N-carbamylglutamate restores nitric oxide synthesis and attenuates high altitudeinduced pulmonary hypertension in Holstein heifers ascended to high altitude

Background: High-altitude pulmonary hypertension(HAPH) is a life-threating condition for animals in high altitude,and disturbance of endothelial nitric oxide(NO) synthesis contributes to its pathogenesis. N-carbamylglutamate(NCG), which enhances arginine synthesis, promotes endogenous synthesis of NO. In this study, we determined the effects of NCG on alleviating HAPH in Holstein heifers that ascended to Tibet(Lhasa, 3,658 m).Methods: Exp. 1, 2,000 Holstein heifers were transported from low elevation(1,027 m) to Lhasa. After being exposed to hypoxia for 1 yr, Holstein heifers were assigned to a healthy group(Control, n = 6) with mean pulmonary hypertension(mPAP) < 41 mmHg, and an HAPH affected group(HAPH, n = 6) with mPAP > 49 mmHg.Lung tissues were collected to evaluate histopathological changes and the expression of endothelial nitric oxide synthase(eNOS). Exp. 2, ten healthy heifers and 10 HAPH affected heifers were supplemented with NCG(20 g/d per heifer) for 4 wk. Physiological parameters were determined and blood samples were collected on d-1 and d 28 of the feeding trial.Results: Expression of e NOS in small pulmonary arteriole intima was higher in the healthy than HAPH group(P = 0.006), whereas HAPH group had significantly thicker media and adventitia than healthy group(all P < 0.05). The mRNA of eNOS and protein level of eNOS were higher in the lungs of heifers in the healthy group than in the HAPH group(both P < 0.001), whereas endothelin-1 protein levels were higher in HAPH group than in the healthy group(P = 0.025). NCG supplementation decreased mPAP and ammonia(both P = 0.001), whereas it increased the expression of eNOS, arginine, and plasma NO(all P < 0.05).Conclusions: The expression of eNOS was decreased in Holstein heifers with HAPH. NCG supplementation decreased m PAP through the restoration of eNOS and endogenous NO synthesis.

Synthesis, Crystal Structure and Spectroscopic Properties of Zn(Ⅱ) with N-carbamylglutamate Ligand

ZnC6HsN2O5H2O （NCGZn） has been synthesized, and its functional groups present in the title compound were confirmed by elemental analysis, TG and IR spectral studies. Meanwhile, the crystal of NCGZn was obtained by slow evaporation of a saturated aqueous solution at room temperature and confirmed by single-crystal X-ray diffraction analysis. It crystallizes in triclinic, space group P21 with a = 5.03220（1）, b = 13.3747（4）, c = 12.9944（4） A, β = 92.987（2）°, V= 873.39（4） A3, Z = 2, CI2H20NaOI2Zn2, Mr = 543.10, Dc = 2.065 g/cm3, F（000） = 552,μ（MoKa） = 2.826 mm-1, R = 0,0422 and wR = 0.1142. In NCGZn, Zn（l） and the symmetry formed Zn（l） ions are connected by five atoms from four NCG.2H anions and a water molecule. A NCG ligand has two carboxylate groups, one connecting a Zn ion, and the other bridgiong two Zn ions. The NCG ligand bridges four Zn ions through the amino group and two carboxylate groups with a water molecule to yield a 3D coordination polymer structure with hydrogen bonds.

Effect of N-Carbamylglutamate(NCG) on Growth Performance of ICR Mice

To understand the effects of self synthesized N-carbamylglutamate（NCG） on growth performance of ICR mice,mice were fed with the basal diet adding with 0.025%,0.05% and 0.1% NCG for 28 d.Effects of different levels of NCG on body weight of mice were observed.The re-sults showed that the addition of NCG in the feed could improve the growth performance of mice,and the growth-promoting effect enhanced with the increasing dose.

