Non-alcoholic fatty liver disease (NAFLD) is the most common form of liver disease in the United States. The high prevalence of NAFLD is largely on the basis of relentless increases in the prevalences of obesity and insulin resistance. The basis of progressive histological injury in a proportion of patients with NAFLD is poorly understood. The overall goals of this project are to 1) develop a model to identify patients with more histologically severe grades of NAFLD and 2) better define the pathophysiological basis of histological progression of NAFLD. Two hypotheses and specific aims have been developed to achieve these goals. Our first hypothesis is that indices of insulin sensitivity, hyperinsulinemia and/or subclinical inflammation can be used to generate a model capable of identifying patients at increased risk for more histologically severe grades of NAFLD. The first specific aim is to prospectively screen a panel of biological parameters for their ability to identify patients at risk for histologically severe NAFLD, as measured by NAFLD grade. In a pilot study of patients with varying degrees of severity of NAFLD, we found that more histologically severe NAFLD is independently associated with greater percent body fat, higher C-reactive protein and fasting insulin levels. Strong trends for an association with severity of NAFLD grade were also seen for higher IGFBP-1 levels, % truncal fat, HGH levels, and continuous glucose monitoring profiles. We now propose to carry out detailed assessments of these and other markers of subclinical inflammation, insulin sensitivity, insulin-like growth factor axis and assessments of anthropometry in patients with grades 1-3 of NAFLD and in controls. Our second hypothesis is that the inflammation and progressive fibrosis that can occur in NAFLD are substantially due to increased oxidative stress. The second specific aim is to determine the mechanism and effects of increased oxidative stress in NAFLD. Oxidative stress is thought to be central to hepatocellular injury in NAFLD. Our preliminary data demonstrate a possible pre-transcriptional basis of increased oxidative stress in patients with more histologically progressive NASH. In a logical extension of these experiments, we now propose to use high-throughput mass spectrometry analysis, using an ICAT tagging technique, to measure differential hepatic protein abundance, including of peroxisomal proteins, in patients with NAFLD grades 1-3, and also in controls. We believe that these experiments will generate mechanistic insight into the cause and effects of increased oxidative stress in patients with histologically progressive NAFLD.