Analytical methods for the study of liver cell proliferation.

Various cytometric methods for analysis of regenerating rat liver growth (DNA ploidy distributions, binucleation, and DNA synthesis by in vivo BrdUrd incorporation) were evaluated. The overall hepatocellular growth rate (labeling index), the binucleation rate, and separate indices for mononuclear and binuclear cells could be measured simply by microscope counting of collagenase-isolated hepatocytes immunostained for BrdUrd. Flow cytometry of cells stained for BrdUrd and DNA provided labeling indices for the various hepatocellular DNA ploidy classes as well as for nonparenchymal cells (identified by their size-dependent light scatter), but could not distinguish between mononuclear and binuclear hepatocytes. Image cytometry, using fluorescence or Feulgen staining, was inferior to flow cytometry in terms of speed and DNA resolution, but allowed a complete analysis of all hepatocellular DNA ploidy and nuclearity classes. It may therefore be the method of choice, particularly for analysis of liver cell cultures from which single cells are not easily obtained. Fluorescence staining would seem to be preferable to Feulgen staining, since the latter could not be used simultaneously with BrdUrd staining and therefore required a two-step analysis. A non-immunological method, based on the ability of incorporated BrdUrd to quench DNA staining by a Hoechst dye, could only be applied to isolated nuclei, thus giving no information about binucleation. The latter method may be useful for analysis of tumors which are difficult to dissociate to intact whole cells.     

Binucleation and polyploidization patterns in developmental and regenerative rat liver growth

The hepatocellular binucleation rate, measured as the percentage of binuclear cells amongst newly formed bromodeoxyuridine-labelled and immunostained collage-nase-isolated rat hepatocytes, decreased from 12% to 4% between days 30 and 40 after birth, rose to 20% between days 50 and 60, and then declined again to the adult rate of about 10% at day 80. During regenerative growth following a two-thirds partial hepatectomy, the rate of binucleation declined to about 3%, causing the fraction of binuclear cells to fall from 27% (before hepactectomy) to 5% (at 45 h after hepactectomy) as pre-existing binuclear cells replicated and formed mononuclear daughter cells. Essentially all (97%) hepatocytes replicated at least once, starting their DNA synthesis at around 13 h and reaching a peak at 30 h, irrespective of ploidy and nuclearity. At later time points, the diploid hepatocytes had a higher labelling index than the polyploid cells, suggesting a greater tendency to go through several cell cycles.     

DNA ploidy and autophagic protein degradation as determinants of hepatocellular growth and survival

Hepatocytes have the ability to go through specialized cell cycles, which, during normal developmental liver growth, result in the formation of binuclear and polyploid cells. In the adult rat liver, the majority of the hepatocytes (about 70%) are tetraploid, 15-20% are octoploid, and only 10-15% are diploid (about 50% in humans). One-third of the hepatocytes in either rats or humans are binuclear (with two diploid or two tetraploid nuclei). Among cultured rat hepatocytes stimulated with growth factors (EGF and insulin), one-half of the mitoses are of the binucleating type (suggesting a "quantal" mechanism), causing one-third of the postmitotic cells to become binuclear. In contrast, regenerative liver growth, induced by partial hepatectomy, is predominantly nonbinucleating. During rat liver carcinogenesis, the early populations of phenotypically altered cells (foci) are predominantly diploid, as are the later neoplastic nodules and carcinomas, which can be shown to have a regeneration-like, largely nonbinucleating growth pattern. A negative correlation between growth capacity and ploidy can be demonstrated in cultured hepatocytes, regenerating livers, neoplastic nodules, and hepatocellular carcinomas, suggesting that suppression of binucleation and polyploidization may carry a growth advantage, in addition to helping to maintain a large population of diploid, potential stem cells. Since a diploid genome is less protected against mutagenic change than a polyploid genome, diploid tumor cells may, furthermore, be more prone than polyploid cells to undergo mutation-based progression toward increasing malignancy. The ability of liver tumor promoters like 2-acetylaminofluorene, cyproterone acetate, -hexachlorocyclohexane and methylclofenapate to induce nonbinucleating hepatocyte growth may, therefore, cooperate with the selective growth stimulation of cancer cells and cancer cell precursors to promote liver carcinogenesis.Autophagy, a mechanism for the bulk degradation of cytoplasm, contributes to intracellular protein turnover and serves to restrict cellular growth. Rat liver carcinogenesis is accompanied by a progressive reduction of autophagic capacity, preneoplastic livers having 50% and hepatocellular carcinoma cells only 20% as much autophagy as normal hepatocytes. The ascites hepatoma cell line AH-130 has virtually no autophagy during logarithmic growth, but some autophagy is turned on when the cells become growth-arrested at high cell density. Ascitic fluid from AH-130 cells is able to completely inhibit autophagy in normal hepatocytes, suggesting that the cancer cells may improve their growth ability through an autocrine, autophagy-suppressive mechanism. Hepatocytes from preneoplastic livers similarly maintain a low autophagic activity under restrictive culture conditions, thereby surviving much better than normal hepatocytes, which switch on their autophagy. In the presence of an autophagy inhibitor (3-methyladenine), normal and preneoplastic hepatocytes survive equally well, testifying to the importance of autophagy as a determinant of cell survival and growth.     

