Hepatic Mitochondrial Respiratory Activation, Depolarization and Recovery After Acute Ethanol

急性乙醇后肝线粒体呼吸激活、去极化和恢复

• 批准号: 10607356
• 负责人: Matthew Savoca
• 金额: $ 4.42万
• 依托单位: MEDICAL UNIVERSITY OF SOUTH CAROLINA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10607356
• 关键词: Accidents; Acetaldehyde; Acetates; Acute; Address; Adenosine; Alcohol abuse; Alcohol consumption; Alcohol dependence; Alcoholic Liver Diseases; Behavior Disorders; Biogenesis; Cardiovascular Diseases; Cause of Death; Cessation of life; Chronic; Cirrhosis; Confocal Microscopy; Dangerousness; Development; Disease; DsRed; Dyes; Ethanol; Ethanol Metabolism; Event; Fibrosis; Fluorescence; Genus Hippocampus; Health; Hepatic; Hepatocyte; Hour; In Vitro; Individual; Knowledge; Label; Laboratories; Lead; Liver; Liver Failure; Liver Fibrosis; Liver Mitochondria; Liver diseases; Malignant Neoplasms; Medical; Membrane Potentials; Mental disorders; Mentors; Metabolic; Mitochondria; Mitochondrial DNA; Mitochondrial Proton-Translocating ATPases; Molecular; Mus; Nucleotides; Oxygen Consumption; Oxygen saturation measurement; Pattern; Permeability; Persons; Process; Productivity; Proteins; Protons; Reactive Oxygen Species; Recovery; Research; Risk; Role; Serum; Steatohepatitis; Time; UCP2 protein; United States; Workplace; alcohol response; cost; cost estimate; extracellular; fluorophore; in vivo; inhibitor; liver inflammation; mitochondrial dysfunction; mitochondrial membrane; mitochondrial metabolism; mitochondrial permeability transition pore; multiphoton microscopy; novel therapeutics; oxidation; prevent; respiratory; response; Accidents; Acetaldehyde; Acetates; Acute; Address; Adenosine; Alcohol abuse; Alcohol consumption; Alcohol dependence; Alcoholic Liver Diseases; Behavior Disorders; Biogenesis; Cardiovascular Diseases; Cause of Death; Cessation of life; Chronic; Cirrhosis; Confocal Microscopy; Dangerousness; Development; Disease; DsRed; Dyes; Ethanol; Ethanol Metabolism; Event; Fibrosis; Fluorescence; Genus Hippocampus; Health; Hepatic; Hepatocyte; Hour; In Vitro; Individual; Knowledge; Label; Laboratories; Lead; Liver; Liver Failure; Liver Fibrosis; Liver Mitochondria; Liver diseases; Malignant Neoplasms; Medical; Membrane Potentials; Mental disorders; Mentors; Metabolic; Mitochondria; Mitochondrial DNA; Mitochondrial Proton-Translocating ATPases; Molecular; Mus; Nucleotides; Oxygen Consumption; Oxygen saturation measurement; Pattern; Permeability; Persons; Process; Productivity; Proteins; Protons; Reactive Oxygen Species; Recovery; Research; Risk; Role; Serum; Steatohepatitis; Time; UCP2 protein; United States; Workplace; alcohol response; cost; cost estimate; extracellular; fluorophore; in vivo; inhibitor; liver inflammation; mitochondrial dysfunction; mitochondrial membrane; mitochondrial metabolism; mitochondrial permeability transition pore; multiphoton microscopy; novel therapeutics; oxidation; prevent; respiratory; response

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.

抽象的 肝衰竭是全球死亡的主要原因。肝线粒体沉积（mtdepo）是之一 对乙醇的最早反应，可能是导致随后肝病的一系列事件中的第一个。 最初，响应乙醇的mtdepo促进了乙醇及其有毒代谢物的更快的两步氧化 乙醛（Acald）到乙酸盐。我假设mtdepo是酒精迅速增加的基础 急性乙醇给药后的代谢（暹罗），这是消除乙醇的一种自适应反应 更快。长期以来，这种反应变得不良适应性导致线索和肝脏无序 炎症和纤维化。此外，我假设通过线粒体带来了EtOH后的mtdepo 质子通过腺苷核苷酸易位（ANT1/2）（线粒体）泄漏引起的解偶联 F1FO-ATP合酶，解偶联蛋白（UCPS）或线粒​​体通透性转变（PT）孔。自从 mtdepo导致通过线粒体消除线粒体，我还假设从mtdepo中恢复 乙醇涉及线粒体生物发生后。在两个具体目标中，我将：1）表征肝细胞 线粒体氧的消耗率（OCR）与线粒体沉积有关 急性乙醇后的重钙化。我将评估OCR和线粒体膜的时间依赖性变化 使用海马从乙醇处理和未处理的小鼠中新鲜分离的肝细胞的电位（Δψ） 细胞外通量分析，Hansatech Oximetry和荧光Δψ指示器的共聚焦显微镜。经过 在进行肝细胞分离之前注射体内米托拉克染料，荧光团仅标记极化 线粒体，我将在体内与mtdepo相关的MTDEPO进行识别肝细胞 体外重钙化。为了解决我的机械假设，我将评估ANT，ATP的特定抑制剂 合成酶，UCP和PT孔反向/预防乙醇诱导的mTDEPO并增加OCR。 2）评估 线粒体生物发生和线粒体在急性乙醇后从mtdepo恢复中的作用。作为老鼠 代谢消除乙醇，肝细胞从mtdepo中恢复。在mitotimer小鼠中，新合成的dsred- E5荧光呈绿色，但随着时间的流逝（12-24 h）会不可逆转地转移到红色荧光。如果先前存在重复的 线粒体发生在乙醇之后，然后线粒体中的线粒体荧光应为红色 从mtdepo恢复。如果将线粒体生物发生引起重校正，则恢复线粒体 将荧光绿色。我期望已经存在的线粒体和新线粒体生物发生的复制 急性乙醇后，两者都会有助于从mtdepo中恢复。 AIMS 1和2在一起将表征密钥 肝脏线粒体对乙醇的反应中的机制，并确定恢复的基础。这 基本知识可能导致新的治疗剂的发展，以治疗和预防酒精肝 疾病。