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.”
Highlights
Glucagon is the major glucose counter-regulatory hormone, and maintains euglycemia by enhancing hepatic gluconeogenesis and glycogenolysis [1]
We have identified a dynamic “glucagon interactome” within secretory granules of alpha cells that is altered in response to glucose levels and the paracrine effectors GABA and insulin
We identified a network of 392 proteins within the secretory granules that interact with glucagon and showed a direct interaction with glucose regulated protein 78 KDa (GRP78) and Histone H4
Summary
Glucagon is the major glucose counter-regulatory hormone, and maintains euglycemia by enhancing hepatic gluconeogenesis and glycogenolysis [1] Both type 1 and type 2 diabetes are characterized by varying levels of hyperglucagonemia [2], which paradoxically exacerbates the hyperglycemia of diabetes [3, 4]. Glucagon secretion is tightly regulated by nutritional, hormonal, and neural effectors to maintain normal glucose homeostasis. In diabetes, this tight coupling is disrupted [6], resulting in dysfunctional glucagon secretion, which may be a factor in the development of type 2 diabetes [7]. A preferable strategy may be to control the secretion, rather than the action, of glucagon for improved glycemic control in diabetes
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
