Endothelial nitric oxide synthase (eNOS) variants were previously demonstrated in cardiovascular disease. To evaluate whether eNOS gene variants are associated with insulin resistance and type 2 diabetes, we evaluated polymorphisms in Exon7 (E298D), intron 18 (IVS18 ؉ 27A3 C), and intron 23 (IVS23 ؉ 10G3 T) in 159 type 2 diabetic patients without macrovascular complications and in 207 healthy control subjects. Samples for all hormonal and metabolic variables were obtained after an overnight fast. The D298 and IVS18 ؉ 27C alleles, but not the IVS23 ؉ 10G3 T variant, were significantly more frequent in type 2 diabetic patients than in control subjects. The two-and three-loci haplotype analysis showed that there is a statistically significant association between the eNOS variants and type 2 diabetes. No significant differences were observed in the clinical characteristics of type 2 diabetic patients according to genotypes (except for visceral obesity [waist-to-hip ratio], which was significantly more present in D298 homozygotes). Healthy control subjects homozygous for both D298 and IVS18 ؉ 27C presented higher insulin, C-peptide, and nitric oxide levels, as well as higher HOMA (homeostasis model assessment) values than the double wild-type homozygotes, with values superimposable on those found in type 2 diabetic patients. In conclusion, we described a significant association between eNOS gene polymorphisms and type 2 diabetes, suggesting a new genetic susceptibility factor for hyperinsulinemia, insulin resistance, and type 2 diabetes. Diabetes 52:1270 -1275, 2003 T ype 2 diabetes is associated with a marked increase in coronary heart disease (CHD) (1), and increased risk factors for CHD before the onset of type 2 diabetes have been shown in several populations (2,3). In addition, type 2 diabetic patients with clinical cardiovascular disease sustain a worse prognosis for survival than cardiovascular disease patients without diabetes (4). This seems correlated to the high atherogenic state already present in a prediabetic state, which might be partly related to insulin resistance (5).Recently, endothelial dysfunction, such as nitric oxide (NO) impairment, is regarded as an early step in the development of insulin resistance, atherosclerosis, and type 2 diabetes (6 -8). Studies evaluating the relation between CHD and endothelial dysfunction have clearly demonstrated that reduced NO-dependent endothelial vasodilation is an early functional disturbance in the development of atherosclerotic lesions (6 -8). In addition, among the many activities of NO, it has demonstrated its ability to modulate peripheral and hepatic glucose metabolism and insulin secretion, according to Pieper (9), who suggested that NO alterations play an important role in the evolution of insulin resistance and type 2 diabetes. Moreover, our group has found high NO levels and endothelial dysfunction in type 2 diabetic patients and first-degree relatives of subjects with type 2 diabetes (10), as well as in subjects with the insulin resistance syndrome...

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OBJECTIVE -The aim of this study was to evaluate whether long-term administration of L-arginine acting through a normalization of NO/cyclic-guanosine-3Ј,5Ј-cyclic monophosphate (cGMP) pathway was able to ameliorate peripheral and hepatic insulin sensitivity in 12 lean type 2 diabetic patients. RESEARCH DESIGN AND METHODS-A double-blind study was performed for 3 months. In the first month, patients were treated with their usual diet. Then they were randomly allocated into two groups. In group 1, patients were treated with diet plus placebo (orally three times per day) for 2 months. In group 2 patients were treated for 1 month with diet plus placebo (orally, three times per day) and then for 1 month with diet plus L-arginine (3 g three times per day). At the end of the first and the second month of therapy, patients underwent a euglycemichyperinsulinemic clamp combined with [6, H 2 ]glucose infusion. A total of 10 normal subjects underwent the same test as control subjects.RESULTS -In group 1, no changes in basal cGMP levels, systolic blood pressure, forearm blood flow, glucose disposal, and endogenous glucose production were observed throughout. In group 2, L-arginine normalized basal cGMP levels and significantly increased forearm blood flow by 36% and glucose disposal during the clamp by 34%, whereas it decreased systolic blood pressure and endogenous glucose production by 14 and 29%, respectively. However, compared with normal subjects, L-arginine treatment was not able to completely overcome the defect in glucose disposal.CONCLUSIONS -L-Arginine treatment significantly improves but does not completely normalize peripheral and hepatic insulin sensitivity in type 2 diabetic patients. Diabetes Care 24:875-880, 2001C ontradictory results have been found concerning the influence of insulin on nitric oxide (NO), a potent molecule with vasodilatory function. Baron et al. (1,2) showed that insulinmediated vasodilation is largely dependent on the action of insulin on NO release, whereas Petrie et al. (3) have shown that endothelial NO synthesis and insulin sensitivity are positively correlated in healthy individuals. In addition, in obese patients and patients with type 2 diabetes, the insulin-mediated vasodilatory response seems blunted (4). However, Yki-Jarvinen et al. (5) were unable to find any correlation between insulin action and increment in blood flow in normal and obese subjects, although all agree that methacoline-induced vasodilation is impaired in insulin-resistant subjects.L-Arginine is a precursor for NO, and both in vitro and in vivo studies have demonstrated that L-arginine can augment vascular dilation under certain conditions (6). Experimental studies in cholesterol-fed rabbits have shown that dietary supplementation with L-arginine causes attenuation of endothelial dysfunction with increased NO activity, resulting in reduced platelet activation (7), monocyte adhesion (8), and a marked reduction in aortic and coronary atherosclerosis (9). Moreover, in young hypercholesterolemic adults, the administr...

