Albumin mRNA Content Research Articles

Stimulation of human albumin synthesis and gene expression by growth hormone treatment

Background and aims: In this study the effects of acute (5 h) and short-term (5 days) GH treatment on albumin synthesis rates in man were investigated and related to changes in the availability of hepatic albumin mRNA.Methods: 30 patients undergoing elective laparoscopic cholecystectomy were randomized into controls (n=10) or GH-treatment (12 U/dose) for 5 h or 5 days (n=10 in each group). Albumin mRNA levels (in liver biopsy specimens) were measured employing a quantitative polymerase chain reaction assay developed specifically for this purpose, whereas albumin synthesis was measured using [2H5]phenylalanine.Results:The fractional synthesis rate of albumin was 6.0±0.9 %/day in the control group and 8.0±1.8 %/day and 8.3±1.7 %/day in the GH-treated groups, respectively (P<0.05 vs controls in both cases). The corresponding values for the concentration of albumin mRNA were 2.6±1.1 ng/μg total RNA, 2.9±0.8 ng/μg total RNA (NS) and 4.7±1.8 ng/μg total RNA in the ‘GH 5’ group (P<0.01 vs controls). The changes in albumin synthesis were only partly explained by the differences in hepatic albumin mRNA levels (r=0.5, P<0.01).Conclusion: These results suggest that GH may induce a quick, gene expression-independent increase in albumin synthesis, which is sustained by a later-occurring increase in albumin gene expression.

Polyunsaturated fatty acids inhibit fatty acid synthase and spot-14-protein gene expression in cultured rat hepatocytes by a peroxidative mechanism

In vivo, polyunsaturated fatty acids (PUFA) inhibit the expression of hepatic genes related to the lipogenic process such as fatty acid synthase and spot-14-protein (S14) genes. In vitro studies have suggested that this was a direct transcriptional effect of PUFA. In hepatocytes, the inhibition of the lipogenic rate by PUFA is not specific, but is linked to a cytotoxic effect due to peroxidative mechanisms. We have investigated whether peroxidation could also explain the inhibitory effect of PUFA on gene expression. Rat hepatocytes were cultured for 24 h with mono-unsaturated or PUFA. PUFA inhibited the expression of fatty acid synthase and S14 genes, and this inhibition was directly related to the number of unsaturations. However, the beta-actin and albumin mRNA concentrations were also affected by the most unsaturated fatty acids, suggesting a non-specific effect of PUFA on gene expression. Measurement of lactate dehydrogenase released into the medium indicated a cytotoxicity of PUFA. This was associated with their peroxidation as evaluated by the presence of thiobarbituric acid-reactive substances in the culture medium. The addition of high concentrations of antioxidants abolished lipid peroxidation and lactate dehydrogenase leakage and completely reversed the inhibitory effect of PUFA on gene expression. This suggests (i) that the results obtained previously in cultured hepatocytes in the presence of low concentrations of antioxidants must be interpretated cautiously and (ii) that in vivo, the inhibitory effect of PUFA on lipogenesis-related genes could be indirect through hormonal or metabolic changes or that their effect on gene expression is somehow linked to peroxidative mechanisms.

Effects of alimentary whole proteins versus their smallpeptide hydrolysates on liver and skeletal muscle during the acute inflammation phase in the rat

