Changes in the sensitivity of target tissues to hormones, as opposed to changes in the level of circulating hormone, have emerged in the last two decades as major causes of endocrinological disease. This is particularly true for the insulin resistance seen with the secondary forms of diabetes, precipitated by circulating hormones and metabolic factors. The purpose of this project is to study the regulation of hormonal responsiveness in cultured cells in order to better understand the molecular mechanisms of these and similar diseases. We have used 3T3-L1 cultured adipose cells to develop new in vitro models for the insulin resistance seen with glucocorticoid excess and saturated fat feeding. The mechamism of resistance and the metabolic consequences of these models will be explored in detail. We will then determine whether these models can be extended to other cultured cells including fibroblasts and liver cells. We will study the development of catecholamine responsiveness when 3T3-L1 cells differentiate into the fatty phenotype. In vitro models will be developed for the effects of growth hormone and thyroid hormone on the response to insulin and catecholamines. In each of these systems we will assess both the binding of hormone to its receptor and post-receptor metabolic events. The number of receptors and the affinity for hormone will be measured using radioactively labeled hormones in radioreceptor assays. Insulin action will be studied by measuring glucose uptake, glucose oxidation, lipogenesis and inhibition of lipolysis. Catecholamine action will be studied by measuring lipolysis and the generation of cyclic AMP in intact cells. Plasma membranes will be prepared in order to measure the adrenergic receptor and adenylate cyclase. In our model of insulin resistance caused by the feeding of saturated fatty acids, we will determine the spectrum of fatty acids involved and correlate their effects with their actual incorporation into triglyceride and phospholipid. In our models for the regulation of hormonal responsiveness by heterologous hormones, we will study the induction or repression of cellular and membrane proteins by incorporating radioactive precursors into proteins and analysis by gel electrophoresis.