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.     

Density-dependent growth control of adult rat hepatocytes in primary culture

Adult rat hepatocytes in primary culture, which show various liver functions, did not show any mitosis at confluent cell density, although they entered the S phase and remained in the G2 phase, judging by cytofluorometry, when insulin and epidermal growth factor (EGF) were added to 2-day cultures (Tomita, Y., Nakamura, T., & Ichihara, A. (1981) Exp. Cell Res. 135, 363-371). However, when the cell density was decreased by half or one third, the number of nuclei and cell number increased to 1.5-2.0 times that after culture for 35 h with insulin and EGF. Moreover, at these lower densities, DNA synthesis started much earlier, although at the usual high density DNA synthesis with these two hormones did not start until the hepatocytes had been cultured for over 40 h. These results suggest that proliferation of mature rat hepatocytes is regulated by the cell density. First, cells in G0 enter the G1 phase density-dependently; then cells in the G1 phase seem to be stimulated to enter the S phase by insulin and EGF, and a low cell density may permit cells after DNA synthesis to enter the M phase. DNA synthesis of rat hepatocyte cultures at low cell density was strongly inhibited by co-culture with a dense culture. Therefore, the density-dependent mechanism of hepatocyte proliferation seems to involve regulation by a soluble inhibitor(s) secreted by the hepatocytes into the culture medium.     

Differential proliferative response of cultured fetal and regenerating hepatocytes to growth factors and hormones

Upon epidermal growth factor (EGF) stimulation, fetal (20 days of gestation) and regenerating (44-48 h after partial hepatectomy) rat hepatocytes, isolated and cultured under identical conditions, increased DNA synthesis and entered into S-phase and mitosis, measured as [3H]thymidine incorporation and DNA content per nucleus in a flow cytometer, respectively. Fetal hepatocytes consisted of a homogeneous population of diploid (2C) cells. Two different populations of cells were present in regenerating liver, diploid (2C) and tetraploid (4C) cells, that responded to EGF. Glucagon or norepinephrine did not affect EGF stimulation of DNA synthesis in fetal liver cells, but they potentiated EGF response in regenerating hepatocyte cultures. Glucocorticoid hormones (dexamethasone) inhibited DNA synthesis in fetal hepatocyte cultures, an effect potentiated by the presence of glucagon or norepinephrine. In contrast, in regenerating hepatocytes, dexamethasone increased EGF-induced proliferation. EGF-dependent DNA synthesis was inhibited by TGF-beta in both fetal and regenerating cultured hepatocytes. TGF-beta action was partially suppressed by norepinephrine in regenerating hepatocytes, but was without effect in fetal hepatocyte cultures, whereas a synergistic action between TGF-beta and dexamethasone inhibiting growth in fetal but not in regenerating hepatocytes was found. Taken together, these results may suggest that there are significant differences between fetal and regenerating hepatocyte growth in their response to various hormones.     

Temporal requirement for epidermal growth factor and insulin in the stimulation of hepatocyte DNA synthesis

Primary monolayer cultures of adult rat hepatocytes were used to study the temporal interaction of epidermal growth factor (EGF) and insulin in their stimulation of DNA synthesis. The hepatocytes were cultured both under defined conditions and with serum. EGF and insulin interacted synergistically. The entry into S phase (G1 exit) followed first-order kinetics both in untreated and hormone-stimulated cells. Addition of EGF and insulin at the time of plating did not alter the lag period before the DNA synthesis started (25-26 h), but the rate constant for the S phase entry increased five- to sixfold. Experiments where the time of hormone addition was varied indicated that insulin exerted its strongest effect at the time of plating, whereas the cells became more responsive to EGF after being cultured for up to 40-50 h. The responsiveness to EGF at these later stages required an early exposure of the hepatocytes to insulin. When the administration of EGF to insulin-pretreated hepatocytes was postponed for 44 h after plating in serum-free medium, the cellular sensitivity was increased as compared to EGF treatment at 0 h (a one-log shift of the dose-effect curve), the rate of S phase entry was more rapid, and the lag period for the onset of the EGF effect (i.e., shift of rate constant) was shortened (6-7 h vs. 26 h).     

