Alterations in fucosyl oligosaccharides of glycoproteins during rat liver regeneration.

[3H]Fucose-labelled glycopeptides in the slices of liver 24h after partial hepatectomy were fractionated on Sephadex G-50. Glycopeptides from regenerating liver contained a higher proportion of lower-Mr components than did controls. Regenerating liver contained a higher proportion of glycopeptides that were bound to concanavalin A-Sepharose and were subsequently eluted with 20mM-methyl alpha-D-glucopyranoside than did controls. Concanavalin A-bound glycopeptides from each source were entirely bound to a lentil lectin-Sepharose column. Both the concanavalin A-bound and -unbound fractions from regenerating liver were indistinguishable from the respective controls by Bio-Gel P6 column chromatography and neuraminidase digestion. These results show that fucosyl glycopeptides from regenerating liver contain a higher proportion of biantennary species with core fucose residues than do controls. Glycopeptides from regenerating livers 12h, 72h and 144h after partial hepatectomy were also examined; however, the difference was not significant. These observations suggest that the alterations in fucosyl glycopeptides may be related to rapid growth of hepatocytes 24h after partial hepatectomy. No significant difference was found in either [3H]mannose- or [3H]fucose-labelled glycoproteins from regenerating liver and from controls by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, suggesting that the alteration in glycopeptides should depend on some differences in the late stage of oligosaccharide processing.     

Cell-density-dependent changes in cell-surface glycopeptides and in adhesion of cultured intestinal epithelial cells

We studied mannose-containing glycopeptides and glycoproteins of subconfluent and confluent intestinal epithelial cells in culture. Cells were labelled with d-[2-³H]mannose for 24h and treated with Pronase or trypsin to release cell-surface components. The cell-surface and cell-residue fractions were then exhaustively digested with Pronase and the resulting glycopeptides were fractionated on Bio-Gel P-6, before and after treatment with endo-β-N-acetylglucosaminidase H to distinguish between high-mannose and complex oligosaccharides. The cell-surface glycopeptides were enriched in complex oligosaccharides as compared with residue glycopeptides, which contained predominantly high-mannose oligosaccharides. Cell-surface glycopeptides of confluent cells contained a much higher proportion of complex oligosaccharides than did glycopeptides from subconfluent cells. The ability of the cells to bind [³H]concanavalin A decreased linearly with increasing cell density up to 5 days in culture and then remained constant. When growth of the cells was completely inhibited by either retinoic acid or cortisol, no significant difference was observed in the ratio of complex to high-mannose oligosaccharides in the cell-surface glycopeptides of subconfluent cells. Only minor differences were found in total mannose-labelled glycoproteins between subconfluent and confluent cells by two-dimensional gel analysis. The adhesion of the cells to the substratum was measured at different stages of growth and cell density. Subconfluent cells displayed a relatively weak adhesion, which markedly increased with increased cell density up to 6 days in culture. It is suggested that alterations in the structure of the carbohydrates of the cell-surface glycoproteins are dependent on cell density rather than on cell growth. These changes in the glycopeptides are correlated with the changes in adhesion of the cells to the substratum.     

Postnatal changes in N-linked oligosaccharides of glycoproteins in rat liver.

[3H]Mannose-labelled glycopeptides in the slices of livers from neonatal and 1-, 2-, 3- and 5-week-old rats were characterized by column chromatographies on Sephadex G-50 and concanavalin A-Sepharose and by endo-beta-N-acetylglucosaminidase H digestion. The proportion of complex-type glycopeptides was increased with time until 2 weeks post partum and then returned to the neonatal level. This was mainly due to the increased proportion of concanavalin A-bound (biantennary) species. These changes were accompanied by consistent changes in the activities of processing enzymes in liver microsomal fraction, especially of N-acetylglucosaminyltransferase I. Complex-type glycopeptides from neonatal and 2- and 5-week-old rat livers were further characterized by column chromatographies on Bio-Gel P-6 and DE 52 DEAE-cellulose in combination with neuraminidase digestion. No significant difference was found between concanavalin A-bound species from neonatal liver and those from liver 5 weeks post partum, most of which were sialylated. Concanavalin A-bound species 2 weeks post partum were comparatively smaller in size and less sialylated. On the other hand, there was no significant difference among concanavalin A-unbound species from the three different sources, most of which were sialylated. Since glycoproteins from regenerating rat liver also contain a higher proportion of complex-type oligosaccharides, as previously reported, such changes in N-linked oligosaccharides of glycoproteins may be related to control of the growth of liver cells.     

