variations in ICAM-1, VCAM-1, E-selectin, and nitrotyrosine during the tests. This study shows an independent and cumulative effect of postprandial hypertriglyceridemia and hyperglycemia on ICAM-1, VCAM-1, and E-selectin plasma levels, suggesting oxidative stress as a common mediator of such effects. Simvastatin shows a beneficial effect on oxidative stress and the plasma levels of adhesion molecules, which may be ascribed to a direct effect in addition to the lipid-lowering action of the drug.

Topics: Body Mass Index; Cross-Over Studies; Diabetes Complications; Diabetes Mellitus; Double-Blind Method; E-Selectin; Electrocardiography; Female; Humans; Hyperglycemia; Hypertriglyceridemia; Hypolipidemic Agents; Intercellular Adhesion Molecule-1; Male; Middle Aged; Oxidative Stress; Postprandial Period; Simvastatin; Triglycerides; Tyrosine; Vascular Cell Adhesion Molecule-1

Peroxynitrite (ONOO(-)) contributes to coronary microvascular dysfunction in diabetes mellitus (DM). We hypothesized that in DM, ONOO(-) interferes with the function of coronary endothelial caveolae, which plays an important role in nitric oxide (NO)-dependent vasomotor regulation. Flow-mediated dilation (FMD) of coronary arterioles was investigated in DM (n = 41) and non-DM (n = 37) patients undergoing heart surgery. NO-mediated coronary FMD was significantly reduced in DM patients, which was restored by ONOO(-) scavenger, iron-(III)-tetrakis(N-methyl-4'pyridyl)porphyrin-pentachloride, or uric acid, whereas exogenous ONOO(-) reduced FMD in non-DM subjects. Immunoelectron microscopy demonstrated an increased 3-nitrotyrosine formation (ONOO(-)-specific protein nitration) in endothelial plasma membrane in DM, which colocalized with caveolin-1 (Cav-1), the key structural protein of caveolae. The membrane-localized Cav-1 was significantly reduced in DM and also in high glucose-exposed coronary endothelial cells. We also found that DM patients exhibited a decreased number of endothelial caveolae, whereas exogenous ONOO(-) reduced caveolae number. Correspondingly, pharmacological (methyl-β-cyclodextrin) or genetic disruption of caveolae (Cav-1 knockout mice) abolished coronary FMD, which was rescued by sepiapterin, the stable precursor of NO synthase (NOS) cofactor, tetrahydrobiopterin. Sepiapterin also restored coronary FMD in DM patients. Thus, we propose that ONOO(-) selectively targets and disrupts endothelial caveolae, which contributes to NOS uncoupling, and, hence, reduced NO-mediated coronary vasodilation in DM patients.

Topics: Aged; Animals; Arterioles; beta-Cyclodextrins; Caveolae; Caveolin 1; Cells, Cultured; Diabetes Mellitus; Endothelial Cells; Endothelium, Vascular; Female; Humans; Male; Mice; Mice, Knockout; Middle Aged; Nitric Oxide; Nitric Oxide Synthase Type III; Peroxynitrous Acid; Pterins; Regional Blood Flow; Tyrosine; Vasodilation

Genetically diabetic Akita mice, kept on a high-fat and high-cholesterol diet, and treated with the peroxisome proliferator-activated receptor-γ agonist rosiglitazone (10 mg/kg per day during 4 months), displayed rosiglitazone-induced side effects, similar to those observed in patients, including weight and fat gain and early signs of hypertrophic cardiomyopathy. As several cases of hepatotoxicity were reported in patients receiving rosiglitazone treatment, this study evaluated whether rosiglitazone also induced hepatotoxicity in these diabetic animals. Liver structure and function was analysed in wild-type and rosiglitazone-treated and untreated Akita mice, kept for 4 months on the high-fat and high-cholesterol diet. Decreased circulating levels of the liver enzymes aspartate and alanine aminotransferase and increased levels of alkaline phosphatases were observed upon rosiglitazone treatment, whereas liver weight was markedly increased. Rosiglitazone administration was associated with liver steatosis, as demonstrated by triglyceride accumulation. However, gene expression of steatosis markers in liver tissue was not markedly affected by rosiglitazone treatment, while expression of fatty acid transport protein was reduced by rosiglitazone treatment, suggesting an impairment of the fatty acid β-oxidation pathway. mRNA expression of pro- and anti-oxidant enzymes and liver 3-nitrotyrosine content was not affected. Furthermore, gene and protein expression of macrophage markers and of cell adhesion molecules did not indicate progression to steatohepatitis, whereas an unaltered collagen deposition did not suggest steatofibrosis. In conclusion, rosiglitazone treatment of diabetic Akita mice induced liver steatosis without, however, progression to more advanced stages of liver disease.

