Research articlel-Arginine stimulates the mTOR signaling pathway and protein synthesis in porcine trophectoderm cells
Introduction
Intrauterine growth retardation (IUGR), defined as impaired growth and development of the mammalian embryo/fetus or its organs during pregnancy, is a major problem in both human pregnancy [1] and animal production [2]. For example, IUGR infants represent 11% of all newborns in developing countries and also a large number of all newborns in developed nations, e.g., approximately 5% in the United States [1]. In swine, 25% of newborn piglets suffer from IUGR [2], which has a permanent stunting effect on their postnatal growth [3] and efficiency of food utilization [4].
Transfer of nutrients across the placenta is essential for fetal growth and development [5]. Thus, impairment of placental growth is a major factor contributing to IUGR in mammals [6], [7]. The placenta is the organ that transports nutrients, respiratory gases and the products of metabolism between the maternal and fetal circulations [8], [9]. The blastocyst is composed of two distinct cell layers (trophectoderm cells and the inner cell mass), with the trophectoderm accounting for two thirds of the cells. The trophectoderm is the first epithelium formed in embryonic development, leading predominantly to extraembryonic tissues including the placenta [10]. Interestingly, IUGR is associated with a deficiency of l-arginine (Arg; an amino acid with enormous versatility and importance [11]) in the placenta [6] and fetal fluids [12], [13], [14]. Of particular interest, results of recent studies indicate that administration of Arg to gestating pigs [15], sheep [16] and women [17] with IUGR fetuses can enhance fetal growth. However, the underlying mechanisms are largely unknown.
Based on the foregoing, the present study was conducted with the porcine trophectoderm cell line-2 (pTr2) [18] to test the hypothesis that Arg stimulates protein synthesis and growth of placental cells. Because it is now known that certain amino acids activate mammalian target of rapamycin (mTOR), which is the major protein kinase that promotes initiation of polypeptide formation [19], we determined total and phosphorylated (active form) levels of mTOR in the placental cells to identify a molecular mechanism for the effect of Arg on placental protein synthesis. Additionally, because the two downstream targets of mTOR are ribosomal protein S6 kinase 1 (p70S6K) and eukaryotic initiation factor 4E-binding protein-1 (4EBP1) [19], the abundance of total and phosphorylated (active form) forms of these two proteins in pTr2 cells was also quantified in the present study.
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Chemicals
We purchased the following chemicals from Sigma-Aldrich (St. Louis, MO, USA): antibiotic–antimycotic solution (P/S), NG-monomethyl-l-arginine (acetate salt; l-NMMA), d-(+)-α-glucose, Dulbecco's modified Eagle's Ham medium (DMEM)/F-12, fetal bovine serum (FBS, charcoal striped), insulin solution (from bovine pancreas, 10 mg/ml), l-Arg (hydrochloride), l-glutamine, l-leucine, l-phenylalanine and l-proline. Custom-made Arg-free DMEM (Formula #08-5009EF) and 2.5% trypsin solution were obtained from
Effects of Arg on pTr2 cell proliferation
Increasing extracellular concentrations of Arg from 10 to 350 μM dose-dependently increased (P<.05) the number of pTr2 cells on day 4 of culture (Fig. 1). The effects of Arg on stimulating cell proliferation were observed on days 2, 4, and 6 (Fig. 2). Cell numbers did not differ (P>.05) between 350 and 500 μM Arg (Fig. 1).
Effects of Arg on intracellular protein turnover in pTr2 cells
Increasing extracellular concentrations of Arg from 10 to 350 μM dose-dependently increased (P<.05) protein synthesis and decreased (P<.05) protein degradation in the pTr2
Discussion
l-Arginine has been reported to improve embryonic/fetal survival and growth in several mammalian species, including pigs, rats and sheep [29]. Similarly, intravenous administration of Arg ameliorates fetal growth restriction in women [17]. We recently found that dietary supplementation with Arg increased placental growth in gilts (X.L. Li, F.W. Bazer, G.A. Johnson and G. Wu; unpublished data). To our knowledge, results of the present study are the first to provide biochemical, cellular and
Acknowledgments
This work was supported by the National Natural Science Foundation of China (No. 30901040, 30928018, 30371038 and 30528006), the Outstanding Overseas Chinese Scholars Fund of the Chinese Academy of Sciences (No. 2005-1-4), the Chinese Academy of Sciences and Knowledge Innovation Project (KSCX2-YW-N-051), the CAS/SAFEA International Partnership Program for Creative Research Teams, Texas AgriLife Research Hatch Project (No. H-8200) and National Research Initiative Competitive Grants from the
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