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OBJECTIVES: Gender has been shown to impact disease expression in ankylosing spondylitis (AS) and Th17 cells play a key role in AS pathogenesis. To better understand what Th17-associated immune pathways are different between men and women, we compared the transcriptome of IL-17-enriched peripheral blood mononuclear cells (PBMCs) in male and female AS patients, with a particular focus on inflammatory cytokine genes. METHODS: PBMCs were collected from 10 female and 11 male AS patients at the Clinical Research Unit of MetroHealth Medical Center. IL-17-enriched PBMCs were isolated and stimulated with CytoStim. RNA-sequencing (RNA-seq) was performed on the samples, and the data were analysed using iPathwayGuide. Inflammatory markers and genes related to Th17 differentiation and function were identified based on previous studies. RESULTS: RNA-seq identified 12,893 genes with 2,851 genes with p-values <0.05 with distinct patterns of gene expression between male and female AS patients. TGF-ß, PGE2, and S100 proteins were significantly upregulated in males. Levels of IL-12B, a Th17 inducer, were lower in males compared to females. Additionally, receptors of IL-6, 12, 23, TGF-ß, and PGE2 were downregulated in males, except for IL-17RC, which was upregulated. Genes involved in Th17 differentiation showed differential expression between genders, with elevated expression of BATF, SOCS1, NKD2, and ARID5A in men and decreased expression of FOXO1. CONCLUSIONS: Transcriptomic analysis revealed that male AS patients exhibit distinct expression patterns of IL-17 pro-inflammatory genes, which may contribute to the phenotypic differences observed between genders in AS.
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Interleucina-17 , Espondilitis Anquilosante , Células Th17 , Humanos , Espondilitis Anquilosante/genética , Espondilitis Anquilosante/inmunología , Masculino , Femenino , Interleucina-17/genética , Interleucina-17/metabolismo , Células Th17/inmunología , Células Th17/metabolismo , Adulto , Factores Sexuales , Transcriptoma , Persona de Mediana Edad , Leucocitos Mononucleares/metabolismo , Leucocitos Mononucleares/inmunología , Perfilación de la Expresión Génica , RNA-Seq , Mediadores de Inflamación/metabolismoRESUMEN
OBJECTIVE: Seropositive rheumatoid arthritis (RA) is a chronic autoimmune disease that is rarely "cured." Human mesenchymal stem cells (hMSCs) are known to reduce inflammation and restore immune homeostasis. However, methods for predicting therapeutic hMSC potency have not been established. The goal of these studies was to use and refine an ex vivo functional assay that determines potency of hMSCs and can then be validated in clinical trials as a potency measure of hMSCs used therapeutically to treat RA. METHODS: Allogeneic hMSCs were cytokine-stimulated, and a conditioned medium (CM) was harvested. The CM was tested for the potential to attenuate RA CD4+ T cell proliferation using suppression assays. Indoleamine 2, 3-dioxygenase (IDO) mRNA, and protein were quantified in hMSCs as a measure to compare hMSCs across (prior) studies. RESULTS: To mimic a proinflammatory environment that resembles that in RA, interleukin-1(IL1ß), tumor necrosis factor α (TNFα), and interferon γ (IFNγ) (alone or in combination) were used to precondition hMSCs. Treating hMSCs with a combination of these cytokines generated a CM "secretome" that suppressed T cell proliferation between 70 and 83%. Forty-eight hours of cytokine preconditioning hMSCs was required to maximize this effect. T cell suppression positively correlated with increases in hMSC cellular IDO mRNA and protein. CONCLUSION: By standardizing assays to measure hMSC effects, their potency on T cell suppression can be quantified. These studies demonstrate that hMSCs can be compared functionally to identify optimal preparation(s) for therapeutic use in RA and that the potency of hMSC-dependent T cell suppression may differ between hMSC donors. Clinical studies are warranted to validate the hypothesis that ex vivo potency in suppressing T cells will positively correlate with a reduction in RA disease activity and increase in immunological quiescence.
