Causes and Consequences of Reduced Placental Glutamine and Glutamate Uptake into The Human Placenta in Fetal Growth Restriction

Abstract

Fetal growth restriction (FGR) describes a fetus that fails to achieve its genetic growth potential and is a major risk factor for stillbirth. Placental dysfunction is the underlying cause of the majority of cases of FGR that cannot be explained by fetal congenital and genetic abnormalities. In FGR, placental uptake of amino acid has been found to be reduced in both humans and animal models. Glutamine is a vital amino acid for fetal growth and is important for the citric acid cycle, ammonia metabolism and the biosynthesis of purines and pyrimidines. Glutamate is a precursor of neurotransmitters and glutathione and regulates intracellular pH levels. Placental uptake of glutamine and glutamate at initial rate (proxy of transporter activity) is reduced in FGR. This is associated with an increased expression of key proteins that transport glutamine and glutamate into the placenta. Thus, the mechanisms underpinning reduced glutamine and glutamate uptake in FGR remain unknown. Additionally, it is assumed that this reduced activity of glutamine and glutamate transporters will result in a reduction in intracellular levels of glutamine and glutamate within the placenta but this assumption remains unproven. FGR is associated with elevated placental oxidative stress (OS) and an agent that induces OS, tert-Butyl hydroperoxide (tBOOH), inhibits amino acid (alanine and methionine) uptake into BeWo cells (human placental cell line that originates from a choriocarcinoma). However, the possibility that OS underlies the reduced placental uptake of glutamine and glutamate in FGR has not been investigated. The key research questions of this thesis are: (i) does oxidative stress reduce the uptake of glutamine and glutamate into placental villous tissue? (ii) does a reduction in placental uptake of glutamine and glutamate at initial rate in FGR translate to reduced intracellular levels in the placenta? Oxidative damage in placental villous explants from normal pregnancy was induced by hydrogen peroxide (H2O2) and tBOOH (1 mM for a 48 hr period), confirmed by increased lipid peroxidation. tBOOH markedly reduced the transporter mediated 24 hr uptake of 14C glutamine and 14C glutamate into villous explants (<10% of control). tBOOH also significantly reduced explant human chorionic gonadotropin (hCG) secretion, increased interleukin 6 (IL-6) secretion and increased apoptosis, suggestive of cell stress, but did not affect lactate dehydrogenase (LDH) release or syncytiotrophoblast regeneration. Despite elevating OS, H2O2 did not affect glutamine and glutamate uptake or any of the other variables measured. Given the suggestion that 1 mM tBOOH increased cell stress, a follow up study was conducted to assess the effect of tBOOH at varying concentrations (100 μM, 500 μM and 1 mM) on glutamine and glutamate uptake, hCG and IL-6 secretion, LDH release and the expression of intracellular cell stress-related proteins in placental explants. Glutamate uptake was reduced to 79% of control by 100 μM tBOOH and 57 % by 500 μM. Glutamine uptake was unaffected by 100 μM and inhibited to 73 % of control by 500μM tBOOH. The inhibitory effect of tBOOH at 500 μM was not associated with a significant change in IL-6 secretion or LDH release. The expression of intracellular cell stress-related proteins was altered by 100 μM, 500 μM and 1 mM tBOOH to varying degrees. To assess whether the previously reported reduction in glutamine and glutamate transporter activity in FGR resulted in lower intracellular levels at steady state, uptake of 14C glutamine and 14C glutamate into placental villous tissue was assessed at 24 hr in FGR versus normal pregnancies. There was no significant difference in glutamine or glutamate uptake at 24 hr. In summary, tBOOH induced oxidative damage and reduced glutamine and glutamate 24 hr uptake (proxy measure of intracellular levels) into villous explants. However, causal links between elevated oxidative stress and decreased amino acid uptake remain to be proven, especially given the increased cell stress observed following 1 mM tBOOH treatment and the lack of effect with H2O2. In addition, the intracellular levels of glutamine and glutamate in the placenta, measured over 24 hr, was not lower in FGR compared to normal pregnancy. It is unclear whether this lack of reduction is due to methodological differences between the previous study demonstrating a reduced glutamine and glutamate transporter activity in FGR, or whether this challenges the assumption that reduced activity leads to reduced intracellular levels of amino acid over time. This knowledge is key given the importance of amino acid for both placental metabolism and fetal growth.