Francesco Nappo

Inflammatory Cytokine Concentrations Are Acutely Increased by Hyperglycemia in Humans Role of Oxidative Stress

Background—Circulating levels of interleukin-6 (IL-6) and tumor necrosis factor-&agr; (TNF-&agr;) are elevated in diabetic patients. We assessed the role of glucose in the regulation of circulating levels of IL-6, TNF-&agr;, and interleukin-18 (IL-18) in subjects with normal or impaired glucose tolerance (IGT), as well as the effect of the antioxidant glutathione. Methods and Results—Plasma glucose levels were acutely raised in 20 control and 15 IGT subjects and maintained at 15 mmol/L for 5 hours while endogenous insulin secretion was blocked with octreotide. In control subjects, plasma IL-6, TNF-&agr;, and IL-18 levels rose (P <0.01) within 2 hours of the clamp and returned to basal values at 3 hours. In another study, the same subjects received 3 consecutive pulses of intravenous glucose (0.33 g/kg) separated by a 2-hour interval. Plasma cytokine levels obtained at 3, 4, and 5 hours were higher (P <0.05) than the corresponding values obtained during the clamp. The IGT subjects had fasting plasma IL-6 and TNF-&agr; levels higher (P <0.05) than those of control subjects. The increase in plasma cytokine levels during the clamping lasted longer (4 hours versus 2 hours, P <0.01) in the IGT subjects than in the control subjects, and the cytokine peaks of IGT subjects after the first glucose pulse were higher (P <0.05) than those of control subjects. On another occasion, 10 control and 8 IGT subjects received the same glucose pulses as above during an infusion of glutathione; plasma cytokine levels did not show any significant change from baseline after the 3 glucose pulses. Conclusions—Hyperglycemia acutely increases circulating cytokine concentrations by an oxidative mechanism, and this effect is more pronounced in subjects with IGT. This suggests a causal role for hyperglycemia in the immune activation of diabetes.

Postprandial endothelial activation in healthy subjects and in type 2 diabetic patients: role of fat and carbohydrate meals.

OBJECTIVES To compare the effect of a high-fat meal and a high-carbohydrate meal (pizza), with and without antioxidant vitamins, on endothelial activation in healthy subjects and in patients with type 2 diabetes mellitus. BACKGROUND The postprandial state is becoming increasingly acknowledged to affect some early events of atherogenesis. METHODS In a randomized, observer-blinded, crossover study, 20 newly diagnosed type 2 diabetic patients and 20 age- and gender-matched healthy subjects received two meals at one-week intervals: a high-fat meal (760 calories) and an isoenergetic high-carbohydrate meal (non-cheese pizza). In all subjects, the same meals were repeated immediately following ingestion of vitamin E, 800 IU, and ascorbic acid, 1,000 mg. RESULTS In normal subjects, the high-fat meal increased the plasma levels of tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), which were prevented by vitamins. No change in these parameters occurred after pizza ingestion or pizza ingestion with vitamins. In diabetic patients, basal concentrations of glucose, cytokines and adhesion molecules were significantly higher than in nondiabetic controls. Both meals significantly increased cytokine and adhesion molecule levels, but the increase was more sustained following the high-fat meal. There was no significant change from baseline when vitamin supplementation accompanied each meal. There was a relationship between changes in serum triglycerides and changes in TNF-alpha (r = 0.39, p < 0.01), IL-6 (r = 0.28, p < 0.05) and VCAM-1 (r = 0.25, p < 0.05), and between changes in plasma glucose and changes in IL-6 (r = 0.36, p < 0.01) and ICAM-1 (r = 0.31, p < 0.02). CONCLUSIONS An oxidative mechanism mediates endothelial activation induced by post-meal hyperlipidemia and hyperglycemia.

Regression of Carotid Atherosclerosis by Control of Postprandial Hyperglycemia in Type 2 Diabetes Mellitus

Background—Postprandial hyperglycemia may be a risk factor for cardiovascular disease. We compared the effects of two insulin secretagogues, repaglinide and glyburide, known to have different efficacy on postprandial hyperglycemia, on carotid intima-media thickness (CIMT) and markers of systemic vascular inflammation in type 2 diabetic patients. Methods and Results—We performed a randomized, single-blind trial on 175 drug-naive patients with type 2 diabetes mellitus (93 men and 82 women), 35 to 70 years of age, selected from a population of 401 patients who participated in an epidemiological analysis assessing the relation of postprandial hyperglycemia to surrogate measures of atherosclerosis. Eighty-eight patients were randomly assigned to receive repaglinide and 87 patients to glyburide, with a titration period of 6 to 8 weeks for optimization of drug dosage and a subsequent 12-month treatment period. The effects of repaglinide (1.5 to 12 mg/d) and glyburide (5 to 20 mg/d) on CIMT were compared by using blinded, serial assessments of the far wall. After 12 months, postprandial glucose peak was 148±28 mg/dL in the repaglinide group and 180±32 mg/dL in the glyburide group (P <0.01). HbA1c showed a similar decrease in both groups (−0.9%). CIMT regression, defined as a decrease of >0.020 mm, was observed in 52% of diabetics receiving repaglinide and in 18% of those receiving glyburide (P <0.01). Interleukin-6 (P =0.04) and C-reactive protein (P =0.02) decreased more in the repaglinide group than in the glyburide group. The reduction in CIMT was associated with changes in postprandial but not fasting hyperglycemia. Conclusions—Reduction of postprandial hyperglycemia in type 2 diabetic patients is associated with CIMT regression.