Acute inflammation induces changes in liver proteins with an increase in synthesis of positiveacute-phase proteins such as α 1-acid glycoprotein (α 1-AGP) and a decrease in synthesis of negative acute-phase proteins such as albumin. This is associated with muscle wasting, mediated by increased proteolysis and impaired protein synthesis. As protein metabolism can be altered in other situations (malnutrition, growth) by the form of the dietary nitrogen, we studied the effects of the molecular form of nitrogen on liver and skeletal muscle adaptation, looking at gene expression for two acute-phase proteins (albumin and α 1-AGP) and a number of muscle proteins (α 1-actin, ubiquitin and C9 proteasome subunit). Two groups of 24 Wistar rats (250 g) were injected S/C with 0.125 ml turpentine/rat and were fed one of two liquid diets. These diets had caloric, nitrogen, carbohydrate and lipid content but differed in the molecular form of the nitrogen source (whole protein [WP] versus peptide hydrolysate [PH]). Liver and muscle adaptation were studied at 18, 42 or 66 h after turpentine injection. Weight, deoxyribonucleic acid and protein content of the liver were significantly higher with the WP diet than with the PH diet at 42 h and 66 h. There was more α 1-AGP messenger ribonucleic acid (mRNA) at 18 h and less albumin mRNA at 42 h. Thus, the PH diet causes a more rapid increase in α 1-AGP mRNA content and a smaller decrease in albumin mRNA content after turpentine injection than the WP diet. However, the changes in plasma acute-phase proteins (albumin and a,-AGP) were similar with the two diets. In skeletal muscle, there was no change in mRNA levels for the C9 proteasome subunit at any time point with both diets compared to the controls. However, there were greater ubiquitin mRNA levels at 18 h and less α-actin mRNA levels at 18 h, 42 h and 66 h following turpentine injection in the two dietary groups than in the controls. These results suggest that the molecular form of nitrogen ingested regulates hepatic gene transcription or mRNA stability of acute-phase proteins, during the early period of inflammation, but did not affect the expression of muscle proteins, which was altered by turpentine injection. Post-transcriptional control of acute-phase protein genes may contribute to the maintenance of similar plasma levels.

Age-Related Shortening of Poly(A) Tail of Albumin mRNA

To determine the molecular mechanisms of age-related changes in hepatic albumin gene expression, male Fisher 344 rats at 4, 12, and 24 months of age were studied. The hepatic albumin content was modestly increased by 20% and plasma albumin concentration was not altered with age. There were no age-related changes in albumin mRNA content of the liver. Thein vivosynthetic rate of albumin as measured with [14C]leucin incorporation in 4-month-old rats (350 ± 24.5 cpm) was not significantly different from that in 24-month-old rats (393 ± 30.3 cpm). However,in vitrotranslational efficiency of albumin mRNA from 24-month-old rats was only 49% of that in 4-month-old rats. This was correlated with shortening of the poly(A) tail of albumin mRNA from approximately 300–450 bases in 4-month-old rats to only 50–100 bases in 24-month-old rats. The reduced albumin mRNA translational efficiencyin vitroalong with shortening of the poly(A) tail with age is probably an adaptive response since hepatic albumin content was increased and plasma albumin concentrations were not altered with age. The age-related shortening of the poly(A) tail is yet another mechanism of age-related alterations in the expression of specific genes where the changes seen with age are mostly translational.Key Words:aging; albumin synthesis; translational control; poly(A) tail.

Vitamin B6 modulates expression of albumin gene by inactivating tissue-specific DNA-binding protein in rat liver.

The level of albumin mRNA in the liver of vitamin B6-deficient rats was found to be 7-fold higher than that of control rats. Since the transcriptional activity of the albumin gene, as measured by a nuclear run-on assay, was increased 5-fold in vitamin B6 deficiency, the higher concentration of albumin mRNA in the liver of vitamin-deficient rats could be attributed to the enhanced rate of transcription. The promoter proximal sequences of the albumin gene interact with a number of tissue-specific transcription factors including HNF-1 and C/EBP. We determined the binding activities of liver nuclear extracts to the HNF-1- and C/EBP-binding sites by gel mobility-shift assay and found that the activities of the extract prepared from liver of vitamin B6-deficient rats were greater than those of controls. As the concentrations of C/EBP in nuclear extracts from control and vitamin-deficient rats, estimated by Western-blot analysis, were essentially the same, the lower binding activity of the extract from control liver is probably due to inactivation of tissue-specific factors by pyridoxal phosphate and/or its analogues. We therefore examined the effect of pyridoxal phosphate and its analogues on the binding activity of nuclear extract in vitro and found that only pyridoxal phosphate effectively inhibited the binding. These observations indicate that vitamin B6 modulates albumin gene expression through a novel mechanism that involves inactivation of tissue-specific transcription factors by direct interaction with pyridoxal phosphate.

