Castillo Luisa F, Heyden Katarina E, Williamson Abigail R, Ma Wenxia, Malysheva Olga V, Vacanti Nathaniel M, Thalacker-Mercer Anna E, Field Martha S
Division of Nutritional Sciences, Cornell University, Ithaca, NY, USA.
Department of Cell, Developmental and Integrative Biology, University of Alabama at Birmingham, Birmingham, AL, USA.
bioRxiv. 2025 Aug 30:2025.05.19.654973. doi: 10.1101/2025.05.19.654973.
Vitamin B12 plays a vital role in folate-mediated one-carbon metabolism (FOCM), a series of one-carbon transfer reactions that generate nucleotides (thymidylate (dTMP) and purines) and methionine. Inadequate levels of B12 impair FOCM, depressing thymidylate (dTMP) synthesis, which in turn leads to uracil accumulation in DNA. This phenomenon has been well documented in nuclear DNA. Our previous work in liver tissue has shown that mitochondrial DNA (mtDNA) is more sensitive to FOCM impairments in that mtDNA exhibits elevated uracil levels before uracil concentrations in nuclear DNA change. However, the functional consequences of uracil accumulation in mtDNA are largely unknown. The purpose of this study was to determine how a functional B12 deficiency (induced by reduced levels of the B12-dependent enzyme methionine synthase (MTR)) and dietary B12 deficiency affects mtDNA integrity and mitochondrial function in energetic and mitochondria-rich tissues such as skeletal muscle.
Male and mice were weaned to either an AIN93G-based control (C) diet containing 25 μg/kg vitamin B12 or a B12-deficient (-B12) diet containing 0 μg/kg vitamin B12 to explore the effects of functional ( ) and dietary B12 deficiency on muscle weight, uracil content in mtDNA, mtDNA content, and oxidative phosphorylation complex capacity in skeletal muscle. Aged (20-22mo) male C57BL6/N mice were acclimated to an AIN93G control diet four weeks, then received either weekly injections of saline (vehicle control [30 uL 0.9% NaCl]) or B12 (0.65mg per 30uL 0.9% NaCl) in each of two hindleg muscles [1.25 mg B12 total]) for 8 weeks.
The tibialis anterior (TA) muscle from mice exhibited lowered maximal respiratory capacity of complex I, II, and IV of the electron transport chain than did TA from mice. Exposure to the -B12 diet lowered maximal capacity of complex I in red, mitochondrially rich muscle (soleus and mitochondria-rich portions of quadriceps and gastrocnemius) (p=0.02). Levels of uracil accumulation in mtDNA in red muscle and gastrocnemius were elevated ~10 fold with exposure to -B12 diet (p=0.04 and p<0.001, respectively). In aged mice gastrocnemius complex IV activity increased with intramuscular B12 supplementation (p=0.04).
Exposure to a B12-deficient diet led to uracil accumulation in mtDNA and impaired maximal oxidative capacity in two different types of skeletal muscle. B12 supplementation improved complex IV maximal capacity in gastrocnemius from aged mice.
维生素B12在叶酸介导的一碳代谢（FOCM）中起着至关重要的作用，FOCM是一系列生成核苷酸（胸苷酸（dTMP）和嘌呤）和蛋氨酸的一碳转移反应。B12水平不足会损害FOCM，抑制胸苷酸（dTMP）合成，进而导致DNA中尿嘧啶积累。这种现象在核DNA中已有充分记录。我们之前在肝脏组织中的研究表明，线粒体DNA（mtDNA）对FOCM损伤更为敏感，因为在核DNA中的尿嘧啶浓度变化之前，mtDNA中的尿嘧啶水平就已升高。然而，mtDNA中尿嘧啶积累的功能后果在很大程度上尚不清楚。本研究的目的是确定功能性B12缺乏（由依赖B12的酶甲硫氨酸合酶（MTR）水平降低引起）和饮食中B12缺乏如何影响富含能量和线粒体的组织（如骨骼肌）中的mtDNA完整性和线粒体功能。
将雄性小鼠断奶后分别喂食基于AIN93G的对照（C）饮食（含25μg/kg维生素B12）或B12缺乏（-B12）饮食（含0μg/kg维生素B12），以探讨功能性（ ）和饮食中B12缺乏对肌肉重量、mtDNA中尿嘧啶含量、mtDNA含量以及骨骼肌中氧化磷酸化复合体能力的影响。将老龄（20 - 22月龄）雄性C57BL6/N小鼠在AIN93G对照饮食中适应四周，然后在两条后肢肌肉中每周分别注射生理盐水（载体对照[30μL 0.9% NaCl]）或B12（每30μL 0.9% NaCl含0.65mg）（共1.25mg B12），持续8周。
与 小鼠相比， 小鼠的胫前肌（TA）中电子传递链复合体I、II和IV的最大呼吸能力降低。暴露于 -B12饮食会降低红色、富含线粒体的肌肉（比目鱼肌以及股四头肌和腓肠肌富含线粒体的部分）中复合体I的最大能力（p = 0.02）。暴露于 -B12饮食时，红色肌肉和腓肠肌中mtDNA的尿嘧啶积累水平升高了约10倍（分别为p = 0.04和p < 0.001）。在老龄小鼠中，腓肠肌复合体IV活性随着肌肉内补充B12而增加（p = 0.04）。
暴露于B12缺乏饮食会导致mtDNA中尿嘧啶积累，并损害两种不同类型骨骼肌的最大氧化能力。补充B12可提高老龄小鼠腓肠肌中复合体IV的最大能力。
