Leptin Methylation Research Articles

Effects of exposure to environmental factors on obesity-related growth parameters and leptin (LEP) methylation in children.

The prevalence of childhood obesity is rapidly increasing. Therefore, gaining more information on the role of environmental parameters is key. With overexpression of leptin (encoded by LEP) in obesity, LEP methylation might be altered by environmental exposures. This study aims to assess effects of ambient air pollution and nearby greenness on obesity-related growth and LEP methylation in early childhood. We monitored 120 mother-child pairs from conception until the age of five. Buccal swabs and anthropometric measurements of the children were taken at six months, one year, and five years old. Buccal DNA was extracted to determine LEP methylation levels. Estimates of air pollution and nearby greenness were calculated using high-resolution models. Effects of air pollution and nearby greenness on growth or LEP methylation were investigated using linear mixed effects models. Positive associations were shown for air pollution between conception and age one on impedance in six-month-olds and one-year-olds in the crude model. PM with aerodynamic diameter ≤10 (PM10) and ≤2.5 μm (PM2.5) positively associated with waist-hip-ratio and waist circumference at age five in the fully adjusted model. In early childhood, closest distance to forest negatively, and urban green and forest positively associated with weight-for-length, body mass index, and fat percentage in five-year-olds in the fully adjusted model. No significant associations for noise, and walkability on growth were seen. Negative associations were shown for smaller green clusters and positive associations for greater green clusters on LEP methylation in one-year-olds. For forest distance, walkability, noise, or all green on LEP methylation, no significant associations were found. Evidence is provided that ambient air pollution might have a significant effect on impedance and waist-hip-ratio, suggesting an increased risk of childhood obesity. Based on LEP methylation, greater green clusters might associate with a decreased risk of childhood obesity, while smaller green clusters showed the opposite.

Alteration of serum leptin and LEP/LEPR promoter methylation in Prader-Willi syndrome

Prader-Willi syndrome (PWS) is a rare neurodevelopmental disorder based on a loss of paternally expressed but maternally imprinted genes in chromosome region 15q11–13. PWS individuals typically show insatiable appetite with subsequent obesity representing the major mortality factor unless food intake is inhibited. The neurobiological basis of PWS-typical hyperphagia has remained poorly understood. Many PWS-typical abnormalities are based on hypothalamic dysregulation, a region in which hunger and satiety are hormonally regulated, with the hormone leptin being a main long-term regulator of satiety. Previous studies in PWS have inconsistently shown leptin alterations solely in early childhood, without investigating the leptin system on an epigenetic level. The present study investigates serum leptin levels (S-leptin) and DNA methylation of the leptin (LEP) and leptin receptor gene (LEPR) promoter in 24 individuals with PWS compared to 13 healthy controls matched for sex, age, and body mass index (BMI) and relates the results to the extent of hyperphagia in PWS. S-Leptin levels were obtained by Enzyme-linked Immunosorbent Assay. LEP/LEPR-promoter DNA methylation was assessed by bisulfite-sequencing, hyperphagia by Hyperphagia Questionnaire for Clinical Trials (HQ-CT). PWS and control groups differed significantly in S-leptin levels with higher S-leptin in PWS. Methylation analysis showed significant differences in mean promoter methylation rate both for LEP and LEPR with a lower methylation rate in PWS. LEPR, but not LEP methylation correlated significantly with S-leptin levels. S-leptin and both LEP and LEPR methylation did not correlate with HQ-CT scores in PWS. The present study is the first to show significantly elevated S-leptin levels in an adult PWS cohort combined with an altered, downregulated LEP and LEPR promoter methylation status compared to sex-, age- and BMI-matched controls. Analogous to previous studies, no link to the behavioral dimension could be drawn. Overall, the results suggest an increased leptin dysregulation in PWS, whereby the findings partly mirror those seen in non-syndromic obesity.

