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

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.

摘要 肝衰竭是世界范围内的主要死亡原因。肝线粒体去极化（mtDepo）是 对乙醇的最早反应，可能是导致随后肝脏疾病的一连串事件中的第一个。 最初，mtDepo响应于乙醇促进乙醇及其有毒代谢物的更快速的两步氧化 乙醛（AcAld）转化为乙酸盐。我假设mtDepo是酒精迅速增加的基础 急性乙醇给药后的SIAM代谢（SIAM）是消除乙醇的适应性反应 更快。慢性时，这种反应变得适应不良，导致线粒体自噬紊乱和肝细胞凋亡。 炎症和纤维化。此外，我假设EtOH后的mtDepo是通过线粒体 解偶联是由于质子通过腺苷酸转运蛋白（ANT 1/2）、线粒体膜蛋白（线粒体膜蛋白） F1 FO-ATP合酶、解偶联蛋白（UCP）或线粒体通透性转换（PT）孔。以来 mtDepo通过线粒体自噬导致线粒体消除，我还假设从mtDepo中恢复 因为乙醇涉及线粒体生物合成。在两个具体目标中，我将：1）表征肝细胞 线粒体耗氧率（OCR）与线粒体去极化和 急性乙醇后复极。我将评估OCR和线粒体膜的时间依赖性变化， 使用Seahorse从乙醇处理和未处理小鼠中新鲜分离的肝细胞的电位（Δ λ） 细胞外通量分析、Hansatech血氧测定法和荧光Δ λ指示剂的共聚焦显微镜。通过 在肝细胞分离之前体内注射MitoTracker染料，仅标记极化的荧光团 线粒体，我将鉴定与mtDepo相关的体内经历mtDepo的肝细胞， 体外复极化。为了解决我的机制假设，我将评估ANT的特异性抑制剂ATP 合成酶、UCP和PT孔逆转/防止乙醇诱导的mtDepo和增加的OCR。2)评估 线粒体生物发生和线粒体自噬在急性乙醇后从mtDepo恢复中的作用。诸如鼠标 代谢消除乙醇，肝细胞从mtDepo中恢复。在线粒体小鼠中，新合成的DsRed- E5发出绿色荧光，但随着时间的推移（12-24小时）不可逆地转变为红色荧光。如果先前存在的复极化 线粒体在乙醇后发生，那么线粒体中Mitotimer荧光应该主要是红色的 从mtDepo恢复过来如果复极化是线粒体生物发生的结果， 会发出绿色荧光。我认为先存线粒体的复极化和新线粒体的生物合成 都将有助于急性乙醇后从mtDepo中恢复。目标1和目标2将共同构成 肝脏线粒体对乙醇的反应机制，以及确定恢复的基础。这 基础知识可能导致开发新的治疗方法来治疗和预防酒精性肝 疾病