Vascular Effects of Acute Hyperglycemia in Humans Are Reversed by l-Arginine Evidence for Reduced Availability of Nitric Oxide During Hyperglycemia

BACKGROUND Acute hyperglycemia may increase vascular tone in normal humans via a glutathione-sensitive, presumably free radical-mediated pathway. The objective of this study was to investigate whether or not the vascular effects of hyperglycemia are related to reduced availability of nitric oxide. METHODS AND RESULTS Acute hyperglycemia (15 mmol/L, 270 mg/dL) was induced in 12 healthy subjects with an artificial pancreas. Systolic and diastolic blood pressures, heart rate, and plasma catecholamines showed significant increases (P < .05) starting after 30 minutes of hyperglycemia; leg blood flow decreased significantly (15%; P < .05) at 60 and 90 minutes. Platelet aggregation to ADP and blood viscosity also showed significant increments (P < .05). The infusion of L-arginine (n = 7, 1 g/min) but not D-arginine (n = 5, 1 g/min) or L-lysine (n = 5, 1 g/min) in the last 30 minutes of the hyperglycemic clamp completely reversed all hemodynamic and rheological changes brought about by hyperglycemia. Infusion of NG-monomethyl-L-arginine (L-NMMA; 2 mg/min) to inhibit endogenous nitric oxide synthesis in 8 normal subjects produced vascular effects qualitatively similar to those of hyperglycemia but quantitatively higher (P < .05); however, heart rate and plasma catecholamine levels decreased during L-NMMA infusion, presumably as a consequence of baroreflex activation. Infusion of L-NMMA during hyperglycemia produced changes not different from those obtained during infusion of L-NMMA alone. CONCLUSIONS The results show that acute hyperglycemia in normal subjects causes significant hemodynamic and rheological changes that are reversed by L-arginine. Moreover, the effects of hyperglycemia are mimicked to a large extent, but not entirely, by infusion of L-NMMA. This suggests that hyperglycemia may reduce nitric oxide availability in humans.

Acute hyperglycemia induces an oxidative stress in healthy subjects

Recent prospective studies indicate that long-term glycemic control of diabetes is an important predictor not only of microvascular disease but also of macrovascular complications, including coronary heart disease (1). Possible links between glucose and cardiovascular events in the diabetic patient include modifications of important vascular functions of the endothelium with a switch from a quiescent, relaxant, antithrombotic, antioxidant, and antiadhesive state to an activated state displaying a more atherogenetic risk profile (2). Generation of reactive oxygen species could be a common downstream mechanism by means of which multiple byproducts of glucose are exerting their adverse effects on blood vessels (3). Indeed, in April 2001, Pennathur et al. (4) reported in the JCI that hyperglycemia favors oxidative reactions in the microenvironment of the artery wall in vivo. We studied the effect of acute elevations of plasma glucose levels on plasma nitrotyrosine, a marker of oxidative stress, in 20 healthy subjects (11 men, 9 women). Their age was 34 ± 4 years (mean ± SD), and the body mass index was 24 ± 1 kg/m2. None used any medication. After giving informed written consent to participate in the study, each subject underwent a hyperglycemic glucose clamp test in which plasma glucose concentrations were acutely raised at about 15 mmol/l for 120 minutes (Figure ​(Figure1).1). Mean blood pressure and nitrotyrosine (5) rose significantly during the clamp; there was a positive correlation (r = 0.49) between nitrotyrosine and mean blood pressure increases during hyperglycemia. In control studies (n = 6), in which plasma glucose was maintained at normal concentrations (5 mmol/l for 120 minutes), we could detect no variation in nitrotyrosine plasma levels from baseline (baseline: 0.15 ± 0.05 μmol/l; 120-minute values: 0.13 ± 0.05 μmol/l, P = not significant). Figure 1 Twenty healthy subjects were submitted to a hyperglycemic glucose clamp study in which plasma glucose levels were acutely raised to 15 mmol/l (0.33 g/kg as intravenous bolus injection followed by a variable 30% glucose infusion). Mean blood pressure ... We show here that acute hyperglycemia in normal subjects causes an oxidative stress as evidenced by the raised circulating nitrotyrosine levels during the hyperglycemic clamp. However, we cannot exclude the possibility that some nitrotyrosine can be generated via a peroxynitrite-independent mechanism, or that a reduced nitrotyrosine clearance during hyperglycemia could also contribute to its raised plasma concentrations. Since nitrotyrosine is considered a good marker of peroxynitrite formation (6), and since peroxynitrite may account for a considerable portion of the toxic effects previously attributed to nitric oxide or the superoxide anion (7), it is possible that some of the toxic effects of hyperglycemia on the vascular tree may be modulated by peroxynitrite. Acute hyperglycemia in normal subjects may in fact induce vasoconstriction, activate thrombosis, increase the circulating levels of soluble adhesion molecules, and prolong the QT interval (8, 9). The recent demonstration (10) that apoptosis of myocytes, endothelial cells, and fibroblasts in heart biopsies taken from diabetic patients is selectively associated with intracellular levels of nitrotyrosine supports a role for high-energy oxidants (such as peroxynitrite) as mediators of the vascular damage brought about by hyperglycemia.

