Vitamin B12 supports skeletal muscle oxidative phosphorylation capacity in male mice

Background

B12 is an essential cofactor required for de novo biosynthesis of thymidylate (dTMP) and for folate-mediated one carbon metabolism (FOCM). This molecule also acts as a cofactor for the enzymes methyl malonyl CoA mutase (MCM) and methionine synthase (MTR). During B12 deficiency or MTR inhibition fails to synthesize folate cofactors needed for the synthesis of dTMP (1). Constant low levels of dTMP cause uracil misincorporations in nuclear DNA, which can reduce its stability due constant activation of base-excision repair mechanisms (2). Such consequences of impaired FOCM can also affect the mitochondrial DNA (mtDNA) (3–5). Since mtDNA encodes several components involved in oxidative phosphorylation (OXPHOS), in this pre-print the authors tested the hypothesis that impaired FOCM by B12 deficiency or MTR reduced activity causes deleterious effects in energy metabolism of muscle tissues. 

Key Findings

B12 deficiency induces mtDNA uracil misincorporation in muscle tissues

B12-deficient diet increased uracil misincorporation into mtDNA in mouse’s red muscle and gastrocnemius (Figure 4). This suggests defective dTMP synthesis in mitochondria due to impaired folate-mediated one-carbon metabolism (FOCM).

Partial MTR loss of function reduced mitochondrial functionality in glycolytic muscles.

A genetic reduction in Mtr expression and dietary B12 deficiency reduced oxidative phosphorylation (OXPHOS) capacity, particularly affecting complexes I, II, and IV in glycolytic muscle (Figures 2A–C) and complexes I and IV in oxidative skeletal muscle (Figure 3).

B12 deficiency or Mtr reduction does not reduce mitochondrial content

There were no changes in mtDNA copy number, mitochondrial mass, or mtDNA deletion frequency in response to B12 deficiency or Mtr reduction (Fig. 5). This suggests that the functional decline was not due to a loss of mitochondrial content.

Why this preprint is important?

This preprint provides new evidence into the role of vitamin B12 in maintaining mitochondrial function and mtDNA integrity in skeletal muscle. The study demonstrates that both genetic impairment of MTR and dietary B12 deficiency led to uracil misincorporation into mtDNA, a marker of impaired nucleotide synthesis and genome instability. This accumulation of uracil was associated with reduced electron transport system, suggesting a new relationship with broad implications for nutritional science and mitochondrial biology

Comments

C1: The authors should clarify that genetic variables between mouse strains were not controlled across all experiments. The data in Figure 6 was obtained from C57BL/6N mice, while the C57BL/6J strain was reportedly used for the rest of the work. Each strain carries specific mutations that differently affects mitochondrial metabolism (6). Due to underlying genetic variations, the data presented in Figure 6 may not be directly comparable to other datasets in this preprint, and its interpretation requires caution.

C2: The data presented in the preprint indirectly suggest a possible negative correlation between mitochondrial energetic capacity (Figures 2A–C and 3) and uracil content in mtDNA (Figure 4). The discussion could be further enriched by including a statistical analysis of this potential relationship. If possible, the authors could use the already existing data to plot a Spearman correlation between uracil content in mtDNA and the oxygen consumption rate for the individual ETS complexes. This analysis could provide valuable insight into the functional impact of mtDNA uracil accumulation on mitochondrial respiration.

C3: Standard laboratory housing temperatures for mice (typically 20–22°C) are below the murine thermoneutral zone, which imposes a state of chronic, mild cold stress. This condition leads to sustained low-grade adrenergic activation, resulting in an increased metabolic rate, altered energy metabolism, shifts in substrate utilization, and enhanced glucose uptake in various tissues (6–8). Given that housing temperature is a critical variable influencing metabolic parameters, specifying the rearing and housing temperature of the mice in the Methods section is necessary for the accurate interpretation and reproducibility of the findings.

Disclaimer: Google Gemini was used for the grammatical review for this report.

References

1. Shane B, Stokstad ELR. Vitamin B12 -Folate Interrelationships. Annu Rev Nutr. julho de 1985;5(1):115–41. 
2. Chon J, Field MS, Stover PJ. Deoxyuracil in DNA and disease: Genomic signal or managed situation? DNA Repair. maio de 2019;77:36–44. 
3. Field MS, Kamynina E, Chon J, Stover PJ. Nuclear Folate Metabolism. Annu Rev Nutr. 21 de agosto de 2018;38(1):219–43. 
4. Fiddler JL, Xiu Y, Blum JE, Lamarre SG, Phinney WN, Stabler SP, et al. Reduced Shmt2 Expression Impairs Mitochondrial Folate Accumulation and Respiration, and Leads to Uracil Accumulation in Mouse Mitochondrial DNA. J Nutr. outubro de 2021;151(10):2882–93. 
5. Heyden KE, Fiddler JL, Xiu Y, Malysheva OV, Handzlik MK, Phinney WN, et al. Reduced methionine synthase expression results in uracil accumulation in mitochondrial DNA and impaired oxidative capacity. Stover P, organizador. PNAS Nexus. 3 de abril de 2023;2(4):pgad105. 
6. Enríquez JA. Mind your mouse strain. Nat Metab. 7 de janeiro de 2019;1(1):5–7. 
7. Reitman ML. Of mice and men – environmental temperature, body temperature, and treatment of obesity. FEBS Lett. junho de 2018;592(12):2098–107. 
8. Fischer AW, Cannon B, Nedergaard J. Optimal housing temperatures for mice to mimic the thermal environment of humans: An experimental study. Mol Metab. janeiro de 2018;7:161–70. 

Tags: mice, oxidative phosphorylation, skeletal muscle, vitamin b12

(No Ratings Yet)