The long term objective of the proposed research is to gain an understanding of the mechanism by which the glucocorticoids and glucagon regulate hepatic amino acid transport. Amino acids, particularly alanine released from muscle, are the major source of carbon for hepatic gluconeogenesis during fasting. In diabetes, the elevated blood glucose is partially owing to the overproduction of glucose by liver for which amino acids are a major substrate. The transport of amino acids into liver parenchymal cells is subject to regulation by several hormones and by the intracellular level of amino acids. The coordinate control of transport activity by the glucocorticoids and glucagon comprises a portion of the integrated response of the hepatocyte to hormonal signals resulting in the stimulation of gluconeogenesis. The mechanisms by which these two classes of hormones interact to regulate transport have not been defined. The elucidation of these mechanisms will ultimately require working with purified components of the transport system. To this end our specific aims are: 1. Identify the hormonally regulated amino acid transport system(s) in rat liver parenchymal cells and parenchymal cell-derived plasma membrane vesicles. 2. Characterize the hormonally regulated amino acid transport system using isolated plasma membrane vesicles from rat liver parenchymal cells. 3. Carry out the isolation and molecular identification of the hormonally regulated amino acid transport system in rat liver parenchymal cells. Liver parenchymal cells will be isolated by a collagenase perfusion technique. Amino acid transport will be studied in suspension cultures and in cells maintained in primary cultures. Plasma membrane vesicles will be prepared from both sets of cells. The hormonally regulated transport system(s) will be identified by examining the transport of system specific amino acids and will be characterized with respect to temperature, pH, ionic and energy requirements utilizing transport-competent membrane vesicles. Three protocols will be used to label the transport system with radioactive amino acids. The transport protein(s) will be isolated by using selective extraction and chromatographic procedures. The protein will be identified by following radioactivity distribution and confirmed by reconstituting the protein into transport-competent lipid vesicles.