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Biomedical subjects

G E Mortimore

Publications and source records attributed to G E Mortimore.

At least 55 records · Page 3Linked to original sources
PubMed1982

Suppression of cytoplasmic protein uptake by lysosomes as the mechanism of protein regain in livers of starved-refed mice.

The suppression of proteolysis that normally accompanies cytoplasmic growth was investigated in livers of mice that had lost approximately 40% of their protein content during 48 h of starvation. The deficit was fully restored after 24 h of refeeding, and the net regain was linear between 12 and 24 h. Rates of protein breakdown were determined from (a) differences between synthesis and the net change in total liver protein, and (b) rates of valine release during 15-min in situ perfusions in the presence of cycloheximide. With appropriate correction for the turnover of a short lived pool, both procedures gave the same results; rates at 12 and 24 h of refeeding were decreased 90% over values in fed controls, an effect which accounted for 93% of protein regrowth. Measurements of degradable, intralysosomal protein revealed that sequestration of cytoplasmic protein by lysosomes was correspondingly decreased. Because the ratio of intracellular proteolysis to internalized protein was the same during refeeding as in earlier experiments where autophagy was the dominant process, the uptake of cytoplasmic proteins by lysosomes appears to be an obligatory step in proteolysis at all levels of regulation. The 20-fold range in rates of degradation exhibited by the mouse hepatocyte thus provides this cell with an unusual capability for regulating its protein content against relatively small changes in protein synthesis.

Animals↗
PubMed1982

Degradation of intra- and extrahepatic protein by livers of normal and diabetic mice: differential responses to starvation.

Rates of total hepatic proteolysis were measured in normal and streptozotocin-diabetic mice during feeding and over 48 hr of starvation, during which livers in the two groups lost 37% and 54% of their protein content, respectively. Measurements were made in 15-min in situ cyclic perfusions from the linear accumulation of free valine in the presence of cycloheximide; rates were corrected for turnover of short-lived proteins because these components contribute negligibly to alterations in liver protein content. During deprivation, corrected rates, expressed on a per liver basis, remained constant in normal mice but increased markedly in the diabetic group, attaining twice prestarvation values by 48 hr. By contrast, degradation rates of long-lived intracellular proteins, calculated from the sum of their synthesis and the linear decrease in protein content, decreased predictably in both groups and in parallel with absolute rates of protein synthesis. The extra proteolysis, representing the difference between total and long-lived protein degradation, was small in fed animals but increased progressively during starvation. With diabetic mice, however, the increase was approximately 5 times that of the normal and, in absolute terms, roughly equaled the total loss of liver protein. We suggest that this fraction arose from intrahepatic breakdown of proteins that were ultimately derived from sources outside the liver. Acceleration of this novel process could play an important interim role in providing endogenous glucogenic substrate under conditions in which the demand for this substrate is high.

Animals↗
PubMed1981

Quantitative relationship between autophagy and proteolysis during graded amino acid deprivation in perfused rat liver.

Fractional volumes of lysosomal-vacuolar elements and long lived protein degradation were quantitatively correlated in rat livers perfused in the single pass mode with varying levels of plasma amino acids. Volumes were determined stereologically; degradation was measured in a second stage cyclic perfusion from the linear accumulation of valine in the presence of cycloheximide and was corrected for loss of short lived proteins. Livers exhibited a high degree of amino acid responsiveness; total protein degradation decreased sharply from 4.5 to 1.5%/h (basal) over an amino acid range of 0-10 times (10X) normal plasma concentrations; near basal values were achieved at 1X. Vacuoles containing undegraded cytoplasm (AVi) appeared immediately following stringent deprivation and by 7.5 min were converted to degradative forms (AVd); both autophagic populations attained steady state volumes by 20 min. With amino acid additions, AVi formation virtually ceased and AVd regressed rapidly (0.087 min-1). Cytoplasmic turnover, calculated from this rate constant and the increases over basal in fractional volumes of either AVi or degradative components, agreed quantitatively with corresponding rates of protein turnover. Predictions from these findings, together with evidence for intralysosomal protein pools in both deprived and basal states, account fully for the accelerated proteolysis and suggest that cytoplasm is also internalized by lysosomes under basal conditions.

Amino Acids↗
PubMed1981

Internalization of cytoplasmic protein by hepatic lysosomes in basal and deprivation-induced proteolytic states.

