Presence of albumin mRNA precursors in nuclei of analbuminemic rat liver lacking cytoplasmic albumin mRNA.

Analbuminemic rats, which lack serum albumin, were previously found to have no albumin mRNA in the cytoplasm of the liver. In the present study, the existence of nuclear albumin mRNA precursors in the liver of analbuminemic rats was examined by RNA X cDNA hybridization kinetics. Albumin mRNA precursors were present in the nuclei of analbuminemic rat liver at almost normal levels, despite the absence of albumin mRNA from the cytoplasm. Nuclear RNA of analbuminemic rat liver was subjected to electrophoresis on 1% agarose gel in parallel with nuclear RNA of normal rat liver. RNA was transferred from the gel to diazobenzyloxymethyl-paper and hybridized to cloned cDNA. Several bands of putative albumin mRNA precursors were obtained with nuclear RNA of analbuminemic rat liver and some of them were indistinguishable from those of normal rat liver. Nuclear RNA of analbuminemic rats was hybridized to 3'-end-labeled cloned cDNA under the conditions of RNA excess and then digested completely with S1 nuclease and subjected to electrophoresis on polyacrylamide gel. By this technique, nuclear RNA that could hybridized to cDNA was found to have the albumin mRNA sequence in at least the 3' half of the mRNa that was covered by cloned cDNA. For comparison of the structures of the albumin genes of analbuminemic and normal rats, DNAs from rat livers of both types were digested completely with EcoRI, HindIII, and Pst I; the fragments were separated by electrophoresis on 1% agarose gel, transferred to nitrocellulose paper, and hybridized to cloned cDNA. The intensities of the corresponding bands and the digestion patterns of the analbuminemic and normal rat genes were indistinguishable. From these data, it is concluded that analbuminemic rats have a unique type of mutation(s) affecting albumin mRNA maturation.     

A seven-base-pair deletion in an intron of the albumin gene of analbuminemic rats.

Analbuminemic rats, which genetically lack serum albumin, have a mutation affecting albumin mRNA processing. Serum albumin genes were cloned from analbuminemic and normal parental Sprague-Dawley rats. Structural analyses of the two albumin genes showed that the gene from analbuminemic rats had a seven-base-pair deletion in an intron. The deletion extended from base 5 to base 11 from the 5' end of intron HI of the albumin gene. This deletion converted the sequence, G-T-A-G-G-T, which is normally located at the 5' end of intron HI, to G-T-A-G-C-G. RNA blot hybridization of analbuminemic and normal rat liver nuclear RNA using a DNA fragment containing the intron HI as a probe showed that this intron sequence persisted in albumin mRNA precursors of analbuminemic rats.     

Hormonal regulation of total RNA and protein biosynthesis in liver cell cultures of rats in the pre- and postnatal period of development

The effects of several hormones on total RNA and protein biosynthesis were examined in primary cultures of liver cells obtained from rat fetuses on 21-22 days of gestation and from 3 week-old weanling rats. The intensity of biosynthesis processes was estimated by the incorporation of labeled precursors in macromolecules. Insulin, cortisol, triiodothyronine (T3) stimulated RNA and protein biosynthesis in both types of cultures. These hormones enhanced total protein biosynthesis in fetal rat liver cells more efficiently than in hepatocytes of weanling rats. Somatotropin (growth hormone--GH) did not change total protein biosynthesis but notably increased RNA synthesis and the production of immunoreactive serum albumin. Experiments on fetal rat liver cell cultures showed that stimulating action of cortisol on RNA synthesis was synergistic in relation to the effects of insulin and GH. It has been concluded that fetal rat liver cell at the end gestation are able to respond adequately to anabolic action of the hormones.     

