TET1通过SIRT1-PGC1a/FOXO1 调控肝脏能量代谢的机制研究

Abnormal hepatic energy metabolism is an important cause of obesity, type II diabetes mellitus, and liver cancer. The SIRT1-PGC1/FOXO1 pathway plays a central role in hepatic energy metabolism via regulating the transcriptional expression of downstream metabolic genes in hepatic cells. It has found that Tet1 knockout mice showed a significant decrease in body size and body weight relative to normal mice. However, the mechanism by which Tet1 regulates these phenotypes of mice is not clear. In preliminary data, we found that: 1) NAD + levels in both the hepatic tissue of Tet1 deficient mice and in HepG2 hepatocytes after TET1 depletion were significantly reduced; 2) Mass spectrometry experiments showed that TET1 may interact with SIRT1; 3) Our data further revealed that the transcriptional expression of some genes related to metabolism exhibited a significant change in TET1-knockout HepG2 hepatocytes. Therefore, we hypothesize that TET1 can interact physically with SIRT1 and regulate the deacetylase activity of SIRT1 via regulating the level of NAD+ in the hepatic cells; Then, via a series of biochemical experiments, we will further prove that TET1 can regulate the acetylation of PGC1a and/or FOXO1 through modulating the deacetylase activity of SIRT1 in HepG2 hepatocytes, thus affecting the expression of metabolic genes and ultimately regulating the energy metabolism in hepatic cells. It will provide the evidence for understanding the role of TET1 in the energy metabolism and metabolic diseases, such as obesity, type II diabetes mellitus and liver cancer.

肝脏能量代谢异常是引起肥胖、II型糖尿病以及肝癌发生的重要因素因而被广泛关注。而SIRT1-PGC1/FOXO1通路在肝脏细胞能量代谢过程中起着核心作用。以前研究发现，Tet1敲除小鼠表现出体型以及体重相对于正常小鼠偏小的重要特征。然而，Tet1调控小鼠该表型的具体作用机制并不清楚。我们前期发现：1）TET1缺失会导致肝脏细胞NAD+水平明显降低；2）质谱实验发现TET1和SIRT1可能存在着相互作用；3）抑制TET1导致代谢相关的基因表达差异显著。据此，我们假设并验证TET1能够和SIRT1相互作用，且通过调节细胞内NAD+的水平而调控SIRT1去乙酰化酶活性，从而改变其下游转录复合物PGC1a和/或FOXO1的乙酰化水平，进而影响糖代谢相关基因的表达，最终调控肝脏能量代谢这一生物学过程。这一机制的阐明对于了解TET1在能量代谢以及肥胖、II型糖尿病等代谢相关疾病中的角色提供了证据。

以前的研究表明，TET1蛋白参与细胞发育、分化和转录调控的多种生物学功能成为研究的热点之一。在本研究中，我们首次研究了TET1在调节肝脏中的葡萄糖代谢的功能及其潜在机制。在小鼠实验中，我们观察到TET1缺失会影响常规饮食或高脂肪饮食小鼠血液中的葡萄糖代谢水平。随后，我们进一步揭示了肝脏TET1通过其C端结构与SIRT1进行相互作用并激活其去乙酰化酶活性，进而调控转录因子PGC1α和FOXO1的细胞定位，导致肝脏糖异生基因表达的激活。有趣的是，在TET1杂合小鼠肝脏中，禁食诱导的肝脏糖异生基因激活程序受到抑制。使用ΑMPK的激活剂二甲双胍能够显著提高小鼠肝脏糖异生基因的表达。. 具体来说，我们通过实验证明TET1通过其C末端与SIRT1特异性结合而激活SIRT1去乙酰化酶活性，使PGC1α和FOXO1去乙酰化以维持它们的核定位，从而激活包括PPΑRGC1Α、G6PC和SLC2Α4在内的肝糖异生相关基因的表达。进一步通过ΑMPK激活剂二甲双胍或ΑICΑR来模拟禁食证明其依赖于ΑMPK-TET1-SIRT1轴的激活而上调肝糖异生基因的表达。本研究不仅揭示了TET1在肝脏葡萄糖代谢基因转录调控中的新作用，而且揭示了TET1在肝脏葡萄糖代谢中依赖ΑMPK激活的潜在分子机制。综上，我们的研究表明，TET1是SIRT1蛋白的共激活因子，并通过分子细胞水平的实验证明ΑMPK-TET1-SIRT1调节轴可能为糖代谢及其相关代谢疾病的潜在治疗靶点。