Protein Synthesis In Hepatocytes Research Articles

Effect of amino acid deprivation on initiation of protein synthesis in rat hepatocytes

Conditions were defined for maintaining optimal protein synthetic activity in suspensions of freshly isolated rat hepatocytes. Under these conditions, isolated hepatocytes exhibited rates of protein synthesis and levels of polysomal aggregation equivalent to those observed in vivo and in perfused liver. Deprivation of total amino acids or single, essential amino acids resulted in a rapid decrease in the rate of protein synthesis, which was readily reversed by readdition of the deficient amino acid(s). The decrease was accompanied by a disaggregation of polysomes and an inhibition of 43S initiation complex formation, which was indicative of a limitation in the rate of initiation of protein synthesis. Extracts prepared from perfused liver deprived of amino acids were inhibitory to initiation of protein synthesis in reticulocyte lysate. The inhibition in reticulocyte lysate was accompanied by an increase in phosphorylation of the alpha-subunit of eukaryotic initiation factor 2 (eIF-2), suggesting activation of an eIF-2 alpha kinase or inhibition of a phosphatase in amino acid-deprived hepatocytes. This suggestion was confirmed by prelabeling hepatocytes with 32Pi before amino acid deprivation. Incorporation of 32Pi into eIF-2 alpha was two- to threefold higher in lysine-deprived cells than in hepatocytes incubated in fully supplemented medium. Overall, the results indicated that an increase in eIF-2 alpha phosphorylation was responsible for the defect in initiation of protein synthesis caused by amino acid deprivation.

Effect of interleukin 2 on Kupffer cell activation. Interleukin 2 primes and activates Kupffer cells to suppress hepatocyte protein synthesis in vitro.

Interleukin 2 (IL-2) is an essential mediator of the immune response and has also been shown to be protective in experimental models of sepsis. Macrophages have IL-2 receptors but their function is unknown. We investigated the effect of IL-2 on Kupffer cells, the fixed macrophages of the liver, using an in vitro rat hepatocyte-Kupffer cell coculture system. In this model, endotoxin (lipopolysaccharide) triggers Kupffer cells to induce suppression of hepatocyte protein synthesis. We found that pretreatment with 10 U/mL or more of IL-2 primed Kupffer cells, significantly reducing the concentration of lipopolysaccharide necessary to trigger Kupffer cell-mediated suppression of hepatocyte protein synthesis. Higher concentrations of IL-2 (greater than or equal to 1 x 10(4) U/mL) alone were capable of priming and triggering Kupffer cells to suppress hepatocyte protein synthesis. These data show that IL-2 increases Kupffer cell sensitivity to endotoxin, suggesting that IL-2 may play an important role in regulating macrophage responses to septic stimuli.

Endotoxin Modulation of Hepatocyte Secretory and Cellular Protein Synthesis Is Mediated by Kupffer Cells

We hypothesize that alterations of hepatocyte function in sepsis are modulated by endotoxin (lipopolysaccharide)-triggered Kupffer cells. In the present experiments the effect of lipopolysaccharide on secreted and cellular proteins synthesized by hepatocytes cultured alone or cocultured with Kupffer cells was investigated using polyacrylamide gel electrophoresis. Lipopolysaccharide had no direct effect on the types or amounts of secreted proteins synthesized by hepatocytes alone. Kupffer cells coculturing resulted in increased synthesis of some hepatocyte proteins (68k and 23k) whose production was not altered by lipopolysaccharide. In contrast, addition of lipopolysaccharide to the hepatocyte-Kupffer cell coculture substantially decreased synthesis of several proteins (73k, 66k, and 35k). Despite an overall decrease in protein synthesis of hepatocytes cocultured with Kupffer cells after the addition of lipopolysaccharide, there was increased synthesis of several individual proteins (58k and 44k). Similar effects were seen in synthesis of cellular protein. The addition of recombinant interleukin 1 to hepatocytes alone or in coculture had no effect on the amount or type of protein synthesized. We conclude that Kupffer cells regulate the types and amounts of individual proteins synthesized by hepatocytes via mediators other than interleukin 1.

