Hepatic Ribosomes Research Articles

Ethanol and protein metabolism.

This article represents the proceedings of a workshop at the 2000 ISBRA Meeting in Yokohama, Japan. The chairs were Carol C. Cunningham and Victor R. Preedy. The presentations were (1) Ribosomal content, ribosomal localization and the levels of ribosomal protein mRNA and rRNA in rat skeletal muscle exposed to ethanol, by Alistair G. Paice, John E. Hesketh, Timothy J. Peters, and Victor R. Preedy; (2) Altered hepatic mitochondrial ribosome structure after chronic ethanol administration, by Vinood B. Patel and Carol C. Cunningham; (3) Clinical aspects of hepatic protein metabolism and alcohol, by Elena Volpi; and (4) Effects of oral intake of alanine plus glutamine on ethanol metabolism and ethanol-related depression in motor activity, by Kazunori Mawatari, H. Masaki, M. Mori, and Kunio Torii.

Inhibition of the initiation of hepatic protein synthesis during ethionine mediated ATP depletion in vivo: Modification to ribosomal subunits, evidence of impaired ternary complex formation and a subcellular redistribution of eIF-2 alpha

Acute ethionine intoxication is known to induce a reversible hepatic injury in female rats by reducing the level of hepatic ATP. The injury indirectly impairs the initiation of hepatic protein synthesis, with resultant polysome disaggregation. Administration of adenine rapidly restores the ATP levels and protein synthesis. Analysis of liver polysome and ribosomal subunits reveals that polysome disaggregation occurs following 3 h of the intoxication, and reaggregation occurs following the administration of adenine. Inactive hepatic ribosomes accumulate as monomers and disomes when analysed by sucrose gradient sedimentation in low-salt buffers. High-salt buffers dissociate the inactive ribosomes into the component 40 S and 60 S subunits. The level of higher density, 1.48 g/cc, 40 S subunit increases during the inhibition of protein synthesis, while the lower density, 1.41 g/cc, 40 S subunit species does not change significantly. Hepatic micosomal and cytosolic extracts examined for their ability to support the formation of the ternary complex of eIF-2-GTP and [ 35S]Met-tRNA i demonstrate that during acute ethionine intoxication, ternary complex formation in the two extracts decrease 65% and 85%, respectively. These changes are coincident with polysome disaggregation. Administration of adenine to reverse the intoxication restores the ternary complex forming ability of the cytosolic extract, but does not affect the activity of the microsomal salt wash extracts. Mixing experiments indicate the accumulation of an inhibitor of ternary complex formation in the microsomal salt wash fraction. The application of quantitative western blotting demonstrates that the level of antigenic eIF-2 alpha in the microsomal salt wash extract increases 31% during the inhibition. These observations are consistent with the idea that the inhibition of the initiation of hepatic protein synthesis induced by ethionine is mediated by eIF-2 alpha phosphorylation. The latter results in an inhibition of ternary complex formation, redistribution of eIF-2 to the microsome fraction, polysomal disaggregation, and accumulation of inactive ribosomal subunits.

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.

Time-Dependent Changes in Aggregation of Hepatic Ribosomes after Meal Feeding of Rats

The effects of consumption of a meal on changes in aggregation of hepatic ribosomes were studied. Hepatic ribosomes were aggregated very rapidly, within 10–20 min, after meal feeding a 25% caselin diet. This response of hepatic ribosomes is faster than the half-life of mRNAs of some liver proteins. Therefore, when rates are fed, the translational changes in protein synthesis (polysome pattern) might be quickly affected by the dietary protein, and the observed aggregation of ribosomes could indicate the state of protein synthesis in liver in vivo.

Distribution of hepatic ribosomes between different functional states in well-fed and protein-energy-deficient rats.

Electron microscopy of hepatic cells from protein-deficient young rats revealed extensive breakdown of the rough endoplasmic reticulum into fragments and vesicles whose membranes appeared fully studded with ribosomes. Additionally, there was biochemical evidence of marked disaggregation of polysomes (n greater than 2), and this was more prominent in the membrane-bound than in free polysomes. Protein malnutrition increased the proportion of membrane-bound ribosomes which were salt-releasable and therefore temporarily non-functional. It has been concluded that disaggregation of membrane-bound polysomes induced by malnutrition does not necessarily imply detachment of the monomeric ribosomes from the endoplasmic membrane into the pool of free ribosomes, which probably has a separate polyribosomal cycle.

