Isolation and subfractionation on ficoll gradients of adult rat hepatocytes. Size, morphology, and biochemical characteristics of cell fractions

The recirculating perfusion of adult rat liver with a Ca-++-free Hanks' solution produces a release of the adhesiveness of cells and a cleaving of the desmosomes. The addition of collagenase and hyaluronidase to the perfusion medium leads to complete dissociation of the liver tissue into a mixture of isolated cells and cell cords in which the hepatocytes remain connected with specific junctional differentiations, namely the gap and tight junctions. Individual cells are released by submitting the suspension of cell trabeculae to a gentle rolling. The gap junctions are ruptured at least in one of the two adjacent cells and remain generally attached to the other cell taking with them a small portion of cytoplasm. This technique of isolation of hepatocytes yields about 60-65% of the parenchymal cells contained in a liver; endothelial cells and other cells of the connective tissue are not recovered. The ultrastructural preservation of the isolated hepatocytes is excellent and the glucose-6-phosphatase activity, confined to the endoplasmic reticulum, appears unaltered in most cells. Protein, DNA and RNA recovery in the preparations of isolated hepatocytes is satisfactory, amounting to 70% of that found in liver homogenate; glycogen, the most labile component examined, is partly lost or degraded during the manipulations. Cell diameters measured by different methods confirm the preservation of the original volume of the in situ hepatocytes and the presence of more than one type of parenchymal cell. By submitting this heterogeneous cell population to an isopycnic density gradient centrifugation, two types of hepatocytes can be distinguished: the light hepatocytes, with a mean diameter of 20.5 mum and a mean density of 1.10, are characterized by an extended smooth- walled endoplasmic reticulum entrapping dispersed alpha-glycogen particles; the heavy hepatocytes, with a mean diameter of 19.0 mum and a mean density of 1.14, present a relatively reduced compartment of smooth endoplasmic reticulum, but large accumulations of glycogen. It is suggested that the cell fraction of low density is enriched in centrolobular cells and the high density fraction in perilobular hepatocytes.     

Establishment and Characterization of a Nontumorigenic Cell Line Derived from a Human Hepatocellular Adenoma Expressing Hepatocyte-Specific Markers

In the present study the establishment and characterization of a nontumorigenic liver epithelial cell line (HACL-1) derived from a human hepatocellular adenoma is described. The HACL-1 cells have a finite life span (i.e., they proliferate for a period of 2 months and then senesce), show cell–cell contact inhibition, do not grow in soft agar, are not tumorigenic when injected in nude mice, and possess a normal diploid karyotype. The cultured cells resemble hepatocytes, but exhibit some features of dedifferentiation. At the ultrastructural level the cells are endowed with round or oval nuclei, abundant cytoplasmic organelles, and varying amounts of glycogen. The rough endoplasmic reticulum is disorganized, while peroxisomes and matrix granules within mitochondria are lacking. HACL-1 cells are cytokeratin 18-positive as well as (transiently) albumin- and α-fetoprotein-positive, but do not express cytokeratin 19. Furthermore, no mutations were observed in exons 5–8 of the tumor suppressor genep53.Taken together these results show that HACL-1 cells are nontumorigenic proliferating liver epithelial cells, which might prove to be of great value in future studies on diverse aspects of human liver cell biology and carcinogenesis.     

Morphometric analysis of fetal rat hepatocytes cultured in monolayer or following gyratory shaking

The stereological technique was used to quantify glycogen areas and endoplasmic reticulum in fetal rat hepatocytes cultured for 24 hr in monolayer (monolayer cells) or following shaking by gyratory rotation (shaken cells). The volume density and volume per cell of glycogen areas decreased in order of freshly isolated hepatocytes, monolayer cells, and shaken cells. The surface density and area per cell of smooth endoplasmic reticulum increased in order of freshly isolated cells, monolayer cells, and shaken cells. The results show that the decrease of glycogen areas and proliferation of the smooth endoplasmic reticulum are more prominent in shaken cells than in monolayer cells. Prominent proliferation of the smooth endoplasmic reticulum in shaken cells may be due to the consumption of glycogen for energy release as a result of gyratory rotation.     

