Protein Synthesis In Rat Liver Research Articles

Effect of Dietary Restriction on Liver Protein Synthesis in Rats

At 6 wk of age, male Fischer F344 rats were fed a purified, casein-based diet either ad libitum or in the amount of 60% of the diet consumed by the rats fed ad libitum (restricted diet). Hepatocytes were isolated from the rats between 2.5 and 19 mo of age. The protein content of the hepatocytes isolated from the rats fed the restricted amount of diet was significantly lower than that of hepatocytes isolated from rats fed ad libitum. The DNA and RNA content of the hepatocytes were similar for the rats fed the two dietary regimens. The absolute rate of protein synthesis for hepatocytes isolated from rats fed ad libitum decreased 55 % between 2.5 and 19 mo of age. However, the rate of protein synthesis by hepatocytes from rats fed the restricted amount of diet decreased only slightly with increasing age. At 19 mo of age, the rate of protein synthesis by hepatocytes from the rats fed the restricted amount of diet was significantly higher than the rate of protein synthesis for hepatocytes from rats fed ad libitum. Therefore, dietary restriction retards the age-related decline in liver protein synthesis. Reproduced by permission. Maria C. Birchenall-Sparks, Michael S. Roberts, Jeffrey Staeker, James E. Hardwick, Arlan Richardson, Effect of Dietary Restriction on Liver Protein Synthesis in Rats. J. Nutr. 115 , 944-950 (1985).

Effects of aging on the various steps of protein synthesis: fragmentation of elongation factor 2

The possible mechanism responsible for the in vivo protein synthesis decline during aging was studied. In order to determine the effect of aging on the various steps of protein synthesis, we determined the ribosomal state of aggregation and the time of assembly and release of polypeptide chains in the process of protein synthesis in rat liver . The results suggest that elongation is the most sensitive step to aging. A molecular study of the Elongation Factor 2 (EF-2), the main protein involved in the elongation step, shows that this protein has a higher content of carbonyl groups and is less active in old rats. In addition, the molecular mass analysis of EF-2 shows that this protein becomes fragmented in old rats. A similar pattern of fragmentation is found in 3-month-old rats suffering oxidative stress, in that the decline in protein synthesis is similar to that found in old rats. These data suggest that: i) oxidative stress seems to be involved in the modifications of EF-2 observed during aging, and ii) the observed modifications (oxidation and fragmentation) of EF-2 could account for the decline in protein synthesis in old animals.

Effect of Oxidative Stress, Produced by Cumene Hydroperoxide, on the Various Steps of Protein Synthesis: MODIFICATIONS OF ELONGATION FACTOR-2

We have studied the effect of oxidative stress on protein synthesis in rat liver. Cumene hydroperoxide (CH) was used as an oxidant agent. The approach used was to determine the ribosomal state of aggregation and the time for assembly and release of polypeptide chains in the process of protein synthesis in rat liver in vivo. The results suggest that the elongation step is the most sensitive to CH treatment. The measurement of both carbonyl groups content and ADP-ribosylatable elongation factor 2 (EF-2), the main protein involved in the elongation step, indicates that under CH treatment EF-2 is oxidatively modified and a lower amount of active EF-2 is present. These results are corroborated by in vitro oxidation of EF-2 and could explain for the decline in the elongation step.

Effects of albumin-bilirubin complexes with syngeneic or allogeneic albumin on DNA and protein synthesis in liver and spleen of partially hepatectomized rats

