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
