Regulation Of Hepatic Protein Synthesis Research Articles

Short-term changes in diet composition do not affect in vivo hepatic protein synthesis in rats.

Protein synthesis is critical to protein homeostasis (proteostasis), and modifications in protein synthesis influence lifespan and the development of comorbidities associated with obesity. In the present study, we examined the acute response of liver protein synthesis to either high-fat or high-sucrose diets in order to elucidate nutrient-mediated regulation of hepatic protein synthesis in the absence of body fat accumulation. Total and endoplasmic reticulum-associated protein syntheses were assessed by use of the stable isotope, deuterium oxide (2H2O), in rats provided a control diet or diets enriched in polyunsaturated fat, saturated fat, or sucrose for 2, 4, or 7 days. The three experimental diets increased hepatic triglycerides 46-91% on day 7 and fasting insulin levels 83-117% on day 7, but did not result in differences in body weight when compared with control ( n = 6/diet/time). The fraction of newly synthesized proteins in total liver lysates and microsomes was not significantly different among dietary groups ( n = 3/diet/time). To determine whether the experimental diets provoked a transcriptional response to enhance the capacity for protein synthesis, we also measured a panel of genes linked to amino acid transport, synthesis, and processing. There were no significant differences in any of the genes measured among groups. Therefore, dietary treatments that have been linked to impaired proteostasis and that promote hepatic steatosis and insulin resistance, did not result in significant changes in total or ER-associated protein synthesis in the liver over a 7-day period.

The Regulation of Hepatic Protein Synthesis during Fasting in the Rat

We have studied translational control in the model of 48 h of fasting in the rat. Our initial observations showed a paradoxical increase in ribosomal protein S6 (rpS6) phosphorylation and a decrease in eukaryotic initiation factor 2alpha (eIF2alpha) phosphorylation. These effects, which would favor an increase in protein synthesis, could be attributed to increased circulating concentrations of branched-chain amino acids in fasting. To determine what mechanisms might account for decreased hepatic translation in fasting, we examined the cap binding complex. eIF4E-bound 4E-BP1 did not increase. However, eIF4E-bound eIF4G and total cellular eIF4G were profoundly decreased in fasted liver. eIF4G mRNA levels were not lower after fasting. Based on the hypothesis that decreased eIF4G translation might account for the reduced eIF4G content, we fractionated ribosomes by sucrose density centrifugation. Immunoblotting for rpS6 showed modest polysomal disaggregation upon fasting. PCR analysis of polysome profiles revealed that a spectrum of mRNAs undergo different translational regulation in the fasted state. In particular, eIF4G was minimally affected by fasting. This indicated that reduced eIF4G abundance in fasting may be a function of its stability, whereas its recovery upon refeeding is necessarily independent of its own involvement in the cap binding complex. Western immunoblotting of polysome fractions showed that phosphorylated rpS6 was disproportionately present in translating polysomes in fed and fasted animals, consistent with a role in translational control. However, the translation of rpS8, an mRNA with a 5'-oligopyrimidine tract, did not coincide with rpS6 phosphorylation, thus dissociating rpS6 phosphorylation from the translational control of this subset of mRNAs.

Oral Administration of Leucine Stimulates Ribosomal Protein mRNA Translation but Not Global Rates of Protein Synthesis in the Liver of Rats

The objective of the current study was to examine the role of the branched-chain amino acid (BCAA) leucine in the regulation of hepatic protein synthesis and ribosomal protein (rp) mRNA translation in vivo. Food-deprived (18 h) male rats (200 g) were orally administered saline (control) or 270 mg leucine, isoleucine or valine and killed 1 h later. Administration of any BCAA resulted in enhanced phosphorylation of eukaryotic initiation factor (eIF) 4E-binding protein-1 (4E-BP1) compared with controls. However, leucine was the most effective at stimulating phosphorylation of 4E-BP1 as well as the 70-kDa ribosomal protein S6 kinase (S6K1). Despite these effects on components of the translation initiation process, there were no differences in total protein synthesis rates among treatment groups. The distribution of rp (S4, S8, L26) and non-rp (albumin, beta-actin) mRNAs across sucrose density gradients showed that the preponderance of hepatic rp mRNAs in control rats was unloaded from polysomes. Of the BCAA, only leucine was the most effective in causing a shift in the distribution of rp mRNA to polysomes compared with controls. Non-rp transcripts remained mainly polysome-associated under all conditions. These results suggest that leucine is most effective among the BCAA in its ability to stimulate translation of rp mRNA in liver. Furthermore, the translation of rp mRNA is disjointed from rates of total protein synthesis in liver and related to the degree of S6K1 phosphorylation.

