Metabolic Regulation by Lipid Activated Receptors By Maxwell A Ruby A dissertation submitted in partial satisfaction of the requirements for the degree of Doctor of Philosophy In Molecular & Biochemical Nutrition In the Graduate Division Of the University of California, Berkeley Committee in charge: Professor Marc K. Hellerstein, Chair Professor Ronald M. Krauss Professor George A. Brooks Professor Andreas Stahl Fall 2010 Abstract Metabolic Regulation by Lipid Activated Receptors By Maxwell Alexander Ruby Doctor of Philosophy in Molecular & Biochemical Nutrition University of California, Berkeley Professor Marc K. Hellerstein, Chair Obesity and related metabolic disorders have reached epidemic levels with dire public health consequences. Efforts to stem the tide focus on behavioral and pharmacological interventions. Several hypolipidemic pharmaceutical agents target endogenous lipid receptors, including the peroxisomal proliferator activated receptor α (PPAR α) and cannabinoid receptor 1 (CB1). To further the understanding of these clinically relevant receptors, we elucidated the biochemical basis of PPAR α activation by lipoprotein lipolysis products and the metabolic and transcriptional responses to elevated endocannabinoid signaling. PPAR α is activated by fatty acids and their derivatives in vitro. While several specific pathways have been implicated in the generation of PPAR α ligands, we focused on lipoprotein lipase mediated lipolysis of triglyceride rich lipoproteins. Fatty acids activated PPAR α similarly to VLDL lipolytic products. Unbound fatty acid concentration determined the extent of PPAR α activation. Lipolysis of VLDL, but not physiological unbound fatty acid concentrations, created the fatty acid uptake necessary to stimulate PPAR α. Consistent with a role for vascular lipases in the activation of PPAR α, administration of a lipase inhibitor (p-407) prevented PPAR α dependent induction of target genes in fasted mice. Apolipoprotein CIII, an endogenous inhibitor of lipoprotein lipase, regulated access to the lipoprotein pool of PPAR α ligands. Our results support a role for the local generation of PPAR α ligands by lipase activity. The endocannabinoid system regulates diverse physiological functions, including energy balance. While loss of CB1 signaling has more pronounced effects on lipid parameters than expected based on weight loss alone, direct demonstration of endocannabinoid regulation of lipid metabolism is lacking. To test the effects of endogenously produced cannabinoids on lipid metabolism, independent of alterations in food intake, we analyzed tissues from mice treated with IDFP, an organophosphorus inhibitor of endocannabinoid breakdown. IDFP administration inhibited hepatic monoacylglycerol lipase leading to elevated levels of 2-arachidonylglycerol. We found that IDFP administration caused accumulation of apoE depleted VLDL. HDL particles accumulated apoE and failed to transfer it to VLDL in vitro. Importantly, these effects were prevented by pharmacological inhibition of CB1 and absent in plasma from CB1 mice. IDFP also caused a CB1-dependent increase in hepatic triglycerides. Thus, endocannabinoids inhibit the transfer of apoE from HDL to VLDL leading to apoE depletion of triglyceride rich lipoproteins. Microarray analysis allowed us to determine the effects of IDFP on expression of genes involved in lipid metabolism and to discover novel cannabinoid responsive genes. IDFP increased expression of lipogenic and SREBP2 target genes in a CB1-dependent fashion. On a global scale, pre-administration of a CB1 antagonist prevented many of the IDFP induced alterations in gene expression. IDFP decreased expression of genes involved in amino acid metabolism and inflammation. PCR analysis of selected mRNAs confirmed several of the key array findings. Our work indicates that endocannabinoids exert a large influence on hepatic lipid metabolism independent of food intake and suggest that peripherally restricted CB1 antagonists 1 may be of therapeutic value. Overall, these findings shed light on the endogenous mechanisms of PPAR α activation and the hepatic responses to Cb1 activation. This information may help guide the continuing effort to develop treatments for metabolic disease. 