Albumin, a biologically active protein acting on Leydig cells

The objective of the present study was to characterize further the albumin fraction of rat testicular fluid (rTF), which can enhance luteinizing hormone (LH)-stimulated pregnenolone production by immature Leydig cells in vitro. Testicular fluid, obtained from 300 rat testes was fractionated by sequential ammonium sulfate precipitation, dye-affinity, anion-exchange chromatography, chromatofocussing and an additional heat treatment. The final fraction showed a single band when analyzed on a silver-stained sodium dodecyl sulfate-polyacrylamide gel and isoelectric focussing gel. The protein had a molecular weight of 67 kDa, an isoelectric point of 5.0 and was identified as albumin after Western blotting using an antibody against rat serum albumin. Albumin in this fraction gave a dose-dependent (0.1-2% protein, w/v) increase in LH-induced pregnenolone production, up to 4-fold, and the increase in specific bioactivity when compared to rTF was 1.4-fold. Selective depletion of albumin from testicular fluid was used as another approach to confirm that albumin itself is the main biologically active component in rTF. rTF from mutant analbuminemic rats (albumin content 0.02 mg/ml) did not stimulate LH-induced steroid production in our assay, in contrast to rTF from normal rats (albumin content 40 mg/ml). Albumin fractions obtained from rat, bovine and human sera were also effective in stimulation of steroid production in the presence of LH, in contrast to chicken serum albumin which gave no stimulation. The stimulatory effect of albumin is not caused by bound fatty acids, nor by the presence of modified forms of albumin such as testibumin or the albumin-bilirubin complex. Our results indicate that Leydig cells are more active in steroid production when surrounded by high but physiological concentrations of albumin.     

Colonization of albumin-producing hepatocytes derived from transplanted F344 rat bone marrow cells in the liver of congenic Nagase's analbuminemic rats

BACKGROUND/AIMS: We investigated whether bone marrow cells (BMCs) of normal rats can be transformed in albumin-producing hepatocytes in analbuminemic rat livers. METHODS: BMCs (2 x 10(7)) from F344 rats (F344) were infused via the portal vein into the livers of congenic Nagase's analbuminemic rats (F344alb) immediately after 70% hepatectomy (PH). Alternatively, F344alb were hematopoietically reconstituted with F344 BMCs by whole body irradiation and BMC transplantation before PH. The recipients were examined for albumin positive (alb +) hepatocytes and albumin mRNA in the livers as well as serum albumin levels 4 weeks later. Sry3 in situ hybridization was done for the livers of female F344alb that received male F344 BMCs. RESULTS: Livers of untreated F344alb contained a few single and double alb+hepatocytes, but these did not form clusters after PH. Clusters (>3 alb + hepatocytes) were detected in livers of the recipients which were transplanted with BMCs immediately after PH as well as the reconstituted F344alb with or without PH. Normal albumin mRNA was detected in the recipient livers, and serum albumin levels were increased. Sry3 was identified in the alb+clusters in the female recipients. CONCLUSIONS: Transplanted BMCs from normal rats can increase clusters of albumin-producing hepatocytes within the liver of analbuminemic rats.     

Albumin synthesis and effect of betamethasone on albumin synthesis in perfused liver of normal and CCL4-intoxicated rats.

Synthesis of plasma albumin was studied in perfused liver of normal and CCl4-intoxicated rats, using 14C-bicarbonate method. 1. In normal liver, albumin synthesis averaged 5.6 mg/hr/100 g body weight. 2. In CCl4-intoxicated liver, albumin synthesis decreased to 4.6 mg at acute stage, but restored to 5.1 mg at non-septal fibrotic stage. At septal fibrotic, cirrhotic and cirrhotic stage with ascites, the synthesis rate averaged 4.5 mg, 4.1 mg and 3.2 mg, respectively. From these results, it is inferred that albumin synthesis rate decreases in accordance with the progress of the liver injury. 3. Effect of betamethasone on plasma albumin synthesis in perfused rat liver was investigated. Albumin synthesis rate in normal liver averaged 5.4 mg, and after addition of betamethasone, the rate averaged 4.3 mg, 3.3 mg and 2.8 mg, respectively. The rate after addition of betamethasone increased to 7.3 mg, 5.5 mg and 4.5 mg, respectively. From these results obtained, it is inferred that betamethasone increases plasma albumin synthesis in both normal and CCl4-intoxicated liver. 4. In normal liver, actinomycin-D did not inhibit basal albumin synthesis, but inhibited the increase of albumin synthesis by betamethasone, when actinomycin-D was added together with betamethasone. From these facts, it is concluded that betamethasone is able to induce albumin synthesis.     

