Beta-adrenergic control of phosphatidylcholine synthesis by transmethylation in hepatocytes from juvenile, adult and adrenalectomized rats.

Changes in isoprenaline-sensitive phospholipid methyltransferase were studied in hepatocytes isolated from juvenile, mature and adrenalectomized rats. Isoprenaline produced greater stimulation of cyclic AMP accumulation in juvenile and mature adrenalectomized rats than in mature animals. Similarly, isoprenaline stimulated phospholipid methyltransferase in juvenile and mature adrenalectomized rats but had no effect in mature animals. Isoprenaline-mediated activation of phospholipid methyltransferase in adrenalectomized rats was time- and dose-dependent. In hepatocytes isolated from adrenalectomized rats incubated with [Me-3H]methionine or [3H]-ethanolamine the addition of isoprenaline increased the amount of radioactivity incorporated into phosphatidylcholine. The activation by isoprenaline of phospholipid methyltransferase was abolished by the beta-blocker propranolol and by insulin. These results indicate that rat liver the occupation of functional beta-receptors causes a stimulation of phospholipid methylation. It is suggested that, as reported previously, cyclic AMP activates phospholipid methyltransferase.     

Inhibition by insulin of glucagon-dependent phospholipid methyltransferase phosphorylation in rat hepatocytes

Addition of insulin to isolated rat hepatocytes prelabeled with [32P]phosphate inhibited glucagon-dependent phospholipid methyltransferase phosphorylation and activation. Insulin alone had no effect on either the phosphorylation of the enzyme or on its activity. The effect of insulin on glucagon-dependent phospholipid methyltransferase phosphorylation was dose-dependent and occurred at physiological doses of the hormone (10(-11)-10(-10) M). Analysis of 32P-labeled peptides after digestion with trypsin revealed only one site of phosphorylation regulated by glucagon (10(-8) M) in isolated rat hepatocytes. This site, as analyzed by HPLC and thin-layer chromatography, coincided with that phosphorylated by the cAMP-dependent protein kinase using purified rat liver phospholipid methyltransferase.     

Activation of phospholipid methyltransferase by glucagon in rat hepatocytes

Glucagon produces a time- and dose-dependent activation of phospholipid methyltransferase activity in isolated rat hepatocytes. Half-maximal effect is caused by a dose of glucagon of 1 x 10(-10) M. This activation is due to an increase of the Vmax value of the enzyme, without affecting the Km value for S-adenosylmethionine. Exogenous cyclic AMP added to isolated rat hepatocytes mimics the effect of glucagon, and the activation of phospholipid methyltransferase by a nonsaturating concentration of glucagon is spontaneously reversible within 40 min of incubation.     

Vasopressin-stimulated phosphorylation of rat liver phospholipid methyltransferase in isolated hepatocytes

Addition of vasopressin (1 microM) to isolated rat hepatocytes prelabeled with [32P]phosphate was accompanied by a 250% increase in the phosphorylation of phospholipid methyltransferase. Vasopressin-stimulated phospholipid methyltransferase phosphorylation was time- and dose-dependent. 32P-labeled phospholipid methyltransferase was recovered by immunoprecipitation and SDS-polyacrylamide gel electrophoresis. After electrophoresis, phospholipid methyltransferase was electroeluted from the polyacrylamide gel and subjected to tryptic digestion or HCl hydrolysis. Analysis of 32P-labeled peptides reveals only one site of phosphorylation and the analysis of [32P]phosphoamino acids indicates that phosphoserine is the only labeled amino acid.     

Autocrine mechanism of growth of neonatal rat hepatocytes in primary culture

Hepatocytes from neonatal rats of 0 to 3 days old grew actively in primary culture without added serum or growth factors. In these culture conditions, growth of hepatocytes decreased progressively with increase in age of the rats from which they were isolated, and hepatocytes from rats of 2 weeks old showed scarcely any growth. Actively growing hepatocytes were found to secrete a growth factor that promoted their growth and that of Swiss 3T3 cells, but not that of adult hepatocytes. This growth factor in conditioned medium of growing hepatocytes was heat- and acid-stable, but sensitive to trypsin, and had a molecular weight of over 10,000. It did not inhibit the binding of [125I]epidermal growth factor to its receptor, and its growth promoting activity was not inhibited by monoclonal antibody against insulin-like growth factor II. Therefore, it seems to be a new growth factor. These results, together with previous findings (Nakamura, T., Nagao, M., & Ichihara, A. (1987) Exp. Cell Res. 169, 1-14) demonstrated a reciprocal relation between growth and maturation of neonatal hepatocytes during development, like that of adult cells, but indicated that unlike growth of the latter, growth of neonatal cells is induced by an autocrine mechanism.     

