Amino acid control of autophagic sequestration and protein degradation in isolated rat hepatocytes.

Abstract

Sequestration of the inert cytosolic marker [ 14C]sucrose by sedimentable organelles was measured in isolated rat hepatocytes made transiently permeable to sucrose by means of electropermeabilization . Lysosomal integrity, protein degradation, autophagic sequestration, and other cellular functions were not significantly impaired by the electric treatment .Hepatocytes sequestered sucrose at an initial rate of -10%/h, which is threefold higher than the estimated rate of auto phagic-lysosomal protein degradation . Almost one-third would appear to represent mitochondrial fluid uptake ; the rest was nearly completely and specifically inhibited by 3-methyladenine (3MA) and can be regarded as autophagic sequestration . A complete amino acid mixture was somewhat less inhibitory than 3MA, and partially antagonized the effect of the latter . This paradoxical effect, taken together with the high sequestration rate, may suggest heterogeneity as well as selectivity in autophagic sequestration . There was no detectable recycling of sequestered [ 14C]sucrose between organelles and cytosol .Studies of individual amino acids revealed histidine as the most effective sequestration inhibitor . Leucine may have a regulatory function, as indicated by its unique additive/ synergistic effect, and a combination of Leu + His was as effective as the complete amino acid mixture . Asparagine inhibited sequestration only 20%, i .e., its very strong effect on overall (long-lived) protein degradation must partially be due to post-sequestrational inhibition.The lysosomal (amine-sensitive) degradation of short-lived protein was incompletely inhibited by 3MA, indicating a contribution from nonautophagic processes like crinophagy and endocytic membrane influx. The ability of an amino acid mixture to specifically antagonize the inhibition of short-lived protein degradation by AsN + GIN (but not by 3MA) may suggest complex amino acid interactions at the level of fusion between lysosomes and other vesicles in addition to the equally complex interactions at the level of autophagic sequestration .Rat hepatocytes offer a particularly suitable material for the study of intracellular protein degradation . These cells are in positive protein balance in well-fed animals (13) or in a culture medium supplied with insulin and high amino acid concentrations (20, 26), but upon starvation in vivo (or amino acid deprivation in vitro), the hepatocytes rapidly turn on their autophagic-lysosomal degradation pathway and enter a state of negative protein balance in an attempt to supply the organism (or medium) with amino acids (13,14,26,27,33). The overall rate of protein degradation may be as high as 4-5%/h, of which the autophagic-lysosomal pathway accounts for -3%/h on the basis of the extent of inhibition seen with amino acids and lysosome inhibitors (32, 35). The remaining -1 .5% (corresponding to the "basal" degradation generally THE JOURNAL OF CELL BIOLOGY " VOLUME 99 AUGUST 1984 435-444 0 The Rockefeller University Press -0021-9525/84/08/435/10 $1 .00 ...

The Phosphatidylinositol 3‐Kinase Inhibitors Wortmannin and LY294002 Inhibit Autophagy in Isolated Rat Hepatocytes

“…The maximal rate of amino-acid-sensitive sucrose sequestration was 5% of the cytosolic volume in 60 min. Amino-acidinsensitive uptake of ['T]sucrose (60% of total sequestered sucrose) is due to accumulation of sucrose in mitochondria [18]. Values are the means (?…”

“…100 nM wortmannin (W), the 4Xcomplete mixture of amino acids (AA) or 5 mM 3-methyladenine (3MA). After 60 min of incubation, ["C]sucrose sequestration was measured as in [18]. The maximal rate of amino-acid-sensitive sucrose sequestration was 5% of the cytosolic volume in 60 min.…”

Untitled

“…High concentrations of amino acids do not, however, completely inhibit autophagic sequestration of [ 14 C]sucrose (Seglen & Gordon, 1984), even though they completely inhibit overall autophagic proteolytic flux. This indicates that amino acids also interfere with post-sequestrational steps.…”

“…Electron microscopic studies (Schworer & Mortimore, 1979;Kovács et al, 1981) and data obtained with electro-injected cytosolic [ 14 C]sucrose and other sugars (Seglen & Gordon, 1984;Seglen & Bohley, 1992) have led to the conclusion that the inhibition of macroautophagy by amino acids primarily occurs at the sequestration step. High concentrations of amino acids do not, however, completely inhibit autophagic sequestration of [ 14 C]sucrose (Seglen & Gordon, 1984), even though they completely inhibit overall autophagic proteolytic flux.…”

Application and interpretation of current autophagy inhibitors and activators

“…Physiologically, autophagy is induced by amino acid deprivation [18] . Electron microscopy studies have indicated that complete deprivation of serum and amino acids provides a useful model for the further study of cellular autophagy [19] .…”