The effect of acute hyperglycaemia on QTc duration in healthy man.

Aims/hypothesis. Prolongation of heart rate-adjusted QT (QTc) is associated with an increased risk of coronary heart disease and sudden death. The objective of this study was to investigate whether acute increases of plasma glucose concentrations in healthy subjects could influence QTc and QTc dispersion. Methods. Plasma glucose concentrations were quickly raised to 15 mmol/l in 20 healthy subjects (10 men/10 women) and maintained for 2 h. On another occasion, and in random order, all subjects underwent the same hyperglycaemic clamp as above and an infusion of the somatostatin analogue octreotide (25 μg as iv bolus followed by a 0.5 g/min infusion) to block the release of endogenous insulin. Results. Systolic and diastolic blood pressures, heart rate and plasma catecholamine concentrations showed significant increases (p < 0.05) starting after 60 min of hyperglycaemia. QTc, QTc dispersion and PR interval also showed significant increments at 120 min of the hyperglycaemic clamp. The infusion of octreotide did not influence QTc duration, QTc dispersion, PR interval and the haemodynamic effects of acute hyperglycaemia. Conclusion/interpretation. The results show that acute hyperglycaemia produces significant increments of QTc and QTc dispersion in normal subjects. In this context, endogenously released insulin during acute hyperglycaemia seems to play a minor part. [Diabetologia (2000) 43: 571–575]

Erectile and endothelial dysfunction in Type II diabetes: a possible link.

Abstract.Aims/hypothesis: The aim of this study was to evaluate the relation between erectile dysfunction and endothelial functions, coagulation activation, peripheral and autonomic neuropathy in men with Type II (non-insulin-dependent) diabetes mellitus. Methods: We studied 30 Type II diabetic patients with symptomatic erectile dysfunction and 30 potent diabetic patients matched for age and disease. Endothelial functions were assessed with the l-arginine test, plasma thrombomodulin and cell adhesion molecules circulating concentrations. Haemostasis was evaluated with markers of thrombin activation and fibrinolysis. Quantitative sensory testing (vibratory, warming, and heat-pain thresholds), cardiovascular reflex tests and 24-h blood pressure monitoring were used to assess peripheral or autonomic neuropathy. Results: Mean erectile score and HbA1 c were 10.5 ± 5.8 and 8.3 ± 1.6 % in patients with erectile dysfunction, and 24.0 ± 0.7 and 6.8 ± 1.4 % in those without erectile dysfunction, respectively (p < 0.001); there was a significant relation between HbA1 c and erectile function score in patients with erectile dysfunction (r = –0.45, p = 0.02). The decrease in blood pressure and platelet aggregation in response to l-arginine was lower (p < 0.05–0.02) in patients with erectile dysfunction, whereas soluble thrombomodulin, P-selectin and intercellular cell ahhesion molecule-1 concentrations were higher (p < 0.05–0.02). Indices of coagulation activation (F1 + 2 and d-dimers) and reduced fibrinolysis (PAI-1) were also found to be higher in erectile dysfunction patients. Heat-pain and warm perception thresholds, as well as cardiovascular reflex tests, were most commonly abnormal in patients with erectile dysfunction (p < 0.05). In multivariate analysis, HbA1 c, MBP response to l-arginine, P-selectin, indices of coagulation, and quantitative sensory testing were independent predictors of erectile function score. Conclusion/interpretation: Erectile dysfunction in diabetic men correlates with endothelial dysfunction. A reduced nitric oxide activity might provide a unifying explanation. [Diabetologia (2001) 44: 1155–1160]

High glucose induces ventricular instability and increases vasomotor tone in rats