Correlation of albumin production rates and albumin mRNA levels in livers of normal, diabetic, and insulin-treated diabetic rats.

We have studied the effects of alloxan-induced diabetes and subsequent insulin replacement on albumin and total hepatic protein synthesis. Diabetes resulted in a reduction to approximately 20% of normal in albumin synthesis relative to the rate of total protein synthesis in vivo and a reduction to 10% in the absolute rate of albumin secretion by perfused livers. In contrast, the synthesis of total secretory protein and retained hepatic protein was affected to a lesser extent by diabetes. Treatment of diabetic rats with insulin restored rates of albumin and total hepatic protein synthesis to normal levels. The molecular basis of these alterations in albumin synthesis was investigated by examining albumin mRNA levels in livers of normal, diabetic, and insulin-treated diabetic animals. The level of albumin mRNA, whether assayed by cell-free translation or by hybridization to a specific complementary DNA probe, was markedly decreased in livers of diabetic animals and was restored to normal by insulin treatment. These changes occurred in parallel with changes in the rates of albumin secretion observed in perfused liver, suggesting that albumin mRNA content is the primary factor responsible for altering rates of albumin synthesis under these conditions.     

Etiology of increased albumin synthesis in old rats

We have investigated albumin synthesis and excretion of albumin into the urine in young and old rats. Evidence is provided to suggest that the higher rate of excretion of albumin into the urine in old rats is not responsible for the increased synthesis.Albumin synthesis in young and old animals was determined after partial hepatectomy, injection of serum protein into the tail vein, and after aminonucleoside puromycin induced nephrosis. For several weeks after partial hepatectomy, an operation that should not effect nephrosis in old rats, albumin synthesis in old rats is decreased. Injection of serum does not decrease albumin synthesis but causes an increase in the amount of albumin excreted into the urine. Aminonucleoside puromycin induced nephrosis does not stimulate albumin synthesis to a significant extent.It is concluded that the rate of excretion of albumin into the urine is a function of the albumin levels in the serum and that synthesis of albumin is not regulated to a great extent by the rate of excretion.     

Serum bilirubin fractions in analbuminemic rats after bile duct ligation. Albumin requirement in the formation of covalently protein-bound bilirubin

To clarify the role of albumin in the production of covalently protein-bound bilirubin (delta bilirubin, B delta) in the blood and the pathophysiological relevance of B delta, changes in the serum bilirubin level and histological findings in the liver, kidneys and myocardium were studied in Sprague-Dawley (SD) rats and Nagase analbuminemic rats (NAR) after bile duct ligation (BDL). In SD rats, the serum bilirubin level increased until 3 days after BDL, and was followed by the appearance of B delta. The increase in serum bilirubin was smaller in NAR, and no serum B delta was noted. Albumin administration to NAR 1 day after BDL increased serum bilirubin with the appearance of B delta. Serum bilirubin decreased in both SD rats and NAR 7 days after BDL. Marked deposition of bile pigments was noted in NAR in the renal tubular epithelium. The renal bilirubin content was decreased after albumin administration. From these results it is concluded that albumin is necessary for the production of B delta, and that renal deposition of bile pigments progresses in the absence of albumin.     

Synthesis and secretion of insulin-like growth factor and its binding protein by the perfused rat liver: dependence on growth hormone status

Isolated livers of normal and hypophysectomized (hypox) rats with or without GH replacement therapy were perfused in an erythrocyte-free recirculating perfusion system for 4 h in the presence of [35S]cysteine. Albumin secretion and synthesis increased in a parallel and linear fashion over 4 h. The albumin secretion rates were 0.53 and 0.21 mg/g liver h-1 in normal and hypox animals, respectively. Insulin-like growth factor (IGF) secretion, measured as insulin equivalents in the fat cell assay as well as in a competitive protein binding assay, and IGF synthesis, as determined from [35S]cysteine incorporation into immunoprecipitable IGF, likewise increased linearly and in parallel throughout the perfusion time. The IGF secretion rate was 50 microU/g liver h-1. The secreted IGF had a molecular weight of approximately 7700 daltons. Secretion and synthesis of IGF were reduced to 11% in hypox rats and were largely restored by human GH replacement therapy (to 86% of normal). A single specific binding protein with an approximate molecular weight of 35,000 was detected in the perfusate. The binding protein was measured by covalent cross-linkage to [125I]IGF I by dimethylsuberimidate. The secretion of this binding protein was 62% of normal in hypox animals and 79% in GH-treated hypox rats. The data suggest that IGF is continuously synthesized and released by the liver. Assuming a half-life for IGF of 3 h in the normal rat, a plasma volume of 8 ml, and a liver weight of 8.5 g, the rate of IGF production by the perfused normal rat liver (50 microU/g liver h-1) would be sufficient to maintain serum IGF at the concentration determined in normal rat serum (approximately 130 microU/ml). This suggests that the liver is the major site of IGF production in the rat.     