Characterization of glycopeptides labelled from D-[2-3H]mannose and L-[6-3H]fucose in intestinal epithelial cell membranes during differentiation.

The labelled glycopeptides obtained by Pronase digestion of rat intestinal epithelial cell membranes were examined by gel filtration after injection of D-[2-3H]mannose and L-[6-3H]fucose. Three labelled fraction were eluted in the following order from Bio-Gel P-6, Fraction I, which was excluded from the gel, was labelled mostly with [3H]fucose and slightly with [3H]mannose. Fraction II contained "complex" asparagine-linked oligosaccharides since it was labelled with [3H]mannose and [3H]fucose, was stable to mild alkali treatment, and resistant to endo-beta-N-acetyl-glucosaminidase H. Fraction III contained "high-mannose" asparagine-linked oligosaccharides, which were labelled with [3H]mannose, but not with [3H]fucose; these were sensitive to endo-beta-N-acetylglucosaminidase H, and were adsorbed on concanavalin A-Sepharose and subsequently eluted with methyl alpha-D-mannopyranoside. The time course of incorporation of [3H]mannose into these glycopeptides in microsomal fractions showed that high-mannose oligosaccharides were precursors of complex oligosaccharides. The rate of this processing was faster in rapidly dividing crypt cells than in differentiated villus cells. The ratio of radioactively labelled complex oligosaccharides to high-mannose oligosaccharides, 3h after [3H]mannose injection, was greater in crypt than in villus-cell lateral membranes. Luminal membranes of both crypt and villus cells were greatly enriched in labelled complex oligosaccharides compared with the labelling in lateral-basal membranes. These studies show that intestinal epithelial cells are polarized with respect to the structure of the asparagine-linked oligosaccharides on their membrane glycoproteins. During differentiation of these cells quantitative differences in labelled membrane glycopeptides, But no major qualitative change, were observed.     

Sialyltransferase activity in regenerating rat liver

Liver microsomal fractions catalyse the transfer of sialic acid from CMP-N-acetyl-neuraminic acid to various exogenous acceptors such as desialylated fetuin, desialylated human Tamm–Horsfall glycoprotein and desialylated bovine submaxillary-gland mucin. An increase in the rate of incorporation of sialic acid into desialylated glycoproteins was found after a lag period (7h) in regenerating liver. The increase was maximum 24h after partial hepatectomy for all acceptors tested. At later times after operation the sialyltransferase activity remained high only for desialylated fetuin. No soluble factors from liver or serum of partially hepatectomized animals influenced the activity of the sialyltransferases bound to the microsomal fraction. The sensitivity of sialyltransferases to activation by Triton X-100, added to the incubation medium, was unchanged in the microsomal preparation from animals 24h after sham operation or partial hepatectomy. The full activity of sialyltransferases towards the various desialylated acceptors showed some differences. Human Tamm–Horsfall glycoprotein was a good acceptor of sialic acid only when desialylated by mild acid hydrolysis. After this treatment, but not after enzymic hydrolysis, a decrease in molecular weight of human Tamm–Horsfall glycoprotein was observed. Further, the sialyltransferase activity as a function of incubation temperature gave different curves according to the acceptor used. The relationship between the biosynthesis of glycoproteins by regenerating liver and the sialyltransferase activity of microsomal fraction after partial hepatectomy is discussed.     