Influence of nidogen complexed or not with laminin on attachment, spreading, and albumin and laminin B2 mRNA levels of rat hepatocytes.

Nidogen/entactin is a Mr = 150,000 glycoprotein which is present within basement membranes in a noncovalent stable complex with laminin. We have studied the effects of nidogen/entactin complexed or not with laminin on attachment, spreading, and functions of adult rat hepatocytes in primary culture. Freshly isolated hepatocytes attached on either recombinant or EHS-derived nidogen, although to a lesser extent than on laminin/nidogen complex, laminin, and E8 and P1 fragments of laminin. Hepatocytes bound on a nidogen fragment bearing the N-terminal and rod-like domains but not on either the N-terminal globules or the rod-like domain which contains a RGD sequence. Attachment of hepatocytes on nidogen and laminin/nidogen complex was inhibited by anti-beta 1 integrin antibodies. Hepatocytes remained rounded on nidogen and laminin, whereas they rapidly spread on laminin/nidogen complex and collagen IV. Nidogen, laminin, and laminin/nidogen complex transiently maintained high steady-state albumin mRNA levels in cultured hepatocytes, but a decrease in albumin mRNA content was observed after 24 h, independently of the substrates. Actinomycin D and cycloheximide treatment indicated that the transient effect of these substrates on albumin expression was related to post-transcriptional mechanisms. Laminin B2 mRNAs were not detectable in freshly isolated hepatocytes but were expressed in 4 h hepatocyte cultures. After 24 h, a dramatic increase in the steady-state level of laminin B2 mRNA was found in hepatocytes cultured on nidogen and laminin/nidogen complex. This effect was slightly prevented in hepatocytes plated on laminin. These results show that interactions of hepatocytes with nidogen/entactin in vitro result only in a transient modulation of hepatocyte functions.

Differential effects of insulin deficiency on albumin and fibrinogen synthesis in humans.

Insulin deficiency decreases tissue protein synthesis, albumin mRNA concentration, and albumin synthesis in rats. In contrast, insulin deficiency does not change, or, paradoxically, increases estimates of whole body protein synthesis in humans. To determine if such estimates of whole body protein synthesis could obscure potential differential effects of insulin on the synthetic rates of individual proteins, we determined whole body protein synthesis and albumin and fibrinogen fractional synthetic rates using 5-h simultaneous infusions of [14C]leucine and [13C]bicarbonate, in six type 1 diabetics during a continuous i.v. insulin infusion (to maintain euglycemia) and after short-term insulin withdrawal (12 +/- 2 h). Insulin withdrawal increased (P less than 0.03) whole body proteolysis by approximately 35% and leucine oxidation by approximately 100%, but did not change 13CO2 recovery from NaH13CO3 or estimates of whole body protein synthesis (P = 0.21). Insulin deficiency was associated with a 29% decrease (P less than 0.03) in the albumin fractional synthetic rate but a 50% increase (P less than 0.03) in that of fibrinogen. These data provide strong evidence that albumin synthesis in humans is an insulin-sensitive process, a conclusion consistent with observations in rats. The increase in fibrinogen synthesis during insulin deficiency most likely reflects an acute phase protein response due to metabolic stress. These data suggest that the absence of changes in whole body protein synthesis after insulin withdrawal is the result of the summation of differential effects of insulin deficiency on the synthesis of specific body proteins.

