NEUROPEPTIDE Y6 RECEPTORS ARE CRITICAL REGULATORS OF ENERGY METABOLISM Ernie Yulyaningsih A thesis in fulfilment of the requirements for the degree of Doctor of Philosophy St Vincent’s Clinical School Faculty of Medicine February 2014 Table of Contents GENERAL INTRODUCTION ....................................................................... 2 Obesity .......................................................................................................... 3 Regulators of energy homeostasis................................................................ 3 Neuropeptide Y ............................................................................................ 4 The Y1 receptor ......................................................................................... 6 The Y2 receptor ......................................................................................... 9 The Y4 receptor ....................................................................................... 16 The Y5 receptor ....................................................................................... 21 The Y6 receptor ....................................................................................... 25 Hypothesis and overall aims of the study .................................................. 28 EXPERIMENTAL PROCEDURES ............................................................. 29 Generation and genotyping of the Y6-/--LacZ mouse ................................ 30 Animal experiments ................................................................................... 31 RNA extraction .......................................................................................... 31 Reverse transcription PCR ........................................................................ 32 Analysis of body weight and composition ................................................. 33 Spontaneous feeding studies ...................................................................... 34 Fasting and re-feeding studies ................................................................... 35 Indirect calorimetry ................................................................................... 35 Glucose tolerance tests ............................................................................... 36 Tissue collection ......................................................................................... 36 Oil Red O staining ...................................................................................... 37 Serum assays .............................................................................................. 38 i ß-galactosidase staining ............................................................................. 38 Colocalization studies with VIP and AVP ................................................. 40 Analysis of diurnal variations in serum hormone levels ........................... 42 PP, PYY and PYY3-36 injection study ..................................................... 43 Statistical analyses ..................................................................................... 44 RESULTS ....................................................................................................... 46 Generation and validation of the Y6 knockout mouse model .................. 47 Y6-/- mice are viable and have normal fertility ......................................... 49 Y6-/- mice exhibit reduced body weight and lean mass and a late onset increase in adiposity ................................................................................... 51 Y6-/- mice displayed increased liver weight and increased hepatic lipid deposition ................................................................................................... 55 Reduced body weight and preferential fat accumulation in Y6-/- mice is independent of feeding behavior ............................................................... 57 Reduced body weight in Y6-/- mice could be explained by metabolic alterations ................................................................................................... 60 Y6 receptors regulate glucose homeostasis in mice .................................. 68 Y6-/- mice show exacerbated diet-induced obesity .................................... 72 Y6-/- mice showed normal serum testosterone levels ................................. 80 Y6 receptors are co-expressed in VIP neurons in the hypothalamic suprachiasmatic nucleus ............................................................................ 81 Y6-/- mice have reduced serum IGF-1 and blunted corticosterone rhythm .................................................................................................................... 