Plasticity in the Glucagon Interactome Reveals Novel Proteins That Regulate Glucagon Secretion in α-TC1-6 Cells

Abstract

Glucagon is stored within the secretory granules of pancreatic alpha cells until stimuli trigger its release. The alpha cell secretory responses to the stimuli vary widely, possibly due to differences in experimental models or microenvironmental conditions. We hypothesized that the response of the alpha cell to various stimuli could be due to plasticity in the network of proteins that interact with glucagon within alpha cell secretory granules. We used tagged glucagon with Fc to pull out glucagon from the enriched preparation of secretory granules in α-TC1-6 cells. Isolation of secretory granules was validated by immunoisolation with Fc-glucagon and immunoblotting for organelle-specific proteins. Isolated enriched secretory granules were then used for affinity purification with Fc-glucagon followed by liquid chromatography/tandem mass spectrometry to identify secretory granule proteins that interact with glucagon. Proteomic analyses revealed a network of proteins containing glucose regulated protein 78 KDa (GRP78) and histone H4. The interaction between glucagon and the ER stress protein GRP78 and histone H4 was confirmed through co-immunoprecipitation of secretory granule lysates, and colocalization immunofluorescence confocal microscopy. Composition of the protein networks was altered at different glucose levels (25 vs. 5.5 mM) and in response to the paracrine inhibitors of glucagon secretion, GABA and insulin. siRNA-mediated silencing of a subset of these proteins revealed their involvement in glucagon secretion in α-TC1-6 cells. Therefore, our results show a novel and dynamic glucagon interactome within α-TC1-6 cell secretory granules. We suggest that variations in the alpha cell secretory response to stimuli may be governed by plasticity in the glucagon “interactome.”

Stathmin-2 Mediates Glucagon Secretion from Pancreatic α-Cells

“…We have previously identified Stmn2 as part of a network of proteins that associate with glucagon within the secretory granules of αTC1-6 cells (21). Data in the current study show that Stmn2 is localized to the secretory granule and Golgi compartments in aTC1-6 cells and mouse pancreatic alpha cells, respectively.…”

“…Twenty-four hours prior to secretion experiments, media were removed and replaced with DMEM without supplements. To evaluate (21) the regulated secretion of glucagon, cells were washed twice with HBSS, pre-incubated for 2h in DMEM without supplements, then incubated for 15 min with or without KCl (55 mM). These media were collected into tubes containing protease inhibitor (PMSF, 45 mM) and phosphatase inhibitors (sodium orthovanadate, 1mM; and sodium fluoride 5 mM) while tubes were kept on ice.…”

“…The control group was treated with Mission siRNA Universal Negative Control # 1 (Cat# SIC001, Sigma-Aldrich). Gene silencing was done based on a previously published protocol (26) and as we have done previously with some modifications (21). Briefly, αTC1-6 cells were cultured in regular DMEM to 60% confluency.…”

“…Regions of interest (ROI) were manually drawn around distinct single or multicellular bodies, and merged values of glucagon and Stmn2 were taken for analysis. Colocalization of the pixels from each pseudo-colored image was used to calculate Pearson’s correlation coefficient, as we described previously (27) (21).…”

“…We are pursuing the hypothesis that glucagon secretion can also be controlled by proteins within the secretory granule that associate with glucagon. By conducting secretory granule proteomics in αTC1-6 cells, we have recently described a dynamic glucagon interactome, and shown that components of this interactome can play a role in modulating glucagon secretion (21). Of these components, a protein of particular interest is Stathmin-2 (Stmn2 or SCG10), a member of the stathmin family of Golgi proteins (22), that may play a role in the regulation of neuroendocrine secretion (23).…”