Maricela Haghiac

Pregravid obesity associates with increased maternal endotoxemia and metabolic inflammation.

Obese pregnant women develop severe insulin resistance and enhanced systemic and placental inflammation, suggesting associated modifications of endocrine and immune functions. Activation of innate immunity by endotoxins/lipopolysaccharides (LPS) has been proposed as a mechanism for enhancing metabolic alterations in disorders with insulin resistance. The aim of this study was to characterize the immune responses developed by the adipose tissue (AT) and their potential links to maternal endotoxemia in pregnancy with obesity. Blood and subcutaneous abdominal AT were obtained from 120 lean and obese women (term pregnancy) recruited at delivery. Gene expression was assessed in AT and stromal vascular cells isolated from a subset of 24 subjects from the same cohort. Doubling of plasma endotoxin concentrations indicated subclinical endotoxemia in obese compared with lean women. This was associated with significant increase in systemic C‐reactive protein and interleukin‐6 (IL‐6) but not tumor necrosis factor‐α (TNF‐α) concentrations. AT inflammation was characterized by accumulation of CD68+ macrophages with a threefold increased gene expression of the macrophage markers CD68, EMR1, and CD14. Gene expression for cytokines IL‐6, TNF‐α, IL‐8, and monocyte chemotactic protein‐1 (MCP1) and for LPS—sensing CD14, toll‐like receptor 4 (TLR4), translocating chain‐associated membrane protein 2 was 2.5‐5‐fold higher in stromal cells of obese compared to lean. LPS‐treated cultured stromal cells of obese women expressed a 5–16‐fold stimulation of the same cytokines upregulated in vivo. Our data demonstrate that subclinical endotoxemia is associated with systemic and AT inflammation in obese pregnant women. Recognition of bacterial pathogens may contribute to the combined dysfunction of innate immunity and the metabolic systems in AT.

Increased Death of Adipose Cells, a Path to Release Cell‐Free DNA Into Systemic Circulation of Obese Women

Remodeling of adipose tissue is required to support the expansion of adipose mass. In obesity, an increased death of adipocytes contributes to the accelerated cellular turnover. We have shown that obesity in pregnancy is associated with metabolic and immune alterations in the adipose tissue. In this study, we characterized the mechanisms responsible for increased death of adipose cells of pregnant obese women and its functional consequences. We postulated that a higher turnover of dead cells in white adipose tissue of obese women would translate into release of cell‐free DNA (cfDNA) into their systemic circulation. Increase in adipose mass of obese compared to lean women results from a lesser number of hypertrophic adipocytes and an accumulation of macrophages in the stromal vascular fraction (SVF). The adipocytes of obese displayed enhanced necrosis with a loss of perilipin staining at the plasma membrane. Apoptosis was prominent in SVF cells with an increased expression of caspase 9 and caspase 3 and a higher rate of terminal deoxynucleotidyl transferase‐mediated deoxyuridine triphosphate nick end‐labeling (TUNEL) positive CD68 macrophages in obese vs. lean. Whereas circulating fetal cfDNA concentrations were not changed, there was a twofold increase in circulating glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) cfDNA and adipose tissue GAPDH mRNA in obese women. The maternal systemic GAPDH cfDNA was positively correlated with BMI and gestational weight gain. These data suggest that the active remodeling of adipose tissue of obese pregnant women results in an increased release of cfDNA of maternal origin into the circulation.

Patterns of Adiponectin Expression in Term Pregnancy: Impact of Obesity

CONTEXT Adiponectin (adpN) production is down-regulated in several situations associated with insulin resistance. The hypoadiponectinemia, which develops in late pregnancy, suggests a role of adpN in pregnancy-induced insulin resistance. OBJECTIVE In obese pregnancy there is a decreased systemic adpN, which results from down-regulation of gene expression in adipose tissue. SETTING AND DESIGN One hundred and thirty-three women with uncomplicated pregnancies and a wide range in pre-gravid body mass index (18-62 kg/m(2)) were recruited at term for a scheduled cesarean delivery. Maternal blood, placenta, and sc abdominal adipose tissue were obtained in the fasting state. DNA methylation was analyzed by MBD-based genome-wide methylation sequencing and methyl-specific PCR of placenta and maternal adipose tissue. mRNA and protein expression were characterized by real-time RT-PCR and immunodetection. Plasma adpN, leptin, and insulin were assayed by ELISA. RESULTS Maternal adipose tissue was the prominent site of adpN gene expression with no detectable mRNA or protein in placenta. In obese women, adipose tissue adpN mRNA was significantly decreased (P < .01) whereas DNA methylation was significantly increased (P < .001) compared with lean women. The decreased adipose tissue expression resulted in normal-weight women having significantly greater plasma adpN compared with the severely obese (12.8 ± 4.3 ng/mL vs 8.6 ± 3.1, P < .001). Plasma adpN was negatively correlated with maternal body mass index (r = -0.28, P < .001) and homeostasis model assessment indices of insulin sensitivity (r = -0.32, P < .001) but not with gestational weight gain. CONCLUSIONS Maternal adipose tissue is the primary source of circulating adpN during pregnancy. Further, based on our results, the placenta does not synthesize adiponectin at term. Obesity in pregnancy is associated with negative regulation of adpN adipose expression with increase in adpN DNA methylation associated with lower mRNA concentrations and hypoadiponectinemia. Maternal hypoadiponectinemia may have functional consequences in down-regulating biological signals transmitted by adpN receptors in various tissues, including the placenta.

