Temperature-dependent effects of glucose and potassium depolarization on granule number and mobility in the submembrane space of insulin-secreting MIN6 cells

Background and aims: TIRF microscopy has changed our view on insulin granule behaviour. Here, we characterized the number and mobility of submembrane granules when insulinotropic stimuli were applied at physiological and sub-physiological temperatures.

Methods: Submembrane granules of MIN6 cells were labeled by insulin-EGFP and imaged by TIRF microscopy. The cells were perifused with Krebs-Ringer medium at 37 °C, 32 °C or 22 °C. Image sequences (1 sequence = 200 images = 25 seconds) were taken at 8 time points during one experiment and evaluated by an in-house written program.

Results: Under control conditions (3 mM glucose, 37 °C), the number of submembrane granules was 324 ± 21 per cell footprint at the beginning of image acquisition. 29.1 ± 1.1% of these remained visible throughout the sequence (long-term residents). Newly arrived granules made up 10.9 ± 0.5% per image, equivalent to about 35 granules. The number of departing granules closely mirrored that of arriving granules. The short-term residents (present for ≤1 s) amounted to 84.5 ± 0.6% of total (8083 ± 785) in a sequence. 30 mM glucose or 40 mM KCl led to an increase of both, arrivals and departures within 2 min at all temperatures. Consequently, there was no change in the number of short-term residents. The number of long-term residents in contrast decreased at low, but not at physiological temperatures.

Conclusion: The loss of efficacy of insulinotropic stimuli by subphysiological temperatures may primarily involve granules with longer presence at the plasma membrane.