Intestinal gram-negative bacterial overgrowth in vivo augments the in vitro response of Kupffer cells to endotoxin.

A number of disease states and therapeutic maneuvers common to surgical patients can result in changes in the intestinal flora, permitting bacterial overgrowth and translocation of bacteria to gut lymphoid tissue. It is possible that these changes in gut flora increase portal levels of several factors that are capable of altering macrophage activation state, including endotoxin, lymphokines, and eicosanoids. Since Kupffer cells are directly exposed to gut factors via the portal circulation, changes in intestinal flora may influence Kupffer cell responses. Using germfree rats, it has previously been shown that the presence of gut bacterial flora is important in inducing Kupffer cells to respond to endotoxin, and that an overgrowth of gram-negative bacteria can further augment Kupffer cell responses, supporting the above-mentioned hypothesis. The current set of experiments examines how intestinal gram-negative bacterial overgrowth in normal adult rats effects the response of Kupffer cells to septic stimuli. Kupffer cells were obtained from conventional rats with induced intestinal overgrowth with Escherichia coli C25 for 2 or 7 days. After 2 days of overgrowth, Kupffer cells were only slightly less responsive to lipopolysaccharide (LPS) than control Kupffer cells. However, after 7 days of overgrowth, when placed in coculture with normal hepatocytes, Kupffer cells were significantly more responsive to LPS (p less than 0.001), inducing a greater degree of suppression in hepatocyte protein synthesis at lower LPS concentrations. When cultured alone, Kupffer cells from these animals also produced more interleukin-1 (p less than 0.002) and prostaglandin E2 (PGE2) (p less than 0.009) in response to LPS. These results show that intestinal gram-negative bacterial overgrowth in conventional rats can have direct influences on the response of hepatic macrophages to septic stimuli, and provides further support to the hypothesis that imbalances in the intestinal flora can effect the responses of immune cells in other sites of the body.

Recombinant human interleukin-6 (IL-6/BSF-2/HSF) regulates the synthesis of acute phase proteins in human hepatocytes

Recombinant human IL-6 (rhIL-6) is a potent inducer of the synthesis of acute phase proteins in adult human hepatocytes. A wide spectrum of acute phase proteins is regulated by this mediator. After labeling of rhIL-6 stimulated human hepatocytes with [ 35S]methionine acute phase protein synthesis was measured by immunoprecipitation. Serum amyloid A, C-reactive protein, haptoglobin, α 1-antichymotrypsin and fibrinogen were strongly induced (26-, 23-, 8.6-, 4.6- and 3.8-fold increases, respectively). Moderate increases were found for α 1-antitrypsin (2.7-fold) and α 1-acid glycoprotein (2.7-fold). RhIL-6 had no effect on α 1-macroglobulin, whereas fibronectin, albumin and transferrin decreased to 64, 56 and 55% of controls. In the cases of serum amyloid A, haptoglobin, α 1-antichymotrypsin, α 1-antitrypsin and α 1-acid glycoprotein, dexamethasone enhanced the action of rhIL-6. We conclude that rhIL-6 controls the acute phase response in human liver cells.

Phenobarbital induction of cytochrome P-450 b,e genes is dependent on protein synthesis.