Albumin mRNA expression in human liver diseases and its correlation to serum albumin concentration

The expression of albumin mRNA in human liver samples was investigated in order to understand the molecular mechanism of albumin gene expression in various liver diseases. Albumin mRNA in acute hepatic failure and decompensated liver cirrhosis was reduced significantly compared to normal control liver (P less than 0.05). Serum albumin concentration is closely correlated with albumin mRNA content (r = 0.895, P less than 0.01). These data suggest that albumin concentration is mainly regulated at albumin mRNA level in the liver despite the presence of other regulatory mechanisms and that expression of albumin mRNA level is correlated with disease severity. But in several cases there was a discrepancy between albumin mRNA level and severity of liver disease, so further investigation of the regulatory factors of albumin gene expression should be performed.

Transforming growth-factor-β (TGF-β) inhibits albumin synthesis in normal human hepatocytes and in hepatoma HepG2 cells

We explored the effect of transforming growth factor β (TGF-β), a cytokine that appears to play a central role in inflammatory events, on albumin expression by normal adult human hepatocytes and hepatoma cells. Addition of TGF-β to primary human hepatocyte cultures resulted in a dramatic decrease in albumin accumulation and synthesis. This effect was dose-dependent, took place after a 48h incubation period and was maintained over 96h. TGF-β-decreased albumin protein levels were associated with reduced albumin mRNA content. Actin mRNA levels were concomittantly increased. Comparable qualitative effects of TGF-β were observed on human hepatoma HepG2 cells.

A low-protein diet restricts albumin synthesis in nephrotic rats.

High-protein diets increase albumin synthesis in rats with Heymann nephritis but albuminuria increases also, causing serum albumin concentration to be suppressed further than in nephrotic animals eating a low-protein diet. Experiments were designed to determine whether dietary protein augmentation directly stimulates albumin synthesis, or whether instead increased albumin synthesis is triggered by the decrease in serum albumin concentration. Evidence is presented that dietary protein augmentation directly stimulates albumin synthesis, accompanied by a proportional increase in steady-state hepatic albumin mRNA concentration (AlbmRNA) and by an increase in AlbmRNA transcription. When the increased albuminuria resulting from dietary protein augmentation is blunted with enalapril, serum albumin concentration is shown to increase in nephrotic rats. Both albumin synthesis and AlbmRNA increase in these animals despite the greater serum albumin concentration. Albumin synthesis correlates inversely with both serum albumin and serum oncotic pressure in nephrotic rats fed 40% protein, but does not correlate with serum albumin concentration in nephrotic rats fed 8.5% protein (LP), even when serum albumin concentration is reduced. Albumin masses are preserved in LP primarily because of reduced albuminuria. Reduced serum oncotic pressure and dietary protein augmentation combine to stimulate albumin synthesis in nephrotic rats at the level of gene transcription.

Regulation of plasma acute-phase protein and albumin levels in the liver of scalded rats

At 12 h after scalding of rats a doubling of the hepatocyte nuclear DNA content, which arose from the presence of additional complete genomes and not from amplification of genes coding for the major acute-phase proteins or albumin, was observed. Examination of relative transcription rates per control DNA mass revealed that alpha 1-acid-glycoprotein and cysteine-proteinase-inhibitor genes remained constitutive, alpha- and gamma-fibrinogen and haptoglobin genes underwent transcriptional activation for 290 and 339% respectively, whereas the relative transcription rate of albumin decreased to 65% of the control level. Along with these changes, the alpha 1-acid glycoprotein, cysteine-proteinase inhibitor and the fibrinogen mRNA concentrations increased about 500%, haptoglobin mRNA 250%, whereas the albumin mRNA concentration fell to 86% of the control. The regulation of the mRNA levels was assessed by comparing the relative change in transcription rates expressed per control DNA content with the relative changes of mRNA concentrations. We arrived at the conclusion that the concentrations of alpha 1-acid-glycoprotein and cysteine-proteinase-inhibitor mRNAs were predominantly regulated by a post-transcriptional mechanism, albumin mRNA by a transcriptional mechanism, and the fibrinogen and haptoglobin mRNAs by a combination of both. The degree of change of the serum levels of the examined proteins was similar to that of their mRNA concentrations and was the result of the complete use of the available RNA templates in protein synthesis.

