Placental amino acid transport system a - An assessment of SNAT1 and SNAT2 expression in F1 and F2 placentas in a rat model of gestational protein restriction

Background. Gestational protein restriction (GPR) can program a fetal phenotype prone to develop metabolic syndrome in successive generations. Although mechanisms are not well characterized, placental amino acid transport system A (SysA) activity is depressed in the setting of GPR. Objectives. To determine mechanisms of GPR-induced SysA-adaptations in F1 and F2 placentas. Material and Methods. Rats (FO) were pair-fed either a 19% normal protein diet (NPD) or an 8% low protein diet (LPD) through pregnancy and lactation. F1 placentas were studied for SNAT1 and SNAT2 mRNA and SNAT1 protein. Male and female offspring (F1) were bred to control animals and allowed to deliver at term at which time placentas were collected for me same studies. Transient transfection of HEK 293 cells was done using p-CMV-FLAG-SNAT2 or the control vector. After 36 hours of transfection, MeAIB transport, expression of SNAT2 mRNA and proteins were assessed. Results. In F1 placentas, steady-state mRNA content of SNAT1 (140 ± 13 vs. 99 ± 11 arbitrary mRNA units; p ≤ 0.01) and SNAT2 (81 ± 6, n = 10 vs. 104 ± 9 arbitrary mRNA units; p ≤ 0.001) were higher in LPD than NPD group. An opposite but non-significant trend in mRNA expression of both isoforms was evident in F2 placentas. Despite up-regulation of mRNA in F1 placentas, SNAT1 immunoblot bands were comparable from placental-apical-membranes (0.62 ± 0.13 vs. 0.63 ± 0.13 arbitrary units; p = 0.9), basal-membranes (0.9 ± 0.14 vs. 1 ± 0.06 arbitrary units; p = 0.6) and placental-homogenates (0.5 ± 0.16 vs. 0.7 ± 0.1 arbitrary units; p = 0.3) between LPD and NPD group. Similar results were seen in F2 placental SNAT1 protein expressions. SNAT2- mRNA over-expression by transient transfection with pLPCX-FLAG-SNAT2 construct vs. control vector in HEK 293 cells resulted in up-regulation of both SNAT2 protein and Na+ dependent MeAIB transport (1243 ± 137 vs. 390 ± 27 pmole^.1mg^.1 min; p < 0.0008). Conclusions. 1. Although GPR-induced SysA repression is associated with up-regulation of SNAT1 and SNAT2 mRNA in F1 placentas, the protein content is unchanged suggesting post-transcriptional regulation of SysA expression and function. Up-regulation of SNAT2 protein and transport activity following SNAT2-mRNA-over-expression noted in our tissue culture studies support this conclusion. 2. If maternal nutrition is optimized, GPR-induced SysA F1 placental abnormalities are not replicated in F2 placentas.