(VP081) PARACRINE FACTORS REGULATE INTRACELLULAR TRAFFICKING OF GLUCAGON IN PANCREATIC ALPHA CELLS

Background: Patients with diabetes mellitus often present with hyperglycemia. One potential cause of hyperglycemia may be hyperglucagonemia, or excess glucagon secretion, from pancreatic alpha cells. Defective alpha cell secretory responses to glucose and paracrine effectors in both Type 1 and Type 2 diabetes may contribute to the development of hyperglucagonemia. Our lab investigates the intracellular trafficking pathways of the alpha cell to examine cellular mechanisms that underlie hyperglucagonemia. Our previous work has shown that glucagon secretion is: 1) mediated by networks of proteins that respond to glucose and the paracrine effectors insulin and gamma-aminobutyric acid (GABA), and 2) regulated by being directed to the endo-lysosomal pathway by a neuronal protein, Stathmin2(Stmn2). We hypothesize that glucose, insulin and GABA modulate glucagon secretion by increasing Stmn2-mediated lysosomal trafficking of glucagon in alpha cells.

METHODS AND RESULTS: AlphaTC1-6 cells, a glucagon-secreting alpha cell line, were treated with 1 nM insulin and 25 µM GABA in media containing high (25 mM) or low glucose (5.5 mM) for 24 hours. K+-stimulated glucagon secretion was measured by ELISA and intracellular trafficking was determined by confocal immunofluorescence microscopy analyzed by ImageJ Fiji and Pearson correlation. There were no significant differences in K+-stimulated glucagon secretion among treatment groups. Immunofluorescence microscopy showed that glucagon co-localized with the exocytosis marker Syntaxin-1A (Pearson Correlation 0.4 ± 0.01), which significantly decreased (p < 0.01, n=3) upon treatment with insulin and GABA. Co-localization of glucagon and the lysosomal marker LAMP1 did not change with insulin and GABA. There was significantly higher co-localization of glucagon with Syntaxin-1A than with LAMP1 (p < 0.01, n=3). Weak co-localization was observed between Stmn2 and Syntaxin-1A (PCC 0.18± 0.011). To better follow the intracellular trafficking of Stmn2, we generated a Stmn2-EGFP fluorescence reporter and localized it to a subset of glucagon-positive vesicles in transfected alphaTC1-6 cells.

Conclusion: Our results suggest that glucagon exocytosis from the readily-releasable pool of secretory granules was sensitive to the paracrine regulators insulin and GABA, and that the lysosomal compartment containing glucagon may be insensitive to regulation by paracrine factors. These changes could not be detected by ELISA. Our results also suggest that Stmn2 may not play a significant role in the trafficking of glucagon to secretory granules. With the use of Stmn2-GFP reporter generated in our lab, further experiments will determine the role of Stmn2 in the intracellular trafficking of glucagon as mediated by paracrine factors.