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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.
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Proteínas de Unión a Ácidos Grasos/metabolismo , Ácidos Grasos/metabolismo , Obesidad/metabolismo , Placenta/metabolismo , Complicaciones del Embarazo/metabolismo , Estudios de Casos y Controles , Cesárea , Células Endoteliales/metabolismo , Femenino , Humanos , Embarazo , Trofoblastos/metabolismoRESUMEN
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.
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Metabolismo Energético , Mitocondrias/metabolismo , Estrés Oxidativo , Ácido Palmítico/metabolismo , Placenta/metabolismo , Adenosina Trifosfato/metabolismo , Adulto , Carnitina O-Palmitoiltransferasa/genética , Carnitina O-Palmitoiltransferasa/metabolismo , Células Cultivadas , Esterificación , Femenino , Humanos , Peróxido de Hidrógeno/administración & dosificación , Mitocondrias/efectos de los fármacos , Oxidación-Reducción , Estrés Oxidativo/efectos de los fármacos , PPAR alfa/genética , PPAR alfa/metabolismo , Placenta/efectos de los fármacos , Embarazo , Adulto JovenRESUMEN
OBJECTIVE: Adenosine monophosphate-activated protein kinase (AMPK) is a cellular energy sensor whose phosphorylation increases energy production. We sought to evaluate the placenta-specific effect of AMPK activation on the handling of nutrients required for fetal development. METHODS: Explants were isolated from term placenta of 29 women (pregravid body mass index: 29.1 ± 9.9 kg/m2) and incubated for 24 hours with 0 to 100 µmol/L resveratrol or 0 to 1 mmol/L of 5-aminoimidazole-4-carboxyamide ribonucleoside (AICAR). Following treatment, uptake and metabolism of radiolabeled fatty acids and glucose were measured. Phosphorylation of AMPK was measured by Western blotting. Adenosine diphosphate (ATP) production was assessed using the mitochondrial ToxGlo assay kit. P < .05 was considered statistically significant. RESULTS: Resveratrol and AICAR increased AMPK phosphorylation in human placental explants. Exposure to resveratrol decreased the uptake of polyunsaturated fatty acids, arachidonic acid, and docosahexaenoic acid at 100 µmol/L ( P < .0001). Fatty acid oxidation was decreased by 100 µmol/L ( P < .05) resveratrol, while esterification was unchanged. Resveratrol decreased glucose uptake at the 50 and 100 µmol/L doses ( P < .05). Glycolysis was not significantly affected. AICAR had similar effects, decreasing fatty acid uptake and glycolysis ( P < .05). Production of ATP declined at doses found to decrease nutrient metabolism ( P < .05). CONCLUSIONS: Activation of AMPK in the human placenta leads to global downregulation of metabolism, with mitotoxicity induced at the doses of resveratrol and AICAR used to activate AMPK. Although activation of this pathway has positive metabolic effects on other tissues, in the placenta there is potential for harm, as inadequate placental delivery of critical nutrients may compromise fetal development.
