DIFFERENTIALLY EXPRESSED GENES IN ADIPOSE TISSUE AND THEIR ROLE IN THE PATHOPHYSIOLOGY OF THE HUMAN METABOLIC SYNDROME Differenziell exprimierte Gene im Fettgewebe und ihre Rolle in der Pathophysiologie des humanen Metabolischen Syndroms Von der Fakultät für Biowissenschaften, Pharmazie und Psychologie der Universität Leipzig genehmigte D I S S E R T A T I O N zur Erlangung des akademischen Grades Doctor rerum naturalium Dr. rer. nat. vorgelegt von M.Sc. Dorit Schleinitz geboren am 21.03.1981 in Karl-Marx-Stadt Dekan: Prof. Dr. Matthias Müller Gutachter: Prof. Dr. Annette Beck-Sickinger Prof. Dr. Michael Stumvoll Tag der Verteidigung: 07.01.2011 ...und Alice fragte die Katze: „Wohin soll ich gehen?“ & die Katze antwortet: „Das kommt immer ein bisschen darauf an, wo du hinwillst...“ (Alice im Wunderland) BIBLIOGRAPHY 3 Bibliography M.Sc. Dorit Schleinitz Differentially Expressed Genes in Adipose Tissue and their Role in the Pathophysiology of the Human Metabolic Syndrome University of Leipzig, Faculty of Biology, Pharmacy and Psychology Dissertation 142 Pages, 241 References, 14 Figures, 26 Tables, Appendix The human metabolic syndrome is characterized by a heterogenic complex of symptoms, including central obesity. Obesity itself is linked to major features of the metabolic syndrome such as insulin resistance, dyslipidemia or type 2 diabetes mellitus. It has been shown that obesity risk and resulting metabolic alterations are associated with adipose tissue distribution, adipocyte size and secretion of adipocytokines, which are in turn influenced by environmental factors and genetic susceptibility. It might be assumed that currently known genetic variants associated with obesity and/or BMI (body mass index) as well as fat distribution explain up to 20 % of the variability in BMI and so, studies employing novel strategies are inevitable. In addition to the role of genetic variation, mRNA levels of several genes have been shown to be differentially expressed in subcutaneous (SC) and visceral (Vis) adipose tissue and to be correlated with obesity-related traits. It is scarcely investigated whether the obesity risk variants also might account for the variability in mRNA expression. The present thesis deals with novel obesity candidate genes, characterized by a differential mRNA expression in various fat depots. The association of genetic variants in these genes with obesity as part of the metabolic syndrome, and related traits was investigated in well characterized German cohorts. The main method used for genotyping was described in detail in a comprehensive review providing explicit troubleshooting and description of modified protocols for specific experimental needs. Further, the influence of genotypes on the gene expression levels was examined. While the differential expression for FTO could be described for the first time, the variant rs8050136 was shown to be significantly associated with obesity but not with the expression. Genetic variants in FASN were shown to be significantly associated with obesity and related traits in a cohort of European ancestry for the very first time. Moreover, one polymorphism showed effects on the ratio of Vis/SC FASN mRNA expression. While CNR1 is controversially discussed in the literature, the present work showed rather moderate effects of genetic variants on obesity. BMPR2 could be described as a novel obesity candidate gene. Amongst others, one variant was associated with obesity in a case-control design and with BIBLIOGRAPHY 4 BMPR2 mRNA expression in Vis adipose tissue. In conclusion, the present study revealed novel genetic variants promoting obesity, and therefore a metabolic risk, which might be partly explicable through an influence of these variants on the mRNA expression levels of the genes in the adipose tissue depots. These findings contribute to better understanding of the genetic background of obesity which is essential in order to translate experimental data into diagnostic, preventive and treatment strategies. TABLE OF CONTENT 5 TABLE OF CONTENT BIBLIOGRAPHY 3 TABLE OF CONTENT 5 ABBREVIATIONS 9 SUMMARY 12 ZUSAMMENFASSUNG 17 CHAPTER 1 23 Introduction to the topic 23 1.1 Human Metabolic Syndrome 23 1.2 Obesity 23 1.2.1 Adipose tissue – developmental origin and its distribution in the human body 24 1.2.2 Fat depots and their relation to the human metabolic syndrome 26 1.3 Genetics of obesity and adipose tissue distribution 28 1.3.1 Genetics of obesity 28 1.3.2 Genes and gene expression pattern of adipose tissue distribution 29 1.4 Aims of the presented work 31 CHAPTER 2 33 Targeted SNP Genotyping Using the TaqMan® Assay 33 2.1 Abstract 34 2.2 Introduction 34 2.3 Materials 37 2.3.1 Target DNA 37 2.3.2 Plastics 37 2.3.3 Genotyping reaction 37 2.3.4 Instruments and software 37 2.4 Methods 38 2.4.1 Preparation of samples to perform SNP genotyping assays 38 2.4.2 Preparation of master mix for SNP