Effect of feeding or intubation of tryptophan or methionine and threonine on hepatic polysome profiles in rats under meal-feeding or fasting conditions.

Under meal-feeding conditions, supplementary tryptophan in a protein-free diet did not aggregate hepatic ribosomes, while the supplementation of methionine and threonine to the protein-free diet aggregated the ribosomes as compared with that in rats fed the protein-free diet. Under long-fasting conditions, this effect of methionine and threonine on polysomal aggregation disappeared. Intubation of tryptophan caused the aggregation of hepatic ribosomes, especially in fasted rats, and this aggregation with tryptophan disappeared on simultaneous administration of glucose, and then serum glucocorticoid was significantly enhanced but serum insulin was not changed. With single addition of tryptophan, methionine, threonine or leucine, serum glucocorticoid was enhanced in all groups but polysome aggregation was observed with only tryptophan addition. These results suggested that, although the intubation of tryptophan or another amino acid to long-fasted rats induced the increase of serum glucocorticoid which causes the degradation of extrahepatic tissue proteins, tryptophan might be the first limiting amino acid of endogenous amino acids in fasting conditions and consequently enhances the hepatic ribosome aggregation. In meal-feeding or ad libitum feeding of a protein-free diet, methionine and threonine might be the more limiting amino acids.

Relationships between dietary levels of each essential amino acid and hepatic polysome profiles in rats.

The relationships between dietary levels of the essential amino acids and hepatic polysome profiles of rats were investigated with special attention to the amino acid requirement pattern for the maximum rat growth as determined by other investigators. The basal diet contained a 7% essential amino acid mixture and a 3% non-essential amino acid mixture, with appropriate amounts of other nutrients. Rats were fed test diet for 5 hours and then the polysome profile was determined. The amounts of essential amino acids needed for maximum aggregation of polysome were low for methionine-cystine, leucine and tryptophan as compared with requirements for maximum growth. But in other essential amino acids, the amounts were in almost the same range as those reported for maximum growth by others. The differences between the amino acid requirement patterns for maximum aggregation of hepatic ribosomes and for maximum growth of rats might be due to a difference in amino acid requirements of the liver and whole body. Therefore, the hepatic polysome profile might be used to measure the effect of amino acid supplementation on dietary proteins. The requirement pattern of essential amino acids in other organs may be studied by polysome profile determination.

On the binding of concanavalin a to bacterial and mammalian ribösome

1. 1. The interaction of concanavalin A with bacterial and mammalian ribosomes was analyzed comparatively. 2. 2. The lectin was radioactively labelled by iodination then incubated, on the one hand, with total Escherichia coli ribosomes and, on the other hand, with total ribosomes or membrane-free polysemes or separated ribosomal subunits prepared from rat hepatocytes. 3. 3. It was found that bacterial ribosomes do not possess any specific binding site accessible to concanavalin A whereas hepatic ribosomes interact with the lectin in a molar ratio of approximately 1:1.

Effects of amino acid imbalance on polysome profiles and 14C-labeled amino acid incorporation into tissue proteins in rats.

Feeding of rats on a low casein diet supplemented with a small amount of methionine caused an enlargement of liver size and an accumulation of lipids in the liver. Experiments were conducted to examine the effects induced by the amino acid-imbalanced diet on the metabolism of dietary amino acids with special reference to protein synthesis. Protein and DNA contents of the liver per 100 g body weight with rats fed on the imbalanced diet were apparently higher than those of rats fed on the basal diet. Ingestion of the imbalanced diet clearly stimulated hepatic ribosome aggregation but not skeletal muscle ribosome aggregation. Incorporation of [14C]leucine into liver protein was markedly increased in the group consisting of rats fed on the imbalanced diet (imbalanced group) and that into skeletal muscle was similar to the result with the group receiving the basal diet (basal group). Relative content of [14C]leucine in plasma albumin was higher in rats of the imbalanced group, whereas that in plasma of very low-density lipoprotein was reduced. These results indicate that the unbalanced inter-organ or intra-organ utilizations of dietary amino acids for protein synthesis were produced by the condition of the amino acid imbalance. Such metabolic effects of the amino acid imbalance may result in the enlargement of liver size and the accumulation of lipids in the liver.