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Proteínas Quinasas Activadas por AMP/metabolismo , Mitocondrias/metabolismo , Placenta/metabolismo , Adulto , Aminoimidazol Carboxamida/administración & dosificación , Aminoimidazol Carboxamida/análogos & derivados , Inhibidores Enzimáticos/administración & dosificación , Ácidos Grasos/metabolismo , Femenino , Glucosa/metabolismo , Humanos , Fosforilación , Embarazo , Resveratrol/administración & dosificación , Ribonucleótidos/administración & dosificación , Trofoblastos/metabolismoRESUMEN
INTRODUCTION: Placentas of obese women have higher lipid content compared to lean women. We have previously shown that supplementation of overweight and obese women with omega-3 fatty acids decreases placental esterification pathways and total lipid content in a mid-western population (Ohio). We hypothesized that placental lipid accumulation and inflammation would be similar between lean and obese women living in a region of high omega-3 intake, such as Hawaii. METHODS: Fifty-five healthy, normal glucose tolerant women from Honolulu Hawaii, dichotomized based on pre-pregnancy BMI into lean (BMI <25â¯kg/m2, nâ¯=â¯29) and obese (BMI >30â¯kg/m2, nâ¯=â¯26), were recruited at scheduled term cesarean delivery. Maternal plasma DHA levels were analyzed by mass spectrometry. Expression of key genes involved in fatty acid oxidation and esterification were measured in placental tissue using qPCR. Total lipids were extracted from placental tissue via the Folch method. TNF-α concentration was measured by enzyme-linked immunosorbent assay in placental lysates. RESULTS: DHA levels were higher in lean women compared to obese women (Pâ¯=â¯0.02). However, DHA levels in obese women in Hawaii were eight times higher compared to obese Ohioan women (P=<0.0001). Placental lipid content and expression of key genes involved in fatty acid oxidation and esterification were similar (Pâ¯>â¯0.05) between lean and obese women in Hawaii. Furthermore, TNF-α placental lysates were not different between lean and obese women. CONCLUSIONS: Though obese women in Hawaii have lower DHA levels compared to their lean counterparts, these levels remain over eight times as high as obese Ohioan women. These relatively high plasma omega-3 levels in obese women in Hawaii may suppress placental lipid esterification/storage and inflammation to the same levels of lean women, as seen previously in vitro.
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Ácidos Grasos Omega-3/sangre , Lípidos/análisis , Fenómenos Fisiologicos Nutricionales Maternos/fisiología , Obesidad/metabolismo , Placenta/química , Adulto , Índice de Masa Corporal , Ácidos Docosahexaenoicos/sangre , Femenino , Humanos , Metabolismo de los Lípidos , EmbarazoRESUMEN
Obese women, on average, give birth to babies with high fat mass. Placental lipid metabolism alters fetal lipid delivery, potentially moderating neonatal adiposity, yet how it is affected by maternal obesity is poorly understood. We hypothesized that fatty acid (FA) accumulation (esterification) is higher and FA ß-oxidation (FAO) is lower in placentas from obese, compared with lean women. We assessed acylcarnitine profiles (lipid oxidation intermediates) in mother-baby-placenta triads, in addition to lipid content, and messenger RNA (mRNA)/protein expression of key regulators of FA metabolism pathways in placentas of lean and obese women with normal glucose tolerance recruited at scheduled term Cesarean delivery. In isolated trophoblasts, we measured [3H]-palmitate metabolism. Placentas of obese women had 17.5% (95% confidence interval: 6.1, 28.7%) more lipid than placentas of lean women, and higher mRNA and protein expression of FA esterification regulators (e.g., peroxisome proliferator-activated receptor γ, acetyl-CoA carboxylase, steroyl-CoA desaturase 1, and diacylglycerol O-acyltransferase-1). [3H]-palmitate esterification rates were increased in trophoblasts from obese compared with lean women. Placentas of obese women had fewer mitochondria and a lower concentration of acylcarnitines, suggesting a decrease in mitochondrial FAO capacity. Conversely, peroxisomal FAO was greater in placentas of obese women. Altogether, these changes in placental lipid metabolism may serve to limit the amount of maternal lipid transferred to the fetus, restraining excess fetal adiposity in this population of glucose-tolerant women.
