Apoptosis in cultured rat islets of Langerhans and occurrence of Bcl-2, Bak, Bax, Fas and Fas ligand

Isolated Langerhans islets are widely used for diabetic transplantation exp eriments and investigations of the mechanisms leading to the death or survi val of insulin-producing cells in cultured islets. The present study was ai med at investigating programmed cell death and the role of apoptosis-associ ated peptides in insulin and glucagon cells of islets isolated from untreat ed rats and held in cultured suspension. Islets were removed from medium on days 0, 7, 14, 21 and 29, embedded in Epon, and semi-thin serial sections were prepared. At designated intervals, histologic sections were treated wi th the direct fluorescein-labelled TUNEL method and immunostained for pancr eatic hormones (glucagon, insulin) and apoptotic peptides [Bak, Bar, Pas, P as ligand (FasL)], as well as for the anti-apoptotic peptide Bcl-2. All tis sue sections were investigated using confocal laser scanning microscopy und er identical setting for semiquantitative estimation of staining intensity. The percentage of apoptotic cells was between 1.6 and 2.1% and most apopto tic cells were beta-cells. Corresponding cells often contained Bak and Bar. Fas and Fast were mostly detected in islet cells within the first week aft er preparing the cultured suspension. The insulin content was low (1.1 +/- 0.22 ng per islet) directly after isolation. It then increased progressivel y up to day 14!, after which it began to decrease. Glucagon expression, on the other hand, remained high for the entire duration of the investigation. In conclusion, the islet beta-cells may recover after the isolation proced ure, but after 4 weeks in culture, both the insulin content and Bcl-2 stain ing decrease. Moreover, apoptosis is mediated by different mechanisms after the isolation procedure and after culturing the islets for 1 month. The pr esent data may be important for further studies on isolated, cultivated or transplanted islets. Copyright (C) 2001 S. Karger AG, Basel.