Phenobarbital induces liver cytochrome P-450 b,e proteins mainly by increasing the rate of transcription of these genes. The mechanism responsible for the phenobarbital increment in the rate of transcription of cytochrome P-450 b,e genes is unknown. The objective of this study was to assess whether active protein synthesis was needed for phenobarbital to induce the liver cytochrome P-450 b,e genes. Cycloheximide (2 mg per kg, i.p.) was administered 90 min prior to a single inductive dose of phenobarbital (80 mg per kg, i.p.) and mRNAS measured at 3, 6 and 12 hr by dot-blot hybridization. While phenobarbital increased cytochrome P-450 b,e mRNAs about 12-fold at 3 hr, this induction was abolished by cycloheximide. To define whether the absence of protein synthesis in hepatocytes inhibited the phenobarbital induction of cytochrome P-450 at the transcriptional level, in vitro transcription rates using isolated nuclei were measured. After phenobarbital administration, there was about a 20-fold increment in transcriptional rate of cytochrome P-450 b,e genes. This increment was abolished by prior injection of cycloheximide. It is proposed that either preexisting regulatory proteins or transacting factors dependent on active protein synthesis participate in the regulation of liver cytochrome P-450 b,e gene transcription after phenobarbital.

Splenectomy alters Kupffer cell response to endotoxin.

The spleen is a potential source for lymphokines, substances known to prime or activate macrophages. Liver macrophages or Kupffer cells are directly exposed to these products via the portal circulation. To determine whether a loss of splenic factors would alter Kupffer cell responses, we studied the effect of splenectomy or sham operation on Kupffer cell responses to endotoxin lipopolysaccharide (LPS). We determined Kupffer cell activation using an in vitro rat hepatocyte-Kupffer cell coculture system in which Kupffer cells normally mediate a significant decrease in hepatocyte protein synthesis if triggered by LPS. We found that Kupffer cells from splenectomized rats were significantly less responsive to LPS three to 60 days after splenectomy. Kupffer cells from sham-operated animals responded normally to LPS. This was contrasted to an increased sensitivity to LPS in the first two days following splenectomy. These data suggest that both splenectomy and a loss of splenic factors alter Kupffer cell responses to septic stimuli. We propose that the decreased sensitivity to LPS after splenectomy may be due to a loss of the priming effects of splenic lymphokines. This decreased responsiveness might contribute to the increased incidence of overwhelming infection after splenectomy.

Regulation of protein synthesis in intact rat liver by calcium mobilizing agents.

1. Exposure of intact perfused rat liver to EGTA, vasopressin or phenylephrine resulted in a rapid decrease in polysome formation. Pretreatment with phentolamine, an alpha-adrenergic antagonist, blocked the effect of phenylephrine. 2. Hormonal inhibitions of leucine incorporation into protein in isolated hepatocytes and of polysome formation in perfused liver were reversed in the presence of supraphysiologic extracellular Ca2+ concentrations. 3. The beta-adrenergic agonist isoproterenol exerted minimal effects on polysome content. 4. It is proposed that intracellular Ca2+ stores sensitive to hormonal modulation are necessary for maintenance of protein synthesis in hepatocytes.

Protein synthesis in hepatocytes isolated from patients with gastrointestinal malignancy.

To investigate the effect of remote and proximate cancer on hepatic protein metabolism, we determined rates of total protein synthesis by hepatocytes (HPS) isolated from 31 patients undergoing liver wedge biopsy: 7 patients with benign disease, 14 with gastric cancer, and 10 with colorectal cancer (5 of whom had liver metastases). Patients with malignant disease without weight loss had a threefold higher rate of total HPS (4,980 +/- 814 pmol/h per 10(5) viable cells) than patients with benign disease without weight loss (1,278 +/- 318 pmol/h per 10(5) viable cells, P less than 0.001). Among the patients with gastric cancer, eight with preoperative weight loss had lower rates of HPS (380 +/- 90 pmol/h per 10(5) viable cells) than those without weight loss (4,061 +/- 401 pmol/h per 10(5) viable cells, P less than 0.002). The highest rates of HPS were seen in patients with colorectal cancer with liver metastases (8,005 +/- 1,975 pmol/h per 10(5) viable cells) vs. colorectal cancer patients without liver metastases (3,060 +/- 575 pmol/h per 10(5) viable cells, P less than 0.03). These data indicate that modulation of hepatic protein synthesis occurs in malignancy in man. However, the stimulatory influence of the tumor-bearing state may be overridden by the inhibitory effects of cachexia.