Involvement of insulin receptor substrates in epidermal growth factor induced activation of phosphatidylinositol 3-kinase in rat hepatocyte primary culture.

Short-term incubation of adult rat hepatocytes with epidermal growth factor (EGF) caused tyrosine phosphorylation of insulin receptor substrate (IRS)-1 and IRS-2 when the cells had been submitted to primary culture from 1-18 h. Tyrosine-phosphorylated IRS-1 and IRS-2 bound to the regulatory subunit (p85) of phosphatidylinositol (PtdIns) 3-kinase, thereby activating the enzymic activity. Tyrosine phosphorylation of the IRSs and activation of PtdIns 3-kinase in 3 h cultured hepatocytes both proceeded similarly to the same actions of insulin; the activation was rapid and transient, with peak values at 15-30 s and with similar EC(50)s in the nM range in both cases. A possible involvement of insulin receptors in these insulin-like actions of EGF was excluded by the following three lines of evidence. Insulin caused tyrosine phosphorylation of the insulin receptor beta-subunit but EGF did not. In contrast, the EGF receptor was phosphorylated by EGF, but the insulin receptor was not. The actions of EGF, but not those of insulin, were inhibited by AG1478, a selective inhibitor of EGF receptor tyrosine kinase. Cultured hepatocytes exposed to insulin or insulin-like growth factor-I (IGF-I) for a short period responded to the subsequent addition of EGF, whereas EGF-treated cells responded to insulin. The cells, however, displayed receptor desensitization under the same conditions, that is, no response was observed upon repeated addition of the same agonist, EGF, insulin or IGF-I. Thus, the EGF receptor-initiated signalling was mediated by PtdIns 3-kinase associated with tyrosine-phosphorylated IRSs in short-term cultured rat hepatocytes.     

Epidermal growth factor counteracts the glycogenic effect of insulin in parenchymal hepatocyte cultures.

Rat parenchymal hepatocytes in monolayer culture were used to study the metabolic effects of epidermal growth factor (EGF) and insulin on ketogenesis, gluconeogenesis and glycogen metabolism. EGF, unlike insulin, did not inhibit ketogenesis from palmitate or gluconeogenesis from pyruvate in hepatocyte cultures. It also had no effect on these pathways in the presence of insulin. In contrast, EGF potently counteracted the stimulation of [14C]pyruvate incorporation into glycogen by insulin, and also glycogen deposition from both gluconeogenic precursors and glucose. The EGF concentration causing half-maximal effect was about 0.1 nM. The anti-glycogenic effect of EGF was observed after both long-term (24 h) and short-term (1 h) exposure to EGF, and was more marked in the presence of insulin than in its absence. EGF did not displace bound insulin, suggesting that it neither competes for the insulin receptor nor affects the affinity of the receptor for insulin. EGF did not alter cellular cyclic AMP; and inhibition of cyclic AMP phosphodiesterase activity did not prevent the anti-glycogenic effect of EGF. In liver-derived dividing epithelial cells, Hep-G2 cells and fibroblasts, which have no capacity for gluconeogenesis, EGF did not counteract the stimulatory effect of insulin on [14C]glucose incorporation into glycogen, and in the epithelial cells EGF increased [14C]glucose incorporation into glycogen. The counter-effect of EGF on the glycogenic action of insulin in parenchymal hepatocytes may be due to a direct effect on glycogen metabolism or to an interaction with the post-receptor events in insulin action.     