Leptin Methylation and mRNA Expression Associated With Psychopathology in Schizophrenia Inpatients

Leptin involved in the regulation of dopaminergic neurons of the central nervous system may confirm the hypothesis of neurodevelopment in schizophrenic patients. However, specific genetic mechanisms are undefined. Therefore, we aimed to explore the regulation of DNA methylation of leptin promoters and mRNA expression in patients with schizophrenia. A cross-sectional study enrolled 40 patients and 40 healthy controls from the Beijing Huilongguan Hospital in China. The leptin methylation levels and mRNA expression were examined by highly sensitive mass spectrometry based on the MassARRAY System and real-time quantitative polymerase chain reaction (qPCR). The Positive and Negative Symptoms Scale (PANSS) was applied to estimate the clinical symptoms of patients. The LEP-CpG7 and CpG15 methylation in patients were significantly higher than in healthy controls (P < 0.05). The LEP-CpG11, CpG33.34.35, CpG36 methylation, and mRNA expression decreased significantly in patients compared with healthy controls (P < 0.05). After controlling gender, age, BMI, dose of antipsychotic and duration of illness, LEP-CpG7 methylation was negatively associated with PANSS positive symptoms subscore (r = −0.485, P = 0.005). In addition, LEP-mRNA expression was negatively correlated with PANSS total score (r = −0.385, P = 0.03) and positive subscale (r = −0.392, P = 0.026). Nevertheless, only the LEP-CpG7 methylation level remained negatively correlated to the PANSS positive subscore following multiple stepwise regression (β = −17.071, P = 0.037). These results suggest that leptin methylation and mRNA expression might contribute to the pathogenesis of schizophrenia. LEP-CpG7 methylation may be negatively associated with positive symptoms in patients with schizophrenia.

Placental expression of leptin: fetal sex-independent relation with human placental growth.

Leptin (LEP) is a vital placental hormone that is known to affect different aspects of placental function and fetal development. The present study aimed to determine the association of placental LEP transcript abundance with maternal, placental, and newborn parameters. In this retrospective case-control study, placental samples (n = 105) were collected from small (SGA) and appropriate (AGA) for gestational age full-term singleton pregnancies (n = 44 SGA and n = 61 AGA). Placental transcript abundance of LEP was assessed by real-time quantitative PCR after normalization to a reference gene panel. LEP methylation was measured using a quantitative MethyLight assay in a subset of samples (n = 54). Placental LEP transcript abundance was negatively and significantly associated with placental weight (β = -3.883, P = 0.015). This association continued to be significant in the SGA group (β = -10.332, P = 0.001), both in female (β = -15.423, P = 0.021) and male births (β = -10.029, P = 0.007). LEP transcript abundance was not associated with LEP methylation levels (Spearman's ρ = 0.148, P = 0.287). We conclude that placental upregulation of LEP is an integral and fetal sex-independent component of placental growth restriction, which can be potentially targeted through maternal dietary modifications to improve fetoplacental growth.

Associations of maternal diet and placenta leptin methylation

BackgroundMaternal diet is an important factor in prenatal development that also has implications for disease risk later in life. The adipokine leptin is a key regulator of energy homeostasis and may be involved in the association between maternal nutrition, maternal obesity, and infant outcomes. DNA methylation of placenta genes may occur in response to exposures and may program subsequent infant development. This study examined maternal diet, placenta leptin gene DNA methylation, and neonatal growth in a sample of healthy neonates and their mothers. MethodsMothers and their healthy neonates (N = 135) were recruited within 1–2 days following delivery at Women and Infants Hospital in Providence, RI. A structured interview was conducted to assess maternal dietary intake. Maternal pre-pregnancy weight, weight gain during pregnancy, maternal health, medications, and vitamin use were obtained from medical records. Bisulfite pyrosequencing was used to measure methylation of CpG sites in the promoter region of the placenta leptin gene and determine genotype of the leptin single nucleotide polymorphism (SNP) rs2167270, which is known to influence leptin methylation. Bivariate analyses and linear regression models were used to evaluate associations of demographics, diet, and mean leptin methylation. ResultsGenotype was a significant predictor of placenta leptin DNA methylation (p < .01), and after controlling for this and other relevant maternal and infant covariates, lower levels of leptin methylation were significantly associated with greater intake of carbohydrates (p < .05), in particular added sugars (p < .05) and white/refined carbohydrates (p < .05). Total caloric intake was also associated with placenta leptin methylation (p < .05), however after controlling for relevant covariates, significance diminished to trend-level. There were no significant associations of placenta leptin methylation and intake of protein (p > .05) or fat (p > .05). ConclusionThese findings underline the importance of intake of carbohydrate consumption for methylation of the placenta leptin gene. Because methylation reduces gene transcription, lower methylation may indicate a placenta response to high caloric intake and carbohydrate food that would result in higher levels of this hormone during fetal development. Further investigation of the developmental ramifications of epigenetic changes to placenta leptin methylation should be pursued.