Isozyme differentiation of aldolase and pyruvate kinase in fetal,regenerating, preneoplastic,and malignant rat hepatocytes during culture

Summary Aldolase and pyruvate kinase isozymes were investigated in cultured hepatocytes from fetal, regenerating, and 2-acetyl-aminofluorene-fed rat liver as well as in some epithelial liver cell lines. Our results show that: (a) cell proliferation and prolonged expression of specific isozymes were found only in cultured hepatocytes from 17-day old fetuses; (b) the fetal type of pyruvate kinase expressed in regenerating and carcinogen-treated liver was temporarily lost only in cultured hepatocytes from regenerating liver; (c) the adult type of aldolase and pyruvate kinase was absent in one epithelial cell line derived from a carcinogen-treated liver and in the hepatoma tissue cell (HTC) line but was found in the Faza clone of the Reuber H₃₅ cell line during the 50 first passages in vitro; and (d) the isozyme pattern of pyruvate kinase was always more strongly shifted than that of aldolase. The observations suggest that: (a) hepatocytes from carcinogen-treated liver exhibit the same lack of ability to proliferate in primary culture as normal adult hepatocytes; (b) adult hepatocytes can produce fetal isozymes without prior cell division; (c) pyruvate kinase is a stronger marker of dedifferentiation (retrodifferentiation) than aldolase; and (d) regulatory processes of isozyme expression are different during ontogenesis, regeneration, and hepatocarcinogenesis. This work was supported by the “Institut National de la Santé et de la Recherche Médicale” and the “Fondation pour la Recherche Medicale Fran?aise”     

Characterization of a rat liver epithelial cell line to detect inhibitors of metabolic cooperation

Summary A normal rat liver epithelial cell line, with phenotype characteristics of “oval” cells (WB-F344), was examined for its ability to perform gap-junctional intercellular communication as measured by metabolic cooperation. To test for gap-junctional intercellular communication, 6-thioguanine-sensitive cells were cocultivated with 6-thioguanine-resistant cells. It was found that the recovery of 6-thioguanine-resistant cells depended on the densities of the 6-thioguanine-sensitive cells. Higher densities of 6-thioguanine-sensitive cells reduced the recovery of 6-thioguanine-resistant cells. These observations demonstrate that rat liver epithelial cells could metabolically cooperate, implying they could perform gap-junctional intercellular communication. Two tumor-promoting organochlorine pesticides, aldrin and dieldrin, were potent inhibitors of metabolic cooperation for these cells, but 12-0-tetradecanoyl-phorbol-13-acetate and teleocidin, known mouse skin tumor promoters, were not significantly effective in inhibiting metabolic cooperation. The results suggest that these cells might provide the basis for an in vitro assay specifically to study liver tumor promoters. Research was sponsored by a grant from the Air Force Office of Scientific Research, Air Force Systems Command, USAF, under grant AFOSR-86-0084. The U. S. Government is authorized to reproduce and distribute reprints for government purposes notwithstanding any copyright notation thereon.     

Enrichment and characterization of clonogenic epithelial cells from adult rat liver and initiation of epithelial cell strains

Summary A highly efficient method is described for obtaining prolifertive epithelial cells from adult rat livers for the reproducible establishment of liver epithelial cell strains. When cells were isolated from livers of 10-to 15-wk-old male Fischer 344 rats by a collagenase-perfusion method, collected by centrifugation at 50×g for 5 min, and cultured in Williams' medium E containing fetal bovine serum and dexamethasone, colonies of epithelial cells different in size and morphology from hepatocytes were obtained. Sequential perfusion with collagenase and dispase yielded numerous epithelial cell colonies. When isolated cells were fractionated by differential centrifugation, the great majority of hepatocytes were sedimented at 50 ×g for 1 min, whereas many non-hepatocytic cells remiined in the supernatant and could be sedimented by a second centrifugation at 50×g for 5 min. Culture of the two fractions revealed that almost all the epithelial cell colonies were derived from cells in the non-hepatocytic cell fraction. The epithelial cells were cytochemically negative for γ-glutamyl transpeptidase activity, whereas an increase in the activity was detected in hepatocytes with duration in culture. Ultrastructural characteristics of hepatocytes were not found in the cells of newly established cell strains. These results suggest that adult rat liver epithelial cells propagable in culture were derived from a cell type other than the hepatocyte.     

