Protein metabolism and survival of rat hepatocytes in early culture

Rat hepatocytes, cultured in a serum- and hormone-free medium on a substratum of adsorbed fibronectin, are in negative protein balance throughout the first 24 h of culture. The rate of protein loss at all times equals the difference between the high rate of protein degradation and the low rate of protein synthesis. A continuous decline in the rate of protein degradation gradually reduces the loss of protein, probably the result of medium conditioning as well as progressive culture deterioration. Inhibition of lysosomal protein degradation by amino acids and the protease inhibitor, leupeptin, reduces the protein loss considerably, but does not prevent cell death. The protein balance per se thus does not seem to be the limiting factor for the survival of cultured hepatocytes.     

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.     

Responsiveness of RNA degradation to amino acids in cultured rat hepatocytes: comparison with isolated rat hepatocytes.

The role of amino acids in the regulation of RNA degradation was investigated in cultured hepatocytes from fed rats previously labeled in vivo with [6-14C]orotic acid. Rates of RNA degradation were determined between 42 and 48 h of culture from the release of radioactive cytidine in the presence of 0.5 mM unlabeled cytidine. The fractional rate was about 4.4 +/- 0.4%/h in the absence of amino acids (0x). The catabolism of RNA was decreased to basal level (1.5 +/- 0.3%/h) by the addition of amino acids at 10 times normal plasma concentration (10x). The inhibition of RNA degradation, expressed as percentage of maximal deprivation-induced response (0x minus 10x), averaged 60% at normal plasma levels of amino acids. The degree of responsiveness was greatly improved as compared to freshly isolated hepatocytes (20%) and was similar to the sensitivity previously observed with perfused livers. In cultured hepatocytes, the sensitivity of RNA degradation to amino acids was not affected by varying the volume of medium from 1 to 4 ml per dish. In freshly isolated hepatocytes, the inhibitory effect of amino acids was not modified by changing the cell density from 0.5 to 5 x 10(6) cells per ml. In the range of normal plasma concentration of amino acids, the low sensitivity of RNA degradation in isolated hepatocytes persisted with inhibition ranging from 10 to 20%. These findings suggest that the control of RNA degradation in both cultured and isolated hepatocytes is not affected by the total quantity of amino acids available in the medium, but their concentration is crucial. Electron microscopy observations and the inhibitory effect of 3-methyl-adenine in cultured rat hepatocytes partially confirmed the role of the lysosomal system in the increase of RNA degradation and its regulation by amino acids.     

Inhibition of autophagic vacuole formation and protein degradation by amino acids in isolated hepatocytes

An amino acid mixture, specifically developed to suppress endogenous protein degradation in isolated hepatocytes, inhibited lysosornal (propylamine-sensitive) protein degradation by 70–75% and reduced the cytoplasmic volume fraction of the autophagic/lysosomal compartment to a similar extent. Incubation with the amino acid mixture for 1 h reduced the subcompartment of early autophagic vacuoles by 95%. These results support the hypothesis that autophagy is the major route of delivery of endogenous proteins to the lysosomes, and that amino acids exert their regulatory function on protein degradation by controlling the sequestration step of autophagy.     

Vanadate inhibits protein degradation in isolated rat hepatocytes

Vanadate (10 mM) strongly inhibited endogenous protein degradation as well as the degradation of an exogenous, endocytosed protein (asialofetuin) in isolated rat hepatocytes. Protein synthesis and cellular viability were unaffected, but changes in cell morphology suggested some interference with cytoskeletal elements. The effect of vanadate was comparable to the effects of several other degradation inhibitors (lysosomotropic amines, leupeptin, vinblastine, amino acids, dimethylaminopurine riboside) known to inhibit the autophagic/lysosomal pathway of protein degradation. Vanadate inhibited proteolysis in a liver homogenate at pH 5, suggesting a direct effect upon the lysosomal proteinases.     

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.     

Protein turnover and proliferation. Turnover kinetics associated with the elevation of 3T3-cell acid-proteinase activity and cessation of net protein gain.

