Amino acid inhibition of the autophagic/lysosomal pathway of protein degradation in isolated rat hepatocytes

Protein degradation in isolated rat hepatocytes, as measured by the release of [¹⁴C]valine from pre-labelled protein, is partly inhibited by a physiologically balanced mixture of amino acids. The inhibition is largely due to the seven amino acids leucine, phenylalanine, tyrosine, tryptophan, histidine, asparagine and glutamine.When the amino acids are tested individually at different concentrations, asparagine and glutamine are the strongest inhibitors. However, when various combinations are tested, a mixture of the first five amino acids as well as a combination of leucine and asparagine inhibit protein degradation particularly strongly.The inhibition brought about by asparagine plus leucine is not additive to the inhibition by propylamine, a lysosomotropic inhibitor; thus indicating that the amino acids act exclusively upon the lysosomal pathway of protein degradation.Following a lag of about 15 min the effect of asparagine plus leucine is maximal and equal to the effect of propylamine, suggesting that their inhibition of the lysosomal pathway is complete as well as specific.Degradation of endocytosed ¹²⁵I-labelled asialofetuin is not affected by asparagine plus leucine, indicating that the amino acids do not affect lysosomes directly, but rather inhibit autophagy at a step prior to the fusion of autophagic vacuoles with lysosomes.The aminotransferase inhibitor, aminooxyacetate, does not prevent the inhibitory effect of any of the amino acids, i.e. amino acid metabolites are apparently not involved.     

Regulation of RNA degradation in cultured rat hepatocytes: Effects of specific amino acids and insulin

The regulation of RNA degradation by specific amino acids and insulin was investigated in cultured rat hepatocytes from fed rats previously injected in vivo with [6-¹⁴C]orotic acid. The effects of three groups of amino acids were compared to those of a complete amino acid mixture. The first one consisted of the eight amino acids (leucine, proline, glutamine, histidine, phenylalanine, tyrosine, methionine, tryptophan) previously found to be particularly effective in the control of proteolysis. The two other groups were defined from our study with single additions of amino acids, one consisting of proline, asparagine, glutamine, alanine, phenylalanine, and leucine and the other including the latter group with serine, histidine, and tyrosine. The results showed that these three groups were able to strongly inhibit deprivation-induced RNA breakdown at one and ten times normal plasma concentrations but to a lower extent than the complete amino acid mixture. Six amino acids (proline, asparagine, glutamine, alanine, phenylalanine, leucine) inhibited individually RNA degradation by more than 20%. However, the deletions of proline, asparagine, glutamine, or alanine from the group of these six amino acids were not followed by a loss of inhibitory effect. On the contrary, an important loss of inhibition was observed when leucine and phenylalanine were deleted. Furthermore, only these two amino acids exhibited an additive inhibitory effect. Thus leucine and phenylalanine could be considered as important inhibitors of RNA breakdown in cultured rat hepatocytes. Finally, insulin which had no significant effect on RNA degradation in the absence of amino acids, was able to potentiate the inhibitory effect of different amino acid groups. © 1993 Wiley-Liss, Inc.     

Inhibition of hepatocytic protein degradation by methylaminopurines and inhibitors of protein synthesis

Nutritional control of protein degradation in isolated rat hepatocytes can take place in the absence of protein synthesis. Suppression of degradation by amino acids (step-up) is unaffected and the enhanced degradation seen upon amino acid deprivation (step-down) is only partially inhibited by cycloheximide at a concentration (10^?3 M) which inhibits protein synthesis virtually completely. Protein degradation per se is, however, inhibited by cycloheximide as well as by puromycin, apparently at least in part by mechanisms additional or unrelated to their effect on protein synthesis. Several puromycin analogues (methylaminopurines) are stronger inhibitors of protein degradation than of protein synthesis, most notably puromycin aminonucleoside and 6-dimethylaminopurine riboside (N⁶, N⁶-dimethyladenosine). The latter compounds appear to specifically inhibit cellular autophagy, since neither the degradation of endocytosed protein (asialofetuin) nor the extralysosoma (amino acid-, propylamine- and leupeptin-resistant) degradation are affected.     

