Development of a Novel Pseudoislet Culture Model to Characterize IGF-II in Pancreatic Islet Function

Abstract

The pathology of chronic T2D is characterised by the eventual death of pancreatic islet β-cells due to long-term hyperglycaemia. Loss of islet β-cells coupled with insulin resistance within peripheral tissues are the known causes of T2D, which in turn leads to reliance on external insulin sources. Therefore, understanding the processes that maintain β-cell function and survival may deepen our understanding of T2D progression and inform new treatment targets. Researchers at the University of Auckland have identified and conducted preliminary characterisation of vesiculin, a novel IGF-II-derived peptide. Preliminary studies have shown that both IGF-II and vesiculin are secreted from islet β-cell granules in response to glucose, with vesiculin reported to be equally active as IGF-II. This indicates an autocrine/paracrine role in islet β-cell physiology. Although there have been some evidence linking the β-cell specific expression of IGF-II to β-cell mass expansion and survival, we have yet to elucidate the role vesiculin may play in β-cells, and how this activity may differ from IGF-II. In this project, we developed a novel 3D pseudoislet model via the culture of MIN-6 β-cells in endothelial (EOMA) cell-conditioned media. We demonstrate that this pseudoislet model conferred improved glucose-stimulated insulin response from MIN-6 pseudoislets, and hence illustrates a more physiologically relevant culture model for studying pancreatic islet function. To assess vesiculin function in β-cells, we attempted to generate vesiculin knock-out cell lines. This study outlines design strategies and optimisation techniques used for guide RNA design and transfection. We also describe the complexities associated with the knock-out approach, in order to provide an insight to prospective generation of a successful vesiculin knock-out cell model. Lastly, this project discusses the expansion and validation of doxycycline-induced IGFII knock-out cell models. These cell lines were used for functional analyses, where we show that the lack of IGF-II expression resulted in decreased insulin response against glucose and incretin stimulation. Furthermore, we demonstrate the significant reduction in phosphorylated IGF-1 receptor expression in IGF-II knock-out cells compared to wild-type controls. These findings suggest that IGF-II may play a vital role in the activation of the IGF1R signalling pathway, alluding to its influence on β-cell proliferation and metabolism.