The effects of non-covalently bound complexes of allogeneic or syngeneic albumin with bilirubin and of albumin alone on DNA and protein synthesis in rat liver and spleen cells after partial hepatectomy were studied. The assay procedure was based on different intravenous doses of these compounds in rats after partial hepatectomy. The allogeneic albumin-bilirubin complex (at protein doses of 0.9 and 90 micrograms/100 g body weight) stimulated DNA and protein synthesis in liver cells irrespective of the dose. At a dose of 0.9 micrograms the syngeneic albumin-bilirubin complex enhanced DNA synthesis insignificantly and produced no effect on protein synthesis, while at a dose of 90 micrograms, both DNA and protein synthesis were considerably increased. Allogeneic or syngeneic albumin at the above doses stimulated only protein, not DNA, synthesis in the liver, while the highest stimulation was at 90 micrograms allogeneic albumin. It was found also that partial hepatectomy decreased DNA and protein synthesis in spleen cells. Albumin-bilirubin complex with allogeneic or syngeneic albumin and albumin alone either significantly enhanced DNA and protein synthesis in the spleen, compared to controls, or only restored synthesis to control levels. Thus DNA and protein synthesis in the regenerating liver and spleen was significantly enhanced after the injection of small doses of the albumin-bilirubin complex, indicating the existence of small amounts of a similar endogenous complex in the blood stream.

Regulation of protein synthesis by modulation of intracellular calcium in rat liver.

The rate of protein synthesis can be modulated in intact cells by varying the concentration and subcellular distribution of intracellular calcium. Because the biochemical reactions required for the pathway of protein synthesis occur in the cytosol of the cell, it might be expected that protein synthesis would be controlled by free cytosolic calcium rather than the sequestered cation. However, a recent report proposed that maintenance of optimal rates of protein synthesis depends on the amount of calcium sequestered in the endoplasmic reticulum rather than free cytosolic calcium (C.O. Brostrom and M. A. Brostrom, Annu. Rev. Physiol. 52: 577-590, 1990). In the present study, rat livers were perfused with buffer containing various compounds previously shown to alter intracellular calcium concentration and distribution in isolated cells. It was found that conditions designed to cause a rise in free cytosolic calcium had no effect on protein synthesis. In contrast, conditions designed to cause depletion of sequestered calcium resulted in an inhibition of protein synthesis characterized by a reduction in peptide-chain initiation relative to elongation. The inhibition of protein synthesis was further localized to a decrease in the activity of eukaryotic initiation factor (eIF) 2B as measured in extracts from perfused livers. The inhibition of eIF-2B activity was associated with a 2.4-fold increase in the proportion of the alpha-subunit of eIF-2 in the phosphorylated form. In summary, the results of the present study support a model whereby mobilization of calcium sequestered in the endoplasmic reticulum results in an inhibition of protein synthesis in rat liver.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of microsomal calcium sequestration causes an impairment of initiation of protein synthesis in perfused rat liver

The present study examined the effect of 2,5-di-( tert-butyl)-hydroquinone (tBuHQ), an inhibitor of liver microsomal calcium sequestration, on initiation of protein synthesis in perfused rat liver. Perfusion of livers with a concentration of tBuHQ previously shown to completely inhibit microsomal calcium sequestration in isolated hepatocytes caused a 50% inhibition of protein synthesis. The inhibition was characterized by an increase in liver content of free ribosomal particles and a decrease in polysomes indicating that peptide-chain initiation was slowed relative to elongation. Furthermore, the inhibition was associated with a 7.5-fold increase in the proportion of the α-subunit of eukaryotic initiation factor 2 (eIF-2) present in the phosphorylated form and a reduction in the activity of eukaryotic initiation factor 2B (eIF-2B) to 37% of the control value. The results suggest that protein synthesis in rat liver is regulated directly by changes in intracellular calcium concentration through a mechanism involving modulation of the phosphorylation state of eIF-2α.

Methionyl aminopeptidase from rat liver: distribution of the membrane-bound subcellular enzyme

The selective distribution of methionyl aminopeptidase (MAP) among rat liver mitochondria (heavy and light) and microsomes is reported. Several properties of MAP from the three subcellular fractions showed that the enzyme is a typical aminopeptidase able to remove N-terminal methionine from oligopeptides and methionyl-2-naphthylamide but not from Met-Ala-Ser. MAP is a membrane-bound enzyme sensitive to SH-group oxidants and inhibitable by L-methionine but not by usual arylaminopeptidase inhibitors. It is suggested that, MAP may play an important role during protein synthesis in rat liver.

Rat liver protein synthesis after partial hepatectomy.