The effects of bioregulators upon amino acid transport and protein synthesis in isolated rat hepatocytes

Isolated rat hepatocytes prepared by an enzyme perfusion technique possess a functional amino acid transport system and retain the capacity to synthesize protein. Amino acid transport was studied using the non-metabolizable amino acid analog alpha-aminoisobutyric acid. The transport process was time, temperature and concentration dependent. Similarly, leucine incorporation into protein was time and temperature dependent being optimal at 3m degrees C. Amino acid, fetal calf serum, growth hormone and glucose all produced small, reproducible increases in protein synthesis rates. Bovine serum albumin diminished the uptake of alpha-aminoisobutyric acid and leucine incorporation into protein. The amino acid content on either side of the cell membrane was found to affect transport into or out of the cellular compartment (transconcentration effects). High cell concentrations decreased transport and protein synthesis as a result of isotopic dilution of labelled amino acids with those released by the hepatocytes. This was consistent with the capacity of naturally occurring amino aicds to compete with alpha-aminoisobutyric acid for uptake into the hepatocyte. In order to define more precisely the effects of bioregulators on transport and protein synthesis it will be necessary to define and subfractionate cellular compartments and proteins which are the specific targets of cellular regulation.     

Control of proteolysis in perifused rat hepatocytes

The mechanism by means of which amino acids inhibit intrahepatic protein degradation has been studied in perifused rat hepatocytes. Proteolysis was extremely sensitive to inhibition by low concentrations of amino acids. A mixture of 0.5 mM leucine and 1-2 mM alanine, concentrations found in the portal vein of the rat after feeding, inhibited proteolysis to the same extent as a complete physiological mixture of amino acids. Inhibition by these two amino acids was accompanied by a rise in the intracellular concentrations of glutamate and aspartate, and was largely prevented by addition of glucagon, by addition of the transaminase inhibitor aminooxyacetate, or by omission of K+. Acceleration of proteolysis by K+ depletion was accompanied by a fall in intracellular glutamate caused by an increased rate of transport of this amino acid to the extracellular fluid. It is concluded that intracellular leucine, glutamate and aspartate are important elements in the control of hepatic protein degradation.     

Signalling pathways and combinatory effects of insulin and amino acids in isolated rat hepatocytes.

Liver metabolism is influenced by hormones and nutrients. Amino acids such as glutamine or leucine induce an anabolic response, which resembles that of insulin in muscle and adipose tissue. In this work, the signalling pathways and the effects of insulin were compared to those of glutamine and leucine in isolated hepatocytes from normal and streptozotocin-diabetic rats. Glutamine increased cell volume and induced an anabolic response characterized by an activation of acetyl-CoA carboxylase (ACC), glycogen synthase (GS) and p70 ribosomal S6 kinase (p70S6K), the key enzymes in fatty acid, glycogen and protein synthesis, respectively. The effects of glutamine were independent of insulin and did not share its signalling components. Leucine, which is poorly metabolized by the liver and does not modify cell volume, activated ACC and p70S6K, and exerted a synergistic effect on the glutamine-induced activation of ACC and p70S6K. These amino acids did not affect insulin signalling. Insulin alone had no anabolic effect in hepatocytes, despite the activation of protein kinase B. Nevertheless, it enhanced the activation of ACC and p70S6K induced by leucine. However, insulin injected intravenously activated rat liver p70S6K. In hepatocytes from streptozotocin-diabetic animals, the metabolic responses to the amino acids and insulin were similar to those in normal hepatocytes. We conclude that glutamine, insulin and leucine exert different effects that are mediated by different signalling pathways, although their effects are combinatory. The anabolic effect of insulin in hepatocytes was strictly dependent on the permissive action of leucine.     

Amino acid and energy requirements for rat hepatocytes in primary culture

The amino acid and energy requirements of rat hepatocytes in suspension and early culture were investigated. Among a number of potential energy substrates tested, pyruvate (20 mM) was found to be most effective in stimulating hepatocytic protein synthesis. Amino acids stimulated protein synthesis both as energy substrates and as protein precursors. An amino acid mixture was designed to provide maximal inhibition of protein degradation as well as maximal stimulation of protein synthesis. In a defined medium containing amino acids at these concentrations, and supplemented with glucocorticoid hormone and insulin, hepatocytes could be maintained--on a collagen substratum--for at least a week without any significant net loss of cells or cellular protein.     

Protein degradation in cat liver cells.

Body proteins in cats were prelabelled with [14C]valine, and protein degradation was studied in isolated hepatocytes. Amino acids appeared to have a direct inhibitory effect on protein degradation, but the effects were generally smaller than those previously shown in the rat. The amino acid control of protein degradation in the cat differs from that in the rat, as shown by the lack of effects of glutamine, asparagine, arginine or methionine in cat hepatocytes. This may be related to the unique features of protein metabolism of this species. NH4Cl, leupeptin and amino acids, which suppress lysosomal protein degradation by different mechanisms, caused less than 30% inhibition of protein degradation when used at the optimum concentrations reported for the rat. The ability of the lysosomal system to respond to nutritional deprivation is apparently lower in the cat than in the rat.     