Interaction of Osteoarthritis and BMI on Leptin Promoter Methylation in Taiwanese Adults.

Leptin (LEP) regulates glucose metabolism and energy storage in the body. Osteoarthritis (OA) is associated with the upregulation of serum LEP. LEP promoter methylation is associated with obesity. So far, few studies have explored the association of BMI and OA with LEP methylation. We assessed the interaction between body mass index (BMI) and OA on LEP promoter methylation. Data of 1114 participants comprising 583 men and 558 women, aged 30–70 years were retrieved from the Taiwan Biobank Database (2008–2015). Osteoarthritis was self-reported and cases were those who reported having ever been clinically diagnosed with osteoarthritis. BMI was categorized into underweight, normal weight, overweight, and obesity. The mean LEP promoter methylation level in individuals with osteoarthritis was 0.5509 ± 0.00437 and 0.5375 ± 0.00101 in those without osteoarthritis. The interaction between osteoarthritis and BMI on LEP promoter methylation was significant (p-value = 0.0180). With normal BMI as the reference, the mean LEP promoter methylation level was significantly higher in obese osteoarthritic individuals (β = 0.03696, p-value = 0.0187). However, there was no significant association between BMI and LEP promoter methylation in individuals without osteoarthritis, regardless of BMI. In conclusion, only obesity was significantly associated with LEP promoter methylation (higher levels) specifically in osteoarthritic patients.

Duration of breastfeeding is associated with leptin (LEP) DNA methylation profiles and BMI in 10-year-old children

BackgroundBreastfeeding is protective against many long-term diseases, yet the mechanisms involved are unknown. Leptin gene (LEP) is reported to be associated with body mass index (BMI). On the other hand, breastfeeding duration has been found to be associated with DNA methylation (DNAm) of the LEP gene. Therefore, epigenetic regulation of LEP may represent the mechanism underlying the protective effect of breastfeeding duration against obesity.MethodsIn the Isle of Wight Birth Cohort, peripheral blood DNAm at 23 cytosine-phosphate-guanine sites (CpGs) in the LEP locus in 10-year-old (n = 297) samples and 16 CpGs in 18-year-old (n = 305) samples, were generated using the Illumina Infinium MethylationEPIC and HumanMethylation450 Beadchips respectively and tested for association with breastfeeding duration (total and exclusive) using linear regression. To explore the association between breastfeeding durations and genome-wide DNAm, epigenome-wide association studies (EWASs) and differential methylation region (DMR) analyses were performed. BMI trajectories spanning the first 18 years of life were used as the outcome to test the association with breastfeeding duration (exposure) using multi-nominal logistic regression. Mediation analysis was performed for significant CpG sites.ResultsBoth total and exclusive breastfeeding duration were associated with DNAm at four LEP CpG sites at 10 years (P value < 0.05), and not at 18 years. Though no association was observed between breastfeeding duration and genome-wide DNAm, DMR analyses identified five significant differentially methylated regions (Sidak adjusted P value < 0.05). Breastfeeding duration was also associated with the early transient overweight trajectory. Furthermore, DNAm of LEP was associated with this trajectory at one CpG site and early persistent obesity at another, though mediation analysis was not significant.ConclusionsBreastfeeding duration is associated with LEP methylation at age 10 years and BMI trajectory. LEP DNAm is also significantly associated with BMI trajectories throughout childhood, though sample sizes were small. However, mediation analysis did not demonstrate that DNAm of LEP explained the protective effect of breastfeeding against childhood obesity.

