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Updated: Sep 6, 2025
Sample Preparation to Bioinformatics Analysis of DNA Methylation: Association Strategy for Obesity and Related Trait Studies
Published on: May 6, 2022
Mitochondrial DNA in Visceral Adipose Tissue in Severe Obesity: From Copy Number to D-Loop Methylation
Laura Bordoni1, Jessica Perugini2, Irene Petracci3
1Unit of Molecular Biology and Nutrigenomics, School of Pharmacy, University of Camerino, 62032 Camerino, Italy.
Background:
Peripheral alterations of mitochondrial DNA copy number (mtDNAcn) in obesity and associated co-morbidities have been previously shown. Furthermore, the possibility that methylation could occur in the mtDNA (in particular in the displacement loop, D-Loop) and regulate its functions has been raised. However, limited data about mtDNA methylation in adipose tissue are currently available. Since a strict crosstalk between the nucleus and mitochondria exists, especially in terms of the one-carbon cycle (that supports methylation reactions in the cell), we investigated methylation in selected areas of the mitochondrial and nuclear DNA and their expression in visceral adipose tissue (VAT) samples of patients with severe obesity.
Methods:
VAT biopsies were collected from surgery patients to isolate DNA and RNA. Gene expression and mtDNAcn were assessed through qPCR. DNA methylation in both nuclear and mitochondrial areas were determined through bisulfite pyrosequencing.
Results:
Methylation levels of the mtDNA were only marginally associated with the obesity degree (higher D-Loop methylation in severe obesity) and were not correlated with mtDNAcn. A significant correlation between D-Loop methylation and LINE-1 methylation was observed in VAT samples, and this was independent from the obesity degree. A progressive reduction of mtDNAcn and increase in NRF1 expression levels were measured in VAT in severe obesity. NRF1 expression was directly correlated with PPARG and MTHFR expression levels, while mtDNAcn was associated to TFAM expression. The correlation between mtDNAcn and TFAM expression was affected by the obesity status.
Conclusions:
This evidence supports the hypothesis that mtDNA alterations occur in obesity and a complex dynamic correlation between mitochondrial and nuclear DNA methylation exists, highlighting the need for further investigations.
Insights
Mitochondrial DNA (mtDNA) methylation in visceral adipose tissue (VAT) shows a complex relationship with obesity. While D-loop methylation slightly increases with severe obesity, it correlates with nuclear DNA methylation, suggesting crosstalk between cellular and mitochondrial genomes.
Area of Science:
- Metabolomics
- Epigenetics
- Obesity Research
Background:
- Peripheral mitochondrial DNA copy number (mtDNAcn) alterations are known in obesity.
- Mitochondrial DNA (mtDNA) methylation, particularly in the D-loop, is proposed to regulate mtDNA function.
- Limited data exist on mtDNA methylation in adipose tissue, despite nuclear-mitochondrial crosstalk via the one-carbon cycle.
Purpose of the Study:
- To investigate DNA methylation in mitochondrial and nuclear DNA.
- To assess gene expression and mtDNA copy number.
- To explore these factors in visceral adipose tissue (VAT) from patients with severe obesity.
Main Methods:
- DNA and RNA isolation from VAT biopsies.
- Quantitative PCR (qPCR) for gene expression and mtDNA copy number (mtDNAcn).
- Bisulfite pyrosequencing for nuclear and mitochondrial DNA methylation analysis.
Main Results:
- mtDNA methylation showed a marginal association with obesity, with higher D-loop methylation in severe obesity, but no correlation with mtDNAcn.
- A significant, obesity-independent correlation was found between D-loop and LINE-1 methylation in VAT.
- Severe obesity was associated with reduced mtDNAcn and increased NRF1 expression, which correlated with PPARG and MTHFR. mtDNAcn correlated with TFAM expression, influenced by obesity status.
Conclusions:
- mtDNA alterations are implicated in obesity.
- A complex interplay between mitochondrial and nuclear DNA methylation exists.
- Further research is needed to elucidate these dynamics.
