Beta-Oxidation and Susceptibility to Fatty Liver Disease

描述（由申请人提供）：非酒精性脂肪性肝病（NAFLD）被认为是最常见的肝脏疾病，但其潜在的发病机制尚不清楚。有证据表明NAFLD的发病机制涉及游离脂肪酸水平升高、先天免疫基因激活和胰岛素抵抗。虽然线粒体β -氧化是脂肪酸氧化的主要途径，但其在NAFLD发病机制中的作用尚不清楚。我们建立了一个线粒体三功能蛋白（MTP）的小鼠模型，该蛋白催化长链β氧化的最后3个步骤。纯合子小鼠会导致新生儿死亡。我们的初步数据表明，衰老杂合小鼠的肝脏脂肪变性与氧化应激和胰岛素抵抗有关。本研究建议使用这种小鼠模型来研究NAFLD的机制。我们的中心假设是MTP的杂合性通过增加细胞氧化应激导致胰岛素抵抗和肝脂肪变性相关的肝损伤。我们提出了以下具体目标的研究：1)测试MTP缺陷的杂合性导致与年龄相关的脂肪变性，导致超氧化物过量产生，导致抑制剂KB激酶（IKK）的激活，胰岛素抵抗和肝损伤，并测试氧化应激的预防在杂合小鼠中预防胰岛素抵抗和肝损伤。将进行研究以确定氧化应激与肝脂肪变性/损伤、胰岛素抵抗和IKK之间的时间关系。我们还建议进行干入性研究，通过使用Tempol清除超氧化物或将我们的MTP杂合小鼠与过表达超氧化物歧化酶的转基因小鼠杂交来预防氧化应激。2. 为了测试IKK的激活导致a) NFkB激活导致低级别炎症和肝损伤，b)在MTP缺陷杂合小鼠中，Principal Investigator3K-Akt通路的IRS依赖性激活受损导致胰岛素抵抗，并测试IKK的抑制是否逆转胰岛素抵抗和肝损伤。为此，我们将测量肝脏和肌肉中IKK和NFkB的含量和激活状态，并评估肝脏中促炎细胞因子的表达谱。我们还建议测量肝脏和肌肉中Akt和上游信号分子Principal Investigator3K和IRS-1/2的基础和胰岛素刺激含量和磷酸化/激活状态。肝脏和外周胰岛素抵抗对全身胰岛素抵抗的相对贡献将使用高胰岛素-血糖钳来评估。为了测试IKK、胰岛素抵抗和肝脏炎症反应之间的因果关系，我们将用已知的IKK抑制剂乙酰水杨酸（ASA）治疗小鼠，然后评估NFkB激活、肝脏炎症、胰岛素抵抗和Principal Investigator3K/Akt通路的激活状态。

DESCRIPTION (provided by applicant): Nonalcoholic fatty liver disease (NAFLD) is considered the most common form of liver disease, yet its underlying pathogenesis is poorly understood. Evidence suggests that pathogenesis of NAFLD involves increased levels of free fatty acids, activation of innate immunity genes, and insulin resistance. Although mitochondrial beta-oxidation is the major pathway for oxidation of fatty acids, its role in the pathogenesis of NAFLD remains unknown. We generated a mouse model for mitochondrial trifunctional protein (MTP) that catalyzes the last 3 steps in long chain beta-oxidation. Homozygous mice suffer neonatal death. Our preliminary data document that aging heterozygous mice develop hepatic steatosis associated with oxidative stress and insulin resistance. This proposal uses this murine model to investigate mechanisms underlying NAFLD. Our central hypothesis is that heterozygosity for MTP causes insulin resistance and liver injury associated with hepatic steatosis by increasing cellular oxidative stress. We propose studies towards the following specific aims: 1) To test that heterozygosity for an MTP defect results in an age-associated steatosis with superoxide overproduction leading to activation of the inhibitor KB kinase (IKK), insulin resistance, and liver injury, and to test that prevention of oxidative stress prevents both insulin resistance and hepatic injury in the heterozygous mice. Studies will be conducted to determine the temporal relationship between oxidative stress and hepatic steatosis/injury, insulin resistance, and IKK. We also propose interventional studies to prevent oxidative stress by either scavenging superoxide species using Tempol or by crossing our MTP heterozygous mice to transgenic mice overexpressing superoxide dismutases. 2. To test that activation of IKK causes a) NFkB activation leading to a low-grade inflammation and hepatic injury and b) impaired IRS- dependent activation of the Principal Investigator3K-Akt pathway leading to insulin resistance in mice heterozygous for an MTP defect and to test that inhibition of IKK reverses insulin resistance and hepatic injury. For this, we will measure the content and activation status of IKK and NFkB in both liver and muscle and assess the expression profile of proinflammatory cytokines in liver. We also propose to measure basal and insulin- stimulated contents and phosphorylation/activation status of Akt and the upstream signaling molecules Principal Investigator3K and IRS-1/2 in both liver and muscle. The relative contribution of hepatic and peripheral insulin resistance to whole body insulin resistance will be assessed using hyperinsulinemic-euglycemic clamp. To test the causative relationship between IKK, insulin resistance, and hepatic inflammatory response, mice will be treated with acetyl salicylic acid (ASA), a known IKK inhibitor and then NFkB activation, hepatic inflammation, insulin resistance, and activation status of the Principal Investigator3K/Akt pathway will be evaluated.