摘要
精氨酸作为哺乳动物的一种半必需氨基酸,其生物学功能已经超过了其在动物体内的营养价值,在动物营养代谢的调控,和生长发育过程中发挥着重要作用。日粮精氨酸能够促进仔猪生长,促进肠的发育和VEGF基因的表达。但是精氨酸转运情况及转运载体如何影响精氨酸对细胞的生长调控作用?L-Arg-NO代谢途径在调控细胞生长增殖过程中的作用如何?转运载体与调控细胞生长的mTOR信号通路之间有何联系?还不是很清楚。
为此,我们利用IPEC-1细胞模型,通过在含不同浓度精氨酸的DMEM-F12培养基中培养,对IPEC-1细胞的生长、增殖、凋亡以及相关信号通路进行探讨,同时利用L-NAME抑制NOS,探讨Arg-NO途径在精氨酸调控细胞增殖生长中的作用,以期从分子生物学的角度解析精氨酸调控猪肠上皮细胞生长增殖的的分子机制,进而为精氨酸在猪生长过程中的研究奠定基础。研究结果发现,(1)肠上皮细胞培养基中添加350gM精氨酸上调了AKR1C4、PTGFR、GHRL、ITGB2、IL15等306个基因,而下调了SLC25A25、COL4A3、C7、TLR1等208个基因。进行KEGG Pathway分析,精氨酸调控的基因变化多分布在与细胞粘附分子、Ⅰ型糖尿病、细胞通讯、钙信号等通路。(2)添加精氨酸对细胞精氨酸转运率无影响,但提高了转运载体y+LAT1和y+CAT1的基因和蛋白表达水平；利用L-NAME抑制NO代谢途径抑制了转运载体y+LAT1的表达,但对精氨酸转运率和转运载体y+CAT1和y+CAT2表达影响不显著。(3)添加精氨酸促进了细胞增殖、抑制凋亡,S期细胞数量提高,抑制精氨酸的NO代谢途径,能抑制细胞增殖,提高细胞的凋亡率；添加精氨酸促进了NO的合成和eNOS的表达,而L-NAME显著降低了NO的合成和eNOS的表达；添加精氨酸显著提高了p-mTOR、 P-4EBP1、p-p70S6K、p-PI3K、Akt、p-Akt和Bcl-2蛋白表达水平,而L-NAME发挥一定的抑制作用。
以上结果提示,精氨酸能通过mTOR信号通路促进细胞生长增殖,通过PI3K-Akt-Bc12途径调控细胞周期和凋亡；NOS抑制剂L-NAME对肠上皮细胞精氨酸转运不产生竞争性抑制作用,但能抑制mTOR信号通路和促进PI3K-Akt-Bcl2途径调控细胞生长增殖,说明NO代谢途径在精氨酸调控肠上皮细胞增殖生长中发挥重要作用。这些研究结果为精氨酸在仔猪营养的应用提供有效的理论依据,并为利用精氨酸在治疗人类肠道疾病提供参考。
Arginine is a semi-essential amino acid that serve as an important role in diverse physiological function ranging from regulation of animal nutrition metabolism to the process of growth and development. Dietary arginine can promote the growth of piglets as well as the development of intestine. It can also improve the expression of VEGF gene. However, it is not clear that the regulatory mechanism of arginine transporter in the regulation of cell growth, the effect of L-Arg-NO pathway in cell proliferation and the connection between arginine transporter and mTOR signaling pathway.
Therefore, we used a cell model of IPEC-1cells which were grown in DMEM-F12containing different concentrations of arginine, to investigate IPEC-1cell growth, proliferation, apoptosis and related signaling pathway. Meanwhile, we have inhibited NOS in using L-NAME to discuss the role of Arg-NO pathway in cell growth and proliferation. The study was designed to elucidate the molecular mechanism of the regulations of arginine in intestinal porcine epithelial cells growth and proliferation, and then to lay the foundation of the research of arginine functions in pig. The results shows that (1)306genes like AKR1C4、PTGFR、GHRL、ITGB2、 IL15were up-regulation while208genes like SLC25A25、COL4A3、C7、TLR1were down-regulation in supplementation of350μM Arg. Through KEGG Pathway analysis, genetic changes of arginine regulation were mostly found in pathways associated with cell adhesion molecules, type I diabetes, cell communication, calcium signaling pathway.(2)Supplementation of arginine had no effect on cell arginine transfer rate, but increased gene and protein levels of y+LAT1and y+CAT1. Addition of L-NAME to suppress NO metabolic pathway had no difference on arginine transfer rate and the levels of y+CAT1and y+CAT2, but reduced the level of y+LAT1.(3) Supplementation of arginine improved cell proliferation, reduced the cell apoptosis, increased the cell number at S phase, while inhibition of NO pathway decreased cell prolifeartion, increased the cell apopotosis. Compared with arginine, L-NAME significantly decreased NO synthesis and expression of eNOS, Arginine significantly increased relative protein levels of p-mTOR、p-4EBP1、p-p70S6K、p-PI3K、Akt、 p-Akt and Bcl-2, yet L-NAME had inhibitory effect on above proteins.