Early hypomethylation of 2'-O-ribose moieties in hepatocyte cytoplasmic ribosomal RNA underlies the protein synthetic defect produced by CCl4.

Carbon tetrachloride (CCl4) treatment of rats produces an early defect in methylation of hepatocyte ribosomal RNA, which occurs concurrently with a defect in the protein synthetic capacity of isolated ribosomes. The CCl4-induced methylation defect is specific for the 2'-O-ribose position, and a corresponding proportional increase in m7G base methylation occurs in vivo. Undermethylated ribosomal subunits (rRNA) from CCl4-treated preparations can be methylated in vitro to a much greater extent than those from control preparations, and in vitro methylation restores their functional capacity. In vitro methylation of treated ribosomal subunits (which restores functional capacity) occurs at 2'-O-ribose positions (largely G residues). In contrast, in vitro methylation of control ribosomal subunits (which does not affect functional activity) represents base methylation as m7G, sites which are apparently methylated in treated preparations in vivo. Methylation/demethylation of 2'-O-ribose sites in rRNA exposed on the surface of cytoplasmic ribosomal subunits may represent an important cellular mechanism for controlling protein synthesis in quiescent hepatocytes, and it appears that CCl4 disrupts protein synthesis by inhibiting this 2'-O-ribose methylation.

Macrophage-Mediated Modulation of Hepatocyte Protein Synthesis

We have previously reported an in vitro model demonstrating decreased hepatocyte protein synthesis following co-culture with macrophage-rich peritoneal cells (MRPC) in Sprague-Dawley rats. These observations have been proposed by us and others to represent a possible model for macrophage- or Kupffer cell-mediated modulation of hepatocyte function. Such a mechanism may play a role in the etiology of hepatic failure in sepsis. This effect is shown in this investigation to be completely blocked by dexamethasone at concentrations equal to or greater than 10(-9) M. The presence of lymphocyte-activating factor in the MRPC-conditioned medium is suppressed at the same concentration of dexamethasone. Resident MRPC were shown to secrete significant amounts of lymphocyte-activating factor activity without further in vitro activation. These results support the concept that the MRPC mediation of decreased hepatocyte protein synthesis is inflammatory in nature and possibly associated with macrophage activation.

Alterations in the synthesis of proteins in hepatocytes of rainbow trout fed cyclopropenoid fatty acids

Hepatocytes from rainbow trout reared on a diet containing cyclopropenoid fatty acids were analyzed for alterations in protein composition and synthesis by double label experiments. Both cytosolic and micrososmal hepatocyte fractions were investigated. In the cytosolic fraction, the synthesis of proteins in the range of 68000 to 74000 daltons were significantly decreased. The identity of these proteins remains uncertain. A pronounced depression in both the mass and apparent synthesis of a 200000 to 240000 dalton microsomal protein was also observed. Immunoblotting with antibodies raised against goose acetyl-CoA carboxylase and avidin-peroxidase staining suggest that this protein is acetyl-CoA carboxylase. Moreover, synthesis of this protein as well as mass of the protein in cyclopropenoid fatty acid-fed fish were less than 20% of that found in control fish.

Transcriptional regulation of serum amyloid A gene expression.