Control by amino acids of the activity of system A-mediated amino acid transport in isolated rat hepatocytes.

The effect of amino acids, in concentrations corresponding to those found in the portal vein of rats given a high-protein diet, was investigated on the activity of system A amino acid transport in hepatocytes from fed rats. Amino acids counteracted the induction of system A by insulin or glucagon. This effect was observed at all concentrations of hormones tested, up to 1 microM. Amino acids did not affect the basal cyclic AMP concentration in hepatocytes, or the large rise in cyclic AMP elicited by glucagon. The reversal of system-A induction was observed at relatively low concentration of amino acids, corresponding to plasma values reported in rats given a basal diet. Amino acids were separately tested: substrates of system A were particularly efficient, but so were glutamine and histidine. Non-metabolizable substrates of system A, such as 2-aminoisobutyrate, were also inhibitory, suggesting that a part of the effect of amino acids is independent of their cellular metabolism. Provision of additional energy substrates such as lactate and oleate did not affect induction of system A or the inhibitory effects of amino acids. Thus amino acids do not act by serving as an energy source and by maintaining the integrity of hepatocytes. Inhibition of mRNA synthesis by actinomycin practically abolished the effect of amino acids on the induction of system A by glucagon. The results suggest that amino acids may promote the synthesis of protein(s) affecting the activity of system A either directly at the carrier unit or at an intermediate stage of its emergence.     

Amino acid and energy requirements for rat hepatocytes in primary culture

Summary The amino acid and energy requirements of rat hepatocytes in suspension and early culture were investigated. Among a number of potential energy substrates tested, pyruvate (20 mM) was found to be most effective in stimulating hepatocytic protein synthesis. Amino acids stimulated protein synthesis both as energy substrates and as protein precursors. An amino acid mixture was designed to provide maximal inhibition of protein degradation as well as maximal stimulation of protein synthesis. In a defined medium containing amino acids at these concentrations, and supplemented with glucocorticoid hormone and insulin, hepatocytes could be maintained—on a collagen substratum—for at least a week without any significant net loss of cells or cellular protein. The work was supported by grants from The Norwegian Cancer Society and from The Norwegian Council for Science and the Humanities. An erratum to this article is available at .     

Inhibition by amino acids of cytokinin-stimulated betacyanin synthesis inAmaranthus caudatus

A number of amino acids have been tested for their ability to inhibit the cytokinininduced synthesis of betacyanin inAmaranthus caudatus cotyledons. Under the conditions employed there was not any serious inhibition of pigment synthesis at amino acid concentrations belowca. 20 μg ml^-1. Amino acids such as methionine, γ-aminobutyric acid and leucine did not give rise to serious inhibition belowca. 200 μg ml^-1. At amino acid concentrations ofca. 2000 μg ml^-1, inhibitions of pigment synthesis was in all instances complete.     

Determination in vitro of the rate of protein synthesis and degradation in human-skeletal-muscle tissue.

The present studies were aimed to evaluate the possibility to use a system for estimation in vitro of the biosynthesis and degradation rates of human skeletal muscle protein. A previously characterized human skeletal muscle preparation was used. Amino acids and insulin stimulated significantly the incorporation rate of leucine into proteins. The effect of amino acids was more pronounced than that of insulin. The stimulatory effect of insulin could be decreased by amino acids. Insulin did not influence the tissue uptake or the oxidation rate of leucine. The release of [14C]leucine deriving from degradation of prelabelled skeletal muscle fibre proteins was linear for at least 2.5 h of incubation and optimal with leucine at concentrations beyond 12.5 mmol/1 or in the presence of puromycin in the incubation medium. The rate of the release of radioactivity was significantly inhibited by amino acids and at borderline significance by insulin but not by puromycin. The specific radioactivity in prelabelled proteins decreased significantly in the presence of puromycin suggesting that leucine derived from protein degradation was reutilized in vitro. This reutilization was found to be 9 +/- 1% of leucine released from degradation of proteins in 30 subjects. A statistically significant positive correlation between the cathepsin D activity in human skeletal muscle tissue and the degradative rate of prelabelled muscle proteins in vitro was observed. The results indicate that biosynthesis and degradation of skeletal muscle proteins in this system in vitro were subjected to control mechanisms. It is suggested that the release of radioactivity from prelabelled muscle fibre proteins during incubation probably only reflects the degradation of some rapidly-turning-over proteins.     