1. At least 95% of the total protein of A31-3T3 cell cultures undergoes turnover. 2. First-order exponential kinetics were used to provide a crude approximation of averaged protein synthesis, Ks, degradation, Kd, and net accumulation, Ka, as cells ceased growth at near-confluent density in unchanged Dulbecco's medium containing 10% serum. The values of the relationship Ka = Ks - Kd were : 5%/h = 6%/h - 1%/h in growing cells, and 0%/h = 3%/h - 3%/h in steady-state resting cells. 3. As determined by comparison of the progress of protein synthesis and net protein accumulation, the time course of increase in protein degradation coincided with the onset of an increase in lysosomal proteinase activity and decrease in thymidine incorporation after approx. 2 days of exponential growth. 4. After acute serum deprivation, rapid increases in protein degradation of less than 1%/h could be superimposed on the prevailing degradation rate in either growing or resting cells. The results indicate that two proteolytic mechanisms can be distinguished on the basis of the kinetics of their alterations. A slow mechanism changes in relation to proliferative status and lysosomal enzyme elevation. A prompt mechanism, previously described by others, changes before changes in cell-cycle distribution or lysosomal proteinase activity. 5. When the serum concentration of growing cultures was decreased to 1% or 0.25%, then cessation of growth was accompanied by a lower steady-state protein turnover rate of 2.0%/h or 1.5%/h respectively. When growth ceased under conditions of overcrowded cultures, or severe nutrient insufficiency, protein turnover did not attain a final steady state, but declined continually into the death of the culture.     

Differential effects of proteinase inhibitors and amines on the lysosomal and non-lysosomal pathways of protein degradation in isolated rat hepatocytes

Ammonia, which like other lysosomotropic amines inhibits protein degradation in isolated rat hepatocytes by 70–80%, was utilized as a diagnostic tool to distinguish between the relative effects of various proteinase inhibitors on the lysosomal and non-lysosomal pathways of intracellular protein degradation.Leupeptin was found to inhibit lysosomal protein degradation by 80–85%, and non-lysosomal degradation by about 15%. Antipain had a similar, but somewhat weaker effect. Pepstain, bestatin and aprotinin (Traysylol) produced minor inhibitory effects (possibly on both degradation, pathways), whereas bacitracin and soybean trypsin inhibitor wre ineffective.Chymostatin inhibited lysosomal protein degradation by about 45%, whereas the non-lysosomal pathway was inhibited by more than 50%. Chymostatin was unique among the inhibitors tested in causing such a pronounced effect on non-lysosomal protein degradation, and appeared to selectively inhibit the energy-dependent portion of this pathway.The effects of the various inhibitors were additive to the extent expected on the basis of their kwown actions on lysosomal and non-lysosomal protein degradation. Thus, a combination of methylamine, leupeptine and chymostatin inhibited overall protein degradation by about 90%, resulting in a substantial improvement of the cellular nitrogen balance.The degradation inhibitors caused a partial inhibition of protein synthesis, apparently mainly by shutting down the supply of amino acids from the lysosome. The inhibitory effects of leupeptin and antipain were completely reversed by amino acid addition, whereas some inhibition remained in the case of chymostatin and the lysosomotropic amines, possibly reflecting a certain nonspecific toxicity.     

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

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.     

Rates of RNA degradation in isolated rat hepatocytes. Effects of amino acids and inhibitors of lysosomal function.

1. RNA degradation in isolated rat hepatocytes was measured as the release of radioactive cytidine from fed rats previously labeled in vivo for 60 h with [6-14C]orotic acid. Rates were determined from the linear accumulation of [14C]cytidine between 30 and 120 min of incubation in the presence of 0.5 mM unlabeled cytidine to suppress reutilization. 2. In the absence of amino acids, rates of RNA degradation in isolated hepatocytes averaged 3.97%/h. A complete mixture of amino acids added at 10-20 times normal plasma concentration inhibited RNA degradation by 65-70%. However, at physiological concentrations of amino acids, RNA degradation in isolated rat hepatocytes was less responsive as compared to perfused rat livers. 3. Numerous and large autophagic vacuoles at various stages of digestion were identified throughout the cytoplasm of isolated hepatocytes after 2 h of incubation in the absence of amino acids. The addition of amino acids at 20 times normal plasma concentration abolished almost completely the appearance of autophagic vacuoles. Furthermore, prophylamine, which accumulates in lysosomes, suppressed RNA degradation by 65% and the inhibitor of autophagic vacuole formation, 3-methyladenine, inhibited 70-80% of the degradation. Taken together, these results strongly suggest a contribution of the lysosomal system in the increase of RNA degradation rates in isolated rat hepatocytes.     