Albumin metabolism in the nephrotic syndrome: the effect of dietary protein intake.

The nephrotic syndrome is characterized by increased urinary excretion of albumin and other serum proteins, accompanied by hypoproteinemia and edema formation. Nephrotic patients have lower serum albumin concentrations than do patients undergoing continuous ambulatory peritoneal dialysis when albumin and protein losses are the same in both groups, suggesting that nephrotic patients may not maximally adapt to loss of protein. The fractional rate of albumin catabolism is increased in nephrotic patients, possibly as a result of increased albumin catabolism by the kidney, but the absolute albumin catabolic rate is decreased in nephrotic patients. The rate of albumin synthesis may be increased, but not sufficiently to maintain normal serum albumin concentration or albumin pools. Augmentation of dietary protein in nephrotic rats directly stimulates albumin synthesis by increasing albumin mRNA content in the liver, but also causes an increase in glomerular permeability to macromolecules so that much if not all of the excess albumin synthesized is lost in the urine. When dietary protein is restricted, the rate of albumin synthesis is not increased either in nephrotic patients or in rats, despite severe hypoalbuminemia. Although dietary protein supplementation may lead to positive nitrogen balance, dietary protein supplementation alone does not cause an increase in serum albumin concentration or body albumin pools, and may instead cause further albumin pool depletion because of changes induced in glomerular permselectivity. The use of angiotensin-converting enzyme inhibitors may blunt the increased albuminuria caused by dietary protein supplementation and allow albumin stores to be increased.

Action of recombinant human interleukin 6, interleukin 1β and tumor necrosis factor α on the mRNA induction of acute‐phase proteins

The rat hepatoma cell line Fao was used to study the role of three inflammatory mediators on the mRNA regulation of several acute-phase proteins. In the presence of 10(-6) M dexamethasone beta-fibrinogen mRNA levels increased 6-fold after addition of recombinant human IL 6 (rhIL 6). rhIL 1 beta or recombinant human tumor necrosis factor alpha (rhTNF alpha) had essentially no effect on beta-fibrinogen mRNA induction but led to a 20-fold increase in alpha 1-acid glycoprotein mRNA in the presence of dexamethasone. On the other hand, rhIL 6 was a much weaker stimulator of alpha 1-acid glycoprotein mRNA synthesis. All three mediators reduced albumin mRNA concentrations to about 30% of controls. Whereas the induction of beta-fibrinogen mRNA was potentiated by dexamethasone, the synthetic glucocorticoid analog was an absolute requirement for the stimulation of alpha 1-acid glycoprotein mRNA. The mRNA levels of the negative acute-phase protein albumin were induced 5-fold by dexamethasone alone. The beta-fibrinogen mRNA induction started immediately after addition of rhIL 6 and reached a maximum between 12 and 18 h. In contrast, the time-course for alpha 1-acid glycoprotein mRNA synthesis showed a lag phase of 8 h followed by an increase up to 20 h after rhIL 1 beta. rhTNF alpha led to an even more delayed increase in alpha 1-acid glycoprotein mRNA. Whereas in the case of beta-fibrinogen mRNA induction no synergistic effect was observed between various concentrations of the three mediators, the combination of rhIL 6/rhIL 1 beta as well as rhIL 6/rhTNF alpha or rhIL 1 beta/rhTNF alpha regulated synergistically alpha 1-acid glycoprotein and albumin mRNA. It is concluded that discrete acute-phase proteins are regulated differently by the inflammatory mediators IL 6, IL 1 beta and TNF alpha, indicating that the acute-phase response is more complex than previously assumed. The Fao cell line used in this study turned out to be an ideal model for acute-phase protein regulation, suitable for the discrimination between the inflammatory mediators IL 6 and IL 1/TNF alpha.

