Choline Transport and Metabolism in Genetically Deficient and Chronic Disease States by Laila Cigana Schenkel A Thesis presented to The University of Guelph In partial fulfilment of requirements for the degree of Doctor of Philosophy in Human Health and Nutritional Sciences Guelph, Ontario, Canada © Laila Cigana Schenkel, August, 2014 ABSTRACT CHOLINE TRANSPORT AND METABOLISM IN GENETICALLY DEFICIENT AND CHRONIC DISEASE STATES Laila Cigana Schenkel Advisor: University of Guelph, 2014 Marica Bakovic Choline is required for the biosynthesis of phosphatidylcholine (PC) by the CDP-choline Kennedy pathway and of betaine. Choline also plays a role in lipid metabolism and hepatic and muscle function. The availability of intracellular choline is regulated by the choline transporter CTL1/SLC44A1 at the plasma membrane. This thesis aims to elucidate the effect of metabolic altered states, such as lipid overload, choline deficiency and CDP:phopshoethanolamine cytidylyltransferase (Pcyt2) genetic modified models, on the choline transport and metabolism. First, we investigated the effect of high fatty acid supply in C2C12 muscle cells. Palmitic acid (PAM) reduced total and plasma membrane CTL1/SLC44A1 protein by activating lysosomal degradation, and limited the choline uptake while increasing diacylglycerol (DAG) and triacylglycerol (TAG) synthesis. Oleic acid (OLA) maintained total and plasma membrane CTL1/SLC44A1, increasing PC and TAG synthesis more than PAM, which offers a protection mechanism from the excess of intracellular DAG and autophagy. We next characterized the choline metabolic defect in fibroblast cells isolated from a Postural Tachycardia Syndrome (POTS) patient, who had low plasma choline. In the POTS fibroblasts, the CTL1/SLC44A1 expression and choline uptake were decreased. PC synthesis and the phospholipid pool were reduced these cells compared to control. Triacylglycerol formation increased 50% in POTS, which is a feature of choline deficiency. The characteristics of the POTS fibroblasts represent a model of choline transport dysfunction . The role of choline in protecting against lipid accumulation and metabolic disease development was elucidated by choline supplementation of Pcyt2 +/- mice. In Pcyt2 +/- mice the CDP- ethanolamine pathway is downregulated and the TAG formation increased, resulting in adult- onset obesity and liver steatosis. Choline supplementation reverses the Pcyt2 +/- phenotype by facilitating the metabolic flux through the CDP-choline pathway, reducing TAG synthesis and increasing expression of genes involved in TAG and DAG degradation. The cross-regulation between CDP-choline and CDP-ethanolamine pathways was also demonstrated in Pcyt2 siRNA knock-down human fibroblasts (KD5), where the rate of choline uptake and PC synthesis were higher. Altogether, this thesis established the metabolic links between CDP-choline and CDP-ethanolamine pathways and provided a mechanism of how choline could be an important modifier of lipid metabolism under various conditions. iv ACKNOWLEDGEMENTS First I would like to thank my advisor Dr Marica Bakovic for the opportunity to work in her Lab and for her guidance, patience and friendship. I appreciate your support and willingness to teach over the years. I further like to thank my advisory committee, Dr Graham Holloway and Dr Fred Brauer for their guidance during this process. The collaboration of various laboratories made this thesis possible. Thank to Linda Groocock for helping with the mice work. Thank to Dr Steven Zeisel, Dr Kerry-Ann da Costa, Dr Amy Johnson and Dr Harvey Mudd for the collaboration on the POTS project and to Genevieve Anna Tyrrell for the inspiration. Thank to Michael Leadley and the Analytical Facility for Bioactive Molecules at the Sick Kids Centre and to Audric Moses and the Women and Children's Health Research Institute at the University of Alberta in assisting with the spectroscopy analysis. Also, thank to Junzeng Zhang, Institute for Nutrisciences and Health National Research Council Canada, for the NMR analysis. To my friends in the Bakovic lab, thank you for the help and the good times. Specifically, to Dr Ratnesh Singh, Zvezdan Pavlovic, Poulami Basu and Sugas Sivanesan. Lastly, thank to my family, who supported my decision to move so far away to do my PhD and who always gives me strength to succeed. And thanks to destiny for showing my path and bringing my love. v TABLE OF CONTENTES ACKNOWLEDGEMENTS …………………………………………………....……………...…iv TABLE OF CONTENTES …………………………………………………………………….....v LIST OF TABLES ……………………………………………………………………………….ix LIST OF FIGURES…………………………………………………………………………...…..x ABREVIATIONS……………………………………………………………………...………...xii 1 INTRODUCTION .................................................................................................................. 1 1.1 Choline: an essential nutrient ........................................................................................... 1 1.2 Absorption and transport of choline ................................................................................. 3 1.3 Betaine production and methylation pathway .................................................................. 6 1.4 The membrane phospholipids .......................................................................................... 9 1.4.1 Phospholipids in the plasma membrane .................................................................... 9 1.4.2 Mitochondrial phospholipids and their role in bioenergetics ................................. 10 1.4.3 The Kennedy pathway and the biosynthesis of the major membrane phospholipids in the ER ............................................................................................................................... 13 1.4.4 The biosynthesis of mitochondrial phospholipids .................................................. 18 1.4.5 Remodeling of phospholipids and the role of dietary lipids ................................... 20 1.4.6 Degradation of phospholipids ................................................................................. 23 2 Rationale for the studies........................................................................................................ 26 3 Palmitic acid and oleic acid differentially regulate choline transporter-like 1 levels and glycerolipid metabolism in skeletal muscle cells ......................................................................... 29 3.1 Introduction .................................................................................................................... 30 3.2 Materials and Methods ................................................................................................... 32 3.2.1 Cell culture and treatments ..................................................................................... 32 3.2.2 Cell viability............................................................................................................ 32 3.2.3 RT-PCR................................................................................................................... 33 3.2.4 Cell lysates and subcellular fractionation ............................................................... 34 3.2.5 Immunoblotting....................................................................................................... 34 3.2.6 Assays for protein degradation and autophagy ....................................................... 36 3.2.7 Choline uptake ........................................................................................................ 36 3.2.8 Radiolabeling of the CDP-choline pathway with [ 3H-methyl]choline ................... 37 3.2.9 Choline efflux ......................................................................................................... 38 3.2.10 Radiolabeling of DAG and TAG with [ 3H]glycerol ............................................... 38 vi 3.2.11 Measurement of total lipids .................................................................................... 39 3.2.12 Mitochondrial potential ........................................................................................... 39 3.2.13 Statistical analysis ................................................................................................... 40 3.3 Results ............................................................................................................................ 40 3.3.1 Differential effects of OLA and PAM on cell growth and CTL1/SLC44A1 protein content and localization ........................................................................................................ 40 3.3.2 Mechanisms for reduction of total CTL1/SLC44A1 protein content by PAM ...... 45 3.3.3 OLA and PAM alter the cell surface choline uptake diferently.............................. 46 3.3.4 OLA and PAM both reduce the mitochondrial choline uptake .............................. 48 3.3.5 OLA stimulates the CDP-choline pathway but not PC levels ................................ 48 3.3.6 PAM and OLA differently modulate TAG and DAG synthesis and content ......... 51 3.3.7 Lipid content in untreated and treated myotubes .................................................... 52 3.4 Discussion ...................................................................................................................... 54 4 Mechanism of choline deficiency and membrane alteration in postural tachycardia syndrome primary skin fibroblasts................................................................................................ 60 4.1