Changes in the albumin content of mouse hepatocytes in vitro

Using the immunoenzyme method followed by the cytophotometrical analysis the dynamics of albumin content changes in isolated mouse hepatocytes in culture was studied. The data obtained point to cyclic changes of the albumin content in the liver cells with the 1 hour periodicity similar to the changes in the rate of protein synthesis revealed for the same cells.     

Measurement of the albumin content in individual cells

A cytophotometric method is proposed for albumin determination in particular cells based on the immunoenzymatic detection of this protein with immuno-peroxidase complexes. The relative contents of albumin in hepatocytes of mouse in two inbred stocks were measured by two independent methods, and the results obtained well compared.     

Immunocytochemical identification of phenylalanine hydroxylase and albumin in cultured hepatoma cells and isolated rat hepatocytes

Rhodamine-conjugated antibodies specific for phenylalanine hydroxylase and serum albumin were employed as cytochemical probes to identify these two proteins in H4 hepatoma cells and in isolated rat hepatocytes. Each fluorescent antibody stained the cells specifically and in a distinctive manner. In both cell types, albumin staining was discretely localized in cytoplasmic and in H4 cultures varied somewhat from cell to cell. Evidence from cultures of REB15 cells, a strain derived by cloning H4 cells in tyrosine-free medium, suggested that the staining variability of H4 cells could reflect a variability in phenylalanine hydroxylase content. Hydrocortisone-treated H4 cells and REB15 cultures contain increased amounts of phenylalanine hydroxylase; and all cells in the culture appear to be induced by the hormone. Evidence was presented to show that the albumin visualized within the isolated hepatocytes had been synthesized by these cells, and, furthermore, that quantitatively nearly all intracellular albumin in the isolated rat hepatocytes appeared to be entrained in the secretion pathway (analogous data already exist for H4 cells [Baker, R.E., and R. Shiman. 1979. J. Biol. Chem. 254:9633-9639]). By scoring specific fluorescence, 86 and 98% of the H4 cells and 89 and 98% of the isolated hepatocytes were found to contain phenylalanine hydroxylase and albumin, respectively. Therefore, almost all cells in each population appeared to synthesize both proteins. An implication of these findings is that in rat virtually all liver parenchymal cells must synthesize both phenylalanine hydroxylase and albumin.     

Acute suppression of albumin synthesis in systemic inflammatory disease: an individually graded response of rat hepatocytes.

The effects of an inflammatory insult on albumin of the rat liver were investigated at the cellular level and were correlated with serum albumin concentration. After SC injection of turpentine, the livers were perfused and fixed in vivo; serial liver sections were stained using a streptavidin-ABC-immunoperoxidase technique with an antibody to rat albumin. Albumin and total protein were measured at intervals after turpentine injection in whole livers and in serum. Fibrinogen was determined in plasma only. Twenty-four hours after turpentine injection serum albumin had dropped by 25% and was at 50% of its initial value at Day 3. Serum fibrinogen increased 2.4-fold within 24 hr and decreased thereafter. Liver homogenates showed no significant changes in albumin concentration. Immunohistochemically, all hepatocytes stained positive for albumin in normal animals. During inflammation, the immunostainable albumin content vanished entirely in a majority of all hepatocytes while remaining unchanged in other cells, thus producing a strikingly patchy staining pattern. No signs of resumption of albumin accumulation in depleted hepatocytes were seen after 8 days, despite a clear trend towards normalization of serum albumin concentration. These results suggest that individual hepatocytes differ widely in their response to agents that suppress albumin synthesis in an acute-phase reaction.     

Changes in collagen and albumin mRNA in liver tissue of mice infected with Schistosoma mansoni as determined by in situ hybridization

We have employed in situ hybridization to evaluate the molecular mechanisms responsible for hypoalbuminemia and increased liver collagen content in murine schistosomiasis. Results were compared using a simplified method of hybridizing isolated hepatocytes from Schistosoma mansoni-infected and normal mouse liver with mouse albumin (pmalb-2) and chick pro-alpha 2(l) collagen (pCg45) probes. Whereas hepatocytes from infected mice showed significantly less albumin mRNA than hepatocytes from control, there were more grains of procollagen mRNA in hepatocytes from infected as compared with control liver. Hybridization of infected liver tissue sections with the collagen probe showed more grains per field in granulomas than in liver regions, whereas with the albumin probe there was more hybridization in liver tissue than in granulomas. These results suggest that in murine schistosomiasis a reduction in albumin mRNA sequence content may be associated with decreased albumin synthesis and ultimately leads to hypoalbuminemia. In addition, although the granuloma seems to be the primary source of type I collagen synthesis, hepatocytes are also capable of synthesizing collagen, especially under fibrogenic stimulation.     