Cell contact but not junctional communication (dye coupling) with biliary epithelial cells is required for hepatocytes to maintain differentiated functions

Specific differentiated gene expression and the morphology of adult rat hepatocytes can be maintained for as long as 8 weeks in vitro only when they are cultured in the presence of biliary epithelial cells; when primary hepatocytes are cultured alone, they lose these functions within 2 to 3 days. We obtained evidence suggesting that contact between hepatocytes and biliary epithelial cells is necessary for maintaining hepatocyte functions. We examined whether junctional communication between and among hepatocytes and biliary epithelial cells is required for long-term maintenance of hepatocyte functions, using a dye-transfer method, in three co-cultures: (1) hepatocytes and biliary epithelial cells prepared from Sprague-Dawley rats; (2) hepatocytes from Sprague-Dawley rats and epithelial cells of the IAR 20 line, originally established from BDVI rats; and (3) hepatocytes from BDVI rats and IAR 20 epithelial cells. The established epithelial cell line (IAR 20) and early-passage cultures of biliary epithelial cells maintained hepatocyte-specific functions in culture for 40 and 70 days, respectively, but the latter induced more stable maintenance of albumin secretion. Hepatocytes cultured alone lost their characteristic morphology within 5 to 8 days, and almost no dye transfer was observed. In co-cultures, the capacity of biliary epithelial cells to communicate among themselves remained relatively high throughout the culture period, whereas hepatocytes showed almost no junctional communication at an early phase of culture and first began to communicate after 2 weeks, communication capacity increasing for at least the next 10 days of culture. The most notable finding was that there was no dye transfer between hepatocytes and biliary epithelial cells in any co-culture system. These results suggest that the maintenance of hepatocyte-specific functions requires intercellular contact but probably not gap-junctional communication between hepatocytes and biliary epithelial cells. This system is useful for studying heterotypic cell-cell interactions and the control of gene expression.     

Differential expression of ACAT1 and ACAT2 among cells within liver, intestine, kidney, and adrenal of nonhuman primates

Two closely related enzymes with more than 50% sequence identity have been identified that catalyze the esterification of cholesterol using acyl-CoA substrates, namely acyl-CoA:cholesterol acyltransferase 1 (ACAT1) and ACAT2. Both are membrane-spanning proteins believed to reside in the endoplasmic reticulum of cells. ACAT2 has been hypothesized to be associated with lipoprotein particle secretion whereas ACAT1 is ubiquitous and may serve a more general role in cellular cholesterol homeostasis. We have prepared and affinity purified rabbit polyclonal antibodies unique to either ACAT enzyme to identify their cellular localization in liver and intestine, the two main lipoprotein-secreting tissues of the body, and for comparison, kidney and adrenal. In the liver, ACAT2 was identified in the rough endoplasmic reticulum of essentially all hepatocytes whereas ACAT1 was confined to cells lining the intercellular spaces among hepatocytes in a pattern typical of Kupffer cells. In the intestine, ACAT2 signal was strongly present in the apical third of the mucosal cells, whereas ACAT1 staining was diffuse throughout the mucosal cell, but with strong signal in goblet cells, Paneth cells, and villus macrophages. In the kidney, ACAT1 immunostaining was specific for the distal tubules and podocytes within the glomerulus. In the adrenal, ACAT1 signal was strongly present in the cells of the cortex, and absent from other adrenal cell types. No ACAT2 signal was identified in the kidney or adrenal.We conclude that only the cells of the liver and intestine that secrete apolipoprotein B-containing lipoproteins contain ACAT2, whereas ACAT1 is present in numerous other cell types. The data clearly suggest separate functions for these two closely related enzymes, with ACAT2 being most closely associated with plasma cholesterol levels.     