The hypothesis that cytoplasmic protein in rat liver is internalized and degraded intralysosomally during basal or steady state turnover as well as in deprivation-accelerated states was tested by (a) determining the subcellular location of degradable protein in homogenates and (b) comparing the amounts of this protein pool with rates of long lived protein degradation in livers perfused under the above conditions. Greater than 95% of total proteolysis in homogenates, measured at 37 degrees C from free amino acid generation or the release of [14C]valine in previously labeled livers, was associated with particulate fractions, and it paralleled lysosomal markers closely during graded differential centrifugation after Triton WR-1339 loading. Because 14C-labeled cytosolic proteins were not degraded by intact isolated lysosomes at pH 6 or 7, the protein substrate must have been internalized by lysosomes prior to homogenization. Time courses of intralysosomal proteolysis from maximal deprivation down to and including basal were identical in shape, and each leveled off abruptly after 120-150 min. Although the velocities of proteolysis were reduced in vitro, estimates of the degradable protein pools were closely proportional to corresponding rates of long lived degradation in perfused livers. In absolute terms, they fit recent predictions of internalized cytoplasmic protein based on stereological measurements of lysosomal volumes.

Amino Acids↗
PubMed1981

Internalization of cytoplasmic protein by lysosomes as the mechanism of resident protein turnover in liver.

Long lived, resident proteins comprise more than 99% of the cytoplasmic protein content in rat liver. In perfusion experiments, their overall rate of degradation can be varied from 4.5-5.0%/h with glucagon or stringent amino acid depletion to 1.5%/h with additions of 4-10 times normal plasma amino acids. The aggregate volume of degradative lysosomal components and their content of degradable protein, estimated in homogenate experiments, were found to relate directly to rates of protein breakdown from maximal down to and including the basal state. The rate constant of autophagic vacuole regression, estimated stereologically after stopping their formation with amino acids, was 0.087/min (t1/2 = 8.0 min), and the turnover of cytoplasmic volume during deprivation, calculated from this constant and increases over basal in either newly induced autophagic vacuoles (AVi) or secondary degradative forms, agreed quantitatively with corresponding rates of protein turnover. The fact that the time courses of intralysosomal proteolysis in liver homogenates were identical, despite large differences in the quantity of internalized protein degraded, suggests that the rate constant of intralysosomal digestion in intact hepatocytes is invariant over the full range of regulation. Our findings thus support the hypothesis that all resident proteins are sequestered and degraded by the lysosomal-vacuolar system.

Amino Acids↗
PubMed1979

Application of liver perfusion as an in vitro model in studies of intracellular protein degradation.

Amino acids appear to be prime regulators of autophagy and proteolysis in liver. They both attain a maximum rapidly when livers from fed rats are perfused in the single-pass mode without amino acids and are suppressed to basal levels by amino acid additions. The fact that their greatest responsiveness to amino acids occurs slightly below normal plasma levels suggests that these cellular processes could play a role in regulating plasma amino acid concentrations in vivo. Autophagy and proteolysis are also inhibited by insulin and stimulated by glucagon. In the latter instance the hormonal action is not direct but mediated indirectly by depletion of intracellular glutamine, probably as a consequence of enhanced gluconeogenesis. Close correlations among (1) rates of intracellular proteolysis, (2) the aggregate volume of lysosomal elements, and (3) estimates of degradable protein internalized within lysosomes indicate that lysosomal function can explain total intracellular protein degradation (with the possible exception of rapidly turning over fractions) over the full range of proteolysis from maximum down to and including the basal state. Since ratios of degradable intralysosomal protein to corresponding rates of proteolysis in intact liver are constant over this range, protein internalization may be the rate-limiting step in lysosomal proteolysis.

Amino Acids↗
PubMed1979

Glucagon-induced autophagy and proteolysis in rat liver: mediation by selective deprivation of intracellular amino acids.

Amino acid deprivation and glucagon are both potent inducers of autography and proteolysis in liver. Because glucagon enhanced the metabolic utilization of some amino acids, the catabolic response to both of these stimuli could be achieved by a lowering of intracellular amino acid pools. Alternatively, glucagon could act independently of amino acids. To clarify the mode of hormonal action and also the relationship between the two cellular responses, livers from fed rats were perfused, with and without glucagon, with plasma amino acids over a concentration range of 0 to 10 times normal. Individual amino acids constancy at each level was ensured by perfusion in the single-pass mode. Amino acids alone strongly regulated autophagy and proteolysis in a coordinated fashion; maximal suppression was achieved at twice normal concentration; both effects increased rapidly to maximum at less than normal concentration. Corresponding effects of glucagon, however, could be elicited only at intermediate amino acid levels. None was noted at 4 and 10 times normal; at 0, hormonal stimulation was minimal. The amino acid inhibition was selective because it did not block cyclic AMP production or glycogenolysis. Intracellular pool measurements and systematic alteration of perfusate amino acid composition indicated that the autophagic and proteolytic effects of glucagon are mediated by a hormonally induced depletion of glycine, alanine, glutamate, and glutamine; of these, glutamine alone is the most effective. We conclude that the stimulation of intracellular protein degradation in liver is a manifestation of deprivation-induced autophagy which results from a decrease in certain intracellular glucogenic amino acids, notably glutamine.

