Effects of Glucose and Amino Acids on the Biosynthesis of DNA and Insulin in Fetal Rat Islets Maintained in Tissue Culture

Abstract

Islet growth and insulin biosynthesis in fetal rat islets have been studied in a recently developed in vitro culture system. Collagenase-digested pancreases of 22-day-old rat fetuses were maintained for 5 days in culture medium RPMI 1640 (11.1 mM glucose) to allow degeneration of acinar tissue. Intact pure islets, composed of more than 90% B-cells, were then collected and subsequently treated for 3 days in culture with different concentrations of glucose and amino acids. Islets were then labeled for 24 h with 3H-thymidine (DNA synthesis) or for 2 h with 3H-phenylalanine (insulin/protein biosynthesis). The specificity of the incorporation of 3H-thymidine into B-cell DNA has been investigated and characterized by both Chromatographie and autoradiographic studies. Moreover, using a colchicine metaphase arrest technique, a relationship was demonstrated between incorporation of labeled thymidine and mitotic incidence in the B-cells. Incorporation of 3H-thymidine was significantly higher in the fetal than in the adult islets following treatment in all the different concentrations of glucose and amino acids studied. In the presence of 10% fetal calf serum, however, there was no increase in DNA synthesis in the fetal islets in response to high concentrations of glucose or amino acids. In media containing 2.5% fetal calf serum, there was a significant increase in the rates of DNA synthesis after addition of high concentrations of either glucose or amino acids. Fetal islets showed a marked insulin biosynthetic response to an acute glucose challenge. Both the basal and stimulated rates of insulin biosynthesis were increased after treatment in high concentrations of glucose but not after treatment with amino acids. The results suggest an important role of nutrients in the regulation of B-cell growth and insulin biosynthesis and also a dissociation between the regulatory mechanisms of these two processes.