The regulatory role of microRNA in human adipocytes and their link to insulin resistance

Abstract: White adipose tissue (WAT) is a highly plastic organ which secretes a large number of peptide factors termed adipokines. Increased caloric intake increases WAT mass which results in marked alterations in the secretory pattern. This may be mediated via both transcriptional and post-transcriptional mechanisms. Obesity is associated with several complications including type 2 diabetes mellitus (T2DM), hypertension, cardiovascular disease and cancer. The overall aim of this thesis was to identify microRNAs (miRNA) in obese WAT and to determine if they regulate genes, in particular those encoding adipokines that are linked to obesity-associated insulin resistance. In Study I, we investigated miRNA regulation of Chemokine (C-C Motif) Ligand 2 (CCL2) by extending and validating a recently identified transcriptional regulatory network in human adipocytes. The updated subnetwork predicted that miR-126-3p, miR-193b-3p and miR-92a- 3p control CCL2 production through several transcription factors (TFs) such as v-ets erythroblastosis virus E26 oncogene homolog 1 (avian) (ETS1), MYC-associated factor X (MAX), and specificity protein 1 (SP1). Selective down-regulation of ETS1, MAX, or SP1 in human adipocytes attenuated CCL2 production and a concomitant gene silencing of ETS1 and MAX resulted in an additive reduction in CCL2 production. Overexpression of miR-126- 3p, miR-193b-3p and miR-92a-3p in various pairwise combinations reduced CCL2 secretion in an additive manner although the effects of different miRNA combinations were cellspecific in adipocytes and macrophages. These findings add novel insights into the TF and miRNA-mediated regulation of CCL2 production in human adipocytes. In Study II, we identified adipocyte-expressed miRNAs altered by obesity that regulate adiponectin production. We found that miR-193b-3p increased adiponectin secretion and mRNA expression when overexpressed in human adipocytes. The expression of miR-193b- 3p significantly correlated with adiponectin gene expression and insulin resistance measured by homeostasis model assessment of insulin resistance. We identified that miR-193b-3p binds directly to the 3'-UTR of negative adiponectin regulators such as nuclear transcription factor Y α and nuclear receptor interacting protein 1 . This explains how miR-193-3p impacts adiponectin expression and secretion. A WAT phenotype characterized by few but large fat cells (hypertrophy) is linked to metabolic complications. In Study III, we identified miRNAs that were significantly upregulated in hypertrophic WAT. Out of 15 identified miRNA, 10 were predicted to bind to early B cell factor 1 (EBF1), a recently described regulator of WAT morphology. Two miRNAs (miR-361-3p and miR-574-3p) bound directly to the 3'-UTR of EBF1 and reduced EBF1 expression alone as well as in a combinatorial manner. Transcription activity of EBF1 was negatively correlated with the expression of the miRNAs. In Study IV, we identified 11 miRNAs that were differentially expressed between insulin sensitive and insulin resistance obese individuals. MiR-143-3p and miR-652-3p affected 4 insulin-stimulated glucose uptake either directly or indirectly affecting several insulin signaling regulators including Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), Protein Kinase, AMP-activated, Alpha 2 Catalytic Subunit (PRKAA2) as well as posttranslational phosphorylation of AKT2, AMP-activated protein kinase and insulin receptor substrate 1. Expression levels of miR-143-3p and miR-652-3p correlated significantly with insulin-stimulated lipogenesis suggesting that the levels of these miRNAs may also be of clinical importance. In summary, we have defined the regulatory pathways for several miRNAs that are dysregulated in obese WAT and that may have a pathophysiological impact. Our studies describe the involvement of miRNAs in regulating the expression of CCL2, adiponectin, EBF1, ENPP1 and PRKAA2. These results provide a better understanding of the role of miRNAs in the regulation of WAT function in health and disease.