Protective effect of S-adenosylmethionine against galactosamine-induced injury of rat hepatocytes in primary culture

The protective effect of S-adenosylmethionine (SAMe) on D-galactosamine (GalN)-induced damage to rat hepatocytes was tested in primary cultures. SAMe at concentrations of 50 and 1000 mg/l significantly reduced lactate dehydrogenase release from cells injured by 40 mM GalN after 24 h of incubation. There were no significant changes in urea production after 24 h among tested groups, including control hepatocytes. Exposure of hepatocytes to GalN leads to 3.5-fold decrease in urea synthesis after 48 h in comparison with control cell cultures. Addition of the highest dose of SAMe (1000 mg/l) into the culture media attenuated this decrease by 180 %. None of the tested doses of SAMe (5, 25, 50 and 1000 mg/l) affected considerably the reduced activity of mitochondrial dehydrogenases. The content of reduced and oxidized glutathione in GalN-exposed cells was diminished to 1.5 % and 16 %, respectively, of the control values after 24 h. Using only the highest concentration SAMe increased significantly these contents. SAMe had no effect on dramatically decreased albumin synthesis. These findings indicate beneficial effect of SAMe, especially of the highest concentration, on GalN-induced toxicity to rat hepatocytes in primary culture. This action of SAMe seems to be associated with reduction of plasma membrane damage and increased synthesis of glutathione.     

Analysis of Differentiation of Hepatocytes and Bile Duct Cells in Developing Mouse Liver by Albumin Immunofluorescence

Differentiation of endodermal cells in embryonic mouse liver was analyzed by study of their albumin immunofluorescence. Hepatocytes extending from the cranial diverticulum in 9.5-day embryos do not contain albumin. In 10.5-to 11.5-day embryos, hepatocytes are still immature, but they all show albumin immunofluorescence. Most precursor cells of intrahepatic bile-duct cells also give a positive reaction for albumin, like immature hepatocytes. The differentiation of intrahepatic bile-duct cells from immature hepatocytes is suggested.     

The influence of glucocorticoid on albumin synthesis and its messenger RNA in rat in vivo and in hepatocyte suspension culture

Corticosteroids are known to stimulate the synthesis of a number of liver-specific proteins. The reports regarding the effect of glucocorticoid on albumin synthesis in vivo and in vitro are controversial. In an attempt to determine the mechanism by which glucocorticoid exerts its influence on hepatic albumin synthesis and to find an explanation for the conflicting data, we have studied the effect of dexamethasone disodium phosphate on albumin synthesis and albumin messenger RNA as determined by the molecular hybridization technique in hepatocytes in rat in vivo and in suspension culture. In hepatocyte suspension culture, addition of 0.48 μM dexamethasone in medium at zero time led to a significant increase (20%) in incorporation of labeled precursor into albumin as compared to control experiments; this was accompanied by a maintainance of the initial level of full-length albumin mRNA for a 9 h period. In hepatocytes cultured without dexamethasone in the medium there was a progressive loss of albumin mRNA content. Despite this finding, dexamethasone was not able to increase the albumin mRNA content in hepatocyte to a level higher than the initial value. Moreover, administration of this hormone either intraperitoneally or intravenously into rats did not lead to enhanced cell-free albumin synthesis or to an increased level of albumin mRNA. These findings suggest that glucocorticoid does not play an essential role in the regulation of albumin synthesis in vivo. In vitro, however, glucocorticoid leads to a preservation of the initial level of albumin mRNA and thus plays a role in the control of spontaneous dedifferentiation of liver cells in culture.     