Topics: Alanine Transaminase; Alkaline Phosphatase; Animals; Aspartate Aminotransferases; Cholesterol, Dietary; Diabetes Mellitus; Diet, High-Fat; Fatty Liver; Gene Expression; Genotype; Liver; Male; Mice; Mice, Inbred C57BL; Organ Size; PPAR gamma; RNA, Messenger; Rosiglitazone; Thiazolidinediones; Triglycerides; Tyrosine

Hyperglycemia in diabetes causes increased oxidative stress in the vascular endothelium with generation of free radicals such as superoxide. Peroxynitrite, a highly reactive species generated from superoxide and nitric oxide (NO), induces proinflammatory tyrosine nitration of intracellular proteins under such conditions. The female sex hormone estrogen appears to exert protective effects on the nondiabetic endothelium. However, several studies show reduced vascular protection in women with diabetes, suggesting alterations in estrogen signaling under high glucose. In this study, we examined the endothelial effects of estrogen under increasing glucose levels, focusing on nitrotyrosine and peroxynitrite. Human umbilical vein endothelial cells were incubated with normal (5.5 mM) or high (15.5 or 30.5 mM) glucose before addition of estradiol (E2, 1 or 10 nM). Selective NO synthase (NOS) inhibitors were used to determine the role of specific NOS isoforms. Addition of E2 significantly reduced high glucose-induced increase in peroxynitrite and consequently, nitrotyrosine. The superoxide levels were unchanged, suggesting effects on NO generation. Inhibition of neuronal NOS (nNOS) reduced high glucose-induced nitrotyrosine, demonstrating a critical role for this enzyme. E2 increased nNOS activity under normal glucose while decreasing it under high glucose as determined by its phosphorylation status. These data show that nNOS contributes to endothelial peroxynitrite and subsequent nitrotyrosine generation under high glucose, which can be attenuated by E2 through nNOS inhibition. The altered regulation of nNOS by E2 under high glucose is a potential therapeutic target in women with diabetes.

Topics: Diabetes Mellitus; Endothelium, Vascular; Enzyme Inhibitors; Estradiol; Female; Glucose; Human Umbilical Vein Endothelial Cells; Humans; Nitric Oxide; Nitric Oxide Synthase Type I; Nitric Oxide Synthase Type III; Oxidation-Reduction; Oxidative Stress; Peroxynitrous Acid; Signal Transduction; Superoxides; Tyrosine

