l-Arginine stimulates the mTOR signaling pathway and protein synthesis in porcine trophectoderm cells

Abstract

Impairment of placental growth is a major factor contributing to intrauterine growth retardation (IUGR) in both human pregnancy and animal production. Results of recent studies indicate that administration of l-arginine (Arg) to gestating pigs or sheep with IUGR fetuses can enhance fetal growth. However, the underlying mechanisms are largely unknown. The present study tested the hypothesis that Arg stimulates the mammalian target of rapamycin (mTOR) signaling pathway and protein synthesis in porcine conceptus trophectoderm (pTr2) cells. The cells were cultured for 4 days in Arg-free Dulbecco's modified Eagle's Ham medium containing 10, 50, 100, 200, 350 or 500 μM Arg. Cell numbers, protein synthesis and degradation, as well as total and phosphorylated levels of mTOR, ribosomal protein S6 kinase 1 (p70S6K) and eukaryotic initiation factor 4E-binding protein-1 (4EBP1), were determined. The pTr2 cells exhibited time (0–6 days)- and Arg concentration (10–350 μM)-dependent increases in proliferation. Addition of 100 and 350 μM Arg to culture medium dose-dependently increased (a) protein synthesis and decreased protein degradation and (b) the abundance of total and phosphorylated mTOR, p70S6K and 4EBP1 proteins. Effects of 350 μM Arg on intracellular protein turnover were only modestly affected when nitric oxide synthesis was inhibited. Collectively, these results indicate a novel and important role for Arg in promoting growth of porcine placental cells largely via a nitric-oxide-independent pathway. Additionally, these findings help to explain beneficial effects of Arg supplementation on improving survival and growth of embryos/fetuses in mammals.

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.