Growth inhibition and induction of specific hepatic phenotype expression by retinoic acid in HEPG2 cells.

Retinoic acid, the active metabolite of vitamin A, plays a role in the growth and differentiation of a variety of normal and malignant cells. In response to 5 microM retinoic acid the human hepatoma-derived cell line HepG2 underwent significant growth inhibition (not associated with cell death), which reached a level of 80% in comparison with controls, after 12 days of continuous treatment. Retinoic acid also induced morphological changes in these cells, in particular the development of canalicular-like structures, indicating progression to a more differentiated phenotype. In addition, a reduced expression of alpha-fetoprotein was found. We suggest that our results may be important for the design of novel therapeutic approaches using RA for the treatment of liver tumors.

Independent modulation of galactose-specific receptor expression in rat liver cells.

The expression of galactose-specific receptors on liver cells from rats at the end of pregnancy and from estrogen-treated animals was studied. The number and distribution of binding sites were estimated on hepatocytes and Kupffer and endothelial cells in vitro as well as in situ by means of protein-gold complexes. Hepatocytes and endothelial cells from pregnant rats showed an increased binding activity of at least three times for hepatocytes and one and a half times for endothelial cells with respect to normal rat livers. The increase in the hepatocyte receptor expression was paralleled by an increase in the level of its specific messenger RNA (mRNA). On Kupffer cells, a decreased number of binding sites, at least three times less than control values, was measured. The correlation between the altered hormonal level during pregnancy and the expression of galactose binding sites was examined in hepatocytes and Kupffer cells isolated from virgin rats treated with the synthetic estrogen diethylstilbesterol. In estrogen-treated rats both the binding sites and the specific mRNA of hepatocytes increased as compared with vehicle-treated or untreated animals. In contrast, in Kupffer cells both the estrogen treatment as well as vehicle-only injection led to a significant reduction in the expression of binding sites as compared with virgin untreated animals. To establish whether the decrease of galactose binding sites in Kupffer cells was related to the activation of macrophages or to the removal of plasma membrane caused by enhanced nonspecific phagocytosis, in situ binding experiments were performed after lipopolysaccharide (LPS)-stimulation or latex-bead phagocytosis. Nonspecific phagocytosis does not affect the binding activity, which instead appears strongly reduced after LPS injection. These findings suggest an independent response of galactose-specific receptor expression systems in the different types of liver cells to modulating agents.

Receptor-mediated endocytosis of galactose and mannose exposing ligands: an electron microscopic study on adult and neonatal cultured rat hepatocytes.

We compared the receptor-mediated endocytosis for galactose and mannose exposing ligands in primary cultures of hepatocytes from newborn and adult rats. The endocytic pathway was revealed ultrastructurally using colloidal gold particles coupled to lactosylated bovine serum albumin and invertase. The binding activity on the cell surfaces is observed by keeping the cells at 4 degrees C. For both ligands used, the binding capacity for hepatocytes from adult rats was greater than for neonatal cultured cells. Increasing the temperature to 37 degrees C, we observed that the protein-gold complexes entered the intracellular endocytic organelles. Within 5-15 min, the marker was confined in vesicles close to the cell surface and in the endosome, while after 60 min, the marker is found in lysosome-like compartments. We found that the process of endocytosis is similar for galactose and mannose exposing ligands. The organelles involved in the process of endocytosis in newborn cultured hepatocytes are not different in shape from those of cultured cells of adult rats, but the process of internalization is slower.

Binding and uptake of ligands for mannose-specific receptors in liver cells: an electron microscopic study during development and aging in rat.

The binding and uptake of mannose exposing ligands in rat liver cells during development and aging was studied. The mannose-specific receptors are visualized using 5-nm diameter colloidal gold particles coated with invertase or mannan. It was found that the binding sites are present on sinusoidal liver cells since prenatal life but their quantitative and qualitative cell surface expression changes with age. The number of receptors affects the endocytotic capacity of Kupffer cells which is low during perinatal and aging periods and reaches the values of adult animals between the 11th and the 15th day after birth. Our results indicate that the expression and the activity of mannose-specific receptors on sinusoidal rat liver cells is related to the differentiative stage of the organ.

Age-related changes of glycosylation pattern in isolated rat hepatocytes.

The glycosylation pattern in isolated rat hepatocytes during pre- and post-natal development and senescence has been studied by following: the [14C]glucosamine and [3H]galactose incorporation into cellular glycoproteins and glycolipids and the activity of two microsomal enzymes, N-acetyl-glucosaminyl-1-P transferase and galactosyl transferase. The data show a lowered precursor incorporation into lipids and proteins in the fetus, newborn and old rats versus the adult. Only the galactosyl transferase activity is enhanced on the 19th and 22nd day of fetal life. The glucosamine and N-acetyl-glucosamine content in both soluble and protein bound fractions was increased, while the galactose content in lipids and proteins decreased in the fetal stage. The different sugar composition of the proteins, and the decreased glucosamine and galactose incorporation into the proteins, observed in the fetus, newborn and old rat, suggest a post-translational modification which may cause alterations in functions usually mediated by glycoproteins.

Insulin binding and internalization in rat hepatocytes during prenatal and postnatal life.

Insulin binding to isolated rat hepatocytes was studied during prenatal and postnatal life. Results show that in hepatocytes isolated from prenatal, postnatal and adult rat there is a constant increase in the number of insulin binding sites per cell, whereas the affinity of plasma membrane receptors for the hormonal ligand remains unaltered from prenatal to adult hepatocytes. Autoradiographic studies indicate a greater internalization of hormone during prenatal life and, taking into account the increase of cell size, suggest an unchanged surface density of receptor sites before and after birth.