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Probing the Role of Pparα in the Small Intestine

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Probing the Role of Pparα in the Small Intestine Probing the role of PPARα in the small intestine A functional nutrigenomics approach Meike Bünger Promotor Prof. dr. Michael Müller Hoogleraar Nutrition, Metabolism and Genomics Humane Voeding, Wageningen Universiteit Co-promotor Dr. Guido J.E.J. Hooiveld Universitair docent Humane Voeding, Wageningen Universiteit Promotiecommissie Prof. dr. Jaap Keijer Wageningen University Prof. dr. Ulrich Beuers University of Amsterdam Prof. dr. Hannelore Daniel Technical University of Munich Prof. dr. Ivonne Rietjens Wageningen University Dit onderzoek is uitgevoerd binnen de onderzoeksschool VLAG. Probing the role of PPARα in the small intestine A functional nutrigenomics approach Meike Bünger Proefschrift ter verkrijging van de graad van doctor op gezag van de rector magnificus van Wageningen Universiteit, Prof. dr. M.J. Kropff, in het openbaar te verdedigen op vrijdag 12 september 2008 des namiddags te vier uur in de Aula. Meike Bünger. Probing the role of PPARα in the small intestine: A functional nutrigenomics approach. PhD Thesis. Wageningen University and Research Centre, The Netherlands, 2008. With summaries in English and Dutch. ISBN 978-90-8504973-9 Abstract Background The peroxisome proliferator-activated receptor alpha (PPARα) is a ligand- activated transcription factor known for its control of metabolism in response to diet. Although functionally best characterized in liver, PPARα is also abundantly expressed in small intestine, the organ by which nutrients, including lipids, enter the body. Dietary fatty acids, formed during the digestion of triacylglycerols, are able to profoundly influence gene expression by activating PPARα. Since the average Western diet contains a high amount of PPARα ligands, knowledge on the regulatory and physiological role of PPARα in the small intestine is of particular interest. Aim In this thesis the function of PPARα in the small intestine was studied using a combination of functional genomics experiments, advanced bioinformatics tools, and dietary intervention studies. Results Detailed analyses on the expression of PPARα in small intestine showed that PPARα is most prominently expressed in villus cells of the jejunum, coinciding with the main anatomical location where fatty acids are digested and absorbed. Genome-wide transcriptome analysis in combination with feeding studies using the synthetic agonist WY14643 and several nutritional PPARα agonists revealed that PPARα controls processes ranging from fatty acid oxidation and cholesterol-, glucose- and bile acid metabolism to apoptosis and cell cycle. In addition, we connected PPARα with the intestinal immune system. In a more focussed study we showed that PPARα controls the barrier function of the intestine. By comparing the intestinal and hepatic PPARα transcriptome we found that PPARα controls in these two organs the expression of two distinct, but overlapping sets of genes. Finally, by performing a range of functional studies deduced from the transcriptome analysis, we demonstrated that PPARα controls intestinal lipid absorption. Conclusion By maximally utilizing the unique possibilities offered in the post-genome era, the studies described in this thesis reported on the function of PPARα in small intestine. We conclude that intestinal PPARα plays an important role, is relevant for nutrition, and its effects are distinguishable from the hepatic PPARα response. Our results provide a better understanding of normal intestinal physiology, and may be of particular importance for the development of fortified foods, and prevention and therapies for treating obesity and inflammatory bowel diseases. Aim and outline of this thesis The small intestine plays a critical role in nutrition, since it is the primary site of food digestion and nutrient absorption. Another function is to prevent the translocation of bacteria and foreign antigens to extra-intestinal sites by forming a selective barrier. It has been demonstrated that the transcription factor PPARα can be activated by natural fatty acids and their activated derivatives (acyl-CoA esters) [1-4]. PPARα is expressed in a variety of tissues including the small intestine [5, 6]; however, its function has been almost exclusively studied in liver. Little is known about PPARα and PPARα target genes in non-hepatic tissues. Knowledge on the regulatory and physiological function of PPARα in the small intestine is of particular interest, since the average Western diet contains a high amount of triacylglycerols [7] that are hydrolyzed to monoacylglycerol and free fatty acids before entering the enterocyte [8]. Consequently the small intestine is frequently exposed to high levels of PPARα agonists, and therefore an important