genotyping 38 2.4.3 Cycling program for genotyping reaction 39 2.4.4 Plate readout – allelic discrimination 39 2.5 Notes 41 2.6 References 43 2.7 Contribution of Authors 44 TABLE OF CONTENT 6 CHAPTER 3 45 Inverse Relationship between Obesity and FTO Gene Expression in Visceral Adipose Tissue in Humans 45 3.1 Abstract 46 3.2 Introduction 46 3.3 Methods 47 3.3.1 Participants and measurement of participant characteristics 47 3.3.2 Assays and measures of glucose metabolism and body fat content 48 3.3.3 Analysis of human FTO and RPGRIP1L mRNA expression 48 3.3.4 Genotyping of the rs8050136 FTO variant 49 3.3.5 Statistical analyses 49 3.4 Results 49 3.4.1 Participant characteristics 49 3.4.2 Association of FTO expression 51 3.4.3 Adipose tissue FTO mRNA expression in function of rs8050136 53 3.5 Discussion 54 3.6 Acknowledgements 56 3.7 Duality of interest 56 3.8 References 56 3.9 Contribution of Authors 58 CHAPTER 4 59 Effect of Genetic Variation in the Human Fatty Acid Synthase Gene (FASN) on Obesity and Fat Depot-Specific mRNA Expression 59 4.1 Abstract 60 4.2 Introduction 60 4.3 Experimental subjects 61 4.3.1 Leipzig adult cohort 61 4.3.2 Childhood cohorts 62 4.4 Methods and Procedures 63 4.4.1 Analysis of human FASN gene expression 63 4.4.2 Sequencing of the FASN 63 4.4.3 Genotyping of the FASN SNPs 64 4.4.4 Statistical analyses 64 4.5 Results 65 4.5.1 Genetic variation in the FASN 65 4.5.2 Association studies in adults 67 4.5.3 Association studies in children 71 4.5.4 Association of genetic variants in the FASN with fat depot-specific FASN mRNA expression 71 4.6 Discussion 72 4.7 Acknowledgements 75 4.8 Disclosure 75 4.9 References 75 4.10 Supplement 77 4.11 Contribution of Authors 80 TABLE OF CONTENT 7 CHAPTER 5 81 Role of Genetic Variation in the Cannabinoid Type 1 Receptor Gene (CNR1) in the Pathophysiology of Human Obesity 81 5.1 Abstract 82 5.2 Introduction 82 5.3 Materials & methods 83 5.3.1 Leipzig cohort 83 5.3.2 Measurement of obesity & glucose metabolism parameters 84 5.3.3 Sorbian cohort 84 5.3.4 DNA extraction & sequencing 85 5.3.5 Genotyping of CNR1 SNPs 85 5.3.6 Statistical analysis 86 5.4 Results 86 5.4.1 Sequencing 86 5.4.2 Association studies 87 5.4.3 Leipzig cohort 87 5.4.4 Sorbian cohort 90 5.4.5 CNR1 genetic variants & fat-depot-specific CNR1 mRNA expression 90 5.5 Discussion 91 5.6 Acknowledgements 93 5.7 Disclosure 93 5.7.1 Financial & competing interests disclosure 93 5.7.2 Ethical conduct of research 93 5.8 Executive summary 94 5.9 References 94 5.10 Supplement 97 5.11 Contribution of Authors 99 CHAPTER 6 100 Human Bone Morphogenetic Protein Receptor 2 (BMPR2) is Involved in the Pathophysiology of Obesity 100 6.1 Abstract 101 6.2 Introduction 101 6.3 Research design and methods 103 6.3.1 Study subjects 103 6.3.2 Evolutionary analyses 104 6.3.3 Analysis of human BMPR2 mRNA expression 104 6.3.4 Sequencing of the BMPR2 105 6.3.5 Genotyping of BMPR2 SNPs 105 6.3.6 Statistical analyses 105 6.4 Results 106 6.4.1 Evolutionary analyses 106 6.4.2 Visceral and subcutaneous BMPR2 mRNA expression and obesity 107 6.4.3 Correlation of BMPR2 mRNA expression with parameters of obesity, glucose metabolism, and insulin sensitivity 108 6.4.4 Genetic variation in the BMPR2 110 TABLE OF CONTENT 8 6.4.5 BMPR2 variants and association with obesity 110 6.4.6 BMPR2 variants and association with obesity related quantitative traits 112 6.4.7 Association of rs6717924 with BMPR2 mRNA expression 112 6.5 Discussion 113 6.6 Acknowledgements 116 6.7 References 116 6.8 Supplement 119 6.9 Contribution of Authors 125 CHAPTER 7 126 Closing Discussion 126 7.1 References - Introduction and Closing Discussion 131 APPENDIX 138 Curriculum Vitae 138 Publications & Presentations 139 Erklärung über die eigenständige Abfassung der Arbeit 141 Danksagung 142 ABBREVIATIONS 9 ABBREVIATIONS (engl., dt.) 2-AG 2-arachidonylglycerol AU Arbitrary Unit, beliebige Einheit BAT Brown adipose tissue, braunes Fettgewebe BMI Body mass index, Körpermasseindex BMP/R Bone morphogenetic protein/receptor CETP Cholesteryl-ester transfer protein CNR Cannabinoid receptor, Cannabinoidrezeptor CT Computed tomography, Computertomographie CTNNBL1 Catenin beta, like 1 DEXA Dual energy X-ray absorptiometry dNTP Deoxyribonucleotide triphosphate DPI3 Dihydrocyclopyrroloindole tripeptide ECS Endocannabinoid system EGFP Enhanced green fluorescent protein 6-FAM 6-Carboxyfluorescein FASN Fatty acid synthase, Fettsäure-Synthase FRET Fluorescence resonance energy transfer FTO/Fto Fat mass and obesity associated (human/mouse) GWAS Genome wide association study, genomweite Assoziationsstudie HSC Hematopoietic stem cell, hämatopoetische Stammzelle HWE Hardy-Weinberg equlibrium, Hardy-Weinberg Gleichgewicht IGT Impaired glucose tolerance, gestörte Glucose-Toleranz iHS Integrated haplotype score IL Interleukin INSIG2 Insulin induced gene 2 IRS-1 Insulin receptor substrate 1 KFO Klinische Forschergruppe L.D.