Cord blood leptin DNA methylation levels are associated with macrosomia during normal pregnancy.

We previously demonstrated an association between placental leptin (LEP) methylation levels and macrosomia without gestational diabetes mellitus (non-GDM). This study further explored the association between LEP methylation in cord blood and non-GDM macrosomia. We carried out a case-control study of 61 newborns with macrosomia (birth weight ≥4000 g) and 69 newborns with normal birth weight (2500-3999 g). Methylation in the LEP promoter region was mapped by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Average cord blood LEP methylation levels were lower in macrosomia newborns than in control newborns (P < 0.001). Eleven CpG sites were associated with macrosomia. Multivariate logistic regression revealed that low LEP methylation levels [adjusted odds ratio (AOR) = 2.84, 95% confidence interval (CI): 1.72-4.17], high pre-pregnancy body mass index (AOR = 7.44, 95% CI: 1.99-27.75), long gestational age (AOR = 3.18, 95% CI: 1.74-5.79), high cord blood LEP concentration (AOR = 2.25, 95% CI: 1.34-3.77), and male newborn gender (AOR = 3.91, 95% CI: 1.31-11.69) significantly increased the risk of macrosomia. Lower cord blood LEP methylation levels and certain maternal and fetal factors are associated with non-GDM macrosomia.

Regulation of Leptin Methylation Not via Apoptosis by Melatonin in the Rescue of Chronic Programming Liver Steatosis.

We examined the mechanisms of chronic liver steatosis after prenatal dexamethasone exposure and whether melatonin rescues adult offspring with liver steatosis. Melatonin rescued prenatal dexamethasone-exposed livers with steatosis in young rats. Sprague-Dawley rats pregnant at gestational day 14–21 were administered with intraperitoneal dexamethasone (DEX) or prenatal dexamethasone and melatonin between gestational day 14 and postnatal day ~120 (DEX+MEL). Chronic programming effects in the liver were assessed at day ~120. Liver steatosis increased in the DEX compared with that in the vehicle group and decreased in the DEX+MEL group (p < 0.05), with no changes in cellular apoptosis. Expression of leptin and its receptor decreased in the DEX (p < 0.05) and increased in the DEX+MEL group (p < 0.05), as revealed by RT-PCR and Western blotting. Tumor necrosis factor alpha (TNF-α) and interleukin (IL)-6 expression increased in the DEX group compared with that in the vehicle group and decreased in the DEX+MEL group (p < 0.05). Liver DNA methyltransferase activity and leptin methylation increased in the DEX group (p < 0.05) and decreased in the DEX+MEL group (p < 0.05), with no changes in HDAC activity. Thus, prenatal dexamethasone induces liver steatosis at ~120 days via altered leptin expression and liver inflammation without leptin resistance. Melatonin reverses leptin methylation and expression and decreases inflammation and chronic liver steatosis not via apoptosis or histone deacetylation (HDAC).

DNA methylation of genes regulating appetite and prediction of weight loss after bariatric surgery in obese individuals.

