Executive Summary : | A well-functioning pancreas preserves glucose homeostasis and hence prevents diabetes. Diabetes disrupts glucose homeostasis and secretion of insulin. During type-2 diabetes (T2D) the insulin-producing β-cells form islet amyloid polypeptide eventually leading to the death of β-cells and impairing insulin secretion. Most of the cells in the body secrete extracellular vesicles (EVs), pancreatic islet cells are no exception to this phenomenon. This secretion regulates systemic responses to stress in β-cells and such secretion is dysregulated in islets obtained from type-2 diabetic patients reflecting higher islet amyloid. The role of EVs in particular exosomes have been widely implicated in T2D but the mechanism of their secretion remains elusive. The study of exosomes has been limited by ways to label them in islet cells. Recently developed methods of fluorescently labeling tetraspanins in fibroblast cells to image exosomes have opened a new array of possibilities to study exosomes involved in various metabolic diseases including diabetes. These labeling methods have never been evaluated in pancreatic cell types. Here we will use the labeling approaches used in other cell types to employ High-resolution TIRF and confocal imaging to follow single EVs in islet cell types (discussed in the research plan). The technique will provide insights into the localization of EVs, their distribution, size, behavior and release, and the protein machinery involved. The outcomes from this will be applied to understand how the mechanism of EV secretion is affected in the type-2 diabetic setting in single cells and whole islets. The idea is to evaluate how these secretions influence islet amyloid formation leading to cell death. The exosomes from healthy counterpart controls will be treated on the diabetic counterpart if it can revive diabetic cells. Further evaluations on how these factors contribute to pancreatic islet hormone secretion and glucose homeostasis will be monitored. The cargo of islet-derived EVs will be identified in the peripheral blood to utilize them as markers of islet health and for early detection and prognosis of type-2 diabetes. These markers from the cargo of the islets combined with blood glucose and islet hormone secretions detected in the peripheral blood will help in monitoring pre-diabetic and diabetic patients in the long term. Such an idea to understand islet-microenvironment at different levels is unique and capabilities to extensively perform such a study with know-how and expertise is very rare. This study would give an understanding of the EV release mechanisms and how these are linked to pancreatic islet health. Overall, the project aims to understand abnormalities in human diabetic islet cells affect exosomes secretion and in turn lead to decreased secretion of insulin so that new treatment strategies to treat diabetes can be developed. |