Effect of autonomic denervation on DNA synthesis during liver regeneration after partial hepatectomy

The regulatory role of autonomic nerves in liver regeneration after partial hepatectomy was studied in rats by bilateral subdiaphragmatic vagotomy or splanchnicectomy. 1. In control rats the wet weight of the regenerating liver was restored to approximately 80% of the preoperative weight 72 h after partial hepatectomy. Restoration of the liver weight was significantly impaired in vagotomy rats, but not in splanchnicectomy. Increases in the DNA and protein contents of the regenerating liver were also suppressed by vagotomy. 2. Hepatic DNA synthesis, measured as the incorporation of [methyl-3H]thymidine into DNA at various times after partial hepatectomy, was significantly less in vagotomized rats, and slightly more in splanchnicectomized rats than in control rats. The onset of DNA synthesis triggered by partial hepatectomy was also delayed by vagotomy. 3. The increases in activities of hepatic aspartate transcarbamoylase and thymidine kinase, the key enzymes in synthesis of pyrimidine nucleotides via the de novo and salvage pathways respectively, during liver regeneration, were significantly suppressed and retarded in vagotomized rats. Conversely, splanchnicectomy tended to stimulate these enzyme inductions after partial hepatectomy. 4. During starvation the plasma insulin level decreased after partial hapatectomy in control and vagotomized rats, as in sham-operated rats, but showed a transient increase 6 h after partial hepatectomy in splanchnicectomized rats. It is concluded that vagotomy inhibits and delays DNA synthesis and proliferation of liver cells after partial hepatectomy, whereas splanchnicectomy tends to stimulate these processes. The data also suggest that parasympathetic innervation of the liver may play an important regulatory role in liver regeneration.     

Alteration of rat liver proteoglycans during regeneration.

Sepharose CL-6B column chromatography of crude extracts from the slices of regenerating rat livers after partial hepatectomy and sham-operated controls labeled with [35S]sulfuric acid revealed an enhancement of [35S]sulfate incorporation into proteoglycan fractions during regeneration. The 35S-labeled proteoglycans contained heparan sulfate (more than 80% of the total) and chondroitin/dermatan sulfate. The 35S-incorporation into both glycosaminoglycans increased to maxima 3-5 days after partial hepatectomy and decreased thereafter toward the respective control levels. When [35S]sulfuric acid was replaced by [3H]glucosamine, similar results were obtained. These results suggest that the maximal stimulation of proteoglycan synthesis in regenerating rat liver follows the maximal mitosis of hepatic cells 1-2 days after partial hepatectomy. The 35S-labeled proteoglycans from regenerating liver 3 days after partial hepatectomy and control were analyzed further. They were similar in chromatographic behavior on a gel filtration or an anion-exchange column and in glycosaminoglycan composition. Their glycosaminoglycans were indistinguishable in electrophoretic mobility. However, these proteoglycans were slightly but significantly different in their affinity to octyl-Sepharose and in the molecular-weight distribution of their glycosaminoglycans.     

Glucosamine metabolism in regenerating rat liver.

1. Glycoprotein synthesis was investigated with [1-14C]glucosamine in vivo. [14C]Glucosamine was administered intravenously 24h after hepatectomy to rats. 2. Incorporation into the acid-soluble fraction was maximum at 15 min after injection both in sham-operated and hepatectomized rats. 3. Enhancement of incorporation into UDP-N-acetylhexosamine in regenerating liver was observed. However, its specific activity was lower, because of a greater enhancement of synthesis de novo of the amino sugar. 4. In the liver acid-insoluble fraction, maximum incorporation of [14C]glucosamine was at 30 min in sham-operated rats and 2 h in hepatectomized rats respectively. 5. In sham-operated rats, incorporation into the plasma acid-insoluble fraction followed that of the liver acid-insoluble fraction, but hepatectomy resulted in a rapid enchancement of incorporation into plasma. 6. It is concluded that synthesis of liver glycoproteins is stimulated after partial hepatectomy and that glycoproteins synthesized are released rapidly into the plasma.     

Increased glycosylation capacity in regenerating rat liver is paralleled by decreased activities of CMP-N-acetylneuraminate hydrolase and UDP-galactose pyrophosphatase.

