DNA-SYNTHESIS IN CULTURED NEONATAL RAT ISLETS - A COMPARISON OF 2 METHODS

To assess the effect of glucose, foetal calf serum (FCS) and iron-satu rated transferrin (Fe-TRF) on DNA synthesis of fibroblast-free neonata l rat islets cultured in Ham's F-12 medium, we measured [H-3]thymidine uptake in cultures and used immunohistochemistry to quantitate nuclea r 5-bromo-2'-deoxyuridine staining. Addition of glucose to a concentra tion of 26.1 mmol/l resulted in a significant increase from baseline i n DNA synthesis in-islet cells as measured by both methods (22 591 cou nts/min per mu g DNA +/- 3628 S.E.M., vs. 9631 counts/min per mu g DNA +/- 1912 S.E.M., P < 0.002 and 19.19% positive beta-cells +/- 2.72 S. E.M., vs. 11.98 positive beta-cells +/- 0.26 S.E.M., P = 0.05). At a g lucose concentration of 16.1 mmol/l we could demonstrate an increase c ompared with baseline only in [H-3]thymidine uptake (15 700 counts/min per mu g DNA +/- 3323 S.E.M., P < 0.05). Supplementation with 10% or 15% FCS also increased [H-3]thymidine uptake compared with baseline (t o 15 809 counts/min per mu g DNA +/- 136 S.E.M., P < 0.05 and 23 746 c ounts/min per mu g DNA +/- 3114 S.E.M., P < 0.01, respectively) but di d not significantly effect the BrDU labelling index. Addition of Fe-TR F to islet cultures significantly increased [H-3]thymidine uptake at a concentration of 45 mu g/ml compared with baseline (23 149 counts/min per mu g DNA +/- 6387 S.E.M., P < 0.01). A significant increase from baseline in the BrDU labelling index was also observed after addition of 15 mu g/ml Fe-TRF (26.17% positive beta-cells +/- 2.38 S.E.M., P < 0.01), but in contrast to [H-3]thymidine uptake, the BrDU labelling in dex decreased with higher concentrations of Fe-TRF and was not signifi cantly different from baseline at 45 mu g/ml (10.08% positive beta-cel ls +/- 0.79 S.E.M., P = NS). In conclusion, islet cell DNA synthesis w as enhanced in vitro by the addition of glucose, FCS and iron-saturate d transferrin into the culture medium. Progression through the cell cy cle was inhibited at high concentrations of Fe-TRF despite further inc reases in the rate of DNA synthesis, Islet cell growth should be evalu ated independently by several methods to distinguish phases of the cel l cycle effected by growth stimulating agents.