Modulation of cytokeratin and actin gene expression and fibrillar organization in cultured rat hepatocytes.

Intermediate filaments of rat hepatocytes are composed of cytokeratins 8 and 18 (CK8 and CK18, respectively). Recent work from our laboratory has indicated a close relationship between the synthesis of these cytokeratins, their organization into intermediate filaments, and the promotion of growth and differentiation of cultured rat hepatocytes by insulin, epidermal growth factor, and dexamethasone. In the present study, we examined the mRNA expression, level of protein synthesis, and fibrillar distribution of cytokeratins 8 and 18 and actin in hepatocytes, isolated from normal and dexamethasone-injected rats and cultured as monolayers or spheroids in the presence of insulin, or from normal rat hepatocytes, cultured as monolayers in the presence of dexamethasone, insulin, and dimethyl sulfoxide. The CK8 mRNA level was lower in hepatocytes isolated from noninjected rats and cultured as either monolayers or spheroids, than in those from dexamethasone-injected rats. However, the CK18 mRNA level varied in a manner that was different from that of CK8 mRNA, showing that the modes of expression of the two genes were independent. The various changes in hepatocyte culture conditions led to variations in albumin mRNA levels that largely followed those observed in CK8 mRNA levels. In the case of actin, the amount of mRNAs varied from relatively high levels in hepatocyte monolayers to extremely low levels in hepatocyte spheroids, even though in both cases the cells were isolated from dexamethasone-injected rats. These changes in mRNA levels did not necessarily correlate with changes in the synthesis of cytokeratins 8 and 18, and actin. Changes in culture conditions induced a major reorganization in the distribution of cytokeratin intermediate filaments and actin filament between the region near the surface membrane and the cytoplasm. The most divergent patterns in cytokeratin intermediate filaments and actin filament distributions were observed between hepatocytes cultured as spheroidal aggregates and as monolayers in the presence of dimethyl sulfoxide. The former condition resulted in patterns of cytokeratin and actin gene expression and fibrillar organization that best matched those in situ. In the latter condition, inappropriate patterns were obtained, in spite of the fact that dimethyl sulfoxide treated hepatocytes are known to exhibit survival and functional activities equivalent to that of hepatocyte spheroids. These results demonstrate for the first time that the survival and functional activity (i.e., albumin production) of rat hepatocytes in vitro is not necessarily correlated with a particular pattern of cytokeratin and actin gene expression and fibrillar arrangement.     

Use of a monoclonal antibody to distinguish between precursor and mature forms of human lysosomal alpha-glucosidase

The levels of functional mRNA encoding glucose-6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49) were examined in hepatocytes from fasted and fasted/carbohydrate-refed rats and in hepatocytes inoculated into primary culture. Functional G6PDH mRNA was assessed in a cell-free protein synthesis system in vitro. We observed that hepatocytes from fasted/carbohydrate-refed rats had a 12-fold higher level of mRNA than did hepatocytes from fasted rats. The possibility that the adrenal glucocorticoids and insulin were responsible for the increase in G6PDH mRNA in refed rats was examined by studying the effect of insulin and the synthetic glucocorticoid, dexamethasone, on the level of functional G6PDH mRNA in primary cultures of rat hepatocytes maintained in a chemically defined medium. Hepatocytes from fasted rats were inoculated into primary culture and maintained for 48 h either in the absence of hormones or in the presence of insulin alone, dexamethasone alone or both hormones together. We observed that dexamethasone alone caused a fourfold increase in G6PDH mRNA while insulin caused about a twofold increase. Both hormones together elicited an increase that was additive. A comparison of functional G6PDH mRNA levels with the effect of the hormones on G6PDH activity and relative rate of enzyme synthesis suggests that the glucocorticoid elevates the level of G6PDH mRNA within the cell without causing a concommitant increase in the rate of synthesis of the enzyme or the level of G6PDH activity. The results obtained with the primary cultures of hepatocytes indicate that insulin and the glucocorticoids are probably involved with the regulation of hepatic G6PDH mRNA. However, involvement of other hormones, such as thyroid hormone, seems likely since the induced levels of G6PDH mRNA in hepatocytes in culture was one-third of that observed in refed rats.     

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.     

