Immunomagnetic purification of beta cells from rat islets of Langerhans.

AIMS/HYPOTHESIS: The aim of this study was to develop immunomagnetic purification by the Dynabead system to separate insulin-containing beta cells from a mixed rat islet cell population. Functional studies on insulin secretion and a test of the susceptibility of Dynabead-separated beta cells to DNA damage following cytokine exposure were carried out. METHODS: Dynabeads are uniform, paramagnetic particles coated with specific antibodies. Single rat islet cells were initially incubated with the beta-cell surface specific antibody (K14D10 mouse IgG) for 20-60 min. A suspension of Dynabeads coated with a secondary antibody (anti-mouse IgG) was added for a further 15 min, after which the Dynabead-coated cells were instantaneously pelleted by contact between the tube and a magnet (Dynal MPC). Immunocytochemistry was used to confirm that the Dynabead-coated cells contained insulin and to quantify the efficiency of the method. Dynabead-coated and non-coated cells were stained for insulin and glucagon. RESULTS: Dynabead immunopurification yielded 95% pure insulin-containing beta cells, which released insulin in response to isobutylmethylxanthine and glucagon-like polypeptide 1. The insulin content of Dynabead-coated beta cells was significantly higher than that of non-coated cells. Successful separation was achieved using as few as 30 islets as starting material. Using the comet assay, we found that Dynabead-coated beta cells showed equal susceptibility to cytokine-induced DNA damage as non-coated cells. CONCLUSION/INTERPRETATION: We conclude that Dynabead separation of beta cells is simple, rapid, applicable to large or small numbers of islets and can be used to study beta-cell specific function and responsiveness.

Use of the Comet assay to investigate the role of superoxide in glutathione-induced DNA damage.

Although glutathione is an important scavenging molecule within the cell, it can also act as a pro-oxidant and at biological concentrations (1 mM) can induce DNA damage. We have used a sensitive cell-free Comet assay for DNA strand breakage to investigate this damage and to try to determine the active species involved. We show a substantial protection against glutathione-mediated DNA damage by superoxide dismutase (200 U/ml) and complete protection by combined superoxide dismutase and catalase. Damage is also prevented by EDTA but only at 100 mM and is not prevented by the chelating agent diethylenetriamine-pentaacetic acid (100 microM). Although superoxide is known to potentiate DNA damage by other reactive species, none of these indirect mechanisms seem to account for our results and it is possible that superoxide may damage DNA directly. Under the same experimental conditions, S-nitrosoglutathione requires ultraviolet A photolysis to cause DNA strand breakage and superoxide dismutase increases the level of this damage. When intact human lymphocytes are incubated with glutathione (1 mM) in phosphate buffer, DNA damage is also observed, but in this case it is completely preventable by catalase, with no protective effect of superoxide dismutase. Since cellular scavenging systems are not completely protective against reactive species formed from autooxidation of extracellular glutathione and since glutathione and oxygen are ubiquitously present within cells, our results imply that cells may have a mechanism of preventing autooxidation, rather than simply relying on scavenging the reactive species formula.

Insulin secretion, DNA damage, and apoptosis in human and rat islets of Langerhans following exposure to nitric oxide, peroxynitrite, and cytokines.

Cytokine-induced damage may contribute to destruction of insulin-secreting beta-cells in islets of Langerhans during autoimmune diabetes. There is considerable controversy (i) whether human and rat islets respond differently to cytokines, (ii) the extent to which cytokine damage is mediated by induction of nitric oxide formation, and (iii) whether the effects of nitric oxide on islets can be distinguished from those of reactive oxygen species or peroxynitrite. We have analyzed rat and human islet responses in parallel, 48 h after exposure to the nitric oxide donor S-nitrosoglutathione, the mixed donor 3-morpholinosydnonimine, hypoxanthine/xanthine oxidase, peroxynitrite, and combined cytokines (interleukin-1beta, tumor necrosis factor-alpha and interferon-gamma). Insulin secretory response to glucose, insulin content, DNA strand breakage, and early-to-late stage apoptosis were recorded in each experiment. Rat islet insulin secretion was reduced by S-nitrosoglutathione or combined cytokines, but unexpectedly increased by peroxynitrite or hypoxanthine/xanthine oxidase. Effects on human islet insulin secretion were small; cytokines and S-nitrosoglutathione decreased insulin content. Both rat and human islets showed significant and similar levels of DNA damage following all treatments. Apoptosis in neonatal rat islets was increased by every treatment, but was at a low rate in adult rat or human islets and only achieved significance with cytokine treatment of human islets. All cytokine responses were blocked by an arginine analogue. We conclude: (i) Reactive oxygen species increased and nitric oxide decreased insulin secretory responsiveness in rat islets. (ii) Species differences lie mainly in responses to cytokines, applied at a lower dose and shorter time than in most studies of human islets. (iii) Cytokine effects were nitric oxide driven; neither reactive oxygen species nor peroxynitrite reproduced cytokine effects. (iv) Rat and human islets showed equal susceptibility to DNA damage. (v) Apoptosis was not the preferred death pathway in adult islets. (vi) We have found no evidence of human donor variation in the pattern of response to these treatments.