Reagents

Guanabenz and tunicamycin were purchased from Sigma-Aldrich (Madrid, Spain).

Study population

The study cohort consisted of 40 mother-newborn pairs recruited at the Hospital Sant Joan de Déu of Barcelona (Spain) from a prenatal cohort study of mothers and infants. Twenty infants were born AGA (10 girls, 10 boys) and 20 SGA (10 girls, 10 boys). The inclusion criteria were: (i) infants born at term (37–42 weeks) from singleton pregnancies and with a birth weight between -1.0 and + 1.0 SD (range 2.9–3.8 kg) for AGA and below -2 SD (range 1.9–2.6 kg) for SGA; (ii) placenta collected at delivery; and (iii) written informed consent obtained in the third trimester of pregnancy. The exclusion criteria were: (i) maternal disease (hypertension, preeclampsia, gestational diabetes, or preexisting type 1 and type 2 diabetes mellitus), alcohol abuse or drug addiction; and (ii) fetal malformations or complications at birth. The SGA babies included in the present study had normal umbilical flow indices, and none of them had oligohydramnios or neonatal complications.

The study was approved by the Institutional Review Board of the Hospital Sant Joan de Déu at the University of Barcelona.

Clinical, endocrine-metabolic and body composition assessments

Information on maternal age at conception, height, pregestational weight, and gestational weight gain were retrieved from the mother’s clinical records. Gestational age was calculated from the last menses and was confirmed by a first-trimester ultrasound.

The weight and length of the newborns were measured in the delivery room and transformed into Z-scores according to country and sex-specific growth charts [21]. Blood samples were obtained at birth from the umbilical cord, before placental separation.

Serum glucose levels were measured with the glucose oxidase method. Insulin and insulin-like growth factor-1 (IGF-1) were assessed by immunechemiluminescence (DPC, IMMULITE 2500, Siemens, Erlangen, Germany), with the intra- and inter-assay coefficient of variation (CVs) being < 10%.

Homeostatic model assessment for insulin resistance (HOMA-IR) was calculated as fasting insulin (mU/L) x fasting glucose (mmol/L)/22.5. Circulating high molecular weight (HMW)-adiponectin was measured with a specific enzyme-linked immunosorbent assay (R&D Systems, Minneapolis, USA), with the intra- and inter-assay CVs being < 9%. Glucagon-like peptide-1 (GLP-1) was assessed by an enzyme-linked immunosorbent assay (Millipore, Billerica, MA, USA). The antibody pair in the assay binds to GLP-1 (7–36) and (9–36) and has no cross-reactivity with GLP-2, GIP, glucagon or oxyntomodulin. The intra-assay and inter-assay CVs were < 2% and < 10%, respectively, and the detection limit was 1.5 pM.

Body composition was assessed at the age of 15 days by dual X-ray absorciometry with a Lunar Prodigy system coupled to Lunar software (Lunar Corp, Madison, WI, USA) adapted for infants. CVs were < 3% for fat and lean mass [22].

Placenta collection

Placentas were collected after childbirth in the delivery room and weighed immediately. Placental tissue encompassing the decidua and the upper side of the chorionic villous proximal to the decidua was dissected to obtain placental maternal biopsies. Three pieces of 1-cm3 cuboidal sections were collected from the maternal side after removal of the amniotic and chorionic layers. Placental samples were washed three times with physiological saline to remove all maternal blood and immediately frozen in liquid nitrogen and stored at − 80 °C until analysis. The personnel always wore face masks and sterile gloves and used a sterile scalpel and instruments.

For the studies of mRNA and protein expression in the placentas from AGA and SGA infants, only girls were selected (as specified in the Results section).

Cell culture

BeWo cells (kindly donated by Dr. Vicente Andreu Fernández from the Universidad Internacional de Valencia, Valencia) were cultured in Ham’s F12 medium (Gibco-Invitrogen) supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich) and 1% penicillin–streptomycin (Gibco-Invitrogen). Cells were seeded and, 72 h later, differentiated into syncytiotrophoblasts by incubation with 40 μM forskolin (Santa Cruz Biotechnology) for 48 h. Once differentiated, cells were treated with 0.1 μg/ml of tunicamycin (TM) (Sigma-Aldrich) and 5 μM guanabenz (GB) (Sigma-Aldrich), which was added 1 h before tunicamycin, for 24 h.

Reverse transcription-polymerase chain reaction and quantitative polymerase chain reaction

