Tacrolimus介导Calcineurin/CRTC2通路调控移植肝糖代谢稳态的作用机制研究

We have demonstrated that new-onset diabetes after liver transplantation (NODALT) is a very common complication in Chinese liver transplant population and is closely associated with poor prognosis. Tacrolimus (TAC) is the most widely used immunosuppressive agent after liver transplantation and has been considered as the predominant risk factor for NODALT. However, the underlying mechanism of TAC-induced imbalance of glucose metabolism homeostasis has not been clearly elucidated yet. The current studies mainly focused on its toxicity in pancreatic beta cells. In our previous reports, we have revealed that donor liver plays a central role in the glucose metabolism homeostasis. We proposed that donor liver may act as a possible origin of NODALT. Furthermore, we found that TAC could target donor liver calcineurin (CaN) and induce imbalanced hepatic glucose metabolism homeostasis. The newly identified CREB regulated transcription coactivator 2 (CRTC2) is regulated by CaN and plays a key role in hepatic glucose and lipid metabolism. Therefore, we hypothesize that "TAC may regulate hepatic glucose metabolism homeostasis through the CaN/CRTC2 signaling pathway in liver transplantation". We plan to conduct in vitro and in vivo studies to observe the impact of TAC on CRTC2 transcription, translation, phosphorylation and targets expression (e.g. PEPCK, IRS2, SREBP1); to evaluate the role of hepatic CaN/CRTC2 signaling pathway in TAC-induced imbalance of glucose metabolism homeostasis; to investigate the effect of "dual strike" from both transplant surgical stress and TAC on hepatic glucose metabolism. The study will clarify the underlying mechanism of TAC-induced NODALT from the view of donor liver, and provide molecular targets for disease prevention and treatment.

我们发现肝移植术后新发糖尿病(NODALT)在中国人群中发病率高且预后差。免疫抑制剂tacrolimus(TAC)被认为是NODALT的重要诱因，但机制未明，目前研究集中于其胰岛损伤作用。我们前期研究表明移植肝在NODALT发病中起重要作用，TAC能抑制肝钙调磷酸酶(CaN)活性，诱发肝糖代谢稳态失衡。新近发现的CRTC2受CaN调控，是肝脏糖脂代谢重要调节子。因此我们提出“TAC介导CaN/CRTC2通路调控移植肝糖代谢稳态”这一科学假设，拟从细胞和动物模型层面观察TAC对肝CRTC2转录、翻译、磷酸化水平及其下游分子(PEPCK、IRS2、SREBP1等)表达的影响，评估肝CaN/CRTC2通路在TAC诱发糖代谢稳态失衡中的作用，利用小鼠肝移植模型探索移植手术和TAC双重打击对肝糖代谢稳态的影响。本研究将从新的视角阐述TAC诱发NODALT的可能机制，为疾病防治提供分子靶点。

免疫抑制剂他克莫司(TAC)被认为是移植后新发糖尿病（NODAT）的重要诱因，但机制未明。我们发现TAC通过直接影响肝脏代谢稳态而促进NODAT。在小鼠中，tac通过抑制糖异生基因诱导饥饿期间低血糖而不是高血糖，提示空腹血糖在NODAT诊断中的局限性。此外，TAC分别通过胰岛素受体底物(IRS)2/AKT和固醇调节元件结合蛋白(SREBP1)信号通路引起肝脏胰岛素抵抗和甘油三酯积累。此外，我们发现creb调节的转录共激活因子2 (CRTC2)在tac诱导的代谢紊乱中发挥了关键作用。肝脏CRTC2的恢复通过其下游分子(如PCK1、IRS2和SREBP1)减轻代谢紊乱。与实验结果一致，移植肝细胞中CRTC2的低表达是NODAT的独立危险因素。将CRTC2评分纳入临床模型可显著提高预测能力。该研究将从新的视角阐述TAC诱发NODALT的可能机制，为疾病防治提供分子靶点。进一步利用小鼠肝移植模型，我们探索了移植肝代谢稳态重塑过程。首先通过单细胞技术，绘制了移植肝单细胞图谱，发现从术前到术后稳定期96%的移植物来源的免疫细胞被受体来源的细胞所取代。更重要的是，我们发现在术后稳定期CD206+MerTK+巨噬细胞和CD49a+CD49b-自然杀伤细胞仍由移植物和受体双重来源组成。巨噬细胞-肝细胞相互作用分析显示CD206+MerTK+巨噬细胞通过特有的交互模式调控肝细胞糖脂代谢稳态。该研究描绘了移植肝的四维细胞图谱(类型-比例-来源-时间)，并阐明了巨噬细胞-肝细胞交互模式在移植肝代谢重塑中的重要作用。本项目共发表SCI论文12篇，会议论文4篇 ，培养研究生3名，国际大会报告3人次，获得国际肝移植协会1项奖励。