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Metabolismo de los Lípidos/fisiología , Obesidad/metabolismo , Placenta/metabolismo , Adulto , Peso Corporal , Ácidos Grasos , Femenino , Regulación de la Expresión Génica , Humanos , Recién Nacido , Masculino , Embarazo , Adulto JovenRESUMEN
AIMS/HYPOTHESIS: Obesity triggers complex inflammatory networks within the innate immune system. During pregnancy, the placenta amplifies the low-grade inflammation through activation of Toll-like receptor 4 (TLR4) signalling pathways. The purpose of this study was to investigate the impact of obesity on placental TLR4 expression and inflammatory signals. The secondary aim was to analyse the placental cell type responsible for TLR4 activation. METHODS: Thirty-nine women recruited at term-scheduled Caesarean section were grouped according to their pre-gravid BMI (<25 kg/m(2) and >30 kg/m(2)). Placenta, venous maternal and cord blood were obtained at delivery for analysis. Data were analysed with linear regression and Spearman's rank correlation coefficient analysis. RESULTS: TLR4, IL6 and IL8 expression was increased three- to ninefold (p < 0.001) in the placenta of obese vs lean women. There was a positive correlation between placental TLR4 and maternal systemic and placental IL6 and IL8 concentrations. Placental TLR4 expression was correlated with maternal pre-gravid BMI, insulin resistance index, plasma insulin and C-reactive protein (r = 0.57, 0.31, 0.35, 0.53, respectively; p < 0.001) but not with plasma glucose, maternal age, gestational age and gestational weight gain (r < 0.2; p > 0.1). TLR4 was located in both trophoblast and macrovascular endothelial cells lining fetal vasculature. Lipopolysaccharide-induced TLR4 activation was more robust in trophoblasts than in endothelial vascular cells (100-fold vs tenfold; p < 0.001). CONCLUSIONS/INTERPRETATION: Trophoblastic TLR4 is strongly implicated in the propagation of placental inflammation. Placental inflammation is related to maternal metabolic conditions such as pre-gravid BMI, whilst gestational weight gain or gestational age are not. These results implicate the pre-gravid condition as a significant contributor to metabolic inflammation in late pregnancy.
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Placenta/metabolismo , Receptor Toll-Like 4/metabolismo , Proteína C-Reactiva/metabolismo , Femenino , Edad Gestacional , Humanos , Inflamación/sangre , Inflamación/metabolismo , Insulina/sangre , Resistencia a la Insulina , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Obesidad , Embarazo , Transducción de Señal/fisiologíaRESUMEN
BACKGROUND: The placentas of obese women accumulate lipids that may alter fetal lipid exposure. The long-chain omega-3 fatty acids (n3 FAs) docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) alter FA metabolism in hepatocytes, although their effect on the placenta is poorly understood. OBJECTIVE: We aimed to investigate whether n3 supplementation during pregnancy affects lipid metabolism in the placentas of overweight and obese women at term. DESIGN: A secondary analysis of a double-blind randomized controlled trial was conducted in healthy overweight and obese pregnant women who were randomly assigned to DHA plus EPA (2 g/d) or placebo twice a day from early pregnancy to term. Placental FA uptake, esterification, and oxidation pathways were studied by measuring the expression of key genes in the placental tissue of women supplemented with placebo and n3 and in vitro in isolated trophoblast cells in response to DHA and EPA treatment. RESULTS: Total lipid content was significantly lower in the placentas of overweight and obese women supplemented with n3 FAs than in those supplemented with placebo (14.14 ± 1.03 compared with 19.63 ± 1.45 mg lipid/g tissue; P < 0.05). The messenger RNA expression of placental FA synthase (FAS) and diacylglycerol O-acyltransferase 1 (DGAT1) was negatively correlated with maternal plasma enrichment in DHA and EPA (P < 0.05). The expression of placental peroxisome proliferatoractivated receptor γ (r = −0.39, P = 0.04) and its target genes DGAT1 (r = −0.37, P = 0.02) and PLIN2 (r = −0.38, P = 0.04) significantly decreased, with an increasing maternal n3:n6 ratio (representing the n3 status) near the end of pregnancy. The expression of genes that regulate FA oxidation or uptake was not changed. Birth weight and length were significantly higher in the offspring of n3-supplemented women than in those in the placebo group (P < 0.05), but no differences in the ponderal index were observed. Supplementation of n3 significantly decreased FA esterification in isolated trophoblasts without affecting FA oxidation. CONCLUSION: Supplementing overweight and obese women with n3 FAs during pregnancy inhibited the ability of the placenta to esterify and store lipids. This trial was registered at clinicaltrials.gov as NCT00957476.
