| The orphan nuclear receptor hepatocyte nuclear factor 4 alpha (HNF4alpha) is a member of the nuclear receptor superfamily and a key regulator of development and metabolism. Since its initial cloning in 1990, HNF4alpha has been shown to regulate a variety of key enzymes that control various metabolic pathways. Furthermore, point mutations in HNF4alpha have been associated with maturity onset diabetes of the young-1 (MODY1), a rare type of adult onset type 2 diabetes mellitus. Due to the role that it plays in development and disease, HNF4alpha has long been considered a viable drug target. However, despite extensive study, the true endogenous ligand for HNF4alpha has not yet been identified.;In Chapter 2 of this study, we use Affinity Isolation/Mass Spectrometry (AIMS) to demonstrate that full-length HNF4alpha binds to a single fatty acid, linoleic acid (LA) in mammalian cells. LA is an essential fatty acid that mammals obtain from their diet. We also applied the AIMS assay to HNF4alpha purified from livers of mice under various nutritional states to demonstrate that the HNF4alpha-LA status is dependent of feeding state and that LA is an exchangeable ligand for HNF4alpha. To our knowledge, this is the first example of identification of an endogenous ligand for a native nuclear receptor purified from intact tissue.;In Chapter 3, we explore the biological relevance of the HNF4alpha-LA interaction. Addition of exogenous LA failed to stimulate HNF4alpha-mediated transactivation of two known HNF4alpha targets in mammalian cells. Using expression profiling analysis, we identified HNF4alpha target genes that are responsive to LA in the presence and absence of the transcriptional coregulator peroxisome proliferator activated receptor gamma coactivator 1 alpha (PGC1alpha). In general, LA down regulated the expression of a majority of genes activated by HNF4alpha, regardless of PGC1alpha. However the presence of LA seems to promote the degradation of HNF4alpha, possibly accounting for the decrease. These data identify several new HNF4alpha targets and establish that LA may serve as a potential HNF4alpha antagonist, although the exact mechanism is still under investigation.;Finally, in Chapter 4, we summarize our main findings and examine the role of the mammalian HNF4alpha LBD in the context of other species and explore evolutionary changes in the LBD that may effect ligand selectivity and/or function. |