In regenerating rat liver the activities of CMP-N-acetylneuraminate hydrolase and UDP-galactose pyrophosphatase were decreased to 40-50% of control values within 35 h after partial hepatectomy. In the same time period the activities of sialyltransferase and galactosyltransferase were increased, and the initial sharp decrease in the carbohydrate content of liver and serum glycoproteins was largely restored. The antiparallel nature of these events is suggestive of an involvement of nucleotide-sugar-hydrolysis enzymes in rat liver glycoprotein biosynthesis.     

Both acidic-type and neutral-type asparaginyl-oligosaccharides of host-cell glycoproteins are altered in Rous-sarcoma-virus-transformed chick-embryo fibroblasts.

In comparisons of [3H]mannose-labelled glycopeptides from chick-embryo fibroblasts infected and transformed with non-defective Prague C Rous-sarcoma virus and from untransformed fibroblasts infected with a transformation-defective derivative of Prague C Rous-sarcoma virus, we have detected transformation-dependent alterations in both the acidic-type and the neutral-type asparagine-linked oligosaccharides of cellular glycoproteins. Pronase-digested glycopeptides were analysed by the combined techniques of gel filtration, exo- and endo-glycosidase digestion and concanavalin A-agarose affinity chromatography. The transformed cell glycoproteins contained more sialic acid and were enriched for more highly branched (versus biantennary) acidic-type structures compared with the untransformed cell glycoproteins, similarly to previously reported transformation-dependent alterations. In addition, the glycopeptides from the virus-transformed cells contained several neutral-type structures that were apparently absent from the untransformed cells: small neutral-type oligosaccharides (Man3GlcNAc2) that were sensitive to endo-beta-N-acetylglucosaminidase D but resistant to endo-beta-N-acetylglucosaminidase H, and oligosaccharides with the property of 'truncated' precursor oligosaccharides (endoglycosidase-resistant, alpha-mannosidase-sensitive). Endoglycosidase-released oligosaccharides with the properties of hybrid-type structures were derived from the glycoproteins of both transformed and untransformed cells.     

Regulation of DNA primase activity by phosphorylation of histone-H1 in regenerating rat liver

The biological importance of histone H1 was investigated in relation to the cell cycle using liver regeneration in rat. Histone H1 was extracted from the regenerating rat liver at various intervals after partial hepatectomy and the number of phosphate residues was measured. The inhibitory effect of the extracted histone H1 on DNA primase was assayed. The activities of DNA polymerase-alpha, DNA primase and DNA synthesis were also determined in the regenerating rat liver. It was found that: 1) phosphate residue in histone H1 from normal rat liver was between 2-3 mol/mol of histone H1. 2) The number of phosphate residues did not change for the first 16h after partial hepatectomy. 3) A dramatic sudden increase of phosphate residues was detected at 18h after partial hepatectomy. 4) The high levels of phosphate residues remained constant thereafter up to 50h. 5) DNA primase activity was less inhibited by highly phosphorylated than by slightly phosphorylated histone H1. It seems probable that phosphorylation of histone H1 is needed for the releasing of DNA primase activity from its inhibited state, which would start DNA synthesis together with DNA polymerase-alpha.     

Uptake of amino acids and nucleic acid precursors by regenerating rat liver

1. At 0.5-1.0h after partial hepatectomy the intracellular acid-soluble fraction of rat liver took up twice as much radioactivity from [(3)H]orotic acid, [(3)H]uridine and [(3)H]thymidine as did similar fractions from sham-operated animals. This increase in penetration was not prevented by adrenalectomy or actinomycin, both of which decreased precursor uptake into nuclear RNA at this time. The increase in entry was still shown by thymidine at 22h after operation, at the height of the S period. Adenine penetration was not increased 1h after partial hepatectomy. 2. Plasma concentrations of ornithine and possibly methionine, tyrosine and lysine were raised 1.5h after partial hepatectomy. [(3)H]Lysine entry into regenerating liver at this time was increased by 60%; [(3)H]valine uptake was unaffected. Intracellular amounts of tyrosine, phenylalanine and ornithine in the liver were also increased. 3. The relation of these events to the start of liver regeneration is discussed.     

