Abstract
The placenta supplies the foetus with critical nutrients such as essential amino acids (AA, eg leucine) for development and growth. It also represents a cellular barrier which is formed by a polarized, differentiated syncytiotrophoblast (STB) monolayer. Active Na+‐independent leucine transport across the placenta is mainly attributed to the System L transporters LAT1/SLC7A5 and LAT2/SLC7A8. This study explored the influence of trophoblast differentiation on the activity of LAT1/LAT2 and the relevance of LAT1/LAT2 in leucine uptake and transfer in trophoblasts by applying specific small molecule inhibitors (JPH203/JG336/JX009). L‐leucine uptake (total dose = 167 μmol/L) was sensitive to LAT1‐specific inhibition by JPH203 (EC50 = 2.55 µmol/L). The inhibition efficiency of JPH203 was increased by an additional methoxy group in the JPH203‐derivate JG336 (EC50 = 1.99 µmol/L). Interestingly, JX009 showed efficient System L inhibition (EC50 = 2.35 µmol/L) and was the most potent inhibitor of leucine uptake in trophoblasts. The application of JPH203 and JX009 in Transwell®‐based leucine transfer revealed LAT1 as the major accumulative transporter at the apical membrane, but other System L transporters such as LAT2 as rate‐limiting for leucine efflux across the basal membrane. Therefore, differential specificity of the applied inhibitors allowed for estimation of the contribution of LAT1 and LAT2 in materno‐foetal AA transfer and their potential impact in pregnancy diseases associated with impaired foetal growth.
Highlights
Adequate amino acid (AA) supply is vital especially for highly proliferative tissues like the placenta
This study explored the influence of trophoblast differentiation on the activity of LAT1/LAT2 and the relevance of LAT1/LAT2 in leucine uptake and transfer in trophoblasts by applying specific small molecule inhibitors (JPH203/JG336/JX009)
The small molecule inhibitors JPH203, JG336 and JX009 were used to assess the relevance of LAT1 and LAT2 in leucine transport
Summary
Adequate amino acid (AA) supply is vital especially for highly proliferative tissues like the placenta. The SLC43 family members LAT3 (SLC43A1) and LAT4 (SLC43A2), known to be involved in facilitated AA diffusion,[6,7] are expressed at the BM (Figure 1A) Both LAT1 and LAT2 are predominantly localized to the MVM of human term placenta, LAT2 as well as LAT3 and LAT4 are present at the BM and in endothelial cells lining the foetal capillaries.[7,8] In the last decade, increasing evidence suggests a tight link between the reduced activity of placental System L transporters and intrauterine growth restriction (IUGR),[9,10] and their up-regulation in placentae of large for gestational age (LGA) infants.[11] Such altered foetal development has a fundamental impact on lifelong health and wellbeing, and may contribute by foetal programming to an increased prevalence for cardiovascular disease and diabetes/adiposity later in life.[12,13,14,15] Notably, it has been reported that LAT1 or its associated glycoprotein 4F2hc is involved in placenta decidualization and fusogenic trophoblast differentiation.[16] This could imply that the diminished leucine uptake found in knock-down cell models[17] results rather from failure in trophoblast differentiation than from reduced SLC7 transport activity. We (a) tested primary human trophoblast and BeWo (clone b30) cell models for LAT1, LAT2 and 4F2hc expression and leucine uptake capacity under Na+-free conditions, (b) investigated whether the differentiation status of primary trophoblasts and BeWo cells has an impact on leucine uptake and (c) assessed the contribution of LAT1 for the uptake and transfer of leucine by application of small molecule inhibitors
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