Role of Group 1B Phospholipase A2 in Diet-induced Hyperlipidemia and Selected Disorders of Lipid Metabolism A dissertation submitted to the Graduate School at the University of Cincinnati in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Pathobiology and Molecular Medicine of the College of Medicine By Norris Isaac Hollie, II B.S., B.A. Oakwood University, Huntsville, Alabama July 23, 2013 Committee Chair: David Y. Hui, Ph.D. Abstract As obesity rates increase in developed nations, the prevalence of other obesity-related metabolic disorders such as hyperlipidemia, atherosclerosis, and non-alcoholic fatty liver disease (NAFLD) has also increased drastically. A greater understanding of the pathobiology and molecular mechanisms of these disorders becomes increasingly relevant. Recent studies demonstrated that certain alleles of the pancreatic group 1B phospholipase A2 (PLA2G1B) are associated with increased obesity and that Pla2g1b-deficient (Pla2g1b-/-) mice are protected against diet-induced obesity and diabetes. Furthermore, supplying mice with lysophosphatidylcholine (LPC), the enzymatic product of Pla2g1b, causes hyperglycemia and insulin resistance, which are risk factors for hyperlipidemia, atherosclerosis, and NAFLD. Thus, Pla2g1b may play a role in the development of these obesity-related diseases and inhibiting Pla2g1b and/or LPC may change the progression of these metabolic disorders. The goal of the first part of this dissertation was to identify a Pla2g1b-mediated protection against hyperlipidemia. Findings from these studies indicate that Pla2g1b-/- mice are protected against high fat diet-induced hyperlipidemia and gain less weight than Pla2g1b+/+ controls due to decreased very-low-density lipoprotein (VLDL) production and increased postprandial triglyceride-rich lipoprotein clearance. Further results indicate that supplying LPC stimulates VLDL production in both Pla2g1b-/- and Pla2g1b+/+ mice and that LPC is directly used as a substrate to make VLDL-triglyceride (TG). Taken together, these findings indicate that inhibition of luminal Pla2g1b may be a viable strategy to decrease hyperlipidemia in vivo. The goal of the second part of this dissertation was to identify a mechanism of LPC- mediated inhibition of hepatic oxidative function. Pla2g1b-/- mice have decreased plasma LPC and increased hepatic oxidation that is inhibited by intraperitoneal injection of LPC by an Page ii unknown mechanism. Findings indicate that though low micromolar concentrations of LPC decrease the mitochondrial membrane potential, oxidation rate remains equal to controls up to incubation with 80 µM LPC. However, when isolated mitochondria or hepatocytes are supplied with 100 µM LPC, decreased substrate-stimulated oxidation and increased mitochondrial permeability are observed. These findings indicate that LPC plays a major role in the maintenance of hepatic mitochondrial integrity and function and that low micromolar changes in intra- and extracellular LPC concentration can greatly affect hepatic oxidation rate. The goal of the third part of this dissertation was to identify a Pla2g1b-mediated protection against atherosclerosis and NAFLD. Findings indicate decreased obesity, VLDL-TG, VLDL-cholesterol, low-density lipoprotein-cholesterol, and hepatomegaly in Pla2g1b-/-/Ldlr-/- mice compared to Pla2g1b+/+/Ldlr-/- controls. Findings also suggested decreased atherosclerotic lesion size in Pla2g1b-/-/Ldlr-/- mice. Taken together, the findings from this dissertation have three implications in the field of metabolic research. First, these studies indicate a vital role for Pla2g1b and LPC in modulating hepatic function. Regardless of the Pla2g1b status of experimental animals, the mechanisms of LPC action appear intact. Secondly, these studies indicate that inhibition of Pla2g1b provides protection against diet-induced obesity in two diverse murine models. Thirdly, decreased VLDL- TG is described as a hallmark of inhibition of Pla2g1b. The development of a single drug to inhibit the action of PLA2G1B would likely lead to improvements in obesity, diabetes, hyperlipidemia, and potentially atherosclerosis. Page iii Copyright Notice This dissertation is based, in part, on a published manuscript. Part of this research was originally published in the Journal of Lipid Research. Hollie NI and Hui DY. Deficiency of group 1b phospholipase A2 protects against diet-induced hyperlipidemia. J Lipid Res. 2011; 52: (11) 2005-2011. © the American Society for Biochemistry and Molecular Biology. Page iv Acknowledgements This work was supported by Grant DK069967 from the National Institute of Diabetes and Digestive and Kidney Diseases of the National Institute of Health. The author was a fellowship recipient of grants