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病形式，但其潜在的发病机制知之甚少。有证据表明，NAFLD的发病机制涉及游离脂肪酸水平的增加、先天免疫基因的激活和胰岛素抵抗。虽然线粒体β-氧化是脂肪酸氧化的主要途径，但其在NAFLD发病机制中的作用仍不清楚。我们生成了线粒体三功能蛋白（MTP）的小鼠模型，该蛋白催化长链β-氧化的最后3个步骤。纯合子小鼠遭受新生儿死亡。我们的初步数据表明，老龄杂合子小鼠发生与氧化应激和胰岛素抵抗相关的肝脂肪变性。该建议使用这种小鼠模型来研究NAFLD的潜在机制。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与肝脂肪变性相关的肝损伤。我们提出了针对以下具体目标的研究：1）为了测试MTP缺陷的杂合性导致与年龄相关的脂肪变性，其中超氧化物过度产生导致抑制剂KB激酶（IKK）的激活、胰岛素抵抗和肝损伤，并且测试防止氧化应激防止杂合小鼠中的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。我们还提出了干预性研究，以防止氧化应激，无论是清除超氧化物使用Tempol或通过交叉我们的MTP杂合子小鼠过表达超氧化物歧化酶的转基因小鼠。2.在MTP缺陷杂合子小鼠中，检测IKK激活是否导致a）NF κ B激活，导致轻度炎症和肝损伤，以及B）主要研究者3 K-Akt通路的IRS依赖性激活受损，导致胰岛素抵抗，并检测抑制IKK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的含量和活化状态，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 Investigator 3 K和IRS-1/2的基础和胰岛素刺激的含量和磷酸化/活化状态。将使用高胰岛素-正葡萄糖钳夹法评估肝脏和外周胰岛素抵抗对全身胰岛素抵抗的相对贡献。为了检测IKK、胰岛素抵抗和肝脏炎症反应之间的因果关系，将用乙酰水杨酸（阿萨）（一种已知的IKK抑制剂）处理小鼠，然后评价NFkB活化、肝脏炎症、胰岛素抵抗和主要研究者3 K/Akt通路的活化状态。

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.