Modulation of fetal and neonatal rat hepatocyte functional activity by glucocorticoids in co-culture

Fetal and neonatal rat hepatocytes were cultured alone or in association with another liver epithelial cell type, in a medium with or without hydrocortisone. Secretion of albumin and alpha-fetoprotein decreased in pure hepatocyte culture, whereas in co-culture it remained stable for several days. Furthermore, addition of hydrocortisone to the co-culture medium induced a rapid increase in albumin production which was maintained at a high level. In contrast, alpha-fetoprotein production was inhibited. At the same time, an abundant extracellular material was secreted between and around hepatocyte colonies. The results demonstrate that the reciprocal relation between albumin and alpha-fetoprotein production which occurs during in vivo perinatal hepatocyte maturation is also observed in vitro. Both cell-cell contacts and glucocorticoids play a key role in this process. It appears that fetal and neonatal hepatocytes can maturate when maintained in a co-culture system.     

Pretranslational regulation of tyrosine aminotransferase and phosphoenolpyruvate carboxykinase (GTP) synthesis by glucagon and dexamethasone in adult rat hepatocytes.

The regulation of synthesis of the gluconeogenic cytosolic enzyme phosphoenolpyruvate carboxykinase (PEPCK) and of tyrosine aminotransferase (TAT) by glucagon and glucocorticoid hormones was studied in hepatocytes maintained in suspension culture for 7 h. Specific antibodies were used to measure relative rates of enzyme synthesis after pulse-labelling of the cells with [3H]leucine or [35S]methionine. Concomitantly, amounts of mRNA were quantified after translation in vitro in a reticulocyte lysate and specific immunoprecipitation of the proteins. Glucagon stimulated the rate of synthesis of PEPCK by 4-6-fold and that of TAT by 6-8-fold in 2h. In contrast, dexamethasone had little effect on PEPCK synthesis, whereas it increased TAT synthesis by 5-9-fold. When used in combination, the two hormones displayed additive effects on TAT synthesis, whereas the glucocorticoid hormone strongly potentiated stimulation of PEPCK synthesis by glucagon. In every instance, changes in rates of synthesis of the two enzymes were totally accounted for by increases in amounts of the corresponding functional mRNA, suggesting a pretranslational site of action for both glucagon and dexamethasone.     

Effects of glucagon, dexamethasone and triiodothyronine on phosphoenolpyruvate carboxykinase (GTP) synthesis and mRNA level in rat liver cells

Acute hormonal effects on the synthesis rate of the cytosolic form of the gluconeogenic enzyme, phosphoenolpyruvate carboxykinase (GTP), were investigated using rat hepatocytes maintained in short-term suspension culture. Cells were pulse-labeled with [3H]leucine or [35S]methionine and the rate of synthesis of phosphoenolpyruvate carboxykinase was estimated after immunoprecipitation of cell extracts with specific antibodies or following high-resolution two-dimensional gel electrophoresis of cell proteins. Total RNA was also extracted from cultured cells and subsequently translated in a wheat germ cell-free protein-synthesis system, in order to quantify the level of functional mRNA coding for phosphoenolpyruvate carboxykinase. Glucagon, the single most effective inducer, causes a 15--20-fold increase in the level of specific mRNA in 2 h, accompanied by a similar increase in enzyme synthesis rate. The extent of induction is further amplified about threefold when dexamethasone is added to the culture medium. The synergistic action of dexamethasone does not require pre-exposure of the cells to the glucocorticoid, but on the contrary occurs without lag upon simultaneous addition of glucagon and dexamethasone. The induction of phosphoenolpyruvate carboxykinase mRNA by glucagon is markedly depressed in hepatocytes inhibited for protein synthesis by cycloheximide. Cycloheximide-inhibited cells, however, display a considerable induction of the message after joint stimulation with dexamethasone and glucagon. Thus, the synergistic action of dexamethasone does not require concomitant protein synthesis. These data provide indirect evidence for a primary effect of the glucocorticoids on the expression of the phosphoenolpyruvate carboxykinase gene. Besides glucagon and dexamethasone, the thyroid hormones are shown to influence the rate of phosphoenolpyruvate carboxykinase synthesis in isolated liver cells. The stimulatory effect of 3,5,3'-triiodothyronine (T3) is best demonstrated as a twofold increase in relative rate of enzyme synthesis in cells supplied with T3 plus glucagon, as compared to cells challenged with glucagon alone. The effect of T3 relies on a pretranslational mechanism, as shown by a commensurate increase in functional mRNA coding for phosphoenolpyruvate carboxykinase. Dose-response experiments with T3 as well as dexamethasone demonstrate effects at very low hormone levels, consistent with a role for these hormones as physiological modulators of phosphoenolpyruvate carboxykinase expression.     

