Glinide, but not sulfonylurea, can evoke insulin exocytosis by repetitive stimulation: imaging analysis of insulin exocytosis by secretagogue-induced repetitive stimulations

Abstract

To investigate the different effects between sulfonylurea (SU) and glinide drugs in insulin secretion, pancreatic beta-cells were repeatedly stimulated with SU (glimepiride) or glinide (mitiglinide). Total internal reflection fluorescent (TIRF) microscopy revealed that secondary stimulation with glimepiride, but not glucose and mitiglinide, failed to evoke fusions of insulin granules although primary stimulation with glucose, glimepiride, and mitiglinide induced equivalent numbers of exocytotic responses. Glimepiride, but not glucose and mitiglinide, induced abnormally sustained [Ca(2+)](i) elevations and reductions of docked insulin granules on the plasma membrane. Our data suggest that the effect of glinide on insulin secretory mechanisms is similar to that of glucose.

Figure 1

Histogram of the numbers of fusion events (/200 μm²) at 1-minute intervals after stimulation. Cells were perifused with 16.7 mM glucose (N = 16) (a), 0.5 μM mitiglinide (N = 17) (b), and 0.5 μM glimepiride (N = 11) (c) for 12.5 minutes. After the first stimulation, cells were perifused with 4 mM glucose for 15 minutes and stimulated again by the same secretagogues as in the first stimulation. Left and right columns show exocytotic responses during thefirst and second stimulations, respectively. (d) The numbers of total fusion events during the first and second stimuli. Data are represented as mean ± S.E.M per 200 μm².

Figure 1

Histogram of the numbers of fusion events (/200 μm²) at 1-minute intervals after stimulation. Cells were perifused with 16.7 mM glucose (N = 16) (a), 0.5 μM mitiglinide (N = 17) (b), and 0.5 μM glimepiride (N = 11) (c) for 12.5 minutes. After the first stimulation, cells were perifused with 4 mM glucose for 15 minutes and stimulated again by the same secretagogues as in the first stimulation. Left and right columns show exocytotic responses during thefirst and second stimulations, respectively. (d) The numbers of total fusion events during the first and second stimuli. Data are represented as mean ± S.E.M per 200 μm².

Figure 2

[Ca²⁺]_i responses to repetitive stimulations. Cells were perifused with 4 mM glucose and then stimulated with 16.7 mM glucose (a), 0.5 μM mitiglinide (b), or 0.5 μM glimepiride (c) for 12.5 minutes. After the first stimulation, cells were perifused with 4 mM glucose for 15 minutes and stimulated again by the same secretagogues as in the first stimulation. Data are represented as a ratio of the fluorescence intensity expressed as F/F ₀, where F ₀ is the ratio of 340 to 380 nm at the baseline. (mean + S.E.M., N = 9, 7, and 7 for 16.7 mM glucose, mitiglinide, and glimepiride, resp.).

Figure 3

The numbers of docked insulin granules just before the second stimulation. Results are mean ± S.E.M percentage of the numbers of docked insulin granules just before the first stimulation (N = 12, 9, and 10 for 16.7 mM glucose, mitiglinide, and glimepiride, resp.).