A tryptic peptide from beta-casein depresses protein synthesis and degradation and enhances ureogenesis in primary cultures of rat hepatocytes

1. A peptide which enhances ureogenesis in primary cultured hepatocytes of rats was isolated from a tryptic digest of bovine beta-casein. 2. The structure of the peptide was Ala-Val-Pro-Tyr-Pro-Gln-Arg which is located from 177th to 183rd residues from N-terminal of beta-casein. 3. The peptide also showed the activity to inhibit protein synthesis and protein degradation. 4. It also inhibited DNA synthesis of hepatocytes induced by insulin and/or epidermal growth factor.     

Cell density-dependent regulation of albumin synthesis and DNA synthesis in rat hepatocytes by hepatocyte growth factor.

Hepatocyte growth factor (HGF), a potent mitogen for mature hepatocytes, has been considered to act as a hepatotropic factor for liver regeneration. We examined the effect of HGF on albumin synthesis and DNA synthesis of adult rat hepatocytes cultured at various cell densities. HGF stimulated albumin synthesis of hepatocytes by 40-60% when they were cultured at higher cell densities such that there was tight cell-cell contact. But at lower cell densities HGF failed to stimulate albumin synthesis. In contrast, the stimulatory effect of HGF on DNA synthesis of hepatocytes was more potent at lower than at higher cell densities: HGF did not stimulate DNA synthesis of hepatocytes cultured at confluent cell density. Thus, HGF seems to stimulate both albumin synthesis and DNA synthesis of hepatocytes, in a reciprocal relationship depending on cell density. When the effects of various cytokines were examined, epidermal growth factor, transforming growth factor-alpha, and acidic fibroblast growth factor also stimulated albumin synthesis by 20-30%. However, transforming growth factor-beta 1, basic fibroblast growth factor, and interleukin-1 beta had no effect on albumin synthesis, while interleukin-6 inhibited it by 42%. Thus HGF was the most potent in stimulating albumin synthesis in these cytokines. Since HGF is markedly increased in the liver or plasma following various liver insults, HGF may be involved in liver regeneration through the potential to stimulate both cell growth and liver-specific functions such as albumin synthesis in a cell density-dependent manner.     

The polar head group of a novel insulin-sensitive glycophospholipid mimics insulin action on phospholipid methyltransferase

A phospholipid has been purified from rat liver membranes which copurified with an insulin-sensitive glycophospholipid isolated from H35 hepatoma cells. The polar head group of this phospholipid was generated by treatment with a phosphatidylinositol-specific phospholipase C from Staphylococcus aureus and purified through a C18 extraction column. Like insulin, the addition of this polar head group to isolated rat adipocytes inhibited the stimulatory effect of isoproterenol on phospholipid methyltransferase. The polar head group was also active on a subcellular fraction. The addition of the polar head group to microsomes isolated from isoproterenol-treated adipocytes produced a time-dependent inactivation of phospholipid methyltransferase, approaching basal activity. It is proposed that the effects of insulin on phospholipid methyltransferase may be mediated by this polar head group.     

Expression line approach to recombinant human epidermal growth factor into the yeast,Pichia pastoris from Huh-7 cell line

Beta-urogastrone also known as human epidermal growth factor is a key member of epidermal growth factor family having role in cell proliferation and differentiation in vivo as well as in vitro. Human epidermal growth factor gene has been isolated from different tissues but the method of isolation is technically difficult and complicated as it deals with biopsies. Here we isolated mature partial human epidermal growth factor gene from Huh-7 cell line, amplified and abridged toward mature coding region with three steps PCR, sequenced for homology with wild type human epidermal growth factor gene, inbuilt with sites of interest and cloned in Pichia pastoris for expression study. Isolated mature human epidermal growth factor gene from Huh-7 cell line showed 100 % sequence homology to wild type human epidermal growth factor gene and gives the native expression for human epidermal growth factor peptide. In this study we report that Huh-7 cell line is an easy source for the particular gene of human epidermal growth factor isolation and we are also suggesting P. pastoris is an expression system to produce recombinant human epidermal growth factor of the therapeutic importance resembling to the natural human system.     

Nerve growth factor induces rapid redistribution of F-actin in PC12 cells

Nerve growth factor (NGF) induces the redistribution of F-actin in rat pheochromocytoma PC12 cells within 2-10 min, whereas epidermal growth factor (EGF) has no effect on microfilament organization. This redistribution of F-actin in PC12 cells is not protein synthesis dependent, but can be blocked by methyltransferase inhibitors.     

