Kinetic constanis of asialoorosomucoid endocytosis comparison between hepatocytes from normal and streptozotocin-diabetic rats

The initial velocity of asialoorosomucoid internalization is determined for normal and diabetic rat hepatocytes. The analysis of results according to Woolf-Hofstee's method, showed no modification of the endocytosis constant. In contrast, the maximum velocity of asialoorosomucoid internalization is decreased by threefold in diabetic rat hepatocytes as compared to normal rat hepatocytes. No modification of internalization constant is observed between the two groups of rats. This suggests that the decrease of asialoorosomucoid total uptake, previously reported for diabetic rat hepatocytes is directly related to a decrease of total surface receptor number.     

Study of hepatic binding protein activity in jejuno-ileal by-passed rat hepatocytes

The kinetic constants of internalization of asialoorosomucoid were determined for normal and jejuno-ileal by-passed rat hepatocytes. In by-passed rats the maximum velocity of asialoorosomucoid internalization is decreased 3-fold, without any modification of apparant constant of internalization. Moreover, the rate constant of internalization was the same in the two groups of rats. These data suggest that the process of asialoorosomucoid internalization is not altered in by-passed hepatocytes and that the decrease of maximal velocity is only due to a decrease of total uptake receptor number.     

Insulin receptors on erythrocytes and hepatocytes from streptozotocin induced diabetic rats

Insulin receptors on hepatocytes and erythrocytes were studied in rats two and eight weeks after the injection of streptozotocin (50 mg/kg) to see if erythrocyte insulin receptors change parallel with hepatocyte insulin receptors in response to hypoinsulinemia. Insulin binding to hepatocytes increased two (14.0 +/- 2.5% v.s. 7.7 +/- 0.7%; P less than 0.025) and eight weeks (15.9 +/- 1.9% v.s. 6.6 +/- 1.1%; P less than 0.005) after the streptozotocin injection. Scatchard analysis revealed that this increase was due to a rise in both the receptor concentration and affinity. The number of receptors was comparable in the two- and eight-week-streptozotocin rats while the increase in the affinity was more pronounced in the latter group. Insulin binding to the erythrocytes was also increased in both two- (5.0 +/- 0.7% v.s. 4.2 +/- 0.6%) and eight-week- (4.3 +/- 0.6% v.s. 2.7 +/- 1.2%) streptozotocin rats. This increase was due to a rise in the receptor concentration rather than the affinity. However, compared to hepatocytes, these changes were inconsistent and statistically not significant. Furthermore, no correlation was obtained between the binding and plasma insulin concentration. These results indicate that insulin receptors on rat erythrocytes are less sensitive to a change in the plasma insulin concentration and do not always reflect accurately the receptor state on hepatocytes.     

Coated pits and asialoglycoprotein receptors redistribute to the substratum during hepatocyte adhesion to galactoside surfaces

Rat hepatocytes bind in a sugar-specific and concentration-dependent manner to flat polyacrylamide matrices containing covalently attached galactosyl (Gal) groups. Previous studies (Weigel, P.H., J. Cell Biol. 87, 855, 1980) concluded that binding was likely mediated by the asialoglycoprotein receptor. Here we confirm that adhesion is mediated by this receptor, since cell binding is inhibited by antireceptor antibody and a threshold binding response is also observed when hepatocytes adhere to surfaces coated with asialoorosomucoid, a ligand for this receptor. Cells that had bound to a Gal surface and were then sheared from the surface left a membrane patch behind on the substratum. The cytoplasmic side of these plasma membrane patches was visualized on the substratum by indirect immunofluorescence using antireceptor antibody or anticlathrin antibody. The density of punctate coated pits, visualized with the latter antibody, was enriched in a circular membrane region of about 4 microns 2 area that mediated cell binding. This zone also contained concentrated receptors, although the staining pattern with antireceptor antibody was more uniform and less punctate. The results show that both asialoglycoprotein receptors and coated pits are redistributed at the substratum interface on hepatocytes bound to Gal surfaces.     