Maintenance of glucokinase activity in primary hepatocyte cultures

When primary cultures of hepatocytes are maintained for 2 weeks from the time of perfusion, the activity of the enzyme glucokinase decreases rapidly, so that the activity can no longer be detected after the fourth day in culture. Concomitantly, there occurs an increase in the activity of hexokinases, the low-K_M isozymes, which predominate in fetal liver. We have made several modifications of the culture medium in an attempt to prevent the decrease in glucokinase activity. When the medium was supplemented with a mixture of insulin, thyroxine, glucagon, dexamethasone, testosterone, and estradiol, the activity of the enzyme in the hepatocytes was present at approximately 15% of in vivo levels after 2 weeks in culture. When this hormone mixture was present during the first 4 hrs of culture and when the hepatocytes were allowed to attach to the collagen support and were maintained thereafter in medium supplemented with fetal bovine serum, insulin, and dexamethasone, the activity of glucokinase increased after an initial decrease for 3 days and was maintained thereafter at levels comparable to those observed in vivo. This effect of the hormone mixture was found to be the result of the presence of glucagon in the mixture, since the presence of glucagon with no other hormones added, except insulin, during the attachment period produced the same pattern of increased glucokinase activity. Immunoprecipitation of glucokinase from the hepatocytes, using monospecific antibody, indicated that the increase in enzyme activity was the result of increased glucokinase enzyme protein and not an increased synthesis of the other hexokinase isozymes. These studies demonstrate the specific hormonal requirements for the maintenance of glucokinase levels in primary hepatocyte culture at those seen in vivo and lends support to the hypothesis that fetal gene expression in primary hepatocyte cultures is selectively regulated rather than being a general effect with a common regulatory mechanism.     

Cooperative effect of thyroid and glucocorticoid hormones on the induction of hepatic phosphoenolpyruvate carboxykinase in vivo and in cultured hepatocytes

The present study investigates the effect and interaction of glucocorticoid and thyroid hormones on the induction of phosphoenolpyruvate carboxykinase (PEPck) mRNA and enzyme protein under in vivo conditions and in serum-free cultured hepatocytes from hypothyroid rats. In hypothyroid/adrenalectomized rats T3 significantly enhanced the cAMP induced PEPck mRNA activity within 3-6 h. This effect was further enhanced by the presence of glucocorticoids. The half-life of PEPck mRNA, as determined after administration of cordycepin, was not affected by hypothyroidism or hyperthyroidism (t 1/2 approximately equal to 45 min), but considerably prolonged by the absence of glucocorticoid hormones (t 1/2 less than 80 min). In hepatocytes in culture Bt2cAMP (0.2 mM) provoked an increase in translatable PEPck mRNA within 2 h incubation time. Preincubation with either T3 (0.1 microM) or dexamethasone (0.1 microM) for 4 h significantly enhanced the cAMP response on PEPck mRNA. Addition of both, T3 plus dexamethasone further enhanced this Bt2cAMP-mediated effect. By measurement of PEPck synthesis corresponding findings were observed. It is concluded that glucocorticoid and thyroid hormones predominantly enhance the cAMP-provoked induction of hepatic PEPck mRNA and, consequently, of PEPck synthesis. Their effect is rapid, significant and additive, indicating an independent action. While glucocorticoids, in addition, accelerate PEPck mRNA degradation, the PEPck mRNA decay rate is similar in the presence and absence of thyroid hormones.     

