Differential changes in alpha2-macroglobulin and hemopexin in brain and liver in response to acute inflammation.

Changes in serum and cerebrospinal fluid (CSF) proteins following generalized acute inflammation induced by fermented yeast in the rat was examined by concanavalin A-blotting, immunoblotting, and radioimmunoassay. Using alpha2-macroglobulin (alpha2-M) and hemopexin (HPX) as marker proteins, the concentration alpha2-M was found to increase in serum and CSF by 150- and 5-fold, respectively, whereas the concentration of HPX increased by about 4-fold in both fluids following yeast-induced inflammation. The lesser increase in alpha2-M in the CSF versus the systemic circulation is not likely to be the result of changes in the permeability of the blood--brain barrier, since no change in the total protein content of CSF was detected in inflamed rats when compared to control animals. These results, however, illustrate the regulation of the same protein, such as alpha2-M, in two separate organs within the same animal can be drastically different. These results also suggest a possible protective role of alpha2-M in the brain during acute inflammation. Moreover, these observations are consistent with the previous observation that there is a differential response in the level of alpha2-M between the testis and the systemic circulation during inflammation.     

Involvement of the acute phase protein alpha 1-acid glycoprotein in nonspecific resistance to a lethal gram-negative infection

Resistance to gram-negative infection can be induced by pretreating animals with several agents such as turpentine and interleukin (IL)-1. Because these agents are powerful inducers of acute phase proteins, we wondered whether these proteins, more particularly alpha(1)-acid glycoprotein (alpha(1)-AGP), are involved in nonspecific resistance to infection. Turpentine and IL-1 protect completely against a lethal challenge of Klebsiella pneumoniae when given 48 and 12-48 h before the challenge, respectively. alpha(1)-AGP induction in the serum reached peak values 48 h after turpentine and 12-48 h after IL-1 injection. Administration of alpha(1)-AGP, 2 h before a challenge of K. pneumoniae, significantly increased the survival. Numbers of bacteria cultured from blood and organs were significantly lower in mice pretreated with a protective dose of turpentine, IL-1, or alpha(1)-AGP. These data suggest that alpha(1)-AGP is a possible mediator in turpentine- or IL-1-induced protection because time points of maximal induction of alpha(1)-AGP by turpentine or IL-1 and of optimal protection by alpha(1)-AGP coincide. Transgenic overexpression of rat alpha(1)-AGP protected mice from a K. pneumoniae infection. Bacterial counts in blood and organs were significantly lower in transgenic mice, and only in control mice were large necrotic areas, apoptosis, and blood clots observed in the spleen. Our data suggest that alpha(1)-AGP prevents gram-negative infections and may be an essential component in nonspecific resistance to infection.     

Messenger RNA activities of four acute phase proteins during inflammation

Poly(A)+ RNA isolated from the livers of normal rats and of rats suffering from an acute inflammation was translated in a cell-free translation system from rabbit reticulocytes. The translation products were immunoprecipitated with specific antisera against alpha 1-acid glycoprotein, alpha 2-macroglobulin, transferrin, alpha 1-proteinase inhibitor and albumin. 15 to 21 h after intramuscular injection of turpentine 73-, 66-, 2.8-, and 2-fold increases in translatable mRNAs for alpha 1-acid glycoprotein, alpha 2-macroglobulin, transferrin and alpha 1-proteinase inhibitor, respectively, were observed. For albumin a decrease in translatable mRNA to about 30% of controls was measured.     

