Granule mobility, fusion frequency and stimulated insulin secretion are differentially affected by insulinotropic stimuli

Background and aims: The view that insulin granules constitute functionally distinct pools which are sequentially recruited to release insulin has been put into question by new imaging techniques. Here, we characterized the number and mobility of submembrane granules in response to insulinotropic stimuli.

Methods: Insulin secretion, cytosolic Ca²⁺ concentration ([Ca²⁺]_i) and granule mobility in the submembrane space were measured in perifused MIN6 pseudo-islets or single MIN6 cells. Granules were labeled by insulin-EGFP and imaged by TIRF microscopy. The temperature was set at 37 °C, 32 °C or 22 °C.

Results: At 37 °C stimulation of MIN6 pseudo-islets with 30 mM glucose and, after washout, 40 mM K⁺, led to an increased insulin secretion. A reversed sequence of exposure did not change the effect of K⁺, but almost abolished the glucose effect. The same experimental protocol was used to measure [Ca²⁺]_i and granule mobility of perifused single MIN6 cells. In both sequences of stimulation K⁺ produced a marked increase in [Ca²⁺]_i while glucose was nearly inefficient. Independent of the sequence of stimulation K⁺ and glucose led to an increase in granule turnover. The effect of glucose was more sustained and was temperature-dependent. K⁺, but not glucose diminished the lateral mobility. Unexpectedly, the rates of fusion events were affected by the temperature, in parallel with secretion, but not by the stimuli.

Conclusion: Delivery to the plasma membrane, tethering and fusion of granules are differentially affected by insulinotropic stimuli. Fusion rates and secretion do not appear to be tightly coupled.