Studies on the effect of 1-deoxynojirimycin on the release of albumin, sialyltransferase and alpha 1 acid glycoprotein from liver slices from normal and inflamed rats

Liver slices from control and 24hr inflamed rats were incubated for up to 20hr with 5mM 1-deoxynojirimycin (DN), an inhibitor of the processing glucosidases. The amounts of albumin and alpha 1-acid glycoprotein (AGP) and the activities of sialyltransferase were determined in liver and medium. The presence of DN significantly inhibited the release of AGP and sialyltransferase. The inhibitory effect of DN was most pronounced with slices from inflamed rats. Secretion of albumin was not inhibited. Incorporation studies with labelled leucine and mannose showed that the inhibitor did not significantly affect protein synthesis, but it did inhibit mannose incorporation into AGP and sialyltransferase. The results show that DN inhibits the secretion of acute phase AGP and sialyltransferase in liver slices and further suggests that sialyltransferase is a glycoprotein.     

Impairment of glycoprotein secretion by phenobarbital in rat liver slices

The effects of phenobarbital on protein and glycoprotein synthesis and secretion were studied in rat liver slices. Phenobarbital (2 mM) decreased [¹⁴C]-glucosamine and [¹⁴C]leucine incorporation into liver proteins and markedly inhibited their incorporation into medium (secretory) proteins. This inhibitory effect of phenobarbital was dose dependent and not reversible under the conditions of this study. In the presence of cycloheximide, an inhibitor of peptide synthesis, phenobarbital still inhibited the release of glycoproteins into the medium; however, the specific activity of liver glycoproteins was increased. The effects of phenobarbital on hepatic macromolecular secretion, independent of its effects on synthesis, were determined by prelabeling proteins in a liver slice system with either [¹⁴C]leucine of [¹⁴C]glucosamine. When phenobarbital was present, the secretion of these prelabeled proteins into the medium was impaired. 12 h after intraperitoneal injections of phenobarbital, glycoprotein secretion was inhibited from liver slices prepared from the pretreated rats. This inhibition of secretion occurred even though protein synthesis was stimulated and intracellular glycosylations unaffected. The results of this study indicate that phenobarbital impairs the secretion of glycoproteins by the liver.     

Role of prostaglandins in acute phase proteins in inflammation

Prostaglandin E1, a mediator of inflammation, was investigated for its effects on serum acute phase proteins, alpha 2 macroglobulin (alpha 2M). Induction of carrageenin inflammation in rats caused an elevation of alpha 2M to a maximum level (100%) at 1 day. Similarly, administration of PGE1 (1 mg/kg) was found to increase serum alpha 2M levels in normal rats. On the other hand, sc injection of PGE1 into inflamed rats significantly reduced the alpha 2M in serum as well as edema. In vitro studies with liver slices showed increasing rates of incorporation of [14C]leucine into alpha 2M with the addition of PGE1 to the medium. It was followed by the secretion of alpha 2M-bound radioactivity into media. But addition of higher doses (greater than 100 ng/ml) of PGE1 resulted in the suppression of incorporation and secretion of alpha 2M-bound radioactivity. Incubation of inflamed liver slices with PGE1, however, showed only decreased incorporation and secretion of alpha 2M-bound radioactivity. These results indicate that (a) primary prostaglandins, like PGE1, generated during inflammation may be responsible for the increase of alpha 2M in serum, and (b) PGE1 enhanced the synthesis of alpha 2M in liver and its secretion into the medium, so the anti-inflammatory drugs which decrease levels of PGs are likely to alter alpha 2M levels.     

Stimulation by insulin of the formation of ribonucleic acid and protein by mammary tissues in vitro