Molecular inflammation and adipose tissue matrix remodeling precede physiological adaptations to pregnancy

Changes in adipose tissue metabolism are central to adaptation of whole body energy homeostasis to pregnancy. To gain insight into the molecular mechanisms supporting tissue remodeling, we have characterized the longitudinal changes of the adipose transcriptome in human pregnancy. Healthy nonobese women recruited pregravid were followed in early (8-12 wk) and in late (36-38 wk) pregnancy. Adipose tissue biopsies were obtained in the fasting state from the gluteal depot. The adipose transcriptome was examined via whole genome DNA microarray. Expression of immune-related genes and extracellular matrix components was measured using real-time RT-PCR. Adipose mass, adipocyte size, and cell number increased in late pregnancy compared with pregravid measurements (P < 0.001) but remained unchanged in early pregnancy. The adipose transcriptome evolved during pregnancy with 10-15% of genes being differently expressed compared with pregravid. Functional gene cluster analysis revealed that the early molecular changes affected immune responses, angiogenesis, matrix remodeling, and lipid biosynthesis. Increased expression of macrophage markers (CD68, CD14, and the mannose-6 phosphate receptor) emphasized the recruitment of the immune network in both early and late pregnancy. The TLR4/NF-κB signaling pathway was enhanced specifically in relation to inflammatory adipokines and chemokines genes. We conclude that early recruitment of metabolic and immune molecular networks precedes the appearance of pregnancy-related physiological changes in adipose tissue. This biphasic pattern suggests that physiological inflammation is an early step preceding the development of insulin resistance, which peaks in late pregnancy.

Saturated fatty acids enhance TLR4 immune pathways in human trophoblasts

STUDY QUESTION What are the effects of fatty acids on placental inflammatory cytokine with respect to toll-like receptor-4/nuclear factor-kappa B (TLR4/NF-kB)? SUMMARY ANSWER Exogenous fatty acids induce a pro-inflammatory cytokine response in human placental cells in vitro via activation of TLR4 signaling pathways. WHAT IS KNOWN ALREADY The placenta is exposed to changes in circulating maternal fatty acid concentrations throughout pregnancy. Fatty acids are master regulators of innate immune pathways through recruitment of toll-like receptors and activation of cytokine synthesis. STUDY DESIGN, SIZE, DURATION Trophoblast cells isolated from 14 normal term human placentas were incubated with long chain fatty acids (FA) of different carbon length and degree of saturation. The expression and secretion of interleukin-6 (IL-6), IL-8 and tumor necrosis factor-alpha (TNF-α) were measured by reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. Antibodies against TLR4 ligand binding domain, downstream signaling and anti-p65 NFkB-inhibitor were used to characterize the pathways of FA action. PARTICIPANTS/MATERIALS, SETTING, METHODS General approach used primary human term trophoblast cell culture. Methods and end-points used real-time quantitative PCR, cytokine measurements, immunohistochemistry, western blots. MAIN RESULTS AND THE ROLE OF CHANCE The long chain saturated fatty acids, stearic and palmitic (PA), stimulated the synthesis as well as the release of TNF-α, IL-6 and IL-8 by trophoblast cells (2- to 6-fold, P < 0.001). In contrast, the unsaturated (palmitoleic, oleic, linoleic) acids did not modify cytokine expression significantly. Palmitate-induced inflammatory effects were mediated via TLR4 activation, NF-kB phosphorylation and nuclear translocation. LIMITATIONS, REASONS FOR CAUTION TNF-α protein level was close to the limit of detection in the culture medium even when cells were cultured with PA. WIDER IMPLICATIONS OF THE FINDINGS These mechanisms open the way to a better understanding of how changes in maternal lipid homeostasis may regulate placental inflammatory status. STUDY FUNDING/COMPETING INTERESTS X.Y. was recipient of fellowship award from West China Second University Hospital, Sichuan University (NIH HD 22965-19). The authors have nothing else to disclose. TRIAL REGISTRATION NUMBER None.

