De Novo Phosphatidylcholine Synthesis in Intestinal Lipid Metabolism and Disease by John Paul Kennelly A thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Nutrition and Metabolism Department of Agricultural, Food and Nutritional Science University of Alberta © John Paul Kennelly, 2018 Abstract Phosphatidylcholine (PC), the most abundant phospholipid in eukaryotic cells, is an important component of cellular membranes and lipoprotein particles. The enzyme CTP: phosphocholine cytidylyltransferase (CT) regulates de novo PC synthesis in response to changes in membrane lipid composition in all nucleated mammalian cells. The aim of this thesis was to determine the role that CTα plays in metabolic function and immune function in the murine intestinal epithelium. Mice with intestinal epithelial cell-specific deletion of CTα (CTαIKO mice) were generated. When fed a chow diet, CTαIKO mice showed normal lipid absorption after an oil gavage despite a ~30% decrease in small intestinal PC concentrations relative to control mice. These data suggest that biliary PC can fully support chylomicron output under these conditions. However, when acutely fed a high-fat diet, CTαIKO mice showed impaired intestinal fatty acid and cholesterol uptake from the intestinal lumen into enterocytes, resulting in lower postprandial plasma triglyceride concentrations. Impaired intestinal fatty acid uptake in CTαIKO mice was linked to disruption of intestinal membrane lipid transporters (Cd36, Slc27a4 and Npc1l1) and higher postprandial plasma Glucagon-like Peptide 1 and Peptide YY. Unexpectedly, there was a shift in expression of bile acid transporters to the proximal small intestine of CTαIKO mice, which was associated with enhanced biliary bile acid, PC and cholesterol output relative to control mice. Gene expression profiling of small intestinal epithelial cells showed induction of transcripts linked to cellular proliferation and inflammation in CTαIKO mice relative to control mice. Colonic inflammation after loss of intestinal epithelial cell CTα was linked to increased intestinal permeability (as assessed by Fluorescein Isothiocyanate-Dextran gavage), invasion of the intestinal epithelium by microbes, and enhanced inflammatory cytokine secretion. Impaired ii intestinal barrier function in CTαIKO mice was mechanistically linked to induction of endoplasmic reticulum stress and depletion of goblet cells. Antibiotics and 4-phenylbutyric acid both partially ameliorated inflammatory cytokine secretion in CTαIKO mice, suggesting that microbes and endoplasmic reticulum stress are key drivers of the inflammatory phenotype in CTαIKO mice. In further support of a role for de novo PC synthesis in colonic barrier function, feeding C57BL/6J mice a choline-deficient diet increased their susceptibility to Citrobacter rodentium-induced colitis relative to mice fed sufficient dietary choline. In conclusion, de novo PC synthesis in the small intestinal epithelium is required for dietary lipid absorption under certain dietary conditions, and the re-acylation of biliary lyso-PC cannot compensate for loss of CTα under these conditions. Furthermore, CTα activity prevents invasion of the intestinal epithelium by microbes. Accordingly, disruption of CTα in intestinal epithelial cells induces spontaneous colitis in mice. Finally, an adequate supply of dietary choline, the essential nutritional substrate for CTα, is an important factor in protecting the colon from Citrobacter rodentium -induced inflammation. iii Preface This thesis is original work by John Paul Kennelly. The research project, of which this thesis is a part, received research ethics approval from the University of Alberta’s Institutional Animal Care Committee and is listed as ‘Dietary determinants of metabolic disorders’ with protocol number AUP00000175, July 2009. The contributions made by the candidate, John P. Kennelly, and the co-authors of these studies, are described below. A modified version of Chapter 1 has been published as J.N. van der Veen, J. P. Kennelly, S. Wan, J. E. Vance, D. E. Vance, and R. L. Jacobs. 2017. The critical role of phosphatidylcholine and phosphatidylethanolamine metabolism in health and disease. Biochim Biophys Acta 1859: 1558-1572. J. P. Kennelly, J.N van der Veen and S. Wan contributed equally to this work and shared first authorship. J. E Vance, D. E Vance and R.L Jacobs wrote and edited the manuscript. Chapter 3 has been published as J.P. Kennelly, J.N. van der Veen, R.C. Nelson, K. Leonard, R. Havinga, J. Buteau, F. Kuipers, and R.L. Jacobs. 