The Effect of Glucose Concentrations on Functional Maturation of Monolayer-Cultured B Cells of Neonatal Rat Pancreases

Abstract

The effect of glucose concentration in medium on the functional maturation of developing B cells was studied in pancreatic monolayer cultures of the neonatal rat. After a 3-day treatment with 10μM iodoacetic acid in TCM 199 medium containing 5.5mM glucose to selectively delete fibroblasts, the monolayer cultures were kept in the medium with either 5.5mM glucose or 16.7mM glucose for up to a total of 12 days. They were then perifused several times so that the phasic insulin secretion could be examined.
On day 0, untreated B cells showed a monophasic insulin secretion in response to a 16.7mM single dose of glucose, whereas in the presence of 200nM 12-o-tetradecanoyl phorbol-13-acetate, 10μM forskolin, 1mM 3-isobutyl-1-methylxanthine or 40μM lyso-phosphatidylcholine, the same dose of glucose stimulated insulin secretion in a biphasic fashion. Further, 20mg/dl sodium salicylate, 100μM tetracaine or 100μM p-bromophenacylbromide used concurrently with 16.7mM glucose also induced a biphasic insulin secretion, but their ability to stimulate insulin secretion was less than that of the other drugs mentioned above. Also, B cells on day 3 that had been exposed to iodoacetic acid responded to glucose in a similar manner to that of B cells on day 0, and the response to 10mM of either leucine or 2-ketoisocaproate was monophasic.
By contrast, B cells that had been kept in 5.5mM glucose on day 7 responded in a biphasic fashion, not only to 16.7mM glucose but also to 10mM of either leucine or 2-ketoisocaproate. The biphasic pattern evoked by glucose was still preserved in magnitude on day 15, whereas the response to leucine and 2-ketoisocaproate decreased to one-third that of B cells on day 7. On the other hand, when the concentration of 16.7mM glucose was present in the medium, B cells on day 7 showed a biphasic pattern of insulin secretion in response to 16.7mM glucose, although the magnitude of the response was quantitatively less than that of B cells in a physiological concentration of glucose. And again the response to 10mM of either leucine or 2-ketoisocaproate appeared monophasic. On day 15, an increased response to secretagogues with a biphasic pattern of insulin secretion was observed. In conclusion, these results suggest that the functional maturation in vitro of neonatal B cells in a physiological concentration of glucose may be achieved in a shorter period of culture time than in high glucose, and in addition, that high glucose is superior for long-term culture to a physiological concentration of glucose.