Characterization and cell cycle kinetics of hepatocyte populations isolated from adult liver tissue by a nonenzymatic procedure

Suspensions of liver cells enriched in lobular parenchymal hepatocytes were isolated from adult mouse hepatic tissue by nonenzymatic dispersion in chelating buffer and sedimentation of the released cells at unit g. Single cell suspensions so obtained were suitable for flow cytometric measurements of hepatic ploidy class distributions. The more quickly sedimenting cell population consisted of 88% albumin/transferrin-positive epithelial hepatocytes, the nuclei of which were bimodally distributed with respect to RNA content. This dual G1 population was observed in 2C DNA content liver nuclei prepared by several methods and appears to be a general cytochemical characteristic of adult liver parenchymal cells.     

Long-term survival of functional hepatocytes from adult rat in the presence of phenobarbital in primary culture

In the presence of phenobarbital (PB) at 3 mM, hepatocytes isolated from adult rats by a collagenase-perfusion technique survived well on plastic dishes for at least 49 days after initiation of primary culture. PB at concentrations less than 3 mM was ineffective for the maintenance of hepatocytes, and the maintenance of them was attained only in the continuous presence of 3 mM PB. The hepatocytes surviving in the presence of 3 mM PB were morphologically indistinguishable from the hepatocytes after 1-day attachment period, except for the presence of prominent nucleoli in the former. Although both the albumin secretion and tyrosine aminotransferase (TAT) activities of the cells decreased gradually up to day 7 with time in culture, both were thereafter maintained at relatively high levels at least up to day 35 of primary culture. The addition of 10 microM dexamethasone caused a 3-5-fold induction in TAT activity, and the cells were capable of responding to the hormone in this manner at least up to day 28 of primary culture. Furthermore, the cells also had glucose-6-phosphatase activity, even though the level of this enzyme activity was relatively low as compared with that of TAT activity. Survival of hepatocytes in the presence of 3 mM PB was further enhanced by simultaneous addition of dexamethasone (10 microM) and insulin (10 micrograms/ml). The sensitivity of hepatocytes to 3'-methyl-4-dimethylaminoazobenzene (0.24 mM) was remarkably reduced by treatment with PB at 3 mM. PB treatment decreased efficiently the falling rate of total cytochrome P-450 content, but did not induce P-450PB, which is the specific form of cytochrome P-450 induced by PB, in primary cultured hepatocytes. On the other hand, 3-methylcholanthrene (MC, 10 microM) caused an increase of both contents of total cytochrome P-450 and P-450MC, which is the specific form of cytochrome P-450 induced by MC, in primary cultured hepatocytes. However, MC was ineffective for the maintenance of hepatocytes in primary culture. The possible biological actions of PB on primary cultured hepatocytes are discussed on the basis of the experimental data obtained.     

Routine overnight starvation and immunocytochemistry of hepatocyte albumin content

Summary The indirect immunoperoxidase method was used to identify albumin in hepatocytes of rats before and after periods of starvation. All hepatocytes in fed rats contained a relatively large amount of nascent albumin. Overnight fasting reduced the number of hepatocytes with a large amount of albumin to primarily those surrounding terminal hepatic venules. These were estimated to be about 30% of the population. The other cells had only a slight amount of albumin. After 48 h of fasting all hepatocytes contained a low level of albumin.     

The influence of hepatoprotector 2-ethylthiobenzimidazole hydrobromide (bemithyl) on the content of glycogen in cirrhotic rat liver hepatocytes located in various microenvironments

Using absorption and fluorescent cytophotometry methods, glycogen contents were studied in hepatocytes located in liver lobules and in hepatocytes, which make the general population of these cells in normal and cirrhotic rat liver. In cirrhosis, the content of glycogen in hepatocytes located in lobules obviously rises in comparison with the norm, but to a lesser degree, than in hepatocytes making the general population of these cells in cirrhotic liver. The content of glycogen in hepatocytes, located in lobules of pathologically changed liver in bemithyl treated rats, did not differ from the norm. At the same time, the glycogen content in hepatocytes, representing the general population of these cells in cirrhotically altered bemithyl injected rat liver, remained higher than in the norm. The data obtained indicate that distinctions in particular cell microinvironment, obviously present in cirrhotic liver, render essential influence on hepatocyte functional activity.     

