We extracted the serum, placenta and decidua from 12 patients diagnosed with GDM who underwent elective repeat cesarean sections, and 12 gestational week-matched controls who also underwent elective repeat cesarean sections. All sera were collected at one day prior to the operation. The baseline characteristics of the patients are summarized in Table 1. Exclusion criteria were fetal structural anomaly, multiple gestations, preeclampsia, and other maternal medical diseases.

All pregnant women were screened at 24 to 28 weeks of gestation for GDM using a 50-g glucose challenge test. When the result was positive, a 100-g oral glucose tolerance test was performed. The diagnostic criteria of Carpenter and Coustan were used, which define GDM as two values exceeding the threshold for positivity [14].

The current study was approved by the institutional review board of Kangwon National University Hospital. We obtained written informed consent from all patients before collection of any tissue. We collected about 1×1-cm-sized placental tissue on the center of placenta and 0.5×0.5-cm-sized decidua in the placental bed. All samples were collected directly into liquid nitrogen and stored in sealed containers at −70℃ until analysis.

Tissues were washed with cold phosphate buffered saline (PBS) and homogenized with 100 µL of tissue protein extraction reagent (Thermo Scientific, Rockford, IL, USA) containing protease inhibitor cocktail (Roche, Hamburg, Germany). The FABP4 was measured using enzyme-linked immunosorbent assay kit according to the manufacturer's instructions (R&D System, Minneapolis, MN, USA) and normalized to total tissue protein content. The total tissue protein concentration was determined using the Pierce BCA protein assay reagent (Thermo Scientific). All experimental samples were assayed in triplicate.

Primary subcutaneous human pre-adipocytes, a fibroblast growth kit, and an adipocyte differentiation toolkit were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). Culture and differentiation of primary subcutaneous human pre-adipocytes were performed following the ATCC protocol.

The cells were cultured with a fibroblast growth kit (ATCC, PCS-210-041) containing 2% fetal bovine serum at 37℃ in a humidified 5% CO₂ incubator. The cells were seeded and maintained in 6-well plates until they reached more than 90% confluence. After the cells reached confluence, the medium was removed, and the cells were washed with PBS and then cultured using the adipocyte differentiation toolkit (ATCC, PCS-500-050). Briefly, the cells were washed gently with PBS on day 0 (confluence) and incubated with 2 mL adipocyte differentiation initiation medium (ATCC) for 48 hours. On day 2, the medium was replaced with adipocyte differentiation maintenance medium (ATCC) for 48 hours. The adipocyte differentiation maintenance medium was changed every 48 hours.

To investigate the effects of sera from pregnant women with GDM and normal pregnant women on primary human subcutaneous pre-adipocytes, the adipocytes were treated with 5% serum from normal pregnant women or from pregnant women with GDM at day 7 post-induction. We used three serum samples randomly selected from pregnant women with GDM and normal pregnant women, respectively. To observe the effect of the sera in the treatment and control groups, cells from both groups were handled identically. The next day, cells were harvested for gene expression or stained with oil red O. All experiments were performed in triplicate.

Total RNA was extracted from placenta and decidua tissue and adipocytes with TRIzol reagent (Invitrogen, Fostercity, CA, USA), and the quality and concentration of RNA were estimated with a NanoDrop 2000 Spectrophotometer (Thermo Scientific, Wilmington, DE, USA). cDNA was synthesized using QuantiTect reverse transcription kits (Qiagen, Hilden, Germany) according to the manufacturer's protocol. Gene expression levels were determined with QuanStudio Real-Time PCR Software (Applied Biosystems, Foster City, CA, USA) using the Power SYBR Green PCR Master Mix (Applied Biosystems, Warrington, UK) and normalized to Glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The amplification conditions were 95℃ for 10 minutes, followed by 40 cycles at 95℃ for 15 seconds and 60℃ for 1 minute. The expression values were calculated using the 2^−ΔΔCt method according to the manufacturer's instructions.

The primers used were as follows: FABP4 forward primer, 5′-ggcatggccaaacctaacat-3′, and reverse primer, 5′-ttccatcccatttctgcacat-3′; adiponectin forward primer, 5′-ccctctcttacaagcccatca-3′, and reverse primer 5′-gagccagtctggtagtacatca-3′; and GAPDH forward primer, 5′-cgccacagtttcccggaggg-3′, and reverse primer, 5′-ccctccaaaatcaagtgggg-3′.

