DNA synthesis in cultured neonatal rat islets--a comparison of two methods

Abstract

To assess the effect of glucose, foetal calf serum (FCS) and iron-saturated transferrin (Fe-TRF) on DNA synthesis of fibroblast-free neonatal rat islets cultured in Ham's F-12 medium, we measured [3H]thymidine uptake in cultures and used immunohistochemistry to quantitate nuclear 5-bromo-2'-deoxyuridine staining. Addition of glucose to a concentration of 26.1 mmol/l resulted in a significant increase from baseline in DNA synthesis in islet cells as measured by both methods (22,591 counts/min per microgram DNA +/- 3628 S.E.M., vs. 9631 counts/min per microgram 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 glucose concentration of 16.1 mmol/l we could demonstrate an increase compared with baseline only in [3H]thymidine uptake (15,700 counts/min per microgram DNA +/- 3323 S.E.M., P < 0.05). Supplementation with 10% or 15% FCS also increased [3H]thymidine uptake compared with baseline (to 15,809 counts/min per micrograms DNA +/- 136 +/- S.E.M., P < 0.05 and 23,746 counts/min per microgram DNA +/- 3114 S.E.M., P < 0.01, respectively) but did not significantly effect the BrDU labelling index. Addition of Fe-TRF to islet cultures significantly increased [3H]thymidine uptake at a concentration of 45 micrograms/ml compared with baseline (23,149 counts/min per microgram DNA +/- 6387 S.E.M., P < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)