Al-Jabir, MJMH; (2016) Regulation of adipogenesis and inflammation: role(s) of adipose microRNAs. Doctoral thesis , UCL (University College London).

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Abstract

Background: Obesity is associated with elevated risk of premature death and a range of co- morbidities. It is multifactorial and heterogeneous in origin, and, stratification of this disease, depending on the range of associated pathogenicities could help identify mediators and in the design of targeted therapies. Recent research has focused on microRNA (miRs) as potential biomarkers of cardiovascular risk, as well as their role as causative agents in the obesity associated pathologies. / Aims of the project were to: / 1. Stratify obese subjects depending on systemic biomarkers of insulin resistance and inflammation. / 2. Identify and validate specific miRs associated with these phenotypes. / 3. Confirm and validate, in the whole transcriptome, targets for the specific miRs to assign functionality. / Methods: Non-diabetic, morbidly obese subjects of Arab origin were studied. Blood and adipose tissue samples were obtained before and after weight loss, along with anthropometric data. Glucose and lipids levels were determined by conventional methods. Insulin and adipokine concentrations were assayed by commercially available 2-site ELISA (R & D Systems, Oxon, UK). The population was dichotomised according to their serum insulin levels: Metabolically Healthy Obese (MHO) insulin <6.5 miU/ml; Patholigcally Obese (PO) insulin >7.0 miU/ml. Total RNA, including miR, was extracted from peripheral blood cells, whole adipose tissue, the stromal vascular fraction and adipocytes of the abdominal subcutaneous and omental adipose tissues. miR expression was assessed using an inflammatory pathway specific array (Qiagen), and by small RNA sequencing (Ion Torrent). mRNA expression was assessed by whole transcriptome analysis (Ion Proton) and validated by PCR based microarrays (SurePrint G3 Human Gene Expression). / Results: PO, matched for age and BMI, had significantly higher serum insulin levels and HOMA index of IR, compared to MHO. They also had higher leptin, a marker of fat mass and adipocyte hypertrophy, and lower adiponectin, an endogenous insulin sensitizer. However, blood pressure, lipids and inflammatory markers, such as IL-6, MCP-1 and CRP were not significantly different between the groups. Three miRs were significantly down-regulated in the PO; miR-29, miR-144 and miR- 374, and associated with inflammation, along with miR-122, miR-302, miR-200, which were associated with hyperinsulinaemia and insulin resistance. Many of their targets, especially those of miR-29, showed elevated expression in the PO. Following surgical weight loss there was a significant reduction in insulin which correlated with an increase in the levels of expression of nine miRs; miR-9, miR-200c, miR-141, miR-124, miR- 376c, miR-302, while one was downregulated, miR-26b. Whole transcriptome analysis of mRNA in blood and adipose tissue revealed modulation of several genes in the cardiometabolic pathways in the PO compared to MHO, along with genes leading to increased fibrosis. / Conclusions: Significant differences in the expression of specific miR species occured along with insulin resistance and inflammation in PO compared to MHO. The target genes of these miRs, especially miR-29, miR-144 and miR-122, suggested fibrosis, in the presence of IR and inflammation, as a major lesion in these patients. Functional studies to explore the role of these miRs in fibrosis may offer new insights on putative therapeutic targets for this group of patients.

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