Cytophotometric study of nuclear proteins during gametogenesis in Ascaris lumbricoides.

Reduction in the number of nucleoli/nucleus and increase in their size were usually observed in rat liver after partial hepatectomy. These changes of nucleoli were greatest 16–18 h after the operation, when RNA biosynthesis in the nucleoli is reported to be highest. Approx. 50% of the nuclei had one enlarged nucleolus at this time but after the increase in nuclear DNA synthesis less than 15% of the nuclei had one nucleolus, as in normal liver. Before the next peak of nuclear DNA synthesis, nucleolar changes appeared again, though less conspicuously.The enlarged nucleoli of regenerating liver were separated from smaller ones by discontinuous sucrose gradient centrifugation and the contents of nucleic acid and ribosomal cistrons in different-sized nucleoli were measured. The large nucleoli in regenerating liver were found to have increased DNA content, whereas smaller ones had the normal content. The total number of ribosomal cistrons in the enlarged nucleoli from regenerating liver was also increased roughly in proportion to the DNA content. No significant difference was found between the percentages of ribosomal cistrons in whole nuclear DNAs from regenerating and normal liver. Small but reproducible [³H]TdR incorporation into nucleolar DNA was observed and this was similar in normal liver and regenerating liver 12 h after partial hepatectomy. Therefore, the nucleolar changes in regenerating liver were not accompanied by any particular DNA synthesis in the nucleolus itself. These results suggest that in the nuclei of regenerating liver nucleolar chromatins may be redistributed and assembled into large nucleoli, rather than that any amplification of ribosomal cistrons occurs.     

Decreased transfer of oligosaccharide from oligosaccharide-lipid to protein acceptors in regenerating rat liver

The transfer of [14C]glucose from UDP-[14C]glucose to lipid intermediates and glycoproteins was decreased in regenerating rat liver microsomes 24 h after partial hepatectomy. In regenerating liver microsomes, the concentration of free dolichyl phosphate (Dol-P) was significantly decreased. However, it was only about 10% of total Dol-P, which was not significantly changed. On the addition of exogenous Dol-P, the transfer of [14C]glucose to glycoproteins was still decreased, while the decrease of the transfer to lipid intermediates was no longer observed. These results suggest that the glycoprotein synthesis is not regulated by the amount of Dol-P in regenerating liver microsomes. Oligosaccharide obtained from [14C]glucosyl-oligosaccharide-lipid was not distinguishable between regenerating liver and control by paper chromatography. The oligosaccharide transfer to protein in microsomes was compared by using [14C]glucosyl-oligosaccharide-lipid as oligosaccharide donor. The transfer of oligosaccharide to endogenous proteins decreased to 77% of control in regenerating liver and the transfer to exogenously added denatured alpha-lactalbumin decreased to 59% of control. Therefore, it is unlikely that the acceptor capacity of endogenous protein is decreased in regenerating liver. Neither the change in oligosaccharide-lipid under the condition for oligosaccharide transfer assay nor the stability of oligosaccharide transferase was different between regenerating liver and control. These results strongly suggest that the decrease in the activity of the oligosaccharide transferase in microsomes causes the decrease of glycoprotein synthesis in regenerating liver, which was shown in our previous studies.     

UDP-N-acetylglucosamine-glycoproteinN-acetylglucosaminyltransferase in regenerating rat liver

The assay condition for N-acetylglucosaminyltransferase activities in rat liver microsomal fraction was developed. The enzyme activities towards endogenous acceptors within 48 h after partial hepatectomy were lower than in controls, exceeding the control level by 96 h, and then higher than in controls up to 240 h after the operation. The changes in N-acetylglucosaminyltransferase activities towards exogenous acceptor (UPD-2-acetamido-2-deoxy-D-glucose: glycoprotein 2-acetamido-2-deoxy-D-glucosyltransferase, EC 2.4.1.51) were consistent with those in the enzyme activities towards endogenous acceptors at 144 h, but not at 48 h, after the operation. The contents of protein and the levels of protein-bound hexosamine in the liver microsomes were decreased at early period of liver regeneration.These results suggest that the acceptor capacity of liver microsomal proteins is diminished during first 48 h of the regeneration. This may be responsible for the decreased transfer of the amino sugar to nascent glycoproteins. However, the enzyme activity was enhanced at 144 h and the level of endogenous acceptors may increase.     

