Nitric oxide production by peritoneal macrophages from aged rats: A short term and direct modulation by citrulline.

Citrulline has anti-inflammatory properties and exerts beneficial effects on various impaired functions in aging. However, there are few data on citrulline action on immune function in aged populations. The objective of the study was to evaluate citrulline ability, after in vivo and in vitro administration, to modulate macrophage functions in aged rats and the possible pathways involved. Twenty-one-month-old Sprague-Dawley rats (n = 27) received a citrulline supplementation at 5 g/kg/d for 5 days, or an isonitrogenous diet, and peritoneal macrophages were cultured with or without LPS. In the in vitro study, macrophages from 22-month-old rats (n = 16) were cultured with or without LPS, citrulline and inhibitors of different inflammatory pathways (n = 8/conditions). Nitric oxide (NO) and tumor necrosis factor α (TNFα) production were measured in both in vivo and in vitro studies. Citrulline decreased NO production variability by peritoneal macrophages after in vivo administration (p = 0.0034) and downregulated NO production by 22% after in vitro administration (95% CI: [6%; 35%]; p = 0.0394), without any direct effect on TNFα production. None of the transductional pathways explored seem to be involved. Citrulline slightly modulates NO production in vivo and in vitro, suggesting a possible action through modulation of arginine metabolism in macrophages rather than a direct transductional effect. The pleiotropic effects of citrulline in aging could be due, at least in part, to the anti-inflammatory effect of citrulline.

In vitro anti-inflammatory effects of citrulline on peritoneal macrophages in Zucker diabetic fatty rats.

In type 2 diabetes (T2D) macrophage dysfunction increases susceptibility to infection and mortality. This may result from the associated decreased plasma concentration of arginine, an amino acid that plays an important role in immunity. In vitro, increasing arginine availability leads to an improvement in macrophage function; however, arginine supplementation in diabetic obese patients may be detrimental. The aim of the present study was to assess in vitro whether citrulline, an arginine precursor, could replace arginine in the regulation of macrophage function under a condition of diabetes and obesity. Peritoneal macrophages from diabetic obese or lean rats were incubated for 6 h in an arginine-free medium, in the presence of increasing citrulline concentrations (0·1, 0·5, 1 or 2 mmol/l). Cytokine and NO production was determined. Peritoneal macrophages from either lean or diabetic obese rats produced NO, and at higher levels in the cells from lean rats. In diabetic obese rats, TNF-α production decreased with increasing citrulline concentrations, but was higher than that in the cells from lean rats. In contrast, IL-6 production increased with increasing citrulline concentrations. The present experiment shows that citrulline is effectively used for NO production and regulates cytokine production in macrophages from diabetic obese rats. This effect warrants in vivo evaluation in T2D-related inflammation.

Effects of a diabetes-specific enteral nutrition on nutritional and immune status of diabetic, obese, and endotoxemic rats: interest of a graded arginine supply.

OBJECTIVE: Obese and type 2 diabetic patients present metabolic disturbance-related alterations in nonspecific immunity, to which the decrease in their plasma arginine contributes. Although diabetes-specific formulas have been developed, they have never been tested in the context of an acute infectious situation as can be seen in intensive care unit patients. Our aim was to investigate the effects of a diabetes-specific diet enriched or not with arginine in a model of infectious stress in a diabetes and obesity situation. As a large intake of arginine may be deleterious, this amino acid was given in graded fashion. DESIGN: Randomized, controlled experimental study. SETTING: University research laboratory. SUBJECTS: Zucker diabetic fatty rats. INTERVENTIONS: Gastrostomized Zucker diabetic fatty rats were submitted to intraperitoneal lipopolysaccharide administration and fed for 7 days with either a diabetes-specific enteral nutrition without (G group, n=7) or with graded arginine supply (1-5 g/kg/day) (GA group, n=7) or a standard enteral nutrition (HP group, n=10). MEASUREMENTS AND MAIN RESULTS: Survival rate was better in G and GA groups than in the HP group. On day 7, plasma insulin to glucose ratio tended to be lower in the same G and GA groups. Macrophage tumor necrosis factor-α (G: 5.0±1.1 ng/2×106 cells·hr⁻¹; GA: 3.7±0.8 ng/2×106 cells·hr⁻¹; and HP: 1.7±0.6 ng/2×106 cells·hr⁻¹; p<.05 G vs. HP) and nitric oxide (G: 4.5±1.1 ng/2×106 cells·hr⁻¹; GA: 5.1±1.0 ng/2×106 cells·hr⁻¹; and HP: 1.0±0.5 nmol/2×106 cells·hr⁻¹; p<.05 G and GA vs. HP) productions were higher in the G and GA groups compared to the HP group. Macrophages from the G and GA groups exhibited increased arginine consumption. CONCLUSIONS: In diabetic obese and endotoxemic rats, a diabetes-specific formula leads to a lower mortality, a decreased insulin resistance, and an improvement in peritoneal macrophage function. Arginine supplementation has no additional effect. These data support the use of such disease-specific diets in critically ill diabetic and obese patients.

High plasma citrulline and arginine levels ensured by sustained-release citrulline supplementation in rats.

OBJECTIVE: Dietary-supplemented arginine has been shown to have positive effects on cardiovascular disease, but several drawbacks exist and could potentially be avoided by using L-citrulline, since it is recycled to L-arginine. However, citrulline is very rapidly metabolized. We therefore developed a sustained-release form of citrulline and evaluated its metabolic behavior in rats. METHODS: Male Sprague Dawley rats were divided into three groups: receiving "empty microcapsule" (control group), 1 g/kg/d immediate-release citrulline (IR citrulline group), or 1 g/kg/d sustained-release citrulline (SR citrulline group). Citrulline was given each day at 9 a.m. after blood samples for 9 d, and on day 10, blood samples were drawn every 4 h to study the decrease in plasma amino acid concentrations. RESULTS: SR citrulline led to a sustained increase in citrullinemia and argininemia compared to IR citrulline, and on day 6 argininemia was significantly (P < 0.01) higher with SR compared to IR citrulline. Moreover, argininemia was significantly higher in the SR citrulline group than in controls throughout the study and SR citrulline maintained high argininemia and citrullinemia, at least over 12 h. CONCLUSION: This experimental study provides a strong rationale for using this new formulation for atherosclerosis treatment.