Article

ISSN 0006-2979, Biochemistry (Moscow), 2024, Vol. 89, No. 10, pp. 1711-1726 © Pleiades Publishing, Ltd., 2024.

1711

Placental Transport of Amino Acids in Rats

with Methionine-Induced Hyperhomocysteinemia

Yulia P. Milyutina

1,a

*, Gleb O. Kerkeshko

, Dmitrii S. Vasilev

1,2

, Irina V. Zalozniaia

Sergey K. Bochkovskii

, Natalia L. Tumanova

, Anastasiia D. Shcherbitskaia

Anastasiia V. Mikhel

, Gulrukhsor H. Tolibova

, and Alexander V. Arutjunyan

D. O. Ott Research Institute of Obstetrics, Gynecology, and Reproductive Medicine, 199034 St.Petersburg, Russia

I. M. Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences,

194223 St.Petersburg, Russia

e-mail: milyutina1010@mail.ru

Received December 2, 2023

Revised April 11, 2024

Accepted May 2, 2024

Abstract— Maternal hyperhomocysteinemia (HHcy) is a risk factor for intrauterine growth restriction presumably

caused by a decrease in the placental transport of nutrients. We investigated the effect of experimental HHcy

induced by daily methionine administration to pregnant rats on the free amino acid levels in the maternal and

fetal blood, as well as on morphological and biochemical parameters associated with the amino acid transport

through the placenta. HHcy caused an increase in the levels of most free amino acids in the maternal blood on

gestational day 20, while the levels of some amino acids in the fetal blood were decreased. In rats with HHcy,

the maternal sinusoids in the placental labyrinth were narrowed, which was accompanied by aggregation of

red blood cells. We also observed an increase in the neutral amino acid transporters (LAT1, SNAT2) protein

levels and activation of 4E-BP1, a downstream effector of mTORC1 complex, in the labyrinth zone. Maternal

HHcy affected the placental barrier permeability, as evidenced by intensification of the mother-to-fetus transfer

of Evans Blue dye. The imbalance in the free amino acid levels in the maternal and fetal blood in HHcy may be

due to the competition of homocysteine with other amino acids for common transporters, as well as a decrease

in the area of exchange zone between maternal and fetal circulations in the placental labyrinth. Upregulation

of the neutral amino acid transporter expression in the labyrinth zone may be a compensatory response to an

insufficient intrauterine amino acid supply and fetal growth restriction.

DOI: 10.1134/S0006297924100055

Keywords: maternal hyperhomocysteinemia, placenta, placental transport, placental barrier, amino acids, mTOR,

amino acid transporters

Abbreviations: 4E-BP1, eukaryotic translation initiation factor 4E-binding protein 1; 5-HT, serotonin; AA, amino acid;

FPP, fetal-faced placental part; GD,gestational day; HHcy, hyperhomocysteinemia; Hcy, homocysteine; LAT, L-type amino

acid transporter; MPP,maternal-faced placental part; mTOR,mammalian target of rapamycin; mTORC1,mammalian target

of rapamycin complex1; S6, ribosomal proteinS6; SNAT,sodium-coupled neutral amino acid transporter; STB,syncytiotro-

phoblast; TGC,trophoblast giant cell.

* To whom correspondence should be addressed.

INTRODUCTION

One of consequences of the elevated content of

the non-proteinogenic amino acid homocysteine (Hcy),

an intermediate metabolite of methionine, in the moth-

er’s blood during pregnancy is fetal growth restriction

and reduced birth weight [1-3]. The negative effects of

maternal hyperhomocysteinemia (HHcy) on the fetal

growth and development likely associated with the tox-

ic effects of high Hcy levels are still poorly understood.

Placenta plays a key role in the regulation of nu-

trient distribution between mother and fetus during

MILYUTINA et al.1712

BIOCHEMISTRY (Moscow) Vol. 89 No. 10 2024

pregnancy, as it integrates the signals on the maternal

and fetal nutrient availability [4]. The main placental

sensor of the nutrient level in the mother’s blood is

the mTORC1 complex containing mammalian target

of rapamycin (mTOR) as a catalytic unit [5,6]. Mater-

nal nutritional deficiencies, stress, and placental hy-

poxia result in reduction of the mTORC1 activity in

the placenta and cause a corresponding decrease in

the expression of placental nutrient transporters [7].

An excess of nutrients in the maternal blood, on the

contrary, results in the activation of placental mTORC1

accompanied by an increase in the number of placen-

tal transporters [8]. It has been suggested that in ad-

dition to signals from the maternal side, the transport

function of the placenta is regulated by nutrition sta-

tus-related signaling from the fetal side, which allows

to compensate a decrease in the available nutrients in

the fetal blood by increasing expression and activity of

placental transporters [7,9].

An adequate placental transport of amino ac-

ids(AAs) from mother to fetus is vital for fetal growth

[10]. The rate of AA transport through the placen-

ta is determined by the rate of maternal placental

blood flow, surface area of the exchange zone be-

tween the maternal and fetal circulations, and activ-

ity and number of AA transporters in this zone [10].

In rodents, such an exchange zone and at the same

time a placental barrier is a continuous two-layered

syncytiotrophoblast (STB) lining sinusoids filled with

maternal blood in the labyrinthine region of the pla-

centa. The expression and activity of the neutral AA

transporters on the STB membranes are controlled by

the mTORC1 complex [11].

Experimental studies have shown that HHcy is ac-

companied by changes in the free AAs profiles in the

blood and cerebral spinal fluid of male mice, as well

as in the blood of pregnant rats and brains of their

fetuses [12-16]. It has been hypothesized that Hcy can

inhibit the mother-to-fetus neutral AA transport by

competing with AAs for common transporters on the

STB membranes [17, 18], and, consequently, affect the

levels of AAs in the maternal and fetal blood. Methi-

onine and, possibly, Hcy are able to change the activi-

ty of mTORC1 by modulating the intracellular content

of mTORC1 activator S-adenosylmethionine [24, 25].

Thestudies conducted on brain tissues or neural cells

have demonstrated that elevated Hcy levels can influ-

ence the mTORC1 activity [19-23]. At the same time, the

effect of maternal HHcy on the activity of mTORC1 and

levels of AA transporters in the placenta remains poor-

ly studied. No changes in the activity of mTORC1 under

the influence of increasing Hcy concentration were ob-

served in primary human trophoblast cells [26]; on the

other hand, administration of methionine to animals in-

creases the activity of mTORC1 and upregulated expres-

sion of AA transporters in the placental tissues [27,28].

Thus, HHcy can affect the transport of AAs from

mother to fetus via the competition of Hcy with other

AAs for common placental transporters, and through

the change in the activity of the mTORC1 complex and

mTORC1-regulated expression of AA transporters on

the STB membranes. On the other hand, due to the pre-

sumed existence of fetal feedback, the compensatory

increase in the expression and/or activity of placental

AA transporters may occur in response to the decreas-

ing of fetal blood AA concentrations and fetal growth

retardation. Furthermore, the possibility of direct tox-

ic effects of high Hcy levels on placental cells, which

could result in the delay in placental development

and the decrease of its transport and barrier function,

should be also considered. In order to test these hy-

potheses, we investigated the effects of methionine-in-

duced HHcy on the free AAs content in the blood of

pregnant rats and their fetuses, placental mTORC1 sig-

naling and neutral AA transporters expression in the

placental labyrinth, as well as on the morphology of

placenta and placental barrier integrity.

MATERIALS AND METHODS

Animals. Female 3 to 4-month-old Wistar rats

(n = 33; Rappolovo Animal Facility, Russia) with the

initial body weight of 220-290 g and stable estrous

cycle of 4 days, were used in the experiments. Thean-

imals were housed at the animal facility with a forced

ventilation and controlled 12-h light/dark cycle (day-

time, 7.00-19.00; nighttime, 19.00-7.00) at a constant

temperature of 20-21°C and 75-85% humidity. Therats

received a standard feed for laboratory animals

(Provimi, Russia) and filtered water ad libitum. All ex-

periments were conducted in accordance with the Di-

rective of European Union (86/609/EEC) on protection

of animals used in experimental studies.

Model of maternal HHcy. Experimental mater-

nal HHcy was induced by chronic methionine admin-

istration to pregnant animals according to the meth-

od previously developed in our laboratory [29, 30].

The day after detection of spermatozoa in the vaginal

smear was considered as the gestational day1 (GD1).

Pregnant animals were divided into control (n = 15)

and experimental (HHcy; n = 18) groups. Experimen-

tal animals received daily 1ml of aqueous solution of

L-methionine [AJI92, USP30 (TLC); Khimmed, Russia]

prepared ex tempora. L-Methionine was administered

perorally at 0.6 g/kg of animal body weight via oral

gavage from GD4 to GD19. Control animals received

1 ml of water administered the same way for the same

period oftime.

Evaluation of weight gain in pregnant rats.

In order to determine the total weight gain, female

rats were weighted prior to mating and on GDs 4, 10,

PLACENTAL TRANSPORT OF AMINO ACIDS IN HHcy 1713

BIOCHEMISTRY (Moscow) Vol. 89 No. 10 2024

14, and 20. The obtained data were used to plot the

dependence of body weight on the gestation period

for each animal. The slopes of the obtained plots were

compared for the control and experimental groups.

Animals with total number of fetuses between 11 and

16 were included in the analysis. The average number

of fetuses in the animals from the control and experi-

mental groups did not differ.

Tissue collection for analysis. Pregnant rats

from both groups were sacrificed by decapitation with-

out anesthesia using a guillotine for laboratory rodents

(Open Science, Russia) on GD20, 24 h after the last

administration of methionine, and their trunk blood

was collected. Fetuses and placentas were isolated

and weighted. The fetuses were decapitated and their

trunk blood was collected (blood from the entire litter

of each dam was pooled together). Placentas for his-

tology examination were immediately placed into 10%

neutral buffered formalin solution (pH7.2) (Sintakon,

Russia). Blood serum was obtained by centrifugation

(10min at 2300g). Blood serum samples and placenta

tissues for biochemical analysis were stored at –80°C.

