Abstract
Administration of leupeptin to rats induces the accumulation of numerous autophagic vacuoles in the liver. Furuno et al. (Furuno, K., Ishikawa, T., and Kato, K. (1982) J. Biochem. (Tokyo) 91, 1485-1494) have recently devised a method for Percoll density gradient equilibrium fractionation of crude lysosomal fractions to isolate a highly enriched preparation of autophagic vacuoles. This system was used to determine whether cytoplasmic enzymes are normally sequestered into autophagic vacuoles in fed animals. Within 30 min following the administration of leupeptin to fed rats, several cytoplasmic enzymes could be demonstrated in vacuolar fractions heavier than mitochondria and normal lyosomes. The activities of tyrosine aminotransferase and lactic dehydrogenase as well as antigens of fructose-bisphosphate aldolase were detectable in fractions with densities of 1.115 to 1.15 g/ml containing cathepsins and acid phosphatase. The cytoplasmic enzymes in these fractions exhibited latency and were sequestered within membranous organelles. Six hours after the administration of leupeptin, the autophagic vacuoles gradually disappeared from these fractions concurrently with the loss of both cytoplasmic and lysosomal marker enzymes. For 6 h after injection of leupeptin the activities of cathepsin D and acid phosphatase increased in autophagic vacuoles and decreased in the postvacuolar lysosomal fraction. Administration of dexamethasone, which induces the synthesis of tyrosine aminotransferase and cytosolic aspartate aminotransferase, selectively increased the sequestration of these enzymes to proportional degrees. Cycloheximide administered simultaneously with leupeptin rapidly inhibited formation of autophagic vacuoles and the sequestrations of both cytoplasmic and lysosomal enzymes. However, when cycloheximide was administered 1 h after leupeptin, the formation of autophagosomes and the sequestration of cytoplasmic enzymes were inhibited but the vacuolar uptake of acid phosphatase and cathepsin D continued to increase for several hours. When cycloheximide was injected 1 h after leupeptin, losses of lactic dehydrogenase and aldolase proteins were observed in autophagic vacuoles isolated 1 and 2 h later.
Highlights
Administration of leupeptin to rats induces the accumulation of numerous autophagic vacuoles in the liver.Furuno et al
Cycloheximide administered simultaneously with leupeptin rapidly inhibited formation of autophagic vacuoles and the sequestrations of both cytoplasmic and lysosomal enzymesH. owever, when cycloheximide was administered 1 h after leupeptin, the formation of autophagosomes and the sequestrationof cytoplasmic enzymes were inhibited but the vacuolar uptake of acid phosphatase and cathepsin D continued to increase for severhaolurs
Autophagy and proteolysis in the liver are enhanced by deprivation of amino acids and insulin (1-3) and by the administration of glucagon (4-6), and these enhancements are reversed by administration of amino acids and insulin (2, 7,8)
Summary
Furuno et al (9) showed that administration of leupeptin, a nonreversible inhibitor of lysosomal thiol proteinases, caused the appearance and persistence of numerous autophagic vacuoles in the liver. They suggested that because of the retarded digestion of sequestered materials, the autophagic vacuolesconstitute most of the lysosomal population. This paper describes changes in activities and properties of cytoplasmic enzymes sequestered into autophagic vacuoles accumulated in the liver followingthe administration of leupeptin. Percoll was obtained from Pharmacia Fine Chemicals. [I-”C]Valine (40-60 mCi/mmol) was obtained from New England Nuclear. Nitrocellulose membranes were from Bio-Rad. Horseradish peroxidase-conjugated second antibodies (rabbit anti-goatIgG and goat anti-rabbit I&) were obtained from Cappel Laboratories, Inc
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