Isolated RNA was reverse transcribed to obtain 1 μg of complementary DNA (cDNA) using Random Hexamers (Thermo Scientific), 10 mM deoxynucleotide (dNTP) mix, and the reverse transcriptase enzyme derived from the Moloney murine leukemia virus (MMLV, Thermo Fisher). The experiment was run in a thermocycler (BioRad) and consisted of a program with different steps and temperatures: 65 °C for 5 min, 4 °C for 5 min, 37 °C for 2 min, 25 °C for 10 min, 37 °C for 50 min, and 70 °C for 15 min. The relative levels of specific mRNAs were assessed by real-time RT-PCR in a mini 48-well T100™ thermal cycler (Bio-Rad), using the SYBR Green Master Mix (Applied Biosystems), as previously described [23]. Briefly, samples had a final volume of 20 μl, containing 20 ng of total cDNA, 0.9 μM of the primer mix, and 10 μl of 2 × SYBR Green Master Mix. The thermal cycler protocol for real-time PCR included a first step of denaturation at 95 °C for 10 min followed by 40 repeated cycles of 95 °C for 15 s, 60 °C for 30 s, and 72 °C for 30 s for denaturation, primer annealing, and amplification, respectively. Primer sequences were designed using the Primer-BLAST tool (NCBI), based on the full mRNA sequences to find the optimal primers for amplification, and evaluated with the Oligo-Analyzer Tool (Integrated DNA Technologies) to ensure an optimal melting temperature (Tm) and avoid the formation of homo/heterodimers or non-specific structures that can interfere with the interpretation of the results. The primer sequences were designed specifically to span the junction between the exons. The primer sequences used were: CHOP, 5’-GGAAATGAAGAGGAAGAATCAAAAAT-3’ and 5’-GTTCTGGCTCCTCCTCAGTCA-3’; GRP78/BiP, 5’- ACTATTGCTGGCCTAAATGTTATGAG-3’ and 5’-TTATCCAGGCCATAAGCAATAGC-3’; SLC7A5/LAT1, 5’-CAGTACATCGTGGCCCTGGT-3’ and 5’-TGAGCAGCAGCACGCAGAG-3’; SLC38A1/SNAT1, 5’-GTGTATGCTTTACCCACCATTGC-3’ and 5’- GCACGTTGTCATAGAATGTCAAGT-3’; SLC38A2/SNAT2, 5’- ACGAAACAATAAACACCACCTTAA-3’ and 5’-AGATCAGAATTGGCACAGCATA-3’; and TBP, 5’-CCACTCACAGACTCTCACAAC-3’ and 5’-CTGCGGTACAATCCCAGAACT-3’. Values were normalized to the expression levels of TATA-box-binding protein (TBP), and measurements were performed in triplicate. All changes in expression were normalized to the untreated control.

Immunoblotting

The isolation of total protein extracts was performed as described elsewhere [24]. For the isolation of total cell membranes, cell suspensions or placenta tissues were resuspended in 3 ml of ice-cold buffer I (250 mM sucrose, 20 mM HEPES, 5 mM NaN3, 2 mM EGTA, 100 μM phenylmethylsulfonyl fluoride, 10 μM L-trans-epoxysuccinyl-leucylamido(4-guanidino)butane, 1 μM pepstatin A, and 1 μM leupeptin; pH 7.4) and homogenized. The resulting homogenate was centrifuged at 177,000 g for 1 h at 4 °C. The pellet, containing the total membranes, was resuspended in 50 μl of buffer I supplemented with a protease inhibitor and stored at -20 °C.

Immunoblotting was performed with antibodies against β-actin (Sigma, A5441), 4EBP1 (Cell Signalling, 9452), AKT (Cell Signalling, 9272), AMPKα (Cell Signalling, 2532), ATF4 (Santa Cruz Biotechnology, sc-200), ATF6 (Santa Cruz Biotechnology, sc-22799), BiP/GRP78 (Cell Signalling, 3183), CHOP (Cell Signalling, 2895), eIF2α (Cell Signalling, 9722), ERK1/2 (44/42 MAPK) (Cell Signalling, 9102), GADD34 (Cell Signalling, sc-46661), GAPDH (Millipore, MAB374), GRASP55 (Proteintech, 66,627–1-Ig), LAT1 (Cell Signalling, 5347), mTOR (Cell Signalling, 2972), Na–K-ATPase (Santa Cruz Biotechnology, sc-514614), NEDD4-L (Cell Signalling, sc-514954), phospho-4EBP1 Thr37/46 (Cell Signalling, 2855), phospho-AKT Ser473 (Cell Signalling, 9271), phospho-AMPKα Thr172 (Cell Signalling, 2531), phospho-ERK1/2 (44/42 MAPK) Thr202/Tyr204 (Cell Signalling, 9101), phospho-IRE Ser724 (Novus Biologicals, NB100-2323), phospho-mTOR Ser2448 (Santa Cruz Biotechnology, sc-101738), phospho-ribosomal protein S6 (Cell Signalling, 2211), ribosomal protein S6 (Cell Signalling, 2317), SNAT1 (Novus Biologicals, NBP-2–59311), SNAT2 (MBL, BMP081), and vinculin (Santa Cruz Biotechnology, sc-73614). Signal acquisition was conducted using the Bio-Rad ChemiDoc apparatus and the quantification of the immunoblot signal was performed with the Bio-Rad Image Lab software. The results for protein quantification were normalized to the levels of a control protein (GAPDH, β-actin, vinculin, or Na–K-ATPase) to avoid unwanted sources of variation.

Statistical analysis

Results are expressed as the mean ± SEM. Significant differences were assessed by either Student’s t-test or one-way ANOVA, according to the number of groups compared, using the GraphPad Prism program (version 9.0.2) (GraphPad Software Inc., San Diego, CA, USA). When significant variations were found by ANOVA, Tukey’s post-hoc test for multiple comparisons was performed only if F achieved a p value < 0.05. Differences were considered significant at p < 0.05.