In conclusion, these findings indicate that arginine improves cell growth and proliferation through mTOR signaling pathway, while regulates cell cycle and apoptosis through PI3K-Akt-Bcl2pathway. L-NAME, as a NOS inhibitor, has no competitive inhibition with arginine transporters of intestinal porcine epithelial cells, but can regulate cell growth and proliferation by inhibiting mTOR pathway and promoting PI3K-Akt-Bcl2pathway. NO metabolic pathway plays a great important role in arginine regulation of intestinal porcine epithelial cell growth and proliferation. These results provide effective theoretical basis of arginine application in piglets nutrition and also provide reference for using arginine to remedy human intestinal disease.
引文
1. 崔天盆,胡必成,Ghrelin活化人T细胞翻译起始分子通过mTOR信号转导通路[J].中国免疫学杂志,2010,(3).
2. 郑杰,mTOR信号途径与肿瘤[J].生命科学,2006,18(3)：261-263.
3. 褚武英,印遇龙,猪肠道精氨酸转运载体的研究进展[J].饲料研究,2008,(5).
4. 曾黎明,谭碧娥,肖昊,印遇龙,卢向阳,方俊.氨基酸转运载体介导的氨基酸感应信号研究进展[J].中国科学：生命科学,2012,09：699-708.
5. Abdelmagid, S. A., J. A. Rickard, et al. CAT-1-mediated arginine uptake and regulation of nitric oxide synthases for the survival of human breast cancer cell lines[J]. J Cell Biochem, 2011,112(4):1084-1092.
6. Achike, F. I. The L-arginine-nitric oxide pathway:a potential therapeutic target in dengue haemorrhagic fever[J]. Clin Exp Pharmacol Physiol,2008,35(10):1135-1136.
7. Adams, M. R., J. Robinson, et al. Smooth muscle dysfunction occurs independently of impaired endothelium-dependent dilation in adults at risk of atherosclerosis[J]. J Am Coll Cardiol,1998,32(1):123-127.
8. Ahn, C. S., J. A. Han, et al. The PP2A regulatory subunit Tap46, a component of the TOR signaling pathway, modulates growth and metabolism in plants [J]. Plant Cell,2011,23(1): 185-209.
9. Aicher, A., C. Heeschen, et al. The role of NOS3 in stem cell mobilization[J]. Trends Mol Med,2004,10(9):421-425.
10. Anderson, C. M., D. S. Grenade, et al. H+/amino acid transporter 1 (PAT1) is the imino acid carrier:An intestinal nutrient/drug transporter in human and rat[J]. Gastroenterology,2004, 127(5):1410-1422.
11. Becker, T., I. Mevius, et al. The L-arginine/NO pathway in end-stage liver disease and during orthotopic liver and kidney transplantation:biological and analytical ramifications[J]. Nitric Oxide,2009,20(1):61-67.
12. Belhassen, L., R. A. Kelly, et al. Nitric oxide synthase (NOS3) and contractile responsiveness to adrenergic and cholinergic agonists in the heart. Regulation of NOS3 transcription in vitro and in vivo by cyclic adenosine monophosphate in rat cardiac myocytes[J]. J Clin Invest,1996,97(8):1908-1915.
13. Brasse-Lagnel, C. G., A. M. Lavoinne, et al. Amino acid regulation of mammalian gene expression in the intestine[J]. Biochimie,2010,92(7):729-735.
14. Broer, A., B. Friedrich, et al. Association of 4F2hc with light chains LAT1, LAT2 or y+LAT2 requires different domains[J]. Biochem J,2001,355(Pt 3):725-731.
15. Broer, A., C. A. Wagner, et al. The heterodimeric amino acid transporter 4F2hc/y+LAT2 mediates arginine efflux in exchange with glutamine[J]. Biochem J,2000,349 Pt 3: 787-795.
16. Brozic, P., S. Turk, et al. Inhibitors of aldo-keto reductases AKR1C1-AKR1C4[J]. Curr Med Chem,2011,18(17):2554-2565.
17. Bryk, B., K. Hahn, et al. MAP4K3 regulates body size and metabolism in Drosophila[J]. Dev Biol,2010,344(1):150-157.
18. Chen, C. C., J. J. Lee, et al. Platonin attenuates LPS-induced CAT-2 and CAT-2B induction in stimulated murine macrophages[J]. Acta Anaesthesiol Scand,2006,50(5):604-612.
