Subjects for Cord serum Samples
Pregnant Native American or Hispanic women with diabetes (N=42, including 31 gestational diabetes and 11 pre-gestational type 2 diabetes), or non-diabetic controls (N=81) were enrolled into a prospective longitudinal study on the impact of in utero exposure to DM, as previously described (24). Gestational or type 2 diabetes was diagnosed according to ADA guidelines (25). Women with type 2 diabetes were defined as those diagnosed before pregnancy. Women were excluded if they delivered prior to 37 weeks gestation, had type 1 diabetes, pre-eclampsia, chronic hypertension, renal disorders or a smoking history during pregnancy. They were also excluded if the infants were small for gestational age, had a major malformation, or chromosome abnormality. Maternal glucose concentrations measured 2 hours after oral glucose challenge (OGTT, Fig. 2) during the second trimester of pregnancy were obtained from clinical records. Cord blood and maternal blood (if available) were obtained after delivery, and cord and maternal serum resistin levels were measured. The protocol was approved by the Institutional Review Boards of the University of Oklahoma Health Science Center, the Chickasaw Nation, and the Choctaw Nation of Oklahoma. The samples collected under this protocol were not used for isolating cord blood mononuclear cells, mesenchymal stem cells, or placental explant culture.
Studies using human cord blood mononuclear cells (CBMCs), mesenchymal stem cells (MSCs) and placental explant culture
CBMCs, MSCs, and placental explants were isolated respectively from cord blood, cord tissue, or placenta obtained at term from healthy human subjects recruited in a separate study cohort as described previously (26). The protocol was approved by the Institutional Review Board of the University of Oklahoma Health Science Center. CBMCs were isolated from cord blood of non-diabetic healthy individuals by Ficoll density gradient centrifugation. The cord blood was diluted 1: 3 in PBS (without Ca2+ and Mg2+), layered over Ficoll buffer, and centrifuged at 400g for 35 minutes. The interphase cell layer was collected and washed in PBS for 3 times. The CBMCs were plated and cultured in Dulbecco's Modified Eagle Medium with 10% Fetal Bovine Serum followed by treatment with TNFα (100 ng/ml), high glucose (25 mM), palmitate acid (0.6 mM), or 4-hydroxynonenal (4-HNE, 0.6 mM) for 16 hours. Mesenchymal stem cells (MSCs) were isolated from Wharton’s Jelly of cord tissue as previously described (27). For Placental explant culture, two pieces of placental tissue were collected from healthy subjects within 15 minutes after delivery, stripped of connective tissues, and dissected to small pieces (about 2 mm). The placental villous explants were cultured in 6-well plate at 37°C in 5% CO2 in Ham's F-12 medium (Gibco/Life Technologies, Grand Island, NY) supplemented with 10% FBS (Mediatech, Manassas, VA), 100 µM MEM Non-Essential Amino Acids (Gibco/Life Technologies, Grand Island, NY), and 0.5% penicillin/streptomycin/amphotericin B (Gibco/Life Technologies, Grand Island, NY) and were treated with indicated doses of resistin or vehicle for 24 hours in culture.
ELISA
The concentrations of resistin in serum and cell culture media were measured using human Resistin DuoSet ELISA kit (R&D Systems, Minneapolis, MN) according to manufacturer’s protocol. Briefly, ELISA plates were coated with capture antibody overnight at room temperature followed by blocking with Reagent Diluent (DuoSet ELISA Reagent Kit) for 2 hours. 100 ul cell culture media, or diluted fetal or maternal serum (1:40 in PBS), along with serial-diluted standards (0 - 4 ng/ml) were loaded to the plates and incubated overnight at 4 ºC, followed by adding detection antibody and streptavidin-HRP subsequently. Optical density was determined using a microplate reader at 450 nm. The detection range of the assay is 0.0625 ng/ml to 4 ng/ml with intraplate coefficient of variation of the duplicates less than 10% and inter-plate coefficient of variation less than 15%.
RNA extraction
Total RNA was extracted from BeWo cells (a human placental trophoblast cell line derived from a choriocarcinoma) using commercially available kits (miRNeasy, Qiagen, Valencia, CA) according to the manufacturer’s instructions. Isolated total RNA was quantified by a NanoDrop ND-1000 spectrophotometer (Thermo Scientific, Wilmington, DE).
qPCR analysis
Reverse transcription (RT) was done with SuperScript VILO cDNA Synthesis Kit according to the manufacturer’s instructions (Invitrogen). Quantitative real-time PCR was performed using TaqMan Real-Time PCR Probes for PGC-1α or GAPDH (Life Technologies). Results were calculated using the 2−ΔΔCt method normalized to endogenous control GAPDH.
Western Blot Analysis
Western blot analysis was performed as described previously (23). Placental explant samples or BeWo cells were lysed and homogenized in protein lysis buffer containing a protease and phosphatase inhibitor cocktail (Pierce Biotechnology, Rockford, IL). Protein concentrations were measured by BCA assay (Pierce, Rockford, IL). Thirty µg of protein lysate was reduced in laemmli sample buffer with dithiothreitol, and subjected to sodium dodecyl sulfated polyacrylamide gel electrophoresis (SDS-PAGE), then transferred to polyvinylidene fluoride (PVDF) membrane and incubated with antibodies specific for PGC-1α, PDH, or β-actin (Cell Signaling Technology, Danvers, MA). The proteins of interest were detected by enhanced chemiluminescence (Pierce, Rockford, IL) and analyzed by imaging densitometry with Image Lab Software (Bio-Rad, Hercules, CA).
Mitochondrial DNA copy number
DNA was isolated from placental tissue using the GenElute Mammalian Genomic DNA Miniprep Kit (Sigma, St. Louis, MO) with proteinase K and RNase treatment, according to the manufacturer’s instructions. Mitochondrial DNA copy number was estimated by comparing the abundance of the mitochondrial tRNALeu(UUR) gene (determined by quantitative RT-PCR, forward primer: 5’-CACCCAAGAACAGGGTTTGT; reverse: 5’-TGGCCATGGGTATGTTGTTA) and with that of the nuclear β2-microglobulin gene (forward: 5’-TGCTGTCTCCATGTTTGATGTATCT; reverse: 5’-TCTCTGCTCCCCACCTCTAAGT).
ATP measurement:
Cellular ATP levels were measured with Luminescent ATP detection assay kit (Abcam) according to manufacturer’s protocol. The cells were cultured in the medium containing galactose instead of glucose and the readings were normalized to DNA abundance measured by Sybrsafe staining.
Statistical methods
Group descriptive statistics are presented as mean ± SD and group count (percentage). The Kolmogorov-Smirnov test was used to test normality of the parameters (cord resistin, maternal Age, HbA1C, BMI, and gestational age). Among them, cord resistin, maternal age, and HbA1c were not normally distributed. Differences in characteristics between control and diabetic groups were assessed using Student’s t-test for normal distribution and nonparametric Mann-Whitney test for non-normal distribution. Maternal glucose (OGTT-2 hours) and maternal factors which displayed significant difference between control and diabetic groups, including maternal age, HbA1C, BMI, and gestational age, were subjected to correlation analysis with cord resistin. Spearman correlations were used for correlation analysis for non-normal distributions. Multiple regression analysis was conducted to further assess relationships after controlling multiple variables. In the multiple regression model, cord resistin was the dependent variable and study groups (control and diabetes), maternal age, BMI and gestational age were the independent variables. The statistical analysis were performed in Excel, GraphPad Prism, and SPSS. For all analysis, P-values <0.05 were treated as statistically significant.