Role of amino acid‐induced changes in ion fluxes in the regulation of hepatic protein synthesis

Alanine is a powerful stimulator of hepatic protein synthesis whose mechanism of action has not yet been ascertained. The present work aimed to elucidate whether rate changes in ion fluxes accompanying the transport of this amino acid could play a role in the stimulation of protein synthesis. In perfused livers, the utilization of alanine produced a net uptake of K+ of 1.5 mumol/min/liver, a progressively increasing efflux of Ca2+ to reach a maximum of 0.9 mumol/min/liver, and alkalization of the extracellular medium. Inhibition of Na+/K+ exchange by ouabain reversed only the uptake of K+, indicating that this is the main way for the efflux of Na+ cotransported with alanine. In isolated hepatocytes, the uptake of alanine increased the intracellular content of K+ and the cell volume. The following observations suggest that these changes, and not an increased intracellular concentration of Na+, are associated with the stimulation of protein synthesis: 1) Ouabain inhibited the alanine stimulation of L-[3H]-valine incorporation into protein without altering the basal rate of protein labeling; 2) ouabain had no effects on alanine uptake indicating that Na+ influx is not involved in the alanine stimulation of protein synthesis; 3) disruption of Na+ gradient across the plasma membrane by specific ionophores, monensin and gramicidin D, inhibited both basal and alanine-stimulated protein synthesis, but substitution of extracellular Na+ by K+ did not prevent the stimulatory action of alanine. The observation that hypotonic buffer enhanced protein synthesis to the same degree than alanine in liver cells indicates that alanine-induced cell swelling could be sufficient to stimulate protein synthesis.

Regulation of iron metabolism in HepG2 cells: A possible role for cytokines in the hepatic deposition of iron

In chronic inflammation it is reported that serum iron is depleted and hepatic iron is increased because of reticuloendothelial system iron blockade. However, recent studies indicate that hepatic parenchymal cells increase the uptake of transferrin-bound iron after in vivo stimulation with bacterial lipopolysaccharide, suggesting that endotoxemia itself or lipopolysaccharide-induced production of inflammation-related cytokines may also be responsible for this phenomenon. In this study the actions of inflammation-related cytokines on the synthesis of iron-binding proteins (transferrin and ferritin) and transferrin receptor and the uptake of transferrin-bound iron were investigated in a human hepatoblastoma cell line, HepG2, which is the most commonly used cell line for examining the regulation of hepatic protein synthesis by cytokines. The cells were exposed to interleukin-1 beta, interleukin-6 or tumor necrosis factor-alpha separately for 24 hr. In each cytokine treatment group, the level of transferrin, which is secreted into the conditioned medium, was found to be decreased compared with that of untreated cells. On the other hand, the biosynthesis of ferritin was markedly elevated after the same treatment. This increase in ferritin by cytokine treatment was diminished when deferoxamine was used concomitantly to deplete intracellular chelatable iron. After stimulation with interleukin-1 beta, interleukin-6 or tumor necrosis factor-alpha, 59Fe-labeled transferrin uptake into the cells was increased by 36%, 48%, or 18%, respectively, and this uptake was inhibited by the addition of excess unlabeled transferrin. A binding study with 125I-labeled diferric transferrin revealed that the three cytokines increased the number of transferrin receptors on the cell surface by 1.15-fold to 1.35-fold.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in hepatocyte water volume affect protein synthesis.

