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Research ArticleCELLULAR AND MOLECULAR

Neuronal Necrosis Inhibition by Insulin through Protein Kinase C Activation

Wakako Hamabe, Ryousuke Fujita and Hiroshi Ueda
Journal of Pharmacology and Experimental Therapeutics October 2003, 307 (1) 205-212; DOI: https://doi.org/10.1124/jpet.103.053033
Wakako Hamabe
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Ryousuke Fujita
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Hiroshi Ueda
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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.

Footnotes

  • DOI: 10.1124/jpet.103.053033.

  • ABBREVIATIONS: BDNF, brain-derived neurotrophic factor; PI, propidium iodide; TEM, transmission electron microscopy; IGF-I, insulin-like growth factor-I; PKC, protein kinase C; PI3-K, phosphatidyl inositol-3-OH-kinase; MAPKK, mitogen-activated protein kinase kinase; ERK, extracellular signal-activated protein kinase; PLC, phospholipase C; PBS, phosphate-buffered saline; WST-8, 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt; PFA, paraformaldehyde; DTT, dithiothreitol; [3H]2-DG, 2-deoxy-d-[3H]glucose.

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Journal of Pharmacology and Experimental Therapeutics: 307 (1)
Journal of Pharmacology and Experimental Therapeutics
Vol. 307, Issue 1
1 Oct 2003
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Research ArticleCELLULAR AND MOLECULAR

Neuronal Necrosis Inhibition by Insulin through Protein Kinase C Activation

Wakako Hamabe, Ryousuke Fujita and Hiroshi Ueda
Journal of Pharmacology and Experimental Therapeutics October 1, 2003, 307 (1) 205-212; DOI: https://doi.org/10.1124/jpet.103.053033

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Research ArticleCELLULAR AND MOLECULAR

Neuronal Necrosis Inhibition by Insulin through Protein Kinase C Activation

Wakako Hamabe, Ryousuke Fujita and Hiroshi Ueda
Journal of Pharmacology and Experimental Therapeutics October 1, 2003, 307 (1) 205-212; DOI: https://doi.org/10.1124/jpet.103.053033
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