Histogenesis of the endocrine pancreas in newborn rats after destruction by streptozotocin. An immunocytochemical study

Endocrine pancreatic tissue in newborn rats was studied 1 to 17 days after the destruction of B cells by an injection of streptozotocin. Regeneration of insulin cells was observed four days after streptozotocin injection, which was followed by recovery from the diabetic state and an increased pancreatic insulin content. Regeneration was characterised by new islets budding from small ducts. The pancreas of newborn rats, like the embryonic pancreas, thus retains a capacity to form endocrine tissue, although some degree of reduplication of preexisting B cells may also be involved in the process. Newborn rats injected with streptozotocin constitute an interesting model for the study of factors which may act on the regenerative potential of pancreatic endocrine tissue in the diabetic state.     

Inhibitory effect of sodium arsenite and azide on asialoglycoprotein receptor mediated endocytosis in suspended rat hepatocytes

The inhibitory effect of sodium arsenite and azide on asialoorosomucoid endocytosis was tested using isolated rat hepatocytes. Under either continuous flux conditions or a single synchronous wave of ligand endocytosis we confirm that azide inhibits the recycling of the receptors and we provide evidence for the involvement of thiol groups in the internalization step. In addition pretreatment of hepatocytes with azide allows us to demonstrate that receptor endocytosis proceeds independently of the presence of any specific ligand.     

Glycogen distribution in rat hepatocyte populations after a single exposure to 4-dimethylaminoazobenzine and subsequent injections of phenobarbital

7 day after a single interperitoneal injection of carcinogen 4-dimethylaminoazobenzen (DAB), a little number of cells with high glycogen contents was found in parallel with a decreased glycogen content in most isolated hepatocytes. 1.5 months after DAB injection, the normal distribution of glycogen content was seen restored in hepatocytes. The treatment of rats with phenobarbital (6 PhB injections 7 days after DAB application) blocked the restoration of the normal glycogen distribution. 2 months after the last PhB injection (3 months after DAB injection) an increased glycogen content was found in the smallest hepatocytes.     

Detection and isolation of a hepatic membrane receptor for ferritin

A ferritin receptor has been detected on isolated rat hepatocytes and has been partially purified from rat liver using affinity chromatography. Isolated hepatocytes exhibit approximately 30,000 ferritin binding sites/cell with a binding association constant (Ka) of 1 x 10(8) mol-1 liter. A binding assay has been developed which utilizes a hepatic ferritin receptor coupled to a microparticulate support to facilitate separation of bound and free ligand. This method yielded a Ka of 3 x 10(8) mol-1 liter for the purified hepatic ferritin receptor. Binding of ferritin to the insolubilized receptor was partially inhibited by human lactoferrin but unaffected by 200-fold molar excess of bovine albumin, rat transferrin, or human asialoorosomucoid.     

Evidence for targeted gene delivery to Hep G2 hepatoma cells in vitro

We have developed a system for targeting foreign DNA to hepatocytes in vitro using a soluble DNA carrier that takes advantage of receptor-mediated endocytosis to achieve internalization. The idea is based on the fact that hepatocytes possess a unique receptor that binds and internalizes galactose-terminal (asialo)glycoproteins. To create a targetable carrier system that could bind DNA in a nondeforming manner, we used poly(L-lysine) to bind DNA in a strong but noncovalent interaction. An asialoglycoprotein, asialoorosomucoid (AsOR), was chemically coupled to poly(L-lysine) to form an asialoorosomucoid-poly(L-lysine) conjugate. Various proportions of conjugate to DNA were tested to determine conditions that maximized DNA content in a soluble complex and that limited solubility of complexes. To test the targetable gene delivery system, AsOR-poly(L-lysine) conjugate was complexed to the plasmid pSV2 CAT containing the gene for chloramphenicol acetyltransferase (CAT) driven by an SV-40 promoter. We tested this complex using a model system consisting of human hepatoma cell line Hep G2 [asialoglycoprotein receptor (+)], hepatoma SK-Hep 1, IMR-90 fibroblasts, and uterine smooth muscle [receptor (-)] cells. Each cell line was incubated with 0.2 micron filtered AsOR-poly(L-lysine)-DNA complex or controls consisting of DNA plus AsOR, DNA plus poly(L-lysine), or DNA alone. Cells were assayed for the presence of CAT activity as a measure of gene transformation. SK-Hep 1, IMR-90, and smooth muscle [receptor (-)] cells produced no detectable acetylated chloramphenicol derivatives under any of these conditions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of streptozotocin diabetes on sialic acid content and glycoprotein binding of isolated hepatocytes

The possible correlation between sialic acid content of hepatocyte plasma membranes, and their binding capacity was investigated. For that purpose, [3H] asialotransferrin binding was tested with hepatocytes from both normal and streptozotocin treated rats. The observed decrease in binding capacity was parallel to a decrease in the sialic acid content Insulin therapy restored simultaneously normal blood glucose level, hepatic membrane sialic acid content and binding capacity.     