Epigenetic traits are influenced by clinical variables; interaction between DNA methylation (DNAmeth) and bariatric surgery-induced weight loss has been scarcely explored. We investigated whether DNAmeth of genes encoding for molecules/hormones regulating appetite, food intake or obesity could predict successful weight outcome following Roux-en-Y gastric bypass (RYGB). Forty-five obese individuals with no known comorbidities were stratified accordingly to weight decrease one-year after RYGB (excess weight loss, EWL ≥ 50%: good responders, GR; EWL < 50%: worse responders, WR). DNAmeth of leptin (LEP), ghrelin (GHRL), ghrelin receptor (GHSR) and insulin-growth factor-2 (IGF2) was assessed before intervention. Single nucleotide polymorphisms of genes affecting DNAmeth, DNMT3A and DNMT3B, were also determined. At baseline, type 2 diabetes was diagnosed by OGTT in 13 patients. Post-operatively, GR (n = 23) and WR (n = 22) achieved an EWL of 67.7 ± 9.6 vs 38.2 ± 9.0%, respectively. Baseline DNAmeth did not differ between GR and WR for any tested genes, even when the analysis was restricted to subjects with no diabetes. A relationship between GHRL and LEP methylation profiles emerged (r = 0.47, p = 0.001). Searching for correlation between DNAmeth of the studied genes with demographic characteristics and baseline biochemical parameters of the studied population, we observed a correlation between IGF2 methylation and folate (r = 0.44, p = 0.003). Rs11683424 for DNMT3A and rs2424913 for DNMT3B did not correlate with DNAmeth of the studied genes. In severely obese subjects, the degree of DNAmeth of some genes affecting obesity and related conditions does not work as predictor of successful response to RYGB.

36\xa0h fasting of young men influences adipose tissue DNA methylation of LEP and ADIPOQ in a birth weight-dependent manner

BackgroundSubjects born with low birth weight (LBW) display a more energy-conserving response to fasting compared with normal birth weight (NBW) subjects. However, the molecular mechanisms explaining these metabolic differences remain unknown. Environmental influences may dynamically affect epigenetic marks, also in postnatal life. Here, we aimed to study the effects of short-term fasting on leptin (LEP) and adiponectin (ADIPOQ) DNA methylation and gene expression in subcutaneous adipose tissue (SAT) from subjects with LBW and NBW.MethodsTwenty-one young LBW men and 18 matched NBW controls were studied during 36 h fasting. Eight subjects from each group completed a control study (overnight fast). We analyzed SAT LEP and ADIPOQ methylation (Epityper MassARRAY), gene expression (q-PCR), and adipokine plasma levels.ResultsAfter overnight fast (control study), LEP and ADIPOQ DNA methylation levels were higher in LBW compared to those in NBW subjects (p ≤ 0.03) and increased with 36 h fasting in NBW subjects only (p ≤ 0.06). Both LEP and ADIPOQ methylation levels were positively associated with total body fat percentage (p ≤ 0.05). Plasma leptin levels were higher in LBW versus NBW subjects after overnight fasting (p = 0.04) and decreased more than threefold in both groups after 36 h fasting (p ≤ 0.0001).ConclusionsThis is the first study to demonstrate that fasting induces changes in DNA methylation. This was shown in LEP and ADIPOQ promoters in SAT among NBW but not LBW subjects. The altered epigenetic flexibility in LBW subjects might contribute to their differential response to fasting, adipokine levels, and increased risk of metabolic disease.

Lower Placental Leptin Promoter Methylation in Association with Fine Particulate Matter Air Pollution during Pregnancy and Placental Nitrosative Stress at Birth in the ENVIRONAGE Cohort.

Background:Particulate matter with a diameter ≤ 2.5 μm (PM2.5) affects human fetal development during pregnancy. Oxidative stress is a putative mechanism by which PM2.5 may exert its effects. Leptin (LEP) is an energy-regulating hormone involved in fetal growth and development.Objectives:We investigated in placental tissue whether DNA methylation of the LEP promoter is associated with PM2.5 and whether the oxidative/nitrosative stress biomarker 3-nitrotyrosine (3-NTp) is involved.Methods:LEP DNA methylation status of 361 placentas from the ENVIRONAGE birth cohort was assessed using bisulfite-PCR-pyrosequencing. Placental 3-NTp (n = 313) was determined with an ELISA assay. Daily PM2.5 exposure levels were estimated for each mother’s residence, accounting for residential mobility during pregnancy, using a spatiotemporal interpolation model.Results:After adjustment for a priori chosen covariates, placental LEP methylation was 1.4% lower (95% CI: –2.7, –0.19%) in association with an interquartile range increment (7.5 μg/m3) in second-trimester PM2.5 exposure and 0.43% lower (95% CI: –0.85, –0.02%) in association with a doubling of placental 3-NTp content.Conclusions:LEP methylation status in the placenta was negatively associated with PM2.5 exposure during the second trimester, and with placental 3-NTp, a marker of oxidative/nitrosative stress. Additional research is needed to confirm our findings and to assess whether oxidative/nitrosative stress might contribute to associations between PM2.5 and placental epigenetic events. Potential consequences for health during the neonatal period and later in life warrant further exploration.Citation:Saenen ND, Vrijens K, Janssen BG, Roels HA, Neven KY, Vanden Berghe W, Gyselaers W, Vanpoucke C, Lefebvre W, De Boever P, Nawrot TS. 2017. Lower placental leptin promoter methylation in association with fine particulate matter air pollution during pregnancy and placental nitrosative stress at birth in the ENVIRONAGE cohort. Environ Health Perspect 125:262–268; http://dx.doi.org/10.1289/EHP38

