Heyden Katarina E, Fiddler Joanna L, Xiu Yuwen, Malysheva Olga V, Handzlik Michal K, Phinney Whitney N, Stiles Linsey, Stabler Sally P, Metallo Christian M, Caudill Marie A, Field Martha S
Division of Nutritional Sciences, Cornell University, Ithaca, NY 14853, USA.
Department of Bioengineering, University of California, La Jolla, San Diego, CA 92093, USA.
PNAS Nexus. 2023 Mar 27;2(4):pgad105. doi: 10.1093/pnasnexus/pgad105. eCollection 2023 Apr.
Adequate thymidylate [deoxythymidine monophosphate (dTMP) or the "T" base in DNA] levels are essential for stability of mitochondrial DNA (mtDNA) and nuclear DNA (nDNA). Folate and vitamin B12 (B12) are essential cofactors in folate-mediated one-carbon metabolism (FOCM), a metabolic network which supports synthesis of nucleotides (including dTMP) and methionine. Perturbations in FOCM impair dTMP synthesis, causing misincorporation of uracil (or a "U" base) into DNA. During B12 deficiency, cellular folate accumulates as 5-methyltetrahdryfolate (5-methyl-THF), limiting nucleotide synthesis. The purpose of this study was to determine how reduced levels of the B12-dpendent enzyme methionine synthase (MTR) and dietary folate interact to affect mtDNA integrity and mitochondrial function in mouse liver. Folate accumulation, uracil levels, mtDNA content, and oxidative phosphorylation capacity were measured in male and mice weaned onto either a folate-sufficient control (C) diet (2 mg/kg folic acid) or a folate-deficient (FD) diet (lacking folic acid) for 7 weeks. heterozygosity led to increased liver 5-methyl-THF levels. mice consuming the C diet also exhibited a 40-fold increase in uracil in liver mtDNA. mice consuming the FD diet exhibited less uracil accumulation in liver mtDNA as compared to mice consuming the FD diet. Furthermore, mice exhibited 25% lower liver mtDNA content and a 20% lower maximal oxygen consumption rates. Impairments in mitochondrial FOCM are known to lead to increased uracil in mtDNA. This study demonstrates that impaired cytosolic dTMP synthesis, induced by decreased expression, also leads to increased uracil in mtDNA.
充足的胸苷酸（脱氧胸苷单磷酸，即DNA中的“T”碱基）水平对于线粒体DNA（mtDNA）和核DNA（nDNA）的稳定性至关重要。叶酸和维生素B12（B12）是叶酸介导的一碳代谢（FOCM）中的必需辅因子，FOCM是一个支持核苷酸（包括dTMP）和甲硫氨酸合成的代谢网络。FOCM的紊乱会损害dTMP合成，导致尿嘧啶（或“U”碱基）错误掺入DNA。在维生素B12缺乏期间，细胞内叶酸以5-甲基四氢叶酸（5-甲基-THF）的形式积累，限制了核苷酸合成。本研究的目的是确定依赖维生素B12的蛋氨酸合酶（MTR）水平降低与膳食叶酸如何相互作用，以影响小鼠肝脏中的mtDNA完整性和线粒体功能。对断奶后食用叶酸充足的对照（C）饮食（2mg/kg叶酸）或叶酸缺乏（FD）饮食（不含叶酸）7周的雄性和小鼠，测量其叶酸积累、尿嘧啶水平、mtDNA含量和氧化磷酸化能力。杂合性导致肝脏5-甲基-THF水平升高。食用C饮食的小鼠肝脏mtDNA中的尿嘧啶也增加了40倍。与食用FD饮食的小鼠相比，食用FD饮食的小鼠肝脏mtDNA中尿嘧啶积累较少。此外，小鼠肝脏mtDNA含量降低了25%，最大耗氧率降低了20%。已知线粒体FOCM受损会导致mtDNA中尿嘧啶增加。本研究表明，由表达降低诱导的胞质dTMP合成受损也会导致mtDNA中尿嘧啶增加。
