Apoptosis in Cultured Rat Islets of Langerhans and Occurrence of Bcl-2, Bak, Bax, Fas and Fas Ligand

Abstract

Isolated Langerhans islets are widely used for diabetic transplantation experiments and investigations of the mechanisms leading to the death or survival of insulin-producing cells in cultured islets. The present study was aimed at investigating programmed cell death and the role of apoptosis-associated peptides in insulin and glucagon cells of islets isolated from untreated 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 with the direct fluorescein-labelled TUNEL method and immunostained for pancreatic hormones (glucagon, insulin) and apoptotic peptides [Bak, Bax, Fas, Fas ligand (FasL)], as well as for the anti-apoptotic peptide Bcl-2. All tissue sections were investigated using confocal laser scanning microscopy under identical setting for semiquantitative estimation of staining intensity. The percentage of apoptotic cells was between 1.6 and 2.1% and most apoptotic cells were beta-cells. Corresponding cells often contained Bak and Bax. Fas and FasL were mostly detected in islet cells within the first week after preparing the cultured suspension. The insulin content was low (1.1 ± 0.22 ng per islet) directly after isolation. It then increased progressively 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 procedure, but after 4 weeks in culture, both the insulin content and Bcl-2 staining decrease. Moreover, apoptosis is mediated by different mechanisms after the isolation procedure and after culturing the islets for 1 month. The present data may be important for further studies on isolated, cultivated or transplanted islets.

Activation of NFκB dependent apoptotic pathway in pancreatic islet cells by hypoxia

“…On the other hand, Bad showed elevated expression across different time points and Bcl-x L was found to increase only at 6 hr hypoxia. Those observations correlate with other studies that showed islets could recover from the isolation procedure with high expression of Bcl-2 during in vitro culture 19 and Bax expression diminished in hypoxic cells. 20 Those findings may indicate hypoxia triggered rescue machinery in islets at the early stage by decreasing proapoptotic genes Bax and increasing anti-apoptotic gene Bcl-2.…”

A GIP Receptor Agonist Exhibits β-Cell Anti-Apoptotic Actions in Rat Models of Diabetes Resulting in Improved β-Cell Function and Glycemic Control

“…At any one time of tissue sampling, increases in both pro- and anti-apoptotic proteins may be detected, as compensatory responses occur. Similar observations have been previously reported, for example with apoptotic β-cells in cultured and developing rat islets [49], [50] and following serum deprivation in MIN6 β-cells [51]. It is possible, that the milder diabetes that develops in obese VDF rats, when compared to obese ZDF rats, is due to a more robust anti-apoptotic response, since the latter exhibit reduced β-cell bcl-2 levels [35], although we have no direct evidence to support this suggestion.…”

Pro- and Antiapoptotic Proteins Regulate Apoptosis but Do Not Protect Against Cytokine-Mediated Cytotoxicity in Rat Islets and β-Cell Lines

“…The proapoptotic proteins Bak and Bax induce apoptosis by interacting with various cellular proteins via their BH3 domains and perhaps by promoting mitochondrial dysfunction, although the precise mechanism is unclear (24). These proteins are present in the rat islet (25,26), and we therefore hypothesized that suppression of their expression via delivery of siRNA duplexes would protect ␤-cells against apoptosis. siRNA duplexes specific for Bax and Bak were individually transfected or transfected in combination into 832/13 cells, resulting in 86 Ϯ 3 and 84 Ϯ 4% decreases in the levels of these proteins, respectively (Fig.…”