Aims/hypothesis. To investigate cardiac repolarization time in streptozotocin-induced diabetic rats and isolated hearts perfused with high glucose concentration. Methods. We studied the effects of streptozotocin-induced diabetes on the cardiac repolarisation time (Q-T interval) in Sprague-Dawley rats during a 4-day period of hyperglycaemia and a subsequent 4-day period of normoglycaemia. The Q-T interval was also evaluated in isolated hearts of non-diabetic rats, in condition of high glucose concentration. Results. Hyperglycaemia in streptozotocin rats increased mean blood pressure and led to a significant (p < 0.001) prolongation of Q-T values, which normalized after 4 days of normoglycaemia with intravenous insulin infusion. Perfusion of isolated hearts in condition of high glucose concentration caused a significant prolongation of Q-T values and increased coronary perfusion pressure (p < 0.001). The effects of high glucose were completely prevented by glutathione and almost completely by l-arginine, the natural precursor of nitric oxide. In a condition of normal glucose, l-NAME, an inhibitor of endogenous nitric oxide synthesis, increased both Q-T and CPP values to levels similar to those induced by high glucose (p < 0.001). Verapamil completely prevented Q-T lengthening and reduced by about two-thirds CPP values (p < 0.001). Conclusion/Interpretation. Streptozotocin-diabetes in rats produces significant haemodynamic and electric perturbations that are reversed by normoglycaemia. Moreover, high glucose increases Q-T and CPP values in the isolated hearts of non-diabetic rats. The latter effects are reversed by glutathione and l-arginine, partially reversed by verapamil and mimicked by l-NAME. By increasing the production of free radicals, high glucose could reduce nitric oxide availability to target cells inducing a state of increased vasomotor tone and ventricular instability. [Diabetologia (2001) 44: 464–470]

Elevated plasma free fatty acid concentrations do not modify cardiac repolarization in patients treated by electrolyte-glucose-insulin infusion.

Fat emulsion infusion is routinely used as a source of calories and essential fatty acids for critically ill patients who may be at risk for acquired ventricular repolarization alterations due either to drugs or electrolyte disturbances. The aim of this study was to evaluate whether acute elevations of plasma free fatty acid concentrations influence the corrected Q-T interval (Q-Tc), Q-Tc dispersion and sympathetic nervous system activity in patients requiring parenteral nutrition. Thirty hospitalized patients (mean±SD: 62±17 yr of age) requiring total parenteral nutrition received an infusion of 10% (500 ml) triacylglycerol emulsion as a source of calories (450 Kcal); on another occasion, and in random order, the same patients received an infusion of 20% (500 ml) triacylglycerol emulsion (900 Kcal). The infusion lasted 8 h and was preceded by a sc injection of heparin (5000 U). Infusions of both 10% and 20% triacylglycerol emulsion increased plasma free fatty acid (p<0.001) and triacylglycerol (p<0.01) concentrations, and was associated with no significant change in mean BP, heart rate, and plasma catecholamines. At baseline, Q-Tc and Q-Tc dispersion were within the normal range (<440 milliseconds for QTc and <40 ms for QTc-d) and did not show any significant change at any time during infusion of triacylglycerol emulsion at both concentrations. In the setting of a balanced parenteral nutrition, acute elevation of plasma free fatty acid concentrations in critically ill patients do not modify ventricular repolarization.

Hemodynamic and metabolic effects of transdermal clonidine in patients with hypertension and non-insulin-dependent diabetes mellitus.

The aim of this study was to evaluate the effect of transdermal clonidine on hemodynamic and metabolic parameters in patients who have elevated blood pressure and non-insulin-dependent diabetes mellitus (NIDDM). After a 2-week run in placebo period, 20 NIDDM patients who had diastolic blood pressure in the range of 90 to 105 mm Hg underwent a randomized, single blind, placebo controlled, cross-over study of 4 week treatment with clonidine (transdermal patch 2.5 mg/week) or placebo (inactive patch). Compared with placebo, clonidine significantly reduced systolic (153 +/- 6 v 163 +/- 8) and diastolic (88 +/- 2 v 98 +/- 3.5 mm Hg, P = .001) blood pressure, left ventricular mass (94 +/- 11 v 99 +/- 12 g/m2, P < .01) and fasting glucose levels. Total glucose disposal (glucose clamp) was 6.5 +/- 1.5 with placebo and 7.1 +/- 1.6 mg/kg/min with clonidine (P < .01). Oxidative glucose disposal (indirect calorimetry) was also greater after clonidine. Plasma glucose, insulin, and C-peptide responses following oral glucose (75 g) were significantly lower after clonidine, as well as urinary albumin excretion. Transdermal clonidine is effective in reducing blood pressure in hypertensive NIDDM patients and is well tolerated. It may be useful to reduce the cardiovascular impact of hypertension in diabetes mellitus.