1. Insulin is one of the hormones that are essential for successful tissue culture of explants of the mammary glands of pregnant mice. We report here effects of insulin on RNA and protein formation by mammary tissue from pregnant mice and rats incubated in tissue-culture medium 199. 2. The incorporation of [(14)C]adenine over 3hr. into the RNA of explants of the mammary glands of pregnant mice was increased by an average of 68% when the medium contained 5mug. of insulin/ml. Under similar conditions the incorporation into the RNA of slices of the glands of pregnant rats was increased by an average of 61%. Incorporation into the RNA of slices from lactating rats was stimulated to a smaller extent. 3. Adipose tissue was separated from the glands of pregnant mice and the effect of insulin on the incorporation of adenine into its RNA was studied. In whole explants the incorporation of adenine, both with and without insulin, is almost entirely into the RNA of the mammary parenchyma and not of the adipose tissue. 4. Insulin also stimulated by 38% the incorporation of [(14)C]leucine over 3hr. into the proteins of slices of the glands of pregnant rats. It had no significant effect on slices from lactating rats. 5. Actinomycin D (10mug./ml.) decreased the incorporation of [(14)C]adenine into the RNA of slices of the glands of pregnant rats by an average of 97%. Though it also decreased the incorporation of [(14)C]leucine into the proteins by an average of 25%, the percentage stimulation by insulin of this incorporation remained unchanged.     

The induction of alpha 1-acid glycoprotein by methylmercury

The incorporation of [14C]leucine into protein was measured in liver preparations and blood of rats following the s.c. administration of methylmercury hydroxide (24 mg/kg body wt) or turpentine (5.0 ml/kg body wt). The translatability of the RNA obtained from polysomes in an mRNA-dependent reticulocyte lysate was elevated significantly in the preparations derived from the treated rats compared to control rats. Immunoprecipitation of the labelled translation products or of serum proteins showed that the mRNA activity and the synthesis of alpha 1-acid glycoprotein, an acute phase reactant, was elevated by the methylmercury treatment as well as by the turpentine-induced inflammatory response.     

Experimental alcoholism in rats. Effect of acute ethanol intoxication on the in vitro incorporation of ( 3 H)leucine into neuronal and glial proteins

Abstract— Ethanol administered in vivo or in vitro during incubation of brain slices was studied with respect to its effect on brain protein synthesis. In the in vivo series the rats were given a single intraperitoneal injection of ethanol 3 h before death. Slices of cerebral cortex and liver were incubated in isotonic saline media containing [³H]leucine. Amounts of free and protein-bound radioactivity were determined. Subcellular fractions and fractions enriched in neuronal perikarya and in glial cells were prepared from cortical slices subsequent to incubation, and the specific radioactivity determined for each cell type. The incorporation of [³H]leucine into brain proteins was inhibited while incorporation into liver proteins was stimulated in ethanol-treated rats. The levels of TCA-soluble radio-activity, however, did not differ between the ethanol group and the controls. In the fractionated material from cerebral cortex, the specific radioactivity in the neuronal fraction was unaffected by ethanol, while the radioactivity in the glial fraction was significantly depressed. In vitro administration of ethanol induced a non-linear response in both brain and liver, with depression of leucine incorporation into proteins of cerebral cortex at all concentrations used. When brain slices were exposed to ethanol in vitro, in concentrations corresponding to the in vivo experiments, a similar reduction of the leucine incorporation into the glial fraction was obtained. Incorporation of leucine into subcellular fractions from whole brain cortex was also investigated. The specific sensitivity of the glial fraction to ethanol is discussed in relation to the involvement of the different cell types with transport processes in the brain.     

Effect of tannic acid on the synthesis of protein and nucleic acid by rat liver

1. As early as 1hr. after the intraperitoneal administration of tannic acid to rats, it could be demonstrated in the liver. At 3hr. the nuclear fraction contained the largest amount of tannic acid. 2. Nuclear RNA synthesis was inhibited in vivo 2hr. after the administration of tannic acid. Induction by cortisol of tryptophan pyrrolase was 90% inhibited at 24hr. 3. Incorporation of [1-(14)C]leucine into protein by liver slices from treated rats was decreased by 50% after 24hr. Its incorporation into postmitochondrial supernatant from treated animals was not inhibited. Incorporation into slices and postmitochondrial supernatants were inhibited in vitro by tannic acid. 4. The sequence of events: concentration of tannic acid in nuclei, inhibition of nuclear RNA synthesis, inhibition of protein synthesis and production of necrosis, is discussed.     

Studies on the effect of lysosomotropic agents on the release of Gal beta 1-4GlcNAc alpha-2,6-sialytransferase from rat liver slices during the acute-phase response.