肝细胞极性与膜蛋白分选的分子机制

肝细胞是高度特化的极性上皮细胞,细胞质膜蛋白的分选和极性转运对于肝细胞极性的建立与维持至关重要.首先,膜蛋白在内质网中合成,随后经高尔基体加工修饰,再由反面高尔基体进一步分选,最后通过膜泡运输等不同的机制分别转运到胆汁腔面或窦状隙面,行使其特殊的功能.近些年来,细胞内负责转运的细胞器和主要的分选信号已逐步被揭示.特别是循环内体也被证明参与了胆汁腔面和窦状隙面膜蛋白的极性分选和转运.肝细胞的极性一旦遭到破坏,将会引起胆汁分泌障碍以及其他肝脏功能的损伤,从而可能导致肝脏糖脂代谢紊乱,甚至丧失正常的生理功能.因此,深入研究肝脏细胞极性的形成与维持机制,将为多种肝脏疾病的预防和治疗寻找到新的方向和靶点,具有重要的理论和临床实践意义.     

Destructive and restorative processes of the liver in rats under intensive physical loading

The ultrastructure of rat liver cells after running exercise was investigated. When rats were trained for a month and sacrificed immediately after the last exercise it was revealed that the number of liver cells mitochondria increased, but many of them had alterations: mitochondria became swollen, had lucid matrix. There were some variations in degree of alterations between different mitochondria: a) in the same hepatocyte, b) in different hepatocytes of the same animal, that was connected with individual sensitivity of organelles on the levels of the cell and of the organ. Rough endoplasmic reticulum bore few ribosomes. Glycogen was absent. There were abundant vesicles of smooth endoplasmic reticulum, autophagic vacuoles and peroxisomes in the liver cell cytoplasm. Adaptation of rat liver to the exercise programme becomes evident by 1.5 month of exercise. Mitochondria and rough endoplasmic reticulum were numerous and of normal structure. There were many peroxisomes and glycogen granules in the cytoplasm of hepatocyte. The presence of large autophagic vacuoles in the cytoplasm of some hepatocytes were obviously connected with more rapid destruction of some organelles, than in control.     

A biochemical and stereological study of neonatal rat hepatocyte subpopulations. Effect of pre- and postnatal exposure to ethanol

Hepatocytes from 12-day-old rats, pre- and post-natally exposed to alcohol, together with those from pair-fed controls, were isolated and subfractionated in six cell subpopulations on Percoll density gradients. These cells were characterized using a combination of biochemical and stereological methods. The low density cells (F2) mainly showed biochemical and stereological features of perivenous hepatocytes, whereas the heavier cells (F6) were primarily periportal hepatocytes. The alcohol-metabolizing enzymes, alcohol dehydrogenase and aldehyde dehydrogenase (high and low Km) showed more activity in the F2 fraction. Alcohol-altered mitochondria and Golgi apparatus occurred mainly in F2 cells, whereas the endoplasmic reticulum and lysosomes appeared to be more altered in the F6 hepatocytes. Alcohol also induced the appearance of some small hepatocytes, with a well-developed rough endoplasmic reticulum and an increased number of mitochondria. Biochemical data indicated that glutamate dehydrogenase and alanine aminotransferase were more affected in F2 cells from alcohol-treated rats, and that the activity of the ethanol-metabolizing enzymes was alos reduced in these hepatocytes. Our results indicate that alcohol exposure during zonal development in the liver could have a selective effect on specific cell components depending on the acinar zone, and that the perivenous hepatocytes appear to be more damaged under these conditions.     

Stem cells, cell transplantation and liver repopulation

Liver transplantation is currently the only therapeutic option for patients with end-stage chronic liver disease and for severe acute liver failure. Because of limited donor availability, attention has been focused on the possibility to restore liver mass and function through cell transplantation. Stem cells are a promising source for liver repopulation after cell transplantation, but whether or not the adult mammalian liver contains hepatic stem cells is highly controversial. Part of the problem is that proliferation of mature adult hepatocytes is sufficient to regenerate the liver after two-thirds partial hepatectomy or acute toxic liver injury and participation of stem cells is not required. However, under conditions in which hepatocyte proliferation is blocked, undifferentiated epithelial cells in the periportal areas, called "oval cells", proliferate, differentiate into hepatocytes and restore liver mass. These cells are referred to as facultative liver stem cells, but they do not repopulate the normal liver after their transplantation. In contrast, epithelial cells isolated from the early fetal liver can effectively repopulate the normal liver, but they are already traversing the hepatic lineage and may not be true stem cells. Mesenchymal stem cells and embryonic stem cells can be induced to differentiate along the hepatic lineage in culture, but at present these cells are inefficient in repopulating the liver. This review will characterize these various cell types and compare the properties of these cells and the conditions under which they do or do not repopulate the liver following their transplantation.     