19. Chicoine, L. G, M. R. Stenger, et al. Nitric oxide suppression of cellular proliferation depends on cationic amino acid transporter activity in cytokine-stimulated pulmonary endothelial cells[J]. Am J Physiol Lung Cell Mol Physiol,2011,300(4):L596-604.
20. Christie, G. R., E. Hajduch, et al. Intracellular sensing of amino acids in Xenopus laevis oocytes stimulates p70 S6 kinase in a target of rapamycin-dependent manner[J]. J Biol Chem,2002,277(12):9952-9957.
21. Crino, P. B. mTOR:A pathogenic signaling pathway in developmental brain malformations[J]. Trends Mol Med,2011,17(12):734-742.
22. Cui, H., B. Chen, et al. Overexpression of cationic amino acid transporter-1 increases nitric oxide production in hypoxic human pulmonary microvascular endothelial cells[J]. Clin Exp Pharmacol Physiol,2011,38(12):796-803.
23. Czarnik, U. and S. Kaminski. Detection of intronic sequence in the bovine ITGB2 gene[J]. Anim Genet,1997,28(4):320-321.
24. D'Antona, G and E. Nisoli. mTOR signaling as a target of amino acid treatment of the age-related sarcopenia[J]. Interdiscip Top Gerontol,2010,37:115-141.
25. Dantas, V. G, L. Furtado-Alle, et al. Obesity and variants of the GHRL (ghrelin) and BCHE (butyrylcholinesterase) genes[J]. Genet Mol Biol,2011,34(2):205-207.
26. Edinger, A. L. Growth factors regulate cell survival by controlling nutrient transporter expression[J]. Biochem Soc Trans,2005,33(Pt 1):225-227.
27. Edinger, A. L. Controlling cell growth and survival through regulated nutrient transporter expression[J]. Biochem J,2007,406(1):1-12.
28. Ettl, T., S. Schwarz-Furlan, et al. The PI3K/AKT/mTOR signalling pathway is active in salivary gland cancer and implies different functions and prognoses depending on cell localisation[J]. Oral Oncol,2012,48(9):822-830.
29. Evans, K., Z. Nasim, et al. Acidosis-sensing glutamine pump SNAT2 determines amino acid levels and mammalian target of rapamycin signalling to protein synthesis in L6 muscle cells[J]. J Am Soc Nephrol,2007,18(5):1426-1436.
30. Fan, X., D. D. Ross, et al. Impact of system L amino acid transporter 1 (LAT1) on proliferation of human ovarian cancer cells:a possible target for combination therapy with anti-proliferative aminopeptidase inhibitors[J]. Biochem Pharmacol,2010,80(6):811-818.
31. Findlay, G. M., L. Yan, et al. A MAP4 kinase related to Ste20 is a nutrient-sensitive regulator of mTOR signalling[J]. Biochem J,2007,403(1):13-20.
32. Fleissner, F. and T. Thum. Critical role of the nitric oxide/reactive oxygen species balance in endothelial progenitor dysfunction[J]. Antioxid Redox Signal,2011,15(4):933-948.
33. Foltz, M., M. Boll, et al. A novel bifunctionality:PAT1 and PAT2 mediate electrogenic proton/amino acid and electroneutral proton/fatty acid symport[J]. FASEB J,2004,18(14): 1758-1760.
34. Frey, I. M., I. Rubio-Aliaga, et al. Profiling at mRNA, protein, and metabolite levels reveals alterations in renal amino acid handling and glutathione metabolism in kidney tissue of Pept2-/-mice[J]. Physiol Genomics,2007,28(3):301-310.
35. Fuchs, B. C. and B. P. Bode. Amino acid transporters ASCT2 and LAT1 in cancer:partners in crime?[J]. Semin Cancer Biol,2005,15(4):254-266.
36. Galavotti, S., S. Bartesaghi, et al. The autophagy-associated factors DRAM1 and p62 regulate cell migration and invasion in glioblastoma stem cells[J]. Oncogene,2012.
37. Gallinetti, J., E. Harputlugil, et al. Amino acid sensing in dietary-restriction-mediated longevity:roles of signal-transducing kinases GCN2 and TOR[J]. Biochem J,2013,449(1): 1-10.
38. Garcia-Maceira, P. and J. Mateo. Silibinin inhibits hypoxia-inducible factor-1 alpha and mTOR/p70S6K/4E-BP1 signalling pathway in human cervical and hepatoma cancer cells: implications for anticancer therapy [J]. Oncogene,2009,28(3):313-324.
39. Gaskin, F. S., S. A. Farr, et al. Ghrelin-induced feeding is dependent on nitric oxide[J]. Peptides,2003,24(6):913-918.