The mechanism of release of Gal beta 1-4GlcNAc alpha-2,6-sialyltransferase (CMP-N-acetylneuraminate: beta-galactoside alpha-2,6-sialytransferase, EC 2.4.99.1) from rat liver during the acute-phase response is due to the action of a cathepsin D-like proteinase that cleaves the trans-Golgi membrane-bound enzyme from a membrane anchor; this allows a major portion of the enzyme containing the catalytic site to escape into the extracellular space [Lammers & Jamieson (1988) Biochem. J. 256, 623-631]. The release of sialytransferase was most effective at pH 5.6, suggesting that release of sialyltransferase from the Golgi in whole cells is dependent on maintaining an acidic environment in the trans-Golgi compartment of the hepatocyte. Golgi membranes contain a proton pump that maintains the acidic pH in these compartments [Glickman, Croen, Kelly & Al-Awquati (1983) J. Cell Biol. 97, 1303-1308; Yamashiro, Tycko & Maxfield (1984) Cell (Cambridge, Mass.) 37, 789-800; Zhang & Schneider (1983) Biochem. Biophys. Res. Commun. 114, 620-625; Anderson & Pathak (1985) Cell (Cambridge, Mass.) 40, 635-643]. Lysosomotropic agents, such as NH4Cl, chloroquine and methylamine can penetrate acidic compartments of the cell, such as the Golgi complex, raise the pH, and thus affect proteolytic cleavage events. The present paper describes the effect of lysosomotropic agents on the release of sialyltransferase from the hepatocyte using liver slices as a whole-cell system. Slices were prepared from control rats and rats suffering from the acute-phase response, where release of sialyltransferase is increased substantially [Lammers & Jamieson (1988) Biochem. J. 256, 623-631; Kaplan, Woloski, Hellman & Jamieson (1983) J. Biol. Chem. 258, 11505-11509]. Release of sialyltransferase was almost abolished in presence of 50 mM-NH4Cl, 50 mM-methylamine or 1 mM-chloroquine. Inhibition of release of sialyltransferase was reversed when the lysosomotropic agents were removed from the medium, showing that these agents are not cytotoxic to the cells under the conditions used. The secretion of rat alpha 1-acid glycoprotein, which is not subject to proteolytic processing in the Golgi complex, was not found to be substantially affected by the presence of lysosomotropic agents. The results suggest that proteolytic cleavage of the catalytic site of sialyltransferase is a process that is significantly affected by the intra-Golgi pH.     

The acute phase response to inflammation: the role of monokines in changes in liver glycoproteins and enzymes of glycoprotein metabolism

The role of monocyte derived factors in the acute phase response to inflammation is discussed. The kinetics of response of alpha 1-acid glycoprotein, sialyltransferase and albumin to a rat monokine preparation is described. There was an increase in synthesis of alpha 1-acid glycoprotein and sialyltransferase and a decrease in albumin synthesis following administration. However, the kinetics of response of sialyltransferase to the monokine was much slower than was found for the other two proteins. The possibility that sialyltransferase responds to a different monokine compared to the other acute phase proteins is discussed.     

The effect of adjuvant-induced arthritis on rat leukocyte membrane phospholipid fatty acid levels following fish oil feeding.

Inflamed rats with adjuvant-induced polyarthritis showed similar incorporation of dietary eicosapentaenoic acid (EPA) into peritoneal exudate cell (PEC) membrane phospholipids compared to pair fed non-inflamed control rats. Arachidonic acid (AA) content was similar in PEC phospholipids of inflamed and control rats whereas the linoleic acid content was consistently higher in inflamed rats.     

Rat choroid plexus specializes in the synthesis and the secretion of transthyretin (prealbumin). Regulation of transthyretin synthesis in choroid plexus is independent from that in liver

Synthesis of total protein and of transthyretin in rat choroid plexus was studied by measuring the incorporation of radioactive leucine into proteins in choroid plexus tissue incubated in vitro. About 20% of the protein newly synthesized in choroid plexus and about 50% of the newly synthesized protein secreted into the medium was transthyretin. Evidently, the choroid plexus is very active in the biosynthesis of this carrier protein for thyroid hormones and could be an important link in the chemical communication between the body and the central nervous system. Acute inflammation, which leads to a profound rearrangement of the pattern of plasma protein synthesis rates in the liver, produced distinct changes in the levels for plasma protein mRNAs in the liver. The levels of the mRNAs for alpha 1-acid glycoprotein and major acute phase alpha 1-protein increased more than 30-fold, those for transthyretin and albumin decreased to 27 and 57% of normal, respectively. The pattern of the observed changes in the levels of mRNAs for plasma proteins in the liver was independent of whether the acute inflammation was produced by subcutaneous injection of turpentine or intraperitoneal injection of a suspension of talcum. However, levels of transthyretin mRNA in choroid plexus were affected only very slightly, or not at all. Apparently, transthyretin synthesis in liver and choroid plexus is regulated independently during the acute phase response. No mRNA was detected in choroid plexus for albumin, alpha 1-acid glycoprotein, and major acute phase alpha 1-protein under any conditions.     