Synthesis, Crystal Structure and Spectroscopic Properties of Zn(II) with N-carbamylglutamate Ligand

ZnC6HsN2O5·H20 （NCGZn） has been synthesized, and its functional groups present in the title compound were confirmed by elemental analysis, TG and IR spectral studies. Meanwhile, the crystal of NCGZn was obtained by slow evaporation of a saturated aqueous solution at room temperature and confirmed by single-crystal X-ray diffraction analysis. It crystallizes in triclinic, space group P21 with α= 5.03220（1）, b = 13.3747（4）, c = 12.9944（4） A,β= 92.987（2）°, V= 873.39（4） A^3, Z = 2, C12H20N4O12Zn2, Mr = 543.10, Dc = 2.065 g/cm^3, F（000） = 552,μ（MoKα） = 2.826 mm^-1, R = 0.0422 and wR = 0.1142. In NCGZn, Znl and the symmetry formed Znl ions are connected by five atoms from four NCG-2H anions and a water molecule. A NCG ligand has two carboxylate groups, one connecting a Zn ion, and the other bridgiong two Zn ions. The NCG ligand bridges four Zn ions through the amino group and two carboxylate groups with a water molecule to yield a 3D coordination polymer structure with hydrogen bonds.

Dietary rumen-protected L-arginine or N-carbamylglutamate enhances placental amino acid transport and suppresses angiogenesis and steroid anabolism in underfed pregnant ewes

This study aimed to investigate the effects of dietary supplementation of underfed Hu ewes from d 35 to110 of gestation with either rumen-protected L-arginine(RP-Arg)or N-carbamylglutamate(NCG)on placental amino acid(AA)transport,angiogenic gene expression,and steroid anabolism.On d 35 of gestation,32 Hu ewes carrying twin fetuses were randomly divided into four treatment groups,each consisting of eight ewes,and were fed the following diets:A diet providing 100%of NRC’s nutrient requirements for pregnant ewes(CON);A diet providing 50%of NRC’s nutrient requirements for pregnant ewes(RES);RES diet plus 5 g/d NCG(RES+NCG);or RES diet plus 20 g/d RP-Arg(RES+ARG).On the d 110 of pregnancy,blood samples were taken from the mother,and samples were collected from type A cotyledons(COT;the fetal portions of the placenta).The levels of 17β-estradiol and progesterone in the maternal serum and both the capillary area density(CAD)and capillary surface density(CSD)in type A COT were decreased in response to Arg or NCG supplementation when compared to the RES group.The concentrations of arginine,leucine,putrescine and spermidine in type A COT were higher(P<0.05)in the RES+ARG or RES+NCG group than in the RES group.The mRNA expression levels of inducible nitric oxide synthase(iNOS)and solute carrier family 15,member 1(SLC15A1)were increased(P<0.05)while those of progesterone receptor(PGR)and fibroblast growth factor 2(FGF2)were decreased in type A COT by supplementation with either NCG or RP-Arg compared to the RES group.The results suggest that providing underfed pregnant ewes from d 35 to 110 of gestation with a diet supplemented with NCG or RP-Arg improves placental AA transport,and reduces the expression of angiogenic growth factor genes and steroid anabolism,leading to better fetal development.

Arginine, N-carbamylglutamate,and glutamine exert protective effects against oxidative stress in rat intestine

