Investigations in the mechanisms of tolbutamide-induced desensitization of Min6-cells

Background and aims:

Desensitization of insulin secretion is a well-known phenomenon and may contribute to the pathogenesis of type 2 diabetes. Defects in signal recognition and defects in secretion are both involved. Here we have investigated the functional consequences of a prolonged exposure to the KATP channel blocker, tolbutamide.

Methods:

MIN6-cells were transfected with hIns-EGFP and cultured for 48 – 96h in DMEM-medium. Desensitization was induced by 18h exposure to 500µM tolbutamide. Thereafter, tolbutamide-exposed and control cells were perifused with Krebs-Ringer-medium and stimulated by 30 mM glucose or 40 mM KCl. The number and mobility of the secretory granules was analyzed by acquiring image sequences at 8 time points utilizing TIRF-microscopy.

Results:

Unexpectedly, the tolbutamide-desensitized MIN6-cells showed no significant decrease of the submembrane granule number per time point. However, the total number identified per image sequence was decreased as was the number of short-term resident granules and the number of granule arrivals at the plasma membrane. These changes point to a diminished granule turnover at the plasma membrane. Conversely, the number of long-term resident granules at the membrane was increased which was reflected by a diminished caging diameter. The latter parameter indicates a decreased mobility in the x/y-dimension, suggesting that these granules are docked. This difference was still visible after glucose and KCl stimulation.

Conclusion:

Prolonged exposure to tolbutamide diminishes the granulation state of insulin-secreting cells not only quantitatively, but also qualitatively. This may be the underlying defect of depolarization-induced desensitization of insulin secretion.