Long lasting protective effects of early l-arginine treatment on endothelium in an in vitro study.

BACKGROUND & AIMS: Excess nutrient supply, such as high fat and high glucose intake, promotes oxidative stress and advanced glycation end products accumulation. Oxidative stress and AGE accumulation cause pathological elevation of arginase activity and pro-inflammatory signaling implicated in endothelial dysfunction. Several studies showed positive effects of l-arginine supplementation in endothelial function but little is currently known about the role of l-arginine as prevention of endothelial dysfunction caused by excessive nutrient supply (overfeeding). Our aim was to evaluate a possible protective effect of l-arginine on endothelial dysfunction caused by excessive nutrient supply (overfeeding), using human endothelial cells line in an in vitro study. METHODS: Endothelial EA.hy926 cells were pre-treated with 1.72 mM of l-arginine for 24 h and afterwards subjected to nutritional stress (high lipid, high insulin and high glucose concentrations) for further 24 h. After treatment discontinuation, the cells were kept in culture for 48 h, in physiological condition, to evaluate the effects of treatments after normalization. RESULTS: Excess nutrient supply in EA.hy926 cell line showed an increase of oxidative and nitrosative stress, a rise of AGEs production, high arginase activity, leading the cells to acidosis and to cell death. l-arginine pretreatment protects the cells by reducing apoptosis, acidosis, oxidative and nitrosative stress, arginase activity and AGE accumulation. l-arginine pretreatment reduces AGEs generation and accumulation by regulating STAB1 and RAGE gene expression levels. STAB1, acting as receptor scavenger of AGEs, interferes with AGE-RAGE binding and thus prevents activation of intracellular signaling pathways leading to cell damage. Moreover the reduction of oxidative stress promotes a decrease of excessive activation of arginase involved in endothelial dysfunction. The effects of pretreatment with l-arginine last even in the absence of stimuli and despite after treatment discontinuation. CONCLUSIONS: An early l-arginine treatment is able to prevent oxidative stress and AGEs accumulation caused by overfeeding in human endothelial cell line by regulating STAB1/RAGE gene expression and by reducing excess arginase activity. The positive effects of l-arginine pretreatment continue even after treatment discontinuation in normal conditions.

Decreased diabetes risk over 9 year after 18-month oral L-arginine treatment in middle-aged subjects with impaired glucose tolerance and metabolic syndrome (extension evaluation of L-arginine study).

PURPOSE: This study aimed to determine whether L-arginine supplementation lasting for 18 months maintained long-lasting effects on diabetes incidence, insulin secretion and sensitivity, oxidative stress, and endothelial function during 108 months among subjects at high risk of developing type 2 diabetes. METHODS: One hundred and forty-four middle-aged subjects with impaired glucose tolerance and metabolic syndrome were randomized in 2006 to an L-arginine supplementation (6.4 g orally/day) or placebo therapy lasting 18 months. This period was followed by a 90-month follow-up. The primary outcome was a diagnosis of diabetes during the 108 month study period. Secondary outcomes included changes in insulin secretion (proinsulin/c-peptide ratio), insulin sensitivity (IGI/HOMA-IR), oxidative stress (AOPPs), and vascular function. After the 18 month participation, subjects that were still free of diabetes and willing to continue their participation (104 subjects) were further followed until diabetes diagnosis, with a time span of about 9 years from baseline. RESULTS: Although results derived from the 18 month of the intervention study demonstrated no differences in the probability of becoming diabetics, at the end of the study, the cumulative incidence of diabetes was of 40.6% in the L-arginine group and of 57.4% in the placebo group. The adjusted HR for diabetes (L-arginine vs. placebo) was 0.66; 95% CI 0.48, 0.91; p < 0.02). Proinsulin/c-peptide ratio (p < 0.001), IGI/HOMA-IR (p < 0.01), and AOPP (p < 0.05) levels were ameliorated in L-arginine compared to placebo. CONCLUSIONS: These results may suggest that the administration of L-arginine could delay the development of T2DM for a long period. This effect could be mediated, in some extent, by L-arginine-induced reduction in oxidative stress.

