The overall goal of this study is to determine the mechanisms of mitochondrial depolarization caused by acute ethanol. A single dose of alcohol induces a rapid increase of mitochondrial respiration in vivo. Recently, we observed a widespread, reversible mitochondrial depolarization in vivo in livers of alcoholtreated mice. The mechanisms of this novel phenomenon remain unclear. Accordingly, we will identify the pores, channels and pathways causing ethanol-induced mitochondrial depolarization and to characterize their regulatory mechanisms.
In Aim 1, we will characterize the dose-dependency, time course and recovery of mitochondrial depolarization in vivo after ethanol treatment using intravital multiphoton microscopy in relation to hepatic respiration, hypoxia, and hepatic steatosis. Additionally, we will investigate the effect of gender and nutritional status on ethanol-induced mitochondrial depolarization.
In Aim 2, we will evaluate the role of alcohol dehydrogense (ADH)-, CYP2E1- and aldehyde dehydrogenase (ALDH)-dependent alcohol metabolism on ethanol-induced mitochondrial depolarization using pharmacological interventions, CYP2E1 and ALDH knockout mice and ADH-deficient deer mice.
In Aim 3. We will evaluate the role of opening of different channels/pores that may cause mitochondrial depolarization in vivo after alcohol treatment. Since our preliminary studies ruled out the involvement of the mitochondrial permeability transition, mitochondrial KATP channel and uncoupling protein-2, we will focus on Ca2+ cycling via the Ca2+uniporter, proton leak through the adenine nucleotide transporer (ANT), and proton leak through F0 proton channel of F1F0 ATP synthase.
Aim 4 is based on our finding that FK506 (tacrolimus) suppresses ethanol-induced mticohondrial depolarization possibly through interaction with mitochondrial FK binding protein 38 (FKBP38). Using RNAi to knock down FKBP38 and Bcl-xL transgenic mice, we will test the hypothesis that loss of FKBP38 caused by acute alcohol acts to destabilize the mitochondrial ATP synthase and produce a proton leak possibly in a Bcl-xL dependent fashion. We will also further characterize if protection by FK506 against mitochondrial depolarization depends on stabilization of FKBP and ATP synthase or proteasomal degradation of damaged synthase subunits. We expect that these studies, using state-of-the-art intravital multphoton microscopic, biochemical, and molecular biological techniques, will provide relevant new mechanistic information regarding ethanol-induced mitochondrial depolarization that may contribute to alcoholic liver injury.
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