Research On The Mechanism Of L-arginine On The Regulation Of Feed Intake And Tissue Protein Metabolism In Laying Hens

In this study, we study the effects of dietary level of L-arginine (Arg) on feed intake, related regulatory factors and protein metabolismused, and usd an established chick primary intestinal epithelial cell (IEC) as experimental model, to investigate the effects of extracellular concentrations of L-Arg on Arg-uptake, metabolism and expression of related amino acid transporters as well as protein synthesis. The results found that L-Arg increased the expression of hormone-associated proteins in hypothalamus while decreased the expression of fatty acid oxidation associated proteins in the liver. Meanwhile, we found the law of gene expression related to tissue protein metabolism affected by dietary level of L-Arg. Besides, we found the cationic amino acid transporter-1 (CAT-1) plays a role in L-Arg uptake, and L-Arg-mediated elevation of nitric oxide (NO) via inducible nitric oxide synthase (iNOS) promotes the growth of IEC. The study reveals the molecular mechanism of L-Arg on tissue protein and to enrich the theoretical knowledge as well as provide new regulation technique ideas on feed intake of laying hens. Results were shown as below:1 Estimation of L-Arg requirement for Xinyang Black laying hens from 33 to 45 weeks of ageThe present study was conducted to estimatet dietary L-Arg requirement for the hybrid layers from 33 to 45 wk of age. Eight hundred and sixty-four 31-wk-old Xinyang Black commercial layers were randomly allotted to 6 treatments with 4 replicates of 36 birds. The dietary treatments were corn-corn gluten meal based diets containing 0.64,0.86,1.03,1.27,1.42 and 1.66%L-Arg, respectively. Feed intake, egg mass, laying rate and FCR of layers responded quadratically (P< 0.05) to the increasing of dietary L-Arg levels; albumen height in 1.27%L-Arg group was the highest(P< 0.05) compared with that in other groups, and 1.27% L-Arg group had higher (P< 0.05) Haugh units than that in 0.64% and 0.86% L-Arg groups. Based on quadratic broken-line regression analysis, the L-Arg requirement of Xinyang Black laying hens (33 to 45 wk) was 1.26% and 1.28% of the diet for feed intake and egg mass, respectively.2 Research on the regulation function of L-Arg in feed intake of layersBased on the previous feeding trial, this study was conducted to investigate the effects of dietary L-Arg levels on concentrations of neuropeptide Y (NPY) and leptin in hypothalamus as well as ghrelin, insulin and NO in serum. Meanwhile, the isotope tags for relative and abosulte quantitation (iTARQ) assay was applied to evaluate the effects of L-Arg on protein components in the liver and hypothalamus. Results showed that 1.27% L-Arg group had the highest concentration of NPY in hypothalamus and the lowest concentration of ghrelin in serum (P< 0.05). We identified a total of 3715 and 3797 proteins in hypothalamus and liver, respectively; whereby, there were 235 and 373 differential expressed proteins in hypothalamus and liver, respectively, between 0.64% and 1.27% L-Arg groups (P< 0.05). It was conclude that appropriate dietary level of L-Arg can increase the contents of NPY in hypothalamus and serum NO while decrease the contens of serum ghrelin to improve the feed intake of layers, and the underlying mechanism may be in related to the function of L-Arg that increased the expression of hormone-associated proteins in hypothalamus while decreased the expression of fatty acid oxidation associated proteins in the liver.3 Research on the regulation function of L-Arg in the tissue protein metabolism of layersAmino acids are considered to be anabolic factors that affect protein metabolism. The aim of this study was to test the effects of dietary L-Arg levels on protein metabolism in the liver and small intestine of laying hens and its underlying molecular mechanism. Results showed that serum concentrations of total protein and albumin were maximized in the 1.27% L-Arg group, and serum concentration of urea acid was the lowest in the 1.27% L-Arg group (P< 0.05). Compared with 0.64% L-Arg group,1.27% L-Arg group had the highest fractional protein synthesis rate in the liver and jejunum (P< 0.05). Consistent with the data on protein turnover, mRNA abundances of target of rapamycin (TOR) and ribosomal protein S6 kinase 1 (S6K1) increased in the liver of layers fed 1.27% L-Arg, while mRNA abundances of cathepsin B and 20S proteasome decreased at the same dietary L-Arg level (P< 0.05). The expression and phosphorylation levels of TOR protein in jejunum of 