The objective of the current study is to evaluate the effects of dietary supplementation with arginine(ARG), N-carbamylglutamate(NCG), and glutamine(GLN) on rat intestinal morphology and antioxidant status under oxidative stress. Rats were fed for 30 d with one of the following iso-nitrogenous diets:basal diet(BD), BD plus 1% ARG, BD plus 0.1% NCG, and BD plus 1% GLN. On day 28, half of the rats fed BD were intraperitoneally injected with 12 mg/kg body weight of diquat(DT; i.e., the DT group) and the other half was intraperitoneally injected with sterile solution(i,e., the control group). The other diet groups were intraperitoneally injected with 12 mg/kg body weight of DT(i,e., DT + 1% GLN [DT + GLN],DT + 1% ARG [DT + ARG], and DT + 0.1% NCG [DT + NCG]). Rat jejunum samples obtained at 48 h after DT injection were analyzed. Results showed that DT significantly decreased catalase(CAT) activity and glutathione(GSH) content by 58.25% and 56.57%, respectively, and elevated malondialdehyde(MDA)content and crypt depth(CD) by 19.39% and 22.13%, respectively, in the jejunum(P < 0.05, relative to the control group). Compared with the DT group, the DT + GLN group exhibited significantly improved villus height(VH), villus width(VW), villus surface area(VSA), CD and total antioxidant capacity(T-AOC) activity(P < 0.05); the DT + ARG group exhibited significantly increased the ratio of VH to CD(H:D) and TAOC activity(P < 0.05); the DT + GLN, DT + ARG and DT + NCG groups exhibited significantly enhanced CAT activity and GSH content as well as decreased MDA content(P < 0.05). Moreover, VH, VW, VSA, CD and GSH content in the DT + GLN group were higher whereas MDA content was lower compared with the corresponding values observed in both the DT + ARG and the DT + NCG groups(P < 0.05). The H:D ratio in the DT + ARG group significantly increased compared with that in the DT + NCG and DT + GLN groups(P < 0.05). Collectively, this study suggested that dietary supplementation with 1% GLN, 0.1% NCG,and 1% ARG was effective in enhancing the antioxidant status and maintaining the morphological structure of rat jejunum under oxidative stress; of these supplements, 1% GLN exerted the greatest effects on mitigating oxidative stress.

Roles of dietary supplementation with arginine or N-carbamylglutamate in modulating the inflammation, antioxidant property, and m RNA expression of antioxidant-relative signaling molecules in the spleen of rats under oxidative stress

This study evaluated the effects of arginine(Arg) or N-carbamylglutamate(NCG) on inflammation, antioxidant property, and antioxidant-related gene expression in rat spleen under oxidative stress. A total of 52 rats were randomly distributed into 4 treatment groups with 13 replicates per group. Rats were fed a basal diet(BD) or BD supplemented with Arg or NCG for 30 days. On day 28, half of the BD-fed rats were intraperitoneally injected with sterile saline(control group), and the other half with 12 mg/kg body weight of diquat(DT; DT group). The other 2 diet groups were intraperitoneally injected with 12 mg/kg body weight of DT with either Arg(1%)(DT t Arg) or NCG(0.1%)(DT t NCG). Rat spleen samples were collected for analysis at 48 h after DT injection. Results showed that DT damaged the antioxidant defense in rats compared with the control group(P < 0.05). Compared with the DT group, the DT t Arg and DT t NCG groups manifested improved anti-hydroxyl radical, catalase, and total superoxide dismutase(T-SOD) activities, increased glutathione content(P < 0.05), and decreased malondialdehyde content(P < 0.05).Moreover, compared with the DT group, the DT t Arg and DT t NCG groups enhanced mRNA expression of superoxide dismutase(SOD), glutathione peroxidase 1(GPx1), glutathione reductase(GR), nuclear factor erythroid 2-related factor 2(Nrf2), Kelch-like ECH-associated protein 1(Keap-1), and mammalian target of rapamycin(m TOR)(P < 0.05). Both NCG and Arg significantly increased anti-inflammatory cytokine mRNA level but suppressed the pro-inflammatory cytokine mRNA expression under oxidative stress(P < 0.05). In summary, NCG and Arg effectively alleviated oxidative stress, improved the antioxidant capacity and regulated the antioxidant-related signaling molecular expression in rat spleen. N-carbamylglutamate and Arg reduced the inflammation in the spleen by mediating the gene expression of anti-inflammatory and pro-inflammatory cytokines and transforming growth factor-β(TGF-β).

