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

项目总结/摘要 非酒精性脂肪性肝病（NAFLD）现在是最常见的肝病，几乎影响三分之一 成年人。从早期阶段（如脂肪变性）进展为非酒精性脂肪性肝炎（NASH），需要 甘油三酯（TAG）蓄积和肝细胞损伤。NASH是可以逆转的，但有有限的 可用的治疗选择。最近的临床试验表明，靶向核受体FXR 对治疗NASH具有重要治疗前景。FXR激活可能是有益的，因为FXR被认为是 通过FXR SHP SREBP 1C途径减少肝脏中的脂肪生成。在本申请中，我们 挑战这个范式。我们已经使用有效的合成和特异性FXR激动剂GSK 2324来证明， FXR激活降低了普通饲料和西方饮食（WD）喂养的野生型大鼠的肝脏TAG水平和脂质合成。 型，但不是Fxr-/-小鼠。然后，我们表明GSK 2324治疗降低了Shp-/-小鼠肝脏中的TAG水平， Srebp 1c-/-小鼠，表明这两个基因对于肝脏TAG的FXR依赖性降低不是必需的。 水平，至少在Chow喂养的小鼠中。我们已经使用脂质组学分析表明，FXR激活导致选择性的 减少特定TAG种类。我们还使用了体内标记技术来测量新合成的 脂质，并显示FXR激活选择性地改变脂质合成。基因表达分析支持和 解释了TAG物种的这些特定变化，并使我们确定了2种关键的脂质代谢mRNA， 在野生型、Shp-/-和Srebp 1c-/-小鼠中有效降低，但在GSK 2324处理的Fxr-/-小鼠中未降低。我们有 还使用了一种新的非侵入性方法来证明FXR激活减少了膳食脂质的吸收。我们 现在设计了两个具体的目标，以确定参与肝细胞减少的分子机制， FXR激活后的TAG。我们将使用体内标记和脂质组学分析以及各种KO小鼠， 确定肝脏FXR依赖性变化是否需要肠道或肝脏FXR、Shp或Srebp 1c 脂质合成和肠脂质吸收的NAFLD模型。我们还将确定 FXR激活后两个关键脂质代谢基因的抑制机制。我们 初步数据表明，我们最近发现的一种新的FXR靶基因， 编码两个关键脂质基因的mRNA。最后，我们将使用这两个基因的AAV依赖性表达， 以确定过表达是否可以单独或联合预防肝TAG种类的减少， FXR激活后。我们还将确定FXR依赖性的脂质吸收减少是否能 通过增加胆汁酸池大小来减弱。我们的研究将提供新的见解的机制 FXR激活通过其改变脂质代谢。这可能是重要的，因为FXR激动剂目前 在临床上使用。此外，我们的发现表明，靶向FXR下游的特定基因， 单独或联合使用，可能对未来的治疗干预具有重要的前景， 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.