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Background Patients with long-term exposure to high levels of hyperglycaemia remain more susceptible to diabetes-related complications, even with subsequent lower levels of hyperglycaemia. We sought to confirm the hypothesis that exposure to continuous increased glucose results in a memory of cellular stress in isolated endothelial cells, even when switched back to normal glucose, and to investigate the ability of gliclazide to attenuate this phenomenon. Methods Human umbilical vein endothelial cells were incubated for 21 days in normal glucose (5 mmol/L), high glucose (30 mmol/L), or high glucose for 14 days followed by normal glucose for 7 days (memory condition). The effects of gliclazide (10 mu mol/L) and glibenclamide (1 mu mol/L) were evaluated in the memory condition and added to the culture media early (first 14 days), late (last 7 days), or throughout the study. Oxidative stress and cell apoptosis parameters were investigated. Results Continuous high glucose increased reactive oxygen species, 8-OHdG, nitrotyrosine, caspase-3, and reduced Bcl-2 expression. These deleterious effects were also observed in the memory condition. Gliclazide applied early or throughout the study improved all parameters. In contrast, glibenclamide showed no relevant effect on study parameters. Conclusions Our results suggest that gliclazide prevents endothelial cell apoptosis by reducing oxidative stress. The results appear to confirm the hypothesis that exposure of cells to continuous increased glucose results in a hyperglycaemic cellular memory that remains, even when cells are switched back to normal glucose. Gliclazide attenuated this cellular memory, decreasing oxidative stress and protecting vascular endothelial cells from apoptosis. Copyright (C) 2007 John Wiley & Sons, Ltd.

OBJECTIVE— Long-lasting hyperglycemia in type 1 diabetic patients induces permanent alterations of endothelial function by increased oxidative stress, even when glycemia is normalized. RESEARCH DESIGN AND METHODS— In this study, 36 type 1 diabetic patients and 12 control subjects were enrolled. The diabetic patients were divided into three groups. The first group was treated for 24 h with insulin, achieving a near normalization of glycemia. After 12 h of this treatment, vitamin C was added for the remaining 12 h. The second group was treated for 24 h with vitamin C. After 12 h of this treatment, insulin was started, achieving a near normalization of glycemia for the remaining 12 h. The third group was treated for 24 h with both vitamin C and insulin, achieving near normalization of glycemia. The same protocols were performed after 1 month of telmisartan or placebo. RESULTS— Neither normalization of glycemia nor vitamin C treatment alone was able to normalize endothelial dysfunction or oxidative stress. Combining insulin and vitamin C normalized endothelial dysfunction and decreased oxidative stress to normal levels. Telmisartan significantly improved basal endothelial function and decreased nitrotyrosine plasma levels. In patients treated with telmisartan, a near normalization of both flow-mediated vasodilation and oxidative stress was achieved when glycemia was normalized, whereas adding vitamin C infusion did not show further effect on endothelial function or nitrotyrosine plasma levels. CONCLUSIONS— These data indicate that combining the normalization of glycemia with an antioxidant can normalize endothelial function in type 1 diabetic patients and that telmisartan works as an antioxidant like vitamin C.

Evidence suggests that postprandial hyperglycaemia may be a cardiovascular risk factor in diabetes. Oxidative stress and inflammation are involved in the pathogenesis of diabetic complications and previous studies have shown increased oxidative stress and inflammation in the postprandial phase in diabetic patients. The aim of the present study was to evaluate whether controlling postprandial hyperglycaemia with S21403 (mitiglinide) is accompanied by a reduced generation of oxidative stress and inflammation.Forty type 2 diabetic patients participated in the study. Two different breakfast-tests were performed in each patient, with placebo or S21403. Plasma nitrotyrosine, plasma malondialdehyde (MDA), oxidised LDL (oxLDL), plasma total radical-trapping antioxidant parameter (TRAP), IL-6, IL-18, TNF-alpha, plasma glucose and insulin were measured.After the administration of S21403, 40 mg, a rapid stimulation of insulin secretion was observed, accompanied by a reduction of postprandial hyperglycaemia. With S21403, a significant decrease of either nitrotyrosine, MDA and oxLDL levels, and a preservation of plasma TRAP compared with placebo was found. Significant decreases of IL-6, IL-18 and TNF-alpha were also observed with S21403 compared with placebo.This study shows that controlling postprandial hyperglycaemia with S21403 significantly improves the cluster of oxidative stress and inflammation markers that are increased in the postprandial state in diabetic patients.