Prominent features of myocardial remodeling in heart failure with preserved ejection fraction (HFPEF) are high cardiomyocyte resting tension (F(passive)) and cardiomyocyte hypertrophy. In experimental models, both reacted favorably to raised protein kinase G (PKG) activity. The present study assessed myocardial PKG activity, its downstream effects on cardiomyocyte F(passive) and cardiomyocyte diameter, and its upstream control by cyclic guanosine monophosphate (cGMP), nitrosative/oxidative stress, and brain natriuretic peptide (BNP). To discern altered control of myocardial remodeling by PKG, HFPEF was compared with aortic stenosis and HF with reduced EF (HFREF).. Patients with HFPEF (n=36), AS (n=67), and HFREF (n=43) were free of coronary artery disease. More HFPEF patients were obese (P<0.05) or had diabetes mellitus (P<0.05). Left ventricular myocardial biopsies were procured transvascularly in HFPEF and HFREF and perioperatively in aortic stenosis. F(passive) was measured in cardiomyocytes before and after PKG administration. Myocardial homogenates were used for assessment of PKG activity, cGMP concentration, proBNP-108 expression, and nitrotyrosine expression, a measure of nitrosative/oxidative stress. Additional quantitative immunohistochemical analysis was performed for PKG activity and nitrotyrosine expression. Lower PKG activity in HFPEF than in aortic stenosis (P<0.01) or HFREF (P<0.001) was associated with higher cardiomyocyte F(passive) (P<0.001) and related to lower cGMP concentration (P<0.001) and higher nitrosative/oxidative stress (P<0.05). Higher F(passive) in HFPEF was corrected by in vitro PKG administration.. Low myocardial PKG activity in HFPEF was associated with raised cardiomyocyte F(passive) and was related to increased myocardial nitrosative/oxidative stress. The latter was probably induced by the high prevalence in HFPEF of metabolic comorbidities. Correction of myocardial PKG activity could be a target for specific HFPEF treatment.

Topics: Aortic Valve Stenosis; Biopsy; Cohort Studies; Comorbidity; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Diabetes Mellitus; Female; Heart; Heart Failure; Humans; Male; Middle Aged; Myocardium; Natriuretic Peptide, Brain; Obesity; Oxidative Stress; Stroke Volume; Tyrosine

The redox enzyme p66Shc produces hydrogen peroxide and triggers proapoptotic signals. Genetic deletion of p66Shc prolongs life span and protects against oxidative stress. In the present study, we evaluated the role of p66Shc in an animal model of diabetic wound healing.. Skin wounds were created in wild-type (WT) and p66Shc(-/-) control and streptozotocin-induced diabetic mice with or without hind limb ischemia. Wounds were assessed for collagen content, thickness and vascularity of granulation tissue, apoptosis, reepithelialization, and expression of c-myc and beta-catenin. Response to hind limb ischemia was also evaluated.. Diabetes delayed wound healing in WT mice with reduced granulation tissue thickness and vascularity, increased apoptosis, epithelial expression of c-myc, and nuclear localization of beta-catenin. These nonhealing features were worsened by hind limb ischemia. Diabetes induced p66Shc expression and activation; wound healing was significantly faster in p66Shc(-/-) than in WT diabetic mice, with or without hind limb ischemia, at 1 and 3 months of diabetes duration and in both SV129 and C57BL/6 genetic backgrounds. Deletion of p66Shc reversed nonhealing features, with increased collagen content and granulation tissue thickness, and reduced apoptosis and expression of c-myc and beta-catenin. p66Shc deletion improved response to hind limb ischemia in diabetic mice in terms of tissue damage, capillary density, and perfusion. Migration of p66Shc(-/-) dermal fibroblasts in vitro was significantly faster than WT fibroblasts under both high glucose and hypoxia.. p66Shc is involved in the delayed wound-healing process in the setting of diabetes and ischemia. Thus, p66Shc may represent a potential therapeutic target against this disabling diabetes complication.

Topics: Animals; Arginine; Diabetes Mellitus; Diabetes Mellitus, Experimental; Lysine; Mice; Mice, Knockout; Muscle, Skeletal; Oxidation-Reduction; Shc Signaling Adaptor Proteins; Skin; Src Homology 2 Domain-Containing, Transforming Protein 1; Tyrosine; Wound Healing