Dietary rumen-protected L-arginine or N-carbamylglutamate attenuated fetal hepatic inflammation in undernourished ewes suffering from intrauterine growth restriction

This study aimed to explore whether dietary rumen-protected L-arginine(RP-Arg)or N-carbamylglutamate(NCG)supplementation to feed-restricted pregnant ewes counteracts fetal hepatic inflammation and innate immune dysfunction associated with intrauterine growth retardation(IUGR)in ovine fetuses.On d 35 of pregnancy,twin-bearing Hu ewes(n=32)were randomly assigned to 4 treatment groups(8 ewes and 16 fetuses per group)and fed diets containing 100%of the NRC requirements(CON),50%of the NRC requirements(RES),RES+RP-Arg(20 g/d)(RESA),or RES+NCG(5 g/d)(RESN).At 08:00 on d 110 of gestation,fetal blood and liver tissue samples were collected.The levels of triglyceride,free fatty acid,cholesterol andβ-hydroxybutyrate in the fetal blood of RESA and RESN groups were lower(P<0.05)than those of the RES group,but were higher(P<0.05)than those of the CON group.The interleukin(IL)-6 and IL-1 levels in fetal blood and liver tissue as well as the myeloid differentiation primary response 88(MyD88),transforming growth factorβ(TGFβ),and nuclear factor kappa B(NF-κB)mRNA levels in the fetal liver were decreased(P<0.05)by the NCG or RP-Arg supplementation compared to the RES treatment.Similarly,the toll-like receptor(TLR)-4,MyD88,TGFβ,and p-c-Jun N-terminal kinase(JNK)protein levels in the fetal liver were reduced(P<0.05)in the NCG and RP-Arg-supplemented groups compared to the RES group.These results showed that dietary supplementation of RP-Arg or NCG to underfed pregnant ewes could protect against IUGR fetal hepatic inflammation via improving lipid metabolism,down-regulating the TLR-4 and the inflammatory JNK and NF-icB signaling pathways,and decreasing cytokine production in ovine fetal blood and liver tissue.

Dietary N-carbamylglutamate and L-arginine supplementation improves redox status and suppresses apoptosis in the colon of intrauterine growth-retarded suckling lambs

Previous studies have revealed that dietary N-carbamylglutamate(NCG)or L-arginine(Arg)improves small intestinal integrity and immune function in suckling Hu lambs that have experienced intrauterine growth retardation(IUGR).Whether these nutrients alter redox status and apoptosis in the colon of IUGR lambs is still unknown.This study,therefore,aimed at investigating whether dietary supplementation of Arg or NCG alters colonic redox status,apoptosis and endoplasmic reticulum(ER)stress and the underlying mechanism of these alterations in IUGR suckling Hu lambs.Forty-eight 7-d old Hu lambs,including 12 with normal birth weight(4.25±0.14 kg)and 36 with IUGR(3.01±0.12 kg),were assigned to 4 treatment groups(n=12 each;6 males and 6 females)for 3 weeks.The treatment groups were control(CON),IUGR,IUGR+Arg and IUGR+NCG.Relative to IUGR lambs,superoxide dismutase(SOD)and glutathione peroxidase(GSH-Px)content,as well as proliferation index,were higher(P<0.05)whereas reactive oxygen species(ROS),malondialdehyde(MDA)levels and apoptotic cell numbers were lower(P<0.05)in colonic tissue for both IUGR+Arg and NCG lambs.Both m RNA and protein levels of C/EBP homologous protein 10(CHOP10),B-cell lymphoma/leukaemia 2(Bcl-2)-associated X protein(Bax),apoptosis antigen 1(Fas),activating transcription factor 6(ATF6),caspase 3,and glucose-regulated protein 78(GRP78)were lower(P<0.05)while glutathione peroxidase 1(GPx1),Bcl-2 and catalase(CAT)levels were higher(P<0.05)in colonic tissue for IUGR+Arg and IUGR+NCG lambs compared with IUGR lambs.Based on our results,dietary NCG or Arg supplementation can improve colonic redox status and suppress apoptosis via death receptor-dependent,mitochondrial and ER stress pathways in IUGR suckling lambs.