Lower Placental Leptin DNA Methylation through Gestational Exposure to Particulate Matter Air Pollution

Background: Particulate matter with a diameter ≤ 2.5 µm (PM2.5) affects fetal human development during pregnancy. Oxidative stress is one putative mechanism by which PM2.5 may exert its effects. Leptin (LEP) is an energy regulating hormone involved in fetal growth and development. Objectives: We investigated in placental tissue whether DNA methylation of the LEP promoter is associated with PM2.5 and whether the oxidative/nitrosative stress biomarker 3-nitrotyrosine (3-NTp) is involved. Methods: LEP DNA methylation status of 361 placentas from the ENVIRONAGE birth cohort was assessed using bisulfite-PCR-pyrosequencing. 3-NTp (n = 313) was determined with an ELISA assay. Daily PM2.5 exposure levels were calculated for each mother’s residence using a spatiotemporal interpolation model. Results: After adjustment for a priori chosen covariates, we found a 1.4% lower placental LEP methylation status (p = 0.02) for an interquartile range increment of PM2.5 exposure during the second gestational trimester. For a doubling in placental 3-NTp content, the placental LEP methylation lowered by -0.43% (p = 0.04). Placental 3-NTp mediated the association between second trimester PM2.5 exposure and LEP methylation. Conclusions: The methylation status of the placental LEP region was sensitive to PM2.5 exposure of the second gestational window and to placental oxidative/nitrosative stress. These stressors influenced the PM2.5-LEP methylation association, suggesting an underlying role of oxidative/nitrosative stress in PM2.5-associated placental epigenetic events. The pathophysiological outcomes of these alterations as to newborns’ health and consequences later in life warrant further exploration.

Maternal obesity and gestational diabetes are associated with placental leptin DNA methylation

In this study, we aimed to investigate relationships between maternal prepregnancy obesity and gestational diabetes mellitus and placental leptin DNA methylation. This study comprises data on 535 mother-infant dyads enrolled in the Rhode Island Child Health Study, a prospective cohort study of healthy term pregnancies. Prepregnancy body mass index was calculated from self-reported anthropometric measures and gestational diabetes mellitus diagnoses gathered from inpatient medical records. DNA methylation of the leptin promoter region was assessed in placental tissue collected at birth using quantitative bisulfite pyrosequencing. In a multivariable regression analysis adjusted for confounders, infants exposed to gestational diabetes mellitus had higher placental leptin methylation (β = 1.89, P = .04), as did those demonstrating prepregnancy obesity (β = 1.17, P = .06). Using a structural equations model, we observed that gestational diabetes mellitus is a mediator of the effects of prepregnancy obesity on placental leptin DNA methylation (β = 0.81, 95% confidence interval, 0.27-2.71). Our results suggest that the maternal metabolic status before and during pregnancy can alter placental DNA methylation profile at birth and potentially contribute to metabolic programming of obesity and related conditions.