It has been reported that urinary oxidative stress markers are higher in diabetic patients with proteinuria. We performed the present study to elucidate the relationship between urinary excretion of oxidative stress markers, albumin excretion, and histological changes, and to confirm the potential utility of oxidative stress markers for clinical treatment.. Diabetic db/db mice or nondiabetic db/m mice were administered candesartan (10 mg/kg/day) or hydralazine (50 mg/kg/day) for 18 weeks.. Thirty-week-old male db/db mice treated with control vehicle revealed elevated urinary excretion and immunohistological levels of 8-hydroxydeoxyguanosine in glomeruli when compared to db/m mice. Treatment with candesartan, but not hydralazine, reduced these values to levels in db/m mice. Increased mesangial expansion, urinary excretion of albumin and 8-isoprostane, and glomerular immunohistological levels of nitrotyrosine in db/db mice were also decreased markedly by candesartan but not hydralazine. Interestingly, correlations between levels of albumin and oxidative stress markers in urine were very high, even when groups undergoing long-term (44 weeks) treatment were included (correlation coefficient 0.767 with respect to 8-hydroxydeoxyguanosine, 0.888 with respect to 8-isoprostane).. It is anticipated that urinary concentrations of oxidative stress markers will be direct barometers of glomerulus-derived oxidative stress and glomerular injury in diabetic nephropathy.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Albuminuria; Angiotensin II Type 1 Receptor Blockers; Animals; Antihypertensive Agents; Benzimidazoles; Biomarkers; Biphenyl Compounds; Deoxyguanosine; Diabetes Mellitus; Diabetic Nephropathies; Dinoprost; Disease Models, Animal; Hydralazine; Kidney Glomerulus; Male; Mice; Mice, Inbred C57BL; Oxidative Stress; Tetrazoles; Treatment Outcome; Tyrosine

In the course of type 2 diabetes mellitus, insulin resistance has a severe impact on endothelial function leading to decreased synthesis of nitric oxide (NO). Postprandial hyperglycemia leads to the generation of reactive oxygen species, which counteracts the beneficial NO effects. NO and superoxide combine very fast in solution to form peroxynitrite, which is a potent protein-oxidizing agent. The peroxynitrite concentrations can be indirectly monitored by the detection of nitrotyrosine residues in proteins, reflecting the extent of damage caused by oxidative stress.. Four commercially available nitrotyrosine-specific immunoassays were evaluated by parallel measurement of nitrotyrosine in 224 serum samples derived from 16 patients with type 2 diabetes and 12 healthy controls (13 male and 15 female, age: 33+/-11 years) following a standardized meal.. The available ELISA tests were not applicable for nitrotyrosine determination in human plasma samples due to technical issues and implausible results. However, a competitive luminescence assay was able to provide sufficient sensitivity and lead to clinically meaningful results in our test samples.. All three ELISA methods were disqualified and conclusions previously derived from clinical experiments using these tests should be carefully reconsidered or reconfirmed. In the absence of a liquid tandem chromatography-mass spectrometry reference method, the luminescence test appears to be the method of choice for determination of nitrotyrosine in human plasma.

Topics: Diabetes Mellitus; Humans; Immunoassay; Tyrosine

Recently we have shown that diabetes-induced retinal neurodegeneration positively correlates with oxidative stress and peroxynitrite. Studies also show that peroxynitrite impairs nerve growth factor (NGF) survival signaling in sensory neurons. However, the causal role of peroxynitrite and the impact of tyrosine nitration on diabetes-induced retinal neurodegeneration and NGF survival signaling have not been elucidated.. Expression of NGF and its receptors was examined in retinas from human and streptozotocin-induced diabetic rats and retinal ganglion cells (RGCs). Diabetic animals were treated with FeTPPS (15 mg x kg(-1) x day(-1) ip), which catalytically decomposes peroxynitrite to nitrate. After 4 weeks of diabetes, retinal cell death was determined by TUNEL assay. Lipid peroxidation and nitrotyrosine were determined using MDA assay, immunofluorescence, and Slot-Blot analysis. Expression of NGF and its receptors was determined by enzyme-linked immunosorbent assay (ELISA), real-time PCR, immunoprecipitation, and Western blot analyses.. Analyses of retinal neuronal death and NGF showed ninefold and twofold increases, respectively, in diabetic retinas compared with controls. Diabetes also induced increases in lipid peroxidation, nitrotyrosine, and the pro-apoptotic p75(NTR) receptor in human and rat retinas. These effects were associated with tyrosine nitration of the pro-survival TrkA receptor, resulting in diminished phosphorylation of TrkA and its downstream target, Akt. Furthermore, peroxynitrite induced neuronal death, TrkA nitration, and activation of p38 mitogen-activated protein kinase (MAPK) in RGCs, even in the presence of exogenous NGF. FeTPPS prevented tyrosine nitration, restored NGF survival signal, and prevented neuronal death in vitro and in vivo.. Together, these data suggest that diabetes-induced peroxynitrite impairs NGF neuronal survival by nitrating TrkA receptor and enhancing p75(NTR) expression.