Protein synthesis in isolated rat hepatocytes was determined from the incorporation of [3H]leucine (4 mM) into acid-precipitable material in the presence of amino acids at twice their physiological concentration. Protein synthesis increased linearly with time and incubated cell protein, and was inhibited by cycloheximide by more than 95%. In normo-osmotic incubations containing amino acids at twice the physiological concentrations the rate of [3H]leucine incorporation was 5.8 ± 0.2 nmol/h per mg cell protein (n = 26). Hyperosmotic cell shrinkage due to addition of 60 mM-NaCl or 120 mM-raffinose inhibited [3H]leucine incorporation into acid-precipitable material by 60% and 74%, respectively, whereas hypo-osmotic cell swelling was ineffective. Inhibition of protein synthesis by adding 120 mM-raffinose was largely counteracted by simultaneous lowering of the NaCl concentration by 60 mM. Glutamine (10 mM) had no effect on protein synthesis in normo-osmotic incubations (320 mosm), but stimulated protein synthesis in hyperosmotically (440 mosm) pre-shrunken cells almost to rates found in normo-osmotic (320 mosm) control incubations. Cyclic AMP and vasopressin inhibited protein synthesis by 23% and 8%, respectively, whereas insulin and phenylephrine were ineffective. However, inhibition of protein synthesis by cyclic AMP was about twice as strong in the presence of vasopressin or phenylephrine. When protein synthesis was preinhibited by cyclic AMP, [3H]leucine incorporation was stimulated by glutamine (10 mM), insulin or hypoosmotic exposure. There was a close relationship between the inhibition of protein synthesis and the extent of hepatocyte shrinkage induced by the abovementioned effectors, suggesting a role of cell volume in the regulation of hepatic protein synthesis.

Nitric Oxide May Upregulate In Vivo Hepatic Protein Synthesis During Endotoxemia

Nitric oxide (NO) has been implicated as a mediator of hemodynamic and metabolic changes associated with endotoxemia and inflammation. In vitro studies suggest that NO inhibits hepatocyte protein synthesis but the role of NO in the regulation of hepatic protein synthesis in vivo is not known. In this study, rats were given endotoxin or saline after pretreatment with the NO synthase inhibitor NG-nitro-L-arginine or solvent, and plasma levels of nitrite (NO2), nitrate (NO3), and aspartate aminotransferase and hepatic protein synthesis rate in vivo were measured after 4 and 10 hours. The NG-nitro-L-arginine effectively blocked the increase in serum NO2/NO3 seen in endotoxemia and also inhibited the increase in hepatic protein synthesis in endotoxemic rats. The aspartate aminotransferase levels were elevated in endotoxemic rats pretreated with NG-nitro-L-arginine. Results support previous reports of a protective effect of NO on the liver in endotoxemia and suggest that NO may upregulate hepatic protein synthesis in vivo. Further study is needed to clarify the reason for the apparent difference between the effect of NO on hepatic protein synthesis in vivo and in vitro.

Liver cell volume and protein synthesis.

Protein synthesis in isolated rat hepatocytes was determined from the incorporation of [3H]leucine (4 mM) into acid-precipitable material in the presence of amino acids at twice their physiological concentration. Protein synthesis increased linearly with time and incubated cell protein, and was inhibited by cycloheximide by more than 95%. In normo-osmotic incubations containing amino acids at twice the physiological concentration the rate of [3H]leucine incorporation was 5.8 +/- 0.2 nmol/h per mg of cell protein (n = 26). Hyperosmotic cell shrinkage due to addition of 60 mM-NaCl or 120 mM-raffinose inhibited [3H]leucine incorporation into acid-precipitable material by 60 and 74% respectively, whereas hypo-osmotic cell swelling was ineffective. Inhibition of protein synthesis by adding 120 mM-raffinose was largely counteracted by simultaneous lowering of the NaCl concentration by 60 mM. Glutamine (10 mM) had no effect on protein synthesis in normo-osmotic incubations (320 mosM), but stimulated protein synthesis in hyperosmotically (440 mosM) pre-shrunken cells almost to rates found in normo-osmotic (320 mosM) control incubations. Cyclic AMP and vasopressin inhibited protein synthesis by 23% and 8% respectively, whereas insulin and phenylephrine were ineffective. However, inhibition of protein synthesis by cyclic AMP was about twice as strong in the presence of vasopressin or phenylephrine. When protein synthesis was preinhibited by cyclic AMP, [3H]leucine incorporation was stimulated by glutamine (10 mM), insulin or hypo-osmotic exposure. There was a close relationship between the inhibition of protein synthesis and the extent of hepatocyte shrinkage induced by the above-mentioned effectors, suggesting a role of cell volume in the regulation of hepatic protein synthesis.

Regulation of hepatic protein synthesis in chronic inflammation and sepsis.

