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Inhibition of mammalian target of rapamycin (mTOR) signaling in cultured human primary trophoblast cells reduces the activity of key placental amino acid transporters. However, the upstream regulators of placental mTOR are unknown. We hypothesized that glucose, insulin, and IGF-I regulate placental amino acid transporters by inducing changes in mTOR signaling. Primary human trophoblast cells were cultured for 24 h with media containing various glucose concentrations, insulin, or IGF-I, with or without the mTOR inhibitor rapamycin, and, subsequently, the activity of system A, system L, and taurine (TAUT) transporters was measured. Glucose deprivation (0.5 mM glucose) did not significantly affect Thr172-AMP-activated protein kinase phosphorylation or REDD1 expression but decreased S6 kinase 1 phosphorylation at Thr389. The activity of system L decreased in a dose-dependent manner in response to decreasing glucose concentrations. This effect was abolished in the presence of rapamycin. Glucose deprivation had two opposing effects on system A activity: 1) an “adaptive” upregulation mediated by an mTOR-independent mechanism and 2) downregulation by an mTOR-dependent mechanism. TAUT activity was increased after incubating cells with glucose-deprived media, and this effect was largely independent of mTOR signaling. Insulin and IGF-I increased system A activity and insulin stimulated system L activity, effects that were abolished by rapamycin. We conclude that the mTOR pathway represents an important intracellular regulatory link between nutrient and growth factor concentrations and amino acid transport in the human placenta.intrauterine growth restriction (IUGR) and accelerated fetal growth represent two important clinical conditions that occur in 15% of all pregnancies (1, 2). Aberrant fetal growth is associated with an increased risk of perinatal morbidity (7) as well as metabolic abnormalities in adult life, such as obesity, type 2 diabetes, and cardiovascular disease (6, 12, 46). The most important determinant of fetal growth is nutrient availability, which is highly dependent on placental transport capacity. The mechanisms underlying altered fetal growth remain to be established, but accumulating evidence implicates changes in the activity of specific placental amino acid transporters as a critical factor contributing to abnormal fetal growth (27, 54). Experimental evidence supports the hypothesis that changes in placental nutrient transporter activity are a cause of rather than a response to altered fetal growth. For example, in pregnant rats subjected to protein malnutrition, it is likely that downregulation of the placental system A amino acid transporter directly contributes to the development of IUGR (26).

In IUGR, fetuses may be hypoglycemic (15) and have reduced circulating levels of insulin (43) and IGF-I (4, 34). The maternal levels of glucose (15) and IGF-I (40, 41) may also be reduced in this condition. The placenta of the IUGR fetus could therefore be exposed to decreased levels of glucose, hormones, and growth factors. Both insulin and IGF-I stimulate placental system A activity (24, 30, 31). These results suggest that extracellular cues regulate placental nutrient transporters and, as a consequence, fetal nutrient supply, but the cellular mechanisms remain to be fully established.

The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that is regulated by a multitude of intracellular and extracellular signals. For example, mTOR is activated by growth factors and nutrient levels, such as amino acids (59), and inhibited by numerous stress conditions, such as cellular energy depletion (13, 17). Glucose may also regulate mTOR signaling through energy production in the form of ATP (13, 17). The AMP-activated protein kinase (AMPK) is regulated by the AMP-to-ATP ratio, which rises under nutrient deprivation and activates AMPK (10). Activated AMPK can in turn phosphorylate tuberous sclerosis complex 2 (TSC2), leading to mTOR inactivation (23). AMPK is phosphorylated and activated by LKB1 (52), and it has been shown that phosphorylation of LKB1 at Ser428 is essential for AMPK activation by metformin, and the authors speculate that LKB1-Ser428 phosphorylation may be a common pathway required for AMPK activation (60). There might also be additional, AMPK-independent, pathways involved in energy depletion. A recent report has shown that REDD1 (regulated in development and DNA damage responses 1) in mouse embryonic fibroblasts is induced by chronic energy depletion, and this in turn leads to inactivation of mTOR complex 1 (mTORC1) measured as phosphorylation of S6 kinase 1 (S6K1) at Thr389, independent of AMPK (55).

Insulin and IGF-I activate the tyrosine kinase activity of its receptors to phosphorylate the insulin receptor substrate 1, which in turn activates phosphatidylinositol 3-kinase (PI3K) to generate PI(3,4,5)P3. Phosphatidylinositol 3,4,5-trisphosphate (PIP3) binding to Akt leads to the translocation of Akt to the plasma membrane, where it is phosphorylated and activated. The activation of Akt positively modulates mTORC1 function, by phosphorylating, and inhibiting, TSC2 (reviewed in Ref. 59).

We have previously shown that inhibition of mTOR reduces the activity of placental system L, system A, and the taurine transporter (TAUT) (50). Since the activity of these amino acid transporter systems is downregulated in the placenta in association to IUGR (14, 19, 28, 37, 45) and placental mTOR activity has been reported to be decreased in IUGR (49, 62), it is possible that mTOR signaling plays an important role in regulating placental amino acid transporters in vivo. However, the upstream regulators of placental mTOR are unknown. We hypothesized that glucose, insulin, and IGF-I regulate placental amino acid transporter activity by inducing changes in mTOR signaling. To test this hypothesis, human primary trophoblast cells were incubated with media containing various concentrations of glucose, insulin, or IGF-I in the presence or absence of the specific mTOR inhibitor rapamycin. Subsequently, the activity of system L, system A, and the taurine transporter was measured. To investigate whether the AMPK pathway and/or REDD1 is activated in glucose-deprived primary trophoblasts, the protein expression of phosphorylated (P)-Thr172-AMPKα, total AMPK, P-Ser428-LKB1, and REDD1 in control and glucose-deprived cells was also studied.