Figure 3: Effect of the high glucose stimulation with and without Ex-4 on insulin vesicle properties and spatial distribution.

A) Number of Insulin vesicles during the two phases of insulin secretion. There was no significant change in the number of vesicles after 1 minute stimulation with glucose alone or in the presence of Ex-4. A significant increase in vesicle numbers was seen at 30 min post stimulation with glucose alone (787±204, ****P<0.0001) and with glucose+Ex-4 (515±156, **P=0.0062), compared with unstimulated cells (319±93). Fewer vesicles were seen after stimulation with glucose+Ex-4 at both 5 (*P=0.0191) and 30 min (*P=0.0436) than when stimulated by glucose alone. B) Mean vesicle LAC values. Left: Treatment with 25 mM glucose. Insulin vesicle LAC values steadily increase after 1 min stimulation, from 0.37±0.03 μm⁻¹ to 0.42±0.02 (*P=0.0138) and to 0.46±0.02 μm⁻¹ after 5 min stimulation (****P<0.0001); there is also a significant difference in the increase between 1- and 5-min stimulation (*P=0.264). At 30 min post stimulation with glucose, there is a significant decrease in LAC value to 0.40±0.02 μm⁻¹ compared with the LAC seen at 5 min post stimulation (****P<0.0001). Right: Treatment with 25 mM glucose+Ex-4. An increase in LAC values after stimulation is seen after 1 min glucose+Ex4 to 0.44±0.04 μm⁻¹ (*P=0.0226), at 5 min to 0.45±0.03 μm⁻¹ (****P<0.0001), and at 30 min to 0.43±0.03 μm⁻¹ (**P=0.0051) (Tukey’s multiple comparison test). C) Plots representing the position of vesicles in the cell as normalized distance from the PM (0.0 is the closest distance to PM and 1.0 the farthest). With respect to the unstimulated cells, significant differences in vesicle population are found for 25 mM glucose-stimulated cells (left panel) within 0.1 from the PM (1 min stimulation: *P=0.0316, and 5 min: **P=0.0040), 0.2–0.3 (5 min stimulation: ****P<0.0001), and 0.3–0.4 (5 min stimulation: ***P=0.005); the same analysis is performed for 25 mM glucose+Ex-4 stimulated cells (right panel), with differences within 0.1 from the PM (1 min stimulated cells: *P=0.0337), 0.1–0.2 (1 min: *P=0.0129), 0.2–0.3 (5 min stimulated cells: ****P<0.0001), 0.3–0.4 (5 min: ***P=0.006 and *P=0.0488) (Dunnett’s multiple comparison test).