Albumin-synthesizing capacity of hepatocytes isolated from rats fed diets differing in protein and energy content

The rate of albumin synthesis has been estimated in hepatocytes prepared from groups of rats maintained on diets of different protein content. These diets were fed either ad libitum or at 50% restriction of ad libitum consumption. The data show that the physiological capacity of hepatocytes to synthesize albumin varies with dietary intake. Albumin production by cells prepared from animals fed ad libitum was directly related to the protein energy:total energy ratio of the food. Restricting consumption of the control diet to 50% of ad libitum intake did not reduce albumin synthesis rates, and similar restriction of the low protein diets ameliorated some of the depression in albumin production observed in hepatocytes isolated from animals fed the same diets ad libitum. The results are discussed with reference to the occurrence of hypoalbuminaemia in children with protein-energy malnutrition.     

Induction of hepatic synthesis of serum amyloid A protein and actin.

Major changes in the mRNA population of murine liver occur after administration of bacterial lipopolysaccharide, an agent that causes increases in the concentrations of acute-phase serum proteins. The mRNA for one of these, serum amyloid A, is increased at least 500-fold compared to the normal level. It becomes one of the most abundant hepatic mRNAs, and serum amyloid A synthesis comprises about 2.5% of total hepatic protein synthesis in the acute-phase response. Its synthesis is tissue-specific in that amyloid A mRNA was not detected in the kidney, an important site of amyloid fibril accumulation. The protein synthesized in largest amount by acute-phase liver tissue in culture is cytoplasmic actin. Its relative rate of synthesis is increased about 5-fold compared to the normal tissue; that of serum albumin is decreased to about one-third of its normal rate. The concentration of mRNA for serum albumin is decreased by a similar amount. Starting with induced liver RNA, we have constructed a recombinant plasmid containing most of the DNA sequence encoding the serum amyloid A polypeptide.     

Synthesis and secretion of rat pancreatic proteins by Xenopus laevis oocytes

An in vivo translation system, the Xenopus laevis oocyte, was employed to study the synthesis and secretion of pancreatic proteins. RNA was purified from normal and diabetic rat pancreas and normal rat liver by use of guanidine isothiocyanate lysis and cesium chloride gradient centrifugation. The presence of functional mRNA was documented by translation in a reticulocyte lysate that yielded precursors of all major secretory proteins, i.e., slightly higher Mr than proteins synthesized in situ by pancreatic acini. Mature X. laevis oocytes were then microinjected with either total RNA or purified mRNA. When oocytes were subsequently incubated with 35S-methionine, pancreatic secretory proteins or hepatic albumin could be immunoprecipitated from oocyte lysate with specific polyclonal antibodies against amylase, trypsin, ribonuclease, and albumin. Amylase was shown to be enzymatically active. Moreover, oocytes released pancreatic secretory proteins into the medium when injected with pancreatic RNA in a time-dependent manner. Only the mature form of amylase was secreted and secretion was not regulated by secretagogues. When a comparison was made after injection of RNA from diabetic pancreas known to contain altered amounts of individual mRNAs, there was a decrease in amylase and an increase in trypsinogen synthesis in oocytes that was comparable to the results of cell free translation. The oocyte expression system, therefore, should be useful not only for studies of protein synthesis but also for processing and secretion.     

