L-Citrulline Ameliorates Iron Metabolism and Mitochondrial Quality Control via Activating AMPK Pathway in Intestine and Improves Microbiota in Mice with Iron Overload.

SCOPE: Oxidative stress caused by iron overload tends to result in intestinal mucosal barrier dysfunction and intestinal microbiota imbalance. As a neutral and nonprotein amino acid, L-Citrulline (L-cit) has been implicated in antioxidant and mitochondrial amelioration properties. This study investigates whether L-cit can alleviate iron overload-induced intestinal injury and explores the underlying mechanisms. METHODS AND RESULTS: C57BL/6J mice are intraperitoneally injected with iron dextran, then gavaged with different dose of L-cit for 2 weeks. L-cit treatment significantly alleviates intestine pathological injury, oxidative stress, ATP level, and mitochondrial respiratory chain complex activities, accompanied by ameliorating mitochondrial quality control. L-cit-mediated protection is associated with the upregulation of Glutathione Peroxidase 4 (GPX4) expression, inhibition Nuclear Receptor Coactivator 4 (NCOA4)-mediated ferritinophagy and ferroptosis, and improvement of gut microbiota. To investigate the underlying molecular mechanisms, Intestinal Porcine Epithelial Cell line-J2 (IPEC-J2) cells are treated with L-cit or AMP-activated Protein Kinase (AMPK) inhibitor. AMPK signaling has been activated by L-cit. Notably, Compound C abolishes L-cit's protection on intestinal barrier, mitochondrial function, and antioxidative capacity in IPEC-J2 cells. CONCLUSION: L-cit may restrain ferritinophagy and ferroptosis to regulate iron metabolism, and induce AMPK pathway activation, which contributes to exert antioxidation, ameliorate iron metabolism and mitochondrial quality control, and improve intestinal microbiota. L-cit is a promising therapeutic strategy for iron overload-induced intestinal injury.

Oral post-treatment supplementation with a combination of glutamine, citrulline, and antioxidant vitamins additively mitigates jejunal damage, oxidative stress, and inflammation in rats with intestinal ischemia and reperfusion.

INTRODUCTION: Intestinal ischemia and reperfusion (IIR) injury is closely associated with oxidative stress. Evidence shows that oral supplementation with glutamine and citrulline alleviates IIR-induced jejunal damage. We investigated the effects of a combination of glutamine, citrulline, and antioxidant vitamins on IIR-induced jejunal damage, oxidative stress, and inflammation. METHOD: Male Wistar rats that underwent 60 min of superior mesenteric artery occlusion were orally administered glutamine plus citrulline (GC), vitamin C plus E (CE), or a combination of GC and CE 15 min before and 3, 9, and 21 h after reperfusion. Healthy rats without IIR were used as controls. RESULTS: After reperfusion for 24 h, rats with IIR showed lower levels of red blood cells, hemoglobin, serum glucose, and jejunal DNA and increased white blood cell counts compared to controls (1-way ANOVA with the least significant difference, P < 0.05). The IIR-induced decrease in serum albumin and increase in plasma interleukin-6 and jejunal thiobarbituric acid-reactive substances (TBARS) were significantly reversed by GC and/or CE. The results of the 2-way ANOVA indicated that GC was the main factor that increased jejunal villus height and muscularis DNA, and CE was the main factor that increased jejunal muscularis protein and decreased jejunal proinflammatory cytokine levels and myeloperoxidase activity. In addition, GC and CE are the main factors that decrease plasma proinflammatory cytokine levels and the jejunal apoptotic index. CONCLUSION: Oral post-treatment supplementation with glutamine and citrulline, combined with vitamins C and E, may alleviate IIR-induced oxidative stress, inflammation, and jejunal damage.

Dose-response effect of L-citrulline on skeletal muscle damage after acute eccentric exercise: an in vivo study in mice.