The extracted proteins from cells were separated in 12% sodium dodecyl sulphate-polyacrylamide gels, transferred to nitrocellulose membranes (Bio-Rad Laboratories, Hercules, CA, USA), blocked with 5% (w/v) nonfat skim milk in Tris-buffered saline, 0.1% Tween 20 for 1 hour, and incubated overnight at 4℃ with the rabbit ant-FABP4 polyclonal antibody (1:500; Abcam, Cambridge, MA, USA) or mouse anti-adiponectin monoclonal antibody (1:500, Abcam). Immunoreactive proteins were detected using a horseradish peroxidase-conjugated secondary antibody and enhanced chemiluminescence assay kits (Millipore, Billerica, MA, USA).

Cells differentiated in the 6-well plate were washed with PBS and fixed with 4% paraformaldehyde for 1 hour at room temperature. After removing the 4% paraformaldehyde, cells were washed with 60% isopropanol solution and dried completely in a hood. The fixed cells were stained with diluted oil red O solution (Sigma-Aldrich, St. Louis, MO, USA; oil red O:distilled water=6:4) for 2 hours. The stained lipid droplets were observed, and images were obtained from randomly selected fields under a microscope (Olympus, Tokyo, Japan). In addition to determine the accumulation of lipid content, the stain was eluted with 100% isopropanol and measured at 510 nm using a spectrophotometer (Spectra MAX 190, Molecular Device, Sunnyvale, CA, USA).

All data are expressed as mean±standard deviation or mean±standard error. Differences between 2 independent groups in Table 1 and Fig. 1 were analyzed using Mann-Whitney U-test, whereas the differences between 3 groups in Figs. 2 and 3 were performed with Kruskal-Wallis test using SPSS ver. 12.0 (SPSS Inc., Chicago, IL, USA). A P-value <0.05 was considered statistically significant.

We first confirmed expression of FABP4 mRNA by real-time polymerase chain reaction in the placenta and decidua and found that FABP4 mRNA was significantly upregulated in placenta (100.0±25.6 vs. 248.0±26.6, P<0.05, Fig. 1B) and decidua (100.0±19.2 vs. 275.9±71.2,P<0.05, Fig. 1D) from pregnant women with GDM. We then measured the total FABP4 levels in the placenta, decidua and serum by enzyme-linked immunosorbent assay in 24 normal pregnant women and with GDM. The average protein level of FABP4 was significantly higher in the placenta (108.1±9.0 vs. 141.3±11.0, P<0.05, Fig. 1A) decidua (40.1±8.4 vs. 116.0±15.3, P<0.05, Fig. 1C) and serum (2.9±0.6 vs. 5.1±0.7, P<0.05, Fig. 1E) from pregnant women with GDM.

We suggest that excessive production of FABP4 in GDM may result from placental hormones, which may enter the maternal circulation more than they would during a normal pregnancy.

To address our hypothesis that circulating placental hormones in GDM upregulate FABP4 and down-regulate adiponectin in adipocytes, we exposed adipocytes to serum from pregnant women with GDM and normal pregnant women in culture media for 24 hours. The serum from pregnant women with GDM increased the expression of FABP4 mRNA in the adipocytes significantly (100.0±3.4 vs. 44.06±4.89 vs. 69.6±6.6, P<0.05, Fig. 2A). However, the serum from pregnant women with GDM decreased the expression of adiponectin mRNA in the adipocytes significantly (100.0±7.8 vs. 48.6±4.0 vs. 32.6±4.7, P<0.05, Fig. 2B).

In contrast, serum from pregnant women with GDM did not alter FABP4 protein and adiponectin protein expression in the adipocyte (Fig. 2C). However, trends toward increased FABP4 protein expression and decreased adiponectin protein expression in GDM group were demonstrated (Fig. 2C, D).

To investigate whether serum from normal pregnant women and with GDM affects lipid accumulation in adipocytes, we performed oil red O staining after adipocyte culture for 24 hours (Fig. 3). Lipid accumulation in the group receiving serum from pregnant women with GDM was significantly greater than that receiving serum from the normal pregnant women (100.0±8.4 vs. 86.8±11.9 vs. 137.7±9.9, P<0.05, Fig. 3).