Human liver cell transplantation. Prolonged function in athymic-Gunn and athymic-analbuminemic hybrid rats

Isolated cryopreserved human liver cells, attached to collagen-coated microcarriers, were injected intraperitoneally into mutant rat recipients genetically deficient in either bilirubin uridine diphosphoglucuronosyltransferase activity (Gunn rats) or albumin synthesis (Nagase analbuminemic rats). One group of the recipient Gunn and analbuminemic rats were made genetically immunodeficient by interbreeding with athymic rats with inherited T-cell deficiency. Injected microcarriers and cells formed aggregates on the surface of the pancreas. There was no morphologic evidence of rejection in athymic recipients, whereas immunocompetent recipients demonstrated rejection within 5 days of transplantation. Athymic-Gunn rat recipients demonstrated excretion of bilirubin glucuronides in bile for 30 days and reduction in their serum bilirubin levels. In recipient athymic-analbuminemic rats, plasma albumin levels increased from pretransplantation levels of 0.025-0.05 mg/ml to 3.9-4.8 mg/ml 8 days posttransplantation and remained nearly at that level throughout the 30 days of the study. A method of harvesting, attaching to microcarriers, cryopreserving, and in vivo testing of human hepatocytes with prolonged survival and function in athymic-Gunn and athymic-analbuminemic hybrid rats is reported. These rats are excellent animal models for testing xenograft function.     

Conservation of free but not total or non-sex-hormone-binding-globulin-bound testosterone in serum from Nagase analbuminemic rats

Nagase analbuminemic rats have normal reproductive capacity, normal apparent libido, and normal serum concentrations of LH and FSH. Therefore, it is reasonable to assume that intracellular sex steroid hormone concentrations are normal or at least adequate to maintain normal reproductive function in these rats. To test whether intracellular testosterone concentrations in these rats are maintained by the circulating concentration of free or free-plus-weakly-bound testosterone, we measured the concentrations of total testosterone, free testosterone, and non-sex-hormone-binding-globulin-bound testosterone in sera from five adult male Nagase analbuminemic rats and from five age- and sex-matched controls. We found that the analbuminemic rats had markedly decreased serum concentrations of total and non-sex-hormone-binding-globulin-bound testosterone, but normal serum concentrations of free testosterone. These results suggest that intracellular concentrations of testosterone in biologically relevant organs of the rat are maintained by the concentration of free rather than free-plus-weakly-bound testosterone in plasma, in accord with the free hormone hypothesis.     

Minor contribution of hepatocytes to collagen production in normal and early fibrotic rat livers

Hepatocyte contribution to hepatic collagen production in vivo was estimated in rats, based on the fact that ornithine is used for protein synthesis in the liver as arginine after conversion by way of the urea cycle only by hepatocytes. From rats given a mixture of [14C] ornithine and [3H]arginine, hepatic collagen and serum albumin were obtained. The hepatocyte contribution was calculated from the 14C and 3H in arginine purified from collagen and albumin by high performance liquid chromatography. The contribution was less than 10% of total collagen production in normal and early fibrotic livers induced by a single dose of carbon tetrachloride or dimethylnitrosamine. We conclude that hepatocytes may play a minor role in collagen production in normal and early fibrotic rat livers.     

Plasma clearance of sulfobromophthalein and its interaction with hepatic binding proteins in normal and analbuminemic rats: is plasma albumin essential for vectorial transport of organic anions in the liver?