HPLC analysis of the free AAs in the maternal

and fetal blood serum was carried out according to the

method described by Askretkov et al. [31]. An AccQ-

Fluor Reagent Kit (Waters, USA) was used for AA deri-

vatization. Standard solution of the 17 AA (Ala, Arg,

Asp, Cystine, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro,

Ser, Thr, Tyr, Val) (Waters), individual standard solu-

tions of Asn, Gln, Trp, and internal standard alanyl-glu-

tamine peptide were used. Analysis was carried out

with a Dionex Ultimate 3000 HPLC system (Thermo

Fisher Scientific, Germany) equipped with an FLD-3100

fluorescence detector. To analyze biological samples,

50 µl of the internal standard and 50 µl of the blood

serum were mixed. To precipitate proteins, acetonitrile

was added to a sample at a 1 : 1 ratio and the mix-

ture was centrifuged for 5 min at 10,000g. An aliquot

(10 µl) of the supernatant was mixed in a vial with

70 µl of AccQ-Fluor Borate Buffer (Waters) from the

derivatization reagent kit and 20µl of 10mMsolution

of 6-aminoquinoline-N-hyroxysuccineimidyl carbamate

(AQC; Waters) in acetonitrile; the mixture was incubat-

ed for 1min at room temperature and then for 10min

at 50°C. The vials were placed into an autosampler of

the chromatograph and AAs were separated on a re-

versed-phase AccQ-Tag column (3.9 × 150 mm; particle

size, 4 µm; Waters) in a gradient mode at a mobile

phase flow rate of 1 ml/min (injected sample volume,

2 µl; column temperature, 37°C). Detection was car-

ried out based on the fluorescence intensity at 395 nm

(λ

 = 250 nm). The obtained chromatograms were pro-

cessed using the Chromeleon7.0 chromatography data

system (Thermo Scientific, USA) and Microsoft Office

Excel. The content of the following 17 individual AA

in maternal and fetal serum was determined: His, Ile,

Leu, Lys, Met, Phe, Thr, Trp, Val, Asn, Asp, Gln, Glu, Gly,

Ser, Tyr, and Pro (the method used did not allow sepa-

rate quantification of Ala and Arg, hence, a combined

content Ala + Arg was determined). The ratio of fetal

blood content to maternal blood content for each AA

was also calculated.

Immunoblotting was used to investigate the ef-

fect of maternal HHcy on the content of neutral AA

transporters in the placenta and on the activation of

signaling pathways controlled by the mTORC1 complex.

One placenta from each dam in the group was used for

analysis. Prior to analysis, frozen placenta tissues were

washed from blood with cold 10mM phosphate buffer

(pH7.4) and separated into two parts: maternal-faced

placental part (MPP) including the basal zone (spon-

giotrophoblast) with thin layer of decidua basalis, and

fetal-faced placental part (FPP) containing the laby-

rinth zone. Placental tissues were homogenized with a

glass Dounce homogenizer at a 1 : 2 (w/v) ratio in RIPA

buffer [50 mM Tris-HCl (pH8.1), 1% TritonX-100, 0.1%

sodium dodecyl sulfate (SDS), 0.5% sodium deoxycho-

late, 1 mM EDTA, 150 mM NaCl] supplemented with a

protease inhibitor cocktail (S8820; Sigma-Aldrich) and

phosphatase inhibitor cocktail (P5726; Sigma-Aldrich)

and centrifuged for 20 min at 16,000g and 4°C; the re-

sulting supernatant was used for analysis. Total pro-

tein concentration in the samples was determined us-

ing Bradford protein assay[32] with a NanoDrop One

spectrophotometer (Thermo Scientific). The samples

(50µg of protein) were separated by electrophoresis in

10% PAAG under denaturing condition according to the

Laemmli procedure in a Mini-Protean Tetra Cell (Bio-

Rad, USA) with a PowerPac HC power source (Bio-Rad)

and transferred onto a PVDF membrane (Bio-Rad) in a

semi-dry Trans-Blot Turbo Transfer System (Bio-Rad).

The membranes were blocked with 3% bovine serum

albumin (Sigma-Aldrich) in TBST buffer [50 mM Tris-

HCl; 150 mM NaCl; 0.1% (v/v) Tween 20; pH 7.5] and

incubated overnight at 4°C with the specific primary

antibodies against SNAT1 [SNAT1 (H-9) mouse mAb;

sc-137032, Santa Cruz Biotechnology, USA; 1 : 1000]

(55 kDa); SNAT2 [SNAT2 (G-8) mouse mAb; sc-166366,

Santa Cruz Biotechnology; 1 : 1000] (60 kDa); LAT1

[LAT1 (D-10) mouse mAb, sc-374232, Santa Cruz Bio-

technology; 1 : 1000] (45 kDa); LAT2 [LAT2 Rabbit Ab,

ab75610, Abcam, UK; 1 : 1000] (58 kDa); mTOR [mTOR

(7C10) rabbitmAb, 2983S, Cell Signaling, USA; 1 : 1000]

(289 kDa); phospho-mTOR [phospho-mTOR (Ser2448)

rabbit Ab, 2971S, Cell Signaling; 1 : 1000] (289 kDa);

4E-BP1 [4E-BP1 (53H11) rabbitmAb, 9644S, Cell Signal-

ing; 1 : 1000] (15-20 kDa); phospho-4E-BP1 [phospho-

4E-BP1 (Thr37/46) (236B4) rabbit mAb, 2855S, Cell

Signaling; 1 : 1000] (15-20kDa); S6 [S6 ribosomal pro-

tein (5G10) rabbitmAb, 2217S, Cell Signaling; 1 : 1000]

(32 kDa); and phospho-S6 [phospho-S6 ribosomal

protein (Ser235/236) (D57.2.2E) rabbit mAb, 4858S,

MILYUTINA et al.1714

BIOCHEMISTRY (Moscow) Vol. 89 No. 10 2024

Cell Signaling; 1 : 1000] (32 kDa). Next, the membranes

were incubated with the corresponding secondary goat

antibodies conjugated with horseradish peroxidase

(HRP) (1 : 1000; Bio-Rad), and the proteins were visu-

alized using a Clarity Western ECL Substrate (Bio-Rad).

The membranes were scanned with a ChemiDoc™

Touch Imaging system (Bio-Rad); the band intensity

was determined with the ImageLab5.2.1 software. The

obtained data were normalized either to the level of

glyceraldehyde 3-phosphate dehydrogenase (GAPDH)

[GAPDH (14C10) rabbit mAb, 2118S, Cell Signaling;

1 : 1000) or to the total protein content in the gel [33]

determined with the help of stain-free technology (Bio-

Rad) according to the manufacturer’s instructions.

Light microscopy. Placentas were fixed in 10%

neutral formalin solution for 24 h. Tissue dehydra-

tion was carried out with a vacuum infiltration pro-

cessor Histo-Tek VP1 (Sakura, Japan); paraffin blocks

were formed with a TES99 paraffin embedding cen-

ter (Medite, Germany). Tissue sections (3-4µm thick)

were prepared with a Rotary 3002 microtome (PFM,

Germany) and stained with hematoxylin-eosin. Light

microscopy was used for examination of the placen-

tal labyrinth, basal zone (spongiotrophoblast), layer of

trophoblast giant cells (TGCs), and blood cells. Micro-

photographs were acquired at 200× and 400× magnifi-

cation with a system consisting of a light microscope

Olympus CX43 (Olympus, Japan), color digital camera

VideoZavr Standart VZ-18C23-B, and the VideoZavr

Catalog database (ATM-praktika, Russia). Images with

tissue defects, staining defects, and artefacts were

eliminated from analysis. Quantitative morphometric

analysis was carried out with the VideoTest-Morphol-

ogy5.2 program (Videotest, Russia). The trophoblastic

septa (tissue between the maternal sinusoids and fetal

capillaries) cross-sectional area in the placental laby-

rinth, as well as TGCs nuclei cross-sectional area were

determined in each section in 5-7 fields of view, and

the relative area of the investigated component (ratio

of the component area to the total sample area) was

calculated according to equation(1):

S (%) = (S component/S total) × 100. (1)

Analysis of the cross-sectional area of erythrocyte

aggregates in the labirynth zone was carried out in the

areas of 1mm

in 3-4 fields of view. Individual blood

cells were not included in analysis; maternal and fetal

blood spaces were not differentiated.

The thickness of the basal zone and placental la-

birynth was evaluted in the whole placenta cross-sec-

tions performed at the level of umbicial cord attach-

ment. The ratio of the basal zone thickness to the

labirynth zone thickness was determined.

Electron microscopy. Placenta tissues were

washed with physiological saline to remove blood,

immersed into a fixing solution (0.1 M PBS, pH 7.4,

containing 1% glutaraldehyde and 1% formaldehyde)

for2h, and then additionally fixed in 1% OsO

for1 h.

Tissue samples were contrasted with uranyl acetate,

dehydrated, and embedded into an epon resin accord-

ing to the protocol described in our previous studies

[34, 35]. Ultrathin sections (thickness, 50 nm) were

prepared with a Leica ultramicrotome (Leica Microsys-

tems, Germany) and examined with a transmission

electron microscope FEI Tecnai Spirit V2 (FEI, USA).

Two placentas (each from different animal) from each

group (control and experimental) were analyzed.

Placental barrier permeability was evaluated

using a technique based on fluorometric detection of

the amount of Evans Blue dye penetrated into the fetal

tissues [36,37]. Pregnant rats in the control and exper-

imental groups were injected under Zoletil anesthesia

(Zoletil 100; Virbac, France; 30 mg/kg of body weight)

with solution of Evans Blue (45 mg in 0.5 ml of water)

through the tail vein catheter on GD20 (3 h prior to de-

capitation). The animals were decapitated, the fetuses

were taken out, and their brains and abdominal cavity

organs (intestine, stomach, pancreas, etc., with excep-

tion of liver) were isolated for further investigation.