Serum amyloid A (SAA) is a plasma apolipoprotein produced by the liver in response to inflammatory stimuli. The murine SAA gene family is made up of three genes, SAA1, SAA2, and SAA3, plus a pseudogene. The SAA1 and SAA2 genes are highly homologous while the SAA3 gene has diverged substantially from the other two genes. Using small fragments from the cloned genes, we have analyzed the expression of each gene in the SAA family. Within 12 h after endotoxin administration, total liver SAA mRNA increases by 2000-fold, reaching approximately 20,000 transcripts/cell. Each gene accounts for approximately one-third of total SAA mRNA transcripts at this time. The increase is specific, since the levels of the mRNAs encoding albumin and apolipoprotein A-I in liver decrease 2-fold by 24 h. This correlates with a 2-fold decrease of the serum concentrations of these two proteins as well as their in vitro protein synthesis in primary hepatocytes. SAA1+2 mRNAs maintain their maximum levels until 36 h after lipopolysaccharide administration, while SAA3 mRNA is degraded to 20% its maximal level. As assayed by in vitro transcription in isolated hepatocyte nuclei, total SAA gene transcription increases at least 300-fold during the inflammatory response. The transcription rates of the individual SAA genes are similar during the initial stages of this response, reaching peak levels at 3 h. A comparison of the rates of SAA gene transcription and SAA mRNA accumulation suggests that SAA mRNA levels are regulated during the acute phase response by increased transcription and mRNA stabilization.

The macrophage-mediated regulation of hepatocyte synthesis of antithrombin III and α1-proteinase inhibitor

Antithrombin III (ATIII) is an anticoagulant protein which binds and inactivates thrombin and other serine proteinases. Little is known about regulation of its synthesis. We confirm that ATIII is synthesized by isolated rat hepatocytes, and that its synthesis is not altered by direct feedback of its complexes with proteinases. Neither is hepatocyte synthesis of ATIII altered by supernatants from macrophages cultured in the presence of ATIII-proteinase complexes. However, culture of macrophages with fibrinogen fragment D results in production of a factor(s) in the macrophage supernatants which stimulates hepatic fibrinogen synthesis, as previously described, and also stimulates the synthesis of ATIII and al-proteinase inhibitor ( α 1PI). Synthesis of albumin and rat α 2-macroglobulin ( α 2M) is not altered. Culture of macrophages in the presence of bacterial endotoxin also results in release of a factor(s) into the medium which stimulates the same changes in hepatocyte protein synthesis. These results show for the first time a mechanism by which synthesis of ATIII can be regulated during coagulation and fibrinolysis.

The effects of ethanol on collagen synthesis by rat hepatocytes in primary culture

Abstract The effect of ethanol upon protein metabolism was investigated using rat hepatocytes purified by centrifugal elutriation and maintained in culture. Hepatocytes maintained in capped culture flasks allowed long‐term studies of the effects of ethanol without cytotoxic damage to cultured cells and the phenotypic integrity of cultured cells was maintained. Ethanol (25–100 mmol/1) markedly inhibited hepatocyte protein synthesis. This inhibition of protein synthesis was not due to defective uptake of amino acids but to alterations to the intracellular redox state. Extracellular redox systems did not regulate hepatocyte protein synthesis. Protein degradation was not affected by ethanol. Glycosaminoglycans secreted into the the medium and extra and intracellular hepatocyte collagen synthesis were markedly stimulated by ethanol; collagen synthesis representing 6–8% of total protein synthesis at high ethanol concentrations. These studies indicate that ethanol exerts selective effects on protein synthesis by hepatocytes in primary culture and suggest that hepatocytes may contribute significantly to perisinusoidal fibrosis in alcohol‐induced liver disease.

Effect of volatile anesthetics on protein synthesis and secretion by rat hepatocyte suspensions.

The effect of volatile anesthetics on protein synthesis and secretion by isolated rat hepatocytes in suspension was investigated. Halothane and enflurane inhibited protein synthesis in a dose-dependent manner. Diethyl ether had little effect on protein synthesis while isoflurane caused a mild inhibition. This effect was more pronounced in hepatocytes from phenobarbital treated male rats when compared to hepatocytes from control rats. Protein synthesis in hepatocytes from phenobarbital treated female rats was inhibited similar to that seen with control male rat hepatocytes. Isoflurane, enflurane, and halothane also caused a dose-dependent inhibition of protein secretion, while diethyl ether was only mildly inhibitory. From these studies it appears that inhibition of protein synthesis and secretion might be an early and sensitive indicator of cellular injury by volatile anesthetics.

