The isolated, perfused rat liver and livers in vivo will be used to investigate the mechanism and regulation of protein and RNA degradation. One current theory is that the degradation of resident proteins in hepatocytes is accomplished by two types of autophagy--an overt form which is actively regulated by specific amino acids, insulin, and glucagon, and a microautophagic process which appears to be influenced by the state of cytoplasm during starvation and refeeding: the latter, for example, has been shown to decrease gradually during cytoplasmic involution and to fall even further when cytoplasm is rapidly regained. Both types are known to internalize protein, and there is strong electron microscopic evidence indicating that ribosomes are sequestered as well. In continuing our liver perfusion studies on acute proteolytic regulation, we plan to make selective, graded deletions of the 7 regulatory amino acids from a normal plasma mixture and to measure immediate proteolytic responses. Recent evidence indicates that the action of glucagon is mediated by the depletion of intracellular glucogenic amino acids, two of which, glutamine and proline, have regulatory activity. We also plan to investigate the nature of a short lived component in valine incorporation and release and to use this information to devise a procedure for measuring the synthesis of resident proteins. We propose to investigate RNA degradation and its regulation by amino acids, insulin, and glucagon in perfused livers previously labeled with (6-14C)orotic acid and to follow the process by the release of label in Beta-alanine. Studies are planned to localize and measure the quantity of RNA internalized in lysosomes and to correlate the latter with rates of RNA degradation. If successful, we hope to apply this information to determine rates of RNA degradation and synthesis during starvation and refeeding. It is conceivable that the starvation-induced decrease in RNA content and total liver protein synthesis is mediated by lysosomal activity and thus would be subject to regulation by amino acids and hormonal agents that control overt autophagy.
