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, o...