Ethanol impairs insulin-stimulated mitochondrial function in cerebellar granule neurons.
Ethanol impairs insulin-stimulated survival and mitochondrial function in immature proliferating neuronal cells due to marked inhibition of downstream signaling through P13 kinase. The present study demonstrates that, in contrast to immature neuronal cells, the major adverse effect of chronic ethanol exposure (50 mM) in post-mitotic rat cerebellar granule neurons is to inhibit insulin-stimulated mitochondrial function (MTT activity, MitoTracker Red fluorescence, and cytochrome oxidase immunoreactivity). Ethanol-impaired mitochondrial function was associated with increased expression of the p53 and CD95 pro-apoptosis genes, reduced Calcein AM retention (a measure of membrane integrity), increased SYTOX Green and propidium iodide uptake (indices of membrane permeability), and increased oxidant production (dihydrorosamine fluorescence and H2O2 generation). The findings of reduced membrane integrity and mitochondrial function in short-term (24 h) ethanol-exposed neurons indicate that these adverse effects of ethanol can develop rapidly and do not require chronic neurotoxic injury. A role for caspase activation as a mediator of impaired mitochondrial function was demonstrated by the partial rescue observed in cells that were pre-treated with broad-spectrum caspase inhibitors. Finally, we obtained evidence that the inhibitory effects of ethanol on mitochondrial function and membrane integrity were greater in insulin-stimulated compared with nerve growth factor-stimulated cultures. These observations suggest that activation of insulin-independent signaling pathways, or the use of insulin sensitizer agents that enhance insulin signaling may help preserve viability and function in neurons injured by gestational exposure to ethanol.
de la Monte
S M
SM
Department of Medicine, Rhode Island Hospital, Brown University School of Medicine, Providence 02903, USA. delamonte@hotmail.com
Neely
T R
TR
Cannon
J
J
Wands
J R
JR
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000818:Animals; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D002470:Cell Survival / Q000502:physiology; D002478:Cells, Cultured; D002531:Cerebellum / Q000378:metabolism* / Q000648:ultrastructure; D003576:Electron Transport Complex IV / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D000431:Ethanol / Q000494:pharmacology*; D005456:Fluorescent Dyes / Q000378:metabolism; D007150:Immunohistochemistry; D007328:Insulin / Q000378:metabolism* / Q000494:pharmacology; D008928:Mitochondria / Q000187:drug effects* / Q000378:metabolism; D020932:Nerve Growth Factor / Q000494:pharmacology; D009474:Neurons / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D016877:Oxidants / Q000378:metabolism; D051381:Rats; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D019014:fas Receptor / Q000378:metabolism
D000590:Amino Acid Chloromethyl Ketones; D061945:Caspase Inhibitors; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D007328:Insulin; D016877:Oxidants; D016159:Tumor Suppressor Protein p53; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D019014:fas Receptor; D000431:Ethanol; D020932:Nerve Growth Factor; D003576:Electron Transport Complex IV; D020169:Caspases