Amino Acids↗
PubMed1976

Assessment of protein turnover in perfused rat liver. Evidence for amino acid compartmentation from differential labeling of free and tRNA-gound valine.

Total protein synthesis in perfused livers of fed rats was determined by measuring the rate of valine incorporation based on the specific activity of valine attached to tRNA. Rates were not significantly altered when perfusate valine was increased from 0.40 to 5 mM and were similar to values calculated earlier from the specific activity of extracellular valine at a concentration of 15 mM. Overall protein degradation, computed from the sum of the rates of synthesis and the total increase of free intra- and extracellular valine, corresponded closely to the increase of free valine that occurred between 5 and 15 min after the addition of cycloheximide. In the latter experiments advantage was taken of the fact that the previously established suppressive effect of cycloheximide on proteolysis does not begin initially with the inhibition of synthesis, but 15 min later. Thus, the release of valine from 5 to 15 min was assumed to represent rates of protein degradation in effect prior to the addition of cycloheximide. The close agreement found among these independent assessments of protein metabolism thus appears to eliminate much of the previous uncertainty in the quantitation of hepatic protein turnover. In the course of these studies we noted that the specific activity of valyl-tRNA attained steady state values that were intermediate between specific activities of the extracellular and intracellular pools, but appeared to reach a steady state sooner than that of intracellular valine. To evaluate these early events more precisely, the specific activity of valine in tRNA and the intracellular pool was measured in a series of single-pass perfusion experiments where extracellular valine concentration and specific activity were held constant. The intracellular valine specific activity rose with a half-life of 1.2 min. By contrast, the rise in the specific activity of valyl-tRNA was biphasic: the initial phase of the valyl-tRNA curve was rapid, while the second phase had a half-life equal to that of intracellular valine. These data show that at physiological concentrations of valine, valyl-tRNA derives its amino acids from both the extracellular and cytoplasmic pools, and that at least some tRNA is charged by extracellular amino acids before they mix with intracellular amino acid pools, possibly from a precursor pool at or near the cell membrane.

Amino Acids↗
PubMed1976

Behavior of the lysosomal system during organ perfusion. An inquiry into the mechanism of hepatic proteolysis.

In this chapter we have attempted to provide some insight into a mechanism(s) of protein degradation in liver which we believe is implicated in the physiological regulation of free amino acid pools. The association we have observed between regulatory effects of insulin and amino acids in the perfused rat liver and physical alterations of the lysosomal system in similarly treated livers suggests that the final stage of the process is localized within the lysosomal-vacuolar system. The present studies show that perfusion of the isolated rat liver is in itself a potent stimulus to general proteolysis, an effect which is associated with increases in lysosomal size, density and sensitivity to osmotic shock. Since the osmotic and density alterations can be prevented or reversed by the provision of insulin or amino acid mixtures, we believe these agents normally act to restrain some inherent proteolytic process. Although the attendant lysosomal alterations are similar in many respects to those induced by glucagon, they differ sharply from the latter in having no sequestered organelles or evident membrane structures and the fact that they rise in the absence of any increase in the tissue level of cyclic AMP. Aside from the obvious problem of evaluating the quantitative importance of the lysosomal system in hepatic proteolysis, three interesting problems remain to be solved: (1) the nature of the proteins involved, (2) their mode of acquisition by the lysosome, and (3) the relationship of this inherent proteolytic process to glucagon or cyclic AMP-induced cellular autophagy. With regard to the former it seems reasonably clear from our studies and those of others that protein substrate does not gain access to lysosomal proteases by the direct penetration of lysosomal membranes, but more likely by fusion of a preexisting lysosome with some, as yet undefined, membrane-bound element. Whether such an element is formed in a manner analogous to that of the precursor vacuoles in autophagy (Ericsson 1969) or by some other process is not known. Closely related to this is the question of the nature of the substrate and the factors determining its uptake by the lysosome. Space does not permit us to review evidence from a number of laboratories which suggests that the turnover of intracellular protein may be a function of size (Dehlinger and Schimke 1971; Dice et al. 1973), charge (Dice and Goldberg 1975), or conformational stability (Li and Knox 1972; Ballard et al. 1974). We have no idea at the moment whether the mechanism we have outlined will eventually be shown to encompass a wide range of turnover of intracellular proteins or, alternatively, whether it will be limited to a certain class of proteins that turnover as a unit. While there is undoubtedly far more specificity in the regulation of intracellular proteins than our present data indicate, we do not view this as an incompatibility, but rather as another aspect of a complex problem we are only beginning to understand.

Acid Phosphatase↗