Insulin and insulin mRNA are detected in neuronal cell cultures maintained in an insulin-free/serum-free medium.

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Abstract
We investigated the effect of a serum-containing medium, exogenous insulin, and insulin-free/serum-free media in the regulation of the rabbit brain insulin-like peptide (ILP) in neuronal cell cultures. The presence of serum or insulin in the medium resulted in approximately 3-5% of neurons that were positive for the peptide by immunohistochemistry and for insulin mRNA by in situ hybridization. The absence of insulin in the medium resulted in a three- to fourfold increase (p less than 0.001) in the insulin-immunoreactive and insulin mRNA-containing neurons. Additionally, in the presence of exogenous insulin or serum, the amount of insulin present in the medium, as measured by ELISA, decreased with time (approximately 80%), the former slower than the latter when compared with their respective zero time point values. However, an increase (approximately 80% from zero time) was noted in the absence of insulin or serum, and this lasted for 24-48 hours alone. The presence of an increased insulin/ILP content in the medium and the increase in the numbers of insulin-immunoreactive neurons suggests an important role of the peptide in the brain. The observation of increased synthesis and secretion of neuronal ILP in the absence of insulin is indicative of an autocrine effect of exogenous insulin on neuronal production of ILP, which in turn may be important in the growth and maintenance of neuronal and possibly glial cells.