The effect of EGF and the comitogen, norepinephrine, on the proliferative responses of fresh and cryopreserved rat and mouse hepatocytes

The effect of cryopreservation on the proliferative response of fresh and cryopreserved (CP) rat and mouse hepatocytes was studied. Of the parameters measured, incorporation of 3H-thymidine and bromodeoxyuridine (BdrU) incorporation were the most sensitive and LDH content was the least sensitive. The optimal seeding density for epidermal growth factor (EGF)-stimulated proliferative response in fresh rat and mouse hepatocytes was 1.8 x 10(4) cells/cm2 and 2.1 x 10(4) cells/cm2, respectively. 3H-thymidine incorporation by fresh rat and mouse hepatocytes was maximal in cultures treated with 10 and 5 ng/ml EGF, respectively. The cell attachment of fresh rat hepatocytes after 48 h was higher (68%) than CP (42%), therefore, the CP hepatocyte seeding density was increased to 7.1 x 10(4) cells/cm2 so that the cell number after 48 h was the same as fresh hepatocytes. Using the adjusted seeding density, the 3H-thymidine and BdrU incorporation into fresh and CP rat hepatocytes was equivalent. The attachment efficiencies of fresh and CP mouse hepatocytes were the same, therefore, no adjustment was needed. The proliferative response (3H-thymidine incorporation and DNA content) to EGF was the same in fresh and CP mouse hepatocytes. The comitogen, norepinephrine (NE), increased the proliferative response to EGF to the same extent in both fresh and CP rat hepatocytes. In summary, cryopreserved rat and mouse hepatocytes retain their ability to proliferate in culture. Adjustment and monitoring of the seeding density is of high importance, especially with rat hepatocytes, which lose some attachment capacity after cryopreservation. The secondary mitogenic effect of NE is also retained by cryopreserved rat hepatocytes, suggesting that these cells retain alpha1-receptor function.     

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.     

Importance of cell aggregation for expression of liver functions and regeneration demonstrated with primary cultured hepatocytes

Adult rat hepatocytes aggregated to form floating multicellular spheroids when cultured in Primaria dishes, which have a positively charged surface, in serum-free Williams' medium E (WE) supplemented with insulin and epidermal growth factor (EGF). These hormones were essential for maintenance of the spheroids, whereas the size of the spheroids depended on the inoculum cell density. The spheroids retained in vivo levels of expressions of albumin and glucokinase and synthesized scarcely any DNA even in the presence of insulin and EGF. On transfer to type I collagen-coated dishes, the spheroids gradually disaggregated and the cells formed monolayers, in which the expressions of albumin and glucokinase were suppressed and DNA synthesis and hexokinase activity were increased. DNA synthesis of hepatocytes in monolayer culture was maximal 24 hr after transfer of the spheroids, ～80% of the hepatocyte nuclei were labelled with bromodeoxyuridine during culture for 48 hr, and the mitotic index was ～70% after 60 hr. These results suggest that, in spheroids, hepatocytes remained in the G₀ phase, but that when they formed monolayers, they progressed to the G₁ phase and proceeded through the cell cycle in the presence of insulin and EGF. This work shows that the cell cycle of hepatocytes in culture can be manipulated by providing conditions for quiescence as spheroids or growth as monolayers and that the shape of hepatocytes is important for regulating their growth and liver-specific functions. © 1993 Wiley-Liss, Inc.     

Different proliferative potential of rat and pig hepatocytes in pure primary culture and coculture.