Objective Recently, much attention has been focused on the possibility that the post-prandial state may be a cardiovascular risk factor in diabetes. The aim of the present study was to evaluate whether the post-prandial state is associated with endothelial dysfunction in patients with diabetes and to explore the effect on this aspect of managing post-prandial hyperglycaemia by insulin aspart. Research design and methods Twenty-three patients with Type 2 diabetes and 10 normal controls were recruited. In the diabetic patients two different tests were performed in each subject: a standard meal preceded by subcutaneous injection of soluble insulin (0.15 U/kg body weight) or of short-acting insulin aspart (0.15 U/kg body weight). These tests were designed to achieve different levels of post-prandial hyperglycaemia. Controls received a single standard meal test. Immediately before, and 1, 2, 4 and 6 h after each meal, blood glucose, triglycerides, free fatty acids and flow-mediated vasodilation were measured. Results Compared with regular insulin, insulin aspart significantly reduced the area under the curve for post-prandial hyperglycaemia (58.3 ± 17.6 vs. 68.1 ± 17.7; P

Aim Data suggest that 2h hyperglycaemia during an OGTT is less predictive for cardiovascular disease of the glucose "spike" (the difference between the baseline glucose level and the "peak" hyperglycaemia during the test). Methods A euinsulinemic hyperglycaemic clamp at 10 and 15mmol/l glucose, with or without vitamin C, was given in increasing steps in diabetic and normal subjects. Moreover, a hyperglycaemic clamp, 10mmol/l, was performed in two groups of diabetic patients with different levels of fasting glycaemia. In both the experiments flow mediated dilation and nitrotyrosine were measured. Results Glucose at 10 and 15mmol/l resulted in a concentration-dependent induction of endothelial dysfunction and oxidative stress. Vitamin C counterbalanced this effect. The increase of glycaemia to 10mmol/l induced a significant endothelial dysfunction and increased nitrotyrosine in both the groups of diabetics with different fasting glycaemia. However, when the delta (the difference between the basal value and the peak value) for endothelial function and nitrotyrosine was evaluated, patients with lower basal values showed a worse outcome. Conclusions Our data suggest that at the same level of hyperglycaemia the grading of the endothelial dysfunction is almost super imposable, but clearly worse in terms of delta from fasting glycaemia.

OBJECTIVE—To explore the possibility that oscillating glucose may outweigh A1C levels in determining the risk for cardiovascular diabetes complications. RESEARCH DESIGN AND METHODS—A euinsulinemic hyperglycemic clamp at 5, 10, and 15 mmol/l glucose was given in increasing steps as a single “spike” or oscillating between basal and high levels over 24 h in normal subjects and type 2 diabetic patients. Flow-mediated dilatation, a marker of endothelial function, and plasma 3-nitrotyrosine and 24-h urinary excretion rates of free 8-iso PGF2α, two markers of oxidative stress, were measured over 48 h postclamp. RESULTS—Glucose at two different levels (10 and 15 mmol/l) resulted in a concentration-dependent fasting blood glucose–independent induction of both endothelial dysfunction and oxidative stress in both normal and type 2 diabetic patients. Oscillating glucose between 5 and 15 mmol/l every 6 h for 24 h resulted in further significant increases in endothelial dysfunction and oxidative stress compared with either continuous 10 or 15 mmol/l glucose. CONCLUSIONS—These data suggest that oscillating glucose can have more deleterious effects than constant high glucose on endothelial function and oxidative stress, two key players in favoring cardiovascular complications in diabetes. Concomitant vitamin C infusion can reverse this impairment.

