Suckling Rat Hepatocytes Research Articles

Dose-dependent biphasic effects of phenobarbital on growth and differentiation of primary culture rat hepatocytes.

The actions of phenobarbital, a liver tumour promoter, on growth and differentiation of primary culture normal rat hepatocytes change biphasically as a function of its concentration. At low concentrations of 0.5-2 mmol/L, phenobarbital enhances DNA synthesis of normal adult rat hepatocytes in the presence of epidermal growth factor (EGF) and/or dexamethasone. This is also true for normal suckling (1-2-week-old) rat hepatocytes, without added growth factor(s), in serum-free primary culture. Contrarily, phenobarbital at high concentrations (3-4 mmol/L) suppresses DNA synthesis of suckling rat hepatocytes. Furthermore, phenobarbital inhibits DNA synthesis of transforming growth factor-a-stimulated primary hepatocytes from normal adult rats in a dose-dependent manner within a concentration range of 3-6 mmol/L. When normal adult rat hepatocytes are led to undergo multiple proliferative cycles upon stimulation with hepatocyte growth factor (HGF) and EGF in the chemically defined hepatocyte growth medium (HGM), 3 mmol/L phenobarbital also remarkably suppresses DNA synthesis. Phenobarbital at 3 mmol/L effectively keeps these hepatocytes morphologically differentiated and accelerates restoration of the expression of markers characteristic of differentiated cells after the initial cellular growth phase. In addition, phenobarbital efficiently supports prolonged survival of the hepatocytes.

Effects of human neonatal serum on DNA synthesis in suckling and adult rat hepatocytes in primary culture

The effect of human neonatal serum on DNA synthesis in suckling and adult rat hepatocytes in primary culture was investigated to characterize growth regulating factors of the liver in neonates and to confirm whether the stimulatory factor is human hepatocyte growth factor (hHGF). Neonatal serum stimulated DNA synthesis of both adult and suckling rat hepatocytes. The stimulatory effect was dose-dependent up to 20% in volume. The molecular weight of the stimulatory substance in neonatal serum was between 12,500 and 25,000, as estimated by gel filtration. Its activity was stable after heating at 56 degrees C for 20 min, but was lost after heating at 90 degrees C for 30 s, and easily passed through S- or heparin-Sepharose columns. The concentration of hHGF quantified by ELISA was too low to stimulate DNA synthesis in vitro. Biological and biochemical properties of the growth stimulatory activity in neonatal serum differed from that of hHGF. The presence of other growth factors in human neonatal serum for suckling and adult hepatocytes was suggested.

Cytotoxicity of galactose, tyrosine and methionine in cultured suckling rat hepatocytes: relation to liver immaturity.

Monolayers of suckling rat hepatocytes cultured for 24 hours were treated with galactose, I-tyrosine and I-methionine. The purpose was to study the reasons for the clinical improvement of patients with neonatal hepatitis after dietary restriction of these nutrients. Galactose, tyrosine, and methionine was cytotoxic on suckling rat hepatocytes, yet had no effect on adult rat hepatocytes. Furthermore, the pretreatment of suckling rat hepatocytes with dexamethasone ameliorated the cytotoxicity and induced a differentiation of the cells. These results suggested that the cytotoxicity resulted from the immaturity of suckling rat hepatocytes and therefore dietary restriction of galactose, tyrosine and methionine might be a useful treatment for patients with neonatal hepatitis.

Epidermal growth factor-induced selective phosphorylation of cultured rat hepatocyte 55-kD cytokeratin before filament reorganization and DNA synthesis.