DNA synthesis in primary cultures of adult rat hepatocytes in a defined medium: effects of epidermal growth factor, insulin, glucagon, and cyclic-AMP

Epidermal growth factor (EGF) especially in combination with insulin and glucagon, has been shown to stimulate DNA synthesis in liver cells, both in the whole animal and in cell cultures. As a further development we have found that in primary monolayer cultures of freshly isolated adult rat liver parenchymal cells, in which contamination with nonparenchymal cells was negligible, DNA synthesis was substantially stimulated by these substances. In control cultures, incorporation of [3H]thymidine into DNA and labeling of nuclei in autoradiographs was low. The stimulation by EGF was enhanced by insulin and glucagon, whereas these hormones by themselves exhibited only limited activity. These observations were made in cultures of hepatocytes that were never exposed to serum, even during cell isolation and plating. Hence for stimulation of DNA synthesis under these conditions neither serum factors nor interactions with other types of cells or their products were required. The effects of glucagon were reproduced by substances that elevate intracellular concentration of cyclic-AMP, including cholera toxin, isoproterenol, and methylisobutylxanthine. These various substances, especially EGF, glucagon, or cyclic-AMP, altered the morphological characteristics of the cultures during early stages, promoting cellular spreading and aggregation.     

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)     

Activation and glucagon regulation of mitogen-activated protein kinases (MAPK) by insulin and epidermal growth factor in cultured rat and human hepatocytes

Many hepatocellular activities may be proximally regulated by intracellular signalling proteins including mitogen-activated protein kinases (MAPK). In this study, signalling events from epidermal growth factor (EGF) and insulin were examined in primary cultured human and rat hepatocytes. Using Western immunoblots, rat and human hepatocytes were found to produce a rapid tyrosine phosphorylation of the EGF receptor and MAPK following 0·5–1 min exposure to EGF. Phosphorylation of p42 and p44 MAPK was observed following 2·5 min exposure to EGF. Insulin treatment produced phosphorylation of the insulin receptor β subunit; shc phosphorylation was not observed. MAPK phosphorylation corresponded with a shift in molecular weight and an increase in kinase activity. Insulin-dependent activation of MAPK was unequivocally observed only in human hepatocytes, though a slight activation was detected in rat. Co-treatment with insulin and EGF produced phosphorylation and complete electrophoretic shift in molecular weight of MAPK, with an additive or synergistic increase in enzyme activity in rat but not human hepatocytes; human hepatocyte MAPK was maximally stimulated by EGF alone. Glucagon pretreatment blocked phosphorylation, gel mobility shift and kinase activity of MAPK induced by insulin but only partially blocked EGF-induced MAPK activation in human hepatocytes. Glucagon also reduced the activation of MAPK by EGF in rat hepatocytes. Pre-treatments with forskolin or cyclic AMP analogues diminished in the insulin-, EGF- and insulin plus EGF-dependent activation of MAPK in rat hepatocytes without effecting phosphorylation of receptors or MAPK. These results indicate that although EGF and insulin may both signal through the MAPK/ras/raf/MAPK pathway, the response for MAPK differs between these ligands and between species. Further, in both rat and human, glucagon exerts its effects through a cyclic AMP-dependent mechanism at a level in the insulin and EGF signal transduction pathways downstream of MAPK but promixal to MAPK. The partial inhibition of EGF-induced MAPK phosphorylation by glucagon in human hepatocytes provides further evidence for a raf-1-independent pathway for activation of MAPK. © 1998 John Wiley & Sons, Ltd.     

Expression of exogenous c-myc oncogene does not initiate DNA synthesis in primary rat hepatocyte cultures.

Cultured hepatocytes from adult rats stimulated with combinations of growth factors enter into S phase but do not undergo multiple rounds of DNA synthesis nor mitosis. We have examined the potential of an introduced oncogene to induce alterations in the DNA synthetic activity of the cultured hepatocytes in response to epidermal growth factor (EGF). Overexpression of c-myc did not initiate significant DNA synthesis in rat hepatocyte cultures alone, although it cooperated with added EGF to super-induce thymidine incorporation into DNA. From our results, it is suggested that EGF is also necessary to initiate hepatocyte DNA synthesis probably by inducing a battery of cell cycle-related genes if incubated with c-myc transfected cultures for only 5 hours. Hepatocyte polypeptides reacting with anti-MYC antisera were found to migrate between 55-67 KDa in SDS-PAGE; only the 64-67 KDa species were found to be phosphorylated, and the observed size heterogeneity may be due to proteolytic degradation or may reflect presently unknown posttranslational modifications.     

