To Investigate the Physiological Role of Arcuate Nucleus Cocaine- and Amphetamine- Regulated Transcript in Energy Homeostasis. Thesis submitted for the degree of Doctor of Philosophy in Imperial College London Daniel Charles Campbell 2010 Department of Investigative Medicine Faculty of Medicine Imperial College London 1 For my mum and dad, brothers and sisters 2 Abstract Cocaine- and amphetamine- regulated transcript (CART) was originally identified as a mRNA transcript upregulated in rats in response to administration of cocaine and amphetamine. CART is widely expressed in the central nervous system (CNS) with high levels of expression in hypothalamic nuclei such as the arcuate nucleus (ARC). CART was initially thought to act as an anorectic peptide since it is coexpressed with the anorectic neuropeptide pro-opiomelanocortin (POMC) in the ARC. In addition, intracerebroventricular (ICV) administration of CART (55-102) peptide inhibits feeding and administration of anti-CART antibody results in stimulation of feeding. However, subsequent studies have suggested CART may also act as an orexigen since injection of CART (55-102) specifically into the ARC and ventromedial nucleus (VMN) of the hypothalamus results in a significant increase in food intake. These data suggest CART acts through both anorectic and orexigenic circuits. Given the importance of the hypothalamus in the regulation of energy homeostasis, and the role of the ARC in integrating peripheral signals, it is essential to elucidate the role of ARC derived CART. In order to elucidate CART’s true physiological role in the ARC I used a combination of genetic approaches. I generated a recombinant Adeno-associated virus (rAAV) expressing CART antisense (CART-AS) and a transgenic mouse model which utilises the POMC promoter to drive expression of CART-AS. In the transgenic CART-AS model mice exhibited a significantly higher body weight relative to control animals, no significant difference in food intake was observed. In addition, mice expressing the CART-AS transgene demonstrated a reduction in uncoupling protein-1 (UCP- 1) mRNA expression in brown adipose tissue (BAT) which is suggestive of decreased thermogenesis. This may explain the observed increase in body weight in the transgenic mice. Bilateral intra-ARC injections of rAAV-CART-AS resulted in a significant increase in cumulative food intake and body weight gain compared to control animals. There was no significant difference in activity or metabolism levels. The data presented in my thesis provides an important contribution to understanding the role of CART within the ARC. The results from my genetic studies appear to suggest that ARC derived CART has an anorectic role. 3 Declaration of Contributors The majority of the research and work described in this thesis was performed by the author. All collaboration and assistance is described below: Chapter 3: The pronuclear injection of transgene constructs was carried out by the MRC Transgenic Core Facility, Hammersmith Hospital. The radioimmunoassays in this chapter were carried out under the supervision of Professor Mohammad Ghatei and Dr Michael Patterson (Department of Investigative Medicine). Chapter 4: Competent bacteria and recombinant AAV were prepared by Dr. J. Gardiner (Department of Investigative Medicine). Studies involving the CLAMS were done with assistance from Dr. N. Semjonous and Dr J. Cooke (Department of Investigative Medicine). Injection of recombinant AAV into the arcuate nucleus was carried out in collaboration with Dr J. Cooke (Department of Investigative Medicine). The radioimmunoassays in this chapter were carried out under the supervision of Professor Mohammad Ghatei and Dr Michael Patterson (Department of Investigative Medicine). All in house radioimmunoassays were established and maintained by Professor M. Ghatei (Department of Investigative Medicine). 4 Acknowledgements I would like to give a special thanks to my supervisors, Dr Gavin Bewick and Dr James Gardiner for their tireless support, help and advice throughout my time in the laboratory and in reading this thesis. I am grateful to Professor Stephen Bloom for giving me the opportunity for working in his department and for providing funding. I would also like to thank all the members of the department for making my time in the laboratory such an enjoyable one. Finally, I would like to thank my family who have been an unending source of support and encouragement. 