Insulin granule number, mobility and exocytosis as compared with insulin content and secretion after the induction of desensitization or beta-cell rest

Background Desensitization of insulin secretion by prolonged stimulation may be the initial phase of beta-cell dysfunction in type-2-diabetes. Here, we have investigated the functional consequences of a prolonged exposure to the KATP-channel-blocker tolbutamide and to the inhibitor of insulin secretion, the α2-adrenoceptor-agonist clonidine.

Methods MIN6-cells were transfected with hIns-EGFP. Desensitization or resting state were induced by 18h exposure to 500µM tolbutamide or 1µM clonidine during cell culture. Exposed and control cells were perifused with 30mM glucose or 40mM KCl. The number, mobility and exocytosis of secretory granules were analyzed by TIRF-microscopy. Insulin secretion was measured using MIN6-pseudoislets and single MIN6 cells.

Results Insulin content was reduced after tolbutamide- and increased after clonidine-culture. However, the number of submembrane granules was not significantly affected by tolbutamide pretreatment, whereas it was increased after clonidine culture. Both types of pretreatment resulted in similar changes in the parameters of the granule mobility, namely a reduction in the z- and x / y-dimension. Perifusion experiments with MIN6-pseudoislets showed that both pretreatments diminished the secretory response to glucose and to KCl, whereas static measurement of single cell secretion showed a stronger response after clonidine- and a weaker response after tolbutamide-pretreatment. The number of exocytotic flashes of insulin-EGFP correlated with the strength of single cell secretion.

Conclusion The characteristics of single granule exocytosis correspond better to the secretion pattern of single MIN6 cells than to the one of pseudo-islets. So, the extrapolation from single cell data to the behavior of entire islets should be done with restraint.

Publication History

Article published online:
06 May 2021