Abstract
The transport of nutrients across the placenta involves trophoblast cell specific transporters modulated through the mammalian target of rapamycin (mTOR). The vasoactive intestinal peptide (VIP) has embryotrophic effects in mice and regulates human cytotrophoblast cell migration and invasion. Here we explored the effect of VIP on glucose and System A amino acid uptake by human trophoblast-derived cells (Swan 71 and BeWo cell lines). VIP activated D-glucose specific uptake in single cytotrophoblast cells in a concentration-dependent manner through PKA, MAPK, PI3K and mTOR signalling pathways. Glucose uptake was reduced in VIP-knocked down cytotrophoblast cells. Also, VIP stimulated System A amino acid uptake and the expression of GLUT1 glucose transporter and SNAT1 neutral amino acid transporter. VIP increased mTOR expression and mTOR/S6 phosphorylation whereas VIP silencing reduced mTOR mRNA and protein expression. Inhibition of mTOR signalling with rapamycin reduced the expression of endogenous VIP and of VIP-induced S6 phosphorylation. Our findings support a role of VIP in the transport of glucose and neutral amino acids in cytotrophoblast cells through mTOR-regulated pathways and they are instrumental for understanding the physiological regulation of nutrient sensing by endogenous VIP at the maternal-foetal interface.
Highlights
The transport of glucose, amino acids and other nutrients across the placenta involves trophoblast cell specific transporters and its regulation is critical for placental function and foetal growth[1,2,3,4]
We performed knocking down experiments using a vasoactive intestinal peptide (VIP) siRNA in Swan and BeWo cells for h, and after confirming the decrease of VIP expression to less than 50% compared to scramble-transfected cells as previously29,31, 2-NBDG incorporation was measured by flow cytometry
In order to elucidate the mechanisms involved in the effect of VIP for the regulation of D-glucose transport, we studied the main signalling pathways described downstream VIP activation of its specific receptors VPAC1 and VPAC2: protein kinase A (PKA), protein kinase C (PKC) and MAP kinases (MAPK). 2-NBDG incorporation assays showed that the pre-treatment of trophoblast cells with the PKA inhibitor H89 (10 μM) or the MEK inhibitor PD 98059 (50 μM) prevented the increase of 2-NBDG uptake induced by 50 nM VIP, whereas the PKC inhibitor STP did not prevent VIP effect (Fig. 4a)
Summary
The transport of glucose, amino acids and other nutrients across the placenta involves trophoblast cell specific transporters and its regulation is critical for placental function and foetal growth[1,2,3,4]. The uptake of amino acids from maternal circulation is largely mediated by System A and System L transporters expressed by trophoblast cells[1]. VIP deficiency in trophoblast cells but not in maternal tissues adversely affected murine pregnancy outcome with reduced foetal weight, structural placental alterations and immune homeostasis loss[42]. The main goal of the present study was to explore VIP effect on glucose and System A amino acid uptake by human cytotrophoblast cells. Interplay between VIP and mTOR in nutrient sensing and transport in cytotrophoblast cells is proposed
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