Synthesis of alpha-fetoprotein in primary cultures of hepatocytes from adult mice

Alpha-fetoprotein (AFP) and albumin synthesis in primary hepatocyte cultures of adult mice was studied by the immunoperoxidase techniques. Albumin was detectable in all hepatocytes during the cultivation period (from day 1 to day 8). AFP could be found regularly beginning from days 3-4 up to day 8. Attempts to obtain hepatocytes surviving for a longer period of time ended in failure. The number of AFP-containing hepatocytes in the culture ranged within single cells to about half of all the hepatocytes. The proliferating hepatocytes that contained both AFP and albumin were found in some of the experiments.     

Glycogen distribution in rat hepatocyte populations after a single exposure to 4-dimethylaminoazobenzine and subsequent injections of phenobarbital

7 day after a single interperitoneal injection of carcinogen 4-dimethylaminoazobenzen (DAB), a little number of cells with high glycogen contents was found in parallel with a decreased glycogen content in most isolated hepatocytes. 1.5 months after DAB injection, the normal distribution of glycogen content was seen restored in hepatocytes. The treatment of rats with phenobarbital (6 PhB injections 7 days after DAB application) blocked the restoration of the normal glycogen distribution. 2 months after the last PhB injection (3 months after DAB injection) an increased glycogen content was found in the smallest hepatocytes.     

Regulation of albumin gene expression in a series of rat hepatocyte cell lines immortalized by simian virus 40 and maintained in chemically defined medium.

A series of simian virus 40 (SV40)-immortalized hepatocyte cell lines were characterized for albumin production, the regulation of albumin production, and the expression of other liver-specific genes. This series of cell lines is particularly useful for studying the regulation of hepatocyte gene expression because the cell lines express liverlike levels of a number of liver-specific functions and do so while growing in a chemically defined medium. SV40-immortalized hepatocyte cell lines were derived from colonies of albumin-producing epithelial cells that arose after primary hepatocytes maintained in chemically defined medium were transfected with SV40 DNA. Some cell lines secreted albumin at levels equal to or greater than those secreted by freshly plated primary hepatocytes, and all but one line continued to produce albumin for more than 20 passages. The variation in albumin secretion among cell lines reflected differences in the amount of albumin produced per cell and not in the percentage of albumin-producing cells in each line. The characterization of selected cell lines showed that albumin production was regulated by cell density during the growth cycle. Albumin production in most cell lines was also regulated by dexamethasone; however, one cell line continued to produce high levels of albumin when the cells were grown in medium lacking dexamethasone, demonstrating that although glucocorticoid can induce albumin production in some cell lines, it is not required for high levels of albumin production by all cells in culture. Regulation of albumin production measured at the level of protein secretion was paralleled by changes in steady-state levels of a 2.3-kilobase albumin RNA. Albumin-producing SV40-immortalized hepatocytes secreted a variety of other plasma proteins, including transferrin, hemopexin, and the third component of complement. These cells also expressed tyrosine aminotransferase activity that was inducible by dexamethasone. Alpha-fetoprotein production was not detected in any of the cell lines examined.     

Distribution of albumin in normal and regenerating livers of mice. A light microscopic immunohistochemical and autoradiographic study

The conditions affecting the immunohistochemical identification of albumin in livers of male NMRI-mice were investigated by light microscopy. In normal livers albumin is randomly distributed, revealing a pancytoplasmic nearly homogen reaction in groups of hepatocytes or single parenchymal cells. However, combined autoradiographic studies after pulse labelling with 3H-valine and perfusion experiments with human albumin indicate that this distribution is caused by albumin from blood plasma and does not reflect true protein synthesis. After perfusion of the livers followed by immunohistochemical amplification techniques which allowed to dilute the primary antibody up to 1:30,000, albumin could be detected nearly in all liver parenchymal cells as granular deposits decreasing in its density from periportal fields towards the terminal hepatic venules. In regenerating livers due to partial hepatectomy no remarkable differences in granular albumin deposits between G1- and S-phase of the cell cycle could be detected as was demonstrated by combined immunohistochemistry and 3H-dThd-autoradiography. However, during mitosis the content of albumin was often considerably reduced.     

