This is the first time LAT1 and LAT2 mediated transport has been studied in primary trophoblast cells using gene targeting approaches. Consistent with previous competitive inhibition studies [
41], we report that both LAT1 and LAT2 isoforms contribute to System L leucine uptake in primary trophoblast cells from human term placenta. We used si-RNA transfections targeting LAT1 and/or LAT2 isoforms to evaluate the effect of these treatments on System L amino acid transport activity. After specifically targeting LAT1 or LAT2, the decrease in System L transport activity was, in general, proportional to the decreases in LAT1 and LAT2 protein expression following si-RNA treatments. Moreover, silencing both LAT1 and LAT2 transporters resulted in an additive reduction of leucine uptake. These results suggest that both transporters independently contribute to trophoblast System L activity. Isolated human primary trophoblast cells syncytialize spontaneously in culture. During syncytialisation, trophoblast cells become polarized and develop abundant and regular microvilli on the cell surface facing the culture media [
42‐
44]. This is the cell surface that is freely accessible in vitro during transport activity measurements and it is very likely that the measured uptake can be accounted for transport across the MVM of the syncytiotrophoblast layer.
Using Western blot and immunohistochemistry we also show the localisation of LAT1 and LAT2 in the human term placenta. With both methods we demonstrated that LAT1 is mainly localised in the syncytiotrophoblast apical/microvillous plasma membrane, confirming the immunohistochemistry results from Okamoto et al. [
11]. These data are in agreement with the model that LAT1 is mainly involved in the uptake of large neutral amino acids at the maternal side of the syncytiotrophoblast layer [
7]. To the best of our knowledge, the localisation of LAT2 in the placenta has so far been explored only by functional studies. Whereas some studies indicate that LAT2 is polarized to the basal membrane of the syncytiotrophoblast layer [
15], other investigators have suggested that LAT2 is the predominant isoform in the MVM [
16]. Our immunohistochemistry and Western blot data demonstrate that LAT2 is highly expressed in the MVM in vivo and that this isoform contributes to apical leucine transport in primary cytotrophoblast cells in culture. The mechanisms underlying the efflux of amino acids through the BM is still largely unknown, but System L has been indicated as a strong candidate contributing to this process. Our results on the localisation of LAT2 in the BM membranes and in the epithelium of the fetal capillaries support the notion that this exchanger could mediate the efflux of amino acids from the syncytiotrophoblast layer (in analogy to its function in the absorptive epithelia of the digestive tract [
8]) and their transfer to the fetal circulation. Experimental data by Cleal and co-workers in the isolated perfused human placental cotyledon indicate that one System L isoform mediates L-leucine exchange in the fetal-facing BM [
17]. However, the same investigators suggested that LAT1, rather than LAT2, is the main BM exchanger and that non-exchange mechanisms also contribute to efflux of leucine across the BM [
17,
45]. This apparent discrepancy could be explained by the fact that LAT2 appears to mediate facilitated transport of neutral amino acids (in addition to function as a true exchanger [
46,
47]) and LAT2 may therefore also contribute to BM amino acid transport by non-exchange mechanisms.
The link between maternal nutrition, placental nutrient transport function and fetal growth has been studied in various animal models of maternal undernutrition and diabetes, which are often associated with restricted and accelerated fetal growth, respectively [
18]. The studies discussed above support the hypothesis that placental transport capacity is regulated by maternal nutritional cues and fetal signals in order to balance fetal demand with the ability of the mother to support pregnancy and the growing fetus [
18,
48]. Consistent with this model, the activity and/or expression of amino acid transporters System A and System L was previously reported to be decreased in placentas of intrauterine growth restricted infants [
19,
49‐
52] and increased with fetal overgrowth associated with maternal diabetes [
21,
53]. Whether fetal overgrowth could be explained by altered placental nutrient transport capacity is less clear in pregnancies of high BMI women without diabetes. Nevertheless, the endocrine and metabolic perturbations induced by maternal obesity emerge as important factors regulating placental nutrient transport function [
36,
38,
54‐
58]. For example, placental mRNA of Glucose Transporter (GLUT)-4 has been shown to be significantly decreased in obese compared to normal BMI patients [
57], while BM protein expression of GLUT-1, but not glucose transport activity, was positively correlated with birth weight in women with varying pre-pregnancy BMI [
36]. Dube et al. [
58] reported altered fatty acids transport and metabolism in the placenta of obese women. With respect to placental amino acid transport, decreased System A activity and lower expression of the System A isoform SNAT4 was reported in placental villous fragments isolated from term placentas of obese Hispanic women giving birth to AGA babies [
55]. Possibly due to differences in the ethnicity of the study populations and in fetal outcome, System A activity was increased in MVM isolated from placentas of obese women giving birth to large babies, in a cohort of 23 Swedish women [
38]. In the same study the expression of the SNAT2 transporter was positively correlated with maternal BMI and birth weight. Consistent with our current results, System L activity and transporters’ expression were not altered in response to varying maternal BMI in the Swedish cohort, suggesting that System A amino acid transporters may be more sensitive to the metabolic and endocrine changes in maternal obesity. As System A provides the substrates for System L exchange mechanism, it is possible that an increase in placental System A activity in pregnancies complicated by maternal overweight/obesity could enhance System L efficiency in vivo. In this report we established that in our study population the MVM activity of System L amino acid transporters is not modulated by maternal overweight/obesity and does not correlate with birth weight. Notably, by excluding study subjects with diabetes and hypertension, we attempted to isolate maternal overweight/obesity from these two common comorbidities [
59‐
61]. Pre-existing and gestational maternal diabetes have been shown to alter the effect of maternal obesity on pregnancy outcomes [
62‐
65], maternal metabolism [
66] and placental function [
67]. Our data indicate that high maternal BMI
per se does not modify the expression and activity of System L transporters in absence of other aggravating conditions, such as diabetes and hypertension. Nevertheless, the increased placental size in the high BMI group suggests that a larger placental exchange surface area could contribute to increased fetal nutrient supply and birth weight in this group. It also possible that elevated maternal nutrient levels and/or upregulation of placental nutrient transporters not examined in our current study could enhance placental nutrient transfer and promote fetal growth in some obese women.