Molecular aspects of age-related changes in albumin synthesis in female WAG/Rij rats

The rate of albumin gene transcription and the level of albumin RNA sequences in liver nuclei of rats of various ages were determined to investigate the regulation of age-related changes in levels of cytoplasmic albumin mRNA in rat liver. No change was observed with age in both the rate of albumin gene transcription and the amount of nuclear albumin RNA sequences, which suggests that the increase in cytoplasmic albumin mRNA content in the rat liver with age is caused by a decreased turnover of this messenger. The length of poly(A)-tails of rat liver cytoplasmic RNA with age was also examined. Although no change with age was observed in the poly(A)-tail length sedimenting at the peak fraction, a shift towards shorter poly(A)-tails was found with age in the overall poly(A)-tail length distribution.

Low Protein—High Energy Diet Induces Repressed Transcription of Albumin mRNA in Rat Liver

The effects of diet at the molecular level were investigated by feeding rats control or protein-deficient diets. Each of the control and the protein-deficient groups was further divided into three subgroups according to the level of energy intake. Liver DNA, RNA and total cellular protein concentrations and serum albumin and albumin mRNA concentrations were determined. Protein deficiency caused a marked inhibition of liver growth but the size of most cells remained normal. The low protein diet caused concurrent decreases in the levels of serum albumin and serum albumin mRNA. However, this effect was observed only with the combination of a low protein diet and normal energy intake. The low protein-low energy diet failed to induce the low serum albumin level. Our findings suggest that the altered balance between at least two factors of protein and energy intake serves as a trigger for several metabolic changes which ultimately regulate specific genes.

Hepatoma variants (C2) are defective for transcriptional and post-transcriptional actions from both endogenous and viral genomes.

A series of rat hepatoma cell lines was infected with a recombinant adenovirus bearing the rat albumin promoter. Transcription from this promoter was scored directly and was highest in FAO, the differentiated parent, undetectable in C2, a cell variant that has lost almost all hepatocytic characteristics, and high again in C2-Rev7, a 'revertant' cell line derived from C2 that has regained the ability to produce many proteins characteristic of hepatocytes. The endogenous albumin gene is not transcribed in C2 cells, and at a very low rate in C2-Rev7 cells, which accumulate endogenous albumin mRNA at close to normal amounts. Thus the C2-Rev7 'recovery' of albumin mRNA concentration for the endogenous gene is based mainly on post-transcriptional events while the ability of C2-Rev7 to transcribe the albumin promoter in the viral genome is based on a transcriptional factor(s). We also showed that the C2 phenotype included post-transcriptional effects for other genes: transcription of phenylalanine hydroxylase and phosphoenolpyruvate carboxykinase mRNA sequences continue in C2 at rates equivalent to FAO but these C2 cells have no mRNA for these proteins while FAO does. In addition, C2 cells transcribed certain early adenovirus transcription units (E2 and 4) as well as FAO cells but accumulated E2 mRNAs poorly if at all. The changes that led to the C2-Rev7 cell line produced a return to normal of the ability to accumulate these viral mRNAs. Thus a major event in the C2 to C2-Rev7 transition involves post-transcriptional processes as well as the ability to transcribe the albumin promoter positioned in the virus genome.

Transcriptional and posttranscriptional effects of dexamethasone on albumin and procollagen messenger RNAs in murine schistosomiasis.

