Accumulation and Metabolism of Neutral Lipids in Obesity

Accumulation and Metabolism of Neutral Lipids in Obesity By JOHN DAVID DOUGLASS A Dissertation submitted to the Graduate School-New Brunswick Rutgers, The State University of New Jersey in partial fulfillment of the requirements for the degree of Doctor of Philosophy Graduate Program in Nutritional Sciences written under the direction of Judith Storch and approved by ________________________ ________________________ ________________________ ________________________ ________________________ New Brunswick, New Jersey [January, 2014] ABSTRACT OF THE DISSERTATION Accumulation and Metabolism of Neutral Lipids in Obesity by John David Douglass Dissertation Director: Judith Storch The ectopic deposition of fat in liver and muscle during obesity is well established, however surprisingly little is known about the intestine. We used ob/ob mice and wild type (C57BL6/J) mice fed a high-fat diet (HFD) for 3 weeks, to examine the effects on intestinal mucosal triacylglycerol (TG) accumulation and secretion. Obesity and high-fat feeding resulted in higher levels of mucosal TG and markedly decreased rates of chylomicron secretion, accompanied by alterations in intestinal genes related to anabolic and catabolic lipid metabolism pathways. Overall, the results demonstrate that during obesity and a HFD, the intestinal mucosa exhibits metabolic dysfunction. There is indirect evidence that the lipolytic enzyme monoacylglycerol lipase (MGL) may be involved in the development of obesity. We therefore examined the role of MG metabolism in energy homeostasis using wild type and MGL-/- mice fed low-fat or high-fat diets for 12 weeks. Tissue MG species were profoundly increased, as expected. Notably, weight gain was blunted in all MGL-/- mice. MGL null mice were also leaner, and had increased fat oxidation on the low-fat diet. Circulating lipids levels were decreased in high fat-fed MGL-/- mice, as were the levels of several plasma peptides involved in energy homeostasis. Interestingly, MGL-/- mice had a blunted rate of intestinal TG secretion following an oral fat challenge. The leaner phenotype and improved metabolic serum profile in MGL-/- mice suggested that pharmacological inhibition may be a potential treatment for metabolic disease. To further examine this, C57BL6/J mice were fed low-fat and high-fat diets for 12 weeks, and then given daily oral administration of ii vehicle or a novel reversible MGL inhibitor for 4 days or 27 days. No changes in food intake were found, nor were adiposity or glucose intolerance substantially altered by inhibitor treatment; this is likely due to the short-term effectiveness of the inhibitor, as the compound was barely detectable 7 hours following administration. Thus, the effects of transient MGL inhibition on energy metabolism are minimal, in contrast to chronic inhibition secondary to genetic ablation. iii Acknowledgements I would like to foremost thank my advisor Dr. Judy Storch, whose guidance and acute editing skills I have relied upon for these past five years. I also thank my dissertation committee, Dr. Malcolm Watford, Dr. Dawn Brasaemle, Dr. Greg Henderson, and Dr. Marge Connelly, for their feedback and oversight of my research. My beloved Storch lab members, Yin Xiu, Sarala, Leslie, and Angela, must be thanked for contributing their expertise and patience towards the research and my training as a scientist. I would also like to thank the Nutritional Sciences Department and the Nutritional Sciences Graduate Program for providing the necessary structure and resources that made this dissertation possible. My gratitude goes to the Rutgers Center for Lipid Research and its members, who are united under the common goal of furthering lipid research in service of human health. I also thank our collaborators at Janssen R&D who allowed us to pursue novel research ideas and provided key analytical support. The seeds of higher education were planted while watching my father Dr. David Douglass tapping away on his own dissertation on a typewriter in our home basement. Inspiration for pursuing a career in science came from Dr. Dennis Winge and Dr. Lisa Joss-Moore. Finally, I owe a debt of gratitude to my dear wife Dr. Katherine Douglass, an exemplar for momentum in life, and whose ability to push through “analysis paralysis” and the tedious aspects of academia is nothing short of incredible. iv Table of Contents Abstract………………………………………….….….….….….………………………………………………….……..ii Acknowledgements…………….……………………………………………………………….…....…….………...iv List of Tables…………………….….……………………………………………………………….…....….…………..viii List of Figures………………….……………….………………………………………………………………..……..…ix List of Abbreviations……….………………………….…………………………….….….…….…….…….