OBJECTIVE: Chronic inflammation and oxidative stress mediate the pathological progression of diabetic complications, like diabetic retinopathy (DR), peripheral neuropathy (DPN) and impaired wound healing. Studies have shown that treatment with a stable form of arginase 1 that reduces l-arginine levels and increases ornithine and urea limits retinal injury and improves visual function in DR. We tested the therapeutic efficacy of PEGylated arginine deiminase (ADI-PEG20) that depletes l-arginine and elevates l-citrulline on diabetic complications in the db/db mouse model of type 2 diabetes (T2D).
METHODS: Mice received intraperitoneal (IP), intramuscular (IM), or intravitreal (IVT) injections of ADI-PEG20 or PEG20 as control. Effects on body weight, fasting blood glucose levels, blood-retinal-barrier (BRB) function, visual acuity, contrast sensitivity, thermal sensitivity, and wound healing were determined. Studies using bone marrow-derived macrophages (BMDM) examined the underlying signaling pathway.
RESULTS: Systemic injections of ADI-PEG20 reduced body weight and blood glucose and decreased oxidative stress and inflammation in db/db retinas. These changes were associated with improved BRB and visual function along with thermal sensitivity and wound healing. IVT injections of either ADI-PEG20, anti-VEGF antibody or their combination also improved BRB and visual function. ADI-PEG20 treatment also prevented LPS/IFNℽ-induced activation of BMDM in vitro as did depletion of l-arginine and elevation of l-citrulline.
CONCLUSIONS: ADI-PEG20 treatment limited signs of DR and DPN and enhanced wound healing in db/db mice. Studies using BMDM suggest that the anti-inflammatory effects of ADI-PEG20 involve blockade of the JAK2-STAT1 signaling pathway via l-arginine depletion and l-citrulline production.

UNASSIGNED: Palmitic acid (PA), the most abundant saturated free fatty acids, induces apoptosis in bovine mammary epithelial cells. It is suggested that oxidative stress and endoplasmic reticulum (ER) stress are key mechanisms underlying PA-induced cell death. This study aimed to investigate the interaction between ER stress and oxidative stress during PA-induced cell death in MAC-T cells. Additionally, we examined whether L-citrulline can protect against PA-induced damage of MAC-T cells.
UNASSIGNED: MAC-T cells were treated with 4-phenyl butyric acid (4-PBA) or N-acetyl-L-cysteine (NAC) to inhibit PA-induced ER stress and oxidative stress, respectively. MAC-T cells were pretreated with or without L-citrulline for 48 h followed by PA treatment. Cell viability was measured with MTT assays. Intracellular reactive oxygen species (ROS) levels in MAC-T cells were assessed using 5-(and-6)-chloromethyl- 2`,7`-dichlorodihydrofluorescein diacetate acetyl ester dye. Real-time qPCR was used to explore the regulation of genes associated with oxidative stress, and ER stress genes. Western blotting analysis was also carried out.
UNASSIGNED: 4-PBA significantly reduced PA-induced mRNA expressions of activating transcription factor 4 (ATF4), C/EBP homologous protein (CHOP), nuclear factor (erythroid-derived 2)-like 2 (NRF2), and intracellular ROS levels. Furthermore, NAC dramatically reduced PA-induced ROS levels and the mRNA expressions of NRF2, ATF4, and CHOP. L-citrulline pretreatment effectively rescued cell viability decreased by PA. Moreover, L-citrulline pretreatment significantly downregulated the PA-induced upregulation of GRP78, ATF4, and CHOP mRNA expression, and protein expression of p-PERK and cleaved caspase-3. PA increased intracellular ROS levels and NRF2 mRNA expression, whereas L-citrulline pretreatment remarkably reduced these levels.
UNASSIGNED: Both ER and oxidative stresses interact during PA-induced cell death in MAC-T cells, and L-citrulline could attenuate this cell death by inhibiting ER and oxidative stresses. Therefore, L-citrulline may be a promising supplement for protecting against PA-induced cell death in bovine MECs during the lactation period of dairy cows.