Comparison of endogenous and exogenous RNA primers of poly(U) polymerase in rat hepatic ribosomes

The present work indicates that RNA primer requirements for poly(U) polymerase in the free ribosomes of the rat liver depend upon the degree of enzyme purification. The poly(U) polymerase activity obtained from a crude free ribosomal preparation was compared with the enzymic activity of a partially purified enzyme. After preliminary purification, the enzyme was fractionated by chromatography on Sephadex G-150 and CM-cellulose. Our results demonstrate the presence of several forms of poly(U) polymerase activities, some requiring exogenous RNA and others possessing their own endogenous primer RNA.

Free and membrane-bound ribosomes in regenerating rat liver

There has heretofore been little agreement concerning the validity of earlier conjectured shifts in the distribution of free and membrane-bound ribosomes during the precess of hepatic regeneration. In order to provide a quantitative test of these conjectures a simple double-isotope technique is here described which permits a detailed comparison of free and membrane-bound ribosomes isolated from “co-homogenates” of livers from partially hepatectomized and sham-operated animals. It is shown that 48 h after partial hepatectomy, a time at which liver cell proliferation is proceeding at a maximal rate, there is indeed a striking shift in ribosome distribution resulting in about a 40% rise in the proportion of free ribosomes. Over the same interval the absolute concentration of hepatic ribosomes increases markedly with the result that the total number of free ribosomes per unit mass of regenerating tissue rises to nearly double that in normal liver. The observed shift to favor a substantial increase in the concentration of free ribosomes is consistent with an enhanced need in regenerating liver for the synthesis of proteins destined for intracellular use. It is suggested that the method described here, which permits sensitive and precise comparison of the distribution of free and membrane-bound ribosomes in paired tissue samples, may find useful application in other systems as well.

Sparsomycin-induced disaggregation but not detachment of hepatic membrane-bound polyribosomes.

SummaryThe nature of attachment of hepatic ribosomes to membranes after spar-somycin treatment in vivo of rats and mice was investigated. The results based upon the influence of treatment in vitro with different concentrations of KC1 and with puromycin on the detachment of membrane-bound ribosomes from membranes of livers of spar-somycin-treated animals revealed that these ribosomes are attached to membranes by peptides as well as by ionic bridging and are considered as fall-off ribosomes. Similar effects have been described after treatment in vivo with dimethylnitrosamine. In contrast, treatment in vivo with ethionine or CCL produced run-off ribosomes which are attached only by ionic bridging since they become detached in vitro with KC1 treatment alone.

Effect of maternal ethanol consumption on foetal and neonatal rat hepatic protein synthesis

Effects of maternal ethanol consumption were investigated on the rates of protein synthehsis by livers of foetal and neonatal rats both in vivo and in vitro, and on the activities of enzymes involved in protein synthesis and degradation. The rates of general protein synthesis by ribosomes in vitro studied by measuring the incorporation of [14C]leucine into ribosomal protein showed that maternal ethanol consumption resulted in an inhibition of the rates of protein synthesis by both foetal and neonatal livers from the ethanol-fed group. The rates of incorporation of intravenously injected [14C]leucine into hepatic proteins were also significantly lower in the foetal, neonatal and adult livers from the ethanol-fed group. Incubation of adult-rat liver slices with ethanol resulted in an inhibition of the incorporation of [14C]leucine into hepatic proteins; however, this effect was not observed in the foetal liver slices. This effect of externally added ethanol was at least partially prevented by the addition of pyrazole to the adult liver slices. Pyrazole addition to foetal liver slices was without significant effect on the rates of protein synthesis. Cross-mixing experiments showed that the capacity of both hepatic ribosomes and pH5 enzyme fractions to synthesize proteins was decreased in the foetal liver from the ethanol-fed group. Maternal ethanol consumption resulted in a decrease in hepatic total RNA content, RNA/DNA ratio and ribosomal protein content in the foetal liver. Foetal hepatic DNA content was not significantly affected. Ethanol consumption resulted in a significant decrease in proteolytic activity and the activity of tryptophan oxygenase in the foetal, neonatal and adult livers. It is possible that the mechanisms of inhibition of protein synthesis observed here in the foetal liver after maternal ethanol consumption may be responsible for at least some of the changes observed in 'foetal alcohol syndrome'.