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Ácidos Docosahexaenoicos/administración & dosificación , Ácido Eicosapentaenoico/administración & dosificación , Metabolismo de los Lípidos/efectos de los fármacos , Placenta/metabolismo , Trofoblastos/efectos de los fármacos , Adulto , Índice de Masa Corporal , Suplementos Dietéticos , Ácidos Docosahexaenoicos/sangre , Método Doble Ciego , Ácido Eicosapentaenoico/sangre , Femenino , Humanos , Obesidad , Sobrepeso , PPAR gamma/genética , PPAR gamma/metabolismo , Placenta/efectos de los fármacos , Embarazo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Trofoblastos/metabolismo , Adulto JovenRESUMEN
OBJECTIVE: Long-chain omega 3 fatty acids, eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) exert potent anti-inflammatory properties in humans. This study characterized the effects of omega-3 ω-3 fatty acids supplements (ω-3 FA) on the inflammatory status in the placenta and adipose tissue of overweight/obese pregnant women. STUDY DESIGN: A randomized, double-masked controlled trial was conducted in overweight/obese pregnant women that were randomly assigned to receive DHA plus EPA (2 g/day) or the equivalent of a placebo twice a day from week 10-16 to term. Inflammatory pathways were characterized in: 1) adipose tissue and placenta of treated vs. untreated women; and 2) adipose and trophoblast cells cultured with long chain FAs. RESULTS: The sum of plasma DHA and EPA increased by 5.8 fold and ω-3 FA/ω-6 FA ratio was 1.5 in treated vs. untreated women (p< 0.005). Plasma CRP concentrations were reduced (p<0.001). The adipose tissue and placenta of treated women exhibited a significant decrease in TLR4 adipose and placental expression as well as IL6, IL8, and TNFα In vitro, EPA and DHA suppressed the activation of TLR4, IL6, IL8 induced by palmitate in culture of adipose and trophoblast cells. CONCLUSION: Supplementation of overweight/obese pregnant women with dietary ω-3 FAs for >25 weeks reduced inflammation in maternal adipose and the placental tissue. TLR4 appears as a central target of the anti-inflammatory effects at the cellular level. TRIAL REGISTRATION: ClinicalTrials.gov NCT00957476.
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Tejido Adiposo/efectos de los fármacos , Suplementos Dietéticos , Ácidos Docosahexaenoicos/administración & dosificación , Ácido Eicosapentaenoico/administración & dosificación , Obesidad/dietoterapia , Tejido Adiposo/metabolismo , Tejido Adiposo/patología , Adulto , Dieta , Método Doble Ciego , Femenino , Regulación de la Expresión Génica , Humanos , Inflamación , Interleucina-6/genética , Interleucina-6/inmunología , Interleucina-8/genética , Interleucina-8/inmunología , Obesidad/genética , Obesidad/inmunología , Obesidad/patología , Placenta/efectos de los fármacos , Placenta/metabolismo , Placenta/patología , Embarazo , Cultivo Primario de Células , Transducción de Señal , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/inmunología , Trofoblastos/efectos de los fármacos , Trofoblastos/metabolismo , Trofoblastos/patologíaRESUMEN
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.
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Citocinas/metabolismo , Ácidos Grasos/metabolismo , Inflamación/metabolismo , Transducción de Señal/fisiología , Receptor Toll-Like 4/metabolismo , Trofoblastos/metabolismo , Adulto , Femenino , Humanos , Interleucina-6/metabolismo , Interleucina-8/metabolismo , FN-kappa B/metabolismo , Embarazo , Trofoblastos/inmunología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
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.