…......xi Page Chapter 1: Introduction and Review of the Literature 1 1. Introduction 2 2. Intestinal lipid metabolism 3 2.1 The Small Intestine 3 2.2 Digestion of Dietary Lipids 4 2.3 Uptake, Transport, and Absorption of Dietary Fatty Acids and Monoacylglycerol 6 2.4 Intracellular Lipid Metabolism in Enterocytes 7 3. Monoacylglycerols 13 3.1 Formation and Biosynthesis of MG 13 3.2 Anabolism and Catabolism of MG 13 4. Monoacylglycerol Lipase 17 4.1 Monoacylglycerol Lipase: From Gene to Regulation 17 4.2 Monoacylglycerol Lipase: Physiological Roles 21 4.3 Monoacylglycerol Lipase: Transgenic Models 22 4.4 Monoacylglycerol Lipase: Knockout Models 24 v 4.4 Monoacylglycerol Lipase: Pharmacological Inhibition 25 5. The Endocannabinoid System 27 5.1 Overview of the EC System 27 5.2 Components and Mechanisms of the System 27 5.3 Role in Energy Homeostasis 31 5.4 Dysregulation in Metabolic Disorders 33 6. Specific Aims of the Studies 34 Chapter 2: Intestinal mucosal triacylglycerol accumulation secondary to decreased 36 lipid secretion in obese and high fat-fed mice Abstract 37 Introduction 38 Materials and Methods 40 Results 47 Discussion 55 Chapter 3: Monoacylglycerol lipase ablation in mice alters energy homeostasis and 60 diet induced obesity Abstract 61 Introduction 62 Materials and Methods 65 Results 74 Discussion 89 vi Chapter 4: Short-term reversible pharmacological inhibition of monoacylglycerol 95 lipase in mice does not alter food intake or diet induced obesity Abstract 96 Introduction 97 Materials and Methods 100 Results 106 Discussion 117 Chapter 5: General Conclusions and Future Directions 120 Appendix: Metabolism of apically and basolaterally delivered fatty acids in the 128 intestinal mucosa of acyl coA synthetase 5-deficient mice Acknowledgement of Collaborative Efforts and Previous Publications 140 Literature Cited 144 vii List of Tables Chapter 2 Page Table 2-1. Diet Composition 41 Table 2-2. Primer sequences used for qRT-PCR analyses 45 Chapter 3 Table 3-1. Diet Composition 68 Table 3-2. Primer sequences used for RT-PCR analyses 72 Table 3-3. Plasma peptide levels in male mice as analyzed by 87 multiplex immunoassay Chapter 4 Table 4-1. Acute MGL inhibitor dosing protocol 102 Table 4-2. Chronic MGL inhibitor dosing protocol 103 viii List of Figures Chapter 1 Page Figure 1-1. Intestinal lipid absorption 12 Figure 1-2. Hydrolysis of MG by MGL 16 Figure 1-3. Three-dimensional structure of MGL 20 Figure 1-4. Over-expression of intestine MGL in mice (iMGL) depletes mucosal 23 MG levels and induces an obese, hyperphagic phenotype Figure 1-5. Overview of endocannabinoid system in neurons 30 Chapter 2 Figure 2-1. Weight and body fat of mouse models 48 Figure 2-2. Intestinal mucosa TG content in fed and 12 h fasted mice 49 Figure 2-3. Lipid accumulation in fed state proximal intestinal mucosa. 51 Figure 2-4. Relative quantitation of mRNA expression of lipid metabolic and 52 transport genes Figure 2-5. Oral fat tolerance tests 54 Chapter 3 Figure 3-1. Ablation of gene and function in MGL-/- mice 75 Figure 3-2. Tissue content of MG species as determined by HPLC-MS analysis 76 Figure 3-3. Tissue content of acylethanolamide (EA) species as determined by 77 HPLC-MS analysis Figure 3-4. Body weights during 12 weeks of LFD or HFD feeding 79 Figure 3-5. Body composition and tissue weights in MGL-/- mice 80 ix Figure 3-6. Energy consumption and utilization for male mice 81 Figure 3-7. Reduced appearance of TG from an oral fat bolus in the blood of 12 83 week HFD-fed male MGL-/- mice Figure 3-8. Oral glucose tolerance test. 84 Figure 3-9. Plasma lipids in 12 h fasted male mice after 12 weeks of LF or HF 85 feeding Figure 3-10. Tissue gene expression measured by RT-PCR and analyzed by ddCT 88 method. Chapter 4 Figure 4-1. Blood levels of MGL inhibitor JNJ-MGLi taken 1.5 and 7 h after oral 107 administration Figure 4-2. Tissue monoacylglycerol (MG) levels after 4 and 27 days of 108 compound administration Figure 4-3. Tissue acylethanolamide (EA) levels after 4 and 27 days of 109 compound administration Figure 4-4. Body weights during the 4 day (acute) and 27 day (chronic) 111 compound administration Figure 4-5. Body fat for the chronic study 112 Figure 4-6. Food intake during the 5-day (acute) and 28 day (chronic) 113 compound administration Figure 4-7. Food intake in each day of acute compound administration 114 Figure 4-8. Oral glucose tolerance test on day 24 in the chronic study 116 x List of Abbreviations 2-AG 2-arachidonoyl glycerol 2-AG-d8 Deuterated 2-arachidonoyl glycerol 2-MG sn-2-monoacylglycerol 2-OG 2-oleoyl glycerol 2-PG 2-palmitoyl glycerol 2-SG 2-stearoyl glycerol AA Arachidonic acid ABHD α-β hydrolase ACC1 Acetyl-CoA carboxylase 1 ACO Acyl-coA oxidase ACS Acyl-CoA synthetase ACSL Long chain acyl CoA synthetase AEA Arachidonoyl ethanolamide AEA-d8 Deuterated arachidonoylethanolamide AgRP

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