F31HL110527 from the National Heart, Lung, And Blood Institute, Grant Number T32GM063483 from the National Institute of General Medical Sciences, and Grant Number 11PRE7310047 from the American Heart Association during the course of this study. The author would like to thank Dr. David Hui for allowing him to complete doctoral research in his laboratory. The author would also like to thank Kimberly Hollie, M.Ed. (wife) and Noah Hollie (son) for their support and understanding through this busy time. Page v Table of Contents List of Figures ............................................................................................................................... vii List of Abbreviations ..................................................................................................................... ix Chapter 1. Introduction ....................................................................................................................1 1. Significance 2. Three obesity-related disorders 3. Treatments for dyslipidemia 4. Normal lipid metabolism 5. Phospholipase A2 6. Metabolic effects of Pla2g1b Chapter 2. Materials and Methods .................................................................................................22 1. Animals 2. Special diets 3. Hepatic VLDL production 4. Postprandial lipid clearance 5. Plasma lipid determination 6. Mitochondrial isolation 7. Mitochondrial swelling 8. Mitochondrial membrane potential 9. Mitochondrial calcium uptake 10. Mitochondrial oxygen consumption 11. Cellular oxygen consumption 12. Hepatocyte viability and mitochondrial permeability transition 13. Determination of atherosclerosis 14. Liver characterization 15. Statistical analysis Chapter 3. Results ..........................................................................................................................39 1. Deficiency of Pla2g1b protects against diet-induced hyperlipidemia 2. Micromolar changes in LPC concentration cause minor effects on hepatic mitochondrial permeability but major alterations in function 3. Inhibition of Pla2g1b protects against selected disorders of lipid metabolism Chapter 4. Discussion ...................................................................................................................58 1. Overview of findings 2. Discussion of “Deficiency of Pla2g1b protects against diet-induced hyperlipidemia” 3. Discussion of “Micromolar changes in LPC concentration cause minor effects on hepatic mitochondrial permeability but major alterations in function” 4. Discussion of “Inhibition of Pla2g1b protects against selected disorders of lipid metabolism” 5. Clinical considerations for inhibition of Pla2g1b References ......................................................................................................................................74 Figures............................................................................................................................................87 Page vi List of Figures 1. Schematic of lipid metabolism through the gut-circulation-liver-adipose axis 2. Responses of Pla2g1b+/+ and Pla2g1b-/- mice to hypercaloric feeding 3. Hepatic VLDL production by Pla2g1b+/+ and Pla2g1b-/- mice 4. Effect of LPC supplementation on hepatic VLDL production by Pla2g1b-/- mice 5. FPLC analysis of 3H radioactivity distribution in plasma subsequent to [3H]LPC injection 6. Postprandial plasma lipid response to a bolus lipid-rich meal 7. Effect of LPC on mitochondrial swelling 8. Effect of LPC on CaCl2-induced mitochondrial swelling 9. Effect of LPC on mitochondrial Ca2+ uptake 10. Effect of LPC on mitochondrial membrane potential 11. Effect of LPC on mitochondrial respiration 12. Effect of LPC on fatty acid-stimulated oxidation in isolated hepatocytes 13. Effect of LPC on hepatocyte mitochondrial permeability in situ 14. Effect of LPC on hepatocyte cytoplasmic activity 15. Effects of diet on body weight of Pla2g1b+/+/Ldlr-/- and Pla2g1b-/-/Ldlr-/- mice 16. Effects of diet on glucose homeostasis in Pla2g1b+/+/Ldlr-/- and Pla2g1b-/-/Ldlr-/- mice 17. Effects of diet on lipoprotein profile of Pla2g1b+/+/Ldlr-/- and Pla2g1b-/-/Ldlr-/- mice 18. Total plasma lipid levels in Pla2g1b+/+/Ldlr-/- and Pla2g1b-/-/Ldlr-/- mice after high fat diet feeding 19. Effect of diet on liver weight of Pla2g1b+/+/Ldlr-/- and Pla2g1b-/-/Ldlr-/- mice 20. Liver histology in high fat fed Pla2g1b+/+/Ldlr-/- and Pla2g1b-/-/Ldlr-/- mice. Page vii 21. Lipid deposition in aortic arch and brachiocephalic artery of Pla2g1b+/+/Ldlr-/- and Pla2g1b-/-/Ldlr-/- mice 22. Atherosclerotic lesion in aortic root of Pla2g1b+/+/Ldlr-/- and Pla2g1b-/-/Ldlr-/- mice 23. Fed state plasma lipid levels in Pla2g1b+/+/Ldlr-/- and Pla2g1b-/-/Ldlr-/- mice after chronic Western