Protein turnover and proliferation. Failure of SV-3T3 cells to increase lysosomal proteinases, increase protein degradation and cease net protein accumulation

The contrasting control of lysosomal proteinases, protein turnover and proliferation was studied in 3T3 and SV-3T3 (SV-40-virus-transformed 3T3) cells. 1. In 3T3 cells, net protein accumulation proceeded from 5%/h (doubling time, T(d)=14h) in growing cells to 0%/h as cells became quiescent. SV-3T3 cells never ceased to gain protein, but rather decreased their protein accumulation rate from 6-7%/h (T(d)=10-12h) to 2%/h (T(d)=35-40h) just before culture death in unchanged medium. 2. In both cell types the rates of protein synthesis per unit of protein (a) were proportional to the initial serum concentration from 0 to 6%, and (b) declined under progressive depletion of undefined serum growth factors. In depleted growth medium, leucine incorporation per unit of protein in 3T3 and SV-3T3 cells declined to almost equal synthetic rates while the 3T3 cell existed in a steady state of zero net gain, and the SV-3T3 cell continued to gain protein at a rate of 2%/h. 3. Whereas a large fraction of the control of 3T3-cell net protein accumulation can be accounted for by an increase in degradation from 1%/h to 3%/h, the SV-3T3 cell did not exhibit a growth-related increase in degradation appreciably above 1%/h. 4. Thus, by using first-order kinetics, the continued net protein accumulation of the transformed cell can be accounted for by a failure to increase protein degradation, whereas fractional synthesis can be made to decline to a rate similar to that in the quiescent non-transformed cell. 5. Upon acute serum deprivation, both cell types similarly exhibited small rapid increases in proteolysis independent of cell growth state or lysosomal enzyme status. 6. The 3T3 cell increased its lysosomal proteinase activity in conjunction with increase in the growth-state-dependent proteolytic mechanism; however, the SV-3T3 cell failed to increase lysosomal proteinases or the growth-state-dependent proteolytic mechanism.     

Effects of amino acid analogues on protein degradation in isolated rat hepatocytes

Analogues and derivatives of six of the amino acids which most effectively inhibit protein degradation in isolated rat hepatocytes (leucine, asparagine, glutamine, histidine, phenylalanine and tryptophan) were investigated to see if they could antagonize or mimic the effect of the parent compound. No antagonists were found. Amino alcohols and amino acid amides tended to inhibit protein degradation strongly, apparently by a direct lysosomotropic effect as indicated by their ability to cause lysosomal vacuolation. Amino acid alkyl esters and dipeptides inhibited degradation to approximately the same extent as did their parent amino acids, possibly by being converted to free amino acids intracellularly. Of several leucine analogues tested, four (L-norleucine, L-norvaline, D-norleucine and L-allo-isoleucine) were found to be as effective as leucine in inhibiting protein degradation. None of the analogues had any effect on protein synthesis. Since leucine appears to play a unique role as a regulator of bulk autophagy in hepatocytes, the availability of active leucine agonists may help tj elucidate the biochemical mechanism for control of this important process.     

Protein-catabolic state of isolated rat hepatocytes

Isolated rat hepatocytes in suspension are in a protein-catabolic state (negative nitrogen balance), as measured by the continuous release of nitrogen in the form of amino acids and urea. The nitrogen loss corresponds to a protein degradation rate of 3–4% per h, while the rate of protein synthesis is negligible. Cells prepared from fasted, fed ot regenerating livers are all highly protein-catabolic.The nitrogen balance is unaffected by insulin or amino acids (physiological mixture), and various metabolites and sera have only moderate effects. However, incubation of the cells for 2–4 h in a tissue culture medium (Dulbecco's) reduces the nitrogen loss dramatically, suggesting the formation of an anticatabolic factor under these conditions.     

6-substituted purines: a novel class of inhibitors of endogenous protein degradation in isolated rat hepatocytes

