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 ne rvous system in vivo and in vitro. We undertook this study to show a r ole for brain endogenous insulin within the fetal brain. We used neuro n cell cultures (NCC) from 19 days gestational age fetal rat brains in cubated in an insulin free/serum free defined medium. The neurons show ed the presence of preproinsulin I and II mRNA using polymerase chain reaction and insulin immunoreaction employing peroxidase anti-peroxida se and radioimmunoassay techniques. Using an anti-pan neurofilament an tibody (that recognizes non-phosphorylated neurofilaments) neurofilame nt 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 neur ons to become hypertrophic and vacuolated. With PD98059 treatment NFI was only observed within the neuron body. The addition of insulin reve rsed the effects of isoproterenol and PD98059, but not those of the in sulin antibody. Treatment with wortmannin had no effect, Western blot analysis showed that the basal level of mitogen activated protein kina se ((MAPK) phosphorylation was inhibited by the treatment of the NCC w ith isoproterenol or trypsin, but was significantly increased by treat ment with exogenous insulin, demonstrating that brain endogenous insul in phosphorylated MAPK (p < 0.05). Thus, brain endogenous insulin prom otes neurite outgrowth, probably via MAPK and by stimulating neurofila ment distribution via this mechanism participates in neuron differenti ation. (C) 1998 Elsevier Science B.V. All rights reserved.