Background: Eccentric exercise may trigger mechanical stress, resulting in muscle damage that may decrease athletic performance. L-citrulline potentially prevents skeletal muscle damage after acute eccentric exercise. This study aimed to assess the dose-response effect of L-citrulline as a preventive therapy for skeletal muscle damage in mice after acute eccentric exercise. Methods: This is a controlled laboratory in vivo study with a post-test-only design. Male mice (BALB/c, n = 25) were randomized into the following groups: a normal control (C1) (n = 5); a negative control (C2) with downhill running and placebo intervention (n = 5); treatment groups: T1 (n = 5), T2 (n = 5), and T3 (n = 5), were subjected to downhill running and 250, 500, and 1,000 mg/kg of L-citrulline, respectively, for seven days. Blood plasma was used to determine the levels of TNNI2 and gastrocnemius muscle tissue NOX2, IL-6, and caspase 3 using ELISA. NF-κB and HSP-70 expressions were determined by immunohistochemistry. Results: Skeletal muscle damage (plasma TNNI2 levels) in mice after eccentric exercise was lower after 250 and 500 mg/kg of L-citrulline. Further, changes in oxidative stress markers, NOX2, were reduced after a 1,000 mg/kg dose. However, a lower level of change has been observed in levels of cellular response markers (NF-κB, HSP-70, IL-6, and caspase 3) after administration of L-citrulline doses of 250, 500, and 1,000 mg/kg. Conclusion: L-citrulline may prevent skeletal muscle damage in mice after acute eccentric exercise through antioxidant effects as well as inflammatory and apoptotic pathways. In relation to dose-related effects, it was found that L-citrulline doses of 250, 500, and 1,000 mg/kg significantly influenced the expression of NF-κB and HSP-70, as well as the levels of IL-6 and caspase 3. Meanwhile, only doses of 250 and 500 mg/kg had an impact on TNNI2 levels, and the 1,000 mg/kg dose affected NOX2 levels.

Pathophysiology of Preeclampsia and L-Arginine/L-Citrulline Supplementation as a Potential Strategy to Improve Birth Outcomes.

In preeclampsia, the shallow invasion of cytotrophoblast cells to uterine spiral arteries, leading to a reduction in placental blood flow, is associated with an imbalance of proangiogenic/antiangiogenic factors to impaired nitric oxide (NO) production. Proangiogenic factors, such as vascular endothelial growth factor (VEGF) and placental growth factor (PlGF), require NO to induce angiogenesis through antioxidant regulation mechanisms. At the same time, there are increases in antiangiogenic factors in preeclampsia, such as soluble fms-like tyrosine kinase type 1 receptor (sFIt1) and toll-like receptor 9 (TLR9), which are mechanism derivates in the reduction of NO bioavailability and oxidative stress in placenta.Different strategies have been proposed to prevent or alleviate the detrimental effects of preeclampsia. However, the only intervention to avoid the severe consequences of the disease is the interruption of pregnancy. In this scenario, different approaches have been analysed to treat preeclamptic pregnant women safely. The supplementation with amino acids is one of them, especially those associated with NO synthesis. In this review, we discuss emerging concepts in the pathogenesis of preeclampsia to highlight L-arginine and L-citrulline supplementation as potential strategies to improve birth outcomes. Clinical and experimental data concerning L-arginine and L-citrulline supplementation have shown benefits in improving NO availability in the placenta and uterine-placental circulation, prolonging pregnancy in patients with gestational hypertension and decreasing maternal blood pressure.

Characterization of the Pharmacokinetic and Pharmacodynamic Profile of Apraglutide, a Glucagon-Like Peptide-2 Analog, in Healthy Volunteers.

Apraglutide (FE 203799) is a glucagon-like peptide-2 (GLP-2) analog under development for the treatment of intestinal failure associated with short bowel syndrome (SBS-IF) and graft-versus-host disease (GvHD). Compared with native GLP-2, apraglutide has slower absorption, reduced clearance, and higher protein binding, enabling once-weekly dosing. This study evaluated the pharmacokinetic (PK) and pharmacodynamic (PD) profile of apraglutide in healthy adults. Healthy volunteers were randomized to receive 6 weekly subcutaneous administrations of 1, 5, or 10 mg apraglutide or placebo. PK and citrulline (an enterocyte mass PD marker) samples were collected at multiple time points. Kinetic parameters of apraglutide and citrulline were calculated using noncompartmental analysis; repeated PD measures were analyzed with a mixed model of covariance. A population PK/PD model was developed that also included data from a previous phase 1 study in healthy volunteers. Twenty-four subjects were randomized; 23 received all study drug administrations. Mean estimated apraglutide clearance was 16.5-20.7 l/day, and mean volume of distribution was 55.4-105.0 liters. A dose-dependent increase in citrulline plasma concentration was observed, with 5-mg and 10-mg doses inducing higher citrulline levels than 1-mg doses and placebo. PK/PD analysis showed that weekly 5-mg apraglutide induced the maximal citrulline response. Increased plasma citrulline levels were sustained for 10-17 days after the final apraglutide administration. Apraglutide displays predictable dose-dependent PK and PD profiles, with a 5-mg dose showing significant PD effects. Results suggest that apraglutide has early and enduring effects on enterocyte mass and supports the continued development of weekly subcutaneous apraglutide for SBS-IF and GvHD patient populations. SIGNIFICANCE STATEMENT: Once-weekly subcutaneous apraglutide results in dose-dependent elevations of plasma citrulline (an enterocyte mass pharmacodynamic marker) with parameters suggesting that apraglutide has lasting effects on enterocyte mass and the potential to provide therapeutic benefits. This is the first report of a model relating glucagon-like peptide-2 (GLP-2) agonism and its effects in intestinal mucosa, affording not only the ability to predict pharmacologic effects of GLP-2 analogs but also the exploration of optimal dosing regimens for this drug class across populations with different body weights.

