The Biosynthesis of Acute‐Phase Proteins in Primary Cultures of Rat Hepatocytes

Self Cite

186

The regulation of the three major acute-phase proteins a,-macroglobulin, cysteine proteinase inhibitor and a,-antitrypsin by recombinant human interleukin-1 fi, recombinant human interleukin-6 and recombinant human tumor necrosis factor a was studied in rat hepatocyte primary cultures. Synthesis and secretion of the acute-phase proteins was measured after labeling with [3sS]methionine and immunoprecipitation.Incubation of hepatocytes with interleukin-6 led to dose-dependent and time-dependent changes in the synthesis of the three major acute-phase proteins and albumin, similar to those occurring in vivo during experimental inflammation. a2-Macroglobulin and cysteine proteinase inhibitor synthesis was induced 54-fold and %fold, respectively, 24 h after the addition of 100 units/ml interleukin-6. At the same time synthesis of the negative acute-phase protein albumin was reduced to 30% of controls. Half-maximal effects were achieved with 4 units interleukin-6/ml. Interleukin-1fi had only a partial effect on the regulation of the four proteins studied: only a twofold stimulation of a,-macroglobulin and a 60% reduction of albumin synthesis were observed. Tumor necrosis factor a did not alter the synthesis of acute-phase proteins.The stimulation of a2-macroglobulin and cysteine proteinase inhibitor synthesis by interleukin-6 was inhibited by interleukin-1 p in a dose-dependent manner.In pulse-chase experiments the effect of interleukin-lfi, interleukin-6 and tumor necrosis factor CI on the secretion of acute-phase proteins was examined. Interleukin-6 markedly accelerated the secretion of total proteins and a,-macroglobulin, whereas the secretion of cysteine proteinase inhibitor, a,-antitrypsin and albumin was not affected. The inhibition of N-glycosylation by tunicamycin abolished the effect of interleukin-6 on the secretion of a2-macroglobulin, indicating a possible role of interleukin-6 on N-glycosylation.Bacterial or parasitic infections, physical and chemical traumata, malignant tumors and immunologic disorders lead to a series of local and systemic reactions of the organism, the so-called acute-phase response [l, 21. The local reaction includes the dilation of blood vessels, alteration in blood flow and clotting, immigration of neutrophils and macrophages, release of lysosomal proteases and the formation of inflammatory mediators. These mediators cause a systemic reaction characterized by fever and pain, leukocytosis, decrease in plasma iron and zinc levels and increased synthesis of acutephase proteins (reviewed in [l, 21). Many of the acute-phase proteins are proteinase inhibitors involved in the protection of healthy tissue from proteinases such as elastase, collagenase and cathepsin G released from granulocytes and macrophagesIn the rat the proteinase inhibitors a,-macroglobulin (azM), cysteine proteinase inhibitor and al-antitrypsin (a,AT) represent positive acute-phase reactants. During acute inflammation their serum levels increase about 100-fold, 8-fold, and 2-fold respectively 14-61. The increase in seru...

Section: Discussionmentioning

confidence: 99%

Regulation of synthesis and secretion of major rat acute‐phase proteins by recombinant human interleukin‐6 (BSF‐2/1L‐6) in hepatocyte primary cultures

Andus

Geiger

Hirano

et al. 1988

Self Cite

186

“…The cellular form was never detected in the HepG, medium. The two molecular entities differ in their carbohydrate moieties: the intracellular form (39 kDa) contains a high proportion of mannose, the medium form (47-51 kDa) contains complex oligosaccharides [33]. Quantification of the autoradiograms by scanning densitometry indicated that [35S]methionine incorporation into a,-glycoprotein was 2.2-fold higher in thyroid-hormone-treated HepG, cells (4.6 5 0.4 arbitrary units) than in control cells (2.2 5 0.4 arbitrary units).…”

Section: Effect Of Thyroid Hormone On A-glycoprotein Secretion In Hementioning

confidence: 99%

The Modulation of Apolipoprotein E Gene Expression by 3,3′‐5‐triiodothyronine in HepG₂ Cells Occurs at Transcriptional and Post‐transcriptional Levels