40. Ghayad, S. E. and P. A. Cohen. Inhibitors of the PI3K/Akt/mTOR pathway:new hope for breast cancer patients[J]. Recent Pat Anticancer Drug Discov,2010,5(1):29-57.
41. Ghishan, F. K., S. M. Knobel, et al. Molecular cloning, sequencing, chromosomal localization, and tissue distribution of the human Na+/H+ exchanger (SLC9A2)[J]. Genomics,1995,30(1):25-30.
42. Gulati, P., L. D. Gaspers, et al. Amino acids activate mTOR complex 1 via Ca2+/CaM signaling to hVps34[J]. Cell Metab,2008,7(5):456-465.
43. Hammermann, R., G. Brunn, et al. Analysis of the genomic organization of the human cationic amino acid transporters CAT-1, CAT-2 and CAT-4[J]. Amino Acids,2001,21(2): 211-219.
44. Harwood, F. C., L. Shu, et al. mTORC1 signaling can regulate growth factor activation of p44/42 mitogen-activated protein kinases through protein phosphatase 2A[J]. J Biol Chem, 2008,283(5):2575-2585.
45. Hatzoglou, M., J. Fernandez, et al. Regulation of cationic amino acid transport:the story of the CAT-1 transporter[J]. Annu Rev Nutr,2004,24:377-399.
46. Heublein, S., S. Kazi, et al. Proton-assisted amino-acid transporters are conserved regulators of proliferation and amino-acid-dependent mTORC1 activation[J]. Oncogene,2010,29(28): 4068-4079.
47. Hoang, B., A. Benavides, et al. The PP242 mammalian target of rapamycin (mTOR) inhibitor activates extracellular signal-regulated kinase (ERK) in multiple myeloma cells via a target of rapamycin complex 1 (TORC1)/eukaryotic translation initiation factor 4E (eIF-4E)/RAF pathway and activation is a mechanism of resistance[J]. J Biol Chem,2012, 287(26):21796-21805.
48. Hou, G., L. Xue, et al. An activated mTOR/p70S6K signaling pathway in esophageal squamous cell carcinoma cell lines and inhibition of the pathway by rapamycin and siRNA against mTOR[J]. Cancer Lett,2007,253(2):236-248.
49. Huang, C. J., P. S. Tsai, et al. Pulmonary transcription of CAT-2 and CAT-2B but not CAT-1 and CAT-2A were upregulated in hemorrhagic shock rats[J]. Resuscitation,2004,63(2): 203-212.
50. Huang, Q., C. Huang, et al. LPS-stimulated RAW264.7 macrophage CAT-2-mediated 1-arginine uptake and nitric oxide biosynthesis is inhibited by omega fatty acid lipid emulsion[J]. J Surg Res,2012.
51. Huang, Y. H., P. S. Tsai, et al. Lidocaine inhibition of inducible nitric oxide synthase and cationic amino acid transporter-2 transcription in activated murine macrophages may involve voltage-sensitive Na+ channel[J]. Anesth Analg,2006,102(6):1739-1744.
52. Hultstrom, M., F. Helle, et al. AT(1) receptor activation regulates the mRNA expression of CAT1, CAT2, arginase-1, and DDAH2 in preglomerular vessels from angiotensin Ⅱ hypertensive rats[J]. Am J Physiol Renal Physiol,2009,297(1):F163-168.
53. Hundal, H. S. and P. M. Taylor. Amino acid transceptors:gate keepers of nutrient exchange and regulators of nutrient signaling[J]. Am J Physiol Endocrinol Metab,2009,296(4): E603-613.
54. Hyde, R., P. M. Taylor, et al. Amino acid transporters:roles in amino acid sensing and signalling in animal cells [J]. Biochem J,2003,373(Pt 1):1-18.
55. Inoki, K., J. Kim, et al. AMPK and mTOR in cellular energy homeostasis and drug targets[J]. Annu Rev Pharmacol Toxicol,2012,52:381-400.
56. Inoki, K., H. Mori, et al. mTORC1 activation in podocytes is a critical step in the development of diabetic nephropathy in mice[J]. J Clin Invest,2011,121(6):2181-2196.
57. Ip, C. K. and A. S. Wong. Exploiting p70 S6 kinase as a target for ovarian cancer[J]. Expert Opin Ther Targets,2012,16(6):619-630.
58. Ivanis, G, A. J. Esbaugh, et al. Branchial expression and localization of SLC9A2 and SLC9A3 sodium/hydrogen exchangers and their possible role in acid-base regulation in freshwater rainbow trout (Oncorhynchus mykiss)[J]. J Exp Biol,2008,211 (Pt 15): 2467-2477.