Pharmacogenetic influence of eNOS gene variant on endothelial and glucose metabolism responses to L-arginine supplementation: Post hoc analysis of the L-arginine trial.

OBJECTIVE: To evaluate whether variants of the eNOS gene are associated with endothelial and metabolic responses to L-arginine (L-arg) supplementation. MATERIAL AND METHODS: We examined a single nucleotide polymorphism of the eNOS gene (rs753482-A>C) to investigate the effects of this variant on endothelial function (EF), colony-forming unit-endothelial cell (CFU-EC) number, asymmetric-dimethylarginine (ADMA) level, insulin sensitivity index (ISI), and insulin secretion (IS) in a post hoc analysis of the L-arg trial. The L-arg trial (6.4 g/day for 18 months) was a single-center, randomized, double-blind, parallel-group, placebo-controlled, phase III trial in individuals with impaired glucose tolerance and metabolic syndrome. followed by a 12-month extended follow-up period after termination of the study drug (NCT 00917449). RESULTS: At baseline, EF, CFU-EC numbers, ADMA levels, and ISI were impaired in subjects carrying minor allele C (both heterozygotes, AC and homozygotes, CC) as compared to subjects carrying major allele A (homozygotes, AA) (p<0.01). Compared to placebo, L-arg increased EF, CFU-EC numbers, and ISI, and improved ADMA levels and IS (p<0.01). The greatest improvements were found in AA subjects treated with L-arg, while the worst results were found in AC+CC subjects treated with placebo. In the placebo-treated subjects, EF, CFU-EC, ISI, and IS were significantly lower and ADMA was significantly higher in AC+CC subjects than in AA subjects. CONCLUSIONS: Treatment with L-arg induced similar improvements in EF, CFU-EC numbers, ADMA levels, ISI, and IS in both AA subjects and AC+CC subjects. The presence of minor allele resulted in the worst prognosis in terms of EF, CFU-EC numbers, ADMA levels, ISI, and IS during the 30-month observation period.

Beneficial effects of a long-term oral L-arginine treatment added to a hypocaloric diet and exercise training program in obese, insulin-resistant type 2 diabetic patients.

Because chronic L-arginine supplementation improves insulin sensitivity and endothelial function in nonobese type 2 diabetic patients, the aim of this study was to evaluate the effects of a long-term oral L-arginine therapy on adipose fat mass (FM) and muscle free-fat mass (FFM) distribution, daily glucose levels, insulin sensitivity, endothelial function, oxidative stress, and adipokine release in obese type 2 diabetic patients with insulin resistance who were treated with a combined period of hypocaloric diet and exercise training. Thirty-three type 2 diabetic patients participated in a hypocaloric diet plus an exercise training program for 21 days. Furthermore, they were divided into two groups in randomized order: the first group was also treated with L-arginine (8.3 g/day), and the second group was treated with placebo. Although in the placebo group body weight, waist circumference, daily glucose profiles, fructosamine, insulin, and homeostasis model assessment index significantly decreased, L-arginine supplementation further decreased FM (P < 0.05) and waist circumference (P < 0.0001), preserving FFM (P < 0.03), and improved mean daily glucose profiles (P < 0.0001) and fructosamine (P < 0.03). Moreover, change in area under the curve of cGMP (second messenger of nitric oxide; P < 0.001), superoxide dismutase (index of antioxidant capacity; P < 0.01), and adiponectin levels (P < 0.02) increased, whereas basal endothelin-1 levels (P < 0.01) and leptin-to-adiponectin ratio (P < 0.05) decreased in the L-arginine group. Long-term oral L-arginine treatment resulted in an additive effect compared with a diet and exercise training program alone on glucose metabolism and insulin sensitivity. Furthermore, it improved endothelial function, oxidative stress, and adipokine release in obese type 2 diabetic patients with insulin resistance.