functional role of this transcription factor may be envisioned. The aim of the research described in this thesis was therefore to characterize the function of PPARα in the small intestine, with special emphasis on nutritional relevance, utilizing a nutrigenomics approach. In chapter 1 an overview of the literature is given concerning the gastrointestinal tract, the concept of nutrigenomics research, the peroxisome-proliferator-activated receptors (PPARs), and the technology used in this thesis. The results of published microarray studies on PPARα- dependent gene regulation performed in different organs are also summarized in this first chapter. In chapter 2 the genome-wide effect of PPARα activation in the small intestine is reported, with focus on intestinal epithelial cells. The commonalities and differences of PPARα dependent gene regulation in small intestine and liver was the subject of the studies described in chapter 3. In chapter 4 and 5 we focus on two main functions of the small intestine, the nutrient absorption (chapter 5) and the selective barrier function (chapter 4). In chapter 4 the effect of PPARα- activation by three different fatty acids and the synthetic compound WY14643 is examined, with emphasis on effects on intestinal transporters and phase I/II metabolic enzymes. In chapter 5 the effect of PPARα activation on intestinal dietary lipid absorption is investigated. Finally, in chapter 6 the general discussion, conclusions and recommendations are presented. Contents Abstract 5 Aim and outline of this thesis 7 1 General introduction 10 2 Genome-wide analysis of PPARα activation in murine small intestine 32 3 Organ-specific function of PPARα as revealed by gene expression profiling 58 4 PPARα-mediated effects of dietary lipids on intestinal barrier gene expression 82 5 PPARα regulates intestinal lipid absorption 102 6 General discussion 126 Appendix 132 References 134 Summary of this thesis 144 Samenvatting 148 Dankwoord 154 Curriculum Vitae 160 List of publications 162 Educational programme 164 1 General introduction Apated from: Meike Bünger, Guido Hooiveld, Sander Kersten and Michael Müller, “Exploration of PPAR functions by microarray technology – a paradigm for nutrigenomics”,Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids, Volume 1771, Issue 8, pages 1046-1064. PMID: 17632033 The gastrointestinal tract: function and anatomy The gastrointestinal tract is the system of organs which serves two main functions — assimilation of nutrients and elimination of waste. The gut anatomy is organized to serve these functions. A schematic overview of the gastrointestinal tract and accessory organs is given in Figure 1. The gastrointestinal tract starts with the mouth, followed by pharynx, esophagus, stomach, small intestine, colon and ends with the rectum (Figure 1). Hence, anatomically the small intestine is localized in-between stomach and colon and is directly connected with the accessory digestive organs, pancreas and liver via small ducts attached to the upper part of the small intestine. Figure 1. The diges- tive tract. (Adapted from Campbell NA, 1987, Biol- ogy, the Benjamin/Cum- mings Publishing compa- ny, Inc, Redwood City.) The physical and chemical digestion of foods starts in the mouth by chewing and the salivary enzyme amylase. This mixture is conducted to the stomach via the esophagus, where it is mixed with the acidic gastric juice for further degradation. The acid environment in the stomach also kills most bacteria that might have entered together with the food. Proteins are hydrolyzed into 12 polypeptides by the protease pepsin in the stomach. Most of the enzymatic hydrolysis of lipids and other food components occurs in the small in- testine. However, pancreas and liver both contribute to this process. The enzymes secreted by the exocrine pancreatic tissue help break down carbohydrates, fats, and proteins in the small intestine. The liver has a wide variety of functions, among others to produce bile to assist in the digestion of food. Both pancreas and liver release their contents via the greater duodenal papilla (comprised of the ampulla of Vater and the sphincter of Oddi) into the upper small intestine, the duodenum. Next to digestion and nutrient absorption a healthy gut incorporates another vital function. By forming a selective barrier the translocation of bacteria and other foreign antigens to extra-intes- tinal sites is prevented. 1 General introduction This barrier function is part of the innate immune system. Together with the host response, which is triggered through the activation of specific transcription factors controlling chemokine and cy- tokine expression, both innate and adaptive defense mechanisms protect the body against patho- gens. An overview of small intestinal anatomy is shown in Figure 2. The mucosa is the inner lining
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