Analysis of submembrane insulin granule number and behavior during the initial phase of secretion – dynamic measurements using TIRF microscopy

Background and aims: The release of a pool of membrane-adjacent secretory granules which are in a primed and docked state and await one final trigger, a depolarization-induced influx of Ca²⁺, is held responsible for the first phase of glucose-induced insulin secretion. Recently, we observed that 15 mM K⁺ led to a 20 mV depolarization and to a lasting increase of the cytosolic Ca²⁺ concentration ([Ca²⁺]_i), but only to a modest transient increase in secretion, suggesting that the effect of depolarization on insulin secretion is only incompletely understood.

Methods: Insulin secretion was measured by perifusion of mouse islets and MIN6 pseudoislets and ELISA of the fractionated effluate. The [Ca²⁺]_i of islets and MIN6 cells was measured with the Fura technique. Submembrane granules were visualized by transient transfection of MIN6 cells with an insulin-EGFP fusion protein and imaging by TIRF microscopy at 37°C. The images were evaluated by a purpose-made program written in MatLab to achieve a complete observer-independent quantitation.

Results: Islets perifused with 5 mM glucose showed only a transient increase in secretion when K⁺ was raised to 15 mM. A subsequent elevation to 40 mM K⁺ resulted in a prompt overshooting increase in secretion which remained increased as long as 40 mM K⁺ was present. In contrast, [Ca²⁺]_i was continuously elevated by 15 mM K⁺ and increased further when K⁺ was raised to 40 mM. The same secretion and [Ca²⁺]_i pattern could be observed with MIN6 pseudoislets. MIN6 cells transfected with insulin-EGFP were therefore used to analyse the behavior of submembrane granules by TIRF microscopy at 5 time points: beginning, prior to high K⁺, one each during 15 and 40 mM K⁺ and one after washout of high K⁺. At each time point a sequence of 100 images was acquired within 12s. Referring to the first sequence as 100%, the numbers of submembrane granules at the next time points were 109%, 76%, 50% and 56%. More than 50% of the granules were long-term resident (≥12s). The percentage of short-term resident granules (≤1s) increased slightly during 15 mM K⁺, then significantly during 40 mM K⁺ and remained so after washout. This correlated with an increase of the percentage of newly arriving granules from 4% initially to 7% during 15 mM K⁺ and to 15% during 40 mM K⁺ and washout. Very similar percentages applied for departing granules (i.e. return or release). There were no such changes in control experiments. The total and the net distances covered by the submembrane granules were not affected by high K⁺.

Conclusion: 40 mM K⁺ affects arrival, departure and residence time of insulin granules, but not movement parallel to the membrane. 15 mM K⁺ is moderately effective, which concurs with dynamic secretion measurements. There is no indication that long-term resident granules are preferentially departing during stimulation. This puts the idea into question that membrane-adjacent granules make up the initial phase of insulin secretion.

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