Role of Hyperglycemia in Nitrotyrosine Postprandial Generation

A total of 23 type 2 diabetic patients on insulin therapy and 15 matched healthy subjects were recruited for this study. The clinical characteristics of the subjects are shown in Table 1. The mean duration of insulin treatment was 3.0 ± 0.9 years. The mean usual daily insulin dose was 28.4 ± 5.6 units, administered in two or three daily injections. The diabetic patients were in satisfactory metabolic control, as judged by means of their glycated hemoglobin levels (7.3 ± 0.5%). All subjects were normotensive nonsmokers. Each subject had a normal resting electrocardiogram and no personal history of vascular disease.

None of the subjects were taking aspirin, lipid-lowering agents, or supplemental vitamins. All subjects were recommended to consume their usual diet during the entire period of the study (1 week) and to not inject intermediate insulin the night before each test. Written consent was obtained from all subjects, with local ethics committee approval.

After visit 1 (screening and informed consent), patients were randomized (visit 2) on the basis of a computer-generated random table list to the first of the two treatment sequences.

On the day of the study, a blood sample was drawn for fasting glycemia, and each patient started an intravenous infusion of regular insulin (Actrapid; Novo Nordisk, Bagsvaerd, Denmark) to achieve a premeal plasma glucose level ranging from 7.2 and 8.3 mmol/l.

The intravenous insulin infusion was then stopped, and after 30 min, the experiment was started. Actrapid (0.15 units/kg body wt) was administered 30 min before the meal, whereas insulin aspart (NovoRapid; Novo Nordisk) (0.15 units/kg body wt) was injected subcutaneously at the beginning of the meal. The dose of 0.15 units/kg body wt was selected according to the recommendations by Rosenfalck et al. (15). The two tests were performed in each subject within 3 days of each other. The meal test was also performed in healthy control subjects. The cooked meal contained 600 kcal (50% carbohydrate, 30% fat, 20% protein) (16). At 0 min and 1, 2, 4, and 6 h after each meal, glycemia, triglyceride, and NT levels were evaluated.

Plasma glucose was measured by the glucose oxidase method, triglycerides were measured enzymatically by a commercially available method (Roche Diagnostics, Basel), and HbA_1c was measured by high-performance liquid chromatography (Menarini Diagnostics, Pisa, Italy).

NT plasma concentration was assayed by enzyme-linked immunosorbent assay (ELISA), developed according to Ter Steege et al. (17) and recently validated by our laboratory (12).

BSA, tetranitromethane (TNM), IgG secondary antibody, and tetramethil-benzidine (TMB) were purchased from Sigma (St. Louis, MO). Maxisorp ELISA plates were from NUNC, Life Technologies (Grand Island, NY), and IgG monoclonal anti-nitrotyrosine antibody was from Upstate (Lake Placid, NY).

Nitrated protein solution was carried out by incubating 1 mg/ml BSA in 50 mmol/l KH₂PO₄ at pH 7.4 for 30 min at 37°C with 1 mmol/l TNM, an efficient nitrating agent (18). After adjusting the pH to 10 with 3 mol/l NaOH, the amount of NT present in the TNM-treated BSA solution was measured at 430 nm (18).

A standard curve was constructed by incubating in the wells serial dilutions of nitro-BSA in 0.1 mol/l Na₂CO₃-NaHCO₃ coating buffer at a pH of 9.6. Plasma samples assayed for NT were diluted up to five times. Standard and plasma samples were applied to Maxisorp ELISA plates and allowed to bind overnight at 4°C. Afterward, nonspecific binding sites were blocked with 1% BSA in PBS. The wells were incubated at room temperature for 2 h with a mouse IgG monoclonal anti-nitrotyrosine (5 μg/ml) with a peroxidase-conjugated goat anti-mouse IgG secondary antibody diluted 1:4,000. After washing the plates, the peroxidase reaction product was generated using a TMB microwell peroxidase substrate. Plates were incubated for 10 min at room temperature, and the reaction was stopped with 50 μl per well of stopping reagent and read at 492 nm in a microplate reader (19). The concentration of nitrated proteins that inhibited anti-nitrotyrosine antibody binding was estimated from the standard curve and expressed as micromoles per liter NT (17).

Glucose interference was excluded when performing the ELISA of standard solution in the presence of various glucose concentrations (20, 40, and 100 mmol/l). The limit of detection was 10 nmol/l, whereas the intra- and interassay coefficients of variation were 4.5 and 8%, respectively.

All measurements are presented as means ± SD. Analyses of the difference in clinical characteristics between diabetic and nondiabetic subjects were made using unpaired and two-tailed t tests. The changes in variables during each test were assessed by two-way ANOVA with repeated measures followed by post hoc testing with a Scheffe’s test.

Taking into account the design of the study, statistical evaluation of data directly investigated the possibilities of a pure period effect (i.e., the general tendency for patients to do better in one of the periods of the trial), of a period-treatment interaction, or of a treatment effect (i.e., the real efficacy of treatment with insulin aspart in comparison to regular insulin) (20). Therefore, the results of the serial measurements of glucose, triglycerides, and NT were first summarized calculating for each parameter the area under the curve after the administration of aspart insulin (AUCA) or regular insulin (AUCR). To verify the possibility of a period effect, a two-sample Student’s t test (two-tailed) was performed, comparing d1 (mean of individual differences AUCA - AUCP in those patients first treated with insulin aspart) with d2 (mean of individual differences AUCR - AUCA, changed of sign, in those patients first treated with regular insulin) (20). The possibility of a period-treatment interaction was evaluated by a second two-sample Student’s t test (two-tailed), comparing the average of the individual observations [(AUCA + AUCR)/2] in the aforementioned two groups of patients (20). The treatment effect was tested by performing a third two-sample Student’s t test (two-tailed), taking into consideration the average effect of treatment in the two periods, i.e., comparing d1 with d2 (20).