Background The production of oxidative stress as a result of postprandial hyperglycaemia is now recognized as an important contributing factor in the development of diabetes complications. The objective of this study was to examine the effects of pramlintide on plasma concentrations of glucose and several markers of oxidative stress in patients with type 2 diabetes following a standardized meal. Methods This was a randomized, single-blind, placebo-controlled, crossover study conducted at two clinical research centres in the United States. A total of 19 subjects (9 men and 10 women) with type 2 diabetes using mealtime insulin participated in the study. Pramlintide (120 µg), or placebo, and rapid-acting mealtime insulin were administered prior to a standardized meal on two separate study days. Plasma concentrations of glucose, nitrotyrosine (NT), oxidized-LDL cholesterol (OxLDL-C), and total radical trapping parameter (TRAP) were assessed during the 4-h postprandial period. Results Compared to placebo, pramlintide treatment reduced postprandial excursions of glucose, NT, and OxLDL-C and protected TRAP from consumption. Correlation analysis revealed positive associations between placebo-corrected glucose incremental AUC0–4h and both NT and OxLDL-C and a negative association between placebo-corrected glucose incremental AUC0–4h and TRAP. Conclusions The reduction in postprandial glucose excursions achieved with addition of pramlintide to rapid-acting insulin in type 2 diabetes was associated with a reduction in postprandial markers of oxidative stress. Copyright © 2007 John Wiley & Sons, Ltd.

OBJECTIVE—Previous studies have shown that in type 1 diabetes endothelial dysfunction persists even when glycemia is normalized. Moreover, oxidative stress has recently been demonstrated to be the mediator of hyperglycemia-induced endothelial dysfunction. RESEARCH DESIGN AND METHODS—Thirty-six type 1 diabetic patients and 12 control subjects were enrolled. The diabetic patients were divided into three groups. The first group was treated for 24 h with insulin, achieving a near-normalization of glycemia. After 12 h of this treatment, vitamin C was added for the remaining 12 h. The second group was treated for 24 h with vitamin C. After 12 h of this treatment, insulin was started, with achievement of near-normalization of glycemia for the remaining 12 h. The third group was treated for 24 h with both vitamin C and insulin, achieving near-normalization of glycemia. RESULTS—Neither normalization of glycemia nor vitamin C treatment alone was able to normalize endothelial dysfunction or oxidative stress. However, a combination of insulin and vitamin C normalized endothelial dysfunction and decreased oxidative stress to normal levels. CONCLUSIONS—This study suggests that long-lasting hyperglycemia in type 1 diabetic patients induces permanent alterations in endothelial cells, which may contribute to endothelial dysfunction by increased oxidative stress even when hyperglycemia is normalized.

In diabetes the exposure of the vascular endothelium to high glucose levels results in increased oxidative insult and in vascular dysfunction. We have investigated the effects of rosuvastatin on oxidative stress and apoptosis induced in human umbilical vein endothelial cells (HUVECs) by constant and intermittent high glucose levels. HUVECs were incubated for 14 days in either low (5 mM) or high (20 mM) glucose concentrations, or intermittent high and low glucose on a daily basis. Constant high glucose levels increased p47-phox, p67-phox, and p22-phox expression [components of the Nicotinamide adenine dinucleotide phosphate [NAD(P)H] oxidase complex]; endothelial nitric oxide synthase, nitric oxide, and O 2 − production; nitrotyrosine, 8-hydroxy-2′-deoxyguanosine, and caspase-3 expression; and reduced Bcl-2 expression. These effects were significantly greater under intermittent compared to constant high/low glucose conditions. The effect of rosuvastatin (1 μM) in the presence or absence of mevalonate (200 μM) was evaluated in the cells under both constant and intermittent glucose conditions. Rosuvastatin almost normalized all these parameters. These effects of rosuvastatin were prevented when mevalonate was also added, demonstrating the link to inhibition of 3-hydroxy-3-methylglutaryl coenzyme A reductase. These data suggest that rosuvastatin has the potential to prevent damage to and apoptosis of HUVECs induced by high glucose exposure, by reducing oxidative stress. The action of rosuvastatin on antioxidant pathways is related to the inhibition of the overexpression of components of NAD(P)H oxidase induced by the two conditions of high glucose.

