The Journal of Nutrition · Jan 20, 2026

Vitamin B12 Supports Skeletal Muscle Oxidative Phosphorylation Capacity in Male Mice

By Castillo LF, Heyden KE, Williamson AR et al.

BACKGROUND: Vitamin B-12 is a cofactor in folate-mediated 1-carbon metabolism, which generates nucleotides {thymidylate [deoxythymidine monophosphate (dTMP)] and purines} and methionine. Depressed de novo thymidylate (dTMP) synthesis leads to uracil accumulation in DNA. OBJECTIVES: This study aimed to determine how B-12 availability affects mitochondrial DNA (mtDNA) integrity and mitochondrial function in skeletal muscle. B-12 deficiency was modeled in young-adult mice. Intramuscular B-12 injection in aged mice assessed the role of B-12 supplementation in age-related changes in skeletal muscle. METHODS: Male methionine synthase knockdown (Mtr+/-) and wild-type littermates (Mtr+/+) were weaned to either an AIN93G-based control diet containing 25 μg/kg vitamin B-12 (Mtr+/+, n = 8; Mtr+/-, n = 9) or a B-12-deficient (-B-12) diet containing 0 μg/kg vitamin B-12 (n = 9 per genotype) for 7 wk. Aged (20-22 mo) male C57BL/6N mice were acclimated to an AIN93G control diet 4 wk, then received either weekly injections of saline [vehicle control (30 μL 0.9% NaCl; n = 5) or B-12 (0.65 μg per 30 μL 0.9% NaCl; n = 6) in each of 2 hindleg muscles (1.25 μg B-12 total)] for 8 wk. Outcomes measured included maximal oxygen consumption rate, uracil in mtDNA (a biomarker of mtDNA integrity), mtDNA copy number, and mitochondrial mass. Data were analyzed using a 2-way analysis of variance in the Mtr+/- mouse model exposed to -B-12 diets and by a Student's t-test for B-12 supplementation in aged mice. RESULTS: The tibialis anterior (TA) muscle from Mtr+/- mice exhibited 50% lower (P = 0.01) maximal respiratory capacity of the electron transport chain than did TA from Mtr+/+ mice. Exposure to the -B-12 diet lowered the maximal capacity of complex I in mitochondrially rich muscle (soleus and mitochondria-rich portions of quadriceps and gastrocnemius) by 25% (P = 0.02). Uracil in mtDNA in red muscle and gastrocnemius was elevated ∼10 fold with exposure to -B-12 diet (P = 0.04 and P < 0.001, respectively). In aged mice, gastrocnemius complex IV activity was increased 2-fold with intramuscular B-12 supplementation (P = 0.04). CONCLUSIONS: Exposure to a-B-12 diet led to uracil accumulation in mtDNA and impaired maximal oxidative capacity in skeletal muscle. B-12 supplementation improved complex IV maximal capacity in gastrocnemius from aged mice, a model of age-related skeletal muscle decline.

doi.org/10.1016/j.tjnut.2026.101367 Open access, accepted manuscript PubMed 41571037 Interventions