Non-coding RNAs in beta cell insulin secretion - emerging players in Type 2 Diabetes pathogenesis

Type 2 diabetes (T2D) is a complex polygenic disease influenced by both environmental and genetic factors resulting in impaired insulin release from pancreatic beta cells and insulin resistance in target tissues. The incidence of T2D is escalating and it is projected that over 640 million people will be affected by 2040. Therefore, it is imperative to understand molecular processes that cause T2D to provide novel therapeutic avenues. Impaired insulin secretion is an early sign of T2D prior to insulin resistance. The functional implications of non-coding RNAs (ncRNAs) in glucose –stimulated insulin secretion (GSIS) have been widely recognized but the molecular mechanisms of many dysregulated ncRNAs in these processes has not been fully explored.Hence, the aim of this thesis was to investigate the cellular mechanism by which non-coding RNAs influence GSIS from pancreatic beta cells. To achieve our aims, we utilized rat INS-1 832/13 cells, human EndoC-βH1 cells, rodent animal models, and pancreatic islets from deceased human donors. In paper I, we showed elevated levels of miR-130a, miR-130b, and miR-152 expression in islets from hyperglycaemic human donors. Overexpression of miR-152 and miR-130a/b reduced dynamic ATP-levels, GSIS, insulin content, and the secreted proinsulin to insulin ratio in INS-1 832/13 cells. Finally, we confirmed pyruvate dehydrogenase alpha subunit E1 (PDHA1) as a target of miR-152 and knock-down of PDHA1 significantly reduced GSIS, insulin content, and ATP production.In paper II, we presented data from islets from glucose intolerant donors where expression of miR-335 negativley correlated with insulin secretion index. Overexpression of miR-335 in INS1-832/13 cells reduced GSIS and depolarization-evoked exocytosis, measured as changes in membrane capacitance as well as with TIRF microscopy. The effect was directly on the exocytotic process since the Ca2+-current was not reduced whereas levels of the exocytotic proteins SNAP25, syntaxin-binding protein 1 (STXBP1), and synaptotagmin 11 (SYT11) were reduced.In paper III, we demonstrated that confluence of cell cultures does not affect expression of miR-375 and its targets CAV1 and AIFM1 in rat INS-1 832/13 and human EndoC-βH1cells. This was also true for other miRNAs such as miR-200a,miR-152 and miR-130a whereas expression of miR-212 and miR-132 was to some extent influenced by cell density in INS-1 832/13 cells. In general we showed that miRNAs are not influenced by cell confluence at densities close to 100%. In paper IV, we showed that treatment of human beta cells with the glucocortocoid dexamethasone reduced expression of the lncRNA GAS5 and impaired GSIS. The negative effect of glucocortocoids on insulin secretion was confirmed in vivo from patient material. GAS5 knock-down in EndoC-βH1cells significantly reduced insulin secretion, increased apoptosis and perturbed expression of genes important in beta cell function and glucocorticoid signaling. Overexpression of GAS5 using GAS5 HREM was able to rescue the negative effect of dexamethasone on GSIS. Under hyperglycemic condition, expression of GAS5 was elevated, which partially explained increased expression of GAS5 in the islets from GK rats and T2D individuals. In conclusion, non-coding RNAs play signifcant role in regulating pancreatic beta cell insulin secretion through effects on metabolism and exocytosis. Understanding their mechanism of action in beta cells will pave way for the development of RNA-based therapeutics to treat T2D. (Less)