84 DISCUSSION ................................................................................................. 89 FUTURE DIRECTIONS ............................................................................. 102 ii REFERENCES ............................................................................................ 105 iii LIST OF FIGURES FIGURE 1. Generation and validation of the Y6 knockout mouse model..…48 FIGURE 2. Y6-/- mice on a normal chow diet exhibit reduced body weight and lean mass and a late onset increase in adiposity………………...53 FIGURE 3. Y6-/- mice displayed increased liver weight and increased lipid deposition in the liver…………………………………………...56 FIGURE 4. Reduced body weight and preferential fat accumulation in Y6-/- mice on a normal chow diet is independent of feeding behavior…………………………………………………………58 FIGURE 5. Reduced body weight and reduced adiposity in 14-week-old Y6-/- mice on a normal chow diet could be explained by metabolic alterations……………………………………………………….62 FIGURE 6. Preferential fat accumulation in 23-week-old Y6-/- mice on a normal chow diet is independent of feeding behavior but could be explained by metabolic alterations……………………………...66 FIGURE 7. Impact of Y6 receptor deletion on glucose homeostasis in mice on a normal chow diet……………………………………………...70 FIGURE 8. Mice lacking Y6 receptors showed exacerbated diet-induced obesity……………………………………………………….......75 FIGURE 9. Effects of Y6 receptor deletion on food intake in mice maintained on a high-fat diet………………………………………………...77 FIGURE 10. Effects of Y6 receptor deletion on energy metabolism and physical activity in mice maintained on a high-fat diet…………78 FIGURE 11. Glucose metabolism was significantly impaired in Y6-/- mice fed a high-fat diet……………………………………………………..79 iv FIGURE 12. Deletion of Y6 receptors did not alter serum testosterone levels…………………………………………………………….80 FIGURE 13. Y6 receptors are expressed in VIP-expressing neurons in the hypothalamic suprachiasmatic nucleus…………………………82 FIGURE 14. Loss of Y6 receptor signalling is associated with reduced serum IGF-1 levels and blunted rhythms in serum corticosterone and light-phase feeding……………………………………………...85 FIGURE 15. Y6 receptors are activated by pancreatic polypeptide in vivo…..87 FIGURE 16. Schematic representation Y6 receptor signalling in the regulation of energy homeostasis…………………………………………100 v LIST OF TABLES TABLE 1. Genotype frequency, litter size, mortality and percentage of male offspring born from heterozygous (Y6+/-), homozygous (Y6-/-) and WT (Y6+/+) breeding pairs………………………………………50 TABLE 2. Resting metabolic rate in male Y6-/- and wildtype (WT) mice on a normal chow diet………………………………………………..64 vi LIST OF ABBREVIATIONS Abbreviation Description A α-MSH alpha melanocyte stimulating hormone AgRP Agouti related peptide ANOVA analysis of variance AP area postrema ARC arcuate (ARC) AVP vasopression-associated neurophysin B BAT brown adipose tissue BDNF brain-derived neurotrophic factor BSA bovine serum albumin C cAMP cyclic AMP CART cocaine- and amphetamine-regulated transcript CRH corticotropin-releasing hormone CV calorific value D DEPC diethylpyrocarbonate DMH dorsomedial hypothalamic nucleus DXA dual energy X-ray absorptiometry DVC dorsal vagal complex E e epididymal (white adipose tissues) ELISA enzyme-linked immunosorbent assay ES embryonic stem F FLS-ob/ob Shionogy ob/ob mice with fatty liver G G418 geneticin GABA γ-amino butyric acid GAPDH glyceraldehyde 3-phosphate dehydrogenase GHRH growth hormone releasing hormone Gi/Go inhibitory G-proteins GRH gonadotropin-releasing hormone H hPP human PP HPA hypothalamic-pituitary-adrenal axis vii I i inguinal (white adipose tissues) i.c.v. intracerebroventricular IGF-1 insulin-like growth factor 1 i.p. intraperitoneal i.v. intravenous K kcal kilocalories L LHA lateral hypothalamic area M m mesenteric (white adipose tissues) N neoR neomycin resistance NPY Neuropeptide Y NTS nucleus tractus solitarus P PBS phosphate-buffered saline PFA paraformaldehyde pNPY2-36 porcine NPY2-36 POMC pro-opiomelanocortin PP pancreatic polypeptide PVN paraventricular nuclei PYY peptide YY R r retroperitoneal (white adipose tissues) RER respiratory exchange ratio RIA radioimmunoassay rPP rat PP S SCN suprachiasmatic V VCO2 volume of carbon dioxide production VIP vasoactive intestinal peptide VMH ventromedial hypothalamus VO2 volume of oxygen consumption VPAC2 vasoactive intestinal peptide receptor 2 W WT wildtype viii Y Y6-/- Homozygous deletion of Y6 receptor Y6+/- Heterozygous deletion of Y6 receptors ix The contents of this thesis are a modified version of 2 manuscripts: British Journal of Pharmacology (163: 1170–1202, 2011) NPY RECEPTORS AS POTENTIAL TARGETS FOR ANTI-OBESITY DRUG DEVELOPMENT Ernie Yulyaningsih, Lei Zhang, Herbert Herzog and Amanda Sainsbury and Cell Metabolism (19: 58-72, 2014)