Rat liver protein synthesis after partial hepatectomy.

Influence of cyclosporine A on protein synthesis in rat liver

The influence of the immunosuppressive agent cyclosporine A on protein synthesis was investigated in rat liver in vivo and in vitro. Incorporation of [ 14C]leucine into total protein by microsomes was inhibited 36% in the presence of cyclosporine A. Treatment of rats with cyclosporine A also resulted in decreased protein synthesis by microsomes isolated from the same animals. This inhibition was dependent on the dose administered and the duration of treatment. The inhibitory factor in this latter case appeared to be associated with the supernatant fraction. Decreased in vivo incorporation of [ 35S]methionine into proteins of microsomal membranes and peroxisomes, but not into microsomal luminal proteins was also observed after treatment of rats with cyclosporine A. On SDS-polyacrylamide gel electrophoresis certain of the microsomal membrane and peroxisomal proteins displayed decreased labeling. Furthermore, cyclosporine A treatment decreased the inductive effects of phenobarbital, clofibrate, and phthalate ester on microsomal and peroxisomal enzymes. It is suggested that certain of the toxic effects of cyclosporine A are exerted through inhibition of cellular protein synthesis.

Effects of experimentally induced nephrosis on protein synthesis in rat liver.

A nephrotic syndrome was experimentally induced in rats by a single intravenous injection of aminonucleoside of puromycin. Experimental animals were studied 8 days after the injection, at which time they exhibited marked proteinuria and hypoalbuminemia compared with control animals. The experimental animals also exhibited alterations in protein synthesis in liver as evidenced by a marked increase in the rate of albumin synthesis relative to total hepatic protein synthesis, changes in the relative concentrations of several plasma proteins, an increased protein content of plasma, an increased liver weight relative to body weight, and an increased RNA content of liver. Perfused liver preparations derived from nephrotic rats exhibited an increased release of albumin and other secretory proteins compared with control preparations. In contrast, there was no difference in the rate of synthesis of nonexported proteins between the two groups. The elevation in the relative rate of albumin synthesis was accompanied by a relative increase of the same magnitude in albumin mRNA. Furthermore, the relative amounts of several other mRNAs, including those coding for beta-fibrinogen, haptoglobin, metallothionein II, and two unidentified proteins, were increased, whereas the amount of mRNA coding for alpha 1-acid glycoprotein was decreased in livers of nephrotic rats compared to controls. These results indicate that nephrosis leads to marked alterations in the synthesis of albumin and other plasma proteins. Mechanisms responsible for these alterations include changes in the relative abundance of specific mRNAs and an increase in total cellular RNA.

Effect of Dietary Proteins on Time-Dependent Changes in Plasma Amino Acid Levels and on Liver Protein Synthesis in Rats

The effects of four dietary proteins on plasma amino acid (PAA) levels in portal vein and aorta and on hepatic ribosome aggregation were investigated in rats fed 10% protein diets ad libitum. Proteins studied were beef, casein, rapeseed and soybean. Measurements were made at 2000, 2300, 0200, 0500 and 0800 h. Portoaortic differences in essential amino acids (EAA) in plasma were calculated and compared to dietary EAA to verify the time-dependent changes in their intestinal absorption. Each protein generated different variations in portal and aortic PAA levels. Rapeseed produced the lowest plasma concentrations of EAA, but its portoaortic differences were as large as those of the other proteins. With each protein, proportions of EAA in plasma (in contrast to concentrations) tended to remain constant in portal vein and aorta over the 12-h period. Significant correlations between portoaortic differences and dietary EAA were found in all groups, but at varying times, indicating that the kinetics of intestinal absorption were dependent on the protein fed. So were variations in liver polysome distribution. For rapeseed and soybean, times of most active protein synthesis were 0200 and 0500 h; with beef and casein, variations between sampling times were not significant. No direct relationship was found between magnitude and composition of PAA portoaortic differences and ribosome aggregation. Thus these two parameters were both affected by dietary proteins, but no synchrony was evidenced between them.

