title: Neuronal necrosis inhibition by insulin through protein kinase C activation.
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 microg/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 microg/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.
authors.affiliation: Division of Molecular Pharmacology and Neuroscience, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan.
authors.forename: Wakako
authors.initials: W
authors.lastname: Hamabe
authors.forename: Ryousuke
authors.initials: R
authors.lastname: Fujita
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Ueda
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D019208:Brain-Derived Neurotrophic Factor / Q000494:pharmacology; D002540:Cerebral Cortex / Q000166:cytology; D016895:Culture Media, Serum-Free; D004789:Enzyme Activation / Q000187:drug effects; D005947:Glucose / Q000378:metabolism; D007328:Insulin / Q000494:pharmacology*; D008854:Microscopy, Electron; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009336:Necrosis; D009474:Neurons / Q000187:drug effects* / Q000473:pathology / Q000648:ultrastructure; D018696:Neuroprotective Agents / Q000494:pharmacology*; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D051381:Rats
chemical_list: D019208:Brain-Derived Neurotrophic Factor; D016895:Culture Media, Serum-Free; D007328:Insulin; D018696:Neuroprotective Agents; D011518:Proto-Oncogene Proteins; D000255:Adenosine Triphosphate; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D011493:Protein Kinase C; D020928:Mitogen-Activated Protein Kinases; D005947:Glucose