The objective of this study was to evaluate the effects of pre-exercise L-citrulline supplementation on the athletic performance of Yili speed-racing horses during a high-intensity exercise. On the 20th day of the experiment, blood samples were collected at 3 h and 6 h post-supplementation to measure the amino acid and polyamine concentrations. On the 38th day of the experiment, the horses participated in a 2000 m speed race, and three distinct blood samples were gathered for assessing blood gases, hematological parameters, the plasma biochemistry, antioxidant parameters, and NO concentrations. The results indicate that the L-citrulline group showed a significant increase in the plasma citrulline and arginine concentrations. Conversely, the concentrations of alanine, serine, and threonine were significantly decreased. The glycine concentration decreased significantly, while there was a trend towards an increase in the glutamine concentration. Additionally, the levels of putrescine and spermidine in the plasma of the L-citrulline group were significantly increased. In terms of exercise performance, L-citrulline can improve the exercise performance of sport horses, significantly reduce the immediate post-race lactate levels in Yili horses, and accelerate the recovery of blood gas levels after an exercise. Furthermore, in the L-citrulline group of Yili horses, The levels of the total protein of plasma, superoxide dismutase, catalase, and lactate dehydrogenase were significantly increased both 2 h before and 2 h after the race. The total antioxidant capacity showed a highly significant increase, while the malondialdehyde content significantly decreased. In the immediate post-race period, the creatinine content in the L-citrulline group significantly increased. In conclusion, this study demonstrates that L-citrulline supplementation can influence the circulating concentrations of L-citrulline and arginine in Yili horses, enhance the antioxidant capacity, reduce lactate levels, and improve physiological and biochemical blood parameters, thereby having a beneficial effect on the exercise performance of athletic horses.

Endothelial dysfunction decreases exercise limb blood flow (BF) and muscle oxygenation. Acute L-Citrulline supplementation (CIT) improves muscle tissue oxygen saturation index (TSI) and deoxygenated hemoglobin (HHb) during exercise. Although CIT improves endothelial function (flow-mediated dilation [FMD]) in hypertensive women, the impact of CIT on exercise BF and muscle oxygenation (TSI) and extraction (HHb) are unknown. We examined the effects of CIT (10 g/day) and a placebo for 4 weeks on blood pressure (BP), arterial vasodilation (FMD, BF, and vascular conductance [VC]), and forearm muscle oxygenation (TSI and HHb) at rest and during exercise in 22 hypertensive postmenopausal women. Compared to the placebo, CIT significantly (p < 0.05) increased FMD (Δ-0.7 ± 0.6% vs. Δ1.6 ± 0.7%) and reduced aortic systolic BP (Δ3 ± 5 vs. Δ-4 ± 6 mmHg) at rest and improved exercise BF (Δ17 ± 12 vs. Δ48 ± 16 mL/min), VC (Δ-21 ± 9 vs. Δ41 ± 14 mL/mmHg/min), TSI (Δ-0.84 ± 0.58% vs. Δ1.61 ± 0.46%), and HHb (Δ1.03 ± 0.69 vs. Δ-2.76 ± 0.77 μM). Exercise BF and VC were positively correlated with improved FMD and TSI during exercise (all p < 0.05). CIT improved exercise artery vasodilation and muscle oxygenation via increased endothelial function in hypertensive postmenopausal women.

