The isolated, perfused rat liver and liver in vivo will be used to investigate the regulation of protein and RNA degradation in the hepatocyte. There in now convincing evidence that the degradation of resident (long-lived) proteins and probably cytoplasmic RNA as well is accomplished by two types of ongoing autophagy: a macro form, which is actively regulated by specific amino acids, insulin, and glucagon, and microadutophagy, which appears to be passively regulated according to the amount of endoplasmic reticulum. The latter process, for example, decreases during starvation in parallel with cytoplasmic involution. Both types sequester protein and RNA and maintain degradable intralysosomal pools that are directly proportional to rates of degradation in the cell and turn over with a tl/2 of about 8 min. Recent studies have shown that the primary regulation of macroautophagy involves the concerted, multiphasis action of 7 amino acids (Leu, Tyr/Phe, Gln, Pro, His, Trp) in which responses (both positive and negative) are strongly concentration-dependent. Of unusual interest is the fact that alanine, which is not inhibitory itself, is required for the expression of inhibition by the regulatory group at normal plasma concentrations. Insulin selectively eliminates the alanine requirement while glucagon specifically abolishes the initial inhibitory response to amino acids. Since leucine is poorly metabolized, it must act through free leucyl-tRNA. We have shown that L-alpha- hydroxyisocaproate can mimic leucine's regulatory effects, and are proposing to investigate effects of alpha-chloro analogues as well. We hope to determine whether the regulation is mediated through specific recognition sites; if so, appropriate amino acid binding to membrane proteins will be sought. Additional studies will be carried out to determine whether alanine's permissive effect is mediated metabolically or through a specific site of recognition. The liver's sensitivity to alanine is low early in the postabsorptive period and appears to increase abruptly by midday. Studies are planned to explore the possible role of the A system transport in this phenomenon. We also plan to investigate the role of protein feeding on microautophagy and basal protein and RNA turnover and to devise a direct approach to the measurement of RNA synthesis in the isolated liver.
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