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Hipoglucemiantes/farmacología , Insulina/farmacología , Obesidad/genética , Placenta/efectos de los fármacos , Complicaciones del Embarazo/genética , ARN Mensajero/efectos de los fármacos , Transcriptoma/efectos de los fármacos , Trofoblastos/efectos de los fármacos , Aborto Inducido , Adolescente , Adulto , Estudios de Casos y Controles , Células Cultivadas , Femenino , Perfilación de la Expresión Génica , Humanos , Obesidad/metabolismo , Placenta/metabolismo , Embarazo , Complicaciones del Embarazo/metabolismo , Primer Trimestre del Embarazo , ARN Mensajero/metabolismo , Transcriptoma/genética , Trofoblastos/metabolismo , Adulto JovenRESUMEN
CONTEXT: Low concentrations of estradiol and progesterone are hallmarks of adverse pregnancy outcomes as is maternal obesity. During pregnancy, placental cholesterol is the sole source of sex steroids. Cholesterol trafficking is the limiting step in sex steroid biosynthesis and is mainly mediated by the translocator protein (TSPO), present in the mitochondrial outer membrane. OBJECTIVE: The objective of the study was to investigate the effects of maternal obesity in placental sex steroid biosynthesis and TSPO regulation. DESIGN/PARTICIPANTS: One hundred forty-four obese (body mass index 30-35 kg/m(2)) and 90 lean (body mass index 19-25 kg/m(2)) pregnant women (OP and LP, respectively) recruited at scheduled term cesarean delivery. Placenta and maternal blood were collected. SETTING: This study was conducted at MetroHealth Medical Center (Cleveland, Ohio). MAIN OUTCOME MEASURES: Maternal metabolic components (fasting glucose, insulin, leptin, estradiol, progesterone, and total cholesterol) and placental weight were measured. Placenta (mitochondria and membranes separated) and cord blood cholesterol values were verified. The expression and regulation of TSPO and mitochondrial function were analyzed. RESULTS: Plasma estradiol and progesterone concentrations were significantly lower (P < .04) in OP as compared with LP women. Maternal and cord plasma cholesterol were not different between groups. Placental citrate synthase activity and mitochondrial DNA, markers of mitochondrial density, were unchanged, but the mitochondrial cholesterol concentrations were 40% lower in the placenta of OP. TSPO gene and protein expressions were decreased 2-fold in the placenta of OP. In vitro trophoblast activation of the innate immune pathways with lipopolysaccharide and long-chain saturated fatty acids reduced TSPO expression by 2- to 3-fold (P < .05). CONCLUSION: These data indicate that obesity in pregnancy impairs mitochondrial steroidogenic function through the negative regulation of mitochondrial TSPO.
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Regulación hacia Abajo/fisiología , Estradiol/biosíntesis , Obesidad/metabolismo , Placenta/metabolismo , Progesterona/biosíntesis , Receptores de GABA/metabolismo , Adulto , Glucemia/metabolismo , Colesterol/metabolismo , Femenino , Humanos , Insulina/metabolismo , Leptina/metabolismo , Embarazo , Receptores de GABA/genética , Adulto JovenRESUMEN
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.
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Adiponectina/genética , Metilación de ADN/fisiología , Obesidad/genética , Complicaciones del Embarazo/genética , Adiponectina/deficiencia , Adiponectina/metabolismo , Tejido Adiposo/metabolismo , Adulto , Índice de Masa Corporal , Regulación hacia Abajo/genética , Femenino , Regulación de la Expresión Génica/fisiología , Humanos , Recién Nacido , Insulina/metabolismo , Resistencia a la Insulina/fisiología , Leptina/metabolismo , Obesidad/metabolismo , Placenta/metabolismo , Embarazo , Complicaciones del Embarazo/metabolismo , Tercer Trimestre del Embarazo/genética , Tercer Trimestre del Embarazo/metabolismo , ARN Mensajero/metabolismo , Adulto JovenRESUMEN
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.
Asunto(s)
Adipocitos , Tejido Adiposo Blanco/fisiopatología , Inflamación/fisiopatología , Obesidad/fisiopatología , Adipocitos/metabolismo , Adipocitos/patología , Adulto , Apoptosis , Muerte Celular , Diferenciación Celular , Sistema Libre de Células , Femenino , Humanos , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Necrosis , EmbarazoRESUMEN
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.
