Beta-Oxidation and Susceptibility to Fatty Liver Disease

描述(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尚不清楚。有证据表明，NAFLD的发病机制包括游离脂肪酸水平升高、天然免疫基因激活和胰岛素抵抗。尽管线粒体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。纯合子小鼠会遭受新生儿死亡。我们的初步数据证明，老化的杂合子小鼠会患上与氧化应激和胰岛素抵抗相关的肝脏脂肪变性。这项建议使用这个小鼠模型来研究NAFLD的潜在机制。我们的中心假设是，MTP的杂合子通过增加细胞氧化应激而导致胰岛素抵抗和肝脏损伤，并与肝脏脂肪变性相关。我们建议进行以下特定目标的研究：1)测试MTP缺陷的杂合性导致与年龄相关的脂肪变性和超氧化物过量产生，导致抑制KKK(IKK)激活、胰岛素抵抗和肝脏损伤，并测试在杂合子小鼠中预防氧化应激既防止胰岛素抵抗又防止肝脏损伤。将进行研究，以确定氧化应激与肝脏脂肪变性/损伤、胰岛素抵抗和IKK之间的时间关系。我们还建议进行干预性研究，通过使用Tempoll清除超氧化物歧化物质，或将我们的MTP杂合子小鼠与高表达超氧化物歧化酶的转基因小鼠杂交，以防止氧化应激。2.测试IKK的激活是否导致a)NFkB的激活导致轻度炎症和肝损伤，以及b)IRS依赖的主要调查者3K-Akt通路的激活导致MTP缺陷小鼠的胰岛素抵抗，并测试抑制IKK是否逆转胰岛素抵抗和肝损伤。为此，我们将检测肝脏和肌肉中IKK和NFkB的含量和激活状态，并评估促炎症细胞因子在肝脏中的表达情况。我们还建议测量肝脏和肌肉中Akt的基础含量和胰岛素刺激下的含量以及磷酸化/激活状态，以及上游信号分子Prime Investigator 3K和IRS-1/2。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-正常血糖钳夹来评估。为了测试IKK、胰岛素抵抗和肝脏炎症反应之间的致病关系，将用已知的IKK抑制剂乙酰水杨酸(ASA)治疗小鼠，然后评估NFkB的激活、肝脏炎症、胰岛素抵抗和主要研究者3K/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.