Roles of unconventional ion channels and insulin granule structure in the pathogenesis of type-2 diabetes

Sammanfattning: T2D is the most widespread endocrine disease. In conventional stimulus secretion coupling increased blood glucose is metabolized causing an increased intracellular level of ATP and closure of the KATP channels, and this, in turn, depolarizes the cell membrane leading to the opening of voltage-gated Ca2+ channels, the influx of Ca2+ and exocytosis of insulin granules. This model has become almost an axiom in the diabetes research area. However, there are clear weak points that so far remain unclarified. More ion channels than potassium channels are needed to depolarize the membrane. Voltage-gated Ca2+ (Cav) channels have an essential role in beta cell function. The role of high voltage-activated Cav channels is well studied while the role of low voltage-activated T-type channels remain elusive. Moreover, glucose-stimulated insulin secretion (GSIS) causes beta cell swelling, which promotes us to explore mechanotransduction signalling pathways in beta cells represented by recently discovered Piezo1 mechanosensitive channel and aquaporins channels. Moreover, we also employed a dSTORM microscope combined with a single domain (SD) antibody to study the insulin granule cores (IGCs) at nano levels. Results: We find that the T-type Cav3.2 channel is abundantly expressed in human islets, and the gene expression is negatively correlated with HbA1c and strongly positively correlated with the expression of islet-predominantly expressed L-type subunits. CaV3.2 plays a fundamental role in maintaining normal insulin secretion by controlling Ca2+ signalling. Meanwhile, we also show that Piezo1 is expressed in pancreatic alpha and beta cells with heterogeneous distribution and is upregulated in T2D donors. Chronic hyperglycemia induces translocation of Piezo1 into the nucleus. In addition, silencing or inhibiting Piezo1 reduces Ca2+ signalling, membrane depolarization, and GSIS. Interestingly, beta- cell-specific Piezo1-knockout mice show impaired glucose tolerance in vivo and reduced electrical activity in islets. Subsequently, we identify that AQP1 gene expression is downregulated in islets from T2D individuals and silencing AQP1 decreased insulin secretion and insulin content. Whereas AQP1 overexpression significantly increased GSIS, AQP1 silencing elevated Ca2+ signalling due to elevated expression of CaV1.2 and CaV1.3 channels. Moreover, we demonstrate that AqB011, a selective AQP1 inhibitor blocking ion transport, substantially increases insulin secretion. Finally, nanoscale imaging for insulin granule cores exhibit that larger size located in exocytotic granules, the size and shape can be regulated by granule proteins cargo and the size is decreased after glucose stimulation, due to release of the readily releasable pool (RRP) part of insulin cores through incomplete granule fusion. Intriguingly, IGCs size was significantly decreased in pancreatic beta cells from human T2D donors and indicating that the lack of the RRP of the insulin core in the diabetic beta cells may be a primary cause for the impaired exocytosis. Conclusion: In this thesis, we have challenged the a consensus model and explored the importance of insulin granule structure in order to gain knowledge in the field around T2D pathophysiological progression and to facilitate finding new drug targets for the disease.