Trophoblast-specific overexpression of the LAT1 increases transplacental transport of essential amino acids and fetal growth in mice

Abstract

Placental System L amino acid transporter activity is decreased in pregnancies complicated by intrauterine growth restriction (IUGR) and increased in fetal overgrowth. However, it is unknown if changes in the expression/activity of placental Large Neutral Amino Acid Transporter Small Subunit 1 (Slc7a5/LAT1) are mechanistically linked to placental function and fetal growth. We hypothesized that trophoblast-specific Slc7a5 overexpression increases placental transport of essential amino acids, activates the placental mechanistic target of rapamycin (mTOR) signaling, and promotes fetal growth in mice. Using lentiviral transduction of blastocysts with a Slc7a5 transgene, we achieved trophoblast-specific overexpression of Slc7a5 (Slc7a5 OX) with increased fetal (+27%) and placental weights (+10%). Trophoblast-specific Slc7a5 overexpression increased trophoblast plasma membrane (TPM) LAT1 protein abundance and TPM System L transporter (+53%) and System A transporter activity (+ 21%). Slc7a5 overexpression also increased transplacental transport of leucine (+ 85%) but not of the System A tracer, 14C-methylamino isobutyric acid, in vivo. Trophoblast-specific overexpression of Slc7a5 activated placental mTORC1, as assessed by increased (+44%) phosphorylation of S6 ribosomal protein (Ser 235/236), and mTORC2 as indicated by phosphorylation of PKCα-Tyr-657 (+47%) and Akt-Ser 473 (+96%). This is the first demonstration that placental transport of essential amino acids is mechanistically linked to fetal growth. The decreased placental System L activity in human IUGR and the increased placental activity of this transporter in some cases of fetal overgrowth may directly contribute to the development of these pregnancy complications.

Keywords: fetal development; leucine; maternal–fetal exchange; mechanistic target of rapamycin; placenta.

Fig. 1.

Trophoblast-specific overexpression of Slc7a5 increases placental Slc7a5 mRNA expression at E18.5. a–f) Gene expression of Slc7a5 (LAT1), Slc7a8 (LAT2), Slc3a2 (4F2hc), Slc38a1 (SNAT1), Slc38a2 (SNAT2), Slc38a4 (SNAT4) mRNA in NSS and Slc7a5 overexpression placentas, pooled from n = 6 litters/group. Each dot represents individual value; *P < 0.05 vs. NSS; paired Student's t test. All placentas of each genotype in a litter were pooled and homogenized prior to analysis.

Fig. 2.

Trophoblast-specific overexpression of Slc7a5 increases fetal and placental weights at E18.5. a–c) Fetal weight, placental weight, fetal/placental ratio in NSS and Slc7a5 overexpression group, n = 12 litters/group. For fetal and placental data, the means of each genotype in a litter were calculated and used in statistical analysis. Each dot represents individual value; *P < 0.05 vs. NSS; paired Student's t test. d) Frequency distribution of individual fetal weights in NSS (n = 69) and Slc7a5 OX (n = 62) groups. Gaussian curves fitted by least-squares nonlinear regression and compared by extra sum-of-squares F-test (P = 0.001): NSS amplitude 22.57%, mean 940 mg, SD 176; Slc7a5 OX amplitude 20.9%, mean 1069 mg, SD 183 mg. The dotted vertical line indicates the 90th percentile of NSS fetal weights (1159 mg).

Fig. 3.

Protein expression of system L amino acid transporter isoform LAT1. a, b) and activity of system L c) and system A d) amino acid transporters in TPM isolated from NSS and Slc7a5 overexpression placentas. a) Representative western blot is shown for L-type amino acid transporter (LAT1, 42 kDa) in TPM isolated from NSS and Slc7a5 OX mice placenta at E 18.5. b) The histogram summarizes the western blotting data (NSS, n = 6; Slc7a5, n = 6). After normalization to total protein, the mean density of NSS samples was assigned an arbitrary value of 1. Subsequently, individual NSS and Slc7a5 group density values were expressed relative to this mean. c) System L and A d) System transporter activities were determined using isotope-labeled substrates and rapid filtration techniques in TPM isolated from NSS and Slc7a5 OX mice placenta at E 18.5. (NSS, n = 6; Slc7a5 OX, n = 6). Each dot represents individual value; *P < 0.05 vs. NSS; paired Student's t test. All placentas of each genotype in a litter were pooled and homogenized prior to analysis.

Fig. 4.

Trophoblast-specific overexpression of Slc7a5 increases transplacental ³H-Leucine transport. Unidirectional maternal–fetal clearances for a) ³H-Leucine and b) ¹⁴C-MeAIB were measured in anesthetized dams. Each dot represents individual value. *P < 0.05 vs. NSS, paired Student's t test, n = 5/each group.

Fig. 5.

Trophoblast-specific overexpression of Slc7a5 activates ribosomal S6 phosphorylation at serine 235/236. a) Effect of trophoblast-specific overexpression of Slc7a5 on placental a) S6Serine-235/236 and b) total S6 protein expression at E 18.5. Representative western blots of S6Serine-235/236 and total S6 expression in placental homogenates of NSS and Slc7a5 overexpression placentas. Protein abundance was normalized to total capillary protein measured using the total protein detection module (AM-PN01, Protein Simple, San Jose, CA, USA). Equal loading was performed. Summary of the western blot data. Each dot represents individual value, n = 6/each group. *P < 0.05 vs. NSS, paired Student's t test. All placentas of each genotype in a litter were pooled and homogenized prior to analysis.

Fig. 6.

Trophoblast-specific overexpression of Slc7a5 activates phosphorylation of placental PKCα at tyrosine-657 and Akt at serine-473. a) Effect of trophoblast-specific overexpression of Slc7a5 on placental a) PKCαTyrosine-657, b) total PKCα, c) AktSerine-473, d) AktThreonine-308, e) total Akt, f) SGKSerine-422, and g) total SGK protein expression at E 18.5. Representative western blots of PKCαTyrosine-657, total PKCα, AktSerine-473, AktThreonine-308, total Akt, SGKSerine-422, and total SGK expression in placental homogenates of NSS and Slc7a5 overexpressed placentas. Equal loading was performed. Summary of the western blot data. Each dot represents individual value, n = 5–6/each group. *P < 0.05 vs. NSS, paired Student's t test. All placentas of each genotype in a litter were pooled and homogenized prior to analysis.

Fig. 7.

Direct mechanistic link between transplacental transport of essential amino acids and fetal growth. Trophoblast-specific overexpression of system L amino acid transporter Slc7a5 increases the abundance of system L amino acid transporter isoform LAT1 in the TPM, which promotes the transplacental transport of essential amino acids and increases fetal growth.