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. [unreadable] [unreadable] [unreadable]