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 肝功能衰竭是世界范围内死亡的主要原因。肝线粒体去极化(MtDepo)是 对乙醇的最早反应可能是导致后续肝病的一连串事件中的第一个。 最初，mtDepo对乙醇的反应促进乙醇及其有毒代谢物更快的两步氧化。 乙醛(乙醛)转化为乙酸酯。我推测mtDepo是酒精含量迅速增加的原因 急性酒精摄入后的代谢(SIAM)，这是一种排出乙醇的适应性反应 速度更快。慢性地，这种反应变得不适应，导致有丝分裂和肝脏紊乱。 炎症和纤维化。此外，我推测乙醇后的mtDepo是通过线粒体产生的。 由于质子通过腺苷核苷酸转运体(ANT1/2)、线粒体 F1FO-ATP合成酶、解偶联蛋白(UCPs)或线粒体通透性转换(PT)孔。自.以来 MtDepo导致线粒体通过有丝分裂消除，我还假设从mtDepo恢复 乙醇参与了线粒体的生物发生。在两个特定的目标中，我将：1)描述肝细胞 线粒体耗氧率(OCR)与线粒体去极化和 急性酒精中毒后的复极。我将评估OCR和线粒体膜的时间依赖性变化 乙醇处理和未处理小鼠新鲜肝细胞的海马电位(ΔΨ) 细胞外流量分析、Hansatech血氧测定仪和荧光ΔΨ指示剂的共聚焦显微镜。通过 在肝细胞分离前体内注射MitoTracker染料，只标记极化的荧光团 线粒体，我将在体内识别肝细胞与mtDepo和mtDepo的关系 体外复极。为了解决我的机械假说，我将评估特定的ANT、ATP抑制剂 合成酶、UCPs和PT毛孔逆转/防止乙醇诱导的mtDepo和OCR增加。2)评估 线粒体生物发生和有丝分裂在急性酒精中毒后线粒体死亡恢复中的作用。像老鼠一样 代谢排除乙醇，肝细胞从mtDepo恢复。在Mitotimer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)不可逆转地转变为红色荧光。如果先前存在的复极化 线粒体在乙醇之后出现，那么线粒体中的Mitomer荧光应该主要是红色的 正在从mtDepo恢复中。如果复极化是线粒体生物发生的结果，那么恢复线粒体 会发出绿色的荧光。我认为先前存在的线粒体的复极化和新线粒体的生物发生 两者都将有助于急性酒精中毒后mtDepo的恢复。总之，目标1和目标2将成为关键 了解肝脏线粒体对乙醇的反应机制，以及确定恢复的基础。这 基础知识可能导致治疗和预防酒精性肝的新疗法的发展 疾病。