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: 10748288
• 负责人: Matthew Savoca
• 金额: $ 4.51万
• 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/10748288
• 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; 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; Peptide Initiation Factors; Permeability; Persons; Process; Productivity; Proteins; Protons; Reactive Oxygen Species; Recovery; Research; Risk; Role; SLC25A4 gene; 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 肝衰竭是全世界死亡的主要原因之一。 对乙醇的最早反应，很可能是导致随后肝病的一系列事件中的第一个。 最初，mtDepo 对乙醇的反应促进了乙醇及其有毒代谢物更快速的两步氧化 我猜测 mtDepo 是酒精迅速增加的原因。 急性乙醇施用后的代谢（SIAM），这是消除乙醇的适应性反应 长期来看，反应会变得适应不良，导致线粒体自噬和肝脏紊乱。 此外，我敢说 EtOH 后的 mtDepo 是通过线粒体产生的。 由于质子通过腺苷核苷酸易位子 (ANT1/2)、线粒体 F1FO-ATP 合酶、解偶联蛋白 (UCP) 或线粒体通透性转变 (PT) 孔。 mtDepo 导致线粒体自噬消除，我也重新认识到 mtDepo 的恢复 在乙醇涉及线粒体生物发生之后，我将：1）表征肝细胞。 线粒体耗氧率（OCR）与线粒体去极化和 我将评估 OCR 和线粒体膜随时间的变化。 使用 Seahorse 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 血氧测定法和荧光 ΔΨ 指示器的共焦显微镜检查。 在肝细胞分离之前体内注射 MitoTracker 染料，仅标记偏振的荧光团 线粒体，我将识别体内经历过 mtDepo 的肝细胞与 mtDepo 的关系，以及 为了解决我的机制假设，我将评估 ANT、ATP 的特异性抑制剂的作用。 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小鼠急性乙醇后 mtDepo 恢复中的作用。 代谢消除乙醇，肝细胞从 Mitotimer 小鼠中恢复，新合成的 DsRed-。 E5 发出绿色荧光，但随着时间的推移（12-24 小时），如果预先存在的复极化，则不可逆地转变为红色荧光。 线粒体发生在乙醇之后，那么线粒体中的 Mitotimer 荧光应主要为红色 从 mtDepo 中恢复 如果重新极化是由线粒体生物发生引起的，则恢复线粒体。 我预计先前存在的线粒体会重新极化并产生新的线粒体。 目标 1 和 2 将共同促进急性乙醇后 mtDepo 的恢复。 肝线粒体对乙醇的反应机制，以及确定恢复的基础。 基础知识可以促进治疗和预防酒精肝的新疗法的开发 疾病。