Topics: Animals; Cadaver; Cell Death; Diabetes Mellitus; Diabetes Mellitus, Experimental; Diabetic Neuropathies; Humans; Lipid Peroxidation; Middle Aged; Nerve Degeneration; Nerve Growth Factor; Optic Nerve; Peroxynitrous Acid; Rats; Reference Values; Retinal Ganglion Cells; RNA, Messenger; Streptozocin; Tyrosine

Studies on plasma nitrotyrosine (NT) levels, a measure of oxidative injury, in diabetes are limited and discordant; the amount of antioxidants might represent a possible explanation for the discordant results. The aim of this paper is to evaluate the association between plasma NT levels and glucose tolerance status, according to antioxidant vitamin intakes.. In three hundred men randomly selected from a population-based cohort, NT levels were measured and dietary intake assessed by a food-frequency questionnaire. Results NT values were similar in patients with diabetes (n = 34), impaired fasting glucose (n = 77) and normoglycaemic subjects (n = 189). However, in subjects with lower than recommended daily intakes of antioxidant vitamins C and A, NT levels were significantly higher in the diabetic patients. In a multiple regression model, after adjustments for age, body mass index (BMI) and smoking habits, NT levels were significantly associated with fasting glucose in patients with lower intakes of vitamin C (beta = 11.4; 95% CI 1.3-21.5) and vitamin A (beta = 14.9; 95% CI 3.9-25.9), but not in subjects with lower intake of vitamin E.. A significant positive correlation between NT levels and fasting glucose is evident only in the presence of a reduced intake of some antioxidant vitamins. These findings might explain, at least in part, the discrepant results of previous studies and, if confirmed by further studies, suggest a simple measure (a balanced diet) to alleviate the increased oxidative stress of diabetes.

Topics: Age Factors; Antioxidants; Ascorbic Acid; Blood Glucose; Body Mass Index; Cohort Studies; Diabetes Mellitus; Diet; Humans; Hyperglycemia; Male; Middle Aged; Smoking; Tyrosine; Vitamin A; Vitamin E; Vitamins

Although the accelerated atherosclerosis and premature aging of the cardiovascular system in patients with metabolic syndrome have been appreciated, the mechanisms of their development and potential therapeutic interventions remain unresolved. Our previous studies implicated advanced glycosylation end products in development of premature senescence preventable with a peroxynitrite scavenger, ebselen. Therefore, the effect of ebselen on endothelial senescence and vasculopathy in a model of metabolic syndrome--Zucker diabetic rats (ZDF)--was investigated. Ebselen decreased the abundance of 3-nitrotyrosine-modified proteins in ZDF rats. A 6-fold increase in the number of senescent endothelial cells in 22-week-old ZDF was prevented by ebselen. Development of vasculopathy, as collectively judged by the acetylcholine-induced vasorelaxation, NO production, angiogenic competence, and number of circulating microparticles, was almost completely prevented when ebselen was administered from 8 to 22 weeks and partially reversed when the treatment interval was 13 to 22 weeks. In conclusion, premature senescence of endothelial cells is progressively rampant in ZDF rats and is associated with the signs of severe vasculopathy. In addition, prevention of premature senescence of vascular endothelium through controlled decrease in nitrotyrosine formation was chronologically associated with the amelioration of vasculopathy, lending support to the idea of the pathogenetic role of premature senescence of endothelial cells in diabetic macrovasculopathy.

