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.     

Effect of rat liver gangliosides on the interaction of liposomes with rat hepatocytes in vitro

The influence of rat liver GM1, GM2, GD1 and GT1 gangliosides on the interaction of liposomes with rat hepatocytes was investigated. It was shown that liposomes coated with GM1 and GT1 are effectively bound and captured by hepatocytes. Preincubation of hepatocytes with N-acetylglucosamine and D-galactose reduced the binding of GM1- and GT1-liposomes by those cells. The data obtained suggest that there are binding sites for some gangliosides on the surface of rat hepatocytes.     

Genetic polymorphism of rat liver gangliosides

Liver ganglioside patterns of eight rat strains were classified according to two phenotypes: SHR type, characterized by predominance of b-series gangliosides (GD1b, GT1b, GQ1b), and DA type, characterized by predominance of a-series gangliosides (GM1, GD1a). Comparison of ganglioside pattern expressed in the liver of F1 hybrids and backcross F2 hybrids indicated that SHR type is controlled by a single autosomal-dominant gene which probably determines the expression of sialytransferase 2 activity for synthesis of GD3 from GM3.     

Subcellular distribution and biosynthesis of rat liver gangliosides

Gangliosides have generally been assumed to be localized primarily in the plasma membrane. Analysis of gangliosides from isolated subcellular membrane fractions of rat liver indicated that 76% of the total ganglioside sialic acid was present in the plasma membrane. Mitochondria and endoplasmic reticulum fractions, while containing only low levels of gangliosides on a protein basis, each contained approx. 10% of total ganglioside sialic acid. Gangliosides also were present in the Golgi apparatus and nuclear membrane fractions, and soluble gangliosides were in the supernatant. Individual gangliosides were non-homogeneously distributed and each membrane fraction was characterized by a unique ganglioside composition. Plasma membrane contained only 14 and 28% of the total GD1a and GD3, respectively, but 80-90% of the GM1, GD1b, GT1b and GQ1b. Endoplasmic reticulum, when corrected for plasma membrane contamination, contained only trace amounts of GM1, GD1b, GT1b and GQ1b, but 11 and 5% of the total GD1a and GD3, respectively. The ganglioside composition of highly purified endoplasmic reticulum was similar. Ganglioside biosynthetic enzymes were concentrated in the Golgi apparatus. However, low levels of these enzymes were present in the highly purified endoplasmic reticulum fractions. Pulse-chase experiments with [3H]galactose revealed that total gangliosides were labeled first in the Golgi apparatus, mitochondria and supernatant within 10 min. Labeled gangliosides were next observed at 30 min in the endoplasmic reticulum, plasma membrane and nuclear membrane fractions. Analysis of the individual gangliosides also revealed that GM3, GM1, GD1a and GD1b were labeled first in the Golgi apparatus at 10 min. These studies indicate that gangliosides synthesized in the Golgi apparatus may be transported not only to the plasma membrane, but to the endoplasmic reticulum and to other internal endomembranes as well.     

Long-term transgene expression from genetically modified hepatocytes grafted to the rat liver.

Primary cultures of rat hepatocytes embedded and grown to high density in a wafer of reticulated polyurethane have been infected in vitro with a retroviral vector expressing the hepatitis B virus surface antigen (HBsAg). Wafers containing infected cells and implanted directly between the liver lobes of recipient rats became vascularized and firmly attached to the liver parenchyma. Stable levels of HBsAg were detected in the blood circulation for at least 4 months after grafting. Hepatocytes recovered from the recipient rats up to 4 months after implantation were recultured under selective conditions and found to produce high levels of HBsAg.     

Biosynthesis and excretion of gangliosides by the isolated perfused rat liver.

De novo synthesis and excretion into perfusate and bile fluid of hepatic gangliosides were studied in isolated perfused rat livers. Addition of N-acetyl-[6-3H(n)]D-mannosamine to the perfusate resulted in radioactive synthesis of at least eight gangliosides labeled in their sialic acid residues. About 10% of total de novo synthesized gangliosides were excreted into the perfusate, less than 1% into the bile fluid. Labeled gangliosides were tentatively identified by cochromatography with known standards. All of them are known to occur in rat liver and sera. The results indicate that most, if not all, normal serum gangliosides are synthesized in the liver; excretion with bile fluid is negligible. They explain previous observations, and indicate clinical implications, which are discussed.     