L-citrulline attenuates lipopolysaccharide-induced inflammatory lung injury in neonatal rats.

BACKGROUND: Prenatal or postnatal lung inflammation and oxidative stress disrupt alveolo-vascular development leading to bronchopulmonary dysplasia (BPD) with and without pulmonary hypertension. L-citrulline (L-CIT), a nonessential amino acid, alleviates inflammatory and hyperoxic lung injury in preclinical models of BPD. L-CIT modulates signaling pathways mediating inflammation, oxidative stress, and mitochondrial biogenesis-processes operative in the development of BPD. We hypothesize that L-CIT will attenuate lipopolysaccharide (LPS)-induced inflammation and oxidative stress in our rat model of neonatal lung injury. METHODS: Newborn rats during the saccular stage of lung development were used to investigate the effect of L-CIT on LPS-induced lung histopathology and pathways involved in inflammatory, antioxidative processes, and mitochondrial biogenesis in lungs in vivo, and in primary culture of pulmonary artery smooth muscle cells, in vitro. RESULTS: L-CIT protected the newborn rat lung from LPS-induced: lung histopathology, ROS production, NFκB nuclear translocation, and upregulation of gene and protein expression of inflammatory cytokines (IL-1ß, IL-8, MCP-1α, and TNF-α). L-CIT maintained mitochondrial morphology, increased protein levels of PGC-1α, NRF1, and TFAM (transcription factors involved in mitochondrial biogenesis), and induced SIRT1, SIRT3, and superoxide dismutases protein expression. CONCLUSION: L-CIT may be efficacious in decreasing early lung inflammation and oxidative stress mitigating progression to BPD. IMPACT: The nonessential amino acid L-citrulline (L-CIT) mitigated lipopolysaccharide (LPS)-induced lung injury in the early stage of lung development in the newborn rat. This is the first study describing the effect of L-CIT on the signaling pathways operative in bronchopulmonary dysplasia (BPD) in a preclinical inflammatory model of newborn lung injury. If our findings translate to premature infants, L-CIT could decrease inflammation, oxidative stress and preserve mitochondrial health in the lung of premature infants at risk for BPD.

The effects of L-Citrulline and Glutathione on Endurance performance in young adult trained males.

BACKGROUND: Citrulline may amplify the effects of L-arginine and nitric oxide concentration, which may augment vasodilation and blood flow, thereby enhancing aerobic exercise performance. The purpose of this randomized, double-blind, placebo-controlled crossover study was to investigate effects of L-citrulline + Glutathione on aerobic exercise performance and blood flow in well-trained men. METHODS: Twenty-five males (Mean ± SD; Age: 22.2 ± 2.4 yrs; Height: 177.0 ± 4.8 cm; Weight: 75.3 ± 6.9 kg) were randomly assigned to the L-citrulline + Glutathione (Setria Performance Blend: SPB; L-citrulline [2 g] + glutathione [200 mg], 6 capsules) or placebo (PL; 3.1 g cellulose, 6 capsules) group. Participants performed a maximal oxygen consumption treadmill test to determine peak velocity (PV) and returned after eight days of ingesting either PL or SPB. Three timed treadmill runs to exhaustion (TTE) were performed at 90%, 100%, and 110% PV. Brachial artery blood flow and vessel diameter were assessed using ultrasound at 1-hr prior to exercise (1hrPrEX), after each exercise bout, immediately post-exercise (immediate PEX), and 30 minutes post exercise (30minPEX) at visits 2 and 4. Blood analytes were assessed via venous blood draws at visit 1, visit 3, and 1hrPEX, immediate PEX, and 30minPEX at visits 2 and 4. After a 14-day washout, participants repeated the same procedures, ingesting the opposite treatment. Separate repeated measures ANOVAs were performed for TTE, vessel diameter, blood flow, and blood analytes. RESULTS: Blood flow was significantly augmented 30minPEX (p = 0.04) with SPB in comparison with PL. L-citrulline and L-arginine plasma concentrations were significantly elevated immediately PEX (p = 0.001) and 30-minPEX (p = 0.001) following SPB in comparison to PL. CONCLUSION: Acute ingestion of SPB after eight days may enhance blood flow, L-citrulline, and L-arginine plasma concentrations after high-intensity exercise, which may enhance performance. CLINICAL TRIAL REGISTRATION: [https://clinicaltrials.gov/ct2/show/nct04090138], identifier [NCT04090138].

