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Maternal Obesity Programs Mitochondrial and Lipid Metabolism Gene Expression in Infant Umbilical Vein Endothelial Cells

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Maternal Obesity Programs Mitochondrial and Lipid Metabolism Gene Expression in Infant Umbilical Vein Endothelial Cells International Journal of Obesity (2016), 1–8 © 2016 Macmillan Publishers Limited, part of Springer Nature. All rights reserved 0307-0565/16 www.nature.com/ijo ORIGINAL ARTICLE Maternal obesity programs mitochondrial and lipid metabolism gene expression in infant umbilical vein endothelial cells SMR Costa1,2,3,4, E Isganaitis2,3,4, TJ Matthews2, K Hughes2, G Daher2, JM Dreyfuss2, GAP da Silva1 and M-E Patti2,3 BACKGROUND/OBJECTIVES: Maternal obesity increases risk for childhood obesity, but molecular mechanisms are not well understood. We hypothesized that primary umbilical vein endothelial cells (HUVEC) from infants of overweight and obese mothers would harbor transcriptional patterns reflecting offspring obesity risk. SUBJECTS/METHODS: In this observational cohort study, we recruited 13 lean (pre-pregnancy body mass index (BMI) o25.0 kg m − 2) and 24 overweight-obese (‘ov-ob’, BMI ⩾ 25.0 kg m − 2) women. We isolated primary HUVEC, and analyzed both gene expression (Primeview, Affymetrix) and cord blood levels of hormones and adipokines. RESULTS: A total of 142 transcripts were differentially expressed in HUVEC from infants of overweight-obese mothers (false discovery rate, FDRo0.05). Pathway analysis revealed that genes involved in mitochondrial and lipid metabolism were negatively correlated with maternal BMI (FDRo0.05). To test whether these transcriptomic patterns were associated with distinct nutrient exposures in the setting of maternal obesity, we analyzed the cord blood lipidome and noted significant increases in the levels of total free fatty acids (lean: 95.5 ± 37.1 μgml− 1, ov-ob: 124.1 ± 46.0 μgml− 1, P = 0.049), palmitate (lean: 34.5 ± 12.7 μgml− 1, ov-ob: 46.3 ± 18.4 μgml− 1, P =0.03) and stearate (lean: 20.8 ± 8.2 μgml− 1, ov-ob: 29.7 ± 17.2 μgml− 1, P = 0.04), in infants of overweight-obese mothers. CONCLUSIONS: Prenatal exposure to maternal obesity alters HUVEC expression of genes involved in mitochondrial and lipid metabolism, potentially reflecting developmentally programmed differences in oxidative and lipid metabolism. International Journal of Obesity advance online publication, 4 October 2016; doi:10.1038/ijo.2016.142 INTRODUCTION Fewer mechanistic studies have examined metabolic phenotypes Evidence from human populations and animal models indicates in humans, largely owing to the practical and ethical challenges of that environmental exposures during early development are obtaining cells and tissues from infants. However, umbilical cords, critical determinants of disease susceptibility throughout the which are usually discarded after delivery, provide an accessible lifespan, a phenomenon termed ‘developmental programming’.1 source of infant cells for translational studies. Interestingly, the A wide range of prenatal perturbations, including maternal analysis of umbilical cord segments from infants of women with undernutrition, obesity, diabetes, high-fat diet and endocrine- type 1 diabetes identified differences in expression of genes disrupting chemicals, are now recognized as risk factors for related to vascular development and function.11 chronic diseases including diabetes, obesity and cardiovascular Primary human umbilical vein endothelial cells (HUVEC) are disease.2–4 Maternal obesity is of particular concern, as it is a readily isolated, remain viable and metabolically active in culture, potent risk factor for childhood obesity: offspring of mothers and are insulin-responsive, features leading to their wide use in entering pregnancy with body mass index (BMI) 430 kg m − 2 vascular biology for over 40 years,12 and more recently, in studies have a 1.5 to 4-fold higher risk of childhood obesity.5 Studies of of fetal adaptations to maternal diabetes and placental insuffi- siblings born before vs after a mother’s weight loss surgery— ciency. For example, maternal gestational diabetes is associated which minimize the contribution of shared genetics—suggest that with reduced vasodilation13 and increased leukocyte adhesion in in utero exposure to maternal obesity per se can increase risk of HUVEC,14 potentially mediated by specific microRNAs.15 Moreover, childhood obesity 42-fold.6,7 increased eNos promoter methylation has been reported in HUVEC Unfortunately, the molecular mechanisms by which maternal from infants with intrauterine growth restriction.16 obesity increases metabolic risk in offspring remain incompletely We therefore hypothesized that maternal obesity would alter understood. Previous rodent and primate studies indicate that metabolism in HUVEC in a cell-autonomous fashion. We now maternal insulin resistance, which is tightly correlated with demonstrate that maternal obesity is associated with a dramatic maternal obesity,8 may be one contributor to obesity-associated transcriptional response in infant HUVEC, particularly within developmental programming.9,10 Other mediators may include pathways related to lipid metabolism and mitochondrial struc- shared environmental risk factors, epigenetics and/or hormonal ture/function, and is accompanied by increases in cord blood and metabolic adaptations to an ‘obese’ intrauterine environment. insulin, palmitate and stearate. 