Dual effects of glucagon and cyclic AMP on DNA synthesis in cultured rat hepatocytes: stimulatory regulation in early G1 and inhibition shortly before the S phase entry

Although several lines of evidence implicate cyclic AMP in the humoral control of liver growth, its precise role is still not clear. To explore further the role of cyclic AMP in hepatocyte proliferation, we have examined the effects of glucagon and other cyclic AMP-elevating agents on the DNA synthesis in primary cultures of adult rat hepatocytes, with particular focus on the temporal aspects. The cells were cultured in a serum-free, defined medium and treated with epidermal growth factor (EGF), insulin, and dexamethasone. Exposure of the hepatocytes to low concentrations (10 pM-1 nM) of glucagon in the early stages of culturing (usually within 6 h from plating) enhanced the initial rate of S phase entry without affecting the lag time from the plating to the onset of DNA synthesis, whereas higher concentrations inhibited it. In contrast, glucagon addition at later stages (24-45 h after plating) produced only the inhibition. Thus, if glucagon was added at a time when there was a continuous EGF/insulin-induced recruitment of cells to S phase, the rate of G1-S transition was markedly decreased within 1-3 h. This inhibitory effect occurred at low glucagon concentrations (ID50 less than 1 nM) and was mimicked by cholera toxin, forskolin, isobutyl methylxanthine, and 8-bromo cyclic AMP. The results indicate that cyclic AMP has dual effects on hepatocyte proliferation with a stimulatory modulation early in the prereplicative period (G0 or early G1), and a marked inhibition exerted immediately before the transition from G1 to S phase.     

Inhibitory effect of transforming growth factor-beta on DNA synthesis of adult rat hepatocytes in primary culture

A transforming growth factor-beta (TGF-beta) found in platelets strongly inhibited DNA synthesis of adult rat hepatocytes in primary culture stimulated by insulin plus EGF or by hepatocyte growth factor (HGF) from rat platelets, but not the syntheses of secretory and intracellular proteins by the cells. TGF-beta had no cytotoxic effect, as judged by phase-contrast microscopic examination of the cell morphology. The inhibition of DNA synthesis by TGF-beta was correlated with marked decrease in the labeling index. TGF-beta did not inhibit growth of hepatoma cell line. These findings indicate that TGF-beta is a strong growth inhibitor of adult rat hepatocytes and may block their shift from the G1 phase to the S phase. The physiological role of TGF-beta in inhibiting growth of adult hepatocytes during liver regeneration is discussed.     

Hormonal regulation of DNA synthesis in primary cultures of adult rat hepatocytes : Action of glucagon

Primary cultures of adult rat hepatocytes, grown in modified minimal essential medium (Eagle's) containing 10% calf serum, could be induced into DNA replication by combinations of epidermal growth factor (EGF), insulin and glucagon. The three hormones acted synergistically, and cells began entering DNA synthesis 48 h after hormone addition. The ability of the hormones to stimulate DNA synthesis was enhanced by plating cells at high cell concentrations or by conditioned medium, and was diminished by daily medium change. The contribution of glucagon to DNA synthesis was replaced by cAMP plus 1-methyl, 3-isobutyl xanthine or by adrenergic agents. Evidence is presented which suggests that all three hormones are required on the first day of culture, and that EGF and insulin are also required after the first day. This appears to be a useful system for studies on the hormonal initiation of growth in quiescent cells.     

A simple medium for the study of hepatocyte growth in culture under defined conditions

Summary The combination (1∶1) of Dulbecco's modified Eagle's medium and Waymouth's medium MAB 87/3 was found to provide favorable conditions for serum-free culture and growth of adult rat hepatocytes. In this simple medium, a majority of hepatocytes stimulated by epidermal growth factor plus insulin entered S phase and divided, with a normal (13 h) interval between DNA synthesis and cell division. The proliferative response did not require extra substratum or the presence of serum, even during cell isolation and plating. This work was supported by the Norwegian Cancer Society.     

The stimulation by epidermal growth factor (Urogastrone) of the growth of neonatal rat hepatocytes in primary tissue culture and its modulation by serum and associated pancreatic hormones

