Novel FXR-dependent Molecular Mechanisms in the Regulation of Liver Metabolism

项目摘要/摘要 非酒精性脂肪性肝病(NAFLD)现在是最常见的肝病，影响到近三分之一 成年人的生活。从早期阶段，如脂肪变性到非酒精性脂肪性肝炎(NASH)，两者都需要 甘油三酯(TAG)蓄积与肝细胞损伤。纳什可以逆转，但有限 可供选择的治疗方法。最近的临床试验表明，以核受体FXR为靶点 治疗NASH的重大承诺。FXR激活可能是有益的，因为FXR被认为是 通过FXR SHP SREBP1C途径减少肝脏中的脂肪生成。在当前的应用程序中，我们 挑战这一范式。我们已经使用了有效的合成和特异性FXR激动剂GSK2324来表明 FXR的激活降低了以野生和西式饮食(WD)喂养的肝脏TAG水平和脂肪合成 类型，但不是FXR-/-小鼠。然后，我们发现GSK2324治疗降低了SHP-/-和SHP-/-和SHP-/-肝脏中TAG的水平 Srebp1c-/-小鼠，提示这两个基因不是FXR依赖的肝脏TAG降低所必需的 水平，至少在喂食食物的小鼠中是这样。我们已经使用脂组学分析表明，FXR激活导致选择性 特定标签种类的减少。我们还使用了体内标记技术来测量新合成的 脂类，并表明FXR激活选择性地改变了脂质的合成。基因表达分析支持和 解释了标签物种的这些特定变化，并使我们识别出两个关键的脂代谢mRNAs，它们是 用GSK2324治疗的野生型、SHP-/-和Srebp1c-/-小鼠的死亡率显著降低，但FXR-/-小鼠的降幅不明显。我们有 还使用了一种新的非侵入性方法来证明FXR的激活减少了饮食中脂肪的吸收。我们 现在已经设计了两个特定的目标来确定涉及肝脏减少的分子机制 FXR激活后的标签。我们将使用体内标记和脂组分析以及各种KO小鼠来 确定依赖FXR的肝脏改变是否需要肠道或肝脏FXR、SHP或Srebp1c 非酒精性脂肪性肝病模型中的脂肪合成和肠道脂肪吸收。我们还将确定分子 FXR激活后抑制两个关键脂代谢基因的机制。我们的 初步数据表明，我们最近发现的一个新的FXR靶基因，促进了 编码两个关键脂质基因的mRNAs。最后，我们将使用AAV依赖的这两个基因的表达， 为了确定过度表达是否可以单独或联合使用来防止肝脏标签种类的减少 在FXR激活之后。我们还将确定依赖FXR的脂肪吸收减少是否可以 通过增加胆汁酸池的大小来减弱。我们的研究将对这种机制提供新的见解。 通过激活FXR改变脂质代谢。这一点可能很重要，因为FXR激动剂目前 正被用于临床。此外，我们的发现表明，针对FXR下游的特定基因， 无论是单独的还是联合的，都可能为未来的治疗干预带来巨大的希望 NAFLD。

PROJECT SUMMARY/ABSTRACT Non-alcoholic fatty liver disease (NAFLD) is now the most prevalent liver disease, and affects almost one third of adults. Progression from early stages such as steatosis to non-alcoholic steatohepatitis (NASH) requires both the accumulation of triglyceride (TAG) and hepatocyte injury. NASH can be reversed, yet there are limited therapeutic options available. Recent clinical trials have shown that targeting the nuclear receptor FXR holds significant therapeutic promise to treat NASH. FXR activation can be beneficial because FXR is thought to decrease lipogenesis in the liver through an FXR SHP SREBP1C pathway. In the current application, we challenge this paradigm. We have used the potent synthetic and specific FXR agonist GSK2324 to show that FXR activation reduces both liver TAG levels and lipid synthesis in both chow and western diet (WD)-fed wild- type, but not Fxr–/– mice. We then show that GSK2324 treatment reduces TAG levels in the livers of Shp–/– and Srebp1c–/– mice, suggesting these two genes are not essential for the FXR-dependent decrease in hepatic TAG levels, at least in chow-fed mice. We have used lipidomic analysis to show that FXR activation results in selective decreases in specific TAG species. We have also used in vivo labeling techniques to measure newly synthesized lipids, and show that FXR activation selectively alters lipid synthesis. Gene expression analysis supports and explains these specific changes in TAG species, and led us to identify 2 key lipid metabolism mRNAs that are potently reduced in wild-type, Shp–/– and Srebp1c–/– mice but not Fxr–/– mice treated with GSK2324. We have also used a novel non-invasive method to show that FXR activation reduces absorption of dietary lipids. We have now designed two specific aims to determine the molecular mechanisms involved in the reduction of hepatic TAGs following FXR activation. We will use in vivo labeling and lipidomic analysis and various KO mice to determine whether intestinal or hepatic FXR, Shp or Srebp1c are required for FXR-dependent changes in hepatic lipid synthesis and intestinal lipid absorption in a model for NAFLD. We will also determine the molecular mechanism involved in the repression of the two key lipid metabolism genes following FXR activation. Our preliminary data suggest that a new FXR target gene, that we recently identified, promotes the degradation of mRNAs encoding the two key lipid genes. Finally, we will use AAV-dependent expression of these two genes, to determine if overexpression can, either alone or in combination, prevent the decrease in hepatic TAG species following FXR activation. We will also determine whether the FXR-dependent decrease in lipid absorption can be attenuated by increasing the bile acid pool size. Our studies will provide new insights into the mechanisms by which FXR activation alters lipid metabolism. This is likely to be important since FXR agonists are currently being used clinically. Further, our discoveries suggest that targeting specific genes that are downstream of FXR, either individually or in combination, may hold significant promise for future therapeutic intervention to treat NAFLD.