Effectiveness and safety evaluation of graded levels of N-carbamylglutamate in growing-finishing pigs

The aim of this study was as follows:1)to investigate the effects of graded levels of N-carbamylglutamate(NCG)on performance,blood biochemical indexes,carcass traits and related indicators in growingfinishing pigs,and 2)to determine the optimal supplemental level.The toxicity of high-dose(much higher than recommended levels)NCG was assessed by routine blood tests and blood biochemical and histopathologic examinations of the heart,liver,spleen,lung,kidney and stomach.One hundred and forty-four growing-finishing pigs(DurocLarge WhiteLandrace,32.24±1.03 kg)were used in a 74-d experiment and each treatment was replicated 6 times with 4 pigs(2 barrows and 2 gilts)per replicate.The dietary treatments were a corn-soybean meal basal diet supplemented with 0%(control),0.05%,0.1%,0.15%,0.2%or 1%NCG.The first 5 groups were used to explore the optimal supplemental level of NCG,while the control,0.1%and 1%NCG groups were used to explore the safety of high-dose NCG.Compared with the normal control group,the final body weight and average daily gain tended to be higher in the 0.1%group(P=0.08),the lean percentage tended to be higher in the 0.05%group(P=0.07),the levels of free amino acids in the blood significantly increased in the 0.1%group(P<0.05),both 0.1%and 0.15%NCG supplementation increased the levels of nitric oxide(NO)in serum(P=0.07)and muscle growth-and lipid metabolism-related gene expression(P<0.05)and NCG supplementation improved C18:1N9C monounsaturated fatty acids(MUFA)in a dose-dependent manner(P=0.08).In addition,routine blood tests,blood biochemical indexes and histopathological examination revealed no abnormalities.Overall,increasing the levels of NCG did not linearly improve the above indicators;the 0.1%dose showed the best effect,and a high dose(1%)did not pose a toxicity risk.

Dietary N-carbamylglutamate or L-arginine improves fetal intestinal amino acid profiles during intrauterine growth restriction in undernourished ewes

Our previous studies demonstrated that prenatal in utero growth restriction impairs postnatal intestinal function.Thus,improving postpartal intestinal absorption capacity and growth by manipulating the maternal diet prepartum is of importance.This work was conducted to determine whether supplementation of N-carbamylglutamate(NCG)or rumen-protected L-arginine(RP-Arg)increased fetal intestinal amino acid(AA)profiles in intrauterine growth retardation(IUGR)fetuses.On d 35 of gestation,Hu ewes(n=32)carrying twin fetuses were randomized into 4 groups(8 ewes and 16 fetuses in each group),where diets were as follows:100%of nutrient requirements recommended by National Research Council(NRC,2007)(CON);50%of nutrient requirements recommended by NRC(2007)(RES);RES+RPArg(20 g/d),(RES+ARG);and RES+NCG(5 g/d),(RES+NCG).On d 110 of gestation,both fetal and maternal tissues were collected and weighed.Compared with RES,solute carrier family 1,member 5(SLC1A5)was upregulated(P<0.05)within fetal jejunum,duodenum and ileum when supplementing NCG and RP-Arg.Relative to RES,RP-Arg or NCG supplementation to RES resulted in upregulation(P<0.05)of peptide transporter 1 protein abundance within the fetal ileum.NCG or RP-Arg supplementation to RES also upregulated phosphorylated mechanistic target of rapamycin(pmTOR)-to-mTOR ratio in the fetal ileum induced by IUGR(P<0.05).As a result,during IUGR,supplementation of Arg or NCG affected intestinal AA profiles in the fetus in part through controlling mTOR signal transduction as well as AA and peptide transport.Future studies should be conducted to understand the role(if any)of the placenta on the improvement of growth and AA profiles independent of the fetal intestine.This would help demonstrate the relative contribution of intestinal uptake in fetal life.

