Epigenetic modifications of nuclear and mitochondrial DNA are associated with the disturbance of serum iron biomarkers among the metabolically unhealthy obesity school-age children

Abstract

Background: Serum iron biomarkers are disordered on the progression of obesity and its associated metabolic syndrome (MetS). However, limited evidence is explored the interactions between serum iron biomarkers and the incidence of MetS. Thus, the purpose of this study is to discuss whether epigenetic modifications of nuclear and mitochondrial DNA (mtDNA) are associated with the disturbance of serum iron biomarkers among the metabolically unhealthy obesity (MUO) school-age children.

Methods: A representative cross-sectional study was performed using the data from 104 obesity school-age children, while the subjects without obesity were as controls (n = 65). Then, the 104 obesity subjects were defined as metabolically healthy obesity (MHO, n = 60) and MUO (n = 44) subgroups according to whether they were accompanied with MetS. Their serum metabolic indicators, transferrin receptor 1 (TFR1), transferrin (TF) and genome-wide methylation were determined by the Elisa method. Moreover, the methylation levels of TFR1 and TF were measured by the Bisulfite sequencing PCR (BSP-PCR). Furthermore, the copy number (mtDNA-CN) and methylation of mtDNA were detected by the RT-PCR, while the semi-long RT-PCR was then used to estimate the lesions of mtDNA.

Results: Compared with the control and MHO groups, the levels of MetS related indicators, anthropological characteristics and 8-OHdG were higher, and the concentrations of CAT, GSH-Px, TF, TFR1 and genome-wide methylation were lower in the MUO group in a BMI-independent manner (P < 0.05). Then, the contents of serum iron were lower in both the MHO and MUO groups than those in the control group (P < 0.017). Moreover, they were positively related with the contents of serum CAT and GSH-Px, and negatively with 8-OHdG, TF and TFR1 (P < 0.05). Furthermore, the methylation patterns on the TF, TFR1 and mtDNA were higher in the MUO group than those in the MHO and control groups (P < 0.017), which were negatively correlated with their serum contents (P < 0.05). Meanwhile, the ratio of methylated/unmethylated mtDNA was significantly associated with their mtDNA-CN and lesions (P < 0.05).

Conclusions: Our findings suggested that the impairments on the epigenetic modifications of nuclear (genome-wide DNA, TF and TFR1) and mtDNA were associated with the disturbance of serum iron biomarkers to involve in the pathophysiology of MetS among the school-age MUO children.

Trial registration: This study was approved by the Ethics Committee of Beijing Children's Hospital affiliated to Capital Medical University (No. IEC-C-006-A04-V.06), which was also registered at the website of http://www.chictr.org.cn/showproj.aspx?proj=4673 (No: ChiCTR-OCH-14004900).

Keywords: Iron; Metabolic syndrome; Metabolically unhealthy obesity; Methylation; Mitochondrial DNA.

Fig. 1

CpGs sites and sequences in the TF and TFR1. Note: A: TF, B: TFR1. TF: transferrin, TFR1: transferrin receptor 1

Fig. 2

Identification of serum iron biomarkers and their correlations with the metabolic related indicators. Note: A: Concentrations of iron, B: Concentrations of hemoglobin, C: β-coefficients and 95% CIs of the regression models between the contents of serum iron and metabolic related indicators. All related indicators were analyzed by the Bonferroni’s correction tests after the One-way analysis of variance (ANOVA). *Compared with the control group, P < 0.017

Fig. 3

Concentrations of oxidative indicators and their correlations with serum iron biomarkers. Note: A: 8-OHdG, B: CAT, SOD and GSH-Px, C: Correlations between the contents of serum iron/hemoglobin and related oxidative indicators; D: β-coefficients and 95% CIs of the above regression models. All indicators were analyzed by the Bonferroni’s correction tests after the One-way analysis of variance (ANOVA). *Compared with the control group, P < 0.017, *It displayed that the regression equations were significantly different in the Figure 3D, P < 0.05. ^#Compared with the MHO group, P < 0.017

Fig. 4

Identification of significant iron related enzymes and their correlations with serum iron biomarkers in the different groups. Note: A: Contents of serum TF, B: Correlations between the contents of serum TF and iron, C: Contents of serum TFR1, D: Correlations between the contents of serum TFR1 and iron. TF: transferrin, TFR1: transferrin receptor 1. The concentrations of serum TF and TFR1 were analyzed by the Bonferroni’s correction tests after the One-way analysis of variance (ANOVA). *Compared with the control group, P < 0.017, ^#Compared with the MHO group, P < 0.017

Fig. 5

Methylation patters on the genome-wide DNA, TF and TFR1, and their correlations with their expressions in the different obesity groups. Note: A: Methylation levels in the genome-wide DNA, B: Hydroxymethylation levels in the genome-wide DNA, C: Methylation levels of CpGs sites in the TF, D: Methylation levels of CpGs sites in the TFR1, E and F: Correlations between the methylation levels of different CpGs sites in the TF/TFR1 and their contents respectively. G and H: β-coefficients and 95% CIs of the above regression models. TF: transferrin, TFR1: transferrin receptor 1. All indicators were analyzed by the Bonferroni’s correction tests after the One-way analysis of variance (ANOVA). *Compared with the control group, P < 0.017. *It displayed that the regression equations were significantly different in the Fig. 5D, P < 0.05. ^#Compared with the MHO group, P < 0.017

Fig. 6

Assessment of differential copy numbers, lesions and methylation levels of mtDNA among the different obesity groups. Note: A: Relative expressions of ND1, B: Relative expressions of MTF3212/R3319; C: mtDNA lesions; D; β-coefficients and 95% CIs of the regression models between the expressions of ND1/MTF3212/R3319 and mtDNA lesions; E: Correlations between the expressions of ND1 and MTF3212/R3319; F: Correlations between the expressions of ND1 and mtDNA lesions; G: Correlations between the expressions of MTF3212/R3319 and mtDNA lesions; H: Methylation levels in the D-loop region; I: Correlations between the methylation levels of mtDNA and expressions of ND1/MTF3212/R3319 and mtDNA lesions. All above indicators were analyzed by the Bonferroni’s correction tests after the One-way analysis of variance (ANOVA). *Compared with the control group, P < 0.017, ^# Compared with the MHO group, P < 0.017

Fig. 7

Obesity related epigenetic modifications on the nuclear DNA and mtDNA could cause the disturbance of serum iron biomarkers to induce the occurrence of MetS. The MHO was adaptive with the abnormal epigenetic modifications of nuclear (whole-genome, TF and TFR1) and mtDNA to result in the decreases of iron related biomarkers in a BMI-independent manner. Then, it might conversely affect the oxidative stress to aggravate the occurrence of MetS with the increasing serum iron biomarkers