AMPK-PGC1α/NRF1-DNMT1通路调控的mtDNA甲基化在肝脏胰岛素抵抗中的机制研究

Loss of mitochondrial homeostasis which includes the function, biogenesis, degradation and dynamics of mitochondria is a major factor causing the hepatic insulin resistance. Nuclear DNA methylation has been well demonstrated in regulating mitochondrial homeostasis and playing roles in hepatic insulin resistance. However it remains unknown whether the mitochondrial DNA (mtDNA) methylation could regulate mitochondrial homeostasis and hepatic insulin resistance as well. Our preliminary data show that the expression of AMPK-PGC1α/NRF1-DNMT1 pathway was changed, the frequency of total mtDNA 5mC was increased and the mRNA levels of mtDNA-encoded components of respiratory chain were decreased in insulin resistance-mouse liver, indicating that the mtDNA methylation might regulate hepatic insulin resistance via mitochondrial homeostasis. Therefore, we propose that nutritional imbalance-induced alteration of AMPK-PGC1α/NRF1-DNMT1 pathway mediates mtDNA methylation to affect mitochondrial homeostasis, then, leading to hepatic insulin resistance. In the present study, we will employ cellular and animal insulin resistance models to draw the hepatic mtDNA methylation map. Then, we will use gene manipulation techniques to elucidate the molecular mechanisms of mtDNA methylation in regulating mitochondrial homeostasis and hepatic insulin resistance on both the cellular and animal levels. The present project is expected to identify a novel mechanism on how disturbance of mitochondrial homeostasis contributes to hepatic insulin resistance from mitochondrial epigenetics and provide novel therapeutic targets for the prevention and treatment of type 2 diabetes.

线粒体稳态（功能、生成、降解和动态变化）失衡是肝脏胰岛素抵抗发生的关键因素，已知核DNA甲基化可以调控线粒体稳态，但线粒体DNA（mtDNA）甲基化能否调控线粒体稳态并参与胰岛素抵抗尚无报道。近期我们发现胰岛素抵抗小鼠肝脏中AMPK-PGC1α/NRF1-DNMT1通路发生改变，mtDNA总体甲基化频率显著升高，mtDNA编码呼吸链亚基mRNA水平显著降低，提示mtDNA甲基化可能调控线粒体稳态并参与肝脏胰岛素抵抗。我们据此假设：营养失衡下AMPK-PGC1α/NRF1-DNMT1调控的mtDNA甲基化升高，影响线粒体稳态，进而导致肝脏胰岛素抵抗。本课题将利用细胞和动物胰岛素抵抗模型绘制肝脏mtDNA甲基化图谱，通过细胞和动物水平的基因操作阐明mtDNA甲基化调控线粒体稳态和肝脏胰岛素抵抗的分子机制，期望从线粒体表观遗传学角度揭示肝脏胰岛素抵抗形成的新机制，并为2型糖尿病的防治提供新靶点。

近年来表观遗传学在代谢紊乱相关疾病的研究中得到了广泛关注。线粒体作为细胞的能量工厂，其稳态失衡是导致胰岛素抵抗和代谢紊乱发生的关键因素，但线粒体DNA（mtDNA）甲基化能否调控线粒体稳态并参与胰岛素抵抗尚无报道。在本课题中，我们发现mtDNA甲基化能够通过调控线粒体稳态，参与肝脏胰岛素抵抗的发生。本课题取得的主要研究进展如下：（1）建立肝脏胰岛素抵抗条件下mtDNA甲基化修饰图谱，明确mtDNA甲基化能够通过抑制线粒体编码基因的转录进而调控线粒体稳态；（2）明确调控mtDNA甲基化的主要分子为AMPK/DNMT1，发现AMPK可能作用于DNMT1上游，通过促进DNMT1的线粒体转位调控mtDNA甲基化，进一步调节线粒体稳态和胰岛素抵抗的发生；（3）建立DNMT1和携带线粒体导肽的mtDNMT1过表达小鼠模型，发现DNMT1和mtDNMT1过表达小鼠肝脏中mtDNA甲基化升高，出现线粒体稳态失衡和胰岛素抵抗的表型，进一步验证了mtDNA甲基化调控线粒体稳态参与肝脏胰岛素抵抗的研究假设。.综上所述，本课题从线粒体表观遗传学角度揭示了线粒体稳态失衡参与肝脏胰岛素抵抗形成的新机制，并为2型糖尿病、肥胖、脂肪肝等代谢紊乱相关疾病的防治提供了新靶点。

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