Characterization of serum, liver, and intestinal sialyltransferases from rats treated with colchicine

A modified high pressure liquid chromatographic method using lactose (Gal beta 1----4Glc) as an exogenous acceptor has been used to characterize the sialyltransferases known to increase in the serum of colchicine-treated rats. The results show a 10-fold increase of Gal beta 1----4GlcNAc alpha 2----6 sialyltransferase (alpha 2----6 ST), whereas the Gal beta 1----3GlcNAc alpha 2----3 sialyltransferase showed only 1.6-fold increase in the serum after 17 h of colchicine treatment. The sialyltransferase activity in serum using exogenous desialylated, alpha 1-acid glycoprotein as acceptor also showed an eightfold increase. In liver homogenate and Golgi membrane, the sialyltransferase activity when assayed with desialylated alpha 1-acid glycoprotein as acceptor showed a slight decrease after 4 h, but returned to normal level after 17 h. A similar trend was seen when the two transferases were assayed with lactose as acceptor. The antiserum to rat alpha 2----6 ST inhibited the sialyltransferase activity in serum, liver, and jejunal incubation medium. Jejunal sections from rats treated with colchicine for 4 h in presence of heated serum showed a decrease of sialyltransferase, with consequent increase of the alpha 2----6 ST enzyme activity in the medium. This result suggests that intestinal tissue could be a source of increased serum enzyme activity in colchicine treatment.     

Peroxisome proliferator-activated receptor gamma agonists inhibit adipocyte expression of alpha1-acid glycoprotein

alpha1-Acid glycoprotein (alpha1-AGP) is an acute phase protein that can potentiate cytokine secretion by mononuclear cells and may induce thrombosis by stabilizing the inhibitory activity of plasminogen activator inhibitor-1. Thus, alpha1-AGP may promote pathobiologies associated with type 2 diabetes mellitus (T2DM) including insulin resistance and cardiovascular disease. Here, we demonstrate that antidiabetic peroxisome proliferator-activated receptor gamma (PPARgamma) agonists inhibited expression of 3T3-L1 adipocyte alpha1-AGP in a concentration- and time-dependent manner via an apparent PPARgamma-mediated mechanism. As a result, synthesis and secretion of the glycoprotein was reduced. While PPARgamma agonist regulation of genes with functional peroxisome proliferator response elements in their promoter such as phosphoenolpyruvate carboxykinase were unaffected when cellular protein synthesis was inhibited, downregulation of alpha1-AGP mRNA was ablated thereby supporting the proposition that PPARgamma activation inhibits alpha1-AGP expression indirectly. These results suggest a potential novel adipocytic mechanism by which PPARgamma agonists may ameliorate T2DM-associated insulin resistance and cardiovascular disease.     

Changes of acute-phase proteins in streptozotocin-induced diabetic rats

Quantitative and qualitative changes of serum proteins, apart from glycation, have not been sufficiently studied in streptozotocin-induced diabetic rats (D), the most common experimental model for diabetes. Thus, we decided to analyze the serum of diabetic rats by concanavalin A-blotting in comparison with rats with acute inflammation induced by fermented yeast (Y), in which characteristic alterations of serum proteins have been described. Two months after the streptozotocin treatment, the blood glucose levels were highly elevated (456+/-24 vs. 124+/-10 mg/dl, p<0.001, n=12), the body weight was significantly lower than normal (279+/-10 vs. 392+/-6 g, p<0.001, n=12), and serum proteins appeared to be highly glycated (p<0.001) when analyzed by the fructosamine assay, without any significant change in the total serum protein concentration. Analysis by concanavalin A-blotting, revealed a significant decrease of alpha1-inhibitor-3 (alpha1-I3, p<0.05) and an increase of the beta chain of haptoglobin (beta-Hp, p<0.05) in both D and Y rats (n=3) compared with control animals. However, acute inflammation caused a marked rise of two prominent acute phase proteins, alpha2-macroglobulin and hemopexin, which did not change appreciably in diabetic rats. Further work will be necessary to evaluate the physiopathological significance of these phenomena which could result from changes of both concentration and glycosylation of the aforementioned proteins.     