Targeted antagonism of galactosamine toxicity in normal rat hepatocytes in vitro

We present evidence that normal hepatocytes can be specifically protected from galactosamine toxicity in vitro by targeting an antagonist to these cells via receptor-mediated endocytosis. The strategy is based upon the following principles: 1) galactosamine is a highly selective hepatotoxin that causes a dose-dependent depletion of uridine intermediates; 2) galactosamine toxicity can be antagonized by supplemental administration of uridine; 3) normal hepatocytes possess unique cell-surface receptors that can internalize galactose terminal (asialo-)glycoproteins with subsequent degradation of the glycoprotein ligand. Based on these facts, we hypothesized that chemical coupling of a galactosamine antagonist to an asialoglycoprotein could result in cell-specific delivery and protection of normal hepatocytes by targeting the antagonist via asialoglycoprotein receptors. Using a model system consisting of freshly isolated rat hepatocytes (receptor (+)) and Morris 7777 rat hepatoma (receptor (-)) cells, sensitivity to galactosamine in vitro was determined and found to be similar for both types of cells. A targetable antagonist was synthesized by coupling uridine monophosphate to asialoorosomucoid in a molar ratio of 5 to 1. Exposure of Morris 7777 cells to the targetable antagonist in the presence of a toxic concentration of galactosamine did not protect these cells as evidenced by a steady decline in the number of viable cells in a fashion identical to cells treated with galactosamine alone. However, normal hepatocytes that received the conjugate in the presence of galactosamine were protected as their viable cell number remained the same as control (untreated) cells. Competition by an excess of asialoglycoprotein inhibited the protective effect of the conjugate, supporting the concept that the asialoglycoprotein component of the conjugate was responsible for the specific delivery of the antagonist to the target cells.     

Insulin binding and degradation in isolated hepatocytes from streptozotocin injected rats

Isolated hepatocytes from streptozotocin injected rats bound the same amount of [125I]monoiodoinsulin as hepatocytes from control rats. Scatchard analysis confirmed that insulin receptor number and affinity were the same for both groups. Relatively more cell-associated radioactivity was located intracellularly in hepatocytes from streptozotocin injected rats. Pretreatment with chloroquine resulted in a smaller increase in intracellular [125I]monoiodoinsulin in cells isolated from streptozotocin injected rats than for control cells. These results suggest that intracellular insulin processing occurs more slowly in hepatocytes isolated from streptozotocin injected rats than from control rats.     

Hepatic uptake of asialoglycoprotein is different among mammalian species due to different receptor distribution

Isolated hepatocytes of rat, rabbit and guinea pig were found to take up and degrade 125I-labelled asialoorosomucoid at different rates with the rank order: rabbit greater than rat greater than guinea pig. Measurement of 125I-asialoorosomucoid binding at 4 degrees C to these hepatocytes revealed that all these cells had two classes of receptors with a major difference occurring in the number of high-affinity binding sites. The average binding affinity constants (K) and receptor concentration (N) calculated from a least-square analysis of the Scatchard plots were K1 = 1.15.10(9) M-1, K2 = 0.93.10(7) M-1, N1 = 0.049 pmol/mg cell protein and N2 = 0.27 pmol/mg cell protein for the rat; K2 = 3.16.10(7) M-1, N1 = 0.027 pmol/mg cell protein and N2 = 0.13 pmol/mg cell protein for the guinea pig and K1 = 0.74.10(9) M-1, K2 = 3.85.10(7) M-1, N1 = 0.205 pmol/mg cell protein and N2 = 0.37 pmol/mg cell protein for the rabbit hepatocytes, respectively. Measurement of the total number of cellular receptors after solubilization with Triton X-100 also revealed the same receptor concentration rank order of rabbit (5.8 pmol/mg cell protein) greater than rat (0.55 pmol/mg cell protein) greater than guinea pig (0.18 pmol/mg cell protein). Intravenous injection of 125I-asialoorosomucoid into anesthetized animals of matched body weight also indicated that the rate of plasma clearance and the rate of appearance of the degraded product of the tracer were different among these species with the same rank order as that observed with isolated hepatocytes. Thus there is a fundamental difference in the number of asialoglycoprotein receptors both on the cell surface and inside hepatocytes of these mammalian species.     