Samples of isolated organ were immersed into forma-

mide (6 ml per 1 g of organ weight), homogenized in

this solution, and incubated for 18 h at 60°C to extract

Evans Blue. The homogenates were centrifuged for

30 min in at 10,000 rpm using a Microspin12 centri-

fuge (BioSan, Latvia), and the supernatants containing

formamide with extracted Evans Blue were collected.

Fluorescence intensity of each sample was measured

in two replicates with a CLARIOstar plus plate read-

er (BMG Labtech, Germany) at the excitation wave-

length of 620 nm and emission wavelength of 670 nm.

The average fluorescence value for each sample was

corrected for the control sample (pure formamide)

fluorescence. To eliminate the possibility of erroneous

measuring tissue autofluorescence, the homogenates of

the corresponding fetal tissue from animals injected

with 0.5 ml of physiological saline without Evans Blue

were used as a negative control.

Statistical data processing. The differences be-

tween the groups were evaluated with the STATISTICA

10.0 and GraphPad Prism 8 programs. The outliers

were identified using the Grubbs test. Thedata were

examined for the normality of distribution using the

Shapiro–Wilk test; the homogeneity of dispersions

was analyzed with the Levene’s test. Depend ing on

the type of parameter distribution in the datasets,

control and experimental groups were compared

using the Student’s t-test (normal distribution, ho-

mogeneous dispersions), Welch’s t-test (normal dis-

tribution, non- homogeneous dispersions), or non-

parametric Mann–Whitney U-test (non-normal distri-

bution). Significance threshold p-value was set at 0.05.

PLACENTAL TRANSPORT OF AMINO ACIDS IN HHcy 1715

BIOCHEMISTRY (Moscow) Vol. 89 No. 10 2024

The study results inthe figures and tables are present-

ed either as mean ±standard error of mean (SEM) or

median and quartiles (Me(Q

;Q

)).

RESULTS

Effect of methionine-induced HHcy on the body

weight of pregnant rats and weight of their placen-

tas and fetuses. The methionine load did not cause

any significant changes in the weight gain of pregnant

rats. The slopes of the curves describing the depen-

dence of body mass on the gestational period were

5.20 ± 1.17 and 5.30 ± 1.24 in the control and experi-

mental group, respectively. At the same time, maternal

HHcy caused a significant reduction in the weight of

fetuses and placentas on GD20 (Table 1).

Changes in the content of free AAs in the blood

of female rats and their fetuses in methionine-

induced maternal HHcy. Pregnant rats with the me-

thionine-induced HHcy demonstrated an increase in

the serum level of the majority of essential (His, Ile,

Val, Lys, Phe, Trp) and non-essential (Ala+ Arg, Asp,

Gln, Tyr, Pro) AAs on GD20, which was also manifest-

ed as an increase in the total AA content (Table 2).

On the contrary, the concentration of Asn in the ma-

ternal blood under the HHcy conditions is decreased.

Fetal serum levels of some essential (Met, Trp) and

non-essential (Gln, Ser) AAs were reduced in the case

Table 1. Effect of maternal HHcy on the fetal and pla-

cental weight on GD20

Fetal weight (mg) Placental weight (mg)

Control HHcy Control HHcy

4182.2±

±79.7

3491.3±

±101.9***

570.1±

±17.4

472.1±

±8.1***

Note. The data are presented as mean±SEM for the average

mass of all fetuses or placentas in a litter from each dam;

n= 13, dams in the control group, n= 16, dams in the group

with HHcy; ***p<0.001, Student’s t-test.

Fig. 1. Effect of maternal HHcy on the free AAs serum levels in pregnant rats and their fetuses, and on the ratio of fetal to

maternal AAs serum content on GD20. Changes are shown as percent relatively to the control group; the data are present-

ed as Me (Q

; Q

); n = 11, dams in the control group; n = 10, dams in the HHcy group; * p < 0.05, ** p < 0.01, *** p < 0.001

in comparison with the control; t-test or Mann–Whitney U-test.

MILYUTINA et al.1716

BIOCHEMISTRY (Moscow) Vol. 89 No. 10 2024

Table 2. Serum concentrations of free amino acids (µmol/liter) in pregnant rats and their fetuses on GD20 under

normal conditions and in the case of methionine-induced maternal HHcy

Essential AAs

Maternal blood Fetal blood

Control HHcy Control HHcy

His 27.4±3.0 40.4±3.7* 69.0±12.2 84.9±10.1

Ile 90.1±4.5 132.1±7.5*** 255.5±15.2 262.9±15.2

Leu 107.3±8.6 120.3±5.8 249.3±14.9 295.1±11.6*

Val 117.1±9.6 153.5±11.3* 373.7±19.5 445.8±11.6**

Lys 462.1±28.4 552.2±24.8* 867.2±45.1 1023.4±38.5*

Met 44.4±1.6 42.9±1.2 133.9±5.8 102.4±4.5***

Thr 530.5±28.2 619.0±46.3 1063.4±51.7 1133.1±45.1

Phe 58.0±2.8 65.6±2.5* 247.6±11.3 241.5±10.6

Trp 51.1±6.7 74.4±6.7* 143.1±9.9 102.9±5.1**

Branched-chain AAs

(Ile+Leu+Val)

301.2±17.7 405.9±23.1* 878.4±43.7 1003.7±31.7*

Total essential AAs 1434.0±69.7 1800.4±98.8* 3402.5±86.2 3682.9±105.1*

Non-

essential AAs

Ala+Arg 845.0±28.4 993.6±43.3* 1464.0±55.6 1562.2±78.7

Asp 22.2±2.8 36.7±3.0** 68.6±5.6 55.4±3.3

Asn 79.5±2.7 50.3±3.6** 103.2±6.3 123.3±9.4

Glu 120.7±9.8 131.9±7.0 362.4±25.8 339.8±21.6

Gln 859.3±31.9 1069.5±54.6** 1655.9±79.1 1377.3±90.9*

Gly 170.9±11.3 201.2±15.1 356.5±24.2 355.1±24.2

Ser 290.1±12.4 314.5±4.8 441.3±18.4 380.8±13.2*

Tyr 30.2±2.0 40.1±2.7* 171.7±18.7 172.9±7.6

Pro 206.4±9.9 317.0±15.3*** 536.4±18.9 559.7±25.0

Total non-essential AAs 2624.2±68.3 3154±86.5** 5160.0±163.1 4926.5±205.6

Total AA concentration 4109.7±127.5 4955.1±115.5*** 8562.5±203.8 8618.2±245.3

Note. The data are presented as mean± SEM; n= 11, dams in the control group; n=10, dams in the HHcy group; *p<0.05,

**p<0.01, ***p<0.001 in comparison with the control; t-test or Mann–Whitney U-test.

of maternal HHcy, while the content of essential AAs

Val and Lys was increased. The total content of essen-

tial AAs in the serum of these fetuses was higher than

in the control; at the same time, the total amount of

non-essential AAs, as well as the total amount of all

AAs did not change significantly. In the case of ma-

ternal HHcy, the ratio between the fetal and maternal

concentrations of AAs was decreased (Ile, Met, Trp,

Asp, Gln, Pro) or not changed (Asn was an exception,

as the ratio for this AA was increased) (Fig.1).

Effect of methionine-induced maternal HHcy

on the neutral AA transporters content in the la-

birynth zone of rat placenta. In the case of methi-

onine-induced HHcy, we observed an increase in the

content of the sodium-dependent neutral AA transport-

er2 (SNAT2; p < 0.01) and the L-type AA transporter1

PLACENTAL TRANSPORT OF AMINO ACIDS IN HHcy 1717

BIOCHEMISTRY (Moscow) Vol. 89 No. 10 2024

Fig. 2. Effect of maternal HHcy on the neutral AA transporters content in the labyrinth zone of rat placenta on GD20. The

contentof SNAT1, SNAT2, LAT1, and LAT2 proteins in the FPP in the control and HHcy groups(a), and representative immu-

noblot(b); y-axis, intensity of protein bands in immunoblots, arbitrary units; the data are shown as mean±SEM; n=4-8 dams

in each group; *p<0.05, Mann–Whitney U-test; **p<0.01, Student’s t-test.

Fig. 3. Effect of maternal HHcy on mTORC1 signaling in different parts of rat placenta on GD20. The content of mTOR,

4E-BP1, and S6 proteins and their phosphorylated forms p-mTOR

Ser2448

, p-4E-BP1

Thr37/46

, and p-S6

Ser235/236

in the control and

HHcy groups in the MPP (a) and FPP (c), and representative immunoblots/gels for MPP (b) and FPP (d); y-axis, intensity of

protein bands in immunoblots, arbitrary units; the data are shown as mean ± SEM; n = 10 dams in each group; **p < 0.05,

Welch’s t-test; ****p<0.001, Student’s t-test.

of large neutral AAs (LAT1; p < 0.05) in the FPP (la-

birynth zone) in pregnant rats on GD20 in comparison

with the control animals (Fig.2,a andb). At the same

time, the level of the SNAT1 and LAT2 transporters in

the FPP in the experimental and control animals did

not differ significantly(Fig.2,a andb).

MILYUTINA et al.1718

BIOCHEMISTRY (Moscow) Vol. 89 No. 10 2024

Fig. 4. Morphological changes in the placenta in rats with maternal HHcy on GD20. a,b) Labyrinth zone of placenta with

trophoblastic septa (TS) and maternal sinusoids (MS) in the control (a) and HHcy (b) animals. Insets show the components

which relative areas were calculated: TS and erythrocyte aggregates (EA); hematoxylin-eosin staining; magnification, ×200.

c) Relative area (S%) of TS in the controland HHcygroup. d-f) TEM images of placenta in the control (d) and HHcy (e and f)

animals; mb,maternal blood in maternal sinusoid; fc,fetal capillary; er,erythrocytes in maternal sinusoids (d and e) and fetal

capillary (f); en, fetal capillary endothelium. g) Area of EA in the labyrinth zone of placenta in the control and HHcy group.

h, i) Trophoblast giant cells (TGCs) in the control (h) and HHcy (i) animals; hematoxylin-eosin staining; magnification, ×400;

TGCs nuclei are shown with arrows. j) Relative area (S%) of TGCs nuclei in the control and HHcy group. k) Thickness of

the labyrinth zone(LZ) and basal zone (BZ) of the placenta. l) BZ thickness to LZ thickness ratio. All data are presented as

mean±SEM. In panels (c) and (j), n = 3 placentas from 3 dams in the control group, n = 14 placentas from 4 dams in the HHcy

group; in panels (g), (k), and(l), n = 9 placentas from 5 dams in the control group, n = 18 placentas from 6 dams in the HHcy

group; ** p < 0.01, Student’s t-test.