Nucleotide sequence of rat alpha 1-acid glycoprotein messenger RNA

The complete nucleotide sequence of rat alpha 1-acid glycoprotein (alpha 1-AGP) mRNA has been determined from cloned double-stranded cDNA. The coding portion of the mRNA was bounded at the ends by a 5'-untranslated region of 35 nucleotides in length and a 3'-untranslated region of 119 nucleotides in length. The 3'-untranslated region contains the characteristic AAUAAA sequence ending 18 nucleotides from the 3'-terminal poly(A) segment. The 5'-region of the mRNA contains two in-phase AUG codons separated by 12 nucleotides. Comparison with the known NH2-terminal amino acid sequence of serum rat alpha 1-AGP suggests that the primary translation product of the mRNA contains an additional 14 or 18 amino acids that are not present in the mature form of the protein, which contains 187 amino acids. The inferred amino acid sequence of rat alpha 1-AGP and the known amino acid sequence of human alpha 1-AGP have several regions of identity clustered in the NH2-terminal portion of the proteins. The carboxyl-terminal regions show significantly less homology. Six potential asparagine glycosylation sites are found in the rat sequence, and four of these sites are in positions similar to known glycosylation sites in the human protein. Furthermore, three of these potential glycosylation sites are in a region that exhibits extensive amino acid sequence conservation, suggesting that this region may be important for the biological function of alpha 1-AGP.     

Induction of alpha 1-acid glycoprotein by recombinant human interleukin-1 in rat hepatoma cells

The induction of alpha 1-acid glycoprotein mRNA by recombinant murine interleukin-1, recombinant human interleukin-1 alpha, and recombinant human interleukin-1 beta has been studied in the rat hepatoma cell line Fao. Whereas the stimulatory capacities of recombinant human interleukin-1 alpha and recombinant murine interleukin-1 were almost identical, the concentrations of recombinant human interleukin-1 beta needed for half-maximal induction of alpha 1-acid glycoprotein mRNA were lower by three orders of magnitude. A 60-fold increase in alpha 1-acid glycoprotein mRNA levels was observed 18 h after the addition of recombinant interleukin-1 beta. In parallel albumin mRNA levels decreased to about 30%. The alpha 1-acid glycoprotein mRNA induction was strictly dependent on the presence of dexamethasone. For a full stimulation dexamethasone concentrations of greater than 10(-7) M were needed, whereas concentrations of less than 10(-12) M were ineffective. The increase in alpha 1-acid glycoprotein mRNA after recombinant human interleukin-1 beta was followed by a 36-fold stimulation in alpha 1-acid glycoprotein synthesis and secretion. When protein synthesis was blocked by either cycloheximide, puromycin, or emetine, the induction of alpha 1-acid glycoprotein mRNA by recombinant human interleukin-1 beta was impaired suggesting the involvement of a short-lived protein in the induction of alpha 1-acid glycoprotein mRNA.     

Influence of chronic inflammation on the level of mRNA for acute-phase reactants in the mouse liver

Systemic injuries provoke a coordinate change in the hepatic synthesis and circulating concentration of plasma proteins. To assess the effect of a transition from acute to chronic inflammation on the expression of the plasma protein genes, the qualitative and quantitative changes of liver mRNA were measured in males of the inbred strain C57BL/6 of Mus musculus during a 14-day period of inflammation. Inflammation was induced and maintained by repeated injections of the irritants, turpentine, lipopolysaccharides, and celite. At various times, total RNA was extracted from the liver. The concentration of specific mRNA was quantitated by cellfree translation and by blot hybridization with cDNA. The mRNA for the increased plasma proteins serum amyloid A, alpha 1-acid glycoprotein, haptoglobin, and alpha-fibrinogen showed essentially the same time course of changes. The concentrations were maximal 24 hr after initiation of inflammation and then declined gradually during the following days. During the progression from acute to chronic inflammation, there was a switch from a predominant expression of the gene for alpha 1-acid glycoprotein-1 to one of alpha 1-acid glycoprotein-2. The prolonged inflammation had an inverse effect on the two major negative acute-phase reactants, albumin and major urinary proteins. The concentration of albumin mRNA was reduced to 50% after 24 hr of inflammation but returned to the control level within 5 days. The mRNA for major urinary proteins, however, were lowered within 2 to 5 days of inflammation to 10 to 20% and were maintained at that level. These results suggest that different regulatory factors, humoral and/or cellular, are involved in controlling the expression of the genes for positive and negative acute-phase plasma proteins during acute and chronic inflammation.     

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.     

