The epigenome of human adipocytes

The increasing incidence of obesity is a major public health crisis worldwide. Obesity is linked to a number of poor health outcomes such as Type 2 Diabetes (T2D), cardiovascular disease and cancer. Thus, the obesity epidemic threatens to lower life expectancy and reduce health-related life quality of current and future generations. It places a significant economic burden on healthcare systems. A better understanding of the factors that contribute to the development of obesity and how fat tissue contributes to these adverse effects are essential to improve prevention and treatment strategies. In this thesis, I examined the potential role of epigenetics as a mediator of gene-environment interactions in manifesting functional changes in fat cells, and whether these processes could be targeted to prevent and treat obesity. Firstly, I investigated whether exposure of human fat cells (adipocytes) to high glucose induces physiological and metabolic changes (Chapter 1). I then examined whether nuclear processes such as the genomic landscape of transcriptional and epigenetic regulation were involved in the manifestation of these changes. I used genome wide microarray analysis of both DNA methylation and transcription during the differentiation of human adipocytes in vitro. I showed that adipocytes grown in high glucose had significant changes in gene expression, particularly in mitochondrial and oxidative stress pathways, and that some of these changes were associated with epigenetic changes. Secondly, increased lipid storage in adipocytes is a major contributor to the increase in fat in humans, and these lipids are stored as lipid droplets within adipocytes (Chapter 2). I developed a novel label-free lipid droplet quantification technique (LipiD-QuanT) to monitor fat accumulation in live adipocytes. The current techniques used to quantify fat accumulation during in vitro studies are based on either enzymatic digest or lipophilic staining. However, LipiD-Quant is label free and applicable to live cells. Additionally, I demonstrated the application of LipiD-QuanT to measure the effect of four potential pro- or anti-obesogenic iv substances during human pre-adipocyte differentiation: Docosahexaenoic acid (DHA), rosiglitazone, D-glucose and Zinc oxide (ZnO) nanoparticles. Thirdly, since I began my PhD, epigenetics analysis methods have continued to improve. While most common methods target locations of the genome where methylation occurs densely, there is growing evidence that methylation in less dense genomic locations is equally, if not more important. Hence, I developed a new genome wide reduced methylome method (COBRA-Seq) (Chapter 3). This method enriches methylated DNA fractions by digesting the genomic DNA with restriction enzymes that recognize potential methylation sites after bisulfite conversion. The genomic complexity is further reduced by removing DNA fragments without the enzyme recognition site using streptavidin coated magnetic beads. COBRA-seq samples the genome proportionally for the generation and analysis of genomescale DNA methylation profiles with nucleotide resolution. This proved an excellent method to study representative, genome wide DNA methylation profiles at reduced cost. I compared the functionality of our novel method with other methylome sampling methods and reviewed its other potential applications in the field. I co-authored a systematic review on the most recent findings in the research area of human epigenetics and obesity (Appendix 1). I defined critical transcriptional and epigenetic signatures of human visceral adipose tissue (VAT) and purified visceral adipocytes (VA) associated with key parameters of obesity. For this part of my research, I coordinated a clinical study and used biological samples from 26 females across a range of BMIs (19-50 kg/m2) (Chapter 4). In the context of a larger team (the EpiSCOPE project) I coordinated the collection of these samples. This research revealed that transcriptional profiles were best correlated with BMI, waist circumference and hip circumference among the range of anthropometric variables. Additionally, I participated in the multi-institutional EpiSCOPE project to increase understanding of epigenetic changes in the emergence of obesity, and generated the first methylome maps of subcutaneous and visceral adipocytes in lean and obese individuals (Appendix 2). I contributed to two more scientific publications during my PhD that were not directly related to my PhD (Appendices 3 and 4).