We have reported previously that the addition of dexamethasone to cultured quiescent suckling rat hepatocytes in the presence of insulin, a culture condition which does not cause growth activation, induces a selective increase in the synthesis of the 49-kD/55-kD cytokeratin (CK49/CK55) pair over a 24-h period. This increased synthesis coincides with the formation of dense filament networks reminiscent of those observed in situ at the cell periphery (Marceau, N., H. Baribault, and I. Leroux-Nicollet. 1985. Can. J. Biochem. Cell Biol. 63:448-457). We show here for the first time that when EGF is added 48 h after insulin and dexamethasone, there is an early preferential phosphorylation of the CK55 of the CK49/CK55 pair, an induced filament rearrangement from the cell periphery to the cytoplasm, and a subsequent entry into S phase and mitosis after a lag period of 8 h. Indirect immunofluorescence microscopy with monoclonal antibodies to CK49 and CK55 indicate that, while before EGF treatment the cytokeratin filaments were mainly distributed near the cell periphery, the addition of EGF resulted in their reorganization to a predominantly cytoplasmic localization within less than 3 h. Antitubulin and anti-actin antibodies showed no detectable alteration in the distribution of microtubules and microfilaments. Pulse-chase measurements with [35S]methionine showed no apparent change in the turnover of either CK49 or CK55 during the period that precedes the initiation of DNA synthesis. 32P-labeling in vivo followed by SDS-PAGE demonstrated that CK55 was phosphorylated at a much higher level than CK49 in nonstimulated hepatocytes, and that the addition of EGF resulted in a selective stimulation of 32P-CK55 labeling within less than 30 min. Comparative analyses by two-dimensional PAGE of [35S]methionine and 32P-labeled cytokeratins at various times after EGF stimulation demonstrated a rapid increase in a first phosphorylated form of CK55 and the appearance of a second phosphorylated form at 30 min poststimulation. The changes in the relative proportion of nonphosphorylated and phosphorylated forms were confirmed by immunoblotting with the anti-CK55 monoclonal antibody. Determinations of the 32P-labeled phosphoamino acids of CK55 extracted from the gels demonstrated that the radioactivity was mostly in serine residues. Labeling of Triton-permeabilized hepatocytes with gamma 32P-ATP after treatment with EGF for 30 min to 3 h at 37 degrees C, also demonstrated a phosphorylation of CK55 and CK49 as well, implying that the EGF-responsive serine protein kinase is detergent insoluble and probably part of the surface membrane skeleton.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

Perfusion technique of suckling rat liver, and comparison of cytologic and biochemical properties between hepatocytes isolated from suckling and adult rats

Collagenase-liver-perfusion technique, currently used with adult rat liver, was applied to isolation of hepatocytes from suckling rat liver. The total hepatocyte numbers isolated from suckling rats by collagenase-liver-perfusion technique were 9-fold higher than those by non-perfusion technique. The yield and viability of isolated hepatocytes from suckling rats were 18.1 X 10(7) cells per gram liver and 95%, respectively. The cell yield per gram liver and viability from suckling rats were 185% and 112% of those from adult ones, respectively. In comparison with adult rat hepatocytes, suckling rat hepatocytes were smaller and more homogeneous. The percentage (3.1%) of binucleate cells in the hepatocytes isolated from suckling rats was about one tenth of that (30.7%) in the hepatocytes isolated from adult rats. The isolated suckling rat hepatocytes had higher tyrosine aminotransferase (TAT) and glucose-6-phosphate (G6Pase) activities than adult ones. Suckling and adult rat hepatocytes were transferred to primary culture to compare their cell number kinetics and functional longevities. The functional longevities of those hepatocytes were assessed by their capacity to secrete albumin and alpha-fetoprotein (AFP) into the culture medium and to express TAT and G6Pase activities up to day 6 of primary culture.

Dexamethasone can modulate the synthesis and organization of cytokeratins in cultured differentiating rat hepatocytes