Insulin degradation. XXIII. Distribution of glutathione-insulin transhydrogenase in isolated rat hepatocytes as studied by immuno-ferritin and electron microscopy.

The distribution of glutathione-insulin transhydrogenase (glutathione: protein-disulphide oxidoreductase, EC 1.8.4.2) in isolated rat hepatocytes that had been first treated with rabbit antiserum against purified rat liver transhydrogenase and then with ferritin-conjugated goat anti-rabbit gamma-globulin was examined by electron microscopy. In cells with intact plasma membrane, the immunoferritin labeling of glutathione-insulin transhydrogenase was observed on a few external microvillous projections at the outside of the cell. In cells with breaks in the plasma membrane, the immunoferritin labeling appeared extensively on smooth vesicles just inside the plasma membrane and on smooth endoplasmic reticulum extending to and including the outer nuclear membrane, in addition to the external microvillous projections. There was some immunoferritin labeling on rough endoplasmic reticulum and on the inner surface of the plasma membrane. The mitochondria and the outer surface of the plasma membrane of the cell did not show the ferritin labeling. Control parallel samples in which the antiserum was substituted with normal (i.e. non-immune) serum or with neutralized antiserum (prepared by absorption with the transhydrogenase) showed little or no immunoferritin labeling. These results are consistent with the idea that gluthalione-insulin transhydrogenase probably synthesized in the endoplasmic reticulum and that the transhydrogenase accessible to cell surface (or found in the isolated plasma membrane preparations) probably represents a functional continuity between the endoplasmic reticulum and the plasma membrane.     

Regulation of acid phosphatase activity in human promyelocytic leukemic cells induced to differentiate in culture

Induction of differentiation of a human promyelocytic leukemic cell line (HL60) in culture is accompanied by changes in acid phosphatase (Acpase) activity. The increase in activity is less than twofold when the leukemic cells are stimulated by dimethylsulfoxide (DMSO) to differentiate into metamyelocytes and granulocytes but is eightfold when the cells are stimulated by the tumor-promoting agent 12-0- tetradecanoylphorbol 13-acetate (TPA) to differentiate into macrophage- like cells. Five different isozymes of Acpase were separated by acrylamide gel electrophoresis. Isozyme 1, the most anodal isozyme, was found to be present in undifferentiated, DMSO-treated and TPA-treated cells; isozyme 2 was a very faint band observed both in DMSO- and TPA- treated cells, the isoenzymes 3a and 3b were present only in TPA- induced cells; and isozyme 4, the most cathodal isozyme, was present both in TPA- and DMSO-induced cells. A time sequence study on the appearance of the various forms after TPA treatment indicated that the expression of the isozymes is regulated in an uncoordinated fashion. Acpase activity has been shown by ultrastructural cytochemistry to be localized in the entire rough endoplasmic reticulum (RER) and in areas of the smooth endoplasmic reticulum (SER) located near the Golgi complex in differentiating cells but to be extremely weak, if at all detectable, in undifferentiated promyelocytes.     

The origin and role of confronting cisternae in selected fetal mouse and rat tissues

Confronting cisternae (CC) are described for the first time in normal fetal rat and mouse liver and intestinal epithelial cells. In these cells, CC characteristically consist of 2 parallel cisternae which are devoid of ribosomes on their juxtaposed surfaces. The intracisternal spacing is consistently 20 nm. While CC occur in rapidly proliferating tissues such as fetal liver and intestinal epithelium, they do not occur in hepatocytes following partial hepatectomy. Although it has been postulated that the intact CC profiles represent a mechanism of assuring the presence of pre-formed nuclear envelope (NE) or rough endoplasmic reticulum (RER) in cells, it is more likely that the subunits which result from CC degradation serve as a pool of membrane precursors for new NE or RER.     