Topics: Acetylcholine; Animals; Antioxidants; Aorta, Thoracic; Azoles; Cell Cycle Proteins; Cellular Senescence; Diabetes Mellitus; Endothelial Cells; Endothelium, Vascular; In Vitro Techniques; Isoindoles; Microcirculation; Muscle, Skeletal; Neovascularization, Physiologic; Nitric Oxide; Obesity; Organoselenium Compounds; Rats; Rats, Zucker; Tyrosine; Vasodilation; Vasodilator Agents

Oxidative stress plays a major role in disease processes such as atherosclerosis and diabetes. Peroxynitrite is a reaction product of nitric oxide (NO) and superoxide and a potent oxidant. The peroxynitrite-mediated tyrosine nitration, which forms nitrotyrosine (NT), is associated with several pathological conditions.. We measured plasma NT levels using the HPLC method in 40 Mexican Americans with diabetes, but not taking medications, and 40 age- and sex-matched euglycaemic controls.. Plasma-free NT levels were not different between subjects with diabetes (11.0 +/- 1.7 nmol/l, n = 40) and with non-diabetes (10.4 +/- 1.5 nmol/l, n = 40). There was also no association with levels of fasting glucose (r = -0.049, P = 0.663) or 2-h glucose (r = -0.099, P = 0.390). However, females had significantly lower free NT level (7.6 +/- 1.4 nmol/l, n = 40) than males (13.8 +/- 1.7 nmol/l, n = 40, P = 0.005), which were not affected by age, smoking status, BMI and glucose levels.. In contrast to some earlier reports, our study shows that diabetes has no effect on plasma NT levels in Mexican Americans. We have also demonstrated lower free NT levels in females than males, which may partly explain the lower risk profile to vascular disease in women.

Topics: Blood Glucose; Diabetes Mellitus; Female; Humans; Male; Middle Aged; Sex Factors; Smoking; Tyrosine

Clinical and experimental studies have shown that reinstitution of good glycemic control (GC) after a period of poor glycemic control (PC) does not produce immediate benefits on the progression of retinopathy, and hyperglycemia is sufficient to initiate the development of diabetic retinopathy. In this study, the effect of reinstitution of GC on hyperglycemia-induced increased oxidative stress and nitrative stress was evaluated in the retina of rats maintained in PC before initiation of GC. In diabetic rats, 2 or 6 months of PC (GHb >11.0%) was followed by 7 months of GC (GHb <5.5%). Reinstitution of GC after 2 months of PC inhibited elevations in retinal lipid peroxides and NO levels by approximately 50%, but failed to have any beneficial effects on nitrotyrosine formation. However, reversal of hyperglycemia after 6 months of PC had no significant effect on retinal oxidative stress and NO levels (P < 0.02 vs. normal). In the same rats, inducible nitric oxide synthase expression and nitrotyrosine levels remained elevated by >80% compared with normal rats or rats kept in GC for the duration. This suggests that oxidative and nitrative modifications in retina occur early in the course of development of retinopathy in diabetes. These abnormalities are not easily reversed by reinstitution of GC, and the duration of PC before initiation of GC influences the outcome of the reversal. Characterization of the abnormalities responsible for the resistance of retinopathy to arrest after reinstitution of GC will help identify potential future therapies to inhibit progression of diabetic retinopathy.

Topics: Animals; Blood Glucose; Diabetes Mellitus; Diabetic Retinopathy; Glutathione; Hyperglycemia; Insulin; Lipid Peroxides; Male; Nitrates; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Oxidative Stress; Rats; Rats, Wistar; Retina; Tyrosine

Topics: Animals; Blood Vessels; Cells, Cultured; Diabetes Mellitus; Endothelium, Vascular; Enzyme Activation; Glucose; Heart; Humans; Hyperglycemia; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type III; Peroxynitrous Acid; Rats; Superoxides; Tyrosine; Up-Regulation