Effect of methionine-induced maternal HHcy

on the activity of mTORC1 signaling in different re-

gions of rat placenta. The content of mTOR protein

was increased (p < 0.05) in the MPP (contains mainly

the bazal zone of placenta) in rats with the exper-

imental HHcy on GD20; however, no changes in the

phosphorylated mTOR or downstream components of

the mTORC1 signaling cascade were observed (Fig. 3, a

and b). HHcy did not affect the extent of mTOR phos-

phorylation or the content and phosphorylation lev-

el of the ribosomal protein S6 (downstream effector

of the mTORC1 complex) in the FPP (labirynth zone);

however, we observed activation of eukaryotic trans-

lation initiation factor 4E-binding protein 1 (4E-BP1),

another downstream target of mTORC1, which was

manifested as an increase in its phosphorylation at

Thr37/46 (p < 0.001; Fig.3,c andd).

Structural changes in placental tissues in ma-

ternal HHcy. The placental labyrinth (corresponding

to FPP) is an exchange zone between the maternal and

fetal circulations. In control on GD20 it composed of

a branched network of thin intersecting trophoblas-

tic septa which contain the trophoblast cells and fe-

tal blood capillaries, and the wide maternal sinusoids

filled with maternal blood (Fig. 4,a andb). In animals

with HHcy, the trophoblastic septa in the labyrinth

zone were thickened in comparison with the control

(p < 0.01; Fig. 4,b andc); correspondingly, the relative

area of the sinusoids was decreased. Narrowing of ma-

ternal sinusoids was accompanied by the blood stasis

PLACENTAL TRANSPORT OF AMINO ACIDS IN HHcy 1719

BIOCHEMISTRY (Moscow) Vol. 89 No. 10 2024

Fig. 5. Effect of maternal HHcy on the ultrastructure and permeability of rat placental barrier on GD20. a-c) TEM images of the

exchange zone between the maternal and fetal circulatory systems in the placental labyrinth; mb,maternal blood in maternal

sinusoid; fc, fetal capillary; placental barrier layers: I,cytotrophoblast layer in contact with maternal blood, II and III, two

STB layers; bm,basal membrane lining the fetal capillary endothelium(en); er,erythrocyte in the vessel lumen; arrow, tight

contact between endothelial cells; arrowhead,fenestra in the fetal capillary endothelium. d) Fluorescence of Evans Blue dye

in the tissues of fetal abdominal organs and brain 3h after dye injection to rat females; n = 12 placentas from 2 dams in the

control group, n = 15 placentas from 2dams in the HHcy group; *** p < 0.001, Welch’s t-test.

and erythrocyte aggregation (Fig. 4, a and b). Accumu-

lation of erythrocytes in the lumen of the smallest

maternal sinusoids and fetal cappilaries has been also

observed during examination of the labyrinth zone

ultrastructure (Fig. 4, d-f). The increase in the eryth-

rocyte aggregates area in the labyrinth zone was also

confirmed by the morphometric data (p < 0.01; Fig. 4g).

The basal zone of the placenta contains three

types of cells: spongiotrophoblasts, glycogen cells, and

trophoblast giant cells (TGCs) forming a thin layer at

the border between the basal zone and decidua basa-

lis and performing the endocrine function. In the con-

trol group, TGCs formed a layer with a thickness of

2-3 cells with the oxyphilic cytoplasm, roundish-oval

nuclei, and uniform chromatin distribution (Fig. 4h).

In the group with maternal HHcy, the TGCs layer was

thinner and consisted of 1-2 cells that demonstrated

vacuolar degeneration (Fig. 4i). In some samples from

the experimental animals, TGCs lacked the nuclei; some

individual cells displayed karyolysis. The relative area

of TGCs nuclei decreased in HHcy (p < 0.01;Fig.4j).

Animals with maternal HHcy on GD20 demon-

strated reduction in the thickness of the labyrinth zone

(p < 0.05; Fig. 4k), while the thickness of the basal zone,

as well as the ratio of the basal zone thickness to the

thickness of the labyrinth zone did not change signifi-

cantly(Fig.4,kandl).

Effect of maternal HHcy on the permeability of

placental barrier. The ultrastructural studies of the

exchange zone between the maternal and fetal circu-

lation in the placental labyrinth showed that in some

rats with HHcy, there was a disintegration of the three

layers comprising the placental barrier (I,cytotropho-

blast layer in contact with maternal blood; II,first STB

layer, and III, second STB layer in contact with the

basement membrane of fetal capillary endothelium),

which resulted in the formation of gaps between their

membranes (Fig.5,a andb). The number of fenestrae

closed by the diaphragms in the fetal vessels endotheli-

um is increased (Fig. 5c). Penetration of Evans blue dye

through the placental barrier was significantly higher

in animals with HHcy, which was manifested as an in-

creasing of the dye fluorescence in the fetal abdominal

organs and brain in the experimental group in com-

parison with the control (p < 0.001; Fig.5d).

DISCUSSION

Currently, administration of methionine, either

with food and drinking water or via an oral gavage,

is the most popular method for modeling HHcy in ani-

mals [38, 39]. In comparison with direct administration

of Hcy, it ensures higher intracellular levels of Hcy typ-

ical for clinical HHcy cases in humans [40]. However,

an unavoidable feature of this model is that, in addi-

tion to the increasing of the Hcy blood level, it leads

to simultaneous elevation of the methionine blood

content, which can affect the balance of free AAs in

blood [41]. In our previous study, we examined the dy-

namics of Hcy content in the blood of female rats and

their fetuses in our HHcy model that involved daily

administration of methionine through a gastric tube to

rat females [29]. The concentration of Hcy in the blood

of pregnant rats on GD20 (5.7 ± 0.2 µmol/liter before

the treatment) increased to 87.2 ± 24.2 µmol/liter al-

ready 1 h after oral administration of methionine and

was maintained at an approximately same high level

(81.4 ± 31.3 µmol/liter) for 6 h after its administration.

This was followed by a gradual decrease in the Hcy

content, which, however, remained significantly higher

(34.1 ± 14.7 µmol/liter) than in the control up to 18 h

after methionine loading and reached the level close

to the normal one (6.6 ± 0.5 µmol/liter) only after 24 h,

right before the next methionine administration [29].

Similar changes in the Hcy concentration were observed

MILYUTINA et al.1720

BIOCHEMISTRY (Moscow) Vol. 89 No. 10 2024

in the fetal blood after methionine administration to

mothers[29]. An increase in the Hcy level in the blood

after methionine administration should be preceded

by methionine transport into cells, its metabolic trans-

formation into Hcy, and export of the formed Hcy back

into the blood. Due to this, the level of methionine it-

self reaches its peak in the blood and returns to the

normal values prior to the corresponding changes in

the Hcy content [42]. Hence, the absence of increase

in the methionine concentration in the blood of rat fe-

males and their fetuses 24 h after methionine admin-

istration in our study is quite natural. We hypothesize

that in our model, the effects of the increased Hcy con-

tent, as a more long-term acting factor in comparison

with the short-term increase in the methionine level,

overcome the effects of methionine.

Consumption of AAs with food at the doses sig-

nificantly exceeding the requirements for these com-

pounds causes toxic effects in animals [43]. Moreover,

the adverse effect of the methionine excess in the diet

is more pronounced in comparison with the excess of

other AAs and is manifested primarily as a decrease

in food intake and organism growth suppression [43].

The fetuses of pregnant animals receiving food with

the excess of methionine also demonstrated growth

restriction [13, 44]. In our study, the weight gain in fe-

male rats exposed to methionine was the same as in

the control animals, which indicated the absence of sig-

nificant differences in the food consumption between

the groups. The toxic effect of methionine-induced

HHcy was manifested as a reduction in fetal weight,

which was in agreement with the results of our earlier

studies [29, 30].

We found that chronic administration of me-

thionine increased the content of the majority of es-

sential (His, Ile, Val, Lys, Phe, Trp) and non-essential

(Ala + Arg, Asp, Asn, Gln, Tyr, Pro) AAs in the blood of

female rats on GD20, which was also reflected as an in-

crease in the total level of essential and non-essential

AAs. Indeed, it has been reported before that the blood

content of AAs was increased in animals with HHcy

caused by genetic defects in enzymes involved in Hcy

metabolism or in those consuming food enriched with

methionine [12, 14-16]. Methionine-enriched diet con-

taining only 1.6-fold excess of methionine caused an

increase in the Hcy level in the mouse blood, while in

order to increase the concentration of methionine and

other AAs in the blood, the content of methionine in

food had to be increased up to 7-10-fold [16]. This fact

implies the existence of adaptive mechanisms aimed

at eliminating an excess of methionine in the blood,

primarily via its transformation into Hcy, which em-

phasizes the significance of Hcy as a key toxic agent

in the case of methionine loading.

It is possible that the increase of the level of free

AAs during methionine loading is mediated by some

metabolic mechanisms. For example, methionine ca-

tabolism through Hcy involves enzymes (propionyl-

CoA carboxylase) and intermediate products (propi-

branched-chain AAs catabolic pathways [45]. Anexcess

of one or several intermediates formed in the course

of methionine metabolism, could slow down the ca-

tabolism of other AAs sharing these pathways with

methionine. However, in our study, we observed in the

maternal blood increased levels not only of branched-

chain AAs, but also of other essential and non-essen-

tial AAs, whose metabolism is not directly associated

with the metabolism of methionine and Hcy. It has

been assumed that the excess of methionine and Hcy

in the blood could reduce the transport of other AAs

by competing with them for common AA transporters

[16, 17, 46]. Our data supports the hypothesis that me-

thionine-induced HHcy can cause the downregulation

of AA transport to the maternal cells and/or to the fetal

circulation through the placenta resulting in AAs accu-

mulation in the mother’s blood.

