IMMUNOHISTOCHEMICAL AND IN-SITU HYBRIDIZATION STUDY OF AN INSULIN-LIKE SUBSTANCE IN FETAL NEURON CELL-CULTURES

We studied the ability of fetal neuron cell cultures from different ra bbit fetal brain gestational ages to produce and secrete an insulin-li ke substance (ILS). Neurons from 22-day gestation were incubated in se rum-containing medium or insulin-free/serum-free medium, and 18-day ge station fetal rabbit neurons were also incubated in serum-free/insulin containing medium and serum-containing medium. The 22-day cultures su rvived in the serum-containing medium and the insulin-free/serum-free medium. The 18-day cultures died when incubated in the insulin-free/se rum-free or serum-free/insulin-containing medium, but survived when in cubated in serum-containing medium. Using immunohistochemical and in s itu hybridization techniques, ILS and insulin-like mRNA were demonstra ted within the 22-day cultures incubated in all media compositions, bu t not within the 18-day cultures incubated in the serum-containing med ium. Ultrastructural studies of the 22-day cultures demonstrated an IL S in the endoplasmic reticulum, Golgi and cytoplasm. Northern blots sh owed the presence of an insulin-like mRNA within the 22-day gestation neuron cell cultures. Insulin receptor was present in the 22-day cultu res, but was absent in the 18-day cultures. In addition, we characteri zed the ILS from the 22-day cultures incubated in the insulin-free/ser um-free medium employing high-performance liquid chromatography (HPLC) , radioimmunoassay and Western blots. Analysis by HPLC and Western blo ts demonstrated the presence of an ILS in the extract. We have shown t hat while 22-day fetal neuron cultures produce and secrete an insulin- like substance indistinguishable from authentic insulin, neuron cell c ultures from early brain development do not express this capability.