Regulation of cytochrome P-450c by glucocorticoids and polycyclic aromatic hydrocarbons in cultured fetal rat hepatocytes

The actions of polycyclic aromatic hydrocarbons and glucocorticoids to regulate the synthesis of cytochrome P-450c (the major isozyme induced by polycyclic aromatic hydrocarbons) were investigated in fetal rat hepatocytes maintained in primary monolayer culture. Treatment of hepatocytes in culture with 1,2-benzanthracene resulted in a 50-fold increase in 7-ethoxycoumarin O-deethylase activity. The level of P-450c increased in the cells in a time-dependent fashion as determined by immunoelectrophoretic analysis. The inductive effect of BA was potentiated approximately 1.6- to 2.3-fold when 1 microM dexamethasone was included in the culture medium. However, dexamethasone alone had little or no effect on the induction of P-450c. The rate of synthesis of P-450c was examined by immunoisolation of the specific isozyme from total cellular proteins radiolabeled with [35S]methionine and from the protein products formed during in vitro translation of the isolated mRNA. In addition, the amount of mRNA specific for cytochrome P-450c was determined by Northern blot analysis of RNA extracted from cultured cells. The changes in the rates of synthesis and mRNA levels were found to parallel the changes in enzyme activity. The concentration of dexamethasone required to cause a half-maximal increase in P-450c content in the presence of 1,2-benzanthracene was between 10(-8) and 10(-7) M. It is concluded that glucocorticoids act synergistically with polycyclic aromatic hydrocarbons to increase the levels of P-450c expressed in the fetal rat liver, and that this action is likely mediated by the classical type II glucocorticoid receptor.     

Regulation of phosphoenolpyruvate carboxykinase (GTP) synthesis in rat liver cells. Rapid induction of specific mRNA by glucagon or cyclic AMP and permissive effect of dexamethasone

Isolated rat liver cells maintained in suspension culture for 4 to 5 h synthesize the gluconeogenic cytosolic enzyme phosphoenolpyruvate carboxykinase at a rate approximately 5-fold lower than the in vivo hepatic rate. Glucagon rapidly re-induces phosphoenolpyruvate carboxykinase synthesis in such cells. The rate of enzyme synthesis doubles in 40 min and plateaus at a level 6- to 13-fold higher than in control cells 120 min after glucagon addition at maximal concentration. Consistent with the presumed role of cyclic AMP as a mediator of enzyme induction, the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine, added simultaneously with glucagon, shifts the hormone dose-response curve 2 log units to the left. Moreover, cyclic AMP supplied exogenously to the cells mimics the inductive effect of glucagon. Total cellular RNA isolated from hepatocytes induced by glucagon contains an increased level of mRNA coding for phosphoenolpyruvate carboxykinase, as determined by translational assay. The kinetics and extent of the rise in mRNA level are adequate to explain the stimulation of enzyme synthesis. Although glucagon on its own induces a build-up of phosphoenolpyruvate carboxykinase mRNA and a commensurate stimulation of enzyme synthesis, the glucagon induction is very markedly amplified when the cells are first preincubated with dexamethasone. The glucocorticoid by itself, however, does not have any substantial effect on the level of phosphoenolpyruvate carboxykinase mRNA or on the rate of enzyme synthesis. Its role can therefore be characterized as permissive.     

Culture of porcine hepatocytes or bile duct epithelial cells by inductive serum-free media

A serum-free, feeder cell-dependent, selective culture system for the long-term culture of porcine hepatocytes or cholangiocytes was developed. Liver cells were isolated from 1-wk-old pigs or young adult pigs (25 and 63 kg live weight) and were placed in primary culture on feeder cell layers of mitotically blocked mouse fibroblasts. In serum-free medium containing 1% DMSO and 1 μM dexamethasone, confluent monolayers of hepatocytes formed and could be maintained for several wk. Light and electron microscopic analysis showed hepatocytes with in vivo-like morphology, and many hepatocytes were sandwiched between the feeder cells. When isolated liver cells were cultured in medium without dexamethasone but with 0.5% DMSO, monolayers of cholangioctyes formed that subsequently self-organized into networks of multicellular ductal structures, and whose cells had monocilia projecting into the lumen of the duct. Gamma-glutamyl transpeptidase (GGT) was expressed by the cholangiocytes at their apical membranes, i.e., at the inner surface of the ducts. Cellular GGT activity increased concomitantly with the development of ductal structures. Cytochrome P-450 was determined in microsomes following addition of metyrapone to the cultures. In vivo-like levels of P-450s were found in hepatocyte monolayers while levels of P-450 were markedly reduced in cholangiocyte monolayers. Serum protein secretion in conditioned media was analyzed by Western blot and indicated that albumin, transferrin, and haptoglobin levels were maintained in hepatocytes while albumin and haptoglobin declined over time in cholangiocytes. Quantitative RT-PCR analysis showed that serum protein mRNA levels were significantly elevated in the hepatocytes monolayers in comparison to the bile ductule-containing monolayers. Further, mRNAs specific to cholangiocyte differentiation and function were significantly elevated in bile ductule monolayers in comparison to hepatocyte monolayers. The results demonstrate an in vitro model for the study of either porcine hepatocytes or cholangiocytes with in vivo-like morphology and function.     