The total level of all AAs in the fetal blood did

not change in HHcy. Although the concentration of two

essential AAs (Val and Lys) in the fetal blood from the

HHcy group increased (similar to the maternal blood),

the content of Met, Gln, Trp, and Ser in the fetal blood

was significantly lower in comparison with the con-

trol. Moreover, the fetal/maternal blood content ratio

of Pro and Ala + Arg levels in the experimental group

was also decreased in comparison to the control.

The AAs, which levels was reduced in the fetal blood

under the HHcy condition, are the substrates of the

placental transport systems L(Met, Gln, Trp, Ala, Ser),

A (Met, Gln, Ala, Ser, Pro), and y

L (Arg, Gln, Met)

[17, 47]. It has been suggested that Hcy could be the

substrate of transporters of the system A (SNAT1,

SNAT2 and SNAT4), L (LAT1, LAT2), and y

L (y

LAT1,

LAT2) [17, 18], hence, the reduction in the aforemen-

tioned AAs content in the fetal blood is in agreement

with the hypothesis on the competitive inhibition of

their placental transport in maternal HHcy.

Gln and its precursor Glu are vital for fetal growth

and development. The concentration of Gln in mater-

nal and fetal blood, as well as the rate of its mother-

to-fetus transport, are the highest among other AAs

[48, 49]. The required levels of Gln and Glu in the fetal

blood are maintained due to the ability of these AAs to

interconvert and recirculate between the placenta and

fetal liver [48]. The observed decrease in the Gln level

in the fetal blood could be caused by its increased con-

sumption to compensate for the fetal growth restric-

tion in maternal HHcy.

Negatively charged AAs (Asp and Glu) are trans-

ported from mother to fetus by the excitatory amino

acid transporters of the EAAT group that belong to the

transport system X

 [47]. With methionine loading,

an increase in the Asp concentration in the mother’s

PLACENTAL TRANSPORT OF AMINO ACIDS IN HHcy 1721

BIOCHEMISTRY (Moscow) Vol. 89 No. 10 2024

blood was observed, which was accompanied by a de-

crease in the ratio of the Asp level in the fetal blood

to its content in the mother’s blood. Methionine and

Hcy are not the substrates of the EAAT transporters;

hence, changes in the Asp level cannot be caused by

the competition with Met and/or Hcy for the common

transporters. The increase in the Asp level with simul-

taneous decrease in the Asn content in the maternal

blood in HHcy can be associated with the decrease in

the Asn synthesis from Asp. Considering that the syn-

thesis of Asn from Asp requires Gln as the nitrogen

donor[50], the decrease in the Gln content in the fetal

blood in HHcy and simultaneous decrease in Asp fetal/

maternal blood content ratio accompanied by the in-

crease of this ratio for Asn could be interconnected.

As a precursor of serotonin (5-HT), maternal Trp

is very important for the development of the fetal ner-

vous system. In rodents, placenta starts to synthesize

5-HT from maternal Trp on GD10, and up to GD15,

placental 5-HT remains the main and, probably, only

source of this neurotransmitter in fetal brain [51, 52].

The decrease in the Trp concentration in fetal blood

observed in this study could result in the decrease of

5-HT synthesis in fetal brain, thus negatively affect-

ing brain development. Considering that in the rodent

placenta, 5-HT is synthesized predominantly by the

TGCs [53], the observed reduction in the number of

these cells could contribute to the decrease in 5-HT

available for the fetus.

In addition to the possible competition of Hcy

with other AAs for common transporters, HHcy-asso-

ciated morphological changes in the placenta could be

the reason for a decreased mother-to-fetus AA trans-

port [54]. In this study, we observed a decrease in the

placenta weight, thickness of the labyrinth zone, and

relative area of maternal sinusoids accompanied by

the slowing of circulation and increased erythrocyte

aggregation, which is a risk factor for the development

of microthrombosis [55]. The decrease in the area of

sinusoids, where maternal blood comes in contact with

the STB layer, as well as slowing down of placental cir-

culation result in the reduction of nutrient transport

through the transporters located on the STB mem-

branes [10]. Defects and deficiencies in the vascular

tree formation in the labyrinth zone, which mediates

the nutrient transport from a mother to a fetus, often

results in the fetal growth restriction [54]. Morpho-

logical changes detected in the labyrinth zone in our

study could, in part, be the consequences of the pre-

viously reported imbalance between angiogenic and

growth factors in the placenta in the case of maternal

HHcy[30].

As has been mentioned above, the mother-to-fetus

transport of neutral AAs through the placenta in ro-

dents and humans is realized primarily through the

system A and L transporters, whose activity and ex-

pression on the STB membranes are controlled by the

mTOR signaling [7, 17,56]. Low amounts of nutrients

in mother’s diet during pregnancy cause a decrease in

the activity of the mTORC1 complex in the placenta

and downregulation of AA transporters expression and

activity on the STB membranes, which is accompanied

by a reduction in the fetal weight [7]. On the other

hand, an excess of nutrients in the maternal blood

causes activation of the mTORC1 complex and upreg-

ulation of AA transporters expression in the placenta,

resulting in the increase in the fetal weight [57, 58].

The effects of maternal HHcy, which is characterized

by the excess of Hcy and, likely, some other AAs in

the maternal blood, but simultaneously accompanied

by the fetus growth restriction [1, 44], on activity of

placental mTORC1 complex have not been investigated

in detail.

Here, we attempted for the first time to evalu-

ate the effects of maternal HHcy induced by chronic

methionine loading, on activity of the mTORC1 com-

plex in different morphological parts of the placenta

using phosphorylation of the mTORC1 downstream

effectors 4E-BP1 and S6 as functional markers.

In the MPP, which is not directly associated with the

transport of nutrients to the fetus, mTOR acts mostly

as a stimulator of growth and functional maturation

via activation of protein synthesis [59]. The observed

increase in the total level of mTOR protein in the MPP

in animals with HHcy could be a compensatory re-

sponse to the reduction of the endocrine function of

this placental region which requires continuously high

level of protein synthesis [60]. In this study the num-

ber of TGCs cells, which are the main endocrine cells

in the MPP [61], was found to decrease in maternal

HHcy. The level of phosphorylated 4E-BP1 protein in

the labyrinth zone was increased; at the same time, no

changes in the expression of total mTOR protein or its

phosphorylated at Ser2448 form, as well as no activa-

tion of S6 protein (another target of mTOR) were ob-

served. At present, several mTOR phosphorylation sites

have been identified, but their functional significance

is still poorly understood [62]. It was shown that mTOR

is phosphorylated at Ser2448 by the downstream ki-

nase S6K1, which is also responsible for S6 protein

phosphorylation [63]. Hence, the absence of changes

in the levels of p-mTOR

Ser2448

and p-S6 could be due to

the unchanged activity of S6K1 kinase. Activation of

4E-BP1 could be caused by either activation of mTOR

through phosphorylation at other sites or by the action

of mTOR-independent kinases [64]. It should be men-

tioned that unidirectional and simultaneous changes

in the levels of p-mTOR, p-4E-BP1, and p-S6 are not

always detected in studies investigating effects of the

mTOR signaling on the fetal growth [5].

The observed in HHcy increase in the LAT1 and

SNAT2 neutral AA transporters content in the labyrinth

MILYUTINA et al.1722

BIOCHEMISTRY (Moscow) Vol. 89 No. 10 2024

zone could be caused by the 4E-BP1 signaling pathway

activation resulting in the increase in the correspond-

ing proteins synthesis and transport to the STB mem-

branes [11]. On the other hand, it has been suggested

that the increase of the number of AA transporters

in the placenta could be a compensatory response to

the reduction of the mother-to-fetus AA transport [7].

A compensatory increase in the expression of the Gln

transporter LAT1 has been observed in the placentas

with a reduced mass and, subsequently, decreased sur-

face area of exchange between the maternal and fetal

blood [48]. However, this adaptation is not always ca-

pable to fully replenish an insufficient AA supply to

the fetus, which results in the fetal growth restriction

even in the presence of a higher number of AA trans-

porters and increase in the relative transporting capac-

ity of the placenta [48].

The exchange zone between the maternal and fe-

tal blood in the rat placental labyrinth consists of three

trophoblast layers: permeable cytotrophoblast layer in

direct contact with the maternal blood in sinusoids

and two continuous STB layers underneath it, which

comprise the structural basis of the placental barrier

[56]. The integrity of the placental barrier is crucial

for normal fetal growth and development [65]. The ob-

served increase in the penetration of Evans Blue dye,

which is transported by albumin and other proteins

in maternal blood [66], into abdominal organs and

brain of the fetuses could result from the increased

permeability of the placenta for macromolecules [67].

Earlier, we demonstrated an increase in the levels of

the proangiogenic factor VEGF-A, a known inducer of

placental permeability [65, 67], in the FPP on GD20 in

rats with HHcy [30]. The morphological basis of the in-

creased permeability of the placental barrier could be

disintegration of its layers observed in some cases, as

well as increase in the number of fenestrae in the fetal

capillary endothelium. It was shown that some mater-

nal proteins could be transferred across the placental

barrier into the fetal circulation by endocytosis/exocy-

tosis across the STB or via the paracellular diffusion

through the STB nanopores [68, 69]. Proteins internal-

ized by macropinocytosis could be catabolized in STB

lysosomes and serve as additional sources of AAs for

the placenta and the fetus [70]. Such metabolic path-

way was shown for albumin internalization by the STB

membrane in the human placenta; in the process, mol-

ecules transported by this protein are released to the

intracellular STB compartments [71] and can possibly

be transported further to the fetal circulation. There-

fore, an increase in the penetration of Evans blue dye

into fetal tissues could also be a consequence of an

increase in the uptake of albumin and other dye-bind-

ing proteins by the STB to compensate for the require-

ments of the placenta and fetus for AAs in conditions

of its reduced growth.