The induction of α1-acid glycoprotein by methylmercury

• 1.1. The incorporation of [¹⁴C]leucine into protein was measured in liver preparations and blood of rats following the s.c. administration of methylmercury hydroxide (24 mg/kg body wt) or turpentine (5.0 ml/kg body wt).
• 2.2. The translatability of the RNA obtained from polysomes in an mRNA-dependent reticulocyte lysate was elevated significantly in the preparations derived from the treated rats compared to control rats.
• 3.3. Immunoprecipitation of the labelled translation products or of serum proteins showed that the mRNA activity and the synthesis of α₁-acid glycoprotein, an acute phase reactant, was elevated by the methylmercury treatment as well as by the turpentine-induced inflammatory response.

     

Apolipoprotein C-II synthesis as studied in explants of rat liver in culture

Adult male Sprague-Dawley rats (200-250 g) were used to study apolipoprotein C-II synthesis and secretion. Liver slices were prepared and incubated in RPMI 1629-medium (tissue amount and incubation time studies) and in Minimum Essential Medium (Eagle) with Earle's salts (hormone experiments). Incubation was performed in scintillation vials in a 95% O2-5% CO2 atmosphere, at 37 degrees C from 1 to 21 hr (2 and 4 hr with hormones). The hormones used and their amounts per millilitre medium were: oestradiol-17 beta 0.1 microgram, progesterone 3.0 micrograms and dexamethasone 1.5 micrograms. Apolipoprotein C-II was determined by specific double immunoprecipitation technique and TCA-insoluble protein fraction represented total protein. Optimal tissue amount was 100 mg/vial and the results show that liver slices quickly secrete the newly synthesized apo C-II (also total protein) into the surrounding medium. There were only minor differences between apo C-II values with the hormones used. The portion of apo C-II synthesis from total protein synthesis was 0.47-1.50%. After 4 hr incubation the [3H]leucine incorporation was almost equal for controls and hormone treated slices.     

Studies on incorporation of mannose into rat alpha 1-acid glycoprotein: evidence for precursor forms in rough membranes

Rats were given pulse injections of D-[14C]mannose and were killed at various times up to 60 min after injection. Rough, smooth, and Golgi fractions were prepared from liver, and alpha 1-acid glycoprotein was isolated from Lubrol extracts of the fractions. The kinetics of incorporation of D-[14C]mannose into total protein, Lubrol protein, and alpha 1-acid glycoprotein showed that proteins associated with rough fractions had particularly high specific radioactivities at early times of incorporation. One explanation for the kinetic data is that glycoproteins contain a high mannose content at early times of assembly of oligosaccharide chains. This idea was confirmed in the case of alpha 1-acid glycoprotein by isolation of a high mannose containing precursor species of alpha 1-acid glycoprotein from rough fractions of liver. This species contained 56 residues of hexose (mainly mannose) compared with 35 residues of hexose (roughly equal amounts of mannose and galactose) which are found in the native protein. It is proposed that the high mannose precursor is a form of alpha 1-acid glycoprotein that exists at an early stage in assembly of the glycoprotein and which contains largely unprocessed carbohydrate chains. In addition, evidence is presented from amino acid analyses and gel electrophoresis of the high mannose precursor and another fraction from which it is formed by limited tryptic treatment, that pro-forms of alpha 1-acid glycoprotein with extensions of the polypeptide chain may also exist.     

