Dario Giugliano

Oxidative Stress and Diabetic Vascular Complications

Long-term vascular complications still represent the main cause of morbidity and mortality in diabetic patients. Although prospective randomized long-term clinical studies comparing the effects of conventional and intensive therapy have demonstrated a clear link between diabetic hyperglycemia and the development of secondary complications of diabetes, they have not defined the mechanism through which excess glucose results in tissue damage. Evidence has accumulated indicating that the generation of reactive oxygen species (oxidative stress) may play an important role in the etiology of diabetic complications. This hypothesis is supported by evidence that many biochemical pathways strictly associated with hyperglycemia (glucose autoxidation, polyol pathway, prostanoid synthesis, protein glycation) can increase the production of free radicals. Furthermore, exposure of endothelial cells to high glucose leads to augmented production of superoxide anion, which may quench nitric oxide, a potent endothelium-derived vasodilator that participates in the general homeostasis of the vasculature. In further support of the consequential injurious role of oxidative stress, many of the adverse effects of high glucose on endothelial functions, such as reduced endothelial-dependent relaxation and delayed cell replication, are reversed by antioxidants. A rational extension of this proposed role for oxidative stress is the suggestion that the different susceptibility of diabetic patients to microvascular and macrovascular complications may be a function of the endogenous antioxidant status.

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.

Oscillating glucose is more deleterious to endothelial function and oxidative stress than mean glucose in normal and type 2 diabetic patients

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.

The effect of Mediterranean diet on metabolic syndrome and its components: a meta-analysis of 50 studies and 534,906 individuals.

OBJECTIVES The aim of this study was to meta-analyze epidemiological studies and clinical trials that have assessed the effect of a Mediterranean diet on metabolic syndrome (MS) as well as its components. BACKGROUND The Mediterranean diet has long been associated with low cardiovascular disease risk in adult population. METHODS The authors conducted a systematic review and random effects meta-analysis of epidemiological studies and randomized controlled trials, including English-language publications in PubMed, Embase, Web of Science, and the Cochrane Central Register of Controlled Trials until April 30, 2010; 50 original research studies (35 clinical trials, 2 prospective and 13 cross-sectional), with 534,906 participants, were included in the analysis. RESULTS The combined effect of prospective studies and clinical trials showed that adherence to the Mediterranean diet was associated with reduced risk of MS (log hazard ratio: -0.69, 95% confidence interval [CI]: -1.24 to -1.16). Additionally, results from clinical studies (mean difference, 95% CI) revealed the protective role of the Mediterranean diet on components of MS, like waist circumference (-0.42 cm, 95% CI: -0.82 to -0.02), high-density lipoprotein cholesterol (1.17 mg/dl, 95% CI: 0.38 to 1.96), triglycerides (-6.14 mg/dl, 95% CI: -10.35 to -1.93), systolic (-2.35 mm Hg, 95% CI: -3.51 to -1.18) and diastolic blood pressure (-1.58 mm Hg, 95% CI: -2.02 to -1.13), and glucose (-3.89 mg/dl, 95% CI:-5.84 to -1.95), whereas results from epidemiological studies also confirmed those of clinical trials. CONCLUSIONS These results are of considerable public health importance, because this dietary pattern can be easily adopted by all population groups and various cultures and cost-effectively serve for primary and secondary prevention of the MS and its individual components.

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.

Metabolic Syndrome and Risk of Cancer A systematic review and meta-analysis

OBJECTIVE Available evidence supports the emerging hypothesis that metabolic syndrome may be associated with the risk of some common cancers. We did a systematic review and meta-analysis to assess the association between metabolic syndrome and risk of cancer at different sites. RESEARCH DESIGN AND METHODS We conducted an electronic search for articles published through October 2011 without restrictions and by reviewing reference lists from retrieved articles. Every included study was to report risk estimates with 95% CIs for the association between metabolic syndrome and cancer. RESULTS We analyzed 116 datasets from 43 articles, including 38,940 cases of cancer. In cohort studies in men, the presence of metabolic syndrome was associated with liver (relative risk 1.43, P < 0.0001), colorectal (1.25, P < 0.001), and bladder cancer (1.10, P = 0.013). In cohort studies in women, the presence of metabolic syndrome was associated with endometrial (1.61, P = 0.001), pancreatic (1.58, P < 0.0001), breast postmenopausal (1.56, P = 0.017), rectal (1.52, P = 0.005), and colorectal (1.34, P = 0.006) cancers. Associations with metabolic syndrome were stronger in women than in men for pancreatic (P = 0.01) and rectal (P = 0.01) cancers. Associations were different between ethnic groups: we recorded stronger associations in Asia populations for liver cancer (P = 0.002), in European populations for colorectal cancer in women (P = 0.004), and in U.S. populations (whites) for prostate cancer (P = 0.001). CONCLUSIONS Metabolic syndrome is associated with increased risk of common cancers; for some cancers, the risk differs betweens sexes, populations, and definitions of metabolic syndrome.

Diabetes mellitus, hypertension, and cardiovascular disease: Which role for oxidative stress?