Effects of hemin on rat liver cyclic AMP-dependent protein kinases in cell extracts and intact hepatocytes

Cyclic AMP-dependent protein kinases I and II, partially purified from rat liver cytosol, were inhibited 50% by 40 μM hemin and 100 μM hemin, respectively. With the purified catalytic subunit of cyclic AMP-dependent protein kinase, hemin caused non-competitive inhibition with respect to the peptide substrate and mixed inhibition with respect to ATP. Hemin also inhibited purified phosphorylase b kinase, indicating that hemin concentrations above 10 μM markedly inhibit multiple protein kinases. In isolated intact hepatocytes, hemin inhibited the glucagon-dependent activation of cyclic AMP-dependent protein kinases and the activation of glycogen phosphorylase. For both effects, high heme concentrations (40–60 μM) were required for 50% inhibition. Similar high levels of exogenous hemin inhibited total hepatocyte protein synthesis. By contrast, 5 μM hemin or less was sufficient to raise intracellular heme levels, as indicated by the relative heme-saturation of tryptophan oxygenase in hepatocytes. Hemin, 5 μM, completely repressed induction of 5-aminolevulinate synthase by dexamethasone in hepatocyte primary cultures. Such repression is unlikely to be mediated by inhibition of protein kinases.

Killed Escherichia coli stimulates macrophage-mediated alterations in hepatocellular function during in vitro coculture: a mechanism of altered liver function in sepsis.

Hepatic dysfunction is a poorly understood and highly lethal component of multiple-system organ failure. Both in vivo and in vitro studies of "liver" function have generally neglected hepatocyte-Kupffer cell interactions. In the following experiments, isolated hepatocytes were cocultivated with unstimulated peritoneal cells, predominately macrophages, which served as a readily available Kupffer cell analog. Coculture of hepatocytes with peritoneal cells resulted in little or no change in [3H]leucine incorporation into hepatocyte protein. When gentamicin-killed Escherichia coli cells (GKEC) were added to coculture, there was a marked decrease in hepatocyte [3H]leucine incorporation. In contrast, GKEC added to hepatocytes alone had no effect. Kinetic data revealed an 8-h delay before any significant decrease in leucine incorporation into hepatocyte protein after the addition of GKEC to the coculture. The maximal decrease in hepatocyte [3H]leucine incorporation occurred 24 h after GKEC were added. The decrease observed 24 h after GKEC were added disappeared almost completely after 48 h of coculture. Similar alterations in cocultured hepatocyte protein synthesis were observed after the addition of phorbol myristate acetate, lipopolysaccharide, or muramyl dipeptide, a component of bacterial peptidoglycan. Hepatocyte viability by trypan blue exclusion was unchanged, and gross morphology by light or electron microscopy was unaffected. We propose that during sepsis, macrophages (Kupffer cells) respond to circulating microbial products and mediate alterations in hepatocyte function. These experiments underscore the important role of Kupffer cell function in attempts to understand hepatic malfunction in multiple-system organ failure.

Regulation of synthesis and turnover of liver-specific proteins in embryonic hepatocytes: [formula omitted], [formula omitted] and [formula omitted]. Dept. of Anatomy and Embryology, University of Amsterdam, AMC, Amsterdam, The Netherlands

Regulation of synthesis and turnover of liver-specific proteins in embryonic hepatocytes: [formula omitted], [formula omitted] and [formula omitted]. Dept. of Anatomy and Embryology, University of Amsterdam, AMC, Amsterdam, The Netherlands

Multiple systems organ failure—Modulation of hepatocyte protein synthesis by endotoxin activated Kupffer cells

The Journal of Trauma: Injury, Infection, and Critical Care: June 1985 - Volume 25 - Issue 6 - p 568