Rationale: Human cardiac mesenchymal cells (CMSCs) are a therapeutically relevant primary cell population. Diabetes mellitus compromises CMSC function as consequence of metabolic alterations and incorporation of stable epigenetic changes.

Objective: To investigate the role of α-ketoglutarate (αKG) in the epimetabolic control of DNA demethylation in CMSCs.

Methods and Results: Quantitative global analysis, methylated and hydroxymethylated DNA sequencing, and gene-specific GC methylation detection revealed an accumulation of 5-methylcytosine, 5-hydroxymethylcytosine, and 5-formylcytosine in the genomic DNA of human CMSCs isolated from diabetic donors. Whole heart genomic DNA analysis revealed iterative oxidative cytosine modification accumulation in mice exposed to high-fat diet (HFD), injected with streptozotocin, or both in combination (streptozotocin/HFD). In this context, untargeted and targeted metabolomics indicated an intracellular reduction of αKG synthesis in diabetic CMSCs and in the whole heart of HFD mice. This observation was paralleled by a compromised TDG (thymine DNA glycosylase) and TET1 (ten–eleven translocation protein 1) association and function with TET1 relocating out of the nucleus. Molecular dynamics and mutational analyses showed that αKG binds TDG on Arg275 providing an enzymatic allosteric activation. As a consequence, the enzyme significantly increased its capacity to remove G/T nucleotide mismatches or 5-formylcytosine. Accordingly, an exogenous source of αKG restored the DNA demethylation cycle by promoting TDG function, TET1 nuclear localization, and TET/TDG association. TDG inactivation by CRISPR/Cas9 knockout or TET/TDG siRNA knockdown induced 5-formylcytosine accumulation, thus partially mimicking the diabetic epigenetic landscape in cells of nondiabetic origin. The novel compound (S)-2-[(2,6-dichlorobenzoyl)amino]succinic acid (AA6), identified as an inhibitor of αKG dehydrogenase, increased the αKG level in diabetic CMSCs and in the heart of HFD and streptozotocin mice eliciting, in HFD, DNA demethylation, glucose uptake, and insulin response.

Conclusions: Restoring the epimetabolic control of DNA demethylation cycle promises beneficial effects on cells compromised by environmental metabolic changes.

原理：人心脏间充质细胞（CMSC）是与治疗相关的原代细胞群。由于代谢改变和稳定表观遗传学改变的结合，糖尿病损害了CMSC的功能。

目的：研究α-酮戊二酸（αKG）在CMSCs DNA脱甲基的表观代谢控制中的作用。

方法和结果：定量全局分析，甲基化和羟甲基化的DNA测序以及基因特异性GC甲基化检测显示，从糖尿病供体中分离出的人CMSC的基因组DNA中积累了5-甲基胞嘧啶，5-羟甲基胞嘧啶和5-甲酰基胞嘧啶。全心基因组DNA分析显示，在暴露于高脂饮食（HFD），注射链脲佐菌素或二者合用（链脲佐菌素/ HFD）的小鼠中，迭代氧化氧化胞嘧啶修饰积累。在这种情况下，未靶向和靶向的代谢组学表明糖尿病CMSC和HFD小鼠整个心脏中αKG合成的细胞内减少。该观察结果与受损的TDG（胸腺嘧啶脱氧核糖核酸DNA糖基化酶）和TET1（十至十一个易位蛋白1）的结合以及与TET1移出细胞核的功能同时出现。分子动力学和突变分析表明，αKG与Arg275上的TDG结合，提供酶促变构活化。结果，该酶显着提高了其去除G / T核苷酸错配或5-甲酰基胞嘧啶的能力。因此，αKG的外源通过促进TDG功能，TET1核定位和TET / TDG缔合而恢复了DNA去甲基化周期。通过CRISPR / Cas9敲除或TET / TDG siRNA敲除导致的TDG失活诱导了5-甲酰基胞嘧啶的积累，从而部分模拟了非糖尿病起源细胞中糖尿病的表观遗传情况。新型化合物（S）-2-[（（2,6-二氯苯甲酰基）氨基]琥珀酸（AA6）被确定为αKG脱氢酶抑制剂，可增加糖尿病CMSC中以及HFD和链脲佐菌素小鼠心脏中的αKG水平。 ，在HFD中，DNA去甲基化，

结论：恢复DNA去甲基化周期的代谢控制有望对环境代谢变化损害的细胞产生有益的影响。