scholarly journals Effects of amino acids, ammonia and leupeptin on protein synthesis and degradation in isolated rat hepatocytes

1978 ◽  
Vol 174 (2) ◽  
pp. 469-474 ◽  
Author(s):  
P O Seglen
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Protein Degradation ◽  
Specific Radioactivity ◽  
Rat Hepatocytes ◽  
Amino Acid Mixture ◽  
Acid Mixture ◽  
Isolated Rat Hepatocytes ◽  
Lysosomal Protein ◽  
Isolated Rat

Protein synthesis in isolated rat hepatocytes, as measured by the incorporation of [14C]-valine at constant specific radioactivity, proceeded at a rate of 0.3-0.5%/h in an unsupplemented medium, i.e. only about one-tenth the rate of protein degradation (4%/h). Leupeptin, which inhibits lysosomal protein degradation (previously found to be 75% of the total degradation in hepatocytes), had no effect on protein synthesis, showing that endogenous protein degradation supplied amino acids in excess of the substrate requirements for protein synthesis. The inhibition of protein synthesis by NH4Cl (another inhibitor of lysosomal protein degradation) as well as the stimulation by a physiological amino acid mixture must therefore represent indirect effects, either on general energy metabolism, or on unknown regulatory processes.

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

1984 ◽  
Vol 99 (2) ◽  
pp. 435-444 ◽  
Author(s):  
P O Seglen ◽  
P B Gordon
Keyword(s):  
Amino Acid ◽  
Protein Degradation ◽  
Rat Hepatocytes ◽  
Amino Acid Mixture ◽  
Regulatory Function ◽  
Acid Mixture ◽  
Cellular Functions ◽  
Isolated Rat Hepatocytes ◽  
Fluid Uptake ◽  
Isolated Rat

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 approximately 10%/h, which is threefold higher than the estimated rate of autophagic-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.

scholarly journals Use of digitonin extraction to distinguish between autophagic-lysosomal sequestration and mitochondrial uptake of [14C]sucrose in hepatocytes

1985 ◽  
Vol 232 (3) ◽  
pp. 773-780 ◽  
Author(s):  
P B Gordon ◽  
H Tolleshaug ◽  
P O Seglen
Keyword(s):  
Rat Hepatocytes ◽  
Amino Acid Mixture ◽  
Sugar Uptake ◽  
Acid Mixture ◽  
Detectable Effect ◽  
Isolated Rat Hepatocytes ◽  
Assay Procedure ◽  
Low Concentrations ◽  
Extraction Step ◽  
Isolated Rat

In isolated rat hepatocytes, electroinjected [14C]sucrose is sequestered both by mitochondria and by autophagosomes/lysosomes. Radioactivity can be selectively extracted from the latter organelles by low concentrations of digitonin, thereby providing a specific bioassay for autophagic sequestration. By including a digitonin extraction step in the assay procedure, autophagic [14C]sucrose sequestration could be shown to be virtually completely (greater than 90%) suppressed by the autophagy inhibitor 3-methyladenine (10 mM), whereas mitochondrial sugar uptake was unaffected. An amino acid mixture likewise suppressed autophagic sequestration very strongly, while having no detectable effect on the mitochondria.

Effects of temperature on protein turnover in isolated rat skeletal muscle

1984 ◽  
Vol 246 (1) ◽  
pp. C125-C130 ◽  
Author(s):  
V. E. Baracos ◽  
E. J. Wilson ◽  
A. L. Goldberg
Keyword(s):  
Protein Synthesis ◽  
Protein Degradation ◽  
Muscle Protein ◽  
Rat Hepatocytes ◽  
Isolated Rat Hepatocytes ◽  
Body Protein ◽  
Influence Of Temperature On ◽  
Net Protein ◽  
Isolated Rat ◽  
Stimulation Of

To understand the net loss of muscle protein during fever and the possible changes in body protein balance with hyperthermia, we investigated the influence of temperature on protein synthesis and degradation in rat skeletal muscles. In the incubated soleus, extensor digitorum longus, or diaphragm, net protein degradation increased by about 11%/degrees C between 33 and 42 degrees C. This loss of muscle protein resulted from an increase in protein degradation (172% between 33 and 42 degrees C). By contrast, protein synthesis increased by only 25% between 33 and 39 degrees C and fell markedly by 42 degrees C. Unlike muscle, in isolated rat hepatocytes, protein breakdown did not increase significantly between 36 and 39 degrees C. The stimulation of protein degradation between 36 and 39 degrees C was not reduced by leupeptin or Ep-475, which inhibit lysosomal thiol proteases and reduce net protein degradation in the incubated muscles. Prostaglandin E2 (PGE2) has been implicated in the accelerated muscle proteolysis during fever. However, PGE2 release by muscles was unchanged between 33 and 42 degrees C, and inhibition of PGE2 synthesis by indomethacin did not reduce the stimulation of proteolysis at 40 degrees C. This catabolic effect of increased temperature may contribute to the negative nitrogen balance during fever.

An abundant supply of amino acids enhances the metabolic effect of exercise on muscle protein

1997 ◽  
Vol 273 (1) ◽  
pp. E122-E129 ◽  
Author(s):  
G. Biolo ◽  
K. D. Tipton ◽  
S. Klein ◽  
R. R. Wolfe
Keyword(s):  
Amino Acids ◽  
Blood Flow ◽  
Protein Synthesis ◽  
Amino Acid ◽  
Muscle Protein ◽  
Muscle Protein Synthesis ◽  
Amino Acid Mixture ◽  
Acid Mixture ◽  
Protein Kinetics ◽  
Amino Acid Infusion

Six normal untrained men were studied during the intravenous infusion of a balanced amino acid mixture (approximately 0.15 g.kg-1.h-1 for 3 h) at rest and after a leg resistance exercise routine to test the influence of exercise on the regulation of muscle protein kinetics by hyperaminoacidemia. Leg muscle protein kinetics and transport of selected amino acids (alanine, phenylalanine, leucine, and lysine) were isotopically determined using a model based on arteriovenous blood samples and muscle biopsy. The intravenous amino acid infusion resulted in comparable increases in arterial amino acid concentrations at rest and after exercise, whereas leg blood flow was 64 +/- 5% greater after exercise than at rest. During hyperaminoacidemia, the increases in amino acid transport above basal were 30-100% greater after exercise than at rest. Increases in muscle protein synthesis were also greater after exercise than at rest (291 +/- 42% vs. 141 +/- 45%). Muscle protein breakdown was not significantly affected by hyperminoacidemia either at rest or after exercise. We conclude that the stimulatory effect of exogenous amino acids on muscle protein synthesis is enhanced by prior exercise, perhaps in part because of enhanced blood flow. Our results imply that protein intake immediately after exercise may be more anabolic than when ingested at some later time.

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