59. Izycka-Swieszewska, E., E. Drozynska, et al. Analysis of PI3K/AKT/mTOR signalling pathway in high risk neuroblastic tumours[J]. Pol J Pathol,2010,61(4):192-198.
60. Jegg, A. M., T. M. Ward, et al. PI3K independent activation of mTORC1 as a target in lapatinib-resistant ERBB2+ breast cancer cells[J]. Breast Cancer Res Treat,2012,136(3): 683-692.
61. Kagawa, S., S. Takano, et al. Akt/mTOR signaling pathway is crucial for gemcitabine resistance induced by Annexin Ⅱ in pancreatic cancer cells[J]. J Surg Res,2012.
62. Kaira, K., N. Oriuchi, et al. L-type amino acid transporter 1 (LAT1) expression in malignant pleural mesothelioma[J]. Anticancer Res,2011,31(12):4075-4082.
63. Kaira, K., N. Oriuchi, et al. LAT1 expression is closely associated with hypoxic markers and mTOR in resected non-small cell lung cancer[J]. Am J Transl Res,2011,3(5):468-478.
64. Kaira, K., T. Takahashi, et al. Relationship between LAT1 expression and response to platinum-based chemotherapy in non-small cell lung cancer patients with postoperative recurrence[J]. Anticancer Res,2011,31(11):3775-3782.
65. Kempisty, B., M. Jackowska, et al. Zona pellucida glycoprotein 3 (pZP3) and integrin beta2 (ITGB2) mRNA and protein expression in porcine oocytes after single and double exposure to brilliant cresyl blue test[J]. Theriogenology,2011,75(8):1525-1535.
66. Khan, M., S. Meduru, et al. Oxygen cycling in conjunction with stem cell transplantation induces NOS3 expression leading to attenuation of fibrosis and improved cardiac function[J]. Cardiovasc Res,2012,93(1):89-99.
67. Kim, J. W., E. I. Closs, et al. Transport of cationic amino acids by the mouse ecotropic retrovirus receptor[J]. Nature,1991,352(6337):725-728.
68. Kim, S. G, Y. C. Ahn, et al. Expression of amino acid transporter LAT1 and 4F2hc in the healing process after the implantation of a tooth ash and plaster of Paris mixture[J]. In Vivo, 2006,20(5):591-597.
69. Kim, S., S. F. Kim, et al. Amino acid signaling to mTOR mediated by inositol polyphosphate multikinase[J]. Cell Metab,2011,13(2):215-221.
70. Kimura, T., T. Yokoyama, et al. NOS3 genotype-dependent correlation between blood pressure and physical activity[J]. Hypertension,2003,41(2):355-360.
71. Kong, X., B. Tan, et al.1-Arginine stimulates the mTOR signaling pathway and protein synthesis in porcine trophectoderm cells[J]. J Nutr Biochem,2012,23(9):1178-1183.
72. Lang, C. H. and R. A. Frost. Endotoxin disrupts the leucine-signaling pathway involving phosphorylation of mTOR,4E-BP1, and S6K1 in skeletal muscle[J]. J Cell Physiol,2005, 203(1):144-155.
73. Leise, B. S., M. Watts, et al. Laminar Regulation of STAT1 and STAT3 in Black Walnut Extract and Carbohydrate Overload Induced Models of Laminitis[J]. J Vet Intern Med,2012, 26(4):996-1004.
74. Leoncini, G, D. Bruzzese, et al. The L-arginine/NO pathway in the early phases of platelet stimulation by collagen[J]. Biochem Pharmacol,2005,69(2):289-296.
75. Li, W., D. Tan, et al. Activation of Akt-mTOR-p70S6K pathway in angiogenesis in hepatocellular carcinoma[J]. Oncol Rep,2008,20(4):713-719.
76. Liu, M. C., P. S. Tsai, et al. Propofol significantly attenuates iNOS, CAT-2, and CAT-2B transcription in lipopolysaccharide-stimulated murine macrophages[J]. Acta Anaesthesiol Taiwan,2006,44(2):73-81.
77. Loewith, R., E. Jacinto, et al. Two TOR complexes, only one of which is rapamycin sensitive, have distinct roles in cell growth control[J]. Mol Cell,2002,10(3):457-468.
78. Lynch, C. J. Role of leucine in the regulation of mTOR by amino acids:revelations from structure-activity studies[J]. J Nutr,2001,131(3):861S-865S.
79. Marnef, A. and N. Standart. Patl proteins:a life in translation, translation repression and mRNA decay[J]. Biochem Soc Trans,2010,38(6):1602-1607.
80. Matsuura, C., T. M. Brunini, et al. Upregulation of platelet (L)-arginine:nitric oxide pathway after exercise training in hypertension[J]. Can J Physiol Pharmacol,2012,90(4): 501-505.