Statistical significance was defined as P < 0.05. All statistical tests were performed using the SPSS statistical software package.

As reported in Table 1, at the screening evaluation, fasting NT concentration was significantly higher in diabetic patients than in healthy control subjects. On the testing day, because patients were instructed to not take intermediate insulin the night before, fasting glycemia (although higher than at the screening evaluation in diabetic subjects) was not different in the patients taking regular insulin compared with the patients taking insulin aspart (10.8 ± 2.4 and 11.3 ± 2.3 mmol/l, respectively).

Figure 1 shows the changes in the plasma concentrations of glucose, triglycerides, and NT after meals in normal subjects and diabetic patients given either regular or aspart insulin. Glycemia (F = 18.5, P = 0.001, by ANOVA for repeated measures), triglycerides (F = 12.1, P = 0.001), and NT (F = 26.2, P = 0.001) significantly changed over time during the two tests. Compared with regular insulin, insulin aspart administration significantly reduced the area under the curve of both glycemia (58.3 ± 17.6 vs. 68.1 ± 17.7 mmol/l; P < 0.04) and NT (3.60 ± 2.04 vs. 4.49 ± 2.06 μmol/l; P < 0.03), whereas there was no difference in the area under the curve of triglycerides between the two treatments (8.2 ± 4.7 vs. 8.1 ± 4.4 mmol/l; P < 0.8). Glycemia (F = 0.4), triglycerides (F = 1.5), and NT (F = 0.8) postprandial variations did not reach significance in normal subjects.

This study, for the first time, shows that the postprandial state is accompanied by a significant increase of NT in type 2 diabetic patients. The demonstration that lower postprandial glycemic levels, in the presence of the same level of postprandial triglycerides after insulin aspart administration, is associated with less production of NT suggests a specific and direct role of postprandial hyperglycemia in favoring NT formation.

The possibility that hyperglycemia may lead to NT formation is supported by studies showing the presence of NT in the placenta, kidney, heart, and plasma of diabetic patients (12,21, 22) and in aortic tissue from diabetic cynomolgus monkeys (23). The direct role of high blood glucose levels has been substantiated by showing that acute hyperglycemia induces NT overproduction, even in the plasma of healthy subjects and in working hearts from rats (13,24). Therefore, it is not surprising that the postprandial phase, which is accompanied in type 2 diabetic patients by a marked increase of glycemia, will show an acute increase in NT. It is also interesting to underline that NT did not increase after meals in normal subjects, whose postprandial triglyceride and blood glucose levels showed only a slight and nonsignificant increase.

Postprandial hypertriglyceridemia may represent an independent predictor of CVD in nondiabetic patients (25,26) and has also been a predictor of carotid intima-media thickness in patients with type 2 diabetes (27). However, in the multiple regression analysis in the same study, postprandial hyperglycemia was also found to be an independent risk factor (27).

This finding is not surprising because many epidemiological studies substantiate postprandial hyperglycemia as a risk factor for CVD (6,28). In our study, hyperglycemia seems to have more relevance in favoring NT formation than triglyceride variations, possibly because of an insufficient sample size, the composition of the mixed meal, and/or the outweighing role of the glycemic factor in the diabetic population.

The process through which postprandial hyperglycemia facilitates CVD has been suggested to be the production of oxidative stress (5). This hypothesis is supported by our finding that, in the postprandial phase, there is an increase in NT plasma levels, with NT being a marker of peroxynitrite production. The finding that peroxynitrite production in the postprandial phase appears to be increased may have important pathogenetic implications.

Peroxynitrite is a potent oxidant and nitrating agent that leads to a host of potentially harmful events, including VLDL peroxidation (29), depletion of antioxidant defenses (30), and inactivation of enzymes (31). In addition, it can be directly cytotoxic for endothelial cells (32). All these events may convincingly be involved in the pathogenesis of atherosclerosis and CVD. This hypothesis is strongly supported by the recent finding that the increased apoptosis of myocytes, endothelial cells, and fibroblasts in heart biopsies from diabetic patients (33) and in hearts from streptozotocin-induced diabetic rats (34) is selectively associated with the levels of NT found in those cells. Furthermore, the demonstration that NT can induce endothelial dysfunction by itself (35) and that it is present in atherosclerotic lesions in humans and diabetic cynomolgus monkeys (23,36) is additional evidence that peroxynitrite production may be strongly involved in atherogenesis.

In conclusion, the present study demonstrates a strong correlation between postprandial hyperglycemia and NT production in insulin-treated type 2 diabetic patients. Because NT is a marker of the production of peroxynitrite, which in turn is a putative determinant of oxidative stress and CVD, our data reinforce the hypothesis that the postprandial state, particularly in diabetic subjects because of their marked hyperglycemia, may have an important role in the pathogenesis of CVD through the production of oxidative stress.