Different effects of oxytetracycline and doxycycline on mitochondrial protein synthesis in rat liver after long-term treatment

The tetracyclines inhibit specifically mitochondrial (mt) and bacterial protein synthesis when they are present in low concentrations (2–10 μg/ml). There is no difference between the various members of this group of antibiotics in this respect. In the present study, however, it is shown that the inhibitory effect of doxycycline on mt-protein synthesis in rat liver is partially lost after continuous treatment for more than 1 week, whereas oxytetracycline continues to inhibit mt-protein synthesis effectively after 1 week of treatment. To find an explanation for this difference between doxycycline and oxytetracycline, a detailed study was made of the distribution and the effects on mt-protein synthesis of both tetracyclines under various conditions in rat liver. The results of the studies lead to the hypothesis that doxycycline treatment induces the formation of a doxycycline complex, and thus to a reduced amount of free doxycycline. This may explain the loss of effective inhibition of mt-protein synthesis.

Soluble factors of protein synthesis in rat liver during the acute-phase reaction

Cell sap of liver cells from rats undergoing an acute-phase reaction has an increased capacity for binding leucine to tRNA. This increased capacity does not depend on concurrent changes in the leucine pool. The kinetics of activity of leucyl-tRNA synthetase from acute-phase cell sap do not show relevant differences from the normal. Acute-phase cell sap contains more tRNA than its normal counterpart. Experiments performed with increasing amounts of exogenous deacylated tRNA demonstrate that under our experimental conditions the observed higher concentration of tRNA in acute-phase cell sap could explain the increased activity in leucine incorporation into leucyl-tRNA.

Effect of Dietary Restriction on Liver Protein Synthesis in Rats

At 6 wk of age, male Fischer F344 rats were fed a purified, casein-based diet either ad libitum or in the amount of 60% of the diet consumed by the rats fed ad libitum (restricted diet). Hepatocytes were isolated from the rats between 2.5 and 19 mo of age. The protein content of the hepatocytes isolated from the rats fed the restricted amount of diet was significantly lower than that of hepatocytes isolated from rats fed ad libitum. The DNA and RNA content of the hepatocytes were similar for the rats fed the two dietary regimens. The absolute rate of protein synthesis for hepatocytes isolated from rats fed ad libitum decreased 55% between 2.5 and 19 mo of age. However, the rate of protein synthesis by hepatocytes from rats fed the restricted amount of diet decreased only slightly with increasing age. At 19 mo of age, the rate of protein synthesis by hepatocytes from the rats fed the restricted amount of diet was significantly higher than the rate of protein synthesis for hepatocytes from rats fed ad libitum. Therefore, dietary restriction retards the age-related decline in liver protein synthesis.

Influence of fasting and refeeding on the elongation step of protein synthesis in rat liver of young (3M) and aging (18M) animals

The in vitro binding capacity for AA-tRNA of ribosomes, catalysed by EF 1 enzyme, was studied in livers of rats, which had been starved for 5 days and re-fed for 3, 9, and 24 hours. The experiments were carried out with 3 and 18-month-old rats. The results were compared to those obtained with normally fed rats of the same ages. For both age groups, there was a similar change in the binding capacity, including a return to normal values after 24 hours of refeeding. When ribosomes of old control animals were treated with different enzymes, we obtained lower binding activities for all the experimental groups than in homologous cell-free systems (ribosomes and EF 1 isolated from the same group). The ribosomes of young animals, tested in the presence of different enzymes, display an increased binding capacity, when the enzymes used were those of old (control and re-fed) animals. The recovery of the catalytic activity of EF 1 on the binding process was observed after 9 hours in old animals, whereas it was not seen for young re-fed animals at that time of refeeding. EF 1 seems to play an important role in the adaptation of older animals to fasting and refeeding.

