PPARa: master regulator of lipid metabolism im mouse and human : identification of hepatic PPARa target genes by expression profiling

Abstract

The peroxisome proliferator activated receptor alpha (PPARα) is a ligand activated tran- scription factor involved in the regulation of a variety of processes, ranging from inflam- mation and immunity to nutrient metabolism and energy homeostasis. PPARα serves as a molecular target for hypolipidemic fibrates drugs which bind the receptor with high affinity. Furthermore, PPARα binds and is activated by numerous fatty acids and fatty acid derived compounds. PPARα governs biological processes by altering the expression of large number of target genes. Although the role of PPARα as a gene regulator in liver has been well estab- lished, a comprehensive overview of its target genes has been missing so far. Additionally, it is not very clear whether PPARα has a similar role in mice and humans and to what extent target genes are shared between the two species. The aim of the research presented in this thesis was to identify PPARα-regulated genes in mouse and human liver and thereby further elucidate hepatic PPARα function. The applied nutrigenomics approaches are mainly expression microarrays combined with knockout mouse models and in vitro cell culture systems. By combining several independent nutrigenomics studies, we generated a comprehensive overview of PPARα-regulated genes in liver with the focus on lipid metabolism. We identi- fied a large number of PPARα target genes involved in different aspects of lipid metabolism. Furthermore, a major role of PPARα in lipogenesis was detected. Our data pointed to several novel putative PPARα target genes. Next, we compared PPARα-regulated genes in primary mouse and human hepatocytes treated with the PPARα agonist Wy14643 and generated an overview of overlapping and species specific PPARα target genes. A large number of genes were found to be regulated by PPARα activation in human primary hepatocytes, which iden- tified a major role for PPARα in human liver. Interestingly, we could characterize mannose binding lectin 2 (Mbl2) as a novel human specific PPARα target gene. Plasma Mbl2 levels were found to be changed in subjects receiving fenofibrate treatment or upon fasting. Regula- tion of Mbl2 by PPARα suggests that it may play a role in regulation of energy metabolism, although additional research is needed. We also compared the PPARα-induced transcriptome in HepG2 cells versus primary human hepatocytes to investigate the suitability of HepG2 cells in PPARα research. The results re- vealed that the HepG2 cell line poorly reflects the established PPARα target genes and func- tion, specifically with respect to lipid metabolism. Finally, we characterized the transcription factors Klf10 and Klf11 as novel PPARα target genes. Our preliminary findings using in vitro transfection assays and in vivo tail vein injection of plasmid DNA suggested a potential metabolic role of Klf10 and Klf11 in liver. In conclusion, this thesis has extended our understanding of PPARα-regulated genes and function in liver, and has specifically highlightened a major role of PPARα in human hepa- tocytes. This research has also given birth to a possible biomarker of hepatic PPARα activity which is of great interest for future studies. Considering the need for proper biomarkers in the field of nutrigenomics and beyond, the properties of Mbl2 as a biomarker should be further investigated. The identification of other novel putative PPARα target genes offers ample op- portunities for continued research