Combined Citrulline and Glutathione Supplementation Improves Endothelial Function and Blood Pressure Reactivity in Postmenopausal Women.

Postmenopausal women (PMW) may experience endothelial dysfunction associated with arginine (ARG) deficiency relative to asymmetric dimethylarginine (ADMA) caused by oxidative stress. Endothelial dysfunction contributes to increased blood pressure (BP) responsiveness to sympathoexcitation induced by the cold pressor test (CPT). We investigated the effects of citrulline alone (CIT) and combined with the antioxidant glutathione (CIT+GSH) on vascular function. Forty-four healthy PMW were randomized to CIT (6 g), CIT+GSH (2 g + 200 mg: Setria®) or placebo (PL) for 4 weeks. Brachial artery flow-mediated dilation (FMD), aortic stiffness (pulse wave velocity, PWV), brachial and aortic BP reactivity to CPT, and serum fasting blood glucose (FBG), ARG, and ARG/ADMA ratio were measured. Baseline FBG was higher in CIT+GSH vs. PL. FMD increased after CIT+GSH vs. PL (p < 0.05). CIT and CIT+GSH increased ARG/ADMA (p < 0.05), but did not affect aortic PWV. CIT+GSH attenuated the brachial and aortic systolic BP and mean arterial pressure (MAP) responses to CPT vs. PL and CIT (p < 0.05). The improvements in FMD were related to baseline FMD (r = -0.39, p < 0.05) and aortic MAP response to CPT (r = -0.33, p < 0.05). This study showed that CIT+GSH improved FMD and attenuated systolic BP and MAP reactivity in PMW. Although CIT increased ARG/ADMA, it did not improve FMD in healthy PMW.

Citrulline inhibits LPS-induced pyroptosis of RAW264.7 macrophages through NF-κB signaling pathway.

BACKGROUND: The aim of this study was to investigate the effect of citrulline on the pyroptosis of mouse macrophage RAW264.7 and the mechanism. We investigated the effect of citrulline on pyroptosis of RAW264.7 cell induced by lipopolysaccharide (LPS), and the modulation of nuclear factor-kappaB (NF-κB) signaling. METHODS: Pyroptosis was evaluated using flow cytometry and caspase-1/sytox double staining. Cell counting kit-8 assay was performed to evaluate cell viability. RESULTS: Citrulline promoted cell viability and inhibited the pyroptosis of RAW264.7 cell stimulated by LPS. Furthermore, citrulline inactivated NF-κb/p65 signaling pathway by suppressing p65 nuclear translocation induced by LPS. An NF-κb signaling pathway activator, betulinic acid, reversed the inhibition of pyroptosis induced by citrulline. CONCLUSION: Citrulline inhibited LPS-induced pyrophosis, which may be closely related to the inactivation of NF-κB/p65 signaling pathway.

Increased production of natural moisturizing factors and bleomycin hydrolase activity in elderly human skin.