This study was based on the hypothesis that ma-

ternal HHcy can inhibit the placental transport of

some AAs, which are shared with Hcy common trans-

port mechanisms mediated by the placental neutral

AA transport systems A and L. We did not investigate

the effect of HHcy on the expression of the y

L sys-

tem transporters, which presumably can also transport

Hcy [17]. The possible influence of sulfur-containing

compounds formed in the course of Hcy metabolism

on the placental transport of AAs was also left outside

our consideration. The main metabolite formed in the

process of the Hcy catabolism via transsulfuration is

Cys. It has been noted that an increase in the total Hcy

level in the blood could be accompanied with a de-

crease in the total content of Cys [16, 72]. In the blood,

Cys is present mostly in its oxidized dimeric form(cys-

tine), which is transported across the maternal-facing

STB membrane with the help of xCT/SLC7A11 transport-

er of the Xc

−

transport system [73]. In STB, cystine is

reduced to Cys and serves as a precursor for the syn-

thesis of glutathione (GSH), thus enhancing the antiox-

idant defense of the placenta [73]. Although GSH could

be formed in the process of Hcy transsulfuration, the

content of its reduced form and/or the ratio between

its reduced and oxidized forms in the blood and brain

are decreased in HHcy, which has been associated with

GSH oxidation caused by the HHcy-induced oxidative

stress[16,74]. The decrease in the total content of Cys

and GSH level in the maternal blood is accompanied

with the decrease in the GSH level and promotion of

the HHcy-induced oxidative stress in the placenta [30],

which could contribute to the negative effect of mater-

nal HHcy on the placental development and transport

function.

The results obtained in this study indicate that

maternal HHcy in rats causes poor vascular develop-

ment in the labyrinth zone of placenta, decrease in the

content of some AAs in the fetal blood, and increase

in the permeability of the placental barrier. Reduced

levels of some AAs in the fetal blood in HHcy can be

consequence of inhibition of their placental transport

due to competition with Hcy for common transporters,

as well as decreasing in the surface area of exchange

zone between maternal and fetal circulations due to

narrow maternal sinusoids. The signaling from the fe-

tal side about the decrease in the content of some AAs

in the fetal blood and fetal weigh reduction might in-

duce an adaptive response in the placenta manifested

as the activation of 4E-BP, a downstream effector of the

mTORC1 complex, and increase in the number of neu-

tral AA transporters LAT1 and SNAT2 in the labyrinth

zone of the placenta.

Acknowledgments. The authors are grateful to

A. N. Kadenov, A. V. Gorbova, and V. A. Poda for their

help in conducting experiments.

PLACENTAL TRANSPORT OF AMINO ACIDS IN HHcy 1723

BIOCHEMISTRY (Moscow) Vol. 89 No. 10 2024

Contributions. A.V.A. supervised the study and ed-

ited the manuscript; Yu.P.M. and G.O.K. developed the

study concept and wrote the text of the article; Yu.P.M.,

G.O.K., and D.S.V. discussed the study results; I.V.Z.,

S.K.B., A.V.M., G.Kh.T., N.L.T., and D.S.V. conducted the

experiments and collected the data; Yu.P.M., A.D.Sh.,

and D.S.V. analyzed the data.

Funding. This study was supported by the Russian

Science Foundation (project no.22-15-00393).

Ethics declarations. All applicable international,

national, and/or institutional guidelines for the care

and use of animals were followed. The authors of this

work declare that they have no conflicts of interest.

REFERENCES

1. Dai,C., Fei,Y., Li,J., Shi,Y., and Yang,X. (2021) Anov-

el review of homocysteine and pregnancy complica-

tions, Biomed. Res. Int., 2021, 6652231, https://doi.org/

10.1155/2021/6652231.

2. Gaiday, A., Balash, L., and Tussupkaliyev, A. (2022)

The role of high concentrations of homocysteine

for the development of fetal growth restriction, Rev.

Bras. Ginecol. Obstet., 44, 352-359, https://doi.org/

10.1055/s-0042-1743093.

3. Memon, S. I., Acharya, N. S., Acharya, S., Potdar, J.,

Karnik,M., and Mohammad,S. (2023) Maternal hyper-

homocysteinemia as a predictor of placenta-mediated

pregnancy complications: a two-year novel study, Cu-

reus, 15, e37461, https://doi.org/10.7759/cureus.37461.

4. Gaccioli, F., Lager, S., Powell, T. L., and Jansson, T.

(2013) Placental transport in response to altered ma-

ternal nutrition, J.Dev. Orig. Health Dis., 4, 101-115,

https://doi.org/10.1017/S2040174412000529.

5. Dong,J., Shin,N., Chen,S., Lei,J., Burd,I., and Wang,X.

(2020) Isthere a definite relationship between placen-

tal mTOR signaling and fetal growth? Biol. Reprod.,

103, 471-486, https://doi.org/10.1093/biolre/ioaa070.

6. Jansson, T., Aye, I. L., and Goberdhan, D. C. (2012)

The emerging role of mTORC1 signaling in placental

nutrient-sensing, Placenta, 33, e23-e29, https://doi.org/

10.1016/j.placenta.2012.05.010.

7. Chassen,S., and Jansson,T. (2020) Complex, coordinat-

ed and highly regulated changes in placental signal-

ing and nutrient transport capacity in IUGR, Biochim.

Biophys. Acta Mol. Basis Dis., 1866, 165373, https://

doi.org/10.1016/j.bbadis.2018.12.024.

8. Kramer, A.C., Jansson,T., Bale, T.L., and Powell, T.L.

(2023) Maternal-fetal cross-talk via the placenta: in-

fluence on offspring development and metabolism,

Development, 150, dev202088, https://doi.org/10.1242/

dev.202088.

9. Sibley, C. P., Brownbill, P., Dilworth, M., and Glazier,

J. D. (2010) Review: adaptation in placental nutri-

ent supply to meet fetal growth demand: implica-

tions for programming, Placenta, 31, S70-S74, https://

doi.org/10.1016/j.placenta.2009.12.020.

10. Lewis, R. M., Brooks, S., Crocker, I. P., Glazier, J.,

Hanson, M.A., Johnstone, E.D., Panitchob,N., Please,

C. P., Sibley, C. P., Widdows, K. L., and Sengers, B. G.

(2013) Review: modelling placental amino acid trans-

fer – from transporters to placental function, Pla-

centa, 34, S46-S51, https://doi.org/10.1016/j.placenta.

2012.10.010.

11. Rosario, F. J., Dimasuay, K. G., Kanai, Y., Powell, T. L.,

and Jansson, T. (2016) Regulation of amino acid

transporter trafficking by mTORC1 in primary hu-

man trophoblast cells is mediated by the ubiquitin

ligase Nedd4-2, Clin. Sci. (Lond), 130, 499-512, https://

doi.org/10.1042/CS20150554.

12. Matsueda,S., and Niiyama,Y. (1982) Theeffects of ex-

cess amino acids on maintenance of pregnancy and

fetal growth in rats, J.Nutr. Sci. Vitaminol. (Tokyo), 28,

557-573, https://doi.org/10.3177/jnsv.28.557.

13. Mori,M., Yamashita, Y., Hiroi,Y., Shinjo, S., Asato, R.,

Hirai,K., Suzuki, K., and Yamamoto, S. (1999) Effect

of single essential amino acid excess during pregnan-

cy on dietary nitrogen utilization and fetal growth

in rats, Asia Pac. J. Clin. Nutr., 8, 251-257, https://

doi.org/10.1046/j.1440-6047.1999.00094.x.

14. Akahoshi, N., Kamata, S., Kubota, M., Hishiki, T.,

Nagahata, Y., Matsuura,T., Yamazaki, C., Yoshida, Y.,

Yamada, H., Ishizaki, Y., Suematsu, M., Kasahara, T.,

and Ishii,I. (2014) Neutral aminoaciduria in cystathi-

onine beta-synthase-deficient mice; an animal mod-

el of homocystinuria, Am. J. Physiol. Renal. Physiol.,

306, F1462-F1476, https://doi.org/10.1152/ajprenal.

00623.2013.

15. Akahoshi, N., Yokoyama, A., Nagata, T., Miura, A.,

Kamata, S., and Ishii, I. (2019) Abnormal amino acid

profiles of blood and cerebrospinal fluid from cys-

tathionine beta-synthase-deficient mice, an animal

model of homocystinuria, Biol. Pharm. Bull., 42, 1054-

1057, https://doi.org/10.1248/bpb.b19-00127.

16. Ishii,I., Kamata,S., Ito,S., Shimonaga,A., Koizumi,M.,

Tsushima, M., Miura, A., Nagata, T., Tosaka, Y.,

Ohtani,H., Kamichatani, W., and Akahoshi, N. (2022)

A high-methionine diet for one-week induces a high

accumulation of methionine in the cerebrospinal

fluid and confers bipolar disorder-like behavior in

mice, Int. J. Mol. Sci., 23, 928, https://doi.org/10.3390/

ijms23020928.

17. Tsitsiou,E., Sibley, C.P., D’Souza, S.W., Catanescu,O.,

Jacobsen, D.W., and Glazier, J.D. (2011) Homocysteine

is transported by the microvillous plasma membrane

of human placenta, J. Inherit. Metab. Dis., 34, 57-65,

https://doi.org/10.1007/s10545-010-9141-3.

18. Tsitsiou,E., Sibley, C.P., D’Souza, S.W., Catanescu,O.,

Jacobsen, D. W., and Glazier, J. D. (2009) Homocys-

teine transport by systems L, A and y+L across the

microvillous plasma membrane of human placenta,

MILYUTINA et al.1724

BIOCHEMISTRY (Moscow) Vol. 89 No. 10 2024

J. Physiol., 587, 4001-4013, https://doi.org/10.1113/

jphysiol.2009.173393.

