Ethanol impairs insulin-stimulated mitochondrial function in cerebellar granule neurons

Abstract

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.

Insulin Resistance, Cognitive Impairment and Neurodegeneration: Roles of Nitrosamine Exposure, Diet and Lifestyles

“…Cell density was determined by labeling the cultures with 10 µg/ml Hoechst H33342 in Tris-buffered saline (TBS), and measuring fluorescence (Ex360 nm/Em460 nm) in a Spectramax M5 microplate reader (Molecular Dynamics, Inc., Sunnyvale, CA). Mitochondrial function was measured using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay as previously described (de la Monte, Neely et al 2001). Absorbances were measured at 540 nm in Spectramax M5 microplate reader.…”

Ethanol Impairs Insulin-stimulated Neuronal Survival in the Developing Brain

“…Previous experiments demonstrated that neuronal loss following ethanol exposure was mediated by apoptosis (8 -10) or mitochondrial dysfunction (10 -12), and recent studies correlated these adverse effects of ethanol to inhibition of growth factor-stimulated survival signaling (9,(11)(12)(13). In the developing CNS, insulin and insulin-like growth factor type 1 (IGF-1) 1 receptors are abundantly expressed (14 -16), and the corresponding growth factor-stimulated responses are critical mediators of neuronal growth, viability, energy metabolism, and synapse formation.…”

“…In the developing CNS, insulin and insulin-like growth factor type 1 (IGF-1) 1 receptors are abundantly expressed (14 -16), and the corresponding growth factor-stimulated responses are critical mediators of neuronal growth, viability, energy metabolism, and synapse formation. Because insulin and IGF-1 signaling pathways are among the important targets of ethanol neurotoxicity in immature nervous system (9,13,17,18), neuronal loss associated with microencephaly in ethanol-exposed fetuses may be caused, in part, by ethanol inhibition of insulin/IGF-1-stimulated survival mechanisms.…”

Therapeutic targets of brain insulin resistance in sporadic Alzheimer s disease

“…In addition, prefibrillar tau can aggregate into soluble neurotoxic oligomers that cause synaptic disconnection and neuronal death (106). Although the key steps leading to tau hyper-phosphorylation and aggregation, could be explained on the basis of brain insulin/IGF resistance (107)(108)(109)(110), due to the associated decreased signaling through phosphoinositol-3-kinase (PI3K), Akt (28,29), and Wnt/β-catenin (111), and increased activation of GSK-3β (112)(113)(114)(115)(116), tau hyper-phosphorylation mediated by other mechanisms such as increased activation of cyclin-dependent kinase 5 (cdk-5) and c-Abl kinases (117,118), and inhibition of protein phosphatases 1 and 2A (105,118,119), could lead to oxidative stress and neuro-inflammation, which are inhibitory to insulin/IGF signaling.…”