BACKGROUND: Bleomycin hydrolase (BH), which is expressed in the stratum granulosum and lower stratum corneum (SC), is involved in final filaggrin degradation. Furthermore, BH plays an essential role in producing free amino acids, which constitute the majority of natural moisturizing factors (NMF). However, the effects of BH expression and protease activity on human skin aging remain unclear. OBJECTIVE: This study was designed to evaluate the activity and expression patterns of BH in SC extracts from healthy young and elderly individuals. METHODS: SC samples were collected by tape stripping. BH activity was assessed by measuring the citrulline aminopeptidase activity. BH expression was determined by Western blotting, and NMF was quantified by liquid chromatography/mass spectrometry. Skin barrier function was determined by measuring SC hydration, transepidermal water loss (TEWL), and skin pH. RESULTS: The activity and expression of BH were higher in the elderly skin than in young skin, and BH activity was correlated with BH expression levels. Evaluation of the NMF showed that the levels of total amino acids, such as glycine, serine, aspartic acid, citrulline, pyrrolidone carboxylic acid (a metabolite of glutamic acid), and trans-urocanic acid (a metabolite of histidine), were significantly higher in elderly skin than in young skin. Moreover, SC hydration and TEWL were significantly lower in elderly, indicating dry skin, and pH was significantly higher in elderly, indicating greater skin alkalinization. CONCLUSION: These results suggest that BH activity and expression, as well as NMF amino acids, increase in elderly people as compensatory mechanisms against dry skin.

Exposure of anti-infective drugs and the dynamic changes of the gut microbiota during gastrointestinal mucositis in autologous stem cell transplant patients: a pilot study.

Gastrointestinal mucositis could potentially compromise drug absorption due to functional loss of mucosa and other pathophysiological changes in the gastrointestinal microenvironment. Little is known about this effect on commonly used anti-infectives. This study aimed to explore the association between different stages of gastrointestinal mucositis, drug exposure, and gut microbiota. A prospective, observational pilot study was performed in HSCT patients aged ≥ 18 years receiving anti-infectives orally. Left-over blood samples and fecal swabs were collected from routine clinical care until 14 days after HSCT to analyze drug and citrulline concentrations and to determine the composition of the gut microbiota. 21 patients with a median age of 58 (interquartile range 54-64) years were included with 252 citrulline, 155 ciprofloxacin, 139 fluconazole, and 76 acyclovir concentrations and 48 fecal swabs obtained. Severe gastrointestinal mucositis was observed in all patients. Due to limited data correlation analysis was not done for valacyclovir and fluconazole, however we did observe a weak correlation between ciprofloxacin and citrulline concentrations. This could suggest that underexposure of ciprofloxacin can occur during severe mucositis. A follow-up study using frequent sampling rather than the use of left-over would be required to investigate the relationship between gastrointestinal mucositis, drug exposure, and gut microbiome.

The impact of L-citrulline on murine intestinal cell integrity, immune response, and arginine metabolism in the face of Giardia lamblia infection.

Giardiasis is an intestinal protozoal disease caused by Giardia lamblia (G. lamblia) which is a major worldwide health problem due to development of resistance to commonly used drugs. Therefore, it is necessary to identify an effective drug for giardiasis. This study aimed to assess the therapeutic role of L-citrulline against giardiasis in experimental animals. 40 male Swiss Albino weaned rats were used in this study, divided into four groups. Group I: normal control; group II: infected un-treated; group III: infected and treated with L-citrulline and Group IV: infected and treated with metronidazole. The efficacy was evaluated by counting Giardia trophozoites in the intestinal mucosa and cysts in the stool of infected rats. Histopathological analyses, immunohistochemistry expression of inducible nitric oxide synthase (iNOS) in the small intestine tissues were performed. Along with, serum IL6, the intestinal arginase enzyme level and giardial flavohemoglobin (flavoHb) expression were measured. L-citrulline administration reduced the mean number of G. lamblia cysts and trophozoites, serum IL-6, and intestinal arginase enzyme levels. Furthermore, the intestinal brush border was restored, with a reduction in the inflammatory infiltrate and an increase in iNOS activity. Moreover, there was a significant decrease in flavoHb gene expression in both the L-citrulline and metronidazole treated groups. Thus L-citrulline is effective in NO production therefore it has a therapeutic potential in controlling giardiasis.

Effect of citrulline malate supplementation on muscle function and bioenergetics during short-term repeated bouts of fatiguing exercise.