19. Tripathi, M., Zhang, C. W., Singh, B. K., Sinha, R. A.,

Moe, K. T., DeSilva, D. A., and Yen, P. M. (2016) Hy-

perhomocysteinemia causes ER stress and impaired

autophagy that is reversed by VitaminB supplemen-

tation, Cell Death Dis., 7, e2513, https://doi.org/10.1038/

cddis.2016.374.

20. Witucki, L., and Jakubowski, H. (2023) Homocysteine

metabolites inhibit autophagy, elevate amyloid beta,

and induce neuropathy by impairing Phf8/H4K20me1-

dependent epigenetic regulation of mTOR in cystathi-

onine beta-synthase-deficient mice, J. Inherit. Metab.

Dis., 46, 1114-1130, https://doi.org/10.1002/jimd.12661.

21. Khayati,K., Antikainen,H., Bonder, E.M., Weber, G.F.,

Kruger, W. D., Jakubowski, H., and Dobrowolski, R.

(2017) Theamino acid metabolite homocysteine acti-

vates mTORC1 to inhibit autophagy and form abnor-

mal proteins in human neurons and mice, FASEB J.,

31, 598-609, https://doi.org/10.1096/fj.201600915R.

22. Wang, M., Liang, X., Cheng, M., Yang, L., Liu, H.,

Wang, X., Sai,N., and Zhang,X. (2019) Homocysteine

enhances neural stem cell autophagy in in vivo and

invitro model of ischemic stroke, Cell Death Dis., 10,

561, https://doi.org/10.1038/s41419-019-1798-4.

23. Zhao, Y., Huang, G., Chen, S., Gou, Y., Dong, Z., and

Zhang, X. (2016) Homocysteine aggravates cortical

neural cell injury through neuronal autophagy over-

activation following rat cerebral ischemia-reperfu-

sion, Int. J. Mol. Sci., 17, 1196, https://doi.org/10.3390/

ijms17081196.

24. Kitada, M., Xu, J., Ogura, Y., Monno, I., and Koya, D.

(2020) Mechanism of activation of mechanistic target

of rapamycin complex1 by methionine, Front. Cell Dev.

Biol., 8, 715, https://doi.org/10.3389/fcell.2020.00715.

25. Ouyang, Y., Wu, Q., Li, J., Sun, S., and Sun, S. (2020)

S-adenosylmethionine: a metabolite critical to the reg-

ulation of autophagy, Cell Prolif., 53, e12891, https://

doi.org/10.1111/cpr.12891.

26. Rosario, F.J., Powell, T.L., and Jansson,T. (2017) mTOR

folate sensing links folate availability to tropho-

blast cell function, J.Physiol., 595, 4189-4206, https://

doi.org/10.1113/JP272424.

27. Chen, Q., Dai,W., Sun,Y., Zhao, F., Liu,J., and Liu,H.

(2018) Methionine partially replaced by methio-

nyl-methionine dipeptide improves reproductive per-

formance over methionine alone in methionine-defi-

cient mice, Nutrients, 10, 1190, https://doi.org/10.3390/

nu10091190.

28. Batistel, F., Alharthi, A. S., Wang, L., Parys, C., Pan,

Y. X., Cardoso, F.C., and Loor, J. J. (2017) Placentome

nutrient transporters and mammalian target of rapa-

mycin signaling proteins are altered by the methi-

onine supply during late gestation in dairy cows and

are associated with newborn birth weight, J. Nutr.,

147, 1640-1647, https://doi.org/10.3945/jn.117.251876.

29. Arutjunyan, A. V., Milyutina, Y. P., Shcherbitskaia,

A. D., Kerkeshko, G.O., Zalozniaia, I. V., and Mikhel,

A.V. (2020) Neurotrophins of the fetal brain and pla-

centa in prenatal hyperhomocysteinemia, Biochem-

istry (Moscow), 85, 248-259, https://doi.org/10.1134/

S000629792002008X.

30. Arutjunyan, A. V., Kerkeshko, G. O., Milyutina, Y. P.,

Shcherbitskaia, A. D., Zalozniaia, I. V., Mikhel, A. V.,

Inozemtseva, D. B., Vasilev, D. S., Kovalenko, A. A.,

and Kogan, I.Y. (2023) Imbalance of angiogenic and

growth factors in placenta in maternal hyperhomo-

cysteinemia, Biochemistry (Moscow), 88, 262-279,

https://doi.org/10.1134/S0006297923020098.

31. Askretkox, A.D., Klishin, A.A., Zybin, D.I., Orlova, N.V.,

Kholodova, A.V., Lobanova, N.V., and Seregin, Yu. A.

(2020) Determination of twenty proteinogenic amino

acids and additives in cultural liquid by high-per-

formance liquid chromatography, J. Anal. Chem., 75,

1038-1045, https://doi.org/10.1134/S1061934820080031.

32. Bradford, M.M. (1976) Arapid and sensitive method

for the quantitation of microgram quantities of pro-

tein utilizing the principle of protein-dye binding,

Anal. Biochem., 72, 248-254, https://doi.org/10.1006/

abio.1976.9999.

33. Bass, J. J., Wilkinson, D.J., Rankin,D., Phillips, B.E.,

Szewczyk, N. J., Smith, K., and Atherton, P. J. (2017)

An overview of technical considerations for West-

ern blotting applications to physiological research,

Scand. J. Med. Sci. Sports, 27, 4-25, https://doi.org/

10.1111/sms.12702.

34. Shcherbitskaia, A. D., Vasilev, D. S., Milyutina, Y. P.,

Tumanova, N. L., Mikhel, A. V., Zalozniaia, I. V., and

Arutjunyan, A.V. (2021) Prenatal hyperhomocystein-

emia induces glial activation and alters neuroinflam-

matory marker expression in infant rat hippocampus,

Cells, 10, 1536, https://doi.org/10.3390/cells10061536.

35. Shcherbitskaia, A. D., Vasilev, D. S., Milyutina, Y. P.,

Tumanova, N. L., Zalozniaia, I. V., Kerkeshko, G. O.,

and Arutjunyan, A.V. (2020) Maternal hyperhomocys-

teinemia induces neuroinflammation and neuronal

death in the rat offspring cortex, Neurotox. Res., 38,

408-420, https://doi.org/10.1007/s12640-020-00233-w.

36. Wang, H. L., and Lai, T. W. (2014) Optimization of

Evans blue quantitation in limited rat tissue samples,

Sci. Rep., 4, 6588, https://doi.org/10.1038/srep06588.

37. Wick, M.J., Harral, J.W., Loomis, Z.L., and Dempsey,

E. C. (2018) An optimized Evans blue protocol to as-

sess vascular leak in the mouse, J. Vis. Exp., 139,

57037, https://doi.org/10.3791/57037.

38. Dayal,S., and Lentz, S.R. (2008) Murine models of hy-

perhomocysteinemia and their vascular phenotypes,

Arterioscler. Thromb Vasc. Biol., 28, 1596-1605, https://

doi.org/10.1161/ATVBAHA.108.166421.

39. Nieraad, H., Pannwitz, N., Bruin, N., Geisslinger, G.,

and Till, U. (2021) Hyperhomocysteinemia: metabol-

ic role and animal studies with a focus on cognitive

PLACENTAL TRANSPORT OF AMINO ACIDS IN HHcy 1725

BIOCHEMISTRY (Moscow) Vol. 89 No. 10 2024

performance and decline– a review, Biomolecules, 11,

1546, https://doi.org/10.3390/biom11101546.

40. Sauls, D.L., Arnold, E.K., Bell, C.W., Allen, J. C., and

Hoffman, M. (2007) Pro-thrombotic and pro-oxi-

dant effects of diet-induced hyperhomocysteinemia,

Thrombosis Res., 120, 117-126, https://doi.org/10.1016/

j.thromres.2006.08.001.

41. Ditscheid, B., Funfstuck, R., Busch, M., Schubert, R.,

Gerth,J., and Jahreis,G. (2005) Effect of L-methionine

supplementation on plasma homocysteine and other

free amino acids: a placebo-controlled double-blind

cross-over study, Eur.J. Clin. Nutr., 59, 768-775, https://

doi.org/10.1038/sj.ejcn.1602138.

42. Andersson, A., Brattstrom, L., Israelsson, B., Isaks-

son, A., and Hultberg, B. (1990) The effect of ex-

cess daily methionine intake on plasma homocys-

teine after a methionine loading test in humans,

Clin. Chim. Acta, 192, 69-76, https://doi.org/10.1016/

0009-8981(90)90273-u.

43. Benevenga, N. J., and Steele, R. D. (1984) Adverse ef-

fects of excessive consumption of amino acids, Annu.

Rev. Nutr., 4, 157-181, https://doi.org/10.1146/annurev.

nu.04.070184.001105.

44. Terstappen, F., Tol, A. J. C., Gremmels, H., Wever,

K.E., Paauw, N.D., Joles, J.A., Beek, E.M.V., and Lely,

A. T. (2020) Prenatal amino acid supplementation to

improve fetal growth: a systematic review and me-

ta-analysis, Nutrients, 12, 2535, https://doi.org/10.3390/

nu12092535.

45. Stanescu,S., Belanger-Quintana,A., Fernandez-Felix,

B.M., Ruiz-Sala,P., Del Valle,M., Garcia,F., Arrieta,F.,

and Martinez-Pardo,M. (2022) Interorgan amino acid

interchange in propionic acidemia: the missing key to

understanding its physiopathology, Amino Acids, 54,

777-786, https://doi.org/10.1007/s00726-022-03128-6.

46. Benevenga, N. J. (1974) Toxicities of methionine and

other amino acids, J.Agric. Food Chem., 22, 2-9, https://

doi.org/10.1021/jf60191a036.

47. Cleal, J.K., Lofthouse, E.M., Sengers, B.G., and Lewis,

R.M. (2018) Asystems perspective on placental amino

acid transport, J. Physiol., 596, 5511-5522, https://doi.

org/10.1113/JP274883.