2 Acknowledgments First and foremost, I’d like to thank my graduate advisor Dr. Ronald Krauss. His constant support and patience has allowed me to grow as a scientist and as a person. Discussing science with Dr. Krauss reminds me of my reasons for coming to graduate school. His contiguous enthusiasm for science fueled all of the research I’ve done and will do. The collection of amazing individuals that make up the Krauss lab are a reflection of Dr. Krauss himself. Every member of the Krauss lab has been incredibly supportive and kind throughout these five years. I’d especially like to acknowledge Dr. Sally Chiu, Myra Gloria, Casey Geany and Katie Wojnoonski for their constant friendship. A special thanks to my office- mate, Dr. Lara Mangravite. You were a professional and personal compass when I was lost. Thanks to the entire lab! I feel very fortunate to have worked with many amazing collaborators during my time at Berkeley. Thanks to Dr. Daniel Nomura. Carolyn Hudak, Roger Issa, and Dr. John Casida for making “work” such a pleasant experience. Daniel you were a true mentor in the daily life of a scientist. Thanks to Dr. Jorge Plutzky for hosting me in Boston and allowing me to learn several novel techniques. Thanks to Dr. Thomas Johnston for all the conversations, scientific and otherwise. I also owe a debt of gratitude to my friends and family who have helped me navigate the occasionally rocky emotional terrain of graduate school. You guys are my heart and everything I do is yours. i TABLE OF CONTENTS CHAPTER 1 Review of the Literature 1 Section 1 Introduction 2 Section 2 PPAR α 3 Background & Discovery 3 Pharmacology 3 Downstream Effects 4 Regulation 4 The New Fat Hypothesis 6 Lipolysis of Stored Triglyceride 7 Lipolysis of Lipoproteins 7 Section 3 Cannabinoid Receptor 1 8 Background and discovery 8 Regulation 9 Pharmacology 10 Downstream Effects 11 References 13 CHAPTER 2 VLDL hydrolysis by LpL activates PPAR α through generation of unbound fatty acids 25 Abstract 26 Introduction 27 Methods 27 Results 29 Discussion 30 References 34 Figures and Tables 38 CHAPTER 3 Overactive endocannabinoid signaling impairs apolipoprotein E mediated clearance of triglyceride-rich lipoproteins 42 Abstract 43 Introduction 44 Methods 45 Results 46 ii Discussion 48 References 51 Figures and Tables 54 CHAPTER 4 Endogenous cannabinoid signalling induces insulin resistance and fatty liver: Identification of downstream targets 66 Abstract 67 Introduction 68 Methods 69 Results 69 Discussion 71 References 74 Figures and Tables 78 CHAPTER 5 Conclusions and future directions 98 iii CHAPTER 1 Review of the Literature 1 Introduction One of life’s most amazing feats is the maintenance of homeostasis in the face of constant flux. Consider that over the past year I have consumed over a million calories, and yet my body weight has shifted less than a pound, representing only a few thousand calories. However, it would be a mistake to assume my body has remained the same. The body is in constant flux, with perpetual turnover of nutrients and cells. For example, while my body fat mass may have remained constant, its composition has undoubtedly shifted to reflect the increase in dairy-derived saturated fatty acid induced by the introduction of a gourmet ice cream parlor in my neighborhood. Homeostatic mechanisms disguise the reality of flux so seamlessly that mass spectrometry is necessary to uncover it. What constitutes an acceptable error rate in biological homeostasis can best be judged by function. Many humans are experiencing a breakdown in their ability to maintain metabolic homeostasis that has serious functional consequences. Children today are not expected to outlive their parents for the first time in two centuries(1). This is largely attributed to increased prevalence of obesity and its associated diseases, which include two of the three leading causes of death, type II diabetes and heart disease. Has the marvelous machinery of our body been fundamentally modified in the past few decades? Probably not, even if epigenetic effects are considered. More likely the current environment, characterized by excess consumption of calories and lack of physical activity, overwhelms the regulatory mechanisms that are in place. Although chronic caloric excess likely did not exert large evolutionary pressure, the organism still displays remarkable ability to adapt to this environmental condition. For example, energy expenditure will increase in response to overfeeding. Similarly, pancreatic β-cell mass will increase in response to insulin resistance. The current epidemic of metabolic diseases, in spite of these adaptations, is testimony to the extreme conditions under which we have placed our bodies. Efforts to combat metabolic disease focus on altering these conditions and exploring the homeostatic mechanisms that they are assaulting. Driven by curiosity, I have chosen the latter for the past half decade. Aberrant lipid accumulation plays a crucial role in the pathogenesis of metabolic disease. For example, retention of lipoproteins in the sub-endothelial space is necessary for the development of atherosclerotic lesions. This knowledge, combined with a firm understanding of the homeostatic mechanisms governing cholesterol metabolism, improved prevention of cardiovascular