In the present study we have compared the growth potential of hepatocytes from rats and pigs and the influence of cocultivation between these hepatocytes and the rat liver epitheloid cell line RL-ET-14. Proliferation, i.e., DNA synthesis, was detected by autoradiography after exposure to [3H]thymidine. Rat hepatocytes cultured at low cell density showed a very low basal growth and responded to epidermal growth factor (EGF) and insulin by a considerable increase in DNA synthesis after 48 h leading to a labeling index (LI) of 33%. Cocultivation with RL-ET-14 cells almost completely blocked the basal as well as the growth factor stimulated proliferation of the rat hepatocytes. In contrast, pig hepatocytes cultured alone showed a much greater growth potential (basal: LI 11%; insulin/EGF:LI 67%) than rat hepatocytes and were further stimulated by cocultivation (basal: LI 39%; insulin/EGF: LI 89%). Density-dependent inhibition of cell growth was less pronounced with pig hepatocytes. Even after reaching confluency, they showed further strong proliferation in pure as well as in cocultures whereas the LI of the rapidly growing clone RL-ET-14 decreased to 40%. Use of conditioned medium from RL-ET-14 cells did not mimic the growth inhibition of rat hepatocytes in coculture indicating that no soluble growth inhibitors produced by the epitheloid cells are responsible for this effect. In particular, the differences between rat and pig hepatocytes in coculture are not simply due to production of TGF-beta by the epitheloid cells since the hepatocytes from both species were inhibited by TGF-beta to a similar extent.(ABSTRACT TRUNCATED AT 250 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.     

Different proliferative responses of periportal and perivenous hepatocytes to EGF.

Stimulation of DNA synthesis by EGF was compared in cultured periportal and perivenous hepatocyte populations. Periportal hepatocytes responded to EGF more sensitive (IC50-values 20 vs 75 ng/ml) and with a higher maximal stimulation (420 vs 290%) than perivenous hepatocytes with respect to both [3H]thymidine incorporation and labeling index. The glutamine synthetase-positive hepatocytes responded much less to EGF than did the perivenous cells in general. The simultaneous presence of insulin increased the sensitivity for EGF predominantly in the periportal hepatocytes. These inherent differences in the growth potential of hepatocytes from different acinar localizations may contribute to different growth patterns across the lobules in normal and regenerating liver.     

Hepatocyte growth factor is a potent mitogen for cultured rabbit renal tubular epithelial cells.

Hepatocyte growth factor (HGF), which is a potent growth factor of adult rat hepatocytes in primary culture, also strongly stimulated DNA synthesis of rabbit renal tubular epithelial cells in secondary culture. Its mitogenic activity was dose-dependent, being detectable at 3 ng/ml and maximal at 30 ng/ml. Over 20% of the cells were shifted to the S-phase by HGF alone, judging by the labeling index. HGF had additive effects with EGF, acidic fibroblast growth factor (a-FGF), and insulin. Transforming growth factor-beta 1 (TGF-beta 1) strongly inhibited DNA synthesis of renal tubular cells stimulated by HGF. The growth of renal tubular epithelial cells was also regulated by cell density: DNA synthesis stimulated by HGF was high at lower cell density and was strongly suppressed at high cell density. These results suggest that HGF may act as a renotropic factor in compensatory renal growth or renal regeneration in vivo.     

Expression of the human hepatocyte growth factor cDNA in primary cultures of rat hepatocytes

Hepatocyte growth factor (HGF) and epidermal growth factor (EGF) are primary mitogens for hepatocytes in culture. hepatocytes express the HGF-receptor MET but not HGF itself. To investigate the influence of autocrine HGF expression on the proliferative potential of hepatocytes, primary cultures were submitted to retrovirus-mediated transduction of the human hgf (huHGF) cDNA. Expression of the transduced cDNA revealed a minimum 2-fold increase in HGF-mRNA, whereas expression of the Escherichia coli beta-galactosidase gene remained even. Estimation of huHGF copy numbers showed there was a minimum 4-fold increase, suggesting an increase in the population of transduced cells. Immunoprecipitation of excreted huHGF and growth bioassays proofed that HGF was present and functional. HGF is excreted into the medium and therefore, by diffusion, available to transduced and non-transduced cells. The increase in huHGF-transduced cells suggests that the autocrine pathway as opposed to the paracrine pathway, which are both present at the same time, confers a growth advantage to these cells.     