Asunto(s)
Adaptación Fisiológica , Tejido Adiposo/fisiología , Inflamación/fisiopatología , Primer Trimestre del Embarazo/fisiología , Tercer Trimestre del Embarazo/fisiología , Adipoquinas/genética , Adipoquinas/inmunología , Adipoquinas/metabolismo , Tejido Adiposo/inmunología , Antígenos CD/biosíntesis , Antígenos CD/inmunología , Antígenos de Diferenciación Mielomonocítica/biosíntesis , Antígenos de Diferenciación Mielomonocítica/inmunología , Quimiocinas/genética , Quimiocinas/inmunología , Quimiocinas/metabolismo , Femenino , Humanos , Inflamación/genética , Inflamación/inmunología , Metabolismo de los Lípidos/genética , Metabolismo de los Lípidos/inmunología , Metabolismo de los Lípidos/fisiología , Receptores de Lipopolisacáridos/biosíntesis , Receptores de Lipopolisacáridos/inmunología , FN-kappa B/inmunología , FN-kappa B/metabolismo , Neovascularización Fisiológica/genética , Neovascularización Fisiológica/inmunología , Embarazo , Primer Trimestre del Embarazo/genética , Primer Trimestre del Embarazo/inmunología , Tercer Trimestre del Embarazo/genética , Tercer Trimestre del Embarazo/inmunología , Receptor IGF Tipo 2/biosíntesis , Receptor IGF Tipo 2/inmunología , Transducción de Señal/genética , Transducción de Señal/inmunología , Transducción de Señal/fisiología , Receptor Toll-Like 4/inmunología , Receptor Toll-Like 4/metabolismo , Transcriptoma/genética , Transcriptoma/inmunología , Transcriptoma/fisiologíaRESUMEN
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.
Asunto(s)
Tejido Adiposo/inmunología , Endotoxemia/inmunología , Mediadores de Inflamación/metabolismo , Inflamación/inmunología , Obesidad/inmunología , Complicaciones del Embarazo/inmunología , Tejido Adiposo/metabolismo , Adulto , Antígenos CD/metabolismo , Proteína C-Reactiva/metabolismo , Quimiocina CCL2/metabolismo , Citocinas/metabolismo , Endotoxemia/etiología , Endotoxemia/metabolismo , Femenino , Humanos , Inmunidad Innata , Inflamación/etiología , Inflamación/metabolismo , Lipopolisacáridos/metabolismo , Macrófagos/metabolismo , Glicoproteínas de Membrana/metabolismo , Obesidad/complicaciones , Obesidad/metabolismo , Embarazo , Complicaciones del Embarazo/metabolismo , Receptor Toll-Like 4/metabolismo , Adulto JovenRESUMEN
Evidence has accumulated that dietary polyphenols, in particular, flavonoids, have protective effects against oral cancer. In this study, we have examined the effects of quercetin, a major dietary flavonoid, on cell growth and necrosis/apoptosis and cell cycle regulation in human oral squamous carcinoma SCC-9 cells. Quercetin induced dose- and time-dependent, irreversible inhibition of cell growth and cellular DNA synthesis. Light microscopy and lactate dehydrogenase measurements showed modifications in the morphology and membrane integrity of these cells after quercetin treatment. Propidium iodide/annexin V staining showed that quercetin induced necrosis at 24 h and 48 h, whereas at 72 h cells underwent apoptosis, correlating with caspase-3 activation. Flow cytometry studies of the cell cycle distribution showed that quercetin induced mainly S-phase arrest. Thymidylate synthase (TS), a key S-phase enzyme, was inhibited in a time- and dose-dependent fashion by quercetin at the protein level. A lack of effect on TS mRNA suggested that TS down-regulation occurred at the translational level. In conclusion, our data support a view that quercetin initially induces a stress response, resulting in necrosis of these oral epithelial cells. Prolonged exposure of the surviving cells to quercetin causes apoptosis, presumably mediated by inhibition of TS protein.
