IDENTIFICATION OF INTERLEUKIN 1-INDUCED APOPTOSIS IN RAT ISLETS USINGIN-SITU SPECIFIC LABELING OF FRAGMENTED DNA

To study the ability of interleukin 1-beta (IL-1) to induce apoptosis in the endocrine pancreas, rat islets of Langerhans obtained from 14-d ay-old BB.1A rats were exposed to 25 U/ml IL-1 for 40 h. In order to p rove the role of nitric oxide (NO) in this process islets were exposed either to 1 mmol/1 N-nitro-L-arginine methylester (NAME) or to 25 mmo l/l nicotinamide (NA) or to a combination of NA or NAME with IL-1. In dispersed cells oligonucleosomes, resulting from cleavage of nuclear D NA due to apoptosis, were identified by enzymatic labelling the free 3 '-OH-DNA ends with fluorescein-dUTP and quantified by means of flow cy tometry. After exposure to IL-1, the islets were characterized by elev ated basal (in response to 2 mmol/l glucose) insulin release while glu cose-stimulated (20 mmol/l glucose) insulin secretion was nearly compl etely abolished. In contrast, glucose-stimulated insulin secretion was well preserved in NAME-exposed islets, but was markedly inhibited aft er NA treatment. Accordingly, only the IL-1-induced inhibition of gluc ose-stimulated insulin secretion was significantly restored in the pre sence of NAME but was reinforced by NA. IL-1 exposure resulted in a si gnificant increase in the percentage of apoptotic cells (untreated con trols 3.8+/-0.5% IL-1 18.8+/-1.8%, P<0.01). This effect was significan tly reduced in the presence of NA and NAME. Nitrite production which w as assayed as an equivalent of NO generation of islets was highest und er the influence of IL-1 (16.48+/-1.40 versus 2.89+/-0.37 pmol/islet f or control islets) which was correlated with the percentage of apoptot ic cells. IL-1-stimulated nitrite production was reduced to 9.25+/-0.4 8 and 3.41+/-0.36 pmol/islet in the presence of NA or NAME, respective ly. The results demonstrate the potency of IL-1 to induce apoptosis in rat islets. Since inhibition of NO production was always paralleled b y a reduced ability of IL-1 to induce programmed cell death, this radi cal appears to be involved in this process. Remarkably, the near-compl ete prevention of NO generation as demonstrated under the influence of NAME was able to prevent the IL-1-induced deterioration of glucose-st imulated insulin secretion in parallel to the prevention of apoptosis- related appearance of DNA double-strand breaks. It is concluded that t he elimination of damaged beta cells due to IL-1 exposure is partly ac hieved by induction of apoptosis. (C) 1996 Academic Press Limited