幼若ラット膵単層培養B細胞の灌流:--2-Deoxy-2-fluoroglucoseとiodoacetic acidの効果の比較

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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 5.5mM glucose plus 1mM 2-deoxy-2-fluoroglucose or with 5.5mM glucose following a 3-day exposure to a medium with 5.5mM glucose plus 504 iodoacetic acid. The use of 2-deoxy-2fluoroglucose 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 16.7mM glucose included only a small rise in insulin secreted during the first and the second phase, and the response to 10mM 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 5.5mM 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 1mM 3-isobutyl-1-methylxanthine and 10pM 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 16.7mM glucose and 200nM 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.<BR>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.
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幼若ラット膵単層培養B細胞の灌流:--2-Deoxy-2-fluoroglucoseとiodoacetic acidの効果の比較

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