Asunto(s)
Apoptosis/efectos de los fármacos , Carcinoma de Células Escamosas/patología , Ciclo Celular/efectos de los fármacos , Neoplasias de la Boca/patología , Quercetina/farmacología , Timidilato Sintasa/antagonistas & inhibidores , Carcinoma de Células Escamosas/enzimología , Carcinoma de Células Escamosas/metabolismo , Caspasa 3 , Caspasas/metabolismo , Línea Celular Tumoral , ADN/metabolismo , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo , Citometría de Flujo , Humanos , Neoplasias de la Boca/enzimología , Neoplasias de la Boca/metabolismo , Timidilato Sintasa/efectos de los fármacos , Factores de Tiempo , Factores de Necrosis Tumoral/metabolismoRESUMEN
Adenine dinucleotides (ApnA) are extracellular signal molecules that are released from blood platelets, following stress, into the vascular system. The most abundant and best-characterized ApnA (Ap4A) interacts with a unique receptor on bovine aortic endothelial cells (BAEC) where it induces nitric oxide. Ap4A also interacts with P2 purinoceptors on BAEC to modulate Ca2+ mobilization and prostacyclin release; this behavior can be equally well explained by Ap4A being either a partial agonist to these receptors, or an antagonist in the presence of ATP contamination. To discern between these two possibilities, we have investigated the presence of such contaminants in ApnA preparations. The studies herein indicate that ApnAs (n = 3-6) contain ATP impurities; thus, when characterizing the ApnA interaction with ATP-binding sites, investigators must assure that the response elicited is not partly due to an ATP impurity. We here provide a means for detecting and estimating ATP impurities within Ap4A preparations while also eliminating them; the level of this contamination is estimated to be as low as 0.2%. We applied our method to distinguish the true effect of Ap4A at P2 purinoceptors; our findings are consistent with Ap4A acting as a partial agonist to these receptors. We also applied our method to characterizing the ApnA interaction with luciferase, and found that decontaminated ApnA (n = 4-6) are weak substrates for luciferase.
Asunto(s)
Nucleótidos de Adenina/metabolismo , Adenosina Trifosfato/química , Nucleótidos de Adenina/aislamiento & purificación , Adenosina Trifosfato/metabolismo , Fosfatasa Alcalina/metabolismo , Animales , Bovinos , Células Cultivadas , Células Endoteliales/citología , Células Endoteliales/metabolismo , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Epoprostenol/metabolismo , Luciferasas/metabolismo , Mediciones Luminiscentes , Receptores Purinérgicos P2/metabolismo , Transducción de Señal/fisiologíaRESUMEN
Adenine dinucleotides are a group of extracellular modulators involved in maintaining blood vessel tone. We have demonstrated previously that Ap2A and Ap4A induce the synthesis of both nitric oxide (NO) and prostacyclin (PGI2) in bovine aortic endothelial cells (BAEC), whereas Ap3A, Ap5A, and Ap6A do not. In this paper, we report that Ap2A and Ap4A are partial agonists for ATP in terms of Ca2+ mobilization and PGI2 synthesis. The Ap(4)A EC50 values for Ca2+ mobilization and PGI2 synthesis were significantly higher than the corresponding values for ATP, while the Ap4A B(max) values for Ca2+ mobilization and PGI2 synthesis were significantly lower than those for ATP. Ap2A and Ap4A concentration-effect curves for Ca2+ mobilization and PGI2 synthesis demonstrated that Ap2A and Ap4A have antagonistic effects at ATP concentrations that induce responses above the maximal amount of Ca2+ mobilized or PGI2 synthesized by these two dinucleotides. On the other hand, Ap2A and Ap4A have agonistic effects at ATP concentrations that induce PGI2 synthesis below the maximal amount of PGI2 synthesized by these two dinucleotides. We also present evidence that suggests Ap3A, Ap5A, and Ap6A are antagonists for ATP in terms of PGI2 synthesis. All these data are consistent with the adenine dinucleotides being negative modulators for ATP-induced PGI2 synthesis.