Terol (NC) and high cholesterol (HC), with the cutoff point set at 200 mg/dL; n = 12?8. e, f the Mirogabalin web fasting plasma VLDL (e) and mtDNAn (f) in individuals with I-CBP112MedChemExpress I-CBP112 normal VLDL (n-VLDL) and high VLDL (h-VLDL), with the cutoff point set at 32 mg/dL; n = 9?1. ***p < 0.0001; NS, not significantZheng et al. Clinical Epigenetics (2015) 7:Page 5 ofTable 2 Univariate regression analysis of mtDNAn among all subjectsParameters Age BMI Fasting glucose Fasting insulin HOMA-IR Triglyceride HDL LDL VLDL LDL/HDL ratio Total cholesterol Cholesterol/HDL HbA1c coefficient -0.008 -0.026 -0.004 -0.015 -0.703 -0.001 0.010 -0.002 -0.005 -0.130 -0.001 -0.116 0.182 R value 0.212 0.333 0.084 0.320 0.379 0.163 0.187 0.095 0.165 0.133 0.050 0.178 0.115 p value 0.189 0.041 0.604 0.044 0.016 0.314 0.248 0.569 0.310 0.425 0.760 0.272 0.human leukocytes (Fig. 2, Table 2, and Additional file 3: Figure S3).D-loop had higher methylation in obese subjectsin the obese individuals (Figs. 1 and 2) prompted us to ask whether the D-loop region underwent aberrant methylation, the modification that may regulate mtDNA replication and transcription [49]. Regression analysis suggested that mtDNAn was negatively correlated with D-loop methylation (-0.078; p < 0.05). Moreover, DNA methylation in the D-loop region was 5.2-fold higher in the obese group than in the lean group (p < 0.05, Fig. 5a). Interestingly, the increased methylation of D-loop was PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/27362935 phenocopied by insulin-resistant (InR) subjects, and DNA methylation was 4.6-fold higher than that of insulin-sensitive (InS) subjects (p < 0.05, Fig. 5b). However, the difference of D-loop methylation was indiscernible between the NFG and IFG groups (Fig. 5c) or between normal triglyceride (NT) and hypertriglyceridemic (HT) groups (Fig. 5d). Therefore, the increased DNA methylation in the D-loop region was associated with insulin resistance but independent from aberrant glucose and lipid levels. Our data adds new and timely evidence to the potential role of mtDNA methylation in metabolic regulation [18?0, 36].The D-loop region controls the replication of the mitochondrial DNA and organization of the mitochondrial nucleoid [33?5]. The observation of reduced mtDNAnDiscussion The growing epidemic of obesity is largely attributed to the current life style of energy overconsumption with inadequate physical activity [2, 50, 51]. As such, the surplus of nutrients is accumulated and contributes to theFig. 5 Measurement of DNA methylation in the D-loop region of mitochondrial genome. a D-loop methylation in lean and obese subjects; n = 8?2. b D-loop methylation in InS and InR subjects; n = 13?7. c D-loop methylation in NFG and IFG individuals; n = 11?9. d D-loop methylation in NT and HT individuals; n = 10?0. The DNA methylation levels were also compared between NC and HC, n-VLDL, and h-VLDL groups, and there was no significant difference (not shown). *p < 0.05; NS, not significantZheng et al. Clinical Epigenetics (2015) 7:Page 6 ofinteractions between genes and environment [2, 51]. Mitochondrial alterations have been observed in obese individuals, including impaired mitochondrial fatty acid/ lipid oxidation capacity in the skeletal muscle and reduced mtDNAn in adipose tissues and peripheral blood samples [6?, 11]. However, whether an epigenetic mechanism underlies the reduced mtDNAn has not been defined, and how genetic and epigenetic traits in mitochondria are related to altered metabolic parameters remains elusive. In this study, we found that the re.Terol (NC) and high cholesterol (HC), with the cutoff point set at 200 mg/dL; n = 12?8. e, f the fasting plasma VLDL (e) and mtDNAn (f) in individuals with normal VLDL (n-VLDL) and high VLDL (h-VLDL), with the cutoff point set at 32 mg/dL; n = 9?1. ***p < 0.0001; NS, not significantZheng et al. Clinical Epigenetics (2015) 7:Page 5 ofTable 2 Univariate regression analysis of mtDNAn among all subjectsParameters Age BMI Fasting glucose Fasting insulin HOMA-IR Triglyceride HDL LDL VLDL LDL/HDL ratio Total cholesterol Cholesterol/HDL HbA1c coefficient -0.008 -0.026 -0.004 -0.015 -0.703 -0.001 0.010 -0.002 -0.005 -0.130 -0.001 -0.116 0.182 R value 0.212 0.333 0.084 0.320 0.379 0.163 0.187 0.095 0.165 0.133 0.050 0.178 0.115 p value 0.189 0.041 0.604 0.044 0.016 0.314 0.248 0.569 0.310 0.425 0.760 0.272 0.human leukocytes (Fig. 2, Table 2, and Additional file 3: Figure S3).D-loop had higher methylation in obese subjectsin the obese individuals (Figs. 1 and 2) prompted us to ask whether the D-loop region underwent aberrant methylation, the modification that may regulate mtDNA replication and transcription [49]. Regression analysis suggested that mtDNAn was negatively correlated with D-loop methylation (-0.078; p < 0.05). Moreover, DNA methylation in the D-loop region was 5.2-fold higher in the obese group than in the lean group (p < 0.05, Fig. 5a). Interestingly, the increased methylation of D-loop was PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/27362935 phenocopied by insulin-resistant (InR) subjects, and DNA methylation was 4.6-fold higher than that of insulin-sensitive (InS) subjects (p < 0.05, Fig. 5b). However, the difference of D-loop methylation was indiscernible between the NFG and IFG groups (Fig. 5c) or between normal triglyceride (NT) and hypertriglyceridemic (HT) groups (Fig. 5d). Therefore, the increased DNA methylation in the D-loop region was associated with insulin resistance but independent from aberrant glucose and lipid levels. Our data adds new and timely evidence to the potential role of mtDNA methylation in metabolic regulation [18?0, 36].The D-loop region controls the replication of the mitochondrial DNA and organization of the mitochondrial nucleoid [33?5]. The observation of reduced mtDNAnDiscussion The growing epidemic of obesity is largely attributed to the current life style of energy overconsumption with inadequate physical activity [2, 50, 51]. As such, the surplus of nutrients is accumulated and contributes to theFig. 5 Measurement of DNA methylation in the D-loop region of mitochondrial genome. a D-loop methylation in lean and obese subjects; n = 8?2. b D-loop methylation in InS and InR subjects; n = 13?7. c D-loop methylation in NFG and IFG individuals; n = 11?9. d D-loop methylation in NT and HT individuals; n = 10?0. The DNA methylation levels were also compared between NC and HC, n-VLDL, and h-VLDL groups, and there was no significant difference (not shown). *p < 0.05; NS, not significantZheng et al. Clinical Epigenetics (2015) 7:Page 6 ofinteractions between genes and environment [2, 51]. Mitochondrial alterations have been observed in obese individuals, including impaired mitochondrial fatty acid/ lipid oxidation capacity in the skeletal muscle and reduced mtDNAn in adipose tissues and peripheral blood samples [6?, 11]. However, whether an epigenetic mechanism underlies the reduced mtDNAn has not been defined, and how genetic and epigenetic traits in mitochondria are related to altered metabolic parameters remains elusive. In this study, we found that the re.