Effects of brain endogenous insulin on neurofilament and MAPK in fetal rat neuron cell cultures

We demonstrated the ‘de novo' synthesis of insulin within the fetal nervous system in vivo and in vitro. We undertook this study to show a role for brain endogenous insulin within the fetal brain. We used neuron cell cultures (NCC) from 19 days gestational age fetal rat brains incubated in an insulin free/serum free defined medium. The neurons showed the presence of preproinsulin I and II mRNA using polymerase chain reaction and insulin immunoreaction employing peroxidase anti-peroxidase and radioimmunoassay techniques. Using an anti-pan neurofilament antibody (that recognizes non-phosphorylated neurofilaments) neurofilament immunoreaction (NFI) was observed within the neuron body, dendrites and axon. Either insulin antibody or isoproterenol treatment induced the neurites to retract and most of the neurons become round, with NFI confined to the neuron body. The antibody treatments induced the neurons to become hypertrophic and vacuolated. With PD98059 treatment NFI was only observed within the neuron body. The addition of insulin reversed the effects of isoproterenol and PD98059, but not those of the insulin antibody. Treatment with wortmannin had no effect. Western blot analysis showed that the basal level of mitogen activated protein kinase (MAPK) phosphorylation was inhibited by the treatment of the NCC with isoproterenol or trypsin, but was significantly increased by treatment with exogenous insulin, demonstrating that brain endogenous insulin phosphorylated MAPK (p<0.05). Thus, brain endogenous insulin promotes neurite outgrowth, probably via MAPK and by stimulating neurofilament distribution via this mechanism participates in neuron differentiation.