Accelerated atherosclerotic vascular disease is the leading cause of mortality in patients with diabetes mellitus. Endothelium-derived nitric oxide (NO) is a potent endogenous nitrovasodilator and plays a major role in modulation of vascular tone. Selective impairment of endothelium-dependent relaxation has been demonstrated in aortas of both nondiabetic animals exposed to elevated concentrations of glucose in vitro and insulin-dependent diabetic animals. The impaired NO release in experimentally induced diabetes may be prevented by a number of antioxidants. It has been hypothesized that oxygen-derived free radicals (OFR) generated during both glucose autoxidation and formation of advanced glycosylation end products may interfere with NO action and attenuate its vasodilatory activity. The oxidative injury may also be increased in diabetes mellitus because of a weakened defense due to reduced endogenous antioxidants (vitamin E, reduced glutathione [GSH]). A defective endothelium-dependent vascular relaxation has been found in animal models of hypertension and in hypertensive patients. An imbalance due to reduced production of NO or increased production of free radicals, mainly superoxide anion, may facilitate the development of an arterial functional spasm. Treatment with different antioxidants increases blood flow in the forearm and decreases blood pressure and viscosity in normal humans; vitamin E inhibits nonenzymatic glycosylation, oxidative stress, and red blood cell microviscosity in diabetic patients. Long-term randomized clinical trials of adequate size in secondary and primary prevention could support the free-radical hypothesis for diabetic diabetic vascular complications and the use of antioxidants to reduce the risk of coronary heart disease.

Metabolic and Cardiovascular Effects of Carvedilol and Atenolol in Non-Insulin-Dependent Diabetes Mellitus and Hypertension: A Randomized, Controlled Trial