Dexamethasone, added to suckling rat hepatocytes cultured in serum-free medium supplemented with insulin and epidermal growth factor (EGF), caused a selective dose-dependent increase in cytokeratin synthesis. The response was dependent on the initial hepatocyte density. At 5 X 10(4) hepatocytes/cm2 a concentration of 1 or 10 microM dexamethasone, in the presence of insulin, enhanced the synthesis of a 55 000 relative mass (Mr) cytokeratin and to a lesser degree a 49 000 Mr cytokeratin, whereas at 1 X 10(5) hepatocytes/cm2 10 microM dexamethasone preferentially stimulated a 51 000 Mr component. Preferential synthesis of the 51 000 Mr component also occurred when either 1 or 10 microM dexamethasone was added to cultures seeded at 2 X 10(5) hepatocytes/cm2. The inclusion of EGF along with dexamethasone and insulin in cultures at 2 X 10(5) cells/cm2 yielded a differential effect of dexamethasone concentration equivalent to that observed at the lower cell density in absence of EGF. Under conditions where increased cytokeratin synthesis was observed, the hepatocytes maintained a high production of albumin and lost their capacity to produce alpha-fetoprotein, a change in gene expression associated with the normal differentiation of suckling rat hepatocytes. In contrast, no enhancement of cytokeratin synthesis was observed in hepatocytes following addition of EGF and insulin only, a condition that promoted hepatocyte growth and the maintenance of alpha-fetoprotein production. The dexamethasone-induced enhancement of cytokeratin synthesis was still present at 3 days postseeding. At this time, morphological observations by phase-contrast and immunofluorescence microscopy using monoclonal antibodies against the 55 000 and 49 000 Mr components revealed that under growth-promoting conditions the hepatocytes were spread and the cytokeratin filaments were stretched, whereas under differentiation-promoting conditions the cultures constitute a compact monolayer of cells exhibiting highly ordered filaments. These data suggest a close relationship between synthesis and organization of cytokeratins and promotion of differentiation of suckling rat hepatocytes by glucocorticoids.

Differential responsiveness of cultured suckling and adult rat hepatocytes to growth-promoting factors: entry into S phase and mitosis.

The capacity of suckling and adult rat hepatocytes in culture to enter into S phase and mitosis in response to EGF, insulin, and glucagon was measured. Both cell types were isolated in high yield and purity and cultured in the absence of serum under identical conditions. At the time of isolation, suckling rat hepatocytes were all diploid and in the G1 phase of the cell cycle. Adult rat hepatocytes constituted a population of mixed ploidy level, as shown by flow cytometry. Upon stimulation, both suckling and adult rate hepatocytes entered S phase after a minimum lag period of 24 h. For suckling rat hepatocytes EGF was required, but its stimulating action was dependent on insulin and/or glucagon. In contrast, adult rat hepatocytes entered into S phase in response to EGF alone; insulin and glucagon did not significantly potentiate its effect. Under optimal hormonal stimulation for entry into S phase a large proportion of suckling rat hepatocytes underwent mitosis, whereas only a few mitoses were observed in the case of adult rat hepatocytes. Therefore, there is a differential response of suckling and adult rat hepatocytes to growth factors which correlates with ploidy level, and this difference may be associated with the degree of maturation.

Galactose Metabolism in Suckling and Adult Isolated Rat Hepatocytes

The metabolism of galactose during the postnatal developmental period has been examined in isolated parenchymal cells of livers from fasted rats aged 7, 14, 21, 28, and 42 days, by measuring the disappearance of 1 and 4 mM 1-[14C]galactose from the incubation media as well as [14C]galactose conversion to [14C]glucose and [14C]lactate and oxidation to [14CO2]. Cells from 7- and 14-day-old suckling animals consistently utilized galactose more rapidly (4 times greater) than those of adult. Hepatocytes of suckling rats also converted greater amounts of galactose to glucose and oxidized galactose 2-3 times faster than the adult. The conversion to glucose by suckling cells occurred with minor recycling of labeled C-1 galactose to C-6 of glucose. A comparison with [1-14C]glucose as substrate showed that oxidative rates by the young cells were 3 times faster for galactose than for glucose. This was not due to the presence of a direct galactose oxidative pathway as assessed by the [14CO2] yield from C-1 and C-2 labeled galactose. Incubation of hepatocytes with galactose appeared to augment the production of glucose from endogenous precursors, 33 nmoles/mg cells in 30 min. The enhanced glucose output from endogenous sources in isolated suckling hepatocytes incubated with galactose contrasts with the sugar's suppression of glucose output observed in isolated perfused liver even though galactose metabolism in both preparations are similarly greater in the suckling than in the adult.