Objective: The purpose of this study was (a) to study whether a folate and vitamin B12 treatment, aimed at decreasing homocysteine levels, might ameliorate insulin resistance and endothelial dysfunction in patients with metabolic syndrome according to the National Cholesterol Education Program-Adult Treatment Panel-III criteria and (b) to evaluate whether, under these metabolic conditions, there is a relationship between hyperhomocysteinemia and insulin resistance. Design and methods: A double-blind, parallel, identical placebo -drug, randomized study was performed for 2 months in 50 patients. Patients were randomly allocated to two groups. In group 1, patients were treated with diet plus placebo for 2 months. In group 2, patients were treated with diet plus placebo for 1 month, followed by diet plus folic acid (5 mg/day) plus vitamin B12 (500 mg/day) for another month. Results: In group 2, folate treatment significantly decreased homocysteine levels by 27.8% (12.2^1.2 vs 8.8^0.7 mmol/l; P , 0.01). A significant decrement was observed for insulin levels (19.9^1.7 vs 14.8^1.6 mU/ml; P , 0.01) accompanied by a 27% reduction in the homeostasis model assessment levels. A positive relationship was found between the decrement of homocysteine and insulin levels (r ¼ 0.60; P , 0.002). In parallel, endothelial dysfunction significantly improved in the treated group, since post-ischemic maximal hyperemic vasodilation increased by 29.8% and cGMP by 13.6% while asymmetrical dimethylarginine levels decreased by 21.7%. On the contrary, in group 1 patients, treated with placebo, no changes were shown in any of the variables. Conclusions: Folate and vitamin B12 treatment improved insulin resistance and endothelial dysfunction, along with decreasing homocysteine levels, in patients with metabolic syndrome, suggesting that folic acid has several beneficial effects on cardiovascular disease risk factors. European Journal of Endocrinology 151 483-489

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Background-We tested the hypothesis that asymmetric dimethylarginine (ADMA) levels could be elevated and influence endothelin-1 and nitric oxide release and action in patients with cardiac syndrome X (CSX). In addition, we evaluated whether an intravenous infusion of L-arginine would improve endothelial function in these subjects. Methods and Results-Nine patients with CSX and 14 control subjects underwent a continuous infusion of L-arginine (0.125 g/min) or saline for 120 minutes. Sixty minutes after L-arginine or saline infusions, an intravenous insulin bolus (0.1 U/kg) combined with a euglycemic clamp was performed. Basal ADMA and endothelin-1 levels were higher in patients with CSX than in controls. At the end of the first hour of infusion, compared with saline, L-arginine infusion increased basal forearm blood flow, nitrite and nitrate (NOx), and forearm cGMP release and decreased endothelin-1. After insulin bolus, during saline, insulin-induced NOx, endothelin-1, and forearm cGMP release was almost abolished. Conversely, L-arginine restored a physiological profile of all endothelial variables compared with control subjects. In control subjects, compared with saline infusion, L-arginine infusion did not modify any parameter. ADMA levels were positively correlated with basal endothelin-1 levels and negatively correlated with insulin-induced incremental levels of NOx and forearm cGMP release. Conclusions-Plasma ADMA levels are increased in patients with CSX, and they are correlated with increases in endothelin-1 and reductions in insulin-induced increments in plasma NOx and cGMP, effects that are reversed by intravenous L-arginine. These data suggest that increased ADMA levels play a role in the abnormal vascular reactivity that is observed in patients with CSX.

Background-Previously undiagnosed diabetes, impaired glucose tolerance, and insulin resistance are common in patients with acute myocardial infarction and coronary heart disease (CHD) and might be involved in early restenosis after stent implantation. To evaluate whether markers of insulin resistance syndrome, including leptin, and endothelial dysfunction are related to increased rate of early restenosis, we studied nondiabetic patients with CHD after successful coronary stenting. Methods and Results-Both patients with CHD undergoing coronary stenting (120 patients) and control subjects (58 patients) were submitted to an oral glucose tolerance test (OGTT). Fasting leptin levels and fasting and postglucose load insulin sensitivity were assessed. Endothelial function was measured by nitrite and nitrate release (NOx) during OGTT. More than 50% of patients treated with stent implantation presented impaired glucose tolerance or type 2 diabetes, which was previously undiagnosed. These patients also had higher glucose, insulin, and leptin levels than control subjects. Among the stented patients, insulin and leptin levels were higher in patients with restenosis than in patients without restenosis. A significant increase in NOx levels was found during OGTT both in patients without restenosis and in control subjects. On the contrary, NOx profiles were blunted in patients with restenosis. At multiple regression analysis, only ⌬AUC-NOx areas and insulin sensitivity index showed an independent correlation with the minimal lumen diameter at follow-up. Conclusions-We demonstrated that insulin resistance and endothelial dysfunction are independent predictors of early restenosis after coronary stenting.

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