81. McDonald, K. K., S. Zharikov, et al. A caveolar complex between the cationic amino acid transporter 1 and endothelial nitric-oxide synthase may explain the "arginine paradox"[J]. J Biol Chem,1997,272(50):31213-31216.
82. Medeiros, C., M. J. Frederico, et al. Exercise training reduces insulin resistance and upregulates the mTOR/p70S6k pathway in cardiac muscle of diet-induced obesity rats[J]. J Cell Physiol,2011,226(3):666-674.
83. Miguel-Aliaga, I. Nerveless and gutsy:intestinal nutrient sensing from invertebrates to humans[J]. Semin Cell Dev Biol,2012,23(6):614-620.
84. Murai, A., S. Abou Asa, et al. Immunohistochemical Analysis of the Akt/mTOR/4E-BP1 Signalling Pathway in Canine Haemangiomas and Haemangiosarcomas[J]. J Comp Pathol, 2012.
85. Nakayama, K., K. W. Kim, et al. A novel nuclear zinc finger protein EZI enhances nuclear retention and transactivation of STAT3[J]. EMBO J,2002,21(22):6174-6184.
86. Nicklin, P., P. Bergman, et al. Bidirectional transport of amino acids regulates mTOR and autophagy[J]. Cell,2009,136(3):521-534.
87. Ogonowski, A. A., W. H. Kaesemeyer, et al. Effects of NO donors and synthase agonists on endothelial cell uptake of L-Arg and superoxide production[J]. Am J Physiol Cell Physiol, 2000,278(1):C136-143.
88. Pan, M., H. A. Choudry, et al. Arginine transport in catabolic disease states[J]. J Nutr,2004, 134(10 Suppl):2826S-2829S; discussion 2853S.
89. Penning, T. M., M. E. Burczynski, et al. Human 3alpha-hydroxysteroid dehydrogenase isoforms (AKR1C1-AKR1C4) of the aldo-keto reductase superfamily:functional plasticity and tissue distribution reveals roles in the inactivation and formation of male and female sex hormones[J]. Biochem J,2000,351(Pt 1):67-77.
90. Piraud, M., S. Ruet, et al. Amino acid profiling for the diagnosis of inborn errors of metabolism[J]. Methods Mol Biol,2011,708:25-53.
91. Pokall, S., A. R. Maldonado, et al. Compensatory lung growth in NOS3 knockout mice suggests synthase isoform redundancy[J]. Eur J Pediatr Surg,2012,22(2):148-156.
92. Populo, H., J. M. Lopes, et al. The mTOR Signalling Pathway in Human Cancer[J]. Int J Mol Sci,2012,13(2):1886-1918.
93. Prasad, P. D., H. Wang, et al. Human LAT1, a subunit of system L amino acid transporter: molecular cloning and transport function[J]. Biochem Biophys Res Commun,1999,255(2): 283-288.
94. Presneau, N., A. Shalaby, et al. Potential therapeutic targets for chordoma: PI3K/AKT/TSC1/TSC2/mTOR pathway[J]. Br J Cancer,2009,100(9):1406-1414.
95. Puli, S., A. Jain, et al. Effect of combination treatment of rapamycin and isoflavones on mTOR pathway in human glioblastoma (U87) cells[J]. Neurochem Res,2010,.35(7): 986-993.
96. Rafikov, R., F. V. Fonseca, et al. eNOS activation and NO function:structural motifs responsible for the posttranslational control of endothelial nitric oxide synthase activity[J]. J Endocrinol,2011,210(3):271-284.
97. Resnik-Docampo, M. and J. F. de Celis. MAP4K3 is a component of the TORC1 signalling complex that modulates cell growth and viability in Drosophila melanogaster[J]. PLoS One, 2011,6(1):e14528.
98. Rotmann, A., A. Simon, et al. Activation of classical protein kinase C decreases transport via systems y+ and y+L[J]. Am J Physiol Cell Physiol,2007,292(6):C2259-2268.
99. Rotoli, B. M., O. Bussolati, et al. The transport of cationic amino acids in human airway cells:expression of system y+L activity and transepithelial delivery of NOS inhibitors [J]. FASEB J,2005,19(7):810-812.
100. Rotoli, B. M., E. I. Closs, et al. Arginine transport in human erythroid cells:discrimination of CAT1 and 4F2hc/y+LAT2 roles[J]. Pflugers Arch,2009,458(6):1163-1173.
101. Rotoli, B. M., V. Dall'asta, et al. Alveolar macrophages from normal subjects lack the NOS-related system y+ for arginine transport[J]. Am J Respir Cell Mol Biol,2007,37(1): 105-112.
