The long term goal is to elucidate the cellular and molecular basis for the pathological effects of fatty acids. The specific pathologic effect chosen for study is insulin resistance in adipocytes. Insulin resistance is a risk factor associated with non-insulin dependent diabetes, stroke, cardiovascular disease and possibly cancer. Obesity has been identified as a risk factor for insulin resistance, and the strongest association of obesity with insulin resistance is in subjects with upper body obesity. This type of obesity is more likely to be associated with stroke, non- insulin dependent diabetes mellitus, cardiovascular disease and hypertension. Increased dietary fatty acids are strongly associated with obesity and insulin resistance in fat cells, and the development of fatty acid induced insulin resistance in adipocytes is critical to the development of insulin resistance in insulin target cells. However, the conditions required for the induction of fatty acid insulin resistance remain undefined, and the cellular mechanism(s) by which the fatty acids induce insulin resistance are unknown. The strategy of the proposal is to use an integrated approach of increasing complexity in which univariant in vitro molecular studies will form the basis for analysis of interactive effects, analysis of dietary effects in the rat, and finally analysis of fatty acid induced insulin resistance in human adipocytes. In the first stage, the prototypic long chain saturated fatty acid, palmitate, will be used to induce insulin resistance in two established in vitro rat adipocyte models. Initial studies will determine the concentration dependency and time course for induction of insulin resistance. The key issues of the potential reversibility of the fatty acid induced insulin resistance and the role of lipoproteins in the presentation of fatty acids will also be determined at this stage. The conditions defined will be used to determine the effects of palmitate on key elements of the insulin signalling-glucose transport pathway that have been previously implicated in the development of insulin resistance, including the insulin receptor (expression, ligand binding, activation, internalization) and GLUT4 (expression, translocation and activation). The mechanistic role of fatty acids will then be assessed, including acylation and inhibition of phosphorylation of proteins and effects on intracellular calcium levels. The results obtained with palmitate then will be used to prioritize similar experiments designed to determine the comparative effects of different dietary fatty acids and their possibly interactive roles at the cellular level. These in vitro studies will then be correlated with the analysis of the effects of dietary fatty acids in vivo, using a rat model in which predisposing factors and diet can be readily controlled. Finally, the relevance of these findings will be determined by analysis of the effects of fatty acids at the cellular level in abdominal adipocytes obtained from normal human subjects. Obesity is a major health problem in the U.S.A. Approximately 34 million Americans including men, women and children, and all ethnic groups are afflicted with obesity, and the health care costs associated with obesity have been estimated as $39 billion annually. This proposal provides a feasible approach to the analysis of the pathological consequences of obesity and will provide essential information pertinent to both dietary recommendations, determination of risk factors and design of potential therapies to avoid the pathological consequences of high fat diets.