Obesity is a long-standing condition¹ that “results from the interaction between genetic factors with environmental influences”.² An overflow of triglycerides (TGs) in adipose tissue due to an imbalance between energy intake and expenditure causes obesity.³ The World Health Organization estimates that 39% of the world adult population is overweight and 13% is obese. Around 3.0 million individuals die each year from obesity-associated complications. Obesity is now considered a key risk factor for a broad range of diseases such as type 2 diabetes, cardiovascular disease, and cancer.⁴ In 2015, an estimated 1.6 million deaths were caused by diabetes.⁵

Adipose tissue expresses leptin, a 16 kDa protein⁶ that works like a hormone and changes whole body energy homeostasis by controlling appetite. The arcuate nucleus in the hypothalamus has a leptin receptor where leptin acts like a hormone and maintains energy homeostasis by regulating appetite.^7,8 Leptin has a key association between leptinaemia and the chronic sub inflammatory state in obesity, which suggests other possible, peripheral biological effects associated with its cytokine-like structure.⁹ Accordingly, supplementation of leptin in generalized lipodystrophy (absence of adipose tissue) polish up insulin sensitivity and metabolic parameters.¹⁰ Leptin expression¹¹ and its serum levels are directly associated with obesity. Increased adipose tissue size (total fat mass) is significantly associated with increase in leptin, hence leptin is a signature of total fat mass.¹² Leptin is a signaling molecule from peripheral adipose tissue to the brain. Leptin gene-lacking mice were obese and had more complicated type 2 diabetes.⁶ These opinions enlighten further work to uncover the mechanism of regulation for leptin expression in adipocytes.

Gene expression in physiologic and pathologic states is regulated by DNA methylation, which signals major epigenetic changes. Methylation of cytosine in CpG sites is one of the important epigenetic modifications that suppresses gene expression. Methylation usually prevents transcriptional factors from binding to promoters, and recruits transcriptional repressors such as methyl CpG binding protein 2.^13,14 As a result, genes associated with the CpG-rich promoter and their expression level of genes have a tendency to rely on the methylation grade. In fact, the reason for down-regulated activity of leptin is methylated CpG rich promoter.¹⁵

Previous studies have reported that DNA methylation levels at candidate gene loci associated to obesity and metabolic abnormalities are impaired in peripheral blood and fat tissue of obese patients^16,17 and in low weight loss responder to diet and exercise interventions.^18-21 In total, these findings suggest that leptin DNA methylation profiles might be involved in the pathology of obesity and metabolic disorder. Thus, the aim of the present study was to explore the role of DNA methylation in leptin gene expression in obese subjects and its link with metabolic risk factors in a northwest Indian Population.

Study design and participants

The researchers designed a hospital based, cross-sectional study. The study design was approved by the Institutional Human Ethics Committee (IHEC-LOP/2016/EF0026 dated 7th Mar 2016) and a written informed consent was obtained from all participants before inclusion. Individuals of both sexes between the age of 25–45 years and who presented to the outpatient department of All India Institute of Medical Sciences, Jodhpur (a tertiary care hospital in northwestern India) for a routine health-check-up were recruited. A questionnaire was administered to all participants to determine their known morbidities, and anthropometry was performed using standard means to classify them as obese (body mass index [BMI] ≥5 kg/m²) or non-obese (BMI, 18.5–24.9 kg/m²) on the basis of BMI. A fasting blood sample was obtained for biochemical and molecular assays.

The obese subjects were further divided into two sub-groups, those with metabolic syndrome and those without metabolic syndrome, according to the National Cholesterol Education Program’s Adult Treatment Panel III, which requires at least three of the following: central obesity (waist circumference [WC] of ≥102 cm in men and ≥88 cm in women); dyslipidemia (TG levels ≥150 mg/dL; decreased high-density lipoprotein [HDL] cholesterol levels <40 mg/dL [males] and <50 mg/dL [female]); blood pressure of 130/85 mmHg or higher or drug treatment for hypertension; and fasting plasma glucose ≥110 mg/dL.²²

Biochemical estimation

Serum insulin concentrations were determined using the ADVIA Centaur Insulin assay kit (Tarrytown, NY, USA). Plasma glucose and lipid profile (Beckman Coulter kit) concentrations were determined using an autoanalyzer (Beckman Coulter 480).

Nucleic acid (DNA and RNA) extraction

DNA was purified from whole blood samples with the Relia Prep Blood gDNA Miniprep (Promega Corp., Madison, WI, USA). Total RNA was isolated using Trizol reagent (Thermo Fischer Life Science, Carlsbad, CA ,USA). Complementary DNA (cDNA) was synthesized from RNA using high-capacity cDNA RT kit (Thermo Fischer Life Science) according to manufacturer instruction.