Induction of mammotroph development by a combination of epidermal growth factor,insulin, and estradiol-17beta in rat pituitary tumor GH3 cells

Several reports have indicated that prolactin-secreting cells (PRL cells) are generated from growth hormone-secreting cells (GH cells). We have shown that treatment with a combination of epidermal growth factor (EGF), insulin, and estradiol-17beta (E (2)) induces the appearance of PRL cells in pituitary tumor GH3 cells. The aim of the present study was to clarify the involvement of mitosis in the cytogenesis of PRL cells in rat pituitary and GH3 cells. The effects of the treatment with EGF, insulin and E(2) on DNA-replication were studied by detecting the uptake of bromodeoxyuridine (BrdU) into the nucleus. In cultured rat pituitary cells, BrdU-labeled PRL cells were observed irrespective of the hormone treatment. In GH3 cells, BrdU-labeled GH cells and mammosomatotrophs (MS cells) were detected; BrdU-labeled PRL cells were not detected, however, when GH3 cells were treated with BrdU for 3 hr and then immediately examined for BrdU-labeling. BrdU-labeled PRL cells were found only when GH3 cells treated with BrdU were allowed to grow for another 3 days. This finding suggests that during the additional 3-day culture, BrdU-labeled PRL cells were generated from BrdU-labeled cells other than PRL cells. These results indicate that PRL cells are transdifferentiated from GH cells or MS cells in GH3 cells by a combined treatment with EGF, insulin and E(2), while PRL cells in rat pituitaries are able to proliferate in response to the hormone treatment. Thus, there may be two pathways for cytogenesis of PRL cells: the transdifferentiation of GH cells or MS cells, and a self-duplication of PRL cells.     

Hepatocytic Cells Form Bile Duct-like Structures within a Three-Dimensional Collagen Gel Matrix

It has been suggested that hepatocytes have the ability to form bile ductal structures during normal development and in various pathological conditions of the liver. In the present study, we attempted to establish anin vitromodel of ductal morphogenesis of hepatocytic cells by combining an aggregate culture and a type I collagen gel culture. When spheroidal aggregates of rat or mouse primary hepatocytes were embedded within the collagen gel matrix and then cultured with a medium containing a fibroblast-conditioned medium, the aggregates extended many dendritic processes composed of a trabecular arrangement of cells. Dendritic morphogenesis was also seen in embedded aggregates of immortal liver epithelial cell lines, which spontaneously emerged during long-term cultures of mouse primary hepatocytes. A similar morphogenesis was induced by the presence of insulin in the medium. Although epidermal growth factor (EGF) and hepatocyte growth factor (HGF) showed only a small effect on the morphogenesis of most of the hepatocytic cells when used alone, these factors, especially EGF, enhanced the morphogenetic effect of insulin. Electron microscopical observations revealed luminal structures lined by microvilli within these dendritic processes, indicating ductal differentiation. Immunocytochemically, the dendritic processes were positive for cytokeratin 19, a marker for bile duct cells. On the other hand, an H-ras-transformed mouse liver epithelial cell line and rat hepatocellular carcinoma cell lines did not demonstrate the organized morphogenesis. Our results indicate that hepatocytic cells can produce bile duct-like structures in the presence of the type I collagenous matrix and soluble morphogenetic factors.     

Regulation of albumin expression in fetal rat hepatocytes cultured under proliferative conditions: role of epidermal growth factor and hormones.

Sustained production of plasma proteins, notably albumin, is a reliable indicator of the differentiated state of hepatocytes. In this work, we have developed a fetal hepatocyte culture system where studying the regulation of albumin expression in proliferating liver cells. Our results show that under proliferative conditions (i.e., in the presence of EGF) fetal hepatocytes maintain albumin production above control quiescent non-treated cells. Glucagon and noradrenaline have no effect on the proliferation induced by EGF in cultured fetal hepatocytes; however, they act synergistically with the growth factor, increasing intracellular albumin levels. The maximum response is obtained by treatment of cells with EGF and noradrenaline. The stimulatory noradrenergic effect is mimicked by agents that increase cyclic AMP levels (forskolin plus IBMX). However, vasopressin or phorbol esters have no effect on albumin production, neither alone nor in combination with EGF. Dexamethasone, which does not alter the proliferative induction of EGF, increases albumin content. This effect is independent of the proliferative status of the cells and is not enhanced by glucagon, noradrenaline, or cyclic AMP increasing agents. The hormonal changes observed in albumin production partially correlate with changes in mRNA levels. This is the first time that cyclic AMP increasing agents are shown to act synergistically with EGF, increasing the expression of this liver specific gene.     