Vandenbrouck¹,

Janvier²,

Loriette³

et al. 1994

The regulation of the synthesis and secretion of apolipoprotein E (apoE) is incompletely understood. This study examines the mechanisms responsible for regulating apoE gene expression in HepG, cells by thyroid hormone (3,3'-5-triiodothyronine). The secretion rate of apoE was by thyroid hormone increased (1.5 -1 %fold) in pulsekhase experiments. Thyroid hormone doubled apoE mRNA concentration as determined by Northern-blot analysis. Inhibition of protein synthesis by cycloheximide increased the thyroid-hormone-induced stimulation of apoE mRNA. This suggests that the synthesis of new protein is not required for thyroid hormone to stimulate apoE mRNA. Actinomycin D was used to inhibit new transcription; there was a more rapid degradation of mature apoE mRNA in thyroid hormone-treated HepG, cells than in control cells, suggesting that thyroid hormone acts post-transcriptionally to regulate apoE gene expression. Cycloheximide blocked the action of thyroid hormone, suggesting that thyroid hormone regulates the turnover of apoE mRNA via the synthesis of de novo protein. Nuclear run-on transcription assays demonstrated that thyroid hormone stimulated apoE gene transcription threefold in 24 h. These findings indicate that the expression of the apoE gene is controlled at both transcriptional and post-transcriptional loci by the thyroid hormone.Apolipoprotein E (apoE) is a major component of several classes of lipoproteins and plays an important role in cholesterol metabolism. Its principal function is the mediation of the cellular uptake of lipids by serving as a ligand for the apoB,E (low-density lipoprotein) receptor and for the remnant receptor (for review, see [l]). Although the liver appears to be the major site of apoE synthesis, a wide variety of peripheral tissues also produce this protein, in contrast to most apolipoproteins [2 -51.Several dietary and hormonal disturbances have been used to gain insight into the regulation of hepatic apolipoprotein synthesis, transport, lipid association and the secretion of lipoprotein particles. Hormonal stimuli affecting hepatic apolipoprotein synthesis by the rat liver in vivo include insulin 161, corticosteroid [7], sex steroids [8], and thyroid hormones [9, 101. When injected into living rats, thyroid hormones induced an increase in hepatic apoAI mRNA 19, 11, 121 and, to a lesser extent, in apoAIV mRNA [9, 111. The change in the transcription rates of apoAI and apoAIV paralleled those of their mRNA concentrations [ll]. However, Strobl et al. found that the major effect of thyroid hormones on apoAI gene expression was post-transcriptional, leading to increased stability of nuclear apoAI mRNA 1131. The synthesis of apoB48 by hyperthyroid rat liver was also greater than in hypothyroid rat liver, at the expense of apoBlOO due to an enhanced post-transcriptional introduction of a stop codon into apoB mRNA [14].The expression of the apoE gene in the liver is known to be increased in vivo by fasting [15], a sucrose diet [16], injection of glucagon or CAMP [17], but is inhibited by...

“…The suspension was rotated for 2 h at 4"C, then centrifuged for 1 min at 10000 x rpm at 4°C. Immunoprecipitation was carried out by the method of Andus et al [29]. Supernatants were then added with either 20 pl anti-(rat apolipoprotein) antiserum or 20 p1 anti-(rat apo B) antiserum and gently rotated overnight at 4°C.…”

Section: Protein Radioactive Labeling and Immunoprecipitationmentioning

confidence: 99%

Effect of dietary fish oil and corn oil on lipid metabolism and apolipoprotein gene expression by rat liver

Ribeiro

Mangeney

Cardot³

et al. 1991

A 3-week fish oil diet induced in weanling rats a decrease in plasma lipids and liver triacylglycerol, and an increase in insulinemia, compared to a corn oil diet. At the same time, plasma apolipoprotein (apo) A-I was slightly lower and plasma heavy apo Bjlight apo B ratio was higher in fish-oil-fed than in corn-oil-fed rats. Hepatocytes obtained from fish-oil-fed and corn-oil-fed rats were used to examine how fish oil affects lipid and apolipoprotein synthesis and secretion. Primary culture of hepatocytes from fish-oil-fed rats displayed a lower ability to synthesize and secrete triacylglycerol than hepatocytes from corn-oil-fed rats, as measured by mass determination or [U-'4C]glycerol incorporation. Hepatocytes from fish-oil-fed rats exhibited a lower synthesis of cholesterol, measured by [I4C]acetate incorporation, than hepatocytes from corn-oil-fed rats. This impairment was associated with an increase in P-oxidation, a higher channeling of oleic acid into phospholipids, and a lower triacylglycerol/diacylglycerol ratio in hepatocytes from fish-oil-fed rats than in hepatocytes from corn-oil-fed rats. Incorporation of [3 %]methionine into secreted apoB was reduced in hepatocytes from fish-oil-fed rats, but was not paralleled by a decrease in apo B mRNA. The appearance of degradative forms of apo B suggest an increase in apo B degradation in hepatocytes from fish-oil-fed rats. Incorporation of [35S]methionine into cellular and secreted apo A-I was lower in hepatocytes from fish-oil-fed rats than in hepatocytes from corn-oil-fed rats, and was not paralleled by any difference in the apo A-I mRNA level. Finally, [35S]methionine incorporation into cellular and secreted forms of apo E and apo A-I mRNA were reduced in hepatocytes from fish-oil-fed rats, compared with hepatocytes from corn-oil-fed rats. These combined data show that fish oil diet reduces triacylglycerol synthesis and secretion and affects apo B synthesis at a post-transcriptional level, and reduces cholesterol synthesis and affects apo E and apo A-I synthesis at a transcriptional and a post-transcriptional level.Dietary fish oil has been shown to decrease plasma triacylglycerol in humans. Such an effect is related to their high content in n-3 polyunsaturated fatty acids, mainly eicosapentaenoic acid [20 : 5(3)] and docosahexaenoic acid [22:6(3)] [l, 21. The low incidence of coronary heart disease in people consuming fish oil as the main fat source has been attributed to their lowering effect on plasma triacylglycerol [3, 41. Nevertheless, humans exhibit contradictory responses to a fish oil diet with respect with plasma cholesterol, apolipoprotein (apo) B or low-density-lipoprotein (LDL) concentrations [5].In nonhuman primates, enrichment of the diet with polyunsaturated fatty acids of the n-6 series has no effect on hepatic apo B secretion and hepatic apo B mRNA levels [6]. When fish oil is substituted for the standard fat source, plasma cholesterol decreases without any significant decrease in plasma apo B level [7]. Furthermore, liver perfusion of ...