Aminoguanidine inhibits the development of retinopathy in diabetic animals, but the mechanism remains unclear. Inasmuch as aminoguanidine is a relatively selective inhibitor of the inducible isoform of nitric oxide synthase (iNOS), we have investigated the effects of hyperglycemia on the retinal nitric oxide (NO) pathway in the presence and absence of aminoguanidine. In vivo studies utilized retinas from experimentally diabetic rats treated or without aminoguanidine for 2 months, and in vitro studies used bovine retinal endothelial cells and a transformed retinal glial cell line (rMC-1) incubated in 5 mm and 25 mm glucose with and without aminoguanidine (100 microg/mL). NO was detected as nitrite and nitrate, and nitrotyrosine and iNOS were detected using immunochemical methods. Retinal homogenates from diabetic animals had greater than normal levels of NO and iNOS (p < 0.05), and nitrotyrosine was greater than normal, especially in one band immunoprecipitated from retinal homogenates. Oral aminoguanidine significantly inhibited all of these increases. Nitrotyrosine was detected immunohistochemically only in the retinal vasculature of non-diabetic and diabetic animals. Retinal endothelial and rMC-1 cells cultured in high glucose increased NO and NT, and aminoguanidine inhibited both increases in rMC-1 cells, but only NT in endothelial cells. Hyperglycemia increases NO production in retinal cells, and aminoguanidine can inhibit this abnormality. Inhibition of diabetic retinopathy by aminoguanidine might be mediated in part by inhibition of sequelae of NO production.

Topics: Animals; Cattle; Cells, Cultured; Cyclic GMP; Diabetes Complications; Diabetes Mellitus; Diabetic Retinopathy; Endothelium, Vascular; Glucose; Guanidines; Hyperglycemia; Immunohistochemistry; Male; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Oxidative Stress; Peroxynitrous Acid; Rats; Rats, Sprague-Dawley; Retina; Streptozocin; Tyrosine

Taurine is actively transported at the retinal pigment epithelial (RPE) apical membrane in an Na(+)- and Cl(-)-dependent manner. Diabetes may alter the function of the taurine transporter. Because nitric oxide (NO) is a molecule implicated in the pathogenesis of diabetes, we asked whether NO would alter the activity of the taurine transporter in cultured ARPE-19 cells. The activity of the transporter was stimulated in the presence of the NO donor 3-morpholinosydnonimine. The stimulatory effects of 3-morpholinosydnonimine were not observed during the initial 16-h treatment; however, stimulation of taurine uptake was elevated dramatically above control values with 20- and 24-h treatments. Kinetic analysis revealed that the stimulation was associated with an increase in the maximal velocity of the transporter with no significant change in the substrate affinity. The NO-induced increase in taurine uptake was inhibited by actinomycin D and cycloheximide. RT-PCR analysis and nuclear run-on assays provided evidence for upregulation of the transporter gene. This study provides the first evidence of an increase in taurine transporter gene expression in human RPE cells cultured under conditions of elevated levels of NO.

Topics: Animals; Antioxidants; Ascorbic Acid; Carrier Proteins; Cell Line; Diabetes Mellitus; Dose-Response Relationship, Drug; Glutathione; Humans; Immunohistochemistry; Membrane Glycoproteins; Membrane Transport Proteins; Methylene Blue; Mice; Mice, Inbred ICR; Molsidomine; Nitric Oxide; Nitric Oxide Donors; Nitroprusside; Pigment Epithelium of Eye; Protein Synthesis Inhibitors; Taurine; Time Factors; Tyrosine

To evaluate whether an intact respiratory burst exists within the orbit of diabetics with rhinocerebral mucormycosis.. Immunohistochemical detection of nitrotyrosine in the orbital tissue of diabetics requiring exenteration due to rhinocerebral mucormycosis. Nitrotyrosine is the stable product of the nitration of tyrosine residues by peroxynitrite. Peroxynitrite is a potent oxidant produced by the combination of superoxide and nitric oxide during the respiratory burst.. Four specimens were analyzed. All showed focal areas of specific staining against nitrotyrosine of the walls and internal structures of fungal organisms.. An intact respiratory burst is present in the orbit of diabetics during infection with rhinocerebral mucormycosis. Possible mechanisms of peroxynitrite's microbicidal effects and reasons for a deficiency in diabetics are discussed.

Topics: Adolescent; Adult; Aged; Brain Diseases; Diabetes Mellitus; Eye Infections, Fungal; Female; Humans; Immunoenzyme Techniques; Male; Middle Aged; Mucormycosis; Nitrates; Orbital Diseases; Paranasal Sinus Diseases; Respiratory Burst; Tyrosine