Background: The management of erectile dysfunction (ED) shows several grey zones and new treatments are required to reduce the percentage of patients discontinuing treatment. Here, we aim to evaluate the role of a natural mixture named Icarifil® (L-Citrulline, L-Carnitine, Eruca vesicaria, Panax ginseng, Tribulus terrestris, Turnera diffusa, Taurine, Vitamin E, Zinc) in the management of patients with ED. Methods: From September 2022 to March 2023, all patients attending 3 urological institutions due to ED were randomized to receive the following for 3 months: Icarifil® 1 sachet every 24 h (Group 1) or Icarifil® 1 sachet + tadalafil 5 mg 1 tablet every 24 h (Group 2) or tadalafil 5 mg 1 tablet daily (Group 3). All patients underwent urologic visits and dedicated questionnaires (IIEF-5, SEP-2, SEP-3) at enrollment and at the follow-up evaluation (3 months). Patient-Reported Outcomes (PROs) at the follow-up evaluation were used. The primary endpoint was the difference in the questionnaires at the follow-up visit compared to the one at enrollment among the study groups. Results: In the per-protocol analysis, 52 patients in Group 1, 55 in Group 2 and 57 in Group 3 were analyzed. At the follow-up evaluation, IIEF-5 scores improved in all the 3 groups between enrollment and the follow-up evaluation, but a statistically significant difference was reported between Group 2 (+7.4) and Group 1 (+4.1) or Group 3 (+5.1), (p < 0.001; p < 0.001). Moreover, 47 patients (94.0%) in Group 2 showed an improvement in the SEP questionnaires, when compared with the baseline, while 29 in Group 1 (56.9%) and 42 in Group 3 (82.3%) showed a statistically significant difference (p = 0.004; p = 0.003) among the groups. The PRO analysis reported better efficacy and patient satisfaction in Group 2 when compared with Group 1 or Group 3. Conclusions: In conclusion, Icarifil® is able to improve penile erectile function in mild-moderate ED and significantly improve the clinical efficacy of daily used tadalafil 5 mg. Icarifil® could represent an interesting alternative treatment in patients experiencing adverse effects or with contraindications for chronic treatment with PDE5-is.

Although mechanical loading is essential for maintaining bone health and combating osteoporosis, its practical application is limited to a large extent by the high variability in bone mechanoresponsiveness. Here, we found that gut microbial depletion promoted a significant reduction in skeletal adaptation to mechanical loading. Among experimental mice, we observed differences between those with high and low responses to exercise with respect to the gut microbial composition, in which the differential abundance of Lachnospiraceae contributed to the differences in bone mechanoresponsiveness. Microbial production of L-citrulline and its conversion into L-arginine were identified as key regulators of bone mechanoadaptation, and administration of these metabolites enhanced bone mechanoresponsiveness in normal, aged, and ovariectomized mice. Mechanistically, L-arginine-mediated enhancement of bone mechanoadaptation was primarily attributable to the activation of a nitric-oxide-calcium positive feedback loop in osteocytes. This study identifies a promising anti-osteoporotic strategy for maximizing mechanical loading-induced skeletal benefits via the microbiota-metabolite axis.

Arginine deiminase (ADI) has garnered significant interest because of its ability to objectively eradicate cancer cells and produce L-citrulline. To meet the production demands, this study focused on enhancing the enzyme activity and thermal stability of ADI. In this study, 24 ADI mutants were obtained through computer aid site-specific mutation in the ADI of Enterobacter faecalis. Notably, the specific enzyme activities of F44W, N163P, E220I, E220L, N318E, A336G, T340I, and N382F increased, reaching 1.33-2.53 times that of the original enzyme. This study confirmed that site-specific mutations are critical for optimizing enzyme function. Additionally, the F44W, N163P, E220I, T340I, and A336G mutants demonstrated good thermal stability. The optimal pH for mutant F44W increased to 8, whereas mutants E220I, I244V, A336G, T340I, and N328F maintained an optimal pH of 7.5. Conversely, the M109L, N163P, E220L, I244L, and N318E mutants shad an optimal pH of 7. This study revealed that mutant enzymes with increased activity were more likely to contain mutation sites situated near the four loops associated with catalytic residues, whereas mutations at the dimer junction sites had a higher tendency to enhance enzyme stability. These findings contribute to the development of ADI industrial applications and its modifications.

