Long-term perfusion of isolated rats islets in vitro

Abstract

A perfusion system is described for long-term maintenance of isolated rat islets in vitro. This system permits the monitoring of the pattern, rate, and amount of insulin secretion following repeated, acute stimulations with glucose during the period of culture. Fibroblastic proliferation did not occur, thus making is possible to reclaim the islets for biochemical and morphologic studies at the conclusion of the experiments. Maintenance of the islets with a low concentration of glucose (1.0 mg./ml.) resulted in a marked decline in insulin secretion following acute stimulations with glucose (5.0 mg./ml.) during an eight-day interval. Stimulation with 10 mM theophylline and 5.0 mg./ml. glucose on day 9 resulted in enhanced insulin release. The decline in glucose sensitivity occurred even more rapidly when the islets were maintained in the presence of a lower concentration of glucose (0.5 mg./ml.). The pattern of insulin release was altered with an absence of a first phase of secretion. Adenylate cyclase activity of islets maintained with 0.5 mg./ml. glucose for four days was significantly decreased in comparison with islets from fed rats and islets maintained with 2.5 mg./ml. glucose. A means of maintaining the same biphasic pattern and amount of glucose-induced insulin release was achieved by using alternating levels of glucose (1.5 and 2.5 mg./ml) for maintenance of the islets during a 36-day interval.