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

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

Comparative Analysis of Gene Regulation by the Transcription Factor PPARα between Mouse and Human

Studies in mice have shown that PPARalpha is an important regulator of hepatic lipid metabolism and the acute phase response. However, little information is available on the role of PPARalpha in human liver. Here we set out to compare the function of PPARalpha in mouse and human hepatocytes via analysis of target gene regulation

Differential gene expression in mouse primary hepatocytes exposed to the peroxisome proliferator-activated receptor α agonists

BACKGROUND: Fibrates are a unique hypolipidemic drugs that lower plasma triglyceride and cholesterol levels through their action as peroxisome proliferator-activated receptor alpha (PPARα) agonists. The activation of PPARα leads to a cascade of events that result in the pharmacological (hypolipidemic) and adverse (carcinogenic) effects in rodent liver. RESULTS: To understand the molecular mechanisms responsible for the pleiotropic effects of PPARα agonists, we treated mouse primary hepatocytes with three PPARα agonists (bezafibrate, fenofibrate, and WY-14,643) at multiple concentrations (0, 10, 30, and 100 μM) for 24 hours. When primary hepatocytes were exposed to these agents, transactivation of PPARα was elevated as measured by luciferase assay. Global gene expression profiles in response to PPARα agonists were obtained by microarray analysis. Among differentially expressed genes (DEGs), there were 4, 8, and 21 genes commonly regulated by bezafibrate, fenofibrate, and WY-14,643 treatments across 3 doses, respectively, in a dose-dependent manner. Treatments with 100 μM of bezafibrate, fenofibrate, and WY-14,643 resulted in 151, 149, and 145 genes altered, respectively. Among them, 121 genes were commonly regulated by at least two drugs. Many genes are involved in fatty acid metabolism including oxidative reaction. Some of the gene changes were associated with production of reactive oxygen species, cell proliferation of peroxisomes, and hepatic disorders. In addition, 11 genes related to the development of liver cancer were observed. CONCLUSION: Our results suggest that treatment of PPARα agonists results in the production of oxidative stress and increased peroxisome proliferation, thus providing a better understanding of mechanisms underlying PPARα agonist-induced hepatic disorders and hepatocarcinomas

Advances in understanding the regulation of apoptosis and mitosis by peroxisome-proliferator activated receptors in pre-clinical models: relevance for human health and disease

Peroxisome proliferator activated receptors (PPARs) are a family of related receptors implicated in a diverse array of biological processes. There are 3 main isotypes of PPARs known as PPARα, PPARβ and PPARγ and each is organized into domains associated with a function such as ligand binding, activation and DNA binding. PPARs are activated by ligands, which can be both endogenous such as fatty acids or their derivatives, or synthetic, such as peroxisome proliferators, hypolipidaemic drugs, anti-inflammatory or insulin-sensitizing drugs. Once activated, PPARs bind to DNA and regulate gene transcription. The different isotypes differ in their expression patterns, lending clues on their function. PPARα is expressed mainly in liver whereas PPARγ is expressed in fat and in some macrophages. Activation of PPARα in rodent liver is associated with peroxisome proliferation and with suppression of apoptosis and induction of cell proliferation. The mechanism by which activation of PPARα regulates apoptosis and proliferation is unclear but is likely to involve target gene transcription. Similarly, PPARγ is involved in the induction of cell growth arrest occurring during the differentiation process of fibroblasts to adipocytes. However, it has been implicated in the regulation of cell cycle and cell proliferation in colon cancer models. Less in known concerning PPARβ but it was identified as a downstream target gene for APC/β-catenin/T cell factor-4 tumor suppressor pathway, which is involved in the regulation of growth promoting genes such as c-myc and cyclin D1. Marked species and tissue differences in the expression of PPARs complicate the extrapolation of pre-clinical data to humans. For example, PPARα ligands such as the hypolipidaemic fibrates have been used extensively in the clinic over the past 20 years to treat cardiovascular disease and side effects of clinical fibrate use are rare, despite the observation that these compounds are rodent carcinogens. Similarly, adverse clinical responses have been seen with PPARγ ligands that were not predicted by pre-clinical models. Here, we consider the response to PPAR ligands seen in pre-clinical models of efficacy and safety in the context of human health and disease

