Early stages of Alcoholic Liver Disease (ALD) are characterized by the accumulation of lipid within hepatocytes and is collectively referred to as steatosis. It is known that alterations in lipid homeostasis occur in chronic ethanol conditions, and lipid binding proteins that are known to aid in the uptake and trafficking of these lipids have not been investigated. The experiments proposed are designed to test the hypothesis that chronic ethanol consumption leads to the covalent modification of liver fatty acid binding protein (L-FABP) by 4-HNE and 4-ONE, which ultimately leads to the disruption of lipid metabolism and pathogenesis of steatosis in ALD. The first phase of the research plan addresses if L-FABP is covalently modified by 4-HNE and 4-ONE, and what are the physiological and structural consequences ofthis modification. Using in vitro incubation assays, immunoblotting, and-LC-MS/MS and MALDI-TOF-TOF mass spectrometry, covalently modified protein side-chains will be identified. Activity assays will also be conducted to assess the physiological consequences of these modifications on lipid binding. In addition, protein bound lipid will be investigated in the formation of lipid hydroperoxides in an in situ model of oxidative stress. The second phase of the research involves the use of chronic ethanol feeding models in genetically modified stocks of mice, where L-FABP knockout and L-FABP transgenic stocks will be used. Using standard indices of liver injury (ALT, triglyceride content, GSH:GSSG ratios, CYP2E1 activity, and histology), the model will elucidate if L-FABP attributes to the accumulation of lipid in the liver during early phases of ALD. Also, pathways in lipid uptake and trafficking will be evaluated to characterize the model by utilizing techniques involving RT-PCR, immunoblotting, and immunohistochemistry. The data provided by this application will provide further insight into the mechanisms involving 4-HNE and 4-ONE toxicity, and the effects they have on lipid homeostasis in early ALD.
