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Title: Arginine-induced stimulation of protein synthesis and survival in IPEC-J2 cells is mediated by mTOR but not nitric oxide

Author
item BAUCHART-THEVRET, CAROLINE - Children'S Nutrition Research Center (CNRC)
item CUI, LIWEI - Children'S Nutrition Research Center (CNRC)
item WU, GUOYAO - Texas A&M University
item Burrin, Douglas - Doug

Submitted to: American Journal of Physiology - Endocrinology and Metabolism
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/13/2010
Publication Date: 9/14/2010
Citation: Bauchart-Thevret, C., Cui, L., Wu, G., Burrin, D.G. 2010. Arginine-induced stimulation of protein synthesis and survival in IPEC-J2 cells is mediated by mTOR but not nitric oxide. American Journal of Physiology - Endocrinology and Metabolism. 299(6):E899-E909.

Interpretive Summary: A significant number of infants are born premature and have poor intestinal function. Many of these premature infants have been shown to have low levels of the amino acid, arginine, in their blood stream. The low level of arginine may lead to depressed intestine growth, since intestinal cells need this essential amino acid for cells to make new cell proteins, called protein synthesis. The cellular signals that explain how arginine activates cell protein synthesis and growth are unknown. This study used intestinal cells grown in a culture dish to test whether arginine activates cell protein synthesis (development) by activation of a well known cell signal called mammalian target of rapamycin, mTOR. We found that growing cells without arginine caused cell death indicating that arginine is necessary for cell survival as well as growth. We also found that incubating cells in relative low arginine concentrations, similar to those found in the blood of neonatal piglets, increased protein synthesis, and activated mTOR. We next treated cells with a chemical inhibitor of mTOR, and found that about 40% of the arginine-induced increase in protein synthesis was dependent on mTOR. We also treated cells with a chemical inhibitor that blocks the production of nitric oxide from arginine in cells. Nitric oxide has been shown to activate some cell growth signals. We found that the cells produced low levels of nitric oxide and adding nitric oxide to the cell culture did not affect protein synthesis or growth, suggesting that nitric oxide is not involved in arginine-induced cell protein synthesis. Our results show that arginine is required for intestinal cell survival and protein synthesis and that this is dependent on activation of mTOR but not nitric oxide production. We now have a greater understanding of the mechanisms of intestinal cell growth and development.

Technical Abstract: Arginine (ARG) is an indispensable amino acid in neonates and required for growth. Neonatal intestinal epithelial cells (IEC) are capable of ARG transport, catabolism, and synthesis, and express nitric oxide synthase (NOS) to produce NO from ARG. Our aim was to determine whether ARG directly stimulates IEC growth, and protein synthesis, and whether this effect is mediated via mammalian target of rapamycin (mTOR) and NO-dependent. We studied neonatal porcine IEC (IPEC-J2) cultured in serum- and ARG-free medium with increasing ARG concentrations for 4 h or 48 h. Our results show that ARG enhances IPEC-J2 cell survival and protein synthesis with a maximal response at a physiological concentration (0.1-0.5 mM). Addition of ARG increased the activation of mTOR, p70S6 kinase, and 4EBP-1 in a time and dose-dependent manner. The ARG-induced protein synthesis response was not inhibited by the NO inhibitors, L-NAME, or aminoguanidine, despite the presence of inducible NOS expression in IPEC-J2 cells. Moreover, protein synthesis was not increased or decreased in some cases by the addition of an NO donor (SNAP), ARG precursors (proline and citrulline) in absence of ARG, or insulin; SNAP suppressed phosphorylation of mTOR, p70S6 kinase and 4EBP-1. We found a markedly higher arginase activity in IPEC-J2 cells compared to primary pig IEC. Furthermore, mTOR inhibition by rapamycin partially (42%) reduced the ARG-induced protein synthesis response and the phosphorylation of mTOR and 4EBP-1. We conclude that ARG-dependent cell survival and protein synthesis signaling in IPEC-J2 cells is mediated by mTOR but not NO.