Cross-species gene expression analysis of species specific differences in the preclinical assessment of pharmaceutical compounds

Animals are frequently used as model systems for determination of safety and efficacy in pharmaceutical research and development. However, significant quantitative and qualitative differences exist between humans and the animal models used in research. This is as a result of genetic variation between human and the laboratory animal. Therefore the development of a system that would allow the assessment of all molecular differences between species after drug exposure would have a significant impact on drug evaluation for toxicity and efficacy. Here we describe a cross-species microarray methodology that identifies and selects orthologous probes after cross-species sequence comparison to develop an orthologous cross-species gene expression analysis tool. The assumptions made by the use of this orthologous gene expression strategy for cross-species extrapolation is that; conserved changes in gene expression equate to conserved pharmacodynamic endpoints. This assumption is supported by the fact that evolution and selection have maintained the structure and function of many biochemical pathways over time, resulting in the conservation of many important processes. We demonstrate this cross-species methodology by investigating species specific differences of the peroxisome proliferatoractivator receptor (PPAR) a response in rat and human

Exploring the activation and function of PPARa and PPARß/d using genomics

For many tissues fatty acids represent the major source of fuel. In the past few decades it has become evident that in addition to their role as energy substrates, fatty acids also have an important signaling function by modulating transcription of genes. An important group of transcription factors involved in mediating the effects of dietary fatty acids on gene transcription are the Peroxisome Proliferator-Activated Receptors (PPARs). PPARs are members of the superfamily of nuclear hormone receptors and regulate genes involved in numerous important biological processes, ranging from lipid metabolism to inflammation and wound healing. In the liver the dominant PPAR isoform has been show to be PPARα, although PPARβ/δ and PPARγ are expressed in liver as well. The aim of this thesis was to further characterize the role of PPARα and PPARβ/δ in hepatic metabolism and study their activation by fatty acids. Even though PPARα as gene regulator in liver has been well described, a complete overview of its target genes has been lacking so far. By combining several nutrigenomics tools, we succeeded in creating a comprehensive list of PPARα-regulated genes involved in lipid metabolism in liver. Additionally, by using a unique design where mice were fed synthetic triglycerides consisting of one type of fatty acid, we could distinguish between different types of dietary unsaturated fatty acids in their ability to activate PPARα. Although it is well known that PPARα plays an important role in liver during fasting, no direct in vivo evidence exists that circulating free fatty acids are able to ligand activate hepatic PPARα. In our studies, we found that upregulation of gene expression by PPARβ/δ is sensitive to circulating plasma free fatty acids whereas this is not the case for PPARα. Not much is known about the function of PPARβ/δ in the liver. In order to better understand the role of this nuclear receptor, we compared the effects of PPARα and PPARβ/δ deletion on whole genome gene regulation and plasma and liver metabolites. Our results revealed that PPARβ/δ does not mediate an adaptive response to fasting, and pointed to a role for PPARβ/δ in hepatic glucose- and lipoprotein metabolism. In conclusion, this thesis contributes to the important work of mapping the molecular mechanisms dictating lipid metabolism in the liver. By using several nutrigenomics tools, we are able to show that PPARα is a key mediator of the effect of dietary fatty acids on hepatic gene expression. In addition, we better define the roles of PPARα and PPARβ/δ in hepatic metabolism and provide a new concept for functional differentiation between PPARs in liver. <br/

Hepatic PPARs: their role in liver physiology, fibrosis and treatment

Complex molecular and cellular mechanisms are involved in the pathway of liver fibrosis. Activation and transformation of hepatic stellate cells (HSCs) are considered the two main reasons for the cause and development of liver fibrosis. The peroxisome proliferator-activated receptors (PPARs) belonging to the family of ligand-activated transcription factors play a key role in liver homeostasis, regulating adipogenesis and inhibiting fibrogenesis in HSCs. Normal transcriptional function of PPARs contributes to maintain HSCs in quiescent phase. A reduced expression of PPARs in HSCs greatly induces a progression of liver fibrosis and an increased production of collagen. Here, we discuss role and function of PPARs and we take into consideration molecular factors able to reduce PPARs activity in HSCs. Finally, although further validations are needed, we illustrate novel strategies available from in vitro and animal studies on how some PPARs-agonists have been proved effective as antifibrotic substances in liver disease