Background— Postprandial hypertriglyceridemia and hyperglycemia are considered risk factors for cardiovascular disease. Evidence suggests that postprandial hypertriglyceridemia and hyperglycemia induce endothelial dysfunction and inflammation through oxidative stress. Statins and angiotensin type 1 receptor blockers have been shown to reduce oxidative stress and inflammation, improving endothelial function. Methods and Results— Twenty type 2 diabetic patients ate 3 different test meals: a high-fat meal, 75 g glucose alone, and a high-fat meal plus glucose. Glycemia, triglyceridemia, endothelial function, nitrotyrosine, C-reactive protein, intercellular adhesion molecule-1, and interleukin-6 were assayed during the tests. Subsequently, diabetics took atorvastatin 40 mg/d, irbesartan 300 mg/d, both, or placebo for 1 week. The 3 tests were performed again between 5 and 7 days after the start of each treatment. High-fat load and glucose alone produced a decrease in endothelial function and increases in nitrotyrosine, C-reactive protein, intercellular adhesion molecule-1, and interleukin-6. These effects were more pronounced when high-fat load and glucose were combined. Short-term atorvastatin and irbesartan treatments significantly counterbalanced these phenomena, and their combination was more effective than either therapy alone. Conclusions— This study confirms an independent and cumulative effect of postprandial hypertriglyceridemia and hyperglycemia on endothelial function and inflammation, suggesting oxidative stress as a common mediator of such an effect. Short-term treatment with atorvastatin and irbesartan may counterbalance this phenomenon; the combination of the 2 compounds is most effective.

OBJECTIVE—Oxidative stress has been shown to be increased in the postprandial period in patients with diabetes and has been implicated in the pathogenesis of micro- and macrovascular complications. The aim of this post hoc analysis was to assess the effects of pramlintide, an amylin analog shown to reduce postprandial glucose excursions in patients with diabetes, on markers of oxidative stress in the postprandial period. RESEARCH DESIGN AND METHODS—In a randomized, single-blind, placebo-controlled, crossover study, 18 evaluable subjects with type 1 diabetes underwent two standardized breakfast meal tests and received pramlintide or placebo in addition to their preprandial insulin. The plasma concentrations of glucose and markers of oxidative stress (nitrotyrosine, oxidized LDL [ox-LDL], and total radical-trapping antioxidant parameter [TRAP]) were measured at baseline and during the 4-h postprandial period. RESULTS—Compared with placebo, pramlintide treatment significantly reduced postprandial excursions of glucose, nitrotyrosine, and ox-LDL and prevented a decline in TRAP (P < 0.03 for all comparisons). Correlation analyses adjusted for treatment revealed a significant association between postprandial mean incremental area under the curve from 0 to 4 h (AUC0–4 h) for glucose and postprandial mean incremental AUC0–4 h for each measure of oxidative stress (r = 0.75, 0.54, and −0.63 for nitrotyrosine, ox-LDL, and TRAP, respectively; P < 0.001 for all correlations). CONCLUSIONS—These findings indicate that the postprandial glucose-lowering effect of pramlintide in type 1 diabetes is associated with a significant reduction in postprandial oxidative stress.