Effect of estradiol 17-beta on LH subunits and prolactin mRNAs expression in the pituitary of old female rats

OBJECTIVES: The aim of the study was to examine susceptibility of the pituitary gland to estrogenic impulse in old, noncycling rats by measurement of steady state level of mRNAs encoding LH subunits a and b and mRNA for PRL. METHODS: 22-month-old rats were ovariectomized and after one week they were subcutaneously implanted with silastic tubing filled with oil or with estradiol 17-beta. Pituitary alpha, LHbeta and PRL mRNAs content and serum LH and PRL concentration was determined. RESULTS: The effect of E (2)treatment was manifested by the significant increase in the weight of the uterus and pituitary gland as well as by elevation of total pituitary RNA (109%, 60% and 78%, respectively; p<0.001). No significant changes (p>0.05) in serum LH concentration were observed, while levels of mRNAs encoding alpha and LH-beta subunits were lowered by 54% (p<0.05) and 96% (p<0.01), respectively, in the rats subjected to E(2) stimuli. No direct correlation between synthesis and release of LH in E(2) treated old rats was observed. The blood PRL concentration and the pituitary level of PRL mRNA increased up to 2,000% and 1,300%, respectively (p<0.001). Spontaneous pituitary adenoma was observed in about 30% of the rats, irrespective of treatment. CONCLUSIONS: These data show that in old rats estrogenic stimulus can effectively diminish both pituitary LH subunits mRNAs as well as stimulate pituitary PRL mRNA level indicating that the E(2)-dependent processes involved in the regulation of corresponding genes are still functional.     

Cell-free synthesis of rat alpha 2-macroglobulin and induction of its mRNA during experimental inflammation

Poly(A)-rich RNA was isolated from the livers of acutely inflamed rats by extraction with guanidinium HCl and oligo(dT)-cellulose chromatography. After translation in a recticulocyte lysate and immunoprecipitation with a specific antiserum to alpha 2-macroglobulin a polypeptide with an apparent molecular weight of 162000 could be detected. The cell-free synthesis of alpha 2-macroglobulin was stimulated 8-fold by the addition of RNase inhibitor. Full-length alpha 2-macroglobulin polypeptide chains appeared after 35 min in the presence of 1.85 mM Mg2+ and 100 mM K+. A nucleotide number of about 5100 was estimated for alpha 2-macroglobulin by means of sucrose gradient centrifugation of poly(A)-rich RNA followed by translation in vitro and immunoprecipitation of alpha 2-macroglobulin. In normal liver alpha 2-macroglobulin mRNA represented about 0.0007% of total translatable RNA. Acute inflammation generated by intramuscular injection of turpentine led to a 66-fold increase in translatable alpha 2-macroglobulin mRNA after 18 h, followed by a rapid decrease. In accordance to the induction of alpha 2-macroglobulin mRNA serum concentrations of alpha 2-macroglobulin increased to about 2 mg/ml. Unlike alpha 2-macroglobulin mRNA serum alpha 2-macroglobulin levels remained unchanged up to 60 h.     

Changes in the blood level and affinity to concanavalin A of rat plasma glycoproteins during acute inflammation and hepatoma growth.

Plasma concentrations of ten individual proteins were measured by electroimmunoassay in young male Buffalo rats following injection of turpentine oil or implantation of Morris hepatoma 7777. The highest relative responses to inflammation and tumour growth were found for alpha 2-macroglobulin, alpha 1-acute-phase globulin and alpha 1-acid glycoprotein. As shown by crossed immuno-affinoelectrophoresis the concanavalin A-reactive fractions of the latter two glycoproteins were predominantly increased in plasma from injured and tumour-bearing 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.     

