Mitochondria metabolomics reveals a role of β-nicotinamide mononucleotide metabolism in mitochondrial DNA replication

J Biochem. 2022 Mar 3;171(3):325-338. doi: 10.1093/jb/mvab136.


Mitochondrial DNA (mtDNA) replication is tightly regulated and necessary for cellular homeostasis; however, its relationship with mitochondrial metabolism remains unclear. Advances in metabolomics integrated with the rapid isolation of mitochondria will allow for remarkable progress in analyzing mitochondrial metabolism. Here, we propose a novel methodology for mitochondria-targeted metabolomics, which employs a quick isolation procedure using a hemolytic toxin from Streptococcus pyogenes streptolysin O (SLO). SLO isolation of mitochondria from cultured HEK293 cells is time- and labor-saving for simultaneous multi-sample processing and has been applied to various other cell lines in this study. Furthermore, our method can detect the time-dependent reduction in mitochondrial ATP in response to a glycolytic inhibitor 2-deoxyglucose, indicating the suitability to prepare metabolite analysis-competent mitochondria. Using this methodology, we searched for specific mitochondrial metabolites associated with mtDNA replication activation, and nucleotides and NAD+ were identified to be prominently altered. Most notably, treatment of β-nicotinamide mononucleotide (β-NMN), a precursor of NAD+, to HEK293 cells activated and improved the rate of mtDNA replication by increasing nucleotides in mitochondria and decreasing their degradation products: nucleosides. Our results suggest that β-NMN metabolism plays a role in supporting mtDNA replication by maintaining the nucleotide pool balance in the mitochondria.

Keywords: beta-nicotinamide mononucleotide (β-NMN); metabolomics; mitochondrial DNA; nucleotide metabolism; streptolysin O.

MeSH terms

  • DNA, Mitochondrial / genetics
  • DNA, Mitochondrial / metabolism
  • HEK293 Cells
  • Humans
  • Metabolomics
  • Mitochondria / metabolism
  • NAD* / metabolism
  • Nicotinamide Mononucleotide* / metabolism
  • Nicotinamide Mononucleotide* / pharmacology


  • DNA, Mitochondrial
  • NAD
  • Nicotinamide Mononucleotide