Biosynthesis of gangliosides in primary cultures of rat hepatocytes. Determination of the net synthesis of individual gangliosides by incorporation of labeled N-acetylmannosamine

The ganglioside content of rat hepatocytes increases several-fold during the first 6 days in monolayer culture. To correlate increased levels with rates of de novo synthesis, the incorporation of N-acetyl-[6-3H]D-mannosamine into individual gangliosides was determined. The calculation of synthetic rates was made possible by the simultaneous measurement of the specific radioactivity of the immediate sialic-acid donor, CMP-Neu5Ac. The CMP-Neu5Ac content of hepatocytes was found by HPLC analysis to be 30.5 nmol/g of plated cells. The specific radioactivity of this precursor pool reached a constant plateau 5 h after addition of the labeled N-acetyl-mannosamine and remained constant for at least 70 h. The incorporation into individual gangliosides was measured in primary cultures of rat hepatocytes between 72 and 144 h after seeding. During this period, the increase in ganglioside levels was greatest. The highest rates of incorporation were seen in GD1a followed by GM3, GM1, GD3 and the polysialylated compounds. The following rates of synthesis (nmol per 60 h and mg of protein) were calculated: GD1a 0.68, GM3 0.59, GM1 0.36, GD3 0.13 and GT1 0.02. These values are compared with the net increase of the gangliosides as measured by the resorcinol reaction.     

Subcellular biosynthesis and transport of gangliosides formed from exogenous lactosylceramide in rat liver.

In order to clarify the mechanisms of ganglioside biosynthesis and transport we intravenously administered a liposomal dispersion of radiolabelled lactosylceramide (LacCer) to rats and then followed the time course of the individual gangliosides which became radioactive in the Golgi-apparatus and plasma-membrane fractions prepared from the liver. After administration of radiolabelled LacCer the liver retained a substantial amount of radioactivity, which was distributed among an organic phase (mainly residual LacCer), a fraction containing low-Mr substances (mainly 3H2O) and a ganglioside fraction. The hepatocytes were found to provide the bulk of gangliosides biosynthesized from exogenous LacCer. After subcellular fractionation, the total radioactive gangliosides increased in the Golgi apparatus up to 8 h, to then decrease and practically disappear at 24 h; in the plasma membranes they were progressively concentrated, accounting for high absolute values. Ganglioside patterns were greatly modified with time in both the Golgi apparatus and plasma membrane, but without significant differences between them. Biosynthesis in the Golgi apparatus and accumulation in the plasma membrane of each individual ganglioside followed a precursor-product relationship. The obtained results indicated that once a ganglioside is biosynthesized in the Golgi apparatus, it is in part made available for translocation to the plasma membrane, which rapidly occurs, and is in part retained in the Golgi apparatus, where it acts as a precursor for the biosynthesis of more glycosylated gangliosides.     

Alterations in gangliosides of rat liver cells in hyperlipidemic state

The possibility that liver cell membrane is modified in hyperlipidemic state was studied using nephrotic hyperlipidemic rats. Liver cells of normal and nephrotic rats were isolated and subjected to labeling of cell surface components using lactoperoxidase catalyzed radioiodination. The labeling of total surface lipids of hepatocytes of nephrotic rats was about five times higher than that of normal ones and was particularly higher in glycosphingolipids. Cultivation of the isolated hepatocytes as primary cultures reduced drastically labeling of surface lipids in liver cells of both nephrotic and normal rats and abolished the differences observed in liver cells of the two types. Determination of cell associated gangliosides, showed that the level in nephrotic rat hepatocytes was only 35% higher than that of normal rats. Yet, in both types of liver cells 24 h cultivation decreased markedly the ganglioside content. However, similar to the effect observed in hyperlipidemic rats, supplementing the culture medium with very low density lipoproteins (VLDL) increased considerably the ganglioside level of cultured hepatocytes. These treatments did not affect the activity of enzymes involved in the synthesis of gangliosides. It is suggested that ganglioside content in liver cell membrane is modulated in the hyperlipidemic state.     

