Effect of growth hormone and glucose on rat islet cells replication using 5-bromo-2-deoxyuridine incorporation

Islet cell proliferation was quantified by labelling fetal and adult rat islets in vitro with 5-bromo-2-deoxyuridine (BrdU), a thymidine analog that cannot be reutilized by the cell. After incubation with BrdU, cells were dispersed, centrifuged onto slides and DNA labeled with BrdU detected by a fluorescent anti BrdU antibody. The fluorescence was always restricted to the nucleus without any labelling in the cytoplasm. Insulin-containing cells were localized in parallel by immunocytochemistry demonstrating that all BrdU + cells were beta cells. The effects of fetal calf serum (FCS), human recombinant growth hormone (hGH) and glucose on islet cell proliferation were studied. After a 24 hour pulse with BrdU in 10% FCS, the percentage of BrdU+ cells found in adult and fetal islets was respectively 2.3% and 19.5% (p less than 0.001); only 8.8% of fetal islet cells were BrdU+ when fetal islets were incubated in 1% FCS. GH stimulated BrdU incorporation in a dose dependent manner: the threshold concentration of GH for a significant increase in BrdU+ cells was 50 ng/ml, the maximal effect was achieved with 100 ng/ml (+86% increased vs baseline p less than 0.01) and the half maximum response was seen at 50 ng/ml. Glucose stimulated the number of cells in S phase with a maximal response seen at 11 mM.