Effects of inhibitors of protein degradation on the rate of protein synthesis in Chinese hamster ovary cells

In the absence of serum and amino acids, cultured Chinese Hamster Ovary cells released to the medium two thirds of the leucine produced by protein degradation. Because protein synthesis requires all the amino acids, the loss of leucine implies incomplete reincorporation of the other amino acids as well. Leupeptin (0.45 mg/ml) and chloroquine (up to 40 microM) inhibited protein breakdown by 21 and up to 41%, respectively, and resulted in proportional decreases in protein synthesis. Chloroquine abolished the stimulation of protein breakdown by amino acid deprivation. From the values of protein synthesis and leucine output with and without chloroquine, it is estimated that the stimulation of protein degradation not only permitted continuing protein synthesis but also increased amino acid output. In the presence of serum or amino acids protein breakdown was slower than in their absence and less sensitive to inhibition by chloroquine, but proportional effects on synthesis and degradation were still observed. It is suggested that protein degradation may be necessary for the maintenance of optimum intracellular concentrations of amino acids even in the presence of extracellular amino acids.     

The yeaS (leuE) gene of Escherichia coli encodes an exporter of leucine, and the Lrp protein regulates its expression

Overexpression of the yeaS gene encoding a protein belonging to the RhtB transporter family conferred upon cells resistance to glycyl-l-leucine, leucine analogues, several amino acids and their analogues. yeaS overexpression promoted leucine and, to a lesser extent, methionine and histidine accumulation by the respective producing strains. Our results indicate that yeaS encodes an exporter of leucine and some other structurally unrelated amino acids. The expression of yeaS (renamed leuE for "leucine export") was induced by leucine, l-alpha-amino-n-butyric acid and, to a lesser extent, by several other amino acids. The global regulator Lrp mediated this induction.     

Structural determinants in substrate recognition by proton-amino acid symports in plasma membrane vesicles isolated from sugar beet leaves.

Amino acids are actively transported across the plasma membrane of plant cells by proton-coupled symports. Previously, we identified four amino acid symports in isolated plasma membrane vesicles, including two porters for the neutral amino acids. Here we investigated the effect of amino acid analogues on neutral amino acid transport to identify structural features that are important in molecular recognition by Neutral System I (isoleucine) and Neutral System II (alanine and leucine). D-Isomers of alanine and isoleucine were not effective transport antagonists of the L-isomers. These data are characteristic of stereospecificity and suggest that the positional relationship between the alpha-amino and carboxyl groups is an important parameter in substrate recognition. This conclusion was supported by the observation that beta-alanine and analogues with methylation at the alpha-carbon, at the carboxyl group, or at the alpha-amino group were not effective transport inhibitors. Specific binding reactions were also implicated in these experiments because substitution of the alpha-amino group with a space filling methyl or hydroxyl group eliminated transport inhibition. In contrast, analogues with various substitutions at the distal end of the amino acid were potent antagonists. Moreover, the relative activity of several analogues was influenced by the location of sidechain branches and Neutral Systems I and II were resolved based on differential sensitivity to branching at the beta-carbon. The kinetics of azaserine and p-nitrophenylalanine inhibition of leucine transport were competitive. We conclude that the binding site for the carboxyl end of the amino acid is a well-defined space that is characterized by compact, asymmetric positional relationships and specific ligand interactions. Although the molecular interactions associated with the distal portion of the amino acid were less restrictive, this component of the enzyme-substrate complex is also important in substrate recognition because the neutral amino acid symports are able to discriminate between specific neutral amino acids and exclude the acidic and basic amino acids.     

Inhibition of protein degradation in isolated rat hepatocytes

1. Isolated parenchymal cells were prepared by collagenase perfusion of livers from fed rats that had been previously injected with [³H]leucine to label liver proteins. When these cells were incubated in a salts medium containing glucose, gelatin and EDTA, cellular integrity was maintained over a period of 6h. 2. Cells incubated in the presence of 2mm-leucine to minimize radioactive isotope reincorporation released [³H]leucine into the medium at a rate accounting for the degradation of 4.5% of the labelled cell protein per h. 3. Degradation of [³H]protein in these cells was inhibited by insulin and by certain amino acids, of which tryptophan and phenylalanine were the most effective. 4. Protein degradation was decreased by several proteinase inhibitors, particularly those that are known to inhibit lysosomal cathepsin B, and by inhibitors of cell-energy production. 5. Ammonia inhibited degradation, but only at concentrations above 1.8mm. Aliphatic analogues of ammonia were effective at lower concentrations than was ammonia. 6. High concentrations of ammonia inhibited degradation by 50%. The extent of this inhibition could not be increased further by the addition of the cathepsin B inhibitor leupeptin, which by itself inhibited degradation by approx. 30%. 7. The sensitivity of proteolysis in isolated hepatocytes to these various inhibitory agents is discussed in relation to their possible modes of action.     