Association of plutonium with lysosomal, lipofuscin-like granules in Chinese hamster hepatocytes: evidence from electron microscopic and biochemical studies with 241Pu and 239Pu

This electron-microscopic-autoradiographic study was undertaken to identify the cell organelles, which bind plutonium in Chinese hamster hepatocytes at different times after injection. Female Chinese hamsters were injected intraperitoneally with 241Pu and sacrificed at time intervals of between 4 days and 35 weeks. The Chinese hamster was chosen as the experimental animal as it is a species in which there is virtually no elimination of plutonium from the liver. From the 4th day onwards beta-tracks were found over globular electron-dense structures, which were randomly distributed in the cytoplasm of the hepatocytes and strongly resembled lipofuscin bodies. Comparison of the results with those from biochemical experiments showed good agreement between the morphological and biochemical observations. At early times after injection 241Pu was also found in the hepatocyte nuclei. All the evidence suggests that in this species plutonium in hepatocytes becomes bound to lipofuscin-accumulating lysosomes, which cannot be excreted.     

Endocytosis of bovine lactoperoxidase by two carbohydrate-specific receptors in rat liver

A perfused rat liver took up bovine lactoperoxidase (LPO) by a Ca2+-dependent, saturable process. This endocytosis was accomplished by both hepatocytes and Kupffer or other nonparenchymal cells (NPCs). The mediating receptors were the Gal/GalNAc lectin of hepatocytes and the Man/GlcNAc lectin of NPCs. Blocking either one of these receptors caused a large shift in distribution of accumulated LPO into the cell type whose receptor was left unblocked, but the extent of uptake was unaffected and the rate was only moderately reduced. Effective inhibition of overall uptake into the perfused organ required the presence of competitors for both receptors. Conversely, LPO was an effective competitor of other ligands (asialoorosomucoid or mannan) for either of the two receptors. The major clearance capacity for LPO was associated with hepatocytes which in suspension took it up by a process completely inhibitable by asialofetuin (ASF) and at a rate more than three times greater than for ASF. A faster cycling time for Gal/GalNAc receptors when bound to LPO is suggested. The glycoprotein selectively lost its affinity for Man/GlcNAc receptors when digested by endoglycosidase H (endo H), suggesting that LPO contains mannose-rich oligosaccharides.     

Morphometric and cytochemical characteristics of the hepatocytes isolated from the liver of rats after a single exposure to 4,dimethylaminoazobenzene

Hepatocytes have been characterized that were isolated with sodium perchlorate from the livers of rats both intact and given a single injection of a carcinogen--4-dimethylaminoazobenzene. A decreased number of big hepatocytes (600-900 mkm2) and the appearance of small hepatocytes (75-120 mkm2) were observed 7 days after the carcinogen injection. An excessive accumulation of glycogen was shown in certain hepatocytes. By the 30th day the picture was nearly normal.     

Kinetic evidence of a surface membraneous step during the endocytosic process of 3H-labelled asialoorosomucoid and its alteration in diabetic mellitus rats

The apparent internalization rate constant of asialoorosomucoid in normal and diabetic hepatocytes was determined using different experimental processes, either following a synchronous wave of prebound ligand or a continuous flux ligand endocytosis, either alone or simultaneously. In continuous flux conditions, no difference between normal and diabetic hepatocytes appeared (k = 0.15 +/- 0.04 and 0.11 +/- 0.02 min-1, respectively). In contrast, in the one-turn endocytosis of prebound ligand, k was lower for diabetic hepatocytes than for normal ones whether it was measured alone (0.20 +/- 0.03 and 0.59 +/- 0.09 min-1, respectively) or simultaneously with a continuous flux of unlabelled ligand (0.25 +/- 0.03 and 0.70 +/- 0.08 min-1, respectively). These differences are attributed to an impediment or a delay in the preclustering of receptors in coated pits at the cell surface of diabetic cells.     