Duration of breastfeeding and gender are associated with methylation of the LEPTIN gene in very young children

Perinatal environmental factors have been associated with the metabolic programming of children and consequent disease risks in later life. Epigenetic modifications that lead to altered gene expression may be involved. Here, we study early life environmental and constitutional factors in association with the DNA methylation of leptin (LEP), a non-imprinted gene implicated in appetite regulation and fat metabolism. We investigated maternal education, breastfeeding, and constitutional factors of the child at 17 mo of age. We measured the DNA methylation of LEP in whole blood and the concentration of leptin in serum. Duration of breastfeeding was negatively associated with LEP methylation. Low education (≤12 y of education) was associated with higher LEP methylation. Boys had higher birth weight and lower LEP methylation than girls. An inverse association was established between birth weight per SD increase (+584 g) and LEP methylation. High BMI and leptin concentration were associated with lower methylation of LEP. The early life environment and constitutional factors of the child are associated with epigenetic variations in LEP. Future studies must reveal whether breastfeeding and the associated decrease in LEP methylation is an epigenetic mechanism contributing to the protective effect of breastfeeding against obesity.

575: Routine testing for copy number variants (CNVs) by chromosomal microarray analysis in patients with a normal karyotype: a single center's 20 month experience

nancy, and leptin levels have been linked to various pregnancy complications, including gestational diabetes, intrauterine growth restriction and preeclampsia. We sought to characterize methylation patterns in the leptin gene in normal early pregnancy and preeclamptic pregnancies. STUDY DESIGN: The methylation profiles of 2 CpG sites in the leptin gene were characterized in DNA from maternal blood longitudinally in 14 women at 16 gestational weeks, at delivery and postpartum; and in 14 preeclamptics at delivery. All women were non-smokers and the groups were matched for age and BMI. Genomic DNA was derived from buffy coat, purified, and bisulfite modified then run on the Illumina Methylation Assay. Mean methylation levels at each CpG site were compared using a t-test. RESULTS: Leptin methylation patterns at both CpG sites differed significantly between the early pregnancy and postpartum samples (P 0.01, P 10-5). Both leptin CpG sites were hypomethylated during early pregnancy as compared to postpartum. Methylation patterns were similar in normotensive and preeclamptic patients at the time of delivery (P 0.64, P 0.71). CONCLUSION: Our data demonstrates that early pregnancy results in decreased methylation at two CpG sites on the leptin gene. As methylation levels tend to be inversely correlated with gene transcription and resultant serum protein levels, this result suggests that altered methylation may be an epigenetic mechanism to control increased leptin production in normal early pregnancy. The presence of preeclampsia at the time of delivery did not appear to impact methylation of the leptin gene compared to normotensive patients. This suggests that there may be another mechanism controlling the increase in circulating serum leptin seen in pregnancies effected by preeclampsia.

High fat diet-induced obesity modifies the methylation pattern of leptin promoter in rats

Leptin is an adipokine involved in body weight and food intake regulation whose promoter region presents CpG islands that could be subject to dynamic methylation. This methylation process could be affected by environmental (e.g. diet) or endogenous (e.g., adipocyte differentiation, inflammation, hypoxia) factors, and could influence adipocyte leptin gene expression. The aim of this article was to study whether a high-energy diet may affect leptin gene promoter methylation in rats. A group of eleven male Wistar rats were assigned into two dietary groups, one fed on a control diet for 11 weeks and the other on a high-fat cafeteria diet. Rats fed a high-energy diet become overweight and hyperleptinemic as compared to the controls. DNA isolated from retroperitoneal adipocytes was treated with bisulfite and a distal portion of leptin promoter (from -694 to -372 bp) including 13 CpG sites was amplified by PCR and sequenced. The studied promoter portion was slightly more methylated in the cafeteria-fed animals, which was statistically significant (p < 0.05) for one of the CpG sites (located at the position -443). In obese rats, such methylation was associated to lower circulating leptin levels, suggesting that this position could be important in the regulation of leptin gene expression, probably by being a target sequence of different transcription factors. Our findings reveal, for the first time, that leptin methylation pattern can be influenced by diet-induced obesity, and suggest that epigenetic mechanisms could be involved in obesity by regulating the expression of important epiobesigenic genes.