Regulation of alpha 1-acid glycoprotein externalization and intracellular accumulation in glucocorticoid-induced rat hepatoma cells

alpha 1-Acid glycoprotein (alpha 1-AGP) is a glucocorticoid inducible gene product that is synthesized and secreted by certain rat hepatoma tissue culture (HTC) cell lines such as M1.54. Exposure to monensin, a Na+-K+ ionophore, causes a significant redistribution of alpha 1-AGP into two distinct fractions; immunoprecipitation of [35S]methionine-labeled proteins revealed that a 27% decrease in secretion accounts for a sixfold increase in accumulation of a stable intracellular species. The new intracellular alpha 1-AGP is more heterogeneous than normal while the extra-cellular form is 6000 Da smaller than normal. These effects are due to selective alterations in carbohydrate maturation; endo-beta-N-acetylglucosaminidase H (endo H) digestion demonstrated that both alpha 1-AGP species contain variable numbers of endo H-resistant oligosaccharide side chains ranging between zero and five. Ricin affinity chromatography revealed that the attachment of galactose residues is strikingly correlated with alpha 1-AGP externalization while neuraminidase digestions demonstrated that sialic acid attachment appears unessential for its secretion. Taken together, our results suggest that in the presence of monensin the cellular transport of intracellular destined and externalized alpha 1-AGP proceeds in common through the early segments of the Golgi and at a point prior to or at the compartment containing galactosyl transferase, alpha 1-AGP becomes committed for secretion.     

Glycosylation of four acute-phase glycoproteins secreted by rat liver cells in vivo and in vitro. Effects of inflammation and dexamethasone

We have studied the role of the liver in the relative increase of Concanavalin A (Con A)-reactive molecular forms of various positive rat acute-phase glycoproteins (APGPs) occurring in serum during inflammation. Secretion media of hepatocytes isolated from inflamed rats showed a 2 to 5-fold increase of the total amounts of four APGPs studied in comparison to secretion media of control hepatocytes. These changes were in analogy with those observed for corresponding sera, except for alpha 1-antitrypsin. All the different Con A-reactive molecular forms were present in the media, with exception of the most reactive form of ceruloplasmin. In vitro and in vivo, dexamethasone augmented the secretion of three APGPs, and especially of the Con A-most reactive forms. The in vitro effect of dexamethasone--augmented secretion of Con A-reactive molecular forms of alpha 1-acid glycoprotein and haptoglobin--was comparable with the results obtained for hepatocytes isolated from inflamed rats. In vivo, dexamethasone treatment resulted in an even higher increase of the serum concentration of the Con A-most reactive forms of both APGPs than experimental inflammation did. Although an extrahepatic contribution cannot be excluded, these results suggest that alterations in the Con A reactivity of APGPs as observed during the acute-phase of inflammation have their origin in the liver. A change in the Con A reactivity of glycoprotein indicates a modulation of its glycosylation. Since dexamethasone can affect these changes in vivo and in vitro, glucocorticoids most probably are involved in the regulation of the glycosylation of the APGPs during biosynthesis in the liver.     

The early induction of the actin-sequestering peptide thymosin beta 4 in thymocytes depends on the proliferative stimulus

The expression of the actin-sequestering peptide, thymosin beta 4, was analyzed in proliferating rat thymocytes, activated by diverse stimuli, during the early G1 phase and the S phase. In the presence of concanavalin A a 6.3-fold increase of thymosin beta 4 occurred already after 1 h of stimulation without elevation of the corresponding mRNA level. In contrast, during the S phase the increase of thymosin beta 4 (2.5-fold) was accompanied by a higher mRNA level, but did not exceed the growth related increase of total protein. Stimulation with a crosslinked antibody against rat T cell antigen receptor or stimulation with phorbol 12-myristate 13-acetate (PMA) and Ca(2+)-ionophore A23187, separately or in combination, did not lead to the marked increase of the thymosin beta 4 concentration in the early G1 phase but resulted in elevated thymosin beta 4 peptide and mRNA levels during the S phase. It therefore appears that protein kinase C activation and a rise in cytoplasmic Ca(2+)-concentration are not exclusively responsible for the stimulation of thymosin beta 4 specific translation in thymocytes. This assumption was reinforced by the observation that inhibition of the protein kinase C activity by 1-(5-isoquinolinylsulfony)-2-methylpiperazine (H-7) did not affect the cellular thymosin beta 4 content 1 h and 48 h after concanavalin A (Con A) stimulation.