1.27% L-Arg group were higher than those in 0.64% L-Arg group (P< 0.05). In conclusion, the dietary level of L-Arg increased the liver fractional protein synthesis rate and fractional protein gain rate of laying hens, and the action of an appropriate level of dietary L-Arg involves upregulating the expression and phosphorylation of TOR protein accompanied by suppressing the mRNA expression of cathepsin B and 20S proteasome in the liver while improved the expression and phosphorylation levels of TOR protein as well as suppressed the mRNA expression of 20S proteasome in jejunum.4 Primary culture of chick intestinal epithelial cell in vitroThe present study evaluated the effects of embryonic age and proteolytic enzymes on the isolation and primary culture of chick epithelial cell (IEC) to establish an effective method for IEC cultivation.14,16 and 18 d embryos were the source for this experiment, and trypsin, collagenase (Type Ⅰ), thermolysin and combination of collagenase and thermolysin were used for digestion medium. Optimal culture protocols were determiened by qualitative assays of proliferation and immunohistochemistry. Results showed that IEC isolated by using 14 d embryo and collageanse obtain the best attachment and growth in culture, and the production of continuously growing IEC culture. Immunochemical staining and immunofluorescent assay found that the production of continuously growing IEC cultures which retain the ability to be positive to cytokeratin 18. Thus, we conclude that the use of collagenase as a dissociating enzyme and 14 d embryo as a source can be advantageously applied to the isolation of chick IEC and useful for various applications and basic studies of the intestinal tract concerning such objects as physiology, immunology and toxicology.5 Effects of L-Arg on the arginine-uptake, metabolism and related gene expression of chick IECThe present study was conducted to investigate the extracellular concentrations of L-Arg regulating the cationic amino acid transporter (CAT,1 and 4) and iNOS in chick IEC. The cells were cultured for 4 days in Arg-free Dulbecco’s modified Eagle’s medium containing 10,100,200,400, or 600 uM Arg. Cell viability, nitric NO concentrations, uptake and metabolism of L-[3H]-Arg as well as expression of CAT-1, CAT-4 and iNOS were determined. Results showed that L-Arg enhances cell growth with a maximal response at at 10-400 μM L-Arg. Addition of 100,200, or 400μM L-Arg increased the L-[3H]-Arg uptake, which was associated with greater conversion of L-[3H]-citrulline and NO production in comparison with 10 μM L-Arg group (P< 0.05). Increasing extracellular concentrations of L-Arg from 10 to 400 μM dose dependently increased the levels of CAT-1 mRNA and protein, while no effect on CAT-4 mRNA abundance was found. Furthermore, supplementation of 100,200, or 400 μM L-Arg upregulated the expression of iNOS mRNA (P< 0.05), and the relative protein levels for iNOS in 200 and 400 μM L-Arg groups were higher than those in 10 and 100 μM LArg groups (P< 0.05). Collectively, we conclude that the CAT-1 isoform plays a role in L-Arg uptake, and L-Arg-mediated elevation of NO via iNOS promotes the growth of chick IEC.6 Effects of L-Arg on protein metabolism and related gene expression of chick IECL-Arg is an indispensable amino acid in avians and is required for growth. The aim of this study was to investigate the effects of L-Arg on protein synthesis and genes expression involved in TOR signaling pathway in chick enterocytes. Cells were cultured for 4 days in L-Arg-free Dulbecco’s modified Eagle’s medium containing 10, 100,200,400, or 600 μM L-Arg. Cell growth, cell cycle, protein synthesis, and protein degradation as well as mRNA expression levels of TOR, S6K1, and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) were determined. The results showed that cell viability was enhanced by L-Arg with a maximal response at 10 to 400 uM. Increasing extracellular concentrations of L-Arg from 10 to 400 μM increased the cells in S and G2/M phase to a significant extent and decreased cell numbers in G0/G1 phase (P< 0.05). Further more, addition of 100,200, or 400μM L-Arg to culture medium increased protein synthesis and reduced protein degradation in chick IEC (P< 0.05). Consistent with the data on cell growth and protein turnover, supplementation of 100,200, or 400 μM L-Arg increased the mRNA abundances of TOR,4E-BP1, and S6K1(P<0.05). It was concluded that the action of L-Arg involves in upregulating the genes expression of TOR cell signaling pathway which increases protein synthesis and reduces protein degradation.