Modulation of insulin signalling and calcium homeostasis by endosomes in pancreatic beta-cells

Disrupted pancreatic β-cell function is a key event in the pathogenesis of diabetes mellitus, a metabolic disorder resulting in elevated blood sugar levels. β-cells are responsible for the secretion of insulin, which promotes the uptake of blood glucose into peripheral tissue. Additionally, autocrine insulin signalling contributes to the maintenance of properly functioning β-cells. Upon insulin binding, the insulin receptor tyrosine kinase is activated and recruits insulin receptor substrates to its intracellular domain. These substrates can activate two major signalling branches, the Akt branch and the Ras/Erk branch. Both signalling branches are suggested to be involved in the maintenance of β-cell function and survival. Interestingly, results from experiments in adipose-like cell lines demonstrate, that endocytic vesicles can act as signalling hubs potentially directing insulin receptor signals between the Erk and Akt branches. Endosomes have also been suggested as organelles that are capable of buffering the rapid influx of calcium into β-cells following glucose stimulation thus avoiding calcium-induced β-cell death. These findings highlight endosomes as important organelles involved in the maintenance of β-cell function. This thesis examines the role of endosomes in autocrine insulin signalling and their involvement in calcium homeostasis. To observe the impact of endocytosis on autocrine insulin signalling, a novel fluorescent protein-labeled insulin receptor construct was developed and validated, revealing that tyrosine-phosphorylated caveolin-1 (Cav1) participates in insulin receptor internalization in β-cells. Remarkably, this process was found to bias insulin signalling towards the Erk branch in vitro and in vivo. As a functional consequence, reduction of Cav1 activity inhibited Erk signalling and was associated with increased β-cell apoptosis and decreased β-cell mass in mice lacking Cav1. The role of endosomes in β-cell calcium buffering was elucidated by creating a genetically encoded calcium sensor specifically localized to the lumen of endosomes and estimating calcium levels in defined endosome sub-populations. Indeed, endosomes accumulate calcium during glucose stimulation. Together, this work highlights endosomes as hubs for autocrine insulin signalling and contributors to the calcium homeostasis in the glucose response of β-cells.Medicine, Faculty ofGraduat