Studies on the assembly and secretion of a multicomponent protein. The biosynthesis of vitellogenin, an oestrogen-induced glycolipophosphoprotein

1. Incorporation of [(32)P]P(i) and [(3)H]leucine into vitellogenin secreted in vitro by liver slices from oestrogen-treated Xenopus laevis is accompanied by a 2h lag; no lag is apparent for the incorporation into total tissue protein. 2. The addition of cycloheximide was found immediately to inhibit further incorporation of radioactive leucine into total tissue protein. The incorporation into secreted vitellogenin, however, continued for 2h after the addition of cycloheximide. 3. Pulse-labelling of liver slices with [(3)H]leucine for 30min, followed by a chase with a large excess of unlabelled leucine, resulted in the appearance of radioactivity in secreted vitellogenin from 90min after the end of the pulse period. 4. Evidence is presented which suggests that of the radioactivity from [(3)H]leucine incorporated into proteins by the liver of oestrogen-treated Xenopus some 70% is present in the single protein vitellogenin. 5. The incorporation of [(32)P]P(i) into vitellogenin followed a pattern identical with that found for [(3)H]leucine in the pulse-labelling experiments and this indicates that synthesis of the polypeptide chain and incorporation of P(i) are closely linked processes. 6. The cumulative evidence suggests that the 2h lag phase represents the time required for the assembly and secretion of this multicomponent protein.     

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'.     

Studies on the effect of inflammation on rat liver and serum sialyltransferase. Evidence that inflammation causes release of Gal beta 1 leads to 4GlcNAc alpha 2 leads to 6 sialyltransferase from liver

Turpentine induced inflammation has been shown to elevate liver sialyl- and galactosyltransferase activities (Turchen, B., Jamieson, J.C., Huebner, E., and van Caeseele, L. (1977) Can. J. Zool. 55, 1567-1571; Lombart, C., Sturgess, J., and Schachter, H. (1980) Biochem. Biophys. Acta 629, 1-12). We now report that serum sialyl-, but not galactosyltransferase activities are significantly elevated in turpentine inflammation. A liver slice system is used to demonstrate that liver releases large amounts of sialyltransferase activity into medium after inflammation, whereas only a low level of galactosyltransferase activity is released. Studies with rat and human asialo-alpha 1-acid glycoprotein as acceptors, coupled with the use of lactose to confirm the nature of the linkages formed, showed that Gal beta 1 leads to 4GlcNAc alpha 2 leads to 6 sialyltransferase is released from liver in turpentine inflammation and is mainly responsible for the elevated sialyltransferase activity found in serum. The alpha 2 leads to 6 sialyltransferase is exhibiting the properties of a typical acute phase reactant.     

Partial hepatectomy and mediators of inflammation decrease the expression of liver alpha 2-HS glycoprotein gene in rats

Liver mRNA levels of two acute phase reactant (APR) proteins, alpha 2-HS glycoprotein (a major negative APR) and alpha 1-acid glycoprotein (a major positive APR) were measured in male rats at different times after the administration of turpentine, of tumor necrosis factor, or following partial hepatectomy. In every case, a marked decrease in mRNA levels of alpha 2-HS glycoprotein was observed which reached a maximum at 24 h. A concomitant increase of alpha 1-acid glycoprotein mRNA levels was observed under the same conditions. These results indicate that the decreased levels of alpha 2-HS glycoprotein induced by the acute-phase response following inflammatory mediators and partial hepatectomy are due to a down-regulation of the gene expression of this protein in rat liver.     

LEUCINE OXIDATION IN BRAIN SLICES AND NERVE ENDINGS1

—It is generally believed that leucine serves primarily as a precursor for protein synthesis in the central nervous system. However, leucine is also oxidized to CO₂ in brain. The present investigation compares leucine oxidation and incorporation into protein in brain slices and synaptosomes. In brain slices from adult rats, these processes were linear for 90min and ¹⁴CO₂ production from 0·1 mm -l -[l-¹⁴C]leucine was 23 times more rapid than incorporation into protein. The rate of oxidation increased further with greater leucine concentrations. Experiments with l -[U-¹⁴C]leucine suggested that all of the carbons from leucine were oxidized to CO₂ with very little incorporation into lipid. Oxidation of leucine also occurred in synaptosomes. In slices, leucine oxidation and incorporation into protein were inhibited by removal of glucose or Na⁺, or addition of ouabain. In synaptosomes, replacement of Na⁺ by choline also reduced leucine oxidation; and this effect did not appear to be due to inhibition of leucine transport. The rate of leucine oxidation did not change in brain slices prepared from fasted animals. Fasting, however, reduced the incorporation of leucine into protein in brain slices prepared from young but not from adult rats. These findings indicate that oxidation is the major metabolic fate of leucine in brain of fed and fasted animals.