Insulin-like effect of epidermal growth factor in isolated rat hepatocytes. Modulation of the alpha-1-adrenergic stimulation of ureagenesis

Insulin and epidermal growth factor (EGF) inhibit the stimulation of ureagenesis induced by adrenaline (alpha 1-adrenergic effect) in hepatocytes from control rats incubated in medium without calcium and in cells from hypothyroid rats. In hepatocytes from euthyroid rats incubated in normal buffer neither insulin or EGF diminished the alpha 1-adrenergic stimulation of ureagenesis. No effect of EGF or insulin on the alpha 1-adrenergic stimulation of phosphatidylinositol labeling was observed under any conditions. It is suggested that EGF mimics the action of insulin on one of the pathways of the alpha 1-adrenergic action: the calcium-independent, insulin-sensitive pathway which predominates in hepatocytes from hypothyroid rats.     

Epidermal growth factor (EGF) stimulation of ATP citrate lyase activity in isolated rat hepatocytes is age dependent.

1. The effect of epidermal growth factor (EGF) on ATP citrate lyase activity was determined in freshly isolated hepatocytes from rats of different ages as a function of incubation time, EGF concentration and hepatocyte density. 2. The activity of this enzyme was responsive to both dose and time of incubation of EGF with a two-fold increase in ATP citrate lyase activity and a half-maximal effect between 10(-12) and 10(-11) M. 3. EGF effects were detectable by 5 min. 4. The age of the rats had a strong effect on the magnitude of the EGF effect with ATP citrate lyase activity in younger (8 weeks) rats being more responsive than in older (14 weeks) rats.     

Effect of inhibitors of polyamine biosynthesis and of exogenous polyamines on the mitogenic effect of epidermal growth factor in a hepatocyte culture

It was established that difluoromethylornithine (DFMO) and methylglyoxal-bis(guanylhydrazone) blocked the mitogenic response of hepatocytes to epidermal growth factor. This effect of inhibitors, especially DFMO, has been prevented by exogenic putrescine, spermidine or spermine. But neither blocking action of inhibitors nor preventing effect of polyamines associated with changes of epidermal growth factor binding to hepatocytes.     

Different signal transduction by epidermal growth factor may be responsible for the difference in modulation of amino acid transport between fetal and adult hepatocytes

[1-¹⁴C]-2-aminoisobutyric acid (AIB) uptake and signal transduction pattern after epidermal growth factor (EGF) stimulation were examined in freshly isolated hepatocytes from 20-day-old fetuses and 3-month-old rats. EGF induced a transient increase of AIB transport after 10 min only in adult animals; the observed unresponsiveness of fetal liver is not dependent on a lack of EGF receptors which are present though to a lesser extent on the plasma membrane in this period. As far as the production of the second messengers, inositol trisphosphate (IP₃) and calcium, is concerned, substantial differences were found: EGF increased IP₃ production in adult hepatocytes, whereas it had no effect in fetal ones. Moreover, the addition of EGF induced a calcium transient in hepatocytes from adult animals, while there was no increase in fetal cells. The lack of EGF effect on amino acid transport in fetal cells could be due to its inability to produce both IP₃ and calcium transients, suggesting that this transduction pathway is not activated during fetal life.     

New primary culture systems to study the differentiation and proliferation of mouse fetal hepatoblasts

Hepatoblasts have the potential to differentiate into both hepatocytes and biliary epithelial cells through a differentiation program that has not been fully elucidated. With the aim to better define the mechanism of differentiation of hepatoblasts, we isolated hepatoblasts and established new culture systems. We isolated hepatoblasts from E12.5 fetal mouse liver by using E-cadherin. The E-cadherin+ cells expressed alpha-fetoprotein (AFP) and albumin (Alb) but not cytokeratin 19 (CK19). Transplantation of the E-cadherin+ cells into mice that had been subjected to liver injury or biliary epithelial injury led to differentiation of the cells into hepatocytes or biliary epithelial cells, respectively. In a low-cell-density culture system in the absence of additional growth factors, E-cadherin+ cells formed colonies of various sizes, largely comprising Alb-positive cells. Supplementation of the culture medium with hepatocyte growth factor and epidermal growth factor promoted proliferation of the cells. Thus the low-cell-density culture system should be useful to identify inductive factors that regulate the proliferation and differentiation of hepatoblasts. In a high-cell-density system in the presence of oncostatin M+dexamethasone, E14.5, but not E12.5, E-cadherin+ cells differentiated into mature hepatocytes, suggesting that unidentified factors are involved in hepatic maturation. Culture of E-cadherin+ cells derived from E12.5 or E14.5 liver under high-cell-density conditions should allow elucidation of the mechanism of hepatic differentiation in greater detail. These new culture systems should be of use to identify growth factors that induce hepatoblasts to proliferate or differentiate into hepatocytes and biliary epithelial cells.     