5 Abbreviations 5-HT 5-hydroxytryptamine 2-AG 2-Arachidonyl glycerol 2-DG 2-Deoxy-D-Glucose AAV Adeno-Associated Virus ACC Acetyl-CoA Carboxylase aCSF artificial Cerebrospinal Fluid ACTH Adrenocorticotrophic Hormone Ad Adenovirus AEA Anandamide Arg Arginine AgRP Agouti-related Protein AMP Adenosine Mono-Phopshate ANOVA Analysis of Variance AP Area Postrema ARC Arcuate Nucleus AS Antisense ATP Adenosine Triphosphate BAC Bacterial Artificial Chromosome BAT Brown Adipose Tissue BGH Bovine Growth Hormone BMI Body Mass Index bp base pairs BSA Bovine Serum Albumin CAC CART-Antisense-CART cAMP cyclic Adenosine Monophosphate CART Cocaine- and Amphetamine-Regulated Transcript CCK Choleycystokinin cDNA copy DNA CLAMS Comprehensive Laboratory Animals Monitoring System CMV Cytomegalovirus CNS Central Nervous System 6 CRH Corticotropin Releasing Hormone CRE cAMP Response Element CSF Cerebrospinal Fluid Cys Cysteine DA Dopamine DMEM Dubecco's Modified Eagle Medium DMN Dorsomedial Nucleus DMV Dorsal Motor Nucleus of the Vagus DNA Deoxyribonucleic Acid dNTP Deoxynucleotide Triphosphate dsRNA double stranded RNA DTT Dithiothreitol DVC Dorsal Vagal Complex EDTA Ethylenediaminetetraacetic acid eGFP Enhanced Green Fluorescent Protein ELISA Enzyme-Linked ImmunoSorbent Assay ES cell Embryonic stem cell FCS Fetal Calf Serum FMM Formamide GABA γ-Aminobutyric Acid GDW Glass Distilled Water GEE Generalised Estimation Equation GFP Green Fluorescent Protein GHRH Growth Hormone Releasing Hormone GHS-R Growth Hormone Secretagogue Receptor GIT Gastrointestinal Tract GLP-1 Glucagon-Like Peptide-1 GLUT Glucose Transporter Gly Glycine GnRH Gonadotropin-releasing Hormone GTE Glucose Tris EDTA GTT Glucose Tolerance Test HIV Human Immunodeficiency Virus HPT Hypothalamo-Pituitary Thyroid 7 ICV Intracerebroventricular IE gene Immediate early gene IgG Immunoglobulin G i.p. intra-peritoneal IPSC Inhibitory Post Synaptic Current IRS Insulin Receptor Substrate ITR Inverted Terminal Repeat ITT Insulin Tolerance Test JAK Janus Kinase K-ATP Potassium-ATP LB Lysogeny Broth LH Lateral Hypothalamus Lys Lysine MAPK Mitogen Activated Protein Kinase MCH Melanin- Concentrating Hormone MTII Melanotan II MOPS 3-(N-Morpholino)propanesulphonic Acid mRNA messenger RNA MSH Melanocyte Stimulating Hormone NAc Nucleus Accumbens NE Norepinephrine NPY Neuropeptide Y NSE Neurone Specific Enolase NTS Nucleus of the Solitary Tract OC Optic Chiasm OCT Optimum Cutting Temperature OD Optical Density pA Polyadenylation signal PAC P1- Derived Artificial Chromosome PBS Phosphate Buffered Saline PC Prohormone Convertase PCR Polymerase Chain Reaction PDX-1 Pancreatic Duodenal Homeobox 1 PEI Polyethylenimine 8 PeV Periventricular Nucleus PFA Perifornical Area PFC Prefrontal cortex Phe Phenylalanine PI3K Phosphatidylinositol-3-Kinase POMC Proopiomelanocortin PP Pancreatic Polypeptide PVN Paraventricular Nucleus PYY Peptide Tyrosine Tyrosine rAAV Recombinant AAV RER Respiratory Exchange Ratio RIA Radioimmunoassay RNA Ribonucleic Acid RNAi RNA interference RT Reverse Transcriptase SEM Standard Error of the Mean SN Substantia Nigra siRNA small interfering RNA SOB Super Optimal Broth STAT Signal Transducers and Activators of Transcription SSPE Saline Sodium Phosphate EDTA TAE Tris-Acetate EDTA TE Tris EDTA Tfam Mitochondrial Transcription Factor A TRH Thyrotropin Releasing Hormone tRNA Transfer RNA TSH Thyroid Stimulating Hormone Tyr Tyrosine UCP-1 Uncoupling Protein-1 VMN Ventromedial Nucleus VTA Ventral Tegmental Area WAT White Adipose Tissue WT Wildtype WPRE Woodchuck hepatitis virus Post-Transcriptional Regulatory Element 9 YAC Yeast Artificial Chromosome 10 Table of Contents 1. General Introduction ....................................................................................................... 22 1.1 Obesity ............................................................................................................................. 22 1.2 The Hypothalamus .......................................................................................................... 22 1.2.1 The Arcuate Nucleus ......................................................................................... 25 1.2.2 The Paraventricular Nucleus .......................................................................... 26 1.2.3 The Ventromedial Nucleus ............................................................................... 26 1.2.4 The Lateral Hypothalamus ............................................................................. 27 1.2.5 Brainstem .......................................................................................................... 28 1.2.6 Ventral Tegmental Area ................................................................................... 29 1.3 Neuropeptide Signalling .................................................................................................. 29 1.3.1 The Melanocortin System ................................................................................ 30 1.3.2 Neuropeptide Y ................................................................................................