Uptake and degradation of formaldehyde-treated 125I-labelled human serum albumin in rat liver cells in vivo and in vitro

The uptake of formaldehyde-treated 125I-labelled human serum albumin in rat hepatocytes and nonparenchymal liver cells was measured in vivo and in vitro. Isolated liver cells were prepared by treating the perfused liver with collagenase. Purified hepatocytes and nonparenchymal cells were obtained by differential centrifugation. Human serum albumin was found to be taken up exclusively or almost exclusively by nonparenchymal cells in vitro and in vivo (after intravenous injection). The maximal rate of human serum albumin-uptake in vitro was comparable to that in vivo. Nonparenchymal cells degraded human serum albumin in vitro as indicated by release of trichloroacetic acid-soluble radioactivity. Degradation started about 20-30 min after addition of human serum albumin to cells and rate of degradation was proportional to rate of uptake. Human serum albumin-degradation could be studied without interference of concurrent uptake by separating cells that had been preincubated with human serum albumin from the medium and then reincubating them with human serum albumin-free medium. The lag phase before human serum albumin-degradation starts and the inhibitory effect of chloroquine on degradation indicate that human serum albumin is degraded in lysosomes. The data obtained show that enzymatically prepared nonparenchymal liver cells retain their endocytic activity in vitro. Denatured human serum albumin should be useful both as a marker for rat liver macrophages and for the study of intracellular proteolysis in these cells.     

Uptake and degdradation of formaldehyde-treated 125I-labeled human serum albumin in rat liver cells in vivo and in vitro

The uptake of formaldehyde-treated ¹²⁵I-labelled human serum albumin in rat hepatocytes and nonparenchymal liver cells was measured in vivo and in vitro. Isolated liver cells were prepared by treating the perfused liver with collagenase. Purified hepatocytes and nonparenchymal cells were obtained by differential centrifugation. Human serum albumin was found to be taken up exclusively or almost exclusively by nonparenchymal cells in vitro and in vivo (after intravenous injection). The maximal rate of human serum albumin-uptake in vitro was comparable to that in vivo. Nonparenchymal cells degraded human serum albumin in vitro as indicated by release of trichloroacetic acid-soluble radioactivity. Degradation started about 20–30 min after addition of human serum albumin to cells and rate of degradation was proportional to rate of uptake. Human serum albumin-degradation could be studied without interference of concurrent uptake by separating cells that had been preincubated with human serum albumin from the medium and then reincubating them with human serum albumin-free medium. The lag phase before human serum albumin-degradation starts and the inhibitory effect of chloroquine on degradation indicate that human serum albumin is degraded in lysosomes. The data obtained show that enzymatically prepared nonparenchymal liver cells retain their endocytic activity in vitro. Denatured human serum albumin should be useful both as a marker for rat liver macrophages and for the study of intracellular proteolysis in these cells.     

Differentiation of putative hepatic stem cells derived from adult rats into mature hepatocytes in the presence of epidermal growth factor and hepatocyte growth factor

Oval cells, putative hepatic stem cells, can differentiate into a wide range of cell types including hepatocytes, bile epithelial cells, pancreatic cells and intestinal epithelial cells. In this study, we used different growth factor combinations to induce oval cells to differentiate into mature hepatocytes. We isolated and purified oval cells utilizing selective enzymatic digestion and density gradient centrifugation. Oval cells were identified by their morphological characteristics and the strong expressions of OV-6, albumin, cytokeratin (CK)-19 and CK-7. Using a 2-step induction protocol, we demonstrated that oval cells first changed into small hepatocytes, then differentiated into mature hepatocytes. Small hepatocytes were distinguished from oval cells by their morphological features (e.g. round shape and nuclei) and the lack of CK-19 mRNA expression. Mature hepatocytes were identified by their ultrastructural traits and their expressions of albumin, CK-18, tyrosine aminotransferase (TAT), and alpha-1-antitrypsin (alpha-1-AT). Differentiated cells acquired the functional attributes of hepatocytes in that they secreted albumin and synthesized urea at a high level throughout differentiation. Oval cells can thus differentiate into cells with the morphological, phenotypic and functional characteristics of hepatocytes. This 2-step induction procedure could provide an abundant source of hepatocytes for cell transplantation and tissue engineering.