TMAO对机体葡萄糖和脂肪代谢的调控及分子机制

Type 2 diabetes has been reached as epidemics in modern society, while associated cardiovascular diseases cause death frequently, yet few drugs available for clinical treatment and prevention. Previous study revealed that knockdown of FMO3 improves hyperglycemia and atherosclerosis, associating with reduced hepatic FoxO1, one of the key transcriptional regulators involved in energy homeostasis. Our preliminary data show knockdown of FMO3 in vitro causes reduced FoxO1 protein, whereas TMAO (enzymatic product of FMO3) treatment increases FoxO1; in vivo, TMAO intervention worsens glucose intolerance, increases FoxO1 protein and its target gene levels of G6pc and Pck1. These data suggest that TMAO may regulate glucose metabolism via FoxO1. Meanwhile, we found that protein levels of SIRT1, p-AMPK, p-AKT, and p-FoxO1 are decreased after TMAO treatment. Taken together, we hypothesize that TMAO may regulate energy homeostasis via SIRT1 or AMPK mediated AKT-FoxO1 pathway. This project is designed to investigate the role of FoxO1 in mediating TMAO effects on glucose and lipid metabolism via using FoxO1 live-specific gene knockout mice; to specify the role of SIRT1 and AMPK in mediating TMAO effects on FoxO1 via using specific activators or siRNAs; finally, to detect TMAO effects on glucose and lipid metabolism via treating obese mice with FMO3 enzymatic inhibitor in vivo. Overall, we aim to clarify the effects and underlying mechanisms of FMO3/TMAO pathway in regulation of glucose and lipid metabolism, applying it as the novel molecular target for treatment of type 2 diabetes and its complications.

2型糖尿病呈广泛流行性趋势，并发的心血管疾病有高致死率，而防治药物相对缺乏。既往发现胰岛素抵抗小鼠FMO3蛋白降低后可显著改善高血糖和动脉粥样硬化，且下调糖脂代谢关键调控因子FoxO1蛋白。我们发现肝细胞FMO3沉默可降低FoxO1蛋白，而其酶活性产物TMAO则升高之；TMAO干预后小鼠糖耐量降低，肝脏FoxO1蛋白升高；我们同时发现TMAO可下调SIRT1蛋白，AMPK，AKT和FoxO1磷酸化水平。因此我们推测TMAO可能通过作用SIRT1或AMPK而影响AKT-FoxO1通路，进而调控肝脏糖脂代谢。本项目拟验证FoxO1在介导TMAO调控代谢的作用；明确SIRT1和AMPK在介导TMAO调控FoxO1方面作用；探讨FMO3酶活性抑制剂的干预对肥胖小鼠糖脂代谢影响。我们旨在研究FMO3/TMAO调控肝脏糖脂代谢的影响及其分子机制，为防治2型糖尿病及其并发症提供新的干预靶点。

氧化三甲胺(Trimetlylamine oxide，TMAO) 在调控代谢性疾病方面的作用引起广泛关注，但其在调控糖脂代谢紊乱的具体机制尚不清楚。本项目旨在探究TMAO调控糖脂代谢紊乱的具体分子机制：（1）首先利用白藜芦醇（Resveratrol，Res）抑制TMA生成菌以降低TMAO含量，发现Res可促进脂肪棕色化而改善肥胖小鼠糖脂代谢紊乱。此外，可以增加HUVEC细胞常氧和缺氧条件下FoxO1 (Forkhead box transcription factor O1)蛋白磷酸化及血管形成。在肢体缺血模型中，Res增加缺血骨骼肌中FoxO1磷酸化水平。另一方面，我们利用肠道菌的代谢产物GUDCA (Glycoursodeoxycholic acid) 降低Turicibacter丰度减少TMAO生成，发现GUDCA可改善ApoE-/-小鼠糖脂代谢紊乱，减少动脉粥样硬化斑块面积。通过上述实验证实TMAO对调控FoxO1蛋白改善代谢方面具有重要作用。（2）利用血管新生模型探究FoxO1介导FMO3/TMAO通路对代谢的调控作用，敲降FoxO1后HUVEC成管能力增加，但Res不再发挥作用。过表达FoxO1后，HUVEC成管能力降低，与Re共处理后可使成管能力增加。表明Res可通过调控FoxO1改善细胞代谢，提高细胞血管新生能力。（3）为探究TMAO调控 FoxO1的具体分子机制，我们在HUVEC细胞中发现AKT抑制剂干预可降低Res诱导的FoxO1磷酸化。此外，Res激活内脏脂肪中Sirt1表达，结合既往发现Res可以激活缺血骨骼肌Sirt1表达并降低FoxO1表达。据此推测TMAO 可通过AKT通路或Sirt1发挥调控 FoxO1蛋白的功能作用，进一步在肥胖小鼠中发现敲降Sirt1也可削弱Res改善代谢、促进脂肪棕色化的能力。这些研究结果为FMO3/TMAO通路调控糖脂代谢紊乱的分子机制提供了理论依据，为2型糖尿病及并发症的防治提供新的干预靶点。