In vivo analysis of the role of O-glycosylations of von Willebrand factor

The objective of this project was to study the function of O-glycosylations in von Willebrand factor (VWF) life cycle. In total, 14 different murine Vwf cDNAs mutated on one or several O-glycosylations sites were generated: 9 individual mutants, 2 doublets, 2 clusters and 1 mutant with all 9 murine glycosylation sites mutated (Del-O-Gly). We expressed each mutated cDNA in VWF deficient-mice by hydrodynamic injection. An immunosorbent assay with Peanut Agglutinin (PNA) was used to verify the O-glycosylation status. Wild-type (WT) VWF expressed by hepatocytes after hydrodynamic injection was able to bind PNA with slightly higher affinity than endothelial-derived VWF. In contrast, the Del-O-Gly VWF mutant did not bind PNA, demonstrating removal of O-linked glycans. All mutants displayed a normal multimeric pattern. Two mutants, Del-O-Gly and T1255A/T1256A, led to expression levels 50% lower than those induced by WT VWF and their half-life in vivo was significantly reduced. When testing the capacity of each mutant to correct the bleeding time of VWF-deficient mice, we found that S1486A, T1255A, T1256A and the doublet T1255A/T1256A were unable to do so. In conclusion we have shown that O-glycosylations are dispensable for normal VWF multimerization and biosynthesis. It also appears that some O-glycosylation sites, particularly the T1255 and T1256 residues, are involved in the maintenance of VWF plasma levels and are essential for normal haemostasis. As for the S1486 residue, it seems to be important for platelet binding as demonstrated in vitro using perfusion experiments.     

Acute suppression of albumin synthesis in systemic inflammatory disease: an individually graded response of rat hepatocytes.

The effects of an inflammatory insult on albumin of the rat liver were investigated at the cellular level and were correlated with serum albumin concentration. After SC injection of turpentine, the livers were perfused and fixed in vivo; serial liver sections were stained using a streptavidin-ABC-immunoperoxidase technique with an antibody to rat albumin. Albumin and total protein were measured at intervals after turpentine injection in whole livers and in serum. Fibrinogen was determined in plasma only. Twenty-four hours after turpentine injection serum albumin had dropped by 25% and was at 50% of its initial value at Day 3. Serum fibrinogen increased 2.4-fold within 24 hr and decreased thereafter. Liver homogenates showed no significant changes in albumin concentration. Immunohistochemically, all hepatocytes stained positive for albumin in normal animals. During inflammation, the immunostainable albumin content vanished entirely in a majority of all hepatocytes while remaining unchanged in other cells, thus producing a strikingly patchy staining pattern. No signs of resumption of albumin accumulation in depleted hepatocytes were seen after 8 days, despite a clear trend towards normalization of serum albumin concentration. These results suggest that individual hepatocytes differ widely in their response to agents that suppress albumin synthesis in an acute-phase reaction.     

Effect of monensin on receptor recycling during continuous endocytosis of asialoorosomucoid

The binding of asialoglycoproteins to their liver cell receptor results in internalization of the ligand-receptor complex. These complexes rapidly appear in intracellular compartments termed endosomes whose acidification results in ligand-receptor dissociation. Ligand and receptor subsequently segregate: ligand is transported to lysosomes and is degraded while receptor recycles to the cell surface. The proton ionophore monensin prevents acidification of endosomes and reversibly inhibits this acid-dependent dissociation of ligand from receptor. The present study determined the effect of monensin treatment of short-term cultured rat hepatocytes on cell-surface-receptor content, determined both by their binding activity and immunologically, following continuous endocytosis of asialoorosomucoid. Inclusion of 5 microM monensin in the incubation medium reduced the number of immunologically detectable cell-surface receptors by 20% in the absence of ligand. During continuous endocytosis of asialoorosomucoid, inclusion of monensin resulted in a 30-40% reduction of cell-surface receptor detectable either by ligand binding or immunologically. These results suggest that the reduced liver-cell-surface content of receptor in monensin is due to intracellular trapping of ligand-receptor complexes. The reduction of surface receptor during monensin incubation in the absence of ligand suggests that "constitutive recycling" of plasma membrane components also requires intracellular acidification.