Peroxisome Proliferator-Activated Receptor Delta (PPARD) : Molecular studies of regulation and activation

The peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors involved in energy homeostasis. Their natural ligands are fatty acids and there are three different PPAR isoforms; PPARA, PPARG and PPARD. They are encoded by separate genes and have distinct functions, due to different tissue expression and affinity for ligands. PPARA controls genes involved in fatty acid oxidation, PPARG regulates genes important for fatty acid storage, and PPARD controls genes implicated in lipid oxidation and lipoprotein metabolism. Primates and humans treated with a PPARD agonist (GW501516) resulted in improved insulin sensitivity, increased HDL and decreased LDL cholesterol levels, making it a putative drug candidate for treatment of metabolic disease. PPARD has recently been assigned a beneficial role in macrophages, by inducing a switch from proinflammatory (M1) to antiinflammatory (M2) macrophages. To characterize additional target genes of PPARD involved in the lipoprotein metabolism, the effect of PPARD activation on the apolipoprotein A-II (apoA-II) gene was investigated in human hepatoma cells. ApoA-II is one of the major proteins in the HDL particles. Treatment with GW501516 increased apoA-II promoter activity and mRNA levels in hepatoma cell lines. A site located at -737/-717 in the promoter was identified as the functional PPAR response element (PPRE). These results suggest that increased expression of the apoA-II gene is one of the reasons for the beneficial effects on lipoprotein metabolism after treatment with the PPARD agonist. To investigate whether PPARs could regulate the alanine aminotransferase (ALT) genes, the effect of PPARA, G and D agonist treatment was studied. ALT activity in plasma is used as a marker for hepatotoxicity in humans. During a clinical trial with the PPARA ligand, AZD4619, the plasma ALT activity increased in some patients and in vitro studies showed that ALT1 protein and mRNA expression was induced by treatment with PPARA agonists in primary hepatocytes. Similarly, transient transfection of a promoter construct of ALT1 in HuH-7 cells showed increased activity mediated via a PPRE located at -574 after treatment with PPAR agonists. This study shows that the ALT1 gene is regulated by PPARs and that PPAR drugs might contribute to increased ALT activity in serum. To explore regulation of the PPARD gene by posttranscriptional events, 5'- and 3'-RACE were performed on cDNA obtained from placenta, adipose tissue and pancreas. Both 5'- and 3'-alternative splicing of PPARD was identified. Coupled transcription/translation showed that the length and number of upstream AUGs in the 5'-UTR had a major impact on translational efficiency. Further, the promoter located upstream of exon one was verified as the major promoter, using reporter gene assays. A 3'-splice variant encoding a truncated PPARD protein, PPARD2, was shown to be a negative regulator of the full length receptor, PPARD1, in transient transfection assays. To identify whether PPARD is regulated by microRNA (miRNA), the 3'-UTR was analysed in silico. Two putative miRNA target sites were identified in the PPARD 3'-UTR; miR-9 and miR-29. The miR-9 was verified as a functional miRNA targeting PPARD. However, PPARD mRNA levels remained unaffected by miR-9 expression, indicating that only the translation of PPARD was inhibited. Since both miR-9 and PPARD have been shown to play important roles in the inflammatory response of monocytes, the regulation of PPAR expression by miR-9 was investigated in these cells. A suppressive role of miR-9 on PPARD expression was identified in monocytes after LPS treatment but not in M1 or M2 macrophages, suggesting that the regulatory role of miR-9 on PPARD is exerted in monocytes, before differentiating into macrophages. In summary, this thesis describes additional functions and ways of regulation of the ubiquitously expressed transcription factor PPARD with a major role in both health and disease