IN VITRO STUDIES ON THE MAINTENANCE AND FUNCTION OF PANCREATIC ISLETS, WITH SPECIAL REFERENCE TO THE B-CELL.
Item
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Title
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IN VITRO STUDIES ON THE MAINTENANCE AND FUNCTION OF PANCREATIC ISLETS, WITH SPECIAL REFERENCE TO THE B-CELL.
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Identifier
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AAI8203325
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identifier
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8203325
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Creator
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SCHWIZER, RONALD WILLIAM.
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Contributor
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Robert C. McEvoy
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Date
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1982
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Language
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English
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Publisher
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City University of New York.
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Subject
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Biology, Anatomy
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Abstract
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A series of studies was undertaken with the objective of increasing the number and maintenance of function of pancreatic B-cells in culture. An experiment was carried out to quantitate the absolute and relative changes in the B-cell and non B-cell populations from cultures established by the monolayer technique of Lambert et al. (Endocrinology 90: 239, 1972) and maintained for up to 8 days. After decanting, approximately 1/3 of the cells originally plated remained unattached and were transferred to new dishes. By day 4, 10.5 (+OR-) 0.8% (mean (+OR-) SE) of the cells originally plated were present. The total number of cells increased 3-fold by day 8. Insulin-positive B-cells, identified by immunocytochemical staining, made up 3.6% of the cells at plating, 5.5 (+OR-) 0.5% at decanting, 2.8 (+OR-) 0.5% on day 4, and 1.0 (+OR-) 0.2% on day 8. The absolute number of B-cells did not change between days 4 and 8. This study provided quantitative confirmation that B-cell enrichment occurred as a result of the decanting procedure.;The ability of the principal islet cell types to maintain their differentiated function in vitro was studied in neonatal rat pancreata prepared according to Hellerstrom et al. (Diabetes 28: 769, 1979). The effects of the dissociation and culture on the yield of islet tissue was estimated by hormone content. The amount of islet tissue available for culture was reduced 50-70% following dissociation; an additional loss occurred by day 1. Only the insulin content of the tissue increased during culture. The hormone content of the media indicated that B-, A- and D-cell functions were maintained throughout the 8-day period of this study. The levels of insulin and glucagon increased approximately 2-fold and somatostatin increased 3-fold.;The effect of the tumor promoter, 12-o-tetradecanoyl-phorbol-13-acetate (TPA), on B-cell number and maintenance of function was investigated in islets cultured for up to 30 days. The insulin content of the media from control islets remained elevated for approximately 3 weeks. The cumulative insulin content of the media was increased almost 2-fold in the presence of 10('-8) and 10('-6) M TPA. After 30 days, the insulin content of cultures exposed to 10('-8) M TPA was 4-fold greater than that of untreated cultures. The mean number of total cells on days 8 and 15 was greater in the presence of TPA, whereas the number of B-cells decreased in both control and TPA-treated cultures. TPA had no effect on the number of B-cells.;The effect of 10('-7) M TPA on insulin secretion was studied using freshly isolated, perifused adult rat islets. The addition of TPA to low-glucose (40 mg/dl) medium resulted in a 3- to 4-fold increase in insulin release above basal levels within 10 minutes, confirming its ability to act as an initiator of insulin secretion. The rate of insulin release continued to rise for the 100-minute duration of the experiment and reached a mean rate of 13.2 times baseline.;The demonstrated maintenance of B-cell differentiated function as characterized by increases in the release and cell content of insulin in islets cultured in the presence of TPA, as well as the initiation of insulin release in an acute perifusion experiment, suggest that the B-cell may serve as a useful biological model for further investigations into the mechanisms of action of TPA and related tumor-promoting agents.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
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Program
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Biomedical Sciences