Epidermal growth factor (EGF) added in a single dose (between 10^–16 and 1.7 ± 10^–9M) to neonatal rat hepatocytes in primary culture with subsequent incubation for 12 and 24 hours in Eagle's MEM fortified with 10% (v/v) FBS stimulated their entry into S and M phases, as shown by (³H)thymidine labeling and autoradiography and by a 1-hour exposure to colchicine (0.1 mM). Growth stimulation by EGF was detectable after 4 hours, peaking between 12 and 16 hours, and thereafter declining in intensity. Rat hepatocytes exposed for 72 hours (between the fourth and the seventh day in vitro) to no serum or to 10% fresh FBS possessed similar growth rates and absolute numbers in the cultures. A 24-hour exposure to 20 to 50% FBS stimulated hepatocytic DNA synthesis and mitotic activity and resulted (except for the 50% FBS treatment) in increased hepatocytes' numbers, which were relatively greater than the concurrent increases in connective tissue cell numbers. In serum-devoid medium EGF (10^–11M) enhanced hepatocytic mitotic, but not DNA-synthetic activity. To be fully effective EGF required a 10% FBS addition to the medium, then eliciting within 24 hours a marked increase in hepatocytes' number with respect to cultures incubated with 10% serum only. When associated with 20 to 30% FBS, EGF stimulated parenchymal cell growth at rates slightly higher, but not significantly different, than those elicited by the same serum concentrations alone. However, when used in conjunction with 10 to 30% FBS, EGF preferentially increased the number of hepatocytes rather than that of non-parenchymal cells. Moreover, comparative proliferation kinetic studies showed that in the presence of 10% FBS, an equimolar (10^–14M) mixture of EGF, insulin, and glucagon promoted an early and marked increase in the DNA-synthetic and mitotic activities of hepatocytes, which peaked after 8 hours. Within a 24-hour time lag this growth stimulation was as effective in increasing the final hepatocytes' number as was a 1000-fold higher EGF concentration, and was twice as active as either an equimolar (10^–14M) mixture of the two pancreatic hormones or EGF by itself at 10^–14M. These results show that the growth-promoting effect of EGF on primary neonatal rat hepatocytes is modulated by serum factor(s) and can be additively amplified by the simultaneous administration of subphysiological doses of glucagon and insulin.     

Cell cycle parameters of adult rat hepatocytes in a defined medium. A note on the timing of nucleolar DNA replication

Hepatocytes, isolated from adult (250-350 g) rats, attached and survived well in primary culture on highly diluted (less than 1 microgram/cm2) collagen gel in a synthetic medium without serum or hormones. About 20% of the cells "spontaneously" entered S phase during the first 4 days of culturing, and mitoses were easily demonstrated at the near physiological concentration (1.25 mM) of Ca++ prevailing in the medium. Cultures given 9 nM epidermal growth factor (EGF) and 20 nM insulin 20 h after inoculation showed vigorous DNA synthesis and mitotic activity. Autoradiography of such cells exposed to [3H]thymidine allowed the determination of the following cell cycle parameters: Lag period from EGF/insulin stimulation till onset of increased DNA synthesis, 17 h; rate of entry into S phase (kG1/S), 0.028/h; duration of S phase, 8.4 h; duration of G2 phase, 2.7 h. The peak DNA synthesis (pulse labelling index, 24%) and peak mitotic activity (mitotic index, 1.7%) occurred 35 and 43 h, respectively, after the stimulation with EGF/insulin. These values are comparable to those reported during the in vivo compensatory hyperplasia following partial hepatectomy of adult rats. A marked variation of the intranuclear [3H]thymidine pulse labelling pattern was noted: During the first 1.5 h of the S phase, the labelling was extranucleolar and during the last 1.5 h chiefly nucleolar. The cells survived well in the absence of glucocorticoid, whose effect on cell cycle parameters therefore could be studied. Dexamethasone (25-250 nM) did not appreciably affect the durations of S phase and G2 phase or the pattern of preferential extranucleolar and nucleolar DNA synthesis within the S phase.     

Reciprocal effects of epidermal growth factor on key lipogenic enzymes in primary cultures of adult rat hepatocytes. Induction of glucose-6-phosphate dehydrogenase and suppression of malic enzyme and lipogenesis

In primary cultured hepatocytes of adult rats epidermal growth factor (EGF) caused 2- to 3-fold induction of glucose-6-phosphate dehydrogenase (EC 1.1.1.49, G6P dehydrogenase) within 2 days. The effect of EGF was additive with a similar effect of insulin. The half-maximum dose of EGF for the induction was 1 ng/ml. Induction of this enzyme by these hormones was shown by immunotitration to be due to increase of the amount of enzyme. Furthermore, this increase in the amount of enzyme was found to result from increase of syntheses of mRNA and enzyme protein. In contrast, the induction of malic enzyme (EC 1.1.1.40, L-malate:NADP+) oxidoreductase) by insulin plus triiodothyronine was strongly suppressed by the concomitant addition of EGF. Induction of G6P dehydrogenase by EGF, like that by insulin, was not suppressed by either glucagon or dibutyryl cAMP, whereas that of malic enzyme was suppressed additively by EGF and dibutyryl cAMP. EGF also suppressed stimulation of lipogenesis by insulin, measured as incorporation of [1-14C]acetate into triglycerides and phospholipids. Another difference between the inductions of G6P dehydrogenase and malic enzyme was in their dependence on cell density; G6P dehydrogenase induction by insulin and EGF was high at low cell density (3 X 10(4) cells/cm2) and less at higher cell density (13 X 10(4) cells/cm2), whereas induction of malic enzyme was high at higher cell density and less at lower cell density. These results are consistent with the dual role of G6P dehydrogenase in lipogenesis in resting cells and in synthesis of nucleic acid in growing cells. Malic enzyme plays a role only for lipogenesis in mature hepatocytes.     