Effects of cycloheximide and changes in lighting regimen on the diurnal rhythm of rat liver protein synthesis

Abstract Under standard conditions we have demonstrated a highly significant diurnal rhythmicity of translation for both total proteins and integral membrane proteins (IMPs) using a cell‐free system from rat liver. IMPs were prepared from microsomes by treatment with EDTA and 2 M LiCl. Normal rhythmic protein synthesis of untreated rats reaches a maximum at 03.00 h and an intermediate maximum at 12.00 h for both total proteins and IMPs. In the following experiments the animals were treated with cycloheximide (CHX) or were subjected to altered lighting regimens before hepatic protein synthesis was investigated in the in vitro system. Injection of CHX 4 hours prior to sacrificing the rats resulted in a marked modification of rhythmic translation, although CHX was washed out from the microsomal preparations and was not present during incubation. The maximal rate of translation was shifted from 03.00 h to 06.00 h, and the intermediate maximum was suppressed. The temporal shift of the maximum after CHX treatme...

Does lead exposure influence liver protein synthesis in rats?

1. The liver and serum of rats was studied 18 hr following the i.p. administration of 0.5mg Pb 2+/100 g body wt. 2. The incorporation of [ 14C]leucine into proteins was measured in ribosomal and mRNA fractions incubated in vitro and was markedly stimulated in the Pb 2+ treated rats. 3. Similar results were found when the proteins of liver and serum were labelled by the injection in vivo of [ 14C]leucine. 4. The increased mRNA activity appears to be chiefly for acid serum proteins of approx. M r , 44,000, 41,000, 33,000 and 18,000.

In vivo studies on the precursor amino acid pool for liver protein synthesis in rats.

The origin of the amino acids in the precursor pool for hepatic protein synthesis has been studied by following the distribution of administered [3h] Asp and [3h] Val to the tRNA-charged, the extracellular (EC) and the intracellular (IC) amino acid pools of rat liver under two physiological states (fed and starved). Our results indicated that both the EC and IC amino acids contributed to the precursor pool for hepatic protein synthesis. Asp was mainly derived from the IC pool for protein synthesis, but in Val, the contribution of the IC and EC pools was not differentiated in fed rats, because the specific radioactivities of the three Val pools were very close. In starved rats, the difference in the contributions of IC and EC Val was relatively small. The difference in the specific radioactivities between tRNA-charged and hepatic EC or IC free amino acids depended on the kind of amino acids and the physiological states of the animals.

Effect of malotilate (diisopropyl 1,3-dithiol-2-ylidenemalonate) on the protein synthesis in rat liver.

The effect of malotilate (diisopropyl 1,3-dithiol-2-ylidenemalonate) on the protein synthesis in rat liver was studied in vivo and in vitro. Oral administration of malotilate to rats caused an increase in the protein and RNA contents of the liver and lef to an acceleration of 14C-leucine incorporation into microsomal and cytosol proteins. In a cell-free system, the protein synthesis was enhanced by treatment with malotilate, and an unknown factor(s) which participates in the protein synthesis was found in the cytosol fraction prepared from malotilate treated livers. These results suggest that malotilate is a new type of inducer for protein synthesis. On the basis of the observations obtained in the present study, a hypothesis can be formulated that malotilate enhances liver protein synthesis by accelerating RNA synthesis and/or increasing the transport of RNA from nuclei to cytosol in rat liver.

EFFECT OF MALOTILATE (DIISOPROPYL 1,3-DITHIOL-2-YLIDENEMALONATE) ON THE PROTEIN SYNTHESIS IN RAT LIVER

The effect of malotilate (diisopropyl 1,3-dithiol-2-ylidene-malonate) on the protein synthesis in rat liver [was studied in vivo and in vitro. Oral administration of malotilate to rats caused an increase in the protein and RNA contents of the liver and led to an acceleration of 14C-leucine incorporation into microsomal and cytosol proteins. In a cell-free system, the protein synthesis was enhanced by treatment with malotilate, and an unknown factor(s) which participates in the protein synthesis was found in the cytosol fraction prepared from malotilate treated livers. These results suggest that malotilate is a new type of inducer for protein synthesis. On the basis of the observations obtained in the present study, a hypothesis can be formulated that malotilate enhances liver protein synthesis by accelerating RNA synthesis and/or increasing the transport of RNA from nuclei to cytosol in rat liver.