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Pregnancy conditions like maternal pre-pregnancy obesity and gestational diabetes (GDM) have been linked with the development of placenta, a critical organ required for maternal-fetal interface during gestation, and can lead to adverse health outcomes in both mother and fetus. However, the molecular signatures associated with these conditions are not fully understood. Epigenetic clocks, which are based upon DNA methylation of age-related CpGs, are thought to better reflect biological age compared to chronological age and have been used in different tissues as potential biomarkers. In the present study, we hypothesized that altered placental epigenetic gestational aging is associated with pre-pregnancy obesity and GDM. We explored these relationships by leveraging data from three diverse cohorts in the Environmental influences on Child Health Outcomes (ECHO) program (overall N=830) . First, we investigated associations between maternal pre-pregnancy obesity and GDM and estimated placental epigenetic gestational age acceleration (eGAA) . Next, we examined associations between these two conditions and methylation of each CpG site within the robust placental clock (558 CpGs) . Significant associations were not observed between pre-pregnancy obesity and GDM and eGAA. Interestingly, pre-pregnancy obesity was associated with methylation levels at 28 placental clock CpGs (q Disclosure K.Huff: None. C.Marsit: None. S.Niemiec: None. H.Volk: None. T.M.O'shea: None. R.Fry: None. K.Roell: None. C.Bulka: None. K.E.Boyle: None. C.Breton: None. A.Burt: None. D.Dabelea: None. L.Kahn: None. M.R.Karagas: None. Funding National Institutes of Health (UH3OD023348)

An adverse intrauterine environment is associated with the future risk of obesity and type 2 diabetes. Changes in placental function may underpin the intrauterine origins of adult disease, but longitudinal studies linking placental function with childhood outcomes are rare. Here, we determined the abundance and phosphorylation of protein intermediates involved in insulin signaling, inflammation, cortisol metabolism, protein glycosylation, and mitochondrial biogenesis in placental villus samples from healthy mothers from the Healthy Start cohort. Using MANOVA, we tested the association between placental proteins and offspring adiposity (fat mass percentage) at birth (n = 109) and infancy (4–6 months, n = 104), and adiposity, skinfold thickness, triglycerides, and insulin in children (4–6 years, n = 66). Placental IGF-1 receptor protein was positively associated with serum triglycerides in children. GSK3β phosphorylation at serine 9, a readout of insulin and growth factor signaling, and the ratio of phosphorylated to total JNK2 were both positively associated with midthigh skinfold thickness in children. Moreover, peroxisome proliferator–activated receptor γ coactivator (PGC)-1α abundance was positively associated with insulin in children. In conclusion, placental insulin/IGF-1 signaling, PGC-1α, and inflammation pathways were positively associated with metabolic outcomes in 4- to 6-year-old children, identifying a novel link between placental function and long-term metabolic outcomes.

An adverse intrauterine environment is associated with the future risk of obesity and type 2 diabetes. Changes in placental function may underpin the intrauterine origins of adult disease, but longitudinal studies linking placental function with childhood outcomes are rare. Here, we determined the abundance and phosphorylation of protein intermediates involved in insulin signaling, inflammation, cortisol metabolism, protein glycosylation, and mitochondrial biogenesis in placental villus samples from healthy mothers from the Healthy Start cohort. Using MANOVA, we tested the association between placental proteins and offspring adiposity (percent fat mass) at birth (n=109) and infancy (4-6mo, n=104), and adiposity, skinfold thickness, triglycerides, and insulin in children (4-6y, n=66). Placental IGF-1 receptor protein was positively associated with serum triglycerides in children. GSK3β phosphorylation at serine 9, a readout of insulin and growth factor signaling, and the ratio of phosphorylated to total JNK2 were both positively associated with midthigh skinfold thickness in children. Moreover, PGC-1α abundance was positively associated with insulin in children. In conclusion, placental insulin/IGF-1 signaling, PGC-1α, and inflammation pathways were positively associated with metabolic outcomes in 4-6-year-old children, identifying a novel link between placental function and long-term metabolic outcomes.