Compared with the general population, diabetic patients have an approximately twofold increased risk for hypertension and are more susceptible to the vascular consequences of high blood pressure. Indeed, an estimated 35% to 75% of cardiovascular and renal complications in diabetic patients can be attributed to hypertension [1]. Treatment with -adrenergic antagonists has been shown to be associated with an increased risk for impaired glucose tolerance or diabetes; this has been attributed to the worsening of insulin resistance and the deterioration of lipoprotein metabolism caused by the agents [2]. All this has made physicians reluctant to prescribe -blockers for diabetic patients with hypertension, although cardioselective -blockers have reduced mortality associated with cardiovascular causes in secondary prevention trials [3]. Carvedilol is a multiple-action antihypertensive drug with nonselective -adrenoreceptor and selective -adrenoreceptor blocking activity [4]. Its ratio of 1-blocking potency to 1-blocking potency is 7.6:1 for a 50-mg dose [5]. In addition, carvedilol prevents lipid peroxidation and the depletion of endogenous antioxidants [6]. This may be particularly useful in diabetic patients who may have increased free-radical activity (oxidative stress) [7]. We compared the metabolic and cardiovascular effects of carvedilol with those of atenolol in diabetic patients with hypertension in a randomized, double-blind, controlled trial. Methods Participants Our research protocol was approved by our institutional review board, and informed consent was obtained from patients before participation. Men and women who had noninsulin-dependent diabetes mellitus and had a supine diastolic blood pressure of 90 to 105 mm Hg on at least two occasions at the end of a 4- to 6-week placebo run-in period were eligible to participate. All patients were referred from the outpatient department of our institution and were consecutively chosen. A total of 45 patients met the inclusion criteria and were randomly assigned to treatment. All but 3 patients completed the study. Study Design Our study had a randomized, double-blind design for parallel study groups. Patients who had blood pressure greater than 160/90 mm Hg or who were taking antihypertensive drugs entered a 4- to 6-week run-in period, during which placebo was given to replace the previous antihypertensive drug, if any. Routine hematologic and blood chemistry analyses (hematologic indices, serum sodium and potassium concentrations, liver enzyme levels, urea concentrations, and creatinine concentrations) were done during the initial screening and after the treatment period. Patients were randomly assigned to receive either carvedilol (25 mg once daily) or atenolol (50 mg once daily) in the morning for 24 weeks. After 4 weeks, patients whose diastolic blood pressure while seated was more than 90 mm Hg and had not decreased by at least 10 mm Hg had their dose of study medication doubled for the remaining 20 weeks of the study. A person who was not involved in trial management randomly assigned the patients using random numbers derived from published tables. The list of randomization numbers was used to label the drug boxes, which were given to the participants sequentially. Both patients and caregivers were blinded to treatment, and randomization codes were not broken until all laboratory measurements had been done. Cardiovascular and metabolic variables were checked at the end of the placebo period and at the end of the active treatment period. All participants were instructed to follow a weight-maintaining diet (50% carbohydrates, 30% lipids, 20% protein) for 3 days before the experiments were done. Side effects, concomitant diseases, and blood pressure were assessed by interview and physical examination every fourth week during treatment. Clinical and Laboratory Measurements Patients were asked to refrain from smoking and to fast overnight before each metabolic assessment was done. The euglycemic clamp technique was used to estimate insulin sensitivity in vivo by infusing insulin (1 mU/kg of body weight per minute) and glucose to keep plasma glucose levels at the baseline concentration [8]. At the unchanged plasma glucose concentrations, the amount of glucose required to maintain euglycemia equals whole-body glucose disposal and is expressed in mol/kg per minute (M). The insulin sensitivity index (M/insulin level during the clamp procedure) measures how effectively plasma insulin induces glucose uptake in insulin-sensitive tissues, such as muscle and fat. Substrate oxidation was estimated by indirect calorimetry [9]. On the day after calorimetry, the patients had an oral glucose tolerance test (75 g of glucose). On the third day, they had an insulin tolerance test (0.15 U/kg). Blood pressure was measured with appropriate cuff size three times after patients rested for 5 minutes in the supine position. Plasma glucose, insulin, glucagon, and epinephrine levels were measured as described elsewhere [10]; hemoglobin A1c (HbA1c) was measured by column chromatography (Bio-Rad, Milan, Italy); and cholesterol, triglyceride, and high-density lipoprotein cholesterol levels were determined enzymatically [9]. Left ventricular mass normalized by surface area was measured by echocardiography [9]. Serum levels of lipid peroxides were measured as reaction products of malondialdehyde with thiobarbituric acid (thiobarbituric-acid-reactive substances) according to the method of Waravdekar and Sadlaw [11], with slight modifications. Normal lipid peroxide values for our laboratory are 0.34 to 0.86 mol/L. Statistical Analysis All values in the tables are presented as the mean SD unless otherwise noted; 95% CIs are provided where appropriate. The areas under the glucose and insulin curves were calculated by trapezoidal rule [12]. Change was calculated as the value obtained at the end of intervention minus the value obtained at the beginning of intervention. A preliminary analysis of variance was used to assess the significance within and between groups. One-sample t-tests were used to compare values obtained before and after carvedilol or atenolol therapy, and two-sample t-tests were used for between-group comparisons. Results Three patients (two in the atenolol group and one in the carvedilol group) were unavailable for follow-up; they refused to complete the study and did not specify a reason. These patients dropped out early (between weeks 4 and 8) after randomization; analysis of the study results did not differ when the analysis was done according to actual treatment or according to intention to treat (we used the latter method). Compliance, determined by tablet count, was 94.5% in the carvedilol group and 95% in the atenolol group. The baseline characteristics of the 45 patients who completed the study are shown in Table 1. The two groups were similar at baseline. Body mass index did not change in either group after treatment. Approximately one third of patients in each group (32% in the carvedilol group and 35% in the atenolol group) required upward dose titration at week 4 because of inadequate response. At the end of treatment, 91% of patients receiving carvedilol and 85% of those receiving atenolol had a diastolic blood pressure while seated of less than 90 mm Hg or had their diastolic blood pressure decreased by more than 10 mm Hg (P > 0.2 for comparison). Average systolic and diastolic blood pressure and left ventricular mass decreased in both groups, but the differences between the groups were small (P > 0.2) (Table 2). The decrease in heart rate was greater in patients receiving atenolol than in those receiving carvedilol (P < 0.005). The decrease in mean triglyceride level was 0.56 mmol/L greater (P < 0.001) and the increase in high-density lipoprotein cholesterol level was 0.13 mmol/L greater (P < 0.001) with carvedilol than with atenolol. Table 1. Baseline Characteristics of the Study Patients Table 2. Effects of 24-Week Intervention with Carvedilol or Atenolol in 45 Patients with Non-Insulin-Dependent Diabetes Mellitus and Hypertension* Mean fasting plasma glucose and insulin levels decreased during carvedilol treatment and increased during atenolol treatment (Table 2). The HbA1c level decreased by 1.4% in the carvedilol group and increased by 4% in the atenolol group (P < 0.001 for the difference). Mean total glucose disposal and insulin sensitivity index increased during carvedilol treatment and decreased during atenolol treatment (P 0.01 for the difference). Serum levels of thiobarbituric-acid-reactive substances decreased by 0.25 mol/L in the carvedilol group and did not change in the atenolol group (P < 0.001 for the difference). The decreases in plasma glucose and insulin responses to the oral glucose load were 61 mmol/L 180 minutes greater (CI, 101 to 21 mmol/L 180 minutes; P = 0.035) and 6.2 nmol/L 180 minutes greater (CI, 9.8 to 2.6 nmol/L 180 minutes; P = 0.03), respectively, with carvedilol than with atenolol (data not shown). The plasma glucose level nadir occurred 60 minutes after the insulin bolus was administered and was not affected by either drug (P = 0.09) (data not shown). Glucagon and epinephrine responses to hypoglycemia were similar before and after treatment with both drugs (P > 0.08) (data not shown). Discussion Our results show that both carvedilol and atenolol effectively decrease blood pressure and ventricular mass in patients with diabetes and hypertension. They also show that the drug doses administered in this study are equivalent with regard to their ability to decrease blood pressure in these patients, who are particularly at risk for cardiovascular disease. The similarities between the two drugs end with their cardiovascular effects, however; their metabolic effects are different and, to a large extent, divergent. Fasting plasma glucose and insulin levels decreased during carvedilol treatment and increased during atenolol treatment. Th