The regulation of protein synthesis was determined in livers from control, sterile inflammatory, and septic animals. Total liver protein was increased in both sterile inflammation and sepsis. The rate of protein synthesis in vivo was measured by the incorporation of [3H]phenylalanine into liver proteins in a chronic (5 day) intra-abdominal abscess model. Both sterile inflammation and sepsis increased total hepatic protein synthesis approximately twofold. Perfused liver studies demonstrated that the increased protein synthesis rate in vivo resulted from a stimulation in the synthesis of both secreted and nonsecreted proteins. The total hepatic RNA content was increased 40% only in sterile inflammation, whereas the translational efficiency was increased twofold only in sepsis. The increase in translational efficiency was accompanied by decreases in the amount of free 40S and 60S ribosomal subunits in sepsis. Rates of peptide-chain elongation in vivo were increased 40% in both sterile inflammation and sepsis. These results demonstrate that sepsis induces changes in the regulation of hepatic protein synthesis that are independent of the general inflammatory response. In sterile inflammation, the increase in protein synthesis occurs by a combination of increased capacity and translational efficiency, while in sepsis, the mechanism responsible for accelerated protein synthesis is an increased translational efficiency.

Hepatic Protein and Amino-Acid Metabolism in Poultry

Two subjects are discussed: first, the regulation of hepatic protein synthesis; and second, the intermediary metabolism of methionine, particularly with respect to the role of 2-hydroxy-4-(methylthio)-butanoic acid (HMB, Alimer®). In the first section, the regulation of albumin synthesis is reviewed in terms of molecular events associated with the changes in albumin synthesis during fasting and refeeding. The effect of infection or of inflammatory stress on both albumin and total protein synthesis in the liver is also discussed. In the second part, research results are presented which indicate that HMB is a naturally occurring compound in methionine intermediary metabolism. The HMB synthesis by chick liver enzymes is demonstrated, and its role in normal avian methionine metabolism is discussed.

Hormonal regulation of hepatic protein synthesis

Hormonal regulation of hepatic protein synthesis

Key role of L-alanine in the control of hepatic protein synthesis.

We investigated the effects of administration of single amino acids to starved rats on the regulation of protein synthesis in the liver. Of all the amino acids tested, only alanine, ornithine and proline promoted statistically significant increases in the extent of hepatic polyribosome aggregation. The most effective of these was alanine, whose effect of promoting polyribosomal aggregation was accompanied by a decrease in the polypeptide-chain elongation time. The following observations indicate that alanine plays an important physiological role in the regulation of hepatic protein synthesis. Alanine was the amino acid showing the largest decrease in hepatic content in the transition from high (fed) to low (starved) rates of protein synthesis. The administration of glucose or pyruvate is also effective in increasing liver protein synthesis in starved rats, and their effects were accompanied by an increased hepatic alanine content. An increase in hepatic ornithine content does not lead to an increased protein synthesis, unless it is accompanied by an increase of alanine. The effect of alanine is observed either in vivo, in rats pretreated with cycloserine to prevent its transamination, or in isolated liver cells under conditions in which its metabolic transformation is fully impeded.

Rapid changes in glucose metabolism following the administration of ethionine: its role in regulating hepatic protein synthesis.

The relationship between the changes in portal glucagon, insulin, glucose, and hepatic protein synthesis were investigated during ethionine intoxication. There was a 50% decrease in blood glucose, a seven-fold increase in portal glucagon and a 90% increase in portal insulin, all of which coincided temporally with the inhibition of hepatic protein synthesis. When reversal of ethionine intoxication was initiated with adenine it simultaneously restored blood glucose, insulin, glucagon, and protein synthesis. Protein synthesis could not be adequately restored by glucose, but in this case hepatic ATP levels did not increase. In addition, glucose given by stomach tube prior to ethionine did not prevent the action of ethionine, though it did maintain plasma glucose levels and prevented the decrease in plasma insulin and increase in plasma glucagon. These results show that in vivo regulation of hepatic protein synthesis during ethionine intoxication is not likely to be mediated by portal insulin, glucose, and glucagon.

Role of the state of reduction of the nad system on the regulation of hepatic protein synthesis in the rat in vivo

1. 1. The administration of octanoate to rats in vivo increased the state of reduction of the hepatic NAD system and decreased the phosphorylation potential. This effect was accompanied by a 20% inhibition of protein synthesis. 2. 2. The acute administration of ethanol produced similar reduction of the hepatic NAD system; however, in contrast to octanoate no effect on the phosphorylation potential was detected and rates of protein synthesis were unaffected. 3. 3. It is concluded that a rise in the state of reduction of the NAD system is not effective in decreasing hepatic protein synthesis in vivo unless it is accompanied by a decrease in the phosphorylation potential.

Regulation of hepatic protein synthesis in the hibernator

Regulation of hepatic protein synthesis in the hibernator