Distribution of gangliosides in parenchymal and non-parenchymal cells of rat liver

Parenchymal and non-parenchymal cells were isolated from rat liver with purities of more than 90%. Total and ganglioside sialic acid contents were higher in non-parenchymal cells than in parenchymal cells. Thin-layer chromatography of gangliosides showed that the main component in rat liver was ganglioside GM3 and that this was abundant in non-parenchymal cells. Parenchymal cells had ganglioside GD1b as the main component and less GM3 than non-parenchymal cells. These results suggested that the main ganglioside of rat liver, GM3, arises mainly from non-parenchymal cells.     

Biosynthesis of gangliosides from asialogangliosides in rat liver Golgi vesicles

Biosynthesis of glycolipids GA2, GA1, GM1b, and GD1c was studied in Golgi vesicles isolated from rat liver. Sequential addition of N-acetylgalactosamine, galactose and two sialic acid residues to lactosylceramide led to the endproduct GD1c. Activities of the corresponding glycosyltransferases were shown to be present in isolated Golgi vesicles and their respective kinetic data were determined. The products of each reaction were characterized by their mobility on thin-layer chromatography, by enzymic degradation to their respective precursors, and in case of GM1b by FAB mass spectrometry.     

Fibronectin binding to gangliosides and rat liver plasma membranes

Binding of fibronectins to gangliosides was tested directly using several different in vitro models. Using an enzyme-linked immunoabsorbent assay (ELISA), gangliosides were immobilized on polystyrene tubes and relative binding of fibronectin was estimated by alkaline phosphatase activity of conjugated second antibody. Above a critical ganglioside concentration, the gangliosides bound the fibronectin (GT1b congruent to GD1b congruent to GD1a greater than GM1 much greater than GM2 congruent to GD3 congruent to GM3) in approximately the same order of efficiency as they competed for the cellular sites of fibronectin binding in cell attachment assays (Kleinman et al., Proc natl acad sci US 76 (1979) 3367). Alternatively, these same gangliosides bound to immobilized fibronectin. Rat erythrocytes coated with gangliosides GM1, GD1a or GT1b bound more fibronectin than erythrocytes not supplemented with gangliosides. Using fibronectin in which lysine residues were radioiodinated, an apparent Kd for binding to mixed rat liver gangliosides of 7.8 X 10(-9) M was determined. This value compared favorably with the apparent Kd for attachment of fibronectin to isolated plasma membranes from rat liver of 3.7 X 10(-9) M for fibronectin modified on the tyrosine residue, or 6.4 X 10(-9) M for fibronectin modified on lysine residues. As shown previously by Grinnell & Minter (Biochem biophys acta 550 (1979) 92), fibronectin modified on tyrosine residues did not promote spreading and attachment of CHO cells. It did, however, bind to cells. In contrast, lysine-modified fibronectin both bound to cells and promoted cell attachment. Plasma membranes isolated from hepatic tumors in which the higher gangliosides that bind fibronectin were depleted bound 43-75% less [125I]fibronectin than did plasma membranes from control livers. The findings were consistent with binding of fibronectins to gangliosides, including the same gangliosides depleted from cell surfaces during tumorigenesis in the rat.     

Concurrent changes in sinusoidal expression of laminin and affinity of hepatocytes to laminin during rat liver regeneration.