About 100 different purine derivatives and analogs were tested for their effect on protein synthesis and protein degradation in isolated rat hepatocytes. These included 6-aminopurines (adenine and adenosine analogs), 6-mercaptopurines, chloropurines, oxypurines, cytokinins, methylxanthines, methylindoles, benzimidazoles, and benzodiazepines. Most of the compounds were either inactive or inhibited protein synthesis as much as or more than they inhibited protein degradation. However, three methylated 6-aminopurines (3-methyladenine, 6-dimethylaminopurine riboside, and puromycin aminonucleoside) and four 6-mercaptopurines (6-methylmercaptopurine, 6-methylmercaptopurine riboside, 6-mercaptopurine riboside, and 2′,3′,5t$\text{-}\text{?}$triacetyl-6-mercaptopurine riboside) had a markedly stronger effect on protein degradation than on synthesis, and might therefore be potentially useful as selective degradation inhibitors. None of the seven above-mentioned purines had any significant effect on the degradation of the exogenous protein, asialofetuin, and would therefore seem to selectively inhibit endogenous protein degradation. Since the degradation was not further affected by purines in the presence of amino acids or lysosomotropic amines, it is suggested that the purines exert their effect specifically upon the autophagic/lysosomal pathway. All the mercaptopurines significantly depressed cellular ATP levels, whereas the methylated aminopurines did not. For this reason, the latter are probably more useful as degradation inhibitors. 3-Methyladenine had no effect on protein synthesis at a concentration (5 mm) which inhibited protein degradation by more than 60%, and may therefore be regarded as a highly specific inhibitor of autophagy.     

The effect of iron and ethanol on rat hepatocyte collagen synthesis

1. Carbonyl iron (2.5% w/w) in rat chow was used to induce iron loading in rat hepatocytes.2. Acute exposure of cultured hepatocytes from control and iron-loaded rats to ethanol (25–100 mM) resulted in a significant inhibition of protein synthesis.3. Inhibition of protein synthesis in hepatocytes from iron-loaded rats was primarily due to impaired amino acid uptake by these cells.4. High concentrations of ethanol stimulated the rate of protein degradation by hepatocytes from iron-loaded rats.5. Acute administration of ethanol to hepatocytes from control animals did not stimulate the absolute rates of collagen biosynthesis nor induce Type I procollagen mRNA.6. Acute administration of ethanol did not inhibit procollagen synthesis.7. Iron overload induced Type I procollagen mRNA and increased the absolute rates of collagen synthesis in hepatocytes.8. These findings may be relevant for the development of hepatic fibrosis in patients with genetic hemochromatosis who consume excess ethanol.     

The effect of iron and ethanol on rat hepatocyte collagen synthesis.

1. Carbonyl iron (2.5% w/w) in rat chow was used to induce iron loading in rat hepatocytes. 2. Acute exposure of cultured hepatocytes from control and iron-loaded rats to ethanol (25-100 mM) resulted in a significant inhibition of protein synthesis. 3. Inhibition of protein synthesis in hepatocytes from iron-loaded rats was primarily due to impaired amino acid uptake by these cells. 4. High concentrations of ethanol stimulated the rate of protein degradation by hepatocytes from iron-loaded rats. 5. Acute administration of ethanol to hepatocytes from control animals did not stimulate the absolute rates of collagen biosynthesis nor induce Type I procollagen mRNA. 6. Acute administration of ethanol did not inhibit procollagen synthesis. 7. Iron overload induced Type I procollagen mRNA and increased the absolute rates of collagen synthesis in hepatocytes. 8. These findings may be relevant for the development of hepatic fibrosis in patients with genetic hemochromatosis who consume excess ethanol.     

Intracellular protein degradation and autophagy in isolated pancreatic acini of the rat

Simultaneous investigation of protein degradation and autophagy of isolated exocrine pancreatic cells is carried out here for the first time in a systematic way by a complex biochemical, morphological and morphometrical approach. Protein degradation proceeds with a decreasing rate of 4-1.5 per cent per h over a 4-h period indicating a comparatively low degradation capacity. Cells in freshly isolated acini do not contain autophagic vacuoles but the latter appear within an hour in vitro and their quantity remains close to a steady state during the subsequent 3 h. Both traditional inhibitors of the autophagic-lysosomal pathway, e.g. vinblastine, leupeptin, and lysosomotropic amines together with the recently introduced 3-methyladenine, inhibit degradation to a similar maximal extent, offering the possibility of the estimation of the ratio of lysosomal/non-lysosomal degradation. In pancreatic acinar cells autophagic sequestration is unaffected and protein degradation is inhibited inside secondary lysosomes by leupeptin and lysosomotropic amines, while 3-methyladenine prevents the formation of autophagosomes. Vinblastine seems to act by inhibiting the fusion of autophagosomes with lysosomes and there is no evidence for the stimulation of autophagic sequestration by vinblastine in the present system. The effect of inhibitors of protein breakdown on protein synthesis is variable and does not correlate with their influence on degradation. Amino acids strongly stimulate protein synthesis, but in contrast to what is found in liver cells, they do not seem to affect protein degradation or autophagy significantly, thus indicating major regulatory differences of these processes between pancreatic acinar cells and hepatocytes.     