To investigate a possible function of plasma albumin in the vectorial transport of organic anions by the liver, the plasma disappearance of sulfobromophthalein (BSP) and its interaction with plasma and liver cytosolic proteins were studied in normal rats and mutant Nagase analbuminemic rats (NAR). After intravenous administration of BSP, plasma BSP decreased rapidly in both NAR and control animals: plasma clearance values of BSP in NAR and controls were 12.45 and 7.40 ml/min per kg, respectively. Gel exclusion Sephadex G-100 chromatography of BSP with control rat serum revealed a protein peak in the void volume and another in the albumin fraction. BSP chromatographed exclusively with the albumin fraction; binding of BSP to plasma albumin occurred stoichiometrically. Similar studies with NAR serum revealed a single protein peak, in the void volume; a small amount of BSP chromatographed with this protein peak. The amount of BSP that chromatographed with NAR serum protein(s) was 8% of that with control rat serum albumin. Sephadex G-100 chromatography of BSP with control rat liver cytosol revealed four peaks of protein-bound BSP in fractions corresponding to the void volume (fraction X), albumin, glutathione S-transferases (fraction Y, Mr 45,000), and fraction Z (Mr 12,000); fraction Y was the major component of BSP binding. Gel chromatography of NAR liver cytosol with BSP revealed three BSP peaks, fractions X, Y, and Z; fraction X was the major component of BSP binding. Total BSP binding by 30 mg of hepatic cytosolic proteins was 4.5 nmol for controls and 10.4 nmol for NAR. Isoelectric focusing of liver cytosol revealed no quantitative or qualitative differences in glutathione S-transferase isozymes between control and mutant animals. Intravenously administered BSP (5 mumol/kg) rapidly appeared in bile as the free form and the glutathione conjugate in normal rats and NAR; 41% and 57% of injected BSP was excreted within 60 min in NAR and control rat bile, respectively. These results indicate that binding of BSP to plasma albumin is not indispensable to transhepatocyte transport of BSP in vivo.     

The role of the urea cycle and polyamines in albumin synthesis

Albumin synthesis is stimulated by those amino acids which increase urea synthesis and membrane bound polysome aggregation. Ornithine, an amino acid not incorporated into protein and produced from arginine in the urea cycle, is an albumin-stimulating amino acid and is the precursor of the polyamines, and we have shown that the polyamine spermine promotes bound polysome aggregation. To test the concept that ureogenesis with its generation of ornithine might play a key role in albumin synthesis regulation via the polyamine pathway, isolated livers from fasted donors were perfused with ornithine, alpha-difluoromethyl ornithine (DFMO), and spermine. In control experiments, albumin synthesis was 13.4 +/- 0.8 mg per 100 gm liver per hr and polysome aggregation was 47%. These were increased in the presence of ornithine (26.0 +/- 2.6 mg and 59%); if the livers were preperfused with DFMO before the addition of ornithine, then the expected increase in albumin synthesis and polysome reaggregation did not occur (16.3 +/- 1.4 mg and 47%). However, if spermine was present with DFMO during the preperfusion, then the addition of ornithine had the expected effect (albumin synthesis = 26.1 +/- 1.2 mg and polysome aggregation = 62%). This suggests that if the ornithine to putrescine pathway is blocked, ornithine does not stimulate albumin synthesis and offers support to the concepts that (a) ornithine stimulation of albumin production is via polyamine synthesis and (b) that the urea cycle plays a more important role in protein metabolism than simply the pathway for nitrogen disposal.     

The synthesis rates of total liver protein and plasma albumin determined simultaneously in vivo in humans

Although the metabolism of liver-derived plasma proteins such as albumin has been extensively studied, human hepatic protein synthesis as a whole has not been well characterized, because a reproducible model for obtaining human liver tissue has not been available. In this study, the fractional synthesis rates of total liver protein and albumin in vivo were determined simultaneously in nine subjects undergoing elective laparoscopic cholecystectomy. L-[²H₅]phenylalanine (45 mg/kg body wt) was administered for 10 minutes intravenously. Blood samples were collected at regular intervals for 90 minutes and a liver biopsy specimen was taken at 35 +/- 7 minutes. The enrichments of plasma free phenylalanine, plasma albumin, and total liver protein were measured with gas chromatography mass spectrometry (GC-MS). The fractional synthesis rate (FSR) of total liver protein was 24.7 +/- 3.1 %/d (mean +/- SD), and that of albumin was 5.9 +/-1.2%/d. The amount of albumin synthesized per day (absolute synthesis rate, ASR) was 109 +/- 21 mg/kg body wt. No correlation between FSR of total liver protein and ASR of albumin was found. It is concluded that the technique of obtaining liver tissue specimens during laparoscopic surgery may serve as a human in vivo model to study total liver protein synthesis. The fractional synthesis rate of total liver proteins (stationary and exported), equals approximately 25% of the liver protein content daily. Within the range of values of this study, the absolute synthesis rate of albumin was not correlated to the fractional synthesis rate of total liver protein.(Hepatology 1997 Jan;25(1):154-8)