2018. Intestinal De Novo Phosphatidylcholine Synthesis is Required for Dietary Lipid Absorption and Metabolic Homeostasis. JLR (1695-1708). J. P. Kennelly and R.L Jacobs designed experiments, interpreted data, and wrote the manuscript. J.N van der Veen , J. Buteau and F. Kuipers designed experiments and interpreted data. J.P. Kennelly, J.N. van der Veen, R.C. Nelson, K. Leonard and R. Havinga performed experiments. Chapter 4 is in preparation for publication as J.P. Kennelly, J.N. van der Veen, R.C. Nelson, J. Buteau and R.L. Jacobs. Spontaneous colitis in mice with intestinal epithelial cell-specific disruption of the phospholipid synthetic enzyme CTP: phosphocholine cytidylyltransferase-α. J. P. Kennelly and R.L. Jacobs designed experiments, interpreted data, and wrote the manuscript. J.N. van der Veen, R.C. Nelson and J. Buteau designed experiments and interpreted data. J.P. Kennelly, J.N. van der Veen, and R.C. Nelson performed experiments. iv Chapter 5 is in preparation for publication as T. Ju, J.P. Kennelly, R.L. Jacobs and B.P. Willing. Insufficient dietary choline aggravates disease severity in a mouse model of Citrobacter rodentium-induced colitis. T. Ju and J.P. Kennelly contributed equally to this work. T. Ju and J.P. Kennelly designed and performed experiments, interpreted data, and wrote the manuscript. R.L. Jacobs and B.P. Willing. designed experiments, interpreted data and edited the manuscript. v Acknowledgments I would like to thank my supervisor, Dr. René Jacobs, for providing me with an exciting project, outstanding mentorship and a supportive lab environment. I am also grateful to my supervisory committee, Dr. Richard Lehner and Dr. Donna Vine, for excellent mentorship and important contributions towards my project. I would like to recognize the excellent scientific support that I have received from Dr. Jelske van der Veen, Randy Nelson, Kelly-Ann Leonard, Nicole Coursen, Susanne Lingrell, Audric Moses, Amy Barr, Priscilla Gao, Wayne Wang, Dr. Kunimasa Suzuki and Rongjia Liu. I extend my thanks to Alberta Innovates and the Alberta Diabetes Institute for financial support during my program. I thank Stan, Bernie, John and Louise for support in pursuing graduate studies. vi Table of Contents CHAPTER 1: Introduction and literature review 1.1 Phospholipid metabolism.......................................................................................................... 1 1.1.1 Overview of phospholipids.................................................................................................... 1 1.1.2 PC biosynthesis by the CDP-choline pathway…................................................................... 2 1.1.3 PC biosynthesis by phosphatidylethanolamine N-methyltransferase ……………………....5 1.1.4 Phospholipid remodeling by Lands’ cycle…………………………………………………..6 1.1.5 PE biosynthesis……………………………………………………………………………...7 1.2 The subcellular roles of phosphatidylcholine...............................................................……….9 1.2.1 Phospholipids control de novo lipogenesis via regulation of sterol regulatory element-binding proteins (SREBPs)...........................................................................................................................9 1.2.2 Phospholipids in mitochondria……………………………………………………………..12 1.2.3 Phospholipids and lipid droplet formation……………………………………………...….14 1.2.4 Phospholipids and very-low density lipoprotein (VLDL) secretion……………………….17 1.2.5 Phospholipid remodeling and VLDL secretion....................................................................20 1.3 The physiological roles of PC and PE synthesis in mammals……………………………….21 1.3.1 Phospholipids in the liver……………….………………………..………………………...21 1.3.2 Phospholipids in skeletal muscle…………………..………………………………………22 1.4 Overview of the intestinal epithelium......................................................................................23 1.4.1 Intestinal epithelial cell types................................................................................................26 1.5 Overview of chylomicron formation and secretion.................................................................29 1.5.1 Dietary lipid digestion by lipases………............................................................................ .31 1.5.2 Intestinal fatty acid uptake: A role for phospholipid remodeling…………........................ 32 1.5.3 Intestinal cholesterol uptake................................................................................................ 34 1.5.4 Intestinal fatty acid transport…………………………………………………................... 35 1.5.5 Intestinal TG synthesis............................................................................…………………..35 1.5.6 Chylomicron formation at the ER of enterocytes…............................................................ 37 1.5.7 Chylomicron secretion into the lymphatics..........................................................................38 1.5.8