The effect of insulin and intermittent mechanical stretching on rates of protein synthesis and degradation in isolated rabbit muscle.

Tyrosine balance and protein synthesis were studied during the same incubation in isolated rabbit forelimb muscles. From these measurements, protein degradation was calculated. Isolated muscles were usually in a state of negative amino acid balance, principally as a result of the 75% decrease in protein synthesis. Muscles from rabbits starved for 18 h had lower rates of both protein synthesis and degradation compared with muscles from normally fed rabbits. Intermittent mechanical stretching and the addition of insulin at 100 microunits/ml increased rates of both protein synthesis and degradation. Increases in the rate of protein synthesis were proportionately greater in the muscles from starved animals. In muscles from both fed and starved donors, increases in protein-synthesis rates owing to intermittent stretching and insulin were proportionately greater than the increases in degradation rates. For example, insulin increased the rate of protein synthesis in the muscles from starved donors by 111% and the rate of degradation by 31%. Insulin also increased the rate of protein synthesis when added at a higher concentration (100 munits/ml); at this concentration, however, the rate of protein degradation was not increased. The suppressive effect of insulin on high rates of protein degradation in other skeletal-muscle preparations may reflect a non-physiological action of the hormone.     

Inhibition of cardiac proteolysis by colchicine. Selective effects on degradation of protein subclasses

1. The effect of colchicine (2.5 microM) on cardiac protein turnover was tested with foetal mouse hearts in organ culture. 2. Colchicine had no effect on protein synthesis, but inhibited total protein degradation by 12-18%. Lumicolchicine, which lacks colchicine's ability to disaggregate microtubules, but shares its non-specific effects, did not alter protein degradation. 3. The colchicine-induced inhibition of protein degradation was accompanied by significant changes in cardiac lysosomal enzyme activities and distribution. 4. Colchicine inhibited the degradation of organellar proteins, including mitochondrial cytochromes, more than that of cytosolic proteins. 5. Colchicine decreased the rate of myosin degradation and the rate of proteolysis of the total protein pool to a similar extent. Since the regulation of myosin degradation does not involve lysosomes, this suggests that colchicine affects non-lysosomal as well as lysosomal pathways. 6. Release of branched-chain amino acids from colchicine-treated hearts was disproportionately decreased, suggesting that colchicine increased their metabolism. 7. It is concluded that colchicine, via its actions on microtubules, exerts important inhibitory effects on cardiac proteolysis. Colchicine is especially inhibitory to the degradation of organellar proteins, including mitochondrial cytochromes. Its inhibitory effects may be mediated in part via lysosomal mechanisms, but non-lysosomal mechanisms are probably involved as well.     

Dynamics of four rat liver plasma membrane proteins and polymeric IgA receptor. Rates of synthesis and selective loss into the bile.

We have determined the half-lives and amounts per hepatocyte of the polymeric IgA receptor (pIgA-R) and four rat hepatocyte plasma membrane proteins and subsequently have predicted their rates of synthesis and possible routes of degradation. Using in vivo pulse-chase metabolic labeling with L-[35S]cysteine, we found that the pIgA-R had an apparent half-life of 1.1 h. Additional metabolic labeling experiments showed that CE9, HA4, and HA321 had apparent half-lives of 4-5 days, and dipeptidyl peptidase IV had an apparent half-life of 9 days. To quantify the amount of each protein per hepatocyte, homogenates and a standard curve of purified protein were compared by immunoblotting. We found that these proteins were present at 1-8 x 10(6) molecules/hepatocyte. The calculated rate of synthesis for pIgA-R was 1.6 x 10(6) molecules/hepatocyte/h, whereas the others were synthesized at much lower rates (0.9-5 x 10(4) molecules/hepatocyte/h). Using immunoblot analysis, we found that pIgA-R was released into bile at a rate of 30%/h (700%/day), whereas dipeptidyl peptidase IV and HA4 were released at a rate of 2-3%/day. While the majority of the loss of pIgA-R from hepatocytes occurred by release into the bile, less than 30% of the degradation of dipeptidyl peptidase IV and HA4 could be accounted for by this pathway, suggesting that the remaining molecules must be retrieved from the apical surface before degradation.