We have previously shown that dexamethasone increases albumin mRNA and decreases procollagen steady-state mRNA levels in rat hepatocyte cultures. These studies were extended by evaluating an in vivo model of fibrogenesis (murine schistosomiasis) and by determining a more precise level of gene expression responsible for these changes. Control mice and litter mates infected with Schistosomiasis mansoni were evaluated at 8 weeks postinfection when the livers of the infected mice had become fibrotic and their serum albumin levels significantly decreased. The addition of 4 micrograms/mL dexamethasone to the drinking water of half of the infected mice led to a 75% decrease in the liver collagen content as determined by high-performance liquid chromatography. RNA was extracted from the livers of mice under three conditions: control and infected +/- dexamethasone. This RNA was then hybridized with cDNA probes to determine steady-state levels of specific mRNAs. In the infected mice, albumin mRNA levels were decreased compared to control; however, infected mice treated with dexamethasone increased their albumin mRNA content by 3-fold at 8 weeks. Types I and IV procollagen steady-state mRNA levels in infected mice were increased compared to control while dexamethasone suppressed the mRNA level of collagen in infected mice by 50%. The level of gene expression responsible for these steady-state changes was evaluated by nuclear run-on analysis. While the effect of schistosomiasis on these genes was primarily at a transcriptional level, dexamethasone exerted its effect on different genes in the injured liver by diverse mechanisms, i.e., decreasing collagen synthesis at a transcriptional level and increasing albumin by posttranscriptional mechanisms.(ABSTRACT TRUNCATED AT 250 WORDS)

Development of molecular hybridization technology to evaluate albumin and procollagen mRNA content in baboons and man.

We have developed the methodology for evaluating the effects of pathophysiological conditions on the molecular mechanisms of hepatic protein synthesis and fibrogenesis in baboons and man. Total RNA was extracted from percutaneous liver biopsies of five baboons who were chronically fed an ethanol-rich liquid diet, their pair-fed controls and from humans with a variety of liver abnormalities. Chronic alcohol administration in baboons with liver fibrosis and normal serum albumin levels increased in vitro protein synthesis as measured by [35S]methionine incorporation, albumin mRNA content and Type I procollagen mRNA content. There was no difference in the beta-actin (a constitutive protein) mRNA content. In humans, serum albumin levels correlated with albumin mRNA content as indicated by the intensity of dot blot hybridization and Type I procollagen mRNA levels correlated with the activity of liver fibrosis. The use of RNA-DNA hybridization to investigate procollagen mRNA from human biopsies appears to be a valuable tool for evaluating the potential for collagen synthesis and the future course of liver disease. Besides the use of RNA-DNA hybridization, we describe other methodologies which are useful in delineating the levels of gene expression responsible for hepatic mRNA regulation in normal liver and disease states in man. The use of molecular techniques to evaluate human liver disease provides an opportunity to develop clinically relevant information while at the same time offering the additional advantage of providing fundamental knowledge about fibrogenesis.

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.

Increased type I procollagen mRNA levels and in vitro protein synthesis in the baboon model of chronic alcoholic liver disease

We have investigated the molecular mechanisms responsible for the histologic changes induced by ethanol in the baboon model of alcoholic liver disease. Eleven ethanol-fed baboons and their pair-fed controls had histology evaluated and RNA extracted from percutaneous liver biopsy specimens. In 6 of the ethanol-fed animals, fatty liver developed, but no significant differences were found when the RNA from the control and ethanol-fed livers was translated in the reticulocyte lysate system or analyzed with specific cDNA probes. Five of the baboons given ethanol, however, developed significant fibrosis. Molecular evaluation revealed that the RNA from these livers was more active in in vitro protein synthesis, and the type I procollagen mRNA content was significantly higher per microgram of liver RNA as determined by hybridization analysis (183% ± 23% SEM of control, p < 0.02). In addition, there were higher levels of albumin mRNA content in the livers of ethanol-fed baboons that developed fibrosis (180% ± 21% SEM of control, p < 0.05). There was no change, however, in the levels of β-actin mRNA, a representative constitutive protein. These findings in the baboon model of alcoholic fibrosis show that ethanol consumption (a) increases type I procollagen mRNA, which may foster fibrogenesis; (b) increases albumin mRNA content without causing an increase in serum albumin; and (c) induces no change in levels of β-actin mRNA. These studies also show that percutaneous needle biopsy can supply sufficient tissue to evaluate molecular changes in human liver disease.