Sodium bisulfite modification

The isolated DNA was modified with sodium bisulfite, which transforms unmethylated cytosine into uracil without changing methylated cytosine by using the EpiTect Bisufite Kit (Qiagen) according to the manufacturer’s instructions.

Quantitative methylation analysis

Real time PCR was carried out using Methylamp MS-qPCR (Epigenetek). PCR amplification was carried out in Bio-Rad CFX96 Real time PCR (Bio-Rad). A 96-well PCR plate using a temperature profile of 95°C, 15 minutes (initial denaturation) followed by 40 cycles of 94°C, 15 seconds, 59°C, 30 seconds and 72°C, 30 seconds for denaturation, annealing, and extension steps, respectively. The primers were designed by using Meth Primer (Li Lab) software. The PCR primers were synthesized by (Eurofins Scientific). Primer sequence for leptin methylated are forward: 5´-TAGGATTAACGAGGGCGTAGTC-3´ and reverse: 5´-AACCCCTTAAAAAAATACTTCGAA-3´ and for unmethylated are forward: 5´-TTAGGATTAATGAGGGTGTAGTTGT-3´ and reverse: 5´-CAACCCCTTAAAAAAATACTTCAAA-3´.

Real time PCR measurement of leptin mRNA, DNMT1, and DNMT3b

Real time-PCR was carried out using the iTaq Universal SYBR Green Supermix kit (Bio-Rad). PCR amplification was conducted in Bio-Rad CFX96 Realtime PCR (Bio-Rad). A 96 well PCR plate using the temperature profile of 95°C, 15 minutes, (initial denaturation) was followed by 40 cycles of 94°C, 15 seconds, 59°C, 30 seconds and 72°C, 30 seconds for denaturation, annealing, and extension steps, respectively. Primer sequence of human leptin, DNA methyltransferase 1 (DNMT1) and DNA methyltransferase 3 beta (DNMT3b) were 5´-GCTGTGCCCATCCAAAAAGT-3´ (forward) 5´-ACTGCCAGTGTCTGGTCCAT-3´ (reverse), 5´-TACCTGGACCCTGACCTC-3´ (forward), 5´-CGTTGGCATCAAAGATGGACA-3´ (reverse) and 5´-GGCAAGTTCTCCGAGGTC TCTG-3´ (forward) 5´-TGGTACATGGCTTTTCGATAGGA-3´ (reverse). The primer sequence of GAPDH as an internal control with the following sequence was 5′-AGGGCTGCTTTTAACTCTGGT-3′ (forward) and 5′-CCCCACTTGATTTTGGAGGGA-3′ (reverse). The PCR primers were synthesized by Eurofins Scientific (Munich, Germany).

Calculation

Insulin resistance

The homeostatic model assessment for insulin resistance (HOMA-IR) was calculated using the HOMA-IR [HOMA-IR=fasting insulin (μU/L)×fasting glucose (mM)/22.5].²³

Relative gene expression

The relative gene expression was calculated using [(1/2)^ΔCt].

Statistical analysis

Data were summarized as the mean±standard error of the mean. Two independent groups (non-obese and obese; and obese with non-metabolic and metabolic syndrome) were compared using the Student t-test. A Pearson correlation analysis was performed to assess the association of methylation (leptin) with gene expression (DNMT1, DNMT3b and leptin) and metabolic risk factors (demographic, biochemical, and clinical variables). A two-tailed P<0.05 was considered statistically significant. Analyses were performed with STATISTICA software version 6.0 (StatSoft, Tulsa, OK, USA).

Demographic, biochemical, and clinical characteristics

A total of 175 individuals were included in the study; 100 of them were obese and 75 were non-obese. The demographic (age, sex, height, weight, BMI, WC, biochemical (cholesterol, HDL cholesterol, TG, very low density lipoprotein (VLDL) cholesterol, low-density lipoprotein (LDL) cholesterol, and clinical (insulin, glucose, HOMA-IR, systolic blood pressure [SBP], and diastolic blood pressure [DBP]) characteristics of these two groups are summarized in Table 1 and Fig. 1. Age and sex distribution of the participants in the two groups was similar (P>0.05). However, the mean weight, BMI, WC, cholesterol, TG, VLDL, LDL, insulin, HOMA-IR, SBP, and DBP were found to be significantly (P<0.05, P<0.01, or P<0.001) different and higher in obese individuals when compared to non-obese individuals.

Relative gene expression

The relative gene expression of markers (DNMT1, DNMT3b, and leptin) of the two groups (obese and non-obese) is summarized in Table 2. The relative mean gene expression of markers was found to be similar (P>0.05) between the two groups though it was 63.3%, 78.4%, and 96.4% higher, respectively, in obese as compared to non-obese individuals.