Insulin degradation XXIII. Distribution of glutathione-insulin transhydrogenase in isolated rat hepatocytes as studied by immuno-ferritin and electron microscopy

The distribution of glutathione-insulin transhydrogenase (glutathione: protein-disulphide oxidoreductase, EC 1.8.4.2) in isolated rat hepatocytes that had been first treated with rabbit antiserum against purified rat liver transhydrogenase and then with ferritin-conjugated goat anti-rabbit γ-globulin was examined by electron microscopy. In cells with antact plasma membrane, the immunoferritin labeling of glutathione-insulin transhydrogenase was observed on a few external microvillous projections at the outside of the cell. In cells with breaks in the plasma membrane, the immunoferritin labeling appeared extensively on smooth vesicles just inside the plasma membrane and on smooth endoplasmic reticulum extending to and including the outer nuclear membrane, in addition to the external microvillous projections. There was some immunoferritin labeling on rough endoplasmic reticulum and on the inner surface of the plasma membrane. The mitochondria and the outer surface of the plasma membrane of the cell did not show the ferritin labeling. Control parallel samples in which the antiserum was substituted with normal (i.e. non-immune) serum or with neutralized antiserum (prepared by absorption with the transhydrogenase) showed little or no immunoferritin labeling. These results are consistent with the idea that gluthalione-insulin transhydrogenase probably synthesized in the endoplasmic reticulum and that the transhydrogenase accessible to cell surface (or found in the isolated plasma membrane preparations) probably represents a functional continuity between the endoplasmic reticulum and the plasma membrane.     

Effect of benzophenones from Hypericum annulatum on carbon tetrachloride-induced toxicity in freshly isolated rat hepatocytes

Five benzophenones and a xanthone, isolated from Hypericum annulatum Moris, were investigated for their protective effect against carbon tetrachloride toxicity in isolated rat hepatocytes. The benzophenones and the xanthone gentisein were administered alone (100 microM) and in combination with CCl4 (86 microM). CCl4 undergoes dehalogenation in the liver endoplasmic reticulum. This process leads to trichlormethyl radical (*CCl3) formation, initiation of lipid peroxidation, and measurable toxic effects on the hepatocytes. The levels of thiobarbituric acid reactive substances (TBARS) were assayed as an index of lipid peroxidation (LPO). Lactate dehydrogenase (LDH) leakage, cell viability and reduced glutathione (GSH) depletion were used as signs of cytotoxicity. CCl4 significantly decreased hepatocyte viability, GSH level and increased TBARS level and LDH leakage as compared to the control. Our data indicate that 2,3',5',6-tetrahydroxy-4-methoxybenzophenone, 2-O-alpha-L-arabinofuranosyl-3',5',6-trihydroxy-4-methoxybenzophenone and 2-O-alpha-L-3'-acetylarabinofuranosyl-3',5',6-trihydroxy-4-methoxybenzophenone showed weaker toxic effects compared to CCl4 and in combination showed statistically significant protection against the toxic agent.     

Fine Structure of the Adrenocortical Homolog and the Corpuscles of Stannius of Amia calva L.

A comparison is made between the fine structure of yellow corpuscles and white corpuscles located within the kidneys of the holostean fish, Amia calva L. The yellow corpuscles are composed of epithelial cells possessing all the features of steroid-producing tissues, namely an abundance of vacuoles, tubular smooth endoplasmic reticulum, and mitochondria with tubular cristae. The Golgi apparatus is also a conspicuous component of their cytoplasm. These cells are homologous to adrenocortical cells of higher vertebrates and they have cytoplasmic projections which extend into the lumina of surrounding sinusoids. The white corpuscles possess epithelial cells of variable appearance but all cells contain secretory granules and an extensive rough endoplasmic reticulum. The secretory granules appear to originate at the Golgi apparatus and occasionally are observed intact in the intercellular space. However the method of release of these granules was not clearly defined. These corpuscles are similar to the corpuscles of Stannius which have been described in modern bony fish. The presence of multivesicular bodies and smooth endoplasmic reticulum in some cells may reflect the origin of the corpuscles of Stannius from the tubular nephron. A. calva appears to be a suitable organism for comparative studies into the function of the adrenocortical homolog and corpuscles of Stannius in “primitive” fish.