Effect of starvation on the distribution of free and membrane-bound ribosomes in rat liver and on the content of phospholipid and glycogen in purified ribosomes

The distribution of free and membrane-bound ribosomes in liver in response to starvation has not been clearly defined. An investigation has been made of the effects of starvation on the content of DNA, RNA, protein, phospholipid and glycogen in rat liver, on the distribution of free and membrane-bound ribosomes, and on the content of phospholipid and glycogen in free and bound ribosome fractions. The results indicate that starvation can produce up to a 50% reduction in hepatic ribosomes without altering either the fraction of rRNA relative to the total RNA or the distribution of free and membrane-bound ribosomes. In addition, the degree of contamination of isolated ribosomes with membranous material does not fluctuate with changes in the nutritional status of the animal. The results suggest that the relative capacities for protein synthesis among the two ribosome compartments are maintained during the early stages of starvation. Further, co-sedimentation with glycogen is not responsible for the presence of membranous materials in purified ribosomes.

Effect of food deprivation and hypophysectomy on in vitro protein synthesis by membrain-bound and free hepatic ribosomes.

The protein synthetic activities of membrane-bound and free hepatic ribosomes isolated from intact rats fed ad libitum, and normal rats subjected to food restriction to match that of hypophysectomised (Hx) rats were compared to the in vitro protein synthetic capacity of hepatic ribosomes isolated from Hx rats. Hypophysectomy resulted in decreased protein synthetic ability of bound ribosomes, whether protein synthesis was directed by endogenous messenger RNA (mRNA) (p less than 0.05) or by polyuridylic acid (polyU) (p less than 0.01). In contrast, the protein synthetic activity of free hepatic ribosomes from Hx rats was reduced when protein synthesis was directed by endogenous mRNA (p less than 0.05) but, when polyU was substituted as the messenger, the protein synthetic activity of these free ribosomes was equal to that of control rats. On the other hand the effects of food restriction on hepatic ribosomal function could be clearly differentiated from the effects observed following hypophysectomy. Thus, the reduced protein synthetic activity of hepatic bound ribosomes isolated from food restricted normal rats was not demonstrable, when polyU was used to direct protein synthesis. Further, food restriction had no effect on the protein synthetic activity of free hepatic ribosomes, and this was true when protein synthesis was directed by either endogenous or artificial messenger. It is concluded that hypophysectomy reduces the protein synthetic ability of both bound and free hepatic ribosomes, and this change of ribosomal function of Hx rats cannot be attributed to their decreased food intake.

Effect of hypophysectomy on protein synthetic-activity of free and bound hepatic ribosomes from insulin-deficient rats

Acute insulin deficiency in rats results in a decrease in the in vitro protein synthetic activity of isolated hepatic membrane-bound ribosomes and an increase in activity of free ribosomes. These changes are prevented by concomitant insulin treatment and are reversed by the administration of insulin. The current study evaluated the role of the pituitary in the genesis of these changes. The severity of diabetes produced by streptozotocin was less in hypophysectomized (Hx) rats, and in Hx rats receiving hormone replacement, as compared with similarly streptozotocin-treated intact rats. Although acute insulin deficiency in intact rats produced the previously described increase in protein synthetic activity of free hepatic ribosomes and decrease in activity of hepatic bound ribosomes, these changes did not occur in Hx rats, even when Hx rats received replacement doses of thyroxine, ACTH, and growth hormone. Thus, the changes in hepatic protein synthetic activity that occur in rats with acute experimental diabetes mellitus are secondary to the metabolic sequalae of insulin lack and the response of the pituitary gland to insulin deficiency.

Effect of duration of insulin deficiency on membrane-bound and free ribosomes from livers of diabetic rats.

In order to define the effect of duration of diabetes on hepatic protein sysntesis, membrane-bound and free ribosomes were isolated from livers of rats, 3, 7 and 28 days after administration of intravenous streptozotocin (75 mg/kg). Hepatocytes from the same rats were subjected to ultrastructural quantitative analysis. By day 3 there was a significant loss in the amount of rough endoplasmic reticulum (RER) per volume cytoplasm; however, the normal ratio of membrane-bound ribosomes per unit length of membrane was maintained. These hepatocyte ultrastructural changes continued over the ensuing four weeks. In spite of this decrease in amount of RER, in vitro protein synthetic activity of hepatic membrane-bound polyribosomes was unchanged from controls at three days, and by 28 days protein synthetic activity of bound hepatic ribosomes from diabetic rats was almost twice that of normal controls (p less than .01). In contrast to the effect of diabetes on bound ribosomes, there was no change in protein synthetic activity of free polyribosomes isolated from livers of rats, 3, 7 or 28 days after induction of diabetes. Thus, the effect of any given degree of diabetes on hepatic protein synthesis appears to vary with the population of hepatic ribosomes being studied, and with duration of insulin deficiency.