Synthesis in vitro of glycoprotein in regenerating rat liver

The metabolism of glucosamine in regenerating rat liver was studied in liver slices. [1-¹⁴C]Glucosamine was incorporated into acid-soluble fraction, rapidly converted to UDP-N-acetylhexosamine and transferred to acid-insoluble fraction. Electrophoretic analysis revealed that most of the radioactive macromolecules released from the slices to the incubation medium were plasma glycoproteins.The incorporation of [1-¹⁴c]glucosamine into UDP-N-acetylhexosamine significantly increased from 6 h to 48 h after partial hepatectomy. On the contrary, the incorporation into acid-insoluble fractions of slice and medium decreased to about 50% of the control values. The rate of transfer of N-acetylhexosamine from UDP-N-acetylhexosamine to acid-insoluble fractions also decreased at 12 h and 48 h respectively. This indicates that the transfer of N-acetylhexosamine to glycoproteins decreases during 48 h of liver regeneration.The enhancement of [1-¹⁴C]glucosamine incorporation into UDP-N-acetylhexosamine is due to an accumulation of the label in the larger pool of this compound. Evidently, some control mechanism may operate on the transfer of N-acetylhexosamine from UDP-N-acetylhexosamine to glycoproteins in regenerating rat liver.     

Single administration of hepatotoxic chemicals transiently decreases the gap-junction-protein levels of connexin 32 in rat liver.

Effects of a single administration of hepatotoxic chemicals on the major gap-junction protein of liver (connexin 32) were studied in rats. The connexin-32 content was analyzed by immunoblotting and immunohistochemistry using an affinity-purified monoclonal antibody against connexin 32. The connexin-32 content decreased dramatically to less than 10% of the control value 24 h after injection of 25 mg/kg dimethylnitrosamine and returned to the normal level 240 h later. Injection of CCl4 at a dose of 1 ml/kg also caused a transient reduction of connexin-32 content in the liver, as seen after the dimethylnitrosamine injection. The decrease in hepatic connexin-32 content was inversely correlated to the increase in plasmic alanine-aminotransferase activity which has been used as an index of acute liver injury. The changes in connexin 32 were essentially similar to those observed in regenerating liver after partial hepatectomy. However, incorporation of [3H]thymidine into liver DNA after dimethylnitrosamine injection was significantly less than that obtained after partial hepatectomy. The 5'-nucleotidase activity of the plasma-membrane fraction of the liver was not significantly altered by the injection of dimethylnitrosamine. These results suggest that liver gap-junction protein is specifically reduced by acute liver injury and that this kind of decrease in connexin 32 is not simply related to cell proliferation, unlike the decrease after partial hepatectomy.     

Inhibition of glucose production during hepatic nerve stimulation in regenerating rat liver perfused in situ. Possible involvement of gap junctions in the action of sympathetic nerves.

To explore the possible role of gap junctions in neural regulation of hepatic glucose metabolism, the effects of hepatic nerve stimulation on metabolic and hemodynamic changes were examined in normal and regenerating rat liver which was perfused in situ at constant pressure via the portal vein with a medium containing 5 mM glucose, 2 mM lactate and 0.2 mM pyruvate. 1. The content of connexin 32, a major component of gap junctions in rat liver, decreased transiently to about 25% of the control level in regenerating liver 48-72 h after partial hepatectomy and recovered to normal by the 11th day after the operation. 2. In normal liver, electrical stimulation of the hepatic nerves (10 Hz, 20 V, 2 ms) and infusion of noradrenaline (1 microM) both increased glucose and lactate output and reduced perfusion flow. 3. In early stage of regenerating liver 48 h and 72 h after partial hepatectomy, the increase in glucose output in response to nerve stimulation was almost completely inhibited, whereas the change in lactate balance was partially suppressed and the reduction of flow rate was retained. The response of glucose output to nerve stimulation recovered by the 11th day after partial hepatectomy. In contrast, exogenous application of noradrenaline increased glucose output even in the early stage of regenerating liver. 4. The increase in noradrenaline overflow during hepatic nerve stimulation in the early stage of regenerating liver was approximately the same as in normal liver. Liver glycogen was sufficiently preserved in the early stage of regenerating liver. However, noradrenaline infusion could no more increase glucose output both in normal and in regenerating livers after 24 h of fasting that depleted liver glycogen. These results suggest that the impaired effects of sympathetic nerve stimulation on glucose metabolism observed in regenerating liver are derived neither from reduced release of noradrenaline nor from depletion of liver glycogen, but rather from transient reduction of gap junctions which assist signal propagation of the nerve action through intercellular communication in rat liver.     