Introduction: Maternal obesity is associated with greater neonatal adiposity and increased offspring risk for type 2 diabetes (T2D) later in life. We have previously reported that mesenchymal stem cells (MSCs) isolated from umbilical cord tissue maintain similar metabolic phenotype to the infant, particularly when differentiated to adipocytes or myocytes. A well-established driver in the pathogenesis of T2D is skeletal muscle insulin resistance. Therefore, we hypothesized that MSCs from infants born to mothers with obesity would display insulin resistance when differentiated to myocytes. Objectives: The objective was to determine the effect of maternal obesity on offspring MSC insulin sensitivity (Si). Methods: MSCs were isolated from umbilical cord tissue from offspring born to either normal weight (BMI 21.1 ± 0.9, n = 9) or obese (BMI 32.9 ± 2.1, n = 10) mothers enrolled in the Healthy Start pre-birth cohort study in Colorado. Following differentiation into skeletal muscle myocytes, MSCs were serum starved and incubated with 14C-glucose, in the presence or absence of insulin. The rate of insulin-mediated 14C-glucose incorporation into glycogen, was used as an index of offspring Si. Results: Offspring Si did not correlate with maternal BMI (r = 0.26), nor was BMI different between groups when median-stratified by offspring Si. However, maternal free fatty acid levels positively correlated with offspring Si (r = 0.63, p < 0.05) and were significantly elevated in the high Si group (low Si vs. high Si; 332.1 ± 30.9 vs. 483.3 ± 48.7 uEq/L, p < 0.05). Conclusions: Offspring Si was not related to the BMI of the mother. Moreover, offspring Si was higher in cells from infants born to mothers with higher free fatty acids during pregnancy. Considering the role of insulin in somatic growth pathways, this phenomenon may serve to increase available nutrient storage capacity during early development. Disclosure A.B. Chaves: None. J.A. Johnson: None. D. Zheng: None. D. Dabelea: None. J.A. Houmard: None. K.E. Boyle: None. Funding American Heart Association (14PRE18230008); Nutrition Obesity Research Center (P30DK048520); National Institutes of Health (R01DK076648); Colorado National Center for Advancing Translational Sciences (UL1TR001082)

We have reported that umbilical cord mesenchymal stem cells (MSCs) from infants born to mothers with obesity have less AMP-activated protein kinase [AMPK] and less inhibitory phosphorylation of glycogen synthase kinase 3 [GSK3]β, a regulator of MSC differentiation. This is accompanied by a greater capacity for in vitro adipogenesis, which positively correlates with infant adiposity. Here we hypothesized that, independent of maternal pre-pregnancy (pp)BMI, maternal glucose and insulin would be associated with GSK3β and AMPK in undifferentiated infant MSCs. We studied 67 women (ppBMI 18-42 kg/m2) at mid (15-23 weeks) and late (24-32 weeks) gestation without preeclampsia or gestational diabetes, as well as their infants’ MSCs. We ran linear models to test the effect of fasting maternal glucose and insulin on each phosphorylated MSC protein, with infant sex and gestational age as covariates, ± ppBMI as a covariate to account for collinearity with metabolic measures. Linear model p-values and Pearson correlations are reported. Maternal glucose at mid (p = 0.03, r = 0.26) and late (p = 0.009, r = 0.35) gestation had a significant effect on AMPKThr172, with no effect of ppBMI, indicating greater AMPK activity with higher maternal glucose. Mid gestation insulin (p = 0.04, r = -0.21) was associated with GSK3βSer9, with the same trend for HOMA-IR—but not when ppBMI was included as a covariate, as it was also negatively associated with GSK3βSer9 (p = 0.02, r = -0.24), showing collinearity of ppBMI and insulin (r = 0.40). This signifies higher ppBMI and insulin with less GSK3β inhibition, which we have previously linked to greater adipogenesis. These results demonstrate the novel finding that maternal glucose during pregnancy is associated with perturbations to lipid metabolism pathways in infant MSCs across a wide range of ppBMI, giving important clues as to how maternal metabolism may impact infant metabolic disease risk. Disclosure M. Keleher: None. D. Dabelea: None. J.E. Friedman: Consultant; Self; Janssen Research & Development. K.E. Boyle: None. Funding National Institutes of Health (4UH3OD023248-03)