Rapid suppression of protein degradation in skeletal muscle after oral feeding of leucine in rats

A diet containing adequate amounts of protein rapidly suppresses myofibrillar protein degradation in rats and mice. This study determined whether dietary amino acids inhibit postprandial protein degradation in rat skeletal muscle. When rats fed on a 20% casein diet for 1 h after 18 h starvation, the rate of myofibrillar protein degradation measured by N(tau)-methylhistidine release from the isolated extensor digitorum longus muscle was significantly (p < 0.05) decreased at 4 h after refeeding. A diet containing an amino acid mixture which is the same composition as casein also reduced myofibrillar protein degradation at 4 h after refeeding (p < 0.05). An essential amino acid mixture (15.1%, corresponding to casein composition) and a leucine (2.9%) diets reduced the rate of myofibrillar protein degradation after refeeding (p < 0.05), whereas a protein free diet did not. Administration of leucine alone (0.135 g/100 g body weight) by a feeding tube induced a decrease in the rate of myofibrillar protein degradation at 2 h after administration (p < 0.05), whereas the serum insulin concentration was constant after leucine administration. These results suggested that leucine is one of regulating factors of myofibrillar protein degradation after refeeding of a protein diet.     

Age-related growth and protein turnover in the thymus of normal and glucocorticoid-treated rats

Possible derivatives of the amino acids tryptophan, tyrosine and histidine were examined as to their effect on protein metabolism in isolated rat hepatocytes. One of the substances tested, kynurenine (a main product of the catabolism of tryptophan), might be a physiological regulator of the lysosomal degradation of endogenous protein, because of the following. (a) Kynurenine decreased the lysosomal (i.e. methylamine-sensitive) pathway of degradation to a much greater extent than its parent amino acid, without interfering with the non-lysosomal pathway. (b) Kynurenine did not appreciably reduce the (lysosomal) degradation of the endocytosed protein asialo-fetuin, or the rate of protein synthesis, indicating a specificity of action. (c) Electron micrographs revealed a reduction in secondary lysosomes due to kynurenine.     

THE EFFECTS OF PHENYLALANINE ON AMINO ACID METABOLISM AND PROTEIN SYNTHESIS IN BRAIN CELLS IN VITRO1

Incubation of brain cell suspensions with 14 mM-phenylalanine resulted in rapid alterations of amino acid metabolism and protein synthesis. Both thc rate of uptake and the final intracellular concentration of several radioactively-labelled amino acids were decreased by high concentrations oi phenylalanine. By prelabelling cells with radioactive amino acids, phenylalanine was also shown to effect a rapid loss of the labelled amino acids from brain cells. Amino acid analysis after the incubation of the cells with phenylalanine indicated that several amino acids were decreased in their intracellular concentrations with effects similar to those measured with radioisotopic experiments (large neutral > small and large basic > small neutral > acidic amino acids). Although amino acid uptake and efflux were altered by the presence of 14 mwphenylalanine, little or no alteration was detected in the resulting specific activity of the intracellular amino acids. High levels of phenylalanine did not significantly altcr cellular catabolism of either alanine, lysine, leucine or isoleucine. As determined by the isolation of labcllcd aminoacyl-tRNA from cells incubated with and without phenylalanine, there was little or no alteration in the level of this precursor for radioactive alanine and lysine. There was, however, a detectable decrease in thc labelling of aminoacyl-tRNA for leucine and isoleucine. Only aftcr correcting for the changes of the specific activity of the precursors and thcir availability to translational events, could the effects of phenylalanine on protein synthesis be established. An inhibition of the incorporation into protein for each amino acid was approximately 20%.     

Amino acid fermentation and hydrogen transfer in mixed cultures

Abstract The degradation of the following amino acids was investigated in mixed cultures obtained from a waste water purification plant: aspartate, glutamate, serine, alanine, valine and leucine. Inhibition of sulfate-reducing bacteria in these mixed cultures by molybdate was found to inhibit amino acid degradation. The degradation of serine, alanine, valine and leucine was accelerated considerably by active sulfate reduction. The fermentation of aspartate and glutamate was not stimulated by the presence of sulfate-reducing bacteria. The existence of species which are able to ferment valine and leucine by coupling their oxidation to the reduction of exogenous acetate to butyrate was demonstrated.