Abstract

In the serum-free culture of rat embryonic neurons, most neurons rapidly died by necrosis, which was revealed by propidium iodide (PI)-positive staining as early as 3 h after the start of culture and by marked membrane disruption and mitochondrial swelling in transmission electron microscopic (TEM) analysis. However, neither nuclear condensation/fragmentation stained with Hoechst 33342 nor activated caspase-3-like immunoreactivity was observed. In the serum-deprived culture, on the other hand, neurons showed apoptotic features, such as caspase-3 activation and nuclear damages in TEM analysis. Insulin at relatively higher concentrations, up to 100 μg/ml, ameliorated the rapid decrease in survival activity measured with 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt WST-8 assay and PI staining in the serum-free culture, despite the fact that brain-derived neurotrophic factor and insulin-like growth factor-I had no survival effect even at concentrations up to 100 μg/ml. Insulin-induced survival effects were abolished by the protein kinase C (PKC) inhibitor calphostin C but not by the phosphatidyl inositol-3-OH-kinase inhibitor wortmannin or the mitogen-activated protein kinase inhibitors PD98059 or U0126. Insulin significantly stimulated the PKC activity in cell lysates and suppressed the mitochondrial swelling and membrane disruption in TEM analysis in a calphostin C-reversible manner. All of these findings suggest that insulin inhibited the neuronal necrosis resistant to known neurotrophic factors under the serum-free culture through PKC mechanisms.