N-carbamoylglutamate improves lipid metabolism, inflammation, and apoptosis responses in visceral adipocytes of Japanese seabass (Lateolabrax japonicus), in vivo and in vitro

This study applied in vivo and in vitro methods to investigate the effect of dietary N-carbamoylglutamate(NCG)on lipid metabolism,inflammation and apoptosis related-gene expression in visceral adipose tissue and isolated adipocytes of Japanese seabass(Lateolabrax japonicus).A basal diet and a test diet supplemented with 720 mg/kg NCG were fed to the fish for 10 weeks.During the growth trial,no mortality and no significant differences in growth performance were observed in fish between the 2 groups(P>0.05).Plasma Arg content and mRNA level of argininosuccinate synthetase(ASS)in adipose tissue were significantly increased,which indicated that NCG inclusion promoted endogenous Arg synthesis.Thereafter,the potential effects of NCG treatment on lipid metabolism-related genes expression were studied through in vivo and in vitro methods.In the present study,we successfully established a primary adipocytes culture system and isolated pre-adipocytes in vitro of Japanese seabass for the first time.Both the results in vivo and in vitro showed that NCG treatment decreased the mRNA levels of genes related to adipogenesis(fatty acid synthase,FASN),cholesterol synthesis(3-hydroxy-3-methylglutaryl-CoA reductase,HMGCR)and fat deposition(lipoprotein lipase[LPL]and leptin),which revealed the underlying mechanism of NCG on reducing fat deposition.The results of this study demonstrated that NCG inclusion reduced the expression of inflammatory and apoptosis cytokines markedly in vivo and in vitro.In conclusion,NCG did exert beneficial effects on ameliorating adipo-genesis,inflammation and apoptosis via promoting Arg endogenous synthesis in Japanese seabass.

Dietary N-carbamylglutamate or L-arginine supplementation improves hepatic energy status and mitochondrial function and inhibits the AMP-activated protein kinase-peroxisome proliferator- activated receptor γ coactivator-1α-transcription factor A pathway in intrauterine-growth-retarded suckling lambs

The objective of this study was to investigate the effects of dietary administration of L-arginine(Arg)or N-carbamylglutamate(NCG)on hepatic energy status and mitochondrial functions in suckling Hu lambs with intrauterine growth retardation(IUGR).Forty-eight newborn Hu lambs of 7 d old were allocated into 4 treatment groups of 12 lambs each,in triplicate with 4 lambs per replicate(2 males and 2 females)as follows:CON(lambs of normal birth weight,4.25±0.14 kg),IUGR(3.01±0.12 kg),IUGR+1%Arg(2.99±0.13 kg),or IUGR+0.1%NCG(3.03±0.11 kg).The experiment lasted for 21 d,until d 28 after birth,and all lambs were fed milk replacer as a basal diet.Compared with IUGR lambs,NCG or Arg administration increased(P<0.05)the adenosine triphosphate(ATP)level and the activities of com-plexes I/III/IV,isocitrate dehydrogenase and citrate synthase in the liver.Compared with CON lambs,the relative mRNA levels of adenosine monophosphate-activated protein kinaseα1(AMPKα1),peroxisome proliferator-activated receptorγcoactivator-1α(PGC1α)and transcription factor A(TFAM)were increased(P<0.05)in the liver of IUGR lambs,but were decreased(P<0.05)in the liver of NCG-or Arg-treated lambs compared with those in the IUGR lambs.Compared with IUGR lambs,NCG or Arg administration decreased(P<0.05)the total AMPKα(tAMPKα)-to-phosphorylated AMPKα(pAMPKα)ratio and the protein expression of PGC1αand TFAM.The results suggested that dietary Arg or NCG supplements improved hepatic energy status and mitochondrial function and inhibited the AMPK-PGC1α-TFAM pathway in IUGR suckling lambs.