Transglutaminase differentially regulates growth signalling in rat perivenous and periportal hepatocytes

The influence of transglutaminase 2 (TG2) activity on the proliferative effect of epidermal growth factor (EGF) and on EGF receptor affinity in periportal hepatocytes (PPH) and perivenous hepatocytes (PVH) has been investigated using a primary culture system. PPH and PVH subpopulations have been isolated using the digitonin/collagenase perfusion technique. DNA synthesis was assessed by [3H] thymidine incorporation into hepatocytes. The assay for binding of [125I] EGF to cultured hepatocytes was analysed by Scatchard plot analysis. Pretreatment with the TG2 inhibitor monodansylcadaverine (MDC) greatly increased EGF-induced DNA synthesis in both PPH and PVH. Furthermore, [125I] EGF binding studies in PVH treated with MDC indicated that high-affinity EGF receptor expression was markedly up-regulated, whereas in PPH, there was no significant effect. Treatment with retinoic acid (RA), an inducer of TG2 expression, significantly decreased EGF-induced DNA synthesis in both PPH and PVH. Binding studies in the presence of RA revealed that the high-affinity EGF receptor was down-regulated and completely absent in both PPH and PVH. These results suggest that TG2 was involved in the differential growth capacities of PPH and PVH through down-regulation of high-affinity EGF receptors.     

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 regulation of glucose transporter 1 and 2 mRNA expression by epidermal growth factor and transforming growth factor-beta in rat hepatocytes.

We have examined by Northern blot analysis the expression of two members of the glucose transporter family of genes (GLUT-1 and GLUT-2) in regenerating liver and in hepatocytes cultured under various conditions. GLUT-1, although thought to be a growth-associated gene, is not expressed in normal or regenerating liver, whereas GLUT-2, a liver-specific gene, is abundant in normal liver and gradually up-regulated during liver regeneration. Conversely, in hepatocytes cultured conventionally on dried rat tail collagen (RTC) in the presence of EGF and insulin, which potentiate proliferation, GLUT-1 mRNA is rapidly and abundantly expressed, whereas GLUT-2 is depressed. To investigate the causes of this "switch" in glucose transporter expression seen when hepatocytes are removed from the liver and cultured under the conventional proliferative conditions, we examined the effects of specific growth factors and extracellular matrices on cultured hepatocytes. EGF, a potent liver mitogen, although causing a threefold induction of GLUT-1, was found to have no effect on GLUT-2 expression, suggesting that the increase in GLUT-2 seen in regenerating liver is not due to EGF. Inhibition of protein synthesis by cycloheximide in cultured hepatocytes does not prevent the induction of GLUT-1 mRNA. In addition, treatment of cells with cycloheximide appears to stabilize the GLUT-2 mRNA, preventing the usual down-regulation of this gene in cultured hepatocytes. The expression of the two glucose transporter mRNAs also differed when the hepatocytes were adherent to particular cell matrices. Culture of hepatocytes on a reconstituted basement membrane gel matrix (EHS) is known to restrain their growth and mediate high levels of differentiated hepatocytic functions that are lost under conventional culture conditions. Unlike cells on RTC, hepatocytes on EHS expressed low levels of GLUT-1 mRNA, and decreased GLUT-2 mRNA. TGF-beta, an attenuator of DNA synthesis, when added to cultures on RTC, substantially down-regulated GLUT-2 but had no effect on GLUT-1. We propose that the effectors, EGF, TGF-beta and basement membrane components, play a significant role in the regulation of expression of GLUT-1 and GLUT-2 in hepatocytes.