Effects of Ca2+, microRNAs, and rosuvastatin on insulin-secreting beta cell function
Abstract: Type 2 diabetes (T2D) is a condition of high blood glucose levels due to insulin resistance and defective insulin secretion. Impaired insulin secretion plays a major role in the pathophysiology of T2D, it is mainly attributed to beta cell function i.e. failure to secrete insulin or reduced beta cell mass. The exocytotic process is crucial for beta cell function and its dysregulation leads to impaired insulin secretion. Therefore, understanding the central mechanisms involved in the regulation of exocytosis is essential to recognize possible targets for therapeutic intervention and treatment of T2D. In this thesis I have investigated the role of Ca2+, miRNAs and rosuvastatin in the regulation of ion channels, exocytosis and insulin secretion of beta cells. For this purpose, pancreatic rat INS-1 832/13 beta cells, rodent animal models, and islets from human cadaver donors has been used. Whole-cell patch clamp was used to study exocytosis measured as changes in cell membrane capacitance. In beta cells, biphasic exocytotic pattern was previously mainly attributed to insulin granule pool depletion. In paper I, we used the pulse length protocol and mixed-effect modelling; the latter takes care of cellular heterogeneity, to study exocytosis as a function of Ca2+ influx (measured as Q). The data suggests that pool depletion plays a minor role in observed biphasic exocytotic pattern in INS-1 832/13 cells; instead exocytosis is mostly determined by the kinetics of Ca2+ current inactivation. In paper II and III, we have investigated the effects of miRNA modulation on insulin secretion and exocytosis. First we investigated miRNA-regulation of voltage-gated Na+ channels since their role in beta cell function is not yet clear. Down-regulation of miR-375 differentially affected Na+ channel inactivation properties in INS-1 832/13 cells and miR-375 knock-out mice beta cells, suggesting species differences. As steady-state inactivation determines the number of channels available for generation of action potential, this study is a proof of principle that mir-375 could be important in regulating electrical activity in human beta cells. Next, miRNA-regulation of the exocytotic process was investigated. Overexpression of miR-335 reduced exocytosis and thereby insulin secretion through decreased expression of the exocytotic proteins STXBP1, SNAP25 and SYT11. In this paper I also made the novel observation that SYT11 increase basal insulin secretion and decrease rapid exocytosis, two phenomenons associated with T2D. The work on miR-335 emphasizes the importance of miRNAs in the regulation of the exocytotic process. In paper IV and V the effects of the cholesterol-lowering drug rosuvastatin was investigated. Rosuvastatin treatment dose dependently affected Ca2+ influx, exocytosis, basal and glucose-induced insulin secretion in INS-1 832/13 cells. Interestingly, most of this effect was though mevalonate pathway and not from the cholesterol lowering ability of rosuvastatin. In vivo rosuvastatin had an overall positive effect on glucose homeostasis in mice but negative effects on beta cell function such as disturbed Ca2+-signalling. In conclusion, the data in my thesis demonstrate the need for investigations of the mechanisms behind defective insulin secretion and exocytosis in order to understand and treat T2D.
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