1Universidade Federal de Pernambuco, Recife, Pernambuco, Brazil; 2Research Division, Joslin Diabetes Center, Boston, MA, USA and 3Harvard Medical School, Boston, MA, USA. Correspondence: Dr E Isganaitis, Research Division, Department of Genetics and Epidemiology, Joslin Diabetes Center and Pediatrics Division, Harvard Medical School, 1 Joslin Place, Room 607, Boston, MA 02215, USA. E-mail: [email protected] 4These two authors contributed equally to this work. Received 5 April 2016; revised 24 May 2016; accepted 25 June 2016; accepted article preview online 17 August 2016 Effects of maternal obesity on infant HUVEC SMR Costa et al 2 MATERIALS AND METHODS and the effluent collected. The cells were then centrifuged and Human subjects—Recife cohort resuspended in complete growth media. Vascular cell growth media contains vascular endothelial growth factor (5 ng ml − 1), endothelial Pregnant women were recruited during prenatal visits at Instituto − − growth factor (5 ng ml 1), fibroblast growth factor basic (5 ng ml 1) de Medicina Integral Professor Fernando Figueira, Recife, Brazil. Eligibility − 1 − 1 IGF-1 (15 ng ml ), L-glutamine (10 mM), heparin sulfate (0.75 units ml ), criteria included age ⩾ 18 years and known gestational age (based on date − − hydrocortisone (1 μgml 1), ascorbic acid (50 μgml 1) and fetal bovine of last menstrual period or ultrasound before 16 weeks). Exclusion criteria serum (2%; ATCC Endothelial Cell Growth Kit, PCS-100-041). included diabetes diagnosed before or during the current pregnancy, multiple gestation, hypertension, artificial reproductive techniques, HIV, syphilis, maternal disease requiring medications known to affect fetal RNA isolation and microarray analysis growth or glucose metabolism, fetal congenital malformations, premature For each cell line, an aliquot of freshly isolated HUVEC was stored in RNA delivery (o36 weeks) and deliveries occurring outside the study hospital. Later reagent (Life Technologies, Thermo-Fisher Scientific, Waltham, MA, USA) The participants were grouped by early pregnancy BMI, defined as BMI at at − 80 °C until RNA extraction, using Trizol (Life Technologies, Thermo-Fisher − 2 the first prenatal visit (o12 weeks gestation): lean (o25.0 kg m )vs Scientific) according to the manufacturer’s protocol, with glycogen (Cat. No. − 2 overweight and obese (‘ov-ob’, ⩾ 25.0 kg m ). The study protocol was AM9510, Life Technologies, Thermo-Fisher Scientific) added as a carrier. The approved by the Hospital Institutional Review Board and the National complementary RNA was prepared and hybridized to Affymetrix PrimeView Committee for Ethics in Research at National Health Council (CONEP- microarrays (Santa Clara, CA, USA; Cat. No. 901837). The data were Statement #387/2011), Brasilia, Brazil, and by the Committee for Human normalized using Robust Multichip Average19 and log-2 transformed. Subjects at Joslin Diabetes Center, Boston, MA, USA. All the participants Normalized microarray data were analyzed (P, FDR (false discovery rate)) provided written informed consent. using GenePattern Comparative Marker Selection module (Broad Institute, Cambridge, MA, USA). Heatmaps were created using GENE-E (Broad Institute). Assessments during pregnancy—Recife cohort Pathway analyses were performed using Gene Set Enrichment Analysis (Broad Institute)20,21 and DAVID (Bioinformatics Resources v6.7).22 At the first prenatal visit, maternal medical history, including systemic disease, smoking, obstetric history and clinical data (height, weight, blood pressure, date of last menstrual period and fasting glucose values) were Quantitative real time PCR collected. Maternal glucose metabolism was assessed twice: a 1 h 75 g oral Complementary DNA (cDNA) was generated using High-Capacity cDNA glucose screen during the second trimester (24 weeks ± 2 weeks), and a 2 h Reverse Transcription Kit (Life Technologies, Thermo-Fisher Scientific) with 75 g oral glucose tolerance test during the third trimester (glucose and 500 ng input RNA. Expression of selected genes was assessed by insulin at time 0 (fasting), 1 and 2 h (30 weeks ± 2 weeks)). Homeostasis quantitative real time PCR, with expression normalized to cyclophilin E method assessment of insulin resistance (HOMA-IR) was calculated as (PPIE), using an ABI 7900 HT thermocycler. Primer sequences are available [(glucose × insulin)/405], and homeostatic
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