Long-term culture of fetal rat hepatocytes in media supplemented with fetal calf-serum Ultroser SF or Ultroser G

Fetal hepatocytes cultured in medium supplemented with fetal calf serum (FCS) or Ultroser SF do not maintain production of albumin or transferrin beyond one week of culture. When dexamethasone (10(-7) M) is present, secretion of albumin and transferrin can be extended to two weeks, however, levels are extremely low. By three weeks, neither plasma protein can be detected in the culture medium in either conditions of culture. In contrast, hepatocytes maintained in medium supplemented with Ultroser G continue to produce albumin and transferrin at high levels for the entire three week period of this study. The morphology of the cultures are different. In FCS and Ultroser SF supplemented medium there are many more fibroblast and epithelial-like cells and relatively fewer cells which are distinctly hepatocytes when compared with Ultroser G supplemented medium. The level of tyrosine aminotransferase, which is a dexamethasone inducible enzyme, is found to be much higher in Ultroser G cultures, with no further increase demonstrable by addition of dexamethasone. In contrast, dexamethasone induces the enzyme by about eight-fold in cultures maintained in FCS supplemented medium. Therefore it appears that Ultroser G already contains sufficient steroid activity to maximize the level of tyrosine aminotransferase. A comparison between Ultroser C and SF (steroid-free) suggests that the mixture of steroid and steroid derivatives in the G formulation must be important in the maintenance of differentiated functions of hepatocytes in culture. However, supplementation of FCS cultures with dexamethasone, which is known to be present in Ultroser G, does not allow hepatocytes to retain their differentiated functions over an extended period. Therefore it is concluded that other components besides dexamethasone must be important.     

Synthesis and regulation of acute phase plasma proteins in primary cultures of mouse hepatocytes

Adult mouse hepatocytes respond in vivo to experimentally induced acute inflammation by an increased synthesis and secretion of alpha 1-acid glycoprotein, haptoglobin, hemopexin, and serum amyloid A. Concurrently, the production of albumin and apolipoprotein A-1 is reduced. To define possible mediators of this response and to study their action in tissue culture, we established primary cultures of hepatocytes. Various hormones and factors that have been proposed to regulate the hepatic acute phase reaction were tested for their ability to modulate the expression of plasma proteins in these cells. Acute phase plasma and conditioned medium from activated monocytes influenced the production of most acute phase plasma proteins, and the regulation appears to occur at the level of functional mRNA. Purified hormones produced a significant anabolic response in only a few cases: dexamethasone was found to be effective in maintaining differentiated expression of the cells; and glucagon produced a specific inhibition of haptoglobin synthesis. When cells were treated with a combination of conditioned monocyte medium and dexamethasone, secretion of proteins was markedly reduced. The carbohydrate moieties of all plasma glycoproteins were incompletely modified, apparently as a result of decreased intracellular transport of newly synthesized plasma proteins. Although primary hepatocytes were not phenotypically stable in tissue culture, the cells nevertheless retained a broad response spectrum to exogenous signals. We propose this as a useful system to study the production of plasma proteins and thereby pinpoint the nature and activity of effectors mediating the hepatic acute phase reaction.     