Oxidative stress is involved in the pathogenesis of microangiopathic and macroangiopathic diabetic complications. The results of recent trials suggest that type 1 angiotensin II (AT-1) receptor blockers may prevent or delay nephropathy and cardiovascular disease in diabetic patients, independently of their anti-hypertensive action. There is evidence that AT-1 receptor blockers can work as intracellular antioxidants. This study investigated whether the AT-1 receptor blocker irbesartan is able to reduce nitrotyrosine formation in non-hypertensive diabetic patients under fasting conditions and during acute hyperglycaemia.A total of 40 non-hypertensive, non-microalbuminuric Type 2 diabetic patients and 20 healthy, normotensive subjects were recruited for this study. Diabetic patients followed a randomised, double-blind, placebo-controlled, crossover protocol, taking either irbesartan (150 mg orally, twice daily) or placebo for 60 days. Fasting glucose and nitrotyrosine were measured at baseline and at the end of each treatment period. An OGTT was also performed at the same time intervals, during which plasma glucose and nitrotyrosine levels were monitored.Compared with baseline measurements, treatment with irbesartan (0.57+/-0.4 vs 0.35+/-0.3 micromol/l, p0.01) but not placebo (0.58+/-0.3 vs 0.59+/-0.2 micromol/l) significantly reduced fasting nitrotyrosine levels. Irbesartan also significantly reduced nitrotyrosine formation during the OGTT.. This study demonstrates that irbesartan reduces plasma levels of nitrotyrosine in diabetic patients and is effective in counterbalancing nitrotyrosine formation during acute hyperglycaemia. Our results may help to elucidate how AT-1 receptor blockers exert their beneficial effect independently of their BP-lowering activity.

Background— Postprandial hypertriglyceridemia and hyperglycemia are considered risk factors for cardiovascular disease. Evidence suggests that postprandial hypertriglyceridemia and hyperglycemia induce endothelial dysfunction through oxidative stress; however, the distinct role of these two factors is a matter of debate. Methods and Results— Thirty type 2 diabetic patients and 20 normal subjects ate 3 different meals: a high-fat meal; 75 g glucose alone; and high-fat meal plus glucose. Glycemia, triglyceridemia, nitrotyrosine, and endothelial function were assayed during the tests. Subsequently, diabetics took 40 mg/d simvastatin or placebo for 12 weeks. The 3 tests were performed again at baseline, between 3 to 6 days after the start, and at the end of each study. High-fat load and glucose alone produced a decrease of endothelial function and an increase of nitrotyrosine 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 endothelial function and nitrotyrosine observed during each different test. Long-term simvastatin treatment was accompanied by a lower increase in postprandial triglycerides, which was followed by smaller variations of endothelial function and nitrotyrosine during the tests. Conclusions— This study shows an independent and cumulative effect of postprandial hypertriglyceridemia and hyperglycemia on endothelial function, suggesting oxidative stress as common mediator of such effect. Simvastatin shows a beneficial effect on oxidative stress and endothelial dysfunction, which may be ascribed to a direct effect as well as the lipid-lowering action of the drug.

In fact, even being scarcely reactive this molecule has the tendency to ‘radicalize,’ forming incompletely reduced molecules characterized by an uncoupled electron. This highly reactive and short-living class of molecules is known as reactive oxygen species (ROS). ROS production is a side effect of the normal metabolism of the cell, which developed a series of enzymes able to disarm them. Superoxide dismutase, catalase, and glutathione peroxidase are powerful weapons that act together with antioxidant molecules introduced with diet to protect the organism. In healthy subjects there is a balance between ROS formation and elimination. Every time this balance is lost due to an augmented production of reactive species or due to a reduction in antioxidant production or activity there is a condition of oxidative stress. Losing control of ROS is very harmful and almost all the constituents of the cell can be targets of these molecules. DNA, proteins, and lipids can be involved in chain reactions that entail their modification and, in the worst case, the loss of their functionality. Genetic degeneration and physiological dysfunction can lead to cell death and aging of the organism.