Methylation and unmethylation

The methylation and unmethylation levels of leptin in the two groups (obese and non-obese) is summarized in Table 3 and Fig. 2. The mean methylation and unmethylation levels of leptin were also found to be similar (P>0.05) between the two groups though it was lower, 16.5% and 7.3% respectively, in obese as compared to non-obese individuals.

Correlation

The correlation of leptin methylation and unmethylation with metabolic risk factors (demographic, biochemical, and clinical) and relative markers (DNMT1, DNMT3b, and leptin) gene expression in the obese is summarized in Table 4. Leptin methylation and unmethylation showed an insignificant (P>0.05) correlation with metabolic risk factors and relative markers gene expression except leptin. Leptin showed a significant and positive (direct) correlation with age (r=0.560, P<0.001) and a significant and negative (inverse) correlation with weight (r=–0.449, P<0.01).

Methylation and unmethylation in obese groups

The methylation and unmethylation levels of leptin in two obese groups (non-metabolic and metabolic) is summarized in Table 5 and Fig. 3. The mean methylation and unmethylation levels of leptin were also found to be similar (P>0.05) between the two obese groups though it was lower, 55.0% and 62.1% respectively, in the metabolic obese as compared to the non-metabolic obese group.

In obesity and obesity-related metabolic disorders, leptin regulation is profoundly altered,²⁴ and some of these changes are regulated by DNA methylation. The present study reports that leptin DNA methylation levels at the promoter region, investigated in peripheral blood specimens, are associated with obesity-related anthropometric variables, biochemical parameters, and leptin mRNA expression.

Study findings report that the hypomethylation (reduced methylation) of the leptin promoter in the peripheral blood of the obese was like that of the non-obese. Further, it demonstrated reduced DNA methylation in the metabolic obese as compared to the non-metabolic obese subjects. Various authors have reported reduced levels of leptin methylation in the blood cells of children with higher birth weight and BMI,²⁵ in obese and insulin resistant adolescents,²⁶ and obese women.²⁷ Another report stipulates that genetic variation may have a substantial impact on local methylation patterns,^28,29 but as to the extent that methylation is not affected by genetic variation, the mechanisms remain unclear.

This study also found higher leptin gene expression in obese subjects. Interestingly, leptin was also higher in non-metabolic obese subjects. This finding is supported by Tiwari et al.²⁴ who reported higher gene expression in the adipose tissue of obese subjects. Another study involving human subjects showed that the developmental increase in leptin mRNA expression in adipose tissue during childhood, reaching maximal capacity in adulthood.³⁰ Moreover, a study also supported associating leptin with a direct supervision of body fat (adipose tissue) metabolism through inhibition of TG synthesis and stimulation of lipid breakdown.³¹ Reduced leptin methylation could account for obesity-related leptin upregulation. These findings indicate that methylation at the promoter region regulates leptin gene expression. A possible mechanism, the in silico analysis, is in line with a study that noted that methylation surrounding the transcription start site is tightly associated to transcriptional silencing while methylation of more downstream regions is not associated with the magnitude of gene expression.^32,33

Researchers in the current study also observed the significant negative association between methylation levels of leptin promoter and body weight. This finding is consistent with the recent finding of García-Cardona et al.²⁶ who reported a negative correlation between methylation frequency and BMI in obese adolescents.

In addition, the study also found a negative association of methylation levels of leptin promoter with BMI, cholesterol, insulin, HOMA-IR and glucose. However, it was not significant. These insignificant results may be due to small sample size. This finding is also consistent with previous findings.^26,34 Moreover, these results also showed higher expression of DNMT1 and DNMT3b in obese rather than non-obese groups, but there was a lower methylation level at the promoter of leptin. This may be due to changes in DNMT protein levels. Moreover, the study found a higher expression of DNMT1 and DNMT3b in the non-metabolic obese than in the metabolic obese. Thus, these findings support that lower methylation might be the cause of the development of obesity and its associated metabolic risks.

The strength of the current study includes analysis of the importance of adipokine central in energy metabolism regulation in blood cells. Blood is the most clinically within-reach tissue. Limitations of the current study are its small sample size and the fact that it did not include analysis in adipose tissue. In conclusion, the present findings add evidence that leptin DNA methylation levels in blood cells are associated with obesity-related anthropometric measures.

The authors declare no conflict of interest.

This study was funded by Science and Engineering Research Board (SERB), New Delhi, India (Grant No. YSS/2015/000054).

Study concept and design: S; acquisition of data: AM and MK; analysis and interpretation of data: PS; drafting of the manuscript: SSM; critical revision of the manuscript: RJ; statistical analysis: VVB and MPSN; obtained funding: S; administrative, technical, or material support: RB; and study supervision: ST and PKS.