Cell culture and GDM-like in vitro model
The human choriocarcinoma-derived cell line, JEG3 was used as a trophoblast model. The JEG3 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Sigma D2902, Sigma-Aldrich, St. Louis, MO, U.S.A.) supplemented with 10% charcoal-stripped fetal bovine serum (cFBS, Gibco, Invitrogen Co., Carlsbad, CA, USA), 100 IU/mL penicillin, and 100 μg/mL streptomycin, and incubated at 37°C in an atmosphere containing 5% CO2. To establish a GDM-like in vitro model, high glucose conditions were generated by culturing the cells in the presence of 30 mM D-glucose (G6152; Sigma-Aldrich) in a time dependent manner (24, 48, and 72 h).
Experimental animals
This study was reviewed and approved by the Pusan National University-Institutional Animal Care and Use Committee (PNU-IACUC; Approval Number PNU-2019-2409) (Busan, Republic of Korea). The 12-week old female Sprague-Dawely rats were purchased from Samtako (Osan, Korea), housed in standard cages, and fed with basal feed. Rats were maintained on a 12-h light/dark cycle. All rats were handled in the Pusan National University-Laboratory Animal Resources Center, accredited by the Korea Food and Drug Administration. After one week of acclimatization before the experiment, rats were mated overnight, and the presence of sperm in the vagina was checked the following morning (day 0 of pregnancy). Pregnant rats were randomly assigned to the following two groups: control pregnancy (n=5) and GDM group (n=5). Diabetes was induced using a single intraperitoneal injection of streptozotocin (STZ, Sigma-Aldrich) in 0.1 M citrate buffer (pH 4.0) at a dose of 45 mg/kg body weight on day 1 of pregnancy. Control animals received an equal volume of citrate buffer. On gestation day (GD) 6, diabetes was confirmed by measuring fasting (8 h) blood glucose concentrations obtained using a tail prick. The animals with fasting plasma glucose above 300 mg/dL were categorized as having GDM. On GD 18, final fasting blood glucose levels were obtained and euthanasia was performed using CO2 gas to prepare tissue samples. Blood samples were collected in SST tubes (Becton Dickinson company, NJ, USA), and the retro-orbital plexus was collected in EDTA-coated micro tubes (BD Biosciences, Franklin Lakes, NJ). The collected tissue samples were fixed in 10% formalin until analysis or stored at -70°C for further experiments.
Hematology
Blood analysis and serum biochemistry were performed for all collected samples. The levels of white blood cells (WBC), lymphocytes (LYM), monocytes (MON), and neutrophils (NEU) were measured using a VetScan hematology system (Abaxis, Sunnyvale, CA). Serum biochemical components, including total cholesterol (TC), triglyceride (TG), glucose (GLU), high density lipoprotein (HDL), and low density lipoprotein (LDL), were assayed using an automatic serum analyzer (Hitachi 747; Hitachi, Tokyo, Japan). All assays were performed in duplicate using fresh serum.
Human placental tissue collection and processing
The biospecimens and data used for this study were provided by the Biobank of Pusan National University Hospital (PNUH), a member of the Korea Biobank Network. This study was approved by the Institutional Review Board (IRB) of PNUH (H-1703-004-006). Human placental tissues were collected and immediately stored at -80°C; samples were divided into normal (n=20) and GDM (n=18). GDM was defined as a condition wherein glycosylated hemoglobin (HbA1c) > 6.23 for glycemic values. Clinical characteristics of sample groups are shown in Table 1.
Quantitative real-time PCR and western blotting
Total RNA was extracted using TRIzol reagent (Invitrogen Co.) according to the manufacturer’s protocol. Real-time quantitative PCR (Q-PCR) was performed in a single reaction to determine gene expression using SYBR Green Realtime PCR Master Mix (TOYOBO, Osaka, Japan) following the manufacturer’s instructions. Primer sequences used for PCR are shown in Table 2. For western blotting, JEG3 cell and placenta tissue lysates were prepared using the Pro-prep solution (iNtRON Biotechnology, Seoul, Republic of Korea) according to the following the manufacturer's protocol. Proteins were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) on 8–10% gels and immunoblotted using primary antibodies. The following specific antibodies were used: anti-sterol regulatory element-binding protein-1c (anti-SREBP-1c; Santa Cruz Biotechnology, CA, USA; cat no. sc-13551; 1:500), anti-fatty acid synthase (anti-FAS; Santa Cruz Biotechnology; cat no. sc-20110; 1:500), anti-peroxisome proliferator-activated receptor-γ (anti-PPARγ; Santa Cruz Biotechnology; cat no. sc-7273; 1:500), anti-hormone-sensitive lipase (anti-HSL; Cell Signaling Technology, Beverly, MA, USA; cat no. 4107S; 1:500), anti-phosphorylated HSL Ser 660 (anti-p-HSL (Ser 660); Cell Signaling Technology; cat no. 4126L; 1:500), and anti-adipose triglyceride lipase (anti-ATGL; Cell Signaling Technology; cat no. 2138S; 1:500) for immunoblotting overnight at 4°C. This was followed by incubation with horseradish peroxidase-conjugated secondary antibodies (Enzo Life Sciences, Inc., Farmingdale, NY, USA; cat. no. ADI-SAB-300-J, ADI-SAB-100-J; 1:5,000) in 5% skim milk prepared in phosphate-buffered saline containing Tween-20 (PBS-T).
Glycerol release assay
JEG3 cell culture media and amniotic fluid were used to analyze glycerol release. Each media sample (10 μL) was mixed with 200 μL of Free Glycerol Reagent (Sigma-Aldrich) following the manufacturer’s protocol. The absorbance was directly read at 540 nm in the wells using a microplate spectrophotometer (Epoch Biotek, VT, USA).
cAMP assay
Cyclic adenosine monophosphate (cAMP) levels in cell extracts were measured using the cAMP enzyme immunoassay kit (GE Healthcare (RPN2251), Buckinghamshire, UK) following the manufacturer's instructions. To measure the concentration of cAMP in the cell extracts, JEG3 cells were seeded at a density of 5*103 cells/mL in hyperglycemic media and grown at 37°C in an incubator. Cell extracts were collected for analysis after 24, 48, and 72 h. Antiserum was added and the samples were incubated at 4°C for 2 h before adding cAMP-peroxidase conjugate for 1 h at 4°C. The reaction was terminated by adding stop solution, and the optical density was recorded at an absorbance of 450 nm using a microplate spectrophotometer (Epoch Biotek).
Oil red O staining
JEG3 cells and placenta tissues from GDM individuals were stained with Oil red O (Sigma-Aldrich) to examine lipid accumulation in the placenta. For staining, the placenta tissues were frozen, and 12-μm thick of cryosection of placental tissues were obtained. JEG3 cells and placental tissues were washed twice with PBS and fixed using 10% formalin for 1 h. Cells were washed with ddH2O and 60% isopropanol for 5 min before incubating with 0.5% Oil Red O dye in isopropyl alcohol for 10 min at room temperature. Stained JEG3 cells were rinsed four times with ddH2O. The stained fat droplets were observed microscopically (Eclipse Ts2, Nikon, Japan) at 100x and 200x magnification in JEG3 cells and placental tissues from GDM individuals, respectively.
Statistical analysis
The results are presented as the mean ± standard error of the mean (SEM). The data were analyzed using one-way analysis of variance (ANOVA) (SPSS for Windows, 10.10, standard version; SPSS Inc., Chicago, IL, USA) and GraphPad Prism® version 8.0.2 (San Diego, CA). P<0.05 indicates significant differences.