Distribution of fibronectin, laminin, and collagens type I, III, IV, and V in the lobular regions of regenerating rat liver was studied by indirect immunofluorescence. Little or no laminin was detected in sham-operated controls throughout the experimental period, while it was detected in sinusoids of regenerating liver as early as 6 h after partial hepatectomy (PH). After reaching a maximum at 24 h, it decreased and was barely detectable 6 days after PH. Changes in the other extracellular matrix (ECM) proteins were evident 3 days after PH, but not earlier than 24 h. Hepatocytes isolated from regenerating rat livers were tested in a short term assay for attachment to the substrates coated with the ECM proteins. The attachment of hepatocytes to laminin substrates increased 12 h after PH, reached a maximum at 24 h, and decreased to the control level 6 days after PH, while that of the control remained constant. The attachment to fibronectin substrates was not different between regenerating livers and controls at any time point. The attachment to collagen did not change earlier than 24 h after PH, but increased slightly 3 days after PH. Primary rat hepatocytes cultured on the substrates coated with the ECM proteins were determined for replicative DNA synthesis in response to epidermal growth factor. Both in normal liver and in regenerating liver 24 h after PH, laminin was one of the most effective substrates in supporting the responsiveness of hepatocytes to the growth stimulus. Taken together, these results suggest the importance of hepatocyte-laminin interaction during the early stage of liver regeneration possibly in growth stimulation of hepatocytes and/or maintenance of hepatocyte-specific functions.     

Acute-phase-response induction in rat hepatocytes co-cultured with rat liver epithelial cells.

The response of rat hepatocytes co-cultured with rat liver epithelial cells to conditioned medium (CM) from lipopolysaccharide (LPS)-activated monocytes was investigated by measuring the concentration of alpha 2-macroglobulin (alpha 2M), alpha 1-acid glycoprotein (AGP), albumin and transferrin, as well as the changes in glycosylation of alpha 1-acid glycoprotein. During an initial 8-day treatment with CM, concentrations of alpha 2M and AGP increased markedly over those of control culture, whereas concentrations of albumin and transferrin decreased. The glycosylation pattern of AGP indicated an important relative increase of the concanavalin A-strongly-reactive (SR) variant upon treatment. When CM addition to hepatocyte culture medium was stopped, the concentrations of the four proteins and the glycosylation pattern of AGP reverted to those of control cultures. Further addition (on day 15) to cultures of CM increased the concentration of alpha 2M and decreased albumin and transferrin concentrations. Although AGP concentrations did not increase above those of controls, the appearance of the SR variant was again stimulated by CM. These results show that, in co-culture, rat hepatocytes remain able to respond to repeated inflammatory stimuli.     

Cell surface expression by adult rat hepatocytes of a non-collagen glycoprotein present in rat liver biomatrix

A rabbit antiserum raised against ACI rat liver biomatrix was used to identify components common to biomatrix and plasma membranes of adult hepatocytes. Biomatrix was isolated from intact rat livers by reverse perfusion via the inferior vena cava with sodium deoxycholate, nucleases and lipid extracting solvents. Immunoprecipitation analysis of detergent extracts of hepatocytes surface-labeled with 125I indicated that antibodies, purified from anti- biomatrix antiserum by adsorption and desorption from intact hepatocytes, showed reactivity with a single MW 105 kD component, designated Hep 105. Indirect immunofluorescence analysis showed that Hep 105 was expressed in some regions of the perisinusoidal space and in all three domains of the hepatocyte plasma membrane and was present on some but not all of the fibrous elements in frozen sections of biomatrix . The presence of Hep 105 on biomatrix was confirmed by immunoprecipitation analysis which showed that Hep 105 was present in components solubilized from biomatrix by sequential treatment with 0.5 M acetic acid, 0.05% collagenase and 4 M urea. Further characterization using immunoprecipitation analysis in combination with immobilized lectins and two-dimensional polyacrylamide gel electrophoresis (PAGE) indicated that Hep 105 was a non-collagen glycoprotein which showed charge heterogeneity and existed on the cell surface as a disulfide-linked heterodimer of apparent MW 125 kD. Two hybridomas, constructed by fusing P3 X 63Ag8 myeloma cells with spleen cells from mice immunized with intact hepatocytes, were shown by immunodepletion and two-dimensional gel electrophoretic analysis to be secreting monoclonal antibodies (Mab) against Hep 105. Examination of frozen sections of rat liver stained by indirect immunofluorescence showed that reactivity of both Mabs was concentrated in the bile canalicular domain of the hepatocyte plasma membrane, suggesting that the reactive epitopes were not accessible in the sinusoidal and intercellular membrane domains. Taken together, these results suggest that Hep 105 may play a role in the interactions between hepatocytes and extracellular matrix.     