102.Rubio-Aliaga, I., M. Boll, et al. The proton/amino acid cotransporter PAT2 is expressed in neurons with a different subcellular localization than its paralog PAT1[J]. J Biol Chem,2004, 279(4):2754-2760.
103.Sancak, Y, T. R. Peterson, et al. The Rag GTPases bind raptor and mediate amino acid signaling to mTORC1[J]. Science,2008,320(5882):1496-1501.
104. Sarchielli, P., A. Alberti, et al. L-Arginine/nitric oxide pathway in chronic tension-type headache:relation with serotonin content and secretion and glutamate content[J]. J Neurol Sci,2002,198(1-2):9-15.
105. Schwartz, I. F., D. Schwartz, et al. L-Arginine transport is augmented through up-regulation of tubular CAT-2 mRNA in ischemic acute renal failure in rats[J]. Kidney Int,2002,62(5): 1700-1706.
106. Shankarishan, P., P. K. Borah, et al. Prevalence of endothelial nitric oxide synthase (eNOS) gene exon 7 Glu298Asp variant in North Eastern India[J]. Indian J Med Res,2011,133(5): 487-491.
107.Sharma, D., S. Kumar, et al. Identification of novel allelic variants of integrin beta 2 (ITGB2) gene and screening for Bubaline leukocyte adhesion deficiency syndrome in Indian water buffaloes (Bubalus bubalis)[J]. Anim Biotechnol,2009,20(3):156-160.
108. Shennan, D. B., D. T. Calvert, et al. A study of L-leucine, L-phenylalanine and L-alanine transport in the perfused rat mammary gland:possible involvement of LAT1 and LAT2[J]. Biochim Biophys Acta,2002,1564(1):133-139.
109.Shichi, D., E. F. Kikkawa, et al. The haplotype block, NFKBIL1-ATP6V1G2-BAT1-MICB-MICA, within the class Ⅲ-class Ⅰ boundary region of the human major histocompatibility complex may control susceptibility to hepatitis C virus-associated dilated cardiomyopathy[J]. Tissue Antigens,2005,66(3):200-208.
110. Sosunov, A. A., X. Wu, et al. The mTOR pathway is activated in glial cells in mesial temporal sclerosis[J]. Epilepsia,2012,53 Suppl 1:78-86.
111. Stayrook, K. R., P. M. Rogers, et al. Regulation of human 3 alpha-hydroxysteroid dehydrogenase (AKR1C4) expression by the liver X receptor alpha[J]. Mol Pharmacol, 2008,73(2):607-612.
112. Suarez, E., A. Burguete, et al. Microarray data analysis for differential expression:a tutorial[J]. P R Health Sci J,2009,28(2):89-104.
113.Sudar, E., B. Dobutovic, et al. Regulation of inducible nitric oxide synthase activity/expression in rat hearts from ghrelin-treated rats[J]. J Physiol Biochem,2011,67(2): 195-204.
114. Sun, C. H., Y. H. Chang, et al. Activation of the PI3K/Akt/mTOR pathway correlates with tumour progression and reduced survival in patients with urothelial carcinoma of the urinary bladder[J]. Histopathology,2011,58(7):1054-1063.
115.Suryawan, A. and T. A. Davis. The abundance and activation of mTORC1 regulators in skeletal muscle of neonatal pigs are modulated by insulin, amino acids, and age[J]. J Appl Physiol,2010,109(5):1448-1454.
116. Suryawan, A., H. V. Nguyen, et al. Abundance of amino acid transporters involved in mTORC1 activation in skeletal muscle of neonatal pigs is developmentally regulated[J]. Amino Acids,2012.
117. Tan, B., Y. Yin, et al. L-Arginine stimulates proliferation and prevents endotoxin-induced death of intestinal cells[J]. Amino Acids,2010,38(4):1227-1235.
118.Thwaites, D. T. and C. M. Anderson. The SLC36 family of proton-coupled amino acid transporters and their potential role in drug transport[J]. Br J Pharmacol,2011,164(7): 1802-1816.
119.Traber, D. L. Nitric oxide and cationic amino acid transport in sepsis[J]. Clin Sci (Lond), 2002,102(6):651-652.
120. van Sluijters, D. A., P. F. Dubbelhuis, et al. Amino-acid-dependent signal transduction[J]. Biochem J,2000,351 Pt 3:545-550.
121.Waickman, A. T. and J. D. Powell. mTOR, metabolism, and the regulation of T-cell differentiation and function[J]. Immunol Rev,2012,249(1):43-58.
122. Walter, C. T., D. F. Bento, et al. Amino acid changes within the Bunyamwera virus nucleocapsid protein differentially affect the mRNA transcription and RNA replication activities of assembled ribonucleoprotein templates[J]. J Gen Virol,2011,92(Pt 1):80-84.