Identification of early transcriptome signatures in placenta exposed to insulin and obesity

OBJECTIVE The purpose of this study was to investigate the effects of insulin on human placental transcriptome and biological processes in first-trimester pregnancy. STUDY DESIGN Maternal plasma and placenta villous tissue were obtained at the time of voluntary termination of pregnancy (7-12 weeks) from 17 lean (body mass index, 20.9±1.5 kg/m2) and 18 obese (body mass index, 33.5±2.6 kg/m2) women. Trophoblast cells were immediately isolated for in vitro treatment with insulin or vehicle. Patterns of global gene expression were analyzed using genome microarray profiling after hybridization to Human Gene 1.1 ST and real time reverse transcription-polymerase chain reaction. RESULTS The global trophoblast transcriptome was qualitatively separated in insulin-treated vs untreated trophoblasts of lean women. The number of insulin-sensitive genes detected in the trophoblasts of lean women was 2875 (P<.001). Maternal obesity reduced the number of insulin-sensitive genes recovered by 30-fold. Insulin significantly impaired several gene networks regulating cell cycle and cholesterol homeostasis but did not modify pathways related to glucose transport. Obesity associated with high insulin and insulin resistance, but not maternal hyperinsulinemia alone, impaired the global gene profiling of early gestation placenta, highlighting mitochondrial dysfunction and decreased energy metabolism. CONCLUSION We report for the first time that human trophoblast cells are highly sensitive to insulin regulation in early gestation. Maternal obesity associated with insulin resistance programs the placental transcriptome toward refractoriness to insulin with potential adverse consequences for placental structure and function.

Oxidative Stress Impairs Fatty Acid Oxidation and Mitochondrial Function in the Term Placenta

Placental fatty acid oxidation (FAO) is impaired and lipid storage is increased in pregnancy states associated with chronic oxidative stress. The effect of acute oxidative stress, as seen in pregnancies complicated with asthma, on placental lipid metabolism is unknown. We hypothesized that induction of acute oxidative stress would decrease FAO and increase esterification. We assessed [3H]-palmitate oxidation and esterification in term placental explants from lean women after exposure to hydrogen peroxide (H2O2) for 4 hours. Fatty acid oxidation decreased 16% and 24% in placental explants exposed to 200 (P = .02) and 400 µM H2O2 (P = .01), respectively. Esterification was not altered with H2O2 exposure. Neither messenger RNA nor protein expression of key genes involved in FAO (eg, peroxisome proliferator-activated receptor α, carnitine palmitoyl transferase 1b) were altered. Adenosine triphosphate (ATP) levels decreased with induction of oxidative stress, without increasing cytotoxicity. Acute oxidative stress decreased FAO and ATP production in the term placenta without altering fatty acid esterification. As decreases in placental FAO and ATP production are associated with impaired fetal growth, pregnancies exposed to acute oxidative stress may be at risk for fetal growth restriction.

Fatty acid transporter expression and regulation is impaired in placental macrovascular endothelial cells in obese women

Abstract Objective: Fetal fatty acid (FA) delivery is ultimately controlled by placental transport. Focus has been the maternal-placental interface, but regulation at the feto-placental interface is unknown. Methods: Placental macrovascular endothelial cells (EC) (n = 4/group) and trophoblasts (TB) (n = 5/group) were isolated from lean (pregravid BMI <25 kg/m2) and obese (body mass index (BMI) > 30) women. Fatty acid transporters FAT/CD36, FABPpm, FATP4, FABP 3, 4 and 5, PLIN2 and PPARα, δ, γ expression, was measured in EC and TB. Transporter response to 24 h palmitate (PA) was assessed. Results: mRNA expression of FABP3, 4, 5 and PPARγ was 2- to 3-fold reduced in EC of obese versus lean women (p < .03), but not in TB. Protein level of FABPpm was 20% lower in obese (p < .05). Palmitate (PA) up-regulated CD36, FABP3, FABP4, and PLIN2 gene expression by 3- to 4-fold in lean but not obese EC (p < .05), while PA increased FABP4 and PLIN2 in lean and obese TB, and FABP5 in lean (p < .05) EC. PA exposure up-regulated peroxisome proliferator activated receptors (PPARs) 2-fold in lean and obese EC (p < .05), but not in TB. Conclusions: In obese women, FA transporter expression is lower in placental EC, but not TB, and less sensitive to saturated FA, compared to lean women. FA transport may be regulated at the feto-placental interface.