Nutritional and hormonal regulation of the translatable levels of malic enzyme and albumin mRNAs in avian liver cells in vivo and in culture

Relative synthesis of malic enzyme is stimulated 25-to 100-fold by feeding neonatal ducklings or by incubating embryonic chick hepatocytes in culture with triiodothyronine. Synthesis of the enzyme is almost completely blocked when fed birds are starved or when triiodothyronine-treated hepatocytes in culture are also treated with glucagon. Cytoplasmic poly(A)+ RNA was isolated from livers of intact ducklings or hepatocytes in culture treated as described above and translated in an mRNA-dependent rabbit reticulocyte lysate. The identity of malic enzyme synthesized in the cell-free system was confirmed by virtue of its antigenicity, subunit molecular weight, and proteolytic peptide pattern. Translatable levels of malic enzyme mRNA paralleled changes in relative synthesis of malic enzyme in vivo and in hepatocytes in culture. Translatable levels od albumin mRNA were either unaffected or changed in a direction opposite to that of malic enzyme mRNA. Thus, both nutritional and hormonal regulation of malic enzyme synthesis involves regulation of cytoplasmic translatable malic enzyme mRNA levels. The hepatocyte culture system is ideally suited for future studies on the regulation of malic enzyme mRNA synthesis and/or degradation by thyroid hormone and glucagon.     

Glucocorticoid-dependent induction of HMG-CoA reductase and malic enzyme gene expression by polychlorinated biphenyls in rat hepatocytes

Administration of xenobiotics to rats results in hypercholesterolemia and in the induction of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and malic enzyme. To investigate the mechanism of the induction of the enzymes by xenobiotics, the effects of xenobiotics on gene expressions for HMG-CoA reductase, malic enzyme, and cytochrome P-450 in rat liver and in cultured hepatocyte were investigated. The treatment of rats with polychlorinated biphenyls (PCB) as a xenobiotic induced mRNAs for HMG-CoA reductase and malic enzyme as well as CYP2B1/2 (cytochrome P-450b/e). Other xenobiotics, 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT), and chloretone, also increased HMG-CoA reductase mRNA. In an investigation of diurnal rhythm of mRNA for HMG-CoA reductase, the induction by PCB was observed in a dark period. Induced expressions of HMG-CoA reductase gene and malic enzyme gene by PCB were observed in primary cultured rat hepatocytes and showed that the action of PCB on the gene expression relating to lipid metabolism was directed on hepatocytes. The induction was observed only in hepatocytes cultured on Engelbreth-Holm-Swarm sarcoma basement membrane gel (EHS-gel), not on type I collagen, which is usually used for monolayer culture of hepatocytes. The induction of CYP2B1/2 gene expression also was observed only in the cells cultured on EHS-gel. The induction of HMG-CoA reductase and malic enzyme by PCB required dexamethasone. However, the addition of dexamethasone per se to medium containing insulin did not show an inductive effect on levels of mRNA for HMG-CoA reductase and malic enzyme. From the data of diurnal variation and hepatocyte culture experiment, HMG-CoA reductase and malic enzyme are considered to be induced by PCB through the so-called "permissive effect" of glucocorticoid.     

Dexamethasone and dimethylsulfoxide as distinct regulators of growth and differentiation of cultured suckling rat hepatocytes

Dexamethasone can promote the differentiation of different tissues in vivo while dimethylsulfoxide is a commonly used inducer of differentiation in various tumor cell types in culture. In the present study, the effects of dexamethasone and dimethylsulfoxide on growth and functional activities of cultured differentiating suckling rat hepatocytes stimulated with various combinations of EGF, insulin, and glucagon were evaluated. Hepatocytes stimulated with EGF and either insulin or glucagon entered S phase and mitosis after a lag period of 24 h. These hormonal factors thus provide simple combinations of hepatocyte-growth regulators. Dexamethasone in the presence of EGF and glucagon inhibited the initiation of DNA synthesis and mitosis, but it had no effect on EGF-insulin stimulated cultures. Such a differential effect of dexamethasone was observed at concentrations ranging from 4 nM to 200 microM. alpha-Fetoprotein, albumin, and tyrosine aminotransferase were used as typical markers of hepatocyte differentiation status. Irrespective of the combinations of growth-promoting factors used, dexamethasone inhibited alpha 1-fetoprotein production and maintained albumin production and tyrosine aminotransferase inducibility. In contrast, dimethylsulfoxide at 2% inhibited hepatocyte growth and supported the maintenance of the production of both alpha 1-fetoprotein and albumin, independent of the hormonal growth regulators used. On this basis, dexamethasone and dimethylsulfoxide act as distinct modulators of growth and maturation of cultured differentiating suckling rat hepatocytes.