123. Wang, X. Q., P. L. Zeng, et al. Effects of dietary lysine levels on apparent nutrient digestibility and cationic amino acid transporter mRNA abundance in the small intestine of finishing pigs, Sus scrofa[J]. Anim Sci J,2012,83(2):148-155.
124. Wempe, M. F., P. J. Rice, et al. Metabolism and pharmacokinetic studies of JPH203, an L-amino acid transporter 1 (LAT1) selective compound[J]. Drug Metab Pharmacokinet, 2012,27(1):155-161.
125. Wu, X., Z. Ruan, et al. Dietary supplementation with L-arginine or N-carbamylglutamate enhances intestinal growth and heat shock protein-70 expression in weanling pigs fed a corn-and soybean meal-based diet[J]. Amino Acids,2010,39(3):831-839.
126. Wu, X. X., Y. Sun, et al. Rebuilding the balance of STAT1 and STAT3 signalings by fusaruside, a cerebroside compound, for the treatment of T-cell-mediated fulminant hepatitis in mice[J]. Biochem Pharmacol,2012.
127. Wullschleger, S., R. Loewith, et al. TOR signaling in growth and metabolism[J]. Cell,2006, 124(3):471-484.
128. Xie, X., L. Le, et al. Autophagy is induced through the ROS-TP53-DRAM1 pathway in response to mitochondrial protein synthesis inhibition[J]. Autophagy,2012,8(7).
129.Xu, G, W. Zhang, et al. Pharmacogenomic profiling of the PI3K/PTEN-AKT-mTOR pathway in common human tumors[J]. Int J Oncol,2004,24(4):893-900.
130.Xu, J., J. Ji, et al. Cross-talk between AMPK and mTOR in regulating energy balance[J]. Crit Rev Food Sci Nutr,2012,52(5):373-381.
131. Yamashita, K., A. Shiozawa, et al. Involvement of OS-2 MAP kinase in regulation of the large-subunit catalases CAT-1 and CAT-3 in Neurospora crassa[J]. Genes Genet Syst,2007, 82(4):301-310.
132. Yang, Y, X. Zhou, et al. Discovery of chrysoeriol, a PI3K-AKT-mTOR pathway inhibitor with potent antitumor activity against human multiple myeloma cells in vitro[J]. J Huazhong Univ Sci Technolog Med Sci,2010,30(6):734-740.
133. Yao, K., S. Guan, et al. Dietary L-arginine supplementation enhances intestinal development and expression of vascular endothelial growth factor in weanling piglets[J]. Br J Nutr,2011, 105(5):703-709.
134.Zervou, M. I., V. M. Vazgiourakis, et al. TRAF1/C5, eNOS, C1q, but not STAT4 and PTPN22 gene polymorphisms are associated with genetic susceptibility to systemic lupus erythematosus in Turkey[J]. Hum Immunol,2011,72(12):1210-1213.
135.Zhan, H. X., L. Cong, et al. Activated mTOR/P70S6K signaling pathway is involved in insulinoma tumorigenesis[J]. J Surg Oncol,2012.
136. Zhang, P., B. C. McGrath, et al. The GCN2 eIF2alpha kinase is required for adaptation to amino acid deprivation in mice[J]. Mol Cell Biol,2002,22(19):6681-6688.
137. Zhang, Z. and C. Grewer. The sodium-coupled neutral amino acid transporter SNAT2 mediates an anion leak conductance that is differentially inhibited by transported substrates[J]. Biophys J,2007,92(7):2621-2632.
138.Zharikov, S. I., A. A. Sigova, et al. Cytoskeletal regulation of the L-arginine/NO pathway in pulmonary artery endothelial cells[J]. Am J Physiol Lung Cell Mol Physiol,2001,280(3): L465-473.
139. Zheng, L., Y. Du, et al. Critical role of inducible nitric oxide synthase in degeneration of retinal capillaries in mice with streptozotocin-induced diabetes[J]. Diabetologia,2007,50(9): 1987-1996.
140. Zhou, Q., Z. Deng, et al. mTOR/p70S6K signal transduction pathway contributes to osteosarcoma progression and patients' prognosis[J]. Med Oncol,2010,27(4):1239-1245.
141.Zoncu, R., L. Bar-Peled, et al. mTORC1 senses lysosomal amino acids through an inside-out mechanism that requires the vacuolar H(+)-ATPase[J]. Science,2011,334(6056): 678-683.
142.Zeng L M, Tan B E, Xiao H, et al. Amino acid sensing signaling induced by amino acid transporters. SCIENTIA SINICA Vitae,2012,42:699-708, doi:10.1360/052012-195
