An Investigation of Placental Glutamine and Glutamate Transport in Normal Pregnancy and Fetal Growth Restriction

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Abstract

A healthy pregnancy depends upon the delivery of amino acids and other essential nutrients to the fetus via the placenta. Placental dysfunction is a major cause of fetal growth restriction (FGR), which is characterised by poor growth in utero and most often defined as an individualised birth weight ratio (IBR) of <5th centile. Growth restricted babies are at increased risk of stillbirth, postnatal complications and disease in adulthood. There is an unmet need for efficacious treatment options, the development of which would be aided by improved understanding of the relationship between placental nutrient delivery and fetal growth. The amino acids glutamine and glutamate are vital for metabolic processes and fetal growth, and are intrinsically linked by interorgan metabolism in the placenta and fetal liver. Studies in normal pregnancy (in women and wild-type (WT) mice) have shown that in the small placenta of a normally grown fetus there is up-regulation (adaptation) of transport of the non- metabolisable amino acid analogue methylaminoisobutyric acid (MeAIB) (per g placenta). This thesis tested the hypothesis that the small, normal placenta up-regulates glutamine and glutamate transport and that in FGR this relationship is disrupted and/or absent. Unidirectional maternofetal clearance (Kmf) of glutamine and glutamate was assessed in normal (WT) mouse pregnancy at embryonic day (E)15.5 and E18.5 (term=E19-20). A method to assess transporter-mediated uptake of glutamine and glutamate by human placental villous fragments in vitro was also developed. In normal WT pregnancy, Kmf of glutamine and glutamate was significantly higher in the lightest placentas towards term (E18.5), which reinforces the importance of glutamine and glutamate in ensuring appropriate fetal growth is maintained. Contrary to the literature, there was no relationship between placental weight or birth weight and transporter-mediated uptake of glutamine or glutamate in placentas from normal birth weight infants in human pregnancy. However, placentas from male infants had significantly higher glutamine/glutamate uptake compared with females but this was not associated with changes in transporter expression. Placental transport capacity was next investigated in the Igf2P0 (P0) knockout mouse, a well- characterised model of FGR. Kmf of glutamine and glutamate was higher across P0 versus WT placentas at E15.5. At E18.5 Kmf of glutamine remained significantly higher whereas Kmf of glutamate was similar between groups. This finding is surprising and suggests that the P0 placenta attempts to adapt to meet fetal nutrient demands. In human FGR where a small dysfunctional placenta is observed, transporter-mediated glutamine uptake was reduced in comparison with normal pregnancy. Glutamate uptake was no different between groups. In contrast, expression of key glutamine and glutamate transporters was significantly higher in FGR, indicating a potential role of post-translational modifications in amino acid transporter activity. Amino acid concentrations in the maternal vein, umbilical vein (UmV) and artery (UmA) were quantified by high performance liquid chromatography (HPLC), and the abundance of small molecule metabolites in UmV and UmA of normal birth weight and FGR infants measured using gas chromatography-mass spectrometry (GC-MS). Glutamine and glutamate concentrations in the maternal circulation were unaltered between normal pregnancy and FGR but glutamate concentration in the UmA was higher in FGR. Levels of lactic acid, pyruvic acid, urea, and others were differentially altered in the UmV and UmA of FGR infants. In summary, glutamine and glutamate uptake into human placental villous fragments was unrelated to fetal or placental measures, but was influenced by the sex of the fetus. Conversely there is evidence to indicate that Kmf of glutamine/glutamate adapts according to placental size in WT mice and that the P0 mouse model of FGR may attempt to modulate its transport capacity in a bid to maintain appropriate fetal growth. HPLC and GC-MS uncovered distinct metabolic changes in FGR compared with normal human pregnancy. Furthermore, glutamine uptake was reduced in FGR despite increased transporter abundance, which suggests that post-translational modifications and/or signaling pathways modify amino acid transporter activity in this pathology. This thesis provides the foundation for future research to investigate the underlying mechanisms that may drive these changes.

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There are 4 powerpoint documents on a CD attached within the back cover of this thesis. The documents contain the full Western blots presented within this thesis. The first slide in each file contains a table of contents. The documents are labelled as follows: ï‚· Chapter 3 Western blot Appendix ï‚· Chapter 4 Western blot Appendix ï‚· Chapter 5 Western blot Appendix ï‚· Chapter 6 Western blot Appendix

Keyword(s)

Fetal growth restriction; Glutamate; Glutamine; Placenta

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