Transcriptional regulation of miRNA-212 and miRNA-132 and their fine-tuning function of beta cell insulin secretion

Type 2 diabetes is characterized by hyperglycemia due to insulin resistance in the target tissue and insufficient insulin secretion from the beta cells. Finding new mechanisms and pathways involved in the regulation of insulin secretion from the pancreatic beta cells is of great importance. Our group has earlier found, miRNA-212 and miRNA-132 to be upregulated in the non-obese type 2 diabetic GK rat. We hypothesize that the upregulation of these miRNAs is caused by a misregulation of the presumptive promoter of this miRNA-212/132 cluster. In this thesis I have therefore investigated mechanisms involved in transcriptional regulation of the miRNA-212/132-cluster and their regulation of insulin secretion in the pancreatic beta cell through their target proteins. The studies were performed in INS1 832/13 cells and primary rat and human pancreatic islets. Cells were incubated at different glucose concentrations in the absence or prescence of cAMP-stimulation using GLP-1 or a combination of forskolin (FSK) and 3-isobutyl-1-methylxanthine (IBMX). Modulations of expression of different transcriptional factors, miRNA-212 and miRNA-132 were performed using siRNA, LNA and mature miRNA mimics. The rat miRNA212/132 promoter was cloned into a luciferase construct to evaluate the activity of the miRNA-212/132 promoter at different conditions, a DuoLink experiment was performed to elucidate interactions between different proteins and an Ago2-RNA-immunoprecipitation was performed to verify a direct gene target of miRNA-132. Results were evaulated by qPCR, Western blot, immunofluorescene, glucose stimulated insulin secretion, Ca2+ measurements and the patch clamp technique. In paper I, AMPK-related protein kinases Sik1-3 and co-transcriptional factor Crtc1 were shown to regulate miRNA212 and miRNA-132 expression in pancreatic beta cells, possibly through Creb/Atf-1. Moreover, Crtc1 and miRNA212/132 regulated insulin secretion. The effect of Crtc1 on insulin secretion was found to be only partly via miRNA212/132. Also, the expression of SIK1 correlated positively with miRNA-132 levels in human pancreatic islets.In paper II, Camta1, a novel regulator of insulin secretion in pancreatic beta cells, was shown to regulate Ca2+-influx. Further Camta1 influenced miRNA-212/132 expression and interacted with the T2D implicated transcriptional factor Nkx2.2 at non-physiological glucose levels. In paper III, Mapt (tau) was proven to be a conserved direct target of miRNA-132 in beta cells. Overexpression of miRNA-132 was suggested to modulate the microtubule network and earlier stages of the insulin secretion process through Mapt and putative targets Sox6 and Isl1. In this thesis I report Crtc1, Camta1, miRNA-212 and miRNA-132 to be regulators of insulin secretion in insulin secreting beta cells, and that miRNA-212 and miRNA-132 are regulated by cAMP and Ca2+, through Crtc1, Camta1 and Creb/Atf1. While Crtc1 is suggested to generally increase insulin secretion, Camta1 more likely increases the Ca2+-influx of the beta cell and miRNA-212/132 regulate insulin secretion through translocation of insulin granules (e.g. Mapt) and insulin transcription (e.g. Sox6 and Isl1). In this thesis I have shown that miRNA-212 and miRNA-132 are likely to have an important function in the insulin secreting beta cells.This work will help us to a better understanding of how changes in the beta cell lead to the pathogenesis of T2D and hopefully the identified regulating molecules may contribute to the development of new therapeutic drugs.