OBJECTIVE: Anti-diabetic and anti-obesity effects of L-citrulline (Cit) have been reported in male rats. This study determined sex differences in response to Cit in Wistar rats.
METHODS: Type 2 diabetes (T2D) was induced using a high-fat diet followed by low-dose of streptozotocin (30 mg/kg) injection. Male and female Wistar rats were divided into 4 groups (n = 6/group): Control, control+Cit, T2D, and T2D + Cit. Cit (4 g/L in drinking water) was administered for 8 weeks. Obesity indices were recorded, serum fasting glucose and lipid profile were measured, and glucose and pyruvate tolerance tests were performed during the Cit intervention. White (WAT) and brown (BAT) adipose tissues were weighted, and the adiposity index was calculated at the end of the study.
RESULTS: Cit was more effective in decreasing fasting glucose (18 % vs. 11 %, P = 0.0100), triglyceride (20 % vs. 14 %, P = 0.0173), and total cholesterol (16 % vs. 11 %, P = 0.0200) as well as decreasing gluconeogenesis and improving glucose tolerance, in females compared to male rats with T2D. Following Cit administration, decreases in WAT weight (16 % vs. 14 % for gonadal, 21 % vs. 16 % for inguinal, and 18 % vs. 13 % for retroperitoneal weight, all P < 0.0001) and increases in BAT weight (58 % vs. 19 %, for interscapular and 10 % vs. 7 % for axillary, all P < 0.0001) were higher in females than male rats with T2D. The decrease in adiposity index was also higher (11 % vs. 9 %, P = 0.0007) in females.
CONCLUSIONS: The anti-obesity and anti-diabetic effects of Cit in rats are sex-dependent, with Cit being more effective in female than male rats.

Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disorder that causes progressive paralysis. L-Citrulline is a non-essential neutral amino acid produced by L-arginine via nitric oxide synthase (NOS). According to previous studies, the pathogenesis of ALS entails glutamate toxicity, oxidative stress, protein misfolding, and neurofilament disruption. In addition, L-citrulline prevents neuronal cell death in brain ischemia; therefore, we investigated the change in the transport of L-citrulline under various pathological conditions in a cell line model of ALS. We examined the uptake of [14C]L-citrulline in wild-type (hSOD1wt/WT) and mutant NSC-34/ SOD1G93A (MT) cell lines. The cell viability was determined via MTT assay. A transport study was performed to determine the uptake of [14C]L-citrulline. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis was performed to determine the expression levels of rat large neutral amino acid transported 1 (rLAT1) in ALS cell lines. Nitric oxide (NO) assay was performed using Griess reagent. L-Citrulline had a restorative effect on glutamate induced cell death, and increased [14C]L-citrulline uptake and mRNA levels of the large neutral amino acid transporter (LAT1) in the glutamate-treated ALS disease model (MT). NO levels increased significantly when MT cells were pretreated with glutamate for 24 h and restored by co-treatment with L-citrulline. Co-treatment of MT cells with L-arginine, an NO donor, increased NO levels. NSC-34 cells exposed to high glucose conditions showed a significant increase in [14C]L-citrulline uptake and LAT1 mRNA expression levels, which were restored to normal levels upon co-treatment with unlabeled L-citrulline. In contrast, exposure of the MT cell line to tumor necrosis factor alpha, lipopolysaccharides, and hypertonic condition decreased the uptake significantly which was restored to the normal level by co-treating with unlabeled L-citrulline. L-Citrulline can restore NO levels and cellular uptake in ALS-affected cells with glutamate cytotoxicity, pro-inflammatory cytokines, or other pathological states, suggesting that L-citrulline supplementation in ALS may play a key role in providing neuroprotection.

UNASSIGNED: 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.
UNASSIGNED: 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.
UNASSIGNED: 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.
UNASSIGNED: 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.