The calcium-dependence of the stimulation of neonatal rat hepatocyte DNA synthesis and division by epidermal growth factor, glucagon and insulin

A low concentration (10(-11) mol/l) of epidermal growth factor (EGF) and/or an equimolar (10(-14) mol/l) mixture of glucagon and insulin stimulated DNA synthesis in hepatocytes in 4-day-old primary cultures of neonatal rat liver. EGF seems to have acted by inducing quiescent hepatocytes to begin cycling, while the glucagon-insulin combination seems to have acted mainly by shortening the cell cycle time. Incubation in low calcium medium blocked untreated hepatocytes in the G1 phase of their cycle and prevented EGF and the glucagon-insulin mixture from stimulating DNA synthesis. Nevertheless, hepatocytes in calcium-deficient medium did respond to these agents, as they reached a late stage of prereplicative development before being blocked: in fact, they initiated DNA synthesis soon after the addition of calcium. EGF, but not the glucagon-insulin combination, also enabled the already cycling hepatocytes (but not the newly activated ones) to overcome the block imposed by the extracellular calcium deficiency after a delay of several hours.     

Effect of epidermal growth factor on cultured adult rat hepatocytes

When adult rat hepatocytes were cultured in plastic Petri dishes in a medium containing insulin and glucagon, supplementation with epidermal growth factor (EGF) had a pronounced effect on their viability, morphology, and biochemical integrity. Transmission and scanning electron microscopic studies showed that after 1 week cells denied EGF accumulated numerous non-electron-dense bodies and filamentous whorls, had irregular nuclei, and exhibited atypical cell surfaces. In contrast, cells grown for 2-3 weeks in the presence of EGF had well-preserved cellular organelles and remained as an epithelial-like monolayer. After 3 weeks EGF-exposed cultures were still inducible for liver-specific tyrosine aminotransferase, and both rat albumin and rat transferrin were recoverable from the culture medium. Virtually no viable cells were present at 3 weeks in EGF-deprived cultures.     

Growth stimulation and apoptosis induced in cultures of neonatal rat liver cells by repeated exposures to epidermal growth factor/urogastrone with or without associated pancreatic hormones

Summary In untreated primary cultures of neonatal rat liver kept in high-calcium (1.8 mmol/l), foetal bovine serum (10%v/v)-containing minimal essential medium (FBSMEM), the absolute numbers of hepatocytes did not change between day 4 and day 9 because ongoing cell loss was counterbalanced by proliferation of a discrete sub-population of the cells. By contrast, the number of stromal cells increased linearly with time. Growth of hepatocytes and stromal cells was differently affected by the daily addition, between day 4 and day 8 of culture, of fresh medium to which peptide mitogen(s) in concentrations ranging from 10^-14 to 10^-8 mol/l had been added. Epidermal growth factor/urogastrone (EGF/URO) with or without equimolar mixtures of glucagon and insulin, induced first hyperplasia of hepatocytes and stromal cells and then apopotosis (degeneration and death) of the progeny of the stimulated cells. By contrast, equimolar mixtures of glucagon and insulin caused a progressive increase in the number of hepatocytes and stromal cells unbalanced by any increase in cell death. At subphysiological concentrations glucagon, in synergism with EGF/URO and/or some other unknown heat-stable component of serum, acted as a trophic factor for hepatocytes. By contrast, insulin alone did not enhance growth of hepatocytes, but rather blocked the mitogenic effects of EGF/URO. The three hormones exerted neither mitogenic nor apoptotic effects when administered in a low calcium (0.01 mmol/l) FBS-MEM medium.These results reveal that EGF/URO may control the size of cell populations in neonatal liver by calcium-dependent mechanisms that make it unlikely to be a promoter of hepatocyte tumours. They also show that glucagon acts as a positive trophic regulator for hepatocytes.