Hormonal requirements for acute stimulation of rat liver polysome formation by amino acid feeding

An investigation has been made of the hormonal requirements for the stimulation of rat liver polysome formation in vivo which results from administration of an amino acid mixture by stomach tube. The use of Mg 2+ precipitation as a technique for isolation of hepatic ribosomes has been evaluated and the proportion of ribosomes in polysomes prepared by this method has been assessed both by measurement of amino acid incorporation in vitro due to the extension of nascent peptide chains and by direct sucrose density gradient analysis. The results of both amino acid incorporation studies and sucrose gradient analysis indicate that amino acids can stimulate polysome formation in hypophysectomised and adrenalectomised rats without the need for administration of hormones. In hypophysectomised rats, growth hormone also promotes polysome formation, but does not enhance the amino acid effect. In contrast, livers of diabetic rats respond very poorly to amino acid force-feeding unless insulin is also given. Insulin alone has much less effect than insulin plus amino acids. The results suggest that amino acid supply may be rate-limiting for hepatic polysome formation in normal, adrenalectomised and hypophysectomised rats, but that availability of insulin is the rate-limiting factor in diabetic animals.

Characteristics of membrane-bound and free hepatic ribosomes from insulin-deficient rats. I. Acute experimental diabetes mellitus.

Membrane-bound and free ribosomes were prepared by discontinuous density gradient centrifugation from livers of rats 2-3 days after receiving alloxan (75 mg/kg) or streptozotocin (100 mg/kg). Hepatocytes from these animals were also examined by electron microscopy and subjected to quantitative morphometric analysis. The results indicated that the two populations of hepatic ribosomes respond differently to acute insulin deficiency. There was an overall reduction (P < 0.001) in total number of bound ribosomes per volume cytoplasm: the remaining bound ribosomes underwent a shift to smaller-sized ribosomal messenger RNA (mRNA) aggregates (P < 0.02); and the proteinsynthetic activity of these bound ribosomes was less than normal (P < 0.02) when protein synthesis was directed by endogenous mRNA. However, there was no difference between bound ribosomes from livers of normal and diabetic rats when protein synthesis was directed by polyuridylic acid. In contrast, free ribosomes were unchanged in number and degree of ribosomal mRNA aggregation, but displayed a significantly increased rate of in vitro protein synthesis (P < 0.01) as compared to normal controls. This increased protein-synthetic activity occurred when amino acid incorporation was directed by endogenous mRNA or polyuridylic acid. These changes in structure and function of bound and free hepatic ribosomes were prevented by the concomitant administration of insulin. The decrease in protein-synthetic activity of bound hepatic ribosomes from acutely diabetic rats seems to be secondary to marked disruption and disaggregation of the rough endoplasmic reticulum (RER) with production of smaller ribosomal mRNA aggregates which incorporate less amino acids into protein. Increased protein synthetic activity of free ribosome appears to be related to the ability of these ribosomes to copy mRNA more efficiently.

Effect of threonine supplementation on hepatic polysome patterns and protein synthesis of rats fed a threonine-deficient diet

We have investigated changes in hepatic polysomal pattern and protein synthesis in rats fed a threonine-deficient diet for 1 week and the response that occurred subsequent to threonine supplementation. The experiments showed that: 1. 1. The percentage of hepatic ribosomes in free form or small aggregates was elevated in rats fed a threonine-deficient diet. 2. 2. Administration of threonine alone to rats previously fed the deficient diet increased the degree of ribosomal aggregation but administration of tryptophan or other amino acids was without effect. The shift in polyribosomes toward heavier aggregates was accompanied by an increase in [ 14C]leucine incorporation in vivo into liver proteins. 3. 3. The increase in ribosomal aggregation after administration of threonine was only transitory but reaggregation could be initiated by subsequent administration of a mixture of amino acids devoid of threonine. 4. 4. Involvement of corticosteroid hormones in mediating the response induced by threonine was ruled out, since the stimulatory effects could be produced in both intact and adrenalectomized animals. 5. 5. Actinomycin D did not prevent the enhancement of hepatic protein synthesis induced by threonine, nor did it affect the extent of polysome reformation. The responses to threonine administration would thus seem not to require RNA synthesis which suggests that threonine exerts its effect through a cytoplasmic mechanism.