Proto-oncogene expression in regenerating liver is simulated in cultures of primary adult rat hepatocytes

Proto-oncogene fos mRNA levels are rapidly and transiently elevated 12-fold in regenerating liver 10-60 min following partial hepatectomy. This response, and the induction of fos protein synthesis, has been simulated qualitatively and quantitatively in long term primary cultures of quiescent adult rat hepatocytes where proliferative transitions can be initiated directly in serum-free medium by known hepatocyte mitogens like epidermal growth factor. Expression of a second proto-oncogene, c-rasH, in proliferatively activated hepatocyte cultures between 6 and 24 h also simulates the delayed hepatic response that occurs in vivo following partial hepatectomy. These results suggest that sequential proto-oncogene expression during liver regeneration is caused directly by hepatocellular interactions with specific mitogens. In addition, a role for monovalent cations in the regulation of hepatocyte gene expression is implicated from findings that Na+ deprivation inhibits induction of fos expression in cultured hepatocytes by epidermal growth factor under chemically defined conditions.     

The biosynthesis of gangliosides. Labelling of rat brain gangliosides in vivo

1. After injection of [6-(3)H]glucosamine into 8-day-old rats it was found that all the major brain gangliosides and their sialyl groups were labelled at essentially the same rate, except the hematoside, which was the least labelled. In 18-day-old rats it was found that the two major gangliosides with the sialyl (2-->8)-sialyl linkage, and their sialyl groups were more labelled than the hematoside, the Tay-Sachs ganglioside, the other two major gangliosides and their respective sialyl groups. 2. No difference was found in any of the cases studied between the specific radioactivities of the neuraminidase-resistant and -labile sialyl groups belonging to the same ganglioside. The same was found for the specific radioactivities of the galactosyl groups proximal and distal to the ceramide moiety of total brain gangliosides from rats injected with [U-(14)C]glucose. From this it was concluded that partial turnover of the ganglioside molecule does not occur. 3. A model for the synthesis of gangliosides is presented that accounts for results from previous experiments in vitro and the lack of precursor-product relationships observed in experiments in vivo.     

Recycling of glucosylceramide and sphingosine for the biosynthesis of gangliosides and sphingomyelin in rat liver.

It was previously shown that sphingomyelin and gangliosides can be biosynthesized starting from sphingosine or sphingosine-containing fragments which originated in the course of GM1 ganglioside catabolism. In the present paper we investigated which fragments were specifically re-used for sphingomyelin and ganglioside biosynthesis in rat liver. At 30 h after intravenous injection of GM1 labelled at the level of the fatty acid ([stearoyl-14C]GM1) or of the sphingosine ([Sph-3H]) moiety, it was observed that radioactive sphingomyelin was formed almost exclusively after the sphingosine-labelled-GM1 administration. This permitted the recognition of sphingosine as the metabolite re-used for sphingomyelin biosynthesis. Conversely, gangliosides more complex than GM1 were similarly radiolabelled after the two treatments, thus ruling out sphingosine re-utilization for ganglioside biosynthesis. For the identification of the lipid fragment re-used for ganglioside biosynthesis, we administered to rats neutral glycosphingolipids (galactosylceramide, glucosylceramide and lactosylceramide) each radiolabelled in the sphingosine moiety or in the terminal sugar residue. Thereafter we compared the formation of radiolabelled gangliosides in the liver with respect to the species administered and the label location. After galactosylceramide was injected, no radiolabelled gangliosides were formed. After the administration of differently labelled glucosylceramide, radiolabelled gangliosides were formed, regardless of the position of the label. After lactosylceramide administration, the ganglioside fraction became more radioactive when the long-chain-base-labelled precursors were used. These results suggest that glucosylceramide, derived from glycosphingolipid and ganglioside catabolism, is recycled for ganglioside biosynthesis.