Citrulline malate (CM) has been shown to improve muscle performance in healthy participants during a single exercise session. Yet, within the framework of exercises repeated at close time interval, the consequences of CM ingestion on mechanical performance are controversial and the bioenergetics side remains undocumented. The aim of this double-blind placebo-controlled study was to evaluate in vivo the effect of short-term (7 doses in 48 h) oral administration of CM upon gastrocnemius muscle function and bioenergetics using non-invasive multimodal NMR techniques in healthy rats. The experimental protocol consisted of two 6-min bouts of fatiguing exercise spaced by an 8-min recovery period. CM treatment did not affect the basal bioenergetics status and increased the half-fatigue time during the first exercise bout. With exercise repetition, it prevented PCr cost alteration and decreased both the glycolytic ATP production and the contractile ATP cost in working muscle, but these changes were not associated to any improvement in mechanical performance. In addition, CM did not influence the replenishment of high-energy phosphorylated compounds during the post-exercise recovery periods. Therefore, short-term CM administration enhances muscle bioenergetics throughout fatiguing bouts of exercise repeated at close time interval but this enhancement does not benefit to mechanical performance.

Citrulline protects human retinal pigment epithelium from hydrogen peroxide and iron/ascorbate induced damages.

Oxidative stress plays an important role in the ageing of the retina and in the pathogenesis of retinal diseases such as age-related macular degeneration (ARMD). Hydrogen peroxide is a reactive oxygen species generated by the photo-excited lipofuscin that accumulates during ageing in the retinal pigment epithelium (RPE), and the age-related accumulation of lipofuscin is associated with ARMD. Iron also accumulates with age in the RPE that may contribute to ARMD as an important source of oxidative stress. The aim of this work was to investigate the effects of L-Citrulline (CIT), a naturally occurring amino acid with known antioxidant properties, on oxidative stressed cultured RPE cells. Human RPE (ARPE-19) cells were exposed to hydrogen peroxide (H2 O2 ) or iron/ascorbate (I/A) for 4 h, either in the presence of CIT or after 24 h of pretreatment. Here, we show that supplementation with CIT protects ARPE-19 cells against H2 O2 and I/A. CIT improves cell metabolic activity, decreases ROS production, limits lipid peroxidation, reduces cell death and attenuates IL-8 secretion. Our study evidences that CIT is able to protect human RPE cells from oxidative damage and suggests potential protective effect for the treatment of retinal diseases associated with oxidative stress.

Effects of dietary L-citrulline supplementation on nitric oxide synthesis, immune responses and mitochondrial energetics of broilers during heat stress.

L-Citrulline is a non-protein amino acid that promotes arginine recycling and muscle protein synthesis. This study investigated whether L-citrulline can exert these functions in heat-stressed chickens. Arbor acre broilers were fed either basal diets (Control) or basal diets supplemented with 1% L-citrulline (L-Cit). At 28 d old, broilers were subjected to two environmental temperatures, 35 °C for 8 h/d (HS) or 24 °C for 24 h/d (TNZ) for 2 weeks. The experiment was designed as a 2 by 2 factorial arrangement. Results showed that HS increased the core body temperature (CBT) and rectal temperature of broilers compared to the TNZ condition. The CBT and mean CBT decreased by ∼0.5oC in TNZ + L-Cit broilers compared to the TNZ + Control group (P < 0.05). L-Cit supplementation at TNZ significantly (P < 0.05) lowered the plasma malondialdehyde content but this was increased during HS. Exposure to HS significantly (P < 0.05) elevated plasma malondialdehyde compared to TNZ condition. Plasma immunoglobulin A, G, and M were increased (P < 0.05) by TNZ + L-Cit compared to the TNZ + Control group. Plasma nitric oxide and endothelial nitric oxide synthase (NOS) activity was increased during HS, whereas the total and inducible NOS enzymes were decreased. In addition, L-Cit supplementation increased both the inducible and endothelial NOS isoforms (P < 0.05). The mRNA expression of peroxisome proliferator-activated receptor γ coactivator 1-α, avian uncoupling protein, cytochrome C oxidase subunit 3, and ATP synthase F1 subunit beta (ATP5ß) in the breast muscle were downregulated (P < 0.05) during HS. However, L-Cit supplementation upregulated the mitochondrial transcription factor A and during HS, L-Cit increased ATP5ß expression similar to TNZ housed broilers. Therefore, this study demonstrates that dietary L-Cit can lower the body temperature, decrease lipid peroxidation and promote the immune status of broilers under thermoneutrality. Also, L-Cit would act to promote muscle ATP generation during heat stress in broilers.

Antitumor Effects of L-citrulline on Hela Cervical Cancer Cell Lines.

