Perifusion of monolayer-cultured B cells of the infant rat: a comparative study of the effects of 2-deoxy-2-fluoroglucose and iodoacetic acid

Cell culture techniques for monolayer islets of 3-week-old rat pancreases and the responsiveness of B cells are described. In this procedure, whole pancreatic tissues from 3-week-old rats were enzymatically dispersed and then cultured in a medium with 5.5 mM glucose plus 1 mM 2-deoxy-2-fluoroglucose or with 5.5 mM glucose following a 3-day exposure to a medium with 5.5mM glucose plus 5 microM iodoacetic acid. The use of 2-deoxy-2-fluoroglucose or iodoacetic acid allowed a selective deletion of fibroblasts, yielding large clusters that consisted mostly of islet cells. The immunocytochemical evaluation of the islet cells in these cultures showed that approximately 70% are B-cells, 20% A-cells, and 10% D-cells. On day 0, the response to 16.7 mM glucose included only a small rise in insulin secreted during the first and the second phase, and the response to 10 mM of leucine or 2-ketoisocaproate was monophasic. After being cultured for 7 days, all three secretagogues markedly stimulated insulin secretion by B cells cultured in both media, resulting in an enhancement of the biphasic pattern. However, quantitative relationships differed. Thus, the total response from B cells in 2-deoxy-2-fluoroglucose during a 30-min stimulation with glucose and leucine was significantly higher (1.6- and 1.9-fold respectively) than that from B cells in 5.5 mM glucose, although there was no significant difference in insulin secretion evoked by 2-ketoisocaproate. Furthermore, in the former B cells, the amount of insulin secreted during the second phase was 84-94% of the total insulin secretion, and in the latter it was 66-76%. Addition of 1 mM 3-isobutyl-1-methylxanthine and 10 microM forskolin resulted in a significant increase in insulin secretion by B cells in 2-deoxy-2-fluoroglucose, whereas there was no difference in the increase of insulin secretion induced by 16.7 mM glucose and 200 nM 12-o-tetradecanoyl phorbol-13-acetate. In monolayer cultures that had been maintained in both media for 15 days, the second phase of insulin secretion due to the secretagogues was slightly decreased, but the biphasicity in the response was well preserved. In conclusion, the present results suggest that B cells of 3-week-old rats may be still immature, and that the medium with 2-deoxy-2-fluoroglucose is beneficial to the continued maturation of the B-cell function in vitro.