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)     

Expression of c-series gangliosides in rat hepatocytes and liver tissues

C-series gangliosides in rat hepatocytes and liver tissues were analyzed by thin-layer chromatographic (TLC) immunostaining with the specific monoclonal antibody A2B5. Primary cultures of hepatocytes isolated from adult rats were immunostained positively by A2B5. TLC immunostaining with A2B5 of gangliosides from the cells suggested that rat hepatocytes express c-series gangliosides including GT3, GT1c, GQ1c, and GP1c. Expression of c-series gangliosides in cultured hepatocytes was modulated by growth conditions of cells. The amount of GT3 was increased significantly by epidermal growth factor, while the contents of polysialo species such as GT1c, GQ1c, and GP1c were enhanced by higher cell density in culture. Examination of c-series gangliosides in rat liver tissues showed a unique developmental profile with a shift from GT3-dominant to polysialo species-dominant composition in late embryonic stages. These results suggest that the expression of c-series gangliosides in rat hepatocytes is regulated in a growth- and development-dependent manner.     

2,3,7,8-Tetrachlorodibenzo-p-dioxin inhibits DNA synthesis in rat primary hepatocytes

Treatment of Sprague-Dawley (SD) rats with a dosing regimen of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) maintaining a steady-state liver concentration of 150 ng/g results in enhanced hepatocyte proliferation in the periportal region, but reduced proliferation in the remainder of the hepatic lobule (Fox et al. (1993) Cancer Res., 53, 2265–2271). Here, we report an initial characterization of the actions of TCDD on hepatocyte proliferation by monitoring DNA synthesis in primary hepatocytes isolated from SD rats. TCDD caused a dose-dependent inhibition (EC₅₀ = 10 pM) of DNA synthesis in primary hepatocytes isolated from either male or female SD rats in the presence or absence of known hepatocyte mitogens (epidermal growth factor, hepatocyte growth factor, and transforming growth factor ). No change in DNA synthesis was observed at TCDD concentrations less than 1 pM. Initial characterization of the EGF response system in these cells revealed that TCDD did not alter the specific binding of EGF, or the levels of EGF receptor protein measured in intact cells or cell lysates. TCDD-dependent inhibition of DNA synthesis occurred independently of the suppression observed with transforming growth factor-β1. Estradiol did not alter DNA synthesis in the presence or absence of TCDD. Taken together, these findings indicate that TCDD suppresses DNA synthesis via a novel pathway that is non-responsive to estradiol, independent of TGF-β, and does not involve a decreased ability of hepatocytes to recognize (bind) EGF, a prototype mitogen.     

EGF-Induced receptor autophosphorylation in primary hepatocytes isolated from phenobarbitone-treated mice.

Phenobarbitone (PB) treatment of mice causes a decrease in the growth factor responsiveness of hepatocytes. Here, epidermal growth factor receptor (EGFR) expression and receptor autophosphorylation was determined in hepatocytes isolated from control and PB-treated mice. There was a decrease in the level of EGFR expression in hepatocytes isolated from mice following PB administration when compared to controls. EGF caused an approximate 20-fold increase of the 170 kD phosphotyrosine band in control hepatocytes, which was inhibited by the EGFR specific tyrosine kinase inhibitor 4, 5-dianilinopthalamide. Following PB treatment, the degree of basal receptor phosphorylation (in the absence of EGF) was significantly greater and therefore the fold rise in EGFR phosphorylation in isolated hepatocytes was lower than in controls. However, the overall extent of EGF-induced receptor phosphorylation was not diminished in hepatocytes isolated from PB-treated mice. Therefore the reduction in responsiveness to growth factors seen in hepatocytes ex vivo or the cessation of proliferation observed in vivo following PB administration is unlikely to be attributed to a decrease in ligand binding and subsequent receptor autophosphorylation.     

Correlation of membrane phosphorylation and epidermal growth factor binding to hepatic membranes isolated from triiodothyronine-treated rats

The in vivo adminstration of l-triiodothyronine to normal adult rats produced a reduction in the number of binding sites in hepatic membranes for epidermal growth factor; hyperthyroidism had no effect on insulin binding. The decreased receptor number correlated with a decrease in epidermal growth factor-stimulated phosphorylation of isolated hepatic membrane proteins (180 and 165 kDa) with adenosine [γ-³²P]triphosphate.