AIM: Cervical cancer is the deadliest gynecological malignancy. This study aims to examine the anticancer effects of L-citrulline on HeLa cell culture. MATERIALS AND METHODS: HeLa cells were cultured in complete Eagle's minimum essential medium. HeLa cells were seeded in 96-well plates and incubated with L-citrulline. After incubation, MTT dye was added and incubated. Annexin- V technique was used to test the apoptosis. The activated caspases of HeLa cells by L-citrulline exposure were measured with the Caspase 3/7 technique. One-way variance analysis was conducted for statistical analysis by using GraphPad Prism 6.0 for Windows. RESULTS: L-citrulline showed its toxicity on HeLa cells in a dose-dependent manner in application times of 24 and 48 hours. The IC50 dose of L-citrulline was 0.19 and 0.16 mg/mL at 24 and 48 hours, respectively. When HeLa cells were exposed to an IC50 dose of L-citrulline for 24 hours, the percentages of the dead, early apoptotic, and late apoptotic cells were detected to be 0.75%, 23.05%, and 12.75%, respectively. The differences in the wideness of the scratch area were observed at the initial stage and after 24 hours of applying L-citrulline. CONCLUSION: L-citrulline showed its toxicity on HeLa cells in a dose-dependent manner. Based on Annexin and Caspase findings, it can be concluded that L-citrulline exerted a pro-apoptotic effect on HeLa cells in only a short exposure time. L-citrulline also showed a migration inhibitory effect. The findings of this study indicate L-citrulline to be worthy of investigation for its anticancer activities in vitro and in vivo, and as a candidate for cancer therapy.

Effect of L-citrulline supplementation on sperm characteristics and hormonal and antioxidant levels in blood and seminal plasma of rams.

With the aim of providing a theoretical basis for the application of L-citrulline (L-Cit) in animal husbandry, the effects of L-Cit on reproductive hormone levels, antioxidant capacity and semen quality of rams were studied by feeding them varying doses of L-Cit. A total of 32 rams were randomly divided into four groups with eight rams each. After all rams were trained to donate sperm normally, the control group was fed a basic diet, whereas the experimental groups I, II and III were provided with feed supplemented with 4, 8 and 12 g/d of L-Cit respectively. The experiment was conducted for 70 days, during which blood samples were collected from the jugular vein on days 0, 15, 30, 45 and 60, and semen samples were collected on days 0, 20, 40 and 60. In the same group, 100 µl of semen was used to test for quality, The rest of the semen sample and blood samples were centrifuged at 600 g for 15 min, and the supernatant and serum, respectively, were used to determine the levels reproductive hormones and antioxidant indices. Ram semen samples were also collected on day 70 and used to study sperm plasma membrane, substitution and mitochondrial membrane potential. Compared with the control group, the groups receiving L-Cit showed an increase in sperm concentration and number of linear motile sperm (p < .01); a decrease in the number of dead sperm (p < .01); an increase in sperm viability, particularly in groups II and III (p < .01); and an increase in sperm mitochondrial membrane potential (p < .01). Moreover, groups I, II and III showed significantly higher levels of serum gonadotropin-releasing hormone (GnRH), glutathione peroxidase (GSH-Px) and nitric oxide (NO) (p < .01). Luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels increased in groups I (p < .05), II (p < .05) and III (p < .01), whereas testosterone (T), catalase (CAT) and superoxide dismutase (SOD) levels increased in groups I and II (p < .01). Serum total antioxidant capacity (T-A) increased (p < .05), whereas both hydroxyl radical (·OH) and peroxy radical ( O 2 · - ) levels decreased (p < .01). Compared with the control, all groups had significantly higher SOD and GSH-Px in their seminal plasma (p < .01), and groups I, II (p < .05 for both) and III (p < .01) had higher levels of GnRH and FSH. LH, CAT and NO levels increased in group I (p < .05), II and III (p < .01 for both); malondialdehyde levels decreased in groups I, II (p < .05 for both) and group III (p < .01); and O 2 · - levels decreased in groups I, II and III (p < .01). Under our experimental conditions, GnRH, FSH, LH, T, CAT, SOD, T-A, GSH-PX and NO levels in the serum and seminal plasma of rams receiving L-Cit increased, whereas Oestradiol (E2 ), O 2 · - and ·OH levels in the seminal plasma decreased; this improved the semen quality of rams supplemented with L-Cit. Moreover, supplementation with 12 g/d gave the best results.

Beneficial effects of citrulline enteral administration on sepsis-induced T cell mitochondrial dysfunction.

