Thesis Format
Integrated Article
Degree
Doctor of Philosophy
Program
Pathology and Laboratory Medicine
Supervisor
Dhanvantari, Savita
Abstract
Patients with diabetes exhibit hyperglucagonemia, or excess glucagon secretion. The glucagonocentric hypothesis of diabetes states that hyperglucagonemia, rather than hypoinsulinemia, may be the underlying mechanism of hyperglycemia of diabetes. Thus, uncovering mechanisms that regulate glucagon secretion from pancreatic α-cells is crucial for developing treatments for hyperglycemia. One clue to the regulation of glucagon secretion may lie in the proteins that interact with glucagon in α-cell’s secretory pathway, primarily within the secretory granule. The purpose of my work was to identify proteins that interact with glucagon within the secretory granule and characterize a candidate protein within this network that regulates the intracellular trafficking of glucagon to control its secretion.
To identify secretory granule proteins that interact with glucagon, I purified secretory granules from α-TC1-6 cells. I then used affinity purification using tagged glucagon to isolate protein complexes that interact with glucagon, and identified these proteins through liquid chromatography/mass spectrometry. In this way, I identified a glucagon “interactome” within the α-cell secretory granule. I found that components of the interactome changed in response to different glucose concentrations, and to treatment with the paracrine inhibitors insulin and GABA.
Next, I characterized the function of one interactome protein, the neuronal cytoskeletal protein stathmin-2, in glucagon secretion. Through overexpression and siRNA-mediated silencing of stathmin-2 in α-TC1-6 cells, I showed that stathmin-2 is a tonic inhibitor of glucagon secretion. Using confocal high-resolution immunofluorescence microscopy, I found that stathmin-2 exerts its regulatory role by trafficking of glucagon to the endolysosomal system.
Finally, I examined how the trafficking role of stathmin-2 is altered in the hyperglucagonemia of diabetes. Using isolated islets from a mouse model of diabetes, I showed that the increase in cellular glucagon was accompanied by a reduction in stathmin-2 levels. Confocal microscopy analysis indicated that, in diabetes, there is a switch from the anterograde trafficking of glucagon towards the lysosome to retrograde trafficking towards secretory granules, possibility mediated by the endosomal protein Rab7.
In summary, my thesis describes the discovery of a regulatory mechanism for glucagon secretion from α-cells that may operate in hyperglucagonemia. These findings have clinical application for treatment of hyperglycemia of diabetes.
Summary for Lay Audience
People with diabetes have an abnormal amount of a hormone called “glucagon” in their blood, which seriously increases blood sugar. Glucagon is released from a cell called “alpha cell”, which is in the gland of the pancreas. My research was to discover some proteins within a special structure in alpha cells called “secretory granule” that control the release of glucagon. I found that the type and numbers of proteins within the secretory granule changed when I exposed alpha cells to suppressors of glucagon secretion (insulin or GABA). This would mean that those proteins are candidates for decreasing glucagon secretion (stathmin-2 being one of the proteins). I found that both glucagon and stathmin-2 are located within the secretory granule of a mouse’s pancreas in a close distance from each other. By using a genetic technique, I removed stathmin-2 from the alpha cell and found that cells released a lot of glucagon. On the other hand, when alpha cells produced a lot of stathmin-2, they did not secrete glucagon. There is a structure within alpha cells called “endolysosome”. I found that when there is a lot of stathmin-2 within the alpha cell, it directs glucagon into the endolysosome, where it is destroyed. However, when there is a lack of stathmin-2 in alpha cells, glucagon is not destroyed in the endolysosome. These mean that stathmin-2 controls glucagon secretion through endolysosome. I then proposed that, in diabetes, glucagon is not destroyed in the endolysosome. This is the reason for high glucagon secretion from the pancreas. To test this idea, I found that, in diabetic mice, there was very little stathmin-2 versus lots of glucagon in its pancreas. Then, by using advanced microscopic studies, I found that both glucagon and stathmin-2 were not destroyed in the endolysosome of alpha cells in the diabetic mouse. I found that stathmin-2 collected into a structure called “late endosome”, and at the same time, alpha cells showed an increase in glucagon secretion. Therefore, stathmin-2 is a novel molecule that can control glucagon secretion. In future studies, it could be used to decrease high blood sugar in people with diabetes.
Recommended Citation
Asadi Jomnani, Farzad, "Regulation of Glucagon Secretion and Trafficking by Proteins in the Glucagon Interactome" (2020). Electronic Thesis and Dissertation Repository. 7074.
https://ir.lib.uwo.ca/etd/7074
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