48. McIntyre, K. R., Hayward, C. E., Sibley, C. P., Green-

wood, S. L., and Dilworth, M. R. (2019) Evidence of

adaptation of maternofetal transport of glutamine

relative to placental size in normal mice, and in those

with fetal growth restriction, J. Physiol., 597, 4975-

4990, https://doi.org/10.1113/JP278226.

49. Hussain,T., Tan,B., Murtaza,G., Metwally,E., Yang,H.,

Kalhoro, M. S., Kalhoro, D. H., Chughtai, M. I., and

Yin, Y. (2020) Role of dietary amino acids and nutri-

ent sensing system in pregnancy associated disorders,

Front. Pharmacol., 11, 586979, https://doi.org/10.3389/

fphar.2020.586979.

50. Huang, H., Vandekeere, S., Kalucka, J., Bierhansl, L.,

Zecchin, A., Bruning, U., Visnagri,A., Yuldasheva, N.,

Goveia,J., Cruys,B., Brepoels,K., Wyns,S., Rayport,S.,

Ghesquiere,B., Vinckier,S., Schoonjans,L., Cubbon,R.,

Dewerchin,M., Eelen,G., and Carmeliet,P. (2017) Role

of glutamine and interlinked asparagine metabolism

in vessel formation, EMBO J., 36, 2334-2352, https://

doi.org/10.15252/embj.201695518.

51. Bonnin,A., Goeden,N., Chen,K., Wilson, M.L., King,J.,

Shih, J. C., Blakely, R. D., Deneris, E.S., and Levitt, P.

(2011) A transient placental source of serotonin for

the fetal forebrain, Nature, 472, 347-350, https://

doi.org/10.1038/nature09972.

52. Bonnin, A., and Levitt, P. (2011) Fetal, maternal,

and placental sources of serotonin and new im-

plications for developmental programming of the

brain, Neuroscience, 197, 1-7, https://doi.org/10.1016/

j.neuroscience.2011.10.005.

53. Mao,J., Jain,A., Denslow, N.D., Nouri, M.Z., Chen,S.,

Wang,T., Zhu, N., Koh,J., Sarma, S.J., Sumner, B. W.,

Lei, Z., Sumner, L. W., Bivens, N. J., Roberts, R. M.,

Tuteja,G., and Rosenfeld, C.S. (2020) BisphenolA and

bisphenolS disruptions of the mouse placenta and po-

tential effects on the placenta-brain axis, Proc. Natl.

Acad. Sci. USA, 117, 4642-4652, https://doi.org/10.1073/

pnas.1919563117.

54. Woods,L., Perez-Garcia,V., and Hemberger,M. (2018)

Regulation of placental development and its impact on

fetal growth-new insights from mouse models, Front.

Endocrinol. (Lausanne), 9, 570, https://doi.org/10.3389/

fendo.2018.00570.

55. Baskurt, O.K., and Meiselman, H.J. (2012) Iatrogenic hy-

perviscosity and thrombosis, Semin. Thromb. Hemost.,

38, 854-864, https://doi.org/10.1055/s-0032-1325616.

56. Winterhager, E., and Gellhaus, A. (2017) Transpla-

cental nutrient transport mechanisms of intrauter-

ine growth restriction in rodent models and hu-

mans, Front. Physiol., 8, 951, https://doi.org/10.3389/

fphys.2017.00951.

57. Rosario, F. J., Kanai, Y., Powell, T. L., and Jansson, T.

(2015) Increased placental nutrient transport in a

novel mouse model of maternal obesity with fetal

overgrowth, Obesity (Silver Spring), 23, 1663-1670,

https://doi.org/10.1002/oby.21165.

58. Aye, I.L., Rosario, F. J., Powell, T. L., and Jansson, T.

(2015) Adiponectin supplementation in pregnant mice

prevents the adverse effects of maternal obesity on

placental function and fetal growth, Proc. Natl. Acad.

Sci. USA, 112, 12858-12863, https://doi.org/10.1073/

pnas.1515484112.

59. Huang, Z., Huang, S., Song, T., Yin, Y., and Tan, C.

(2021) Placental angiogenesis in mammals: a review

of the regulatory effects of signaling pathways and

functional nutrients, Adv. Nutr., 12, 2415-2434, https://

doi.org/10.1093/advances/nmab070.

60. Yung, H. W., Hemberger, M., Watson, E. D., Senner,

C.E., Jones, C.P., Kaufman, R.J., Charnock-Jones, D.S.,

and Burton, G.J. (2012) Endoplasmic reticulum stress

MILYUTINA et al.1726

BIOCHEMISTRY (Moscow) Vol. 89 No. 10 2024

disrupts placental morphogenesis: implications for

human intrauterine growth restriction, J.Pathol., 228,

554-564, https://doi.org/10.1002/path.4068.

61. Cross, J. C., Hemberger, M., Lu, Y., Nozaki, T., White-

ley, K., Masutani, M., and Adamson, S.L. (2002) Tro-

phoblast functions, angiogenesis and remodeling of

the maternal vasculature in the placenta, Mol. Cell En-

docrinol., 187, 207-212, https://doi.org/10.1016/s0303-

7207(01)00703-1.

62. Wang, Y., Engel, T., and Teng, X. (2024) Post-trans-

lational regulation of the mTORC1 pathway: a

switch that regulates metabolism-related gene ex-

pression, Biochim. Biophys. Acta Gene Regul. Mech.,

1867, 195005, https://doi.org/10.1016/j.bbagrm.2024.

195005.

63. Kavitha, J. V., Rosario, F. J., Nijland, M. J., McDonald,

T.J., Wu,G., Kanai,Y., Powell, T.L., Nathanielsz, P.W.,

and Jansson, T. (2014) Down-regulation of placental

mTOR, insulin/IGF-I signaling, and nutrient trans-

porters in response to maternal nutrient restric-

tion in the baboon, FASEB J., 28, 1294-1305, https://

doi.org/10.1096/fj.13-242271.

64. Qin,X., Jiang,B., and Zhang,Y. (2016) 4E-BP1, a multi-

factor regulated multifunctional protein, Cell Cy-

cle, 15, 781-786, https://doi.org/10.1080/15384101.

2016.1151581.

65. Pang,V., Bates, D.O., and Leach,L. (2017) Regulation

of human feto-placental endothelial barrier integrity

by vascular endothelial growth factors: competitive

interplay between VEGF-A(165)a, VEGF-A(165)b, PIGF

and VE-cadherin, Clin. Sci. (Lond), 131, 2763-2775,

https://doi.org/10.1042/CS20171252.

66. Saunders, N. R., Dziegielewska, K. M., Mollgard, K.,

and Habgood, M. D. (2015) Markers for blood-brain

barrier integrity: how appropriate is Evans blue in

the twenty-first century and what are the alterna-

tives? Front. Neurosci., 9, 385, https://doi.org/10.3389/

fnins.2015.00385.

67. Haghighi Poodeh, S., Salonurmi, T., Nagy, I.,

Koivunen,P., Vuoristo, J., Rasanen, J., Sormunen, R.,

Vainio, S., and Savolainen, M. J. (2012) Alcohol-

induced premature permeability in mouse placen-

ta-yolk sac barriers in vivo, Placenta, 33, 866-873,

https://doi.org/10.1016/j.placenta.2012.07.008.

68. Lewis, R.M., Baskaran,H., Green,J., Tashev,S., Palai-

ologou,E., Lofthouse, E.M., Cleal, J.K., Page,A., Chat-

elet, D.S., Goggin, P., and Sengers, B.G. (2022) 3Dvi-

sualization of trans-syncytial nanopores provides a

pathway for paracellular diffusion across the human

placental syncytiotrophoblast, iScience, 25, 105453,

https://doi.org/10.1016/j.isci.2022.105453.

69. Sibley, C. P., Brownbill, P., Glazier, J. D., and Green-

wood, S. L. (2018) Knowledge needed about the ex-

change physiology of the placenta, Placenta, 64, S9-

S15, https://doi.org/10.1016/j.placenta.2018.01.006.

70. Shao, X., Cao, G., Chen, D., Liu,J., Yu, B., Liu, M., Li,

Y.X., Cao,B., Sadovsky,Y., and Wang, Y.L. (2021) Pla-

cental trophoblast syncytialization potentiates mac-

ropinocytosis via mTOR signaling to adapt to reduced

amino acid supply, Proc. Natl. Acad. Sci. USA, 118,

e2017092118, https://doi.org/10.1073/pnas.2017092118.

71. Cooke, L.D.F., Tumbarello, D.A., Harvey, N.C., Sethi,

J. K., Lewis, R. M., and Cleal, J. K. (2021) Endocytosis

in the placenta: an undervalued mediator of placental

transfer, Placenta, 113, 67-73, https://doi.org/10.1016/

j.placenta.2021.04.014.

72. Elshorbagy, A. K., Graham, I., and Refsum, H.

(2020) Body mass index determines the response

of plasma sulfur amino acids to methionine load-

ing, Biochimie, 173, 107-113, https://doi.org/10.1016/

j.biochi.2020.03.001.

73. Lofthouse, E.M., Manousopoulou,A., Cleal, J.K., O’Kelly,

I.M., Poore, K.R., Garbis, S.D., and Lewis, R.M. (2021)

N-acetylcysteine, xCT and suppression of Maxi-chloride

channel activity in human placenta, Placenta, 110,

46-55, https://doi.org/10.1016/j.placenta.2021.05.009.

74. Da Silva, V. C., Fernandes, L., Haseyama, E. J.,

Agamme, A.L., Guerra Shinohara, E.M., Muniz, M.T.,

and D’Almeida, V. (2014) Effect of vitaminB depriva-

tion during pregnancy and lactation on homocyste-

ine metabolism and related metabolites in brain and

plasma of mice offspring, PLoS One, 9, e92683, https://

doi.org/10.1371/journal.pone.0092683.

Publisher’s Note. Pleiades Publishing remains

neutral with regard to jurisdictional claims in published

maps and institutional affiliations. AI tools may have

been used in the translation or editing of this article.