Severe sepsis induces a sustained immune dysfunction associated with poor clinical behavior. In particular, lymphopenia along with increased lymphocyte apoptosis and decreased lymphocyte proliferation, enhanced circulating regulatory T cells (Treg), and the emergence of myeloid-derived suppressor cells (MDSCs) have all been associated with persistent organ dysfunction, secondary infections, and late mortality. The mechanisms involved in MDSC-mediated T cell dysfunction during sepsis share some features with those described in malignancies such as arginine deprivation. We hypothesized that increasing arginine availability would restore T cell function and decrease sepsis-induced immunosuppression. Using a mouse model of sepsis based on cecal ligation and puncture and secondary pneumonia triggered by methicillin-resistant Staphylococcus aureus inoculation, we demonstrated that citrulline administration was more efficient than arginine in increasing arginine plasma levels and restoring T cell mitochondrial function and proliferation while reducing sepsis-induced Treg and MDSC expansion. Because there is no specific therapeutic strategy to restore immune function after sepsis, we believe that our study provides evidence for developing citrulline-based clinical studies in sepsis.

Regulation of citrulline synthesis in human enterocytes: Role of hypoxia and inflammation.

Intensive care unit patients and chronic airway inflammatory disease are characterized by chronic systemic hypoxia and inflammation inducing a decrease in nitric oxide release due to impaired l-arginine (ARG) homeostasis. As ARG is synthesized from circulating l-citrulline (CIT), an alteration of CIT production in small intestine by ornithine carbamoyltransferase could be involved. Here, we posit that hypoxia and/or inflammation has effects on ornithine carbamoyltransferase regulation in enterocytes. A duodenal explant incubation model was used. Biopsy specimens taken from 25 selected patients were incubated for 6 h in 4 groups: control, inflammation, hypoxia, and hypoxia + inflammation. At the end of the incubation period, we measured CIT concentration in culture media, ornithine carbamoyltransferase activity, ornithine carbamoyltransferase protein and gene expression, protein expression of enzymes involved in the CIT production pathway, and expression of energy status proteins. Inflammation and/or hypoxia conditions did not affect CIT production. Ornithine carbamoyltransferase activity was increased in hypoxia conditions (p = 0.023). Expression of enzymes implicated in the CIT crossroads pathway and enzymes reflecting energy status variation was not affected by inflammation and hypoxia. Data sets were pooled to evaluate the variability of the four quartiles for each parameter. CIT production was found to increase over the quartiles whereas other parameters remained stable. Our results showed that intestinal CIT production is preserved during inflammation and/or hypoxia, thus confirming the significance of this metabolic pathway. This suggests that the CIT deficiency observed in clinical hypercatabolic states could be a consequence of high utilization for ARG synthesis.

l-Citrulline ameliorates pathophysiology in a rat model of superimposed preeclampsia.

BACKGROUND AND PURPOSE: Preeclampsia, characterized by hypertension, proteinuria and restriction of fetal growth, is one of the leading causes of maternal and perinatal mortality. So far, there is no effective pharmacological therapy for preeclampsia. The present study was conducted to investigate the effects of supplementation with l-citrulline in Dahl salt-sensitive rats, a model of superimposed preeclampsia. EXPERIMENTAL APPROACH: Parental Dahl salt-sensitive rats were treated with l-citrulline (2.5 g·L-1 in drinking water) from the day of mating to the end of lactation period. Blood pressure was monitored throughout pregnancy and markers of preeclampsia were assessed. Endothelial function of the pregnant Dahl salt-sensitive rats was assessed by wire myograph. KEY RESULTS: In Dahl salt-sensitive rats, l-citrulline supplementation significantly reduced maternal blood pressure, proteinuria and levels of circulating soluble fms-like tyrosine kinase 1. l-Citrulline improved maternal endothelial function by augmenting the production of nitric oxide in the aorta and improving endothelium-derived hyperpolarizing factor-mediated vasorelaxation in resistance arteries. l-Citrulline supplementation improved placental insufficiency and fetal growth, which were associated with an enhancement of angiogenesis and reduction of fibrosis and senescence in the placentas. In addition, l-citrulline down-regulated genes involved in the TLR4 and NF-κB signalling pathways. CONCLUSION AND IMPLICATIONS: This study shows that l-citrulline supplementation reduced gestational hypertension and improved placentation and fetal growth in a rat model of superimposed preeclampsia. l-Citrulline supplementation may provide an effective and safe therapeutic strategy for preeclampsia that benefits both the mother and the fetus.