Hepatic Mitochondrial Respiratory Activation, Depolarization and Recovery After Acute Ethanol

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ABSTRACT Liver failure is a major cause of death worldwide. Hepatic mitochondrial depolarization (mtDepo) is one of the earliest responses to ethanol and likely is the first in a chain of events leading to subsequent liver disease. Initially, mtDepo in response to ethanol facilitates more rapid two-step oxidation of ethanol and its toxic metabolite acetaldehyde (AcAld) to acetate. I hypothesize that mtDepo underlies a swift increase in alcohol metabolism (SIAM) after acute ethanol administration that is an adaptive response to eliminate ethanol more rapidly. Chronically, the response becomes maladaptive leading to disordered mitophagy and hepatic inflammation and fibrosis. Furthermore, I hypothesize that mtDepo after EtOH is brought about via mitochondrial uncoupling due to proton leaks through either the adenosine nucleotide translocator (ANT1/2), the mitochondrial F1FO-ATP synthase, uncoupling proteins (UCPs) or mitochondrial permeability transition (PT) pores. Since mtDepo leads to elimination of mitochondria by mitophagy, I also hypothesize that recovery from mtDepo after ethanol involves mitochondrial biogenesis. In two Specific Aims, I will: 1) Characterize hepatocyte mitochondrial oxygen consumption rate (OCR) in relation to mitochondrial depolarization and repolarization after acute ethanol. I will assess time-dependent changes in OCR and mitochondrial membrane potential (ΔΨ) of hepatocytes freshly isolated from ethanol-treated and untreated mice using Seahorse extracellular flux analysis, Hansatech oximetry, and confocal microscopy of fluorescent ΔΨ indicators. By injecting in vivo prior to hepatocyte isolation MitoTracker dyes, fluorophores that label only polarized mitochondria, I will identify hepatocytes having undergone mtDepo in vivo in relation to mtDepo and repolarization in vitro. To address my mechanistic hypotheses, I will assess how specific inhibitors of ANT, ATP synthase, UCPs, and PT pores reverse/prevent ethanol-induced mtDepo and increased OCR. 2) Assess the role of mitochondrial biogenesis and mitophagy in recovery from mtDepo after acute ethanol. As mice metabolically eliminate ethanol, hepatocytes recover from mtDepo. In Mitotimer mice, newly synthesized DsRed- E5 fluoresces green but over time (12-24 h) shifts irreversibly to red fluorescence. If repolarization of preexisting mitochondria occurs after ethanol, then Mitotimer fluorescence should be predominantly red in mitochondria recovering from mtDepo. If repolarization results from mitochondrial biogenesis, then recovering mitochondria will fluoresce green. I expect that repolarization of preexisting mitochondria and biogenesis of new mitochondria will both contribute to recovery from mtDepo after acute ethanol. Together, Aims 1 and 2 will characterize key mechanisms in the hepatic mitochondrial response to ethanol, as well as identify the basis for recovery. This fundamental knowledge could lead to development of new therapeutics to treat and prevent alcoholic liver disease.