Accumulation and release of triglycerides by rat liver following partial hepatectomy

Regenerating liver accumulates lipid for about 20 hr following partial hepatectomy. During this time incorporation of intravenously administered palmitate-9, 10-(3)H into beta-lipoprotein increased. 13 hr after partial hepatectomy, there was no change in the level of serum beta-lipoproteins, but the specific activities of the triglycerides in the liver and beta-lipoproteins were significantly diminished. Extension of these studies to the isolated perfused liver system demonstrated that 13 hr after partial hepatectomy the regenerating liver is capable of secreting greater quantities of the lipid, but not the protein, moiety of the beta-lipoproteins in comparison with liver taken immediately from a partially hepatectomized animal, although there was no difference between the weights of the livers. However following addition of palmitate-(3)H and (14)C-labeled amino acids to the perfusate, the specific activity of the hepatic and beta-lipoprotein triglycerides of the liver excised 13 hr after partial hepatectomy was diminished, but that of the protein was not affected. Prelabeling of the accumulated triglyceride with palmitate-1-(14)C in vivo revealed that the proportions of the accumulated triglyceride secreted as beta-lipoproteins by perfused livers excised immediately and 13 hr after partial hepatectomy were identical. It is concluded that regenerating liver rapidly acquires the ability to mobilize triglycerides at a rate equal to that of the much larger normal liver, so that it can handle all free fatty acids presented to it.     

DNA synthesis, mitotic index, drug-metabolising systems and cytogenetic analysis in regenerating rat liver. Comparison with bone marrow test after 'in vivo' treatment with cyclophosphamide

Rat-liver cells can be used to reveal "in vivo" clastogenic activity of indirect mutagens, provided that they are stimulated to divide by partial hepatectomy. In order to characterize the rat-liver metabolic capacity in such experimental conditions, several biochemical parameters were measured during the first 54-66 h of liver regeneration in Sprague-Dawley male rats, subjected to a partial hepatectomy. The levels of cytochrome P-450, the activities of styrene monooxygenase, epoxide hydrolase and glutathione-S-epoxide transferase were chosen as markers. All the enzymatic activities and the level of cytochrome P-450 decreased during the first 12 h after the hepatectomy to about 50% of the activities of the sham-operated rats considered as controls. Subsequent recovery of the metabolic capacity was not observed. DNA synthesis and the mitotic index were measured to find the most suitable time for metaphase analysis. DNA synthesis and the number of metaphases were maximal at, respectively, 22-25 and 28-31 h after partial removal of the liver. The sensitivity to clastogenic damage induced by "in vivo" treatment with cyclophosphamide (CPA) was assayed in regenerating liver cells by chromosome-aberration analysis. Different doses, ranging from 5 to 30 mg/kg b.w., were given i.p. to the rats 17 h before or 7 h after partial hepatectomy. Liver cells were collected 31 h after surgery. Clastogenic damage was greater when the drug was administered to the animals after the hepatectomy (24 h of exposure) than before (48 h of exposure). The sensitivity to CPA-induced damage was compared with a bone marrow cell test carried out on non-hepatectomized rats treated in the same way. The results indicated that in these conditions regenerating liver cells are more sensitive than bone marrow cells to the induction of chromosome aberrations by CPA.