Characteristics of hepatocyte proliferation in the regenerating rat liver with hydroxyurea synchronization of the proliferative processes

5-week old rats underwent a partial hepatectomy (2/3 of the liver was removed). The operated rats were repeatedly injected hydroxyurea, within 9-24 or 12-24 hours following operation. Proliferation of hepatocytes and non-parenchymal cells in the regenerating liver was studied. Five or six injections of hydroxyurea increased the labelling index of hepatocytes up to 62.5% and induced proliferation of bile duct cells significantly.     

The effect of ceruloplasmin on hepatocyte proliferation in the regenerating rat liver

The influence of ceruloplasmin on cell proliferation in regeneration liver of the rat has been studied. Ceruloplasmin stimulates cell proliferation in regeneration liver increasing functional activity of the mononuclear phagocytes.     

Gene expression in regenerating liver in relation to cell proliferation and stress

When hepatocyte proliferation is stimulated in the liver by partial hepatectomy, messenger RNAs coding for fibrinogen, actin, c-myc and topoisomerase I are rapidly accumulated. We distinguish an early phase of accumulation (0-3 h after partial hepatectomy) which is also observed after a sham operation for the four genes, and during inflammation produced by Freund's adjuvant in the case of fibrinogen and c-myc genes. The hepatic response to inflammation appears therefore to mimic events characteristic of the G0/G1 transition, such as the accumulation of the c-myc mRNA. The late phase of mRNA accumulation (beyond 3 h after partial hepatectomy) is typical of liver regeneration. The level of c-myc mRNA is transiently increased (20-fold over normal) 20 h after partial hepatectomy, that is, at the time of DNA synthesis. Topoisomerase-I mRNA level increases between 3 and 24 h after partial hepatectomy (5-10-fold over normal). These results suggest that accumulation of c-myc and topoisomerase-I mRNAs is associated with DNA replication in regenerating liver.     

Analysis of hepatocyte cell membrane antigens in regenerating rat liver]

Antigens of plasma membranes in hepatocytes from regenerating rat liver were studied. Immunochemical investigation with polyvalent rabbits antiserum against plasma membrane proteins in hepatocytes from regenerating and normal rat liver have shown that liver regeneration processes are accompanied by the increase of proteins number with molecular weight of--80 kDa, 62 kDa, 40 kDa and 27 kDa. It is not excluded that protein with molecular weight of 27 kDa is the tissue-specific peripheral protein. The influence of antibodies against proteins of hepatocytes plasmatic membranes on histostructure of pathologically changed liver tissue has been studied. The data obtained testify to a possibility of participation of the above mentioned proteins in the regulation of rat liver regeneration processes.     

Proliferation and cell death of hepatocytes in regenerating fetal rat liver

Proliferation and death of hepatocytes in regenerating liver were studied in 17-day-old fetal white rats. Two days after liver resection (20%), animals were sacrificed every 3 h. In experimental groups, the index of Ki67-positive hepatocytes increased sharply 15 h after liver resection. In all experimental and control groups, the ratio of the index of the metaphase, the longest phase of mitosis, to the mitotic index remained unchanged, indicating the same duration of hepatocyte mitoses in regenerating liver. In regenerating and intact liver, hepatocytes labeled with antibodies to caspase 3 were not detected. Thus, resection of 20% fetal rat liver did not promote enhancement of apoptosis of hepatocytes.     

Proliferation and cell death of hepatocytes in regenerating rat fetal liver

Proliferation and death of hepatocytes in regenerating liver of 17-day white rat fetuses were investigated. During 2 days after liver resection (20%), animals were sacrificed every 3 h. In experimental groups, the index of Ki67-positive hepatocytes increased sharply in 15 h after liver resection. In all experimental and control groups, the ratio of the metaphase, the longest phase of mitosis, and index to mitotic index remained unchanged, indicating identical duration of hepatocytes mitoses in regenerating liver. In the regenerating and intact liver hepatocytes labeled with antibodies to caspase 3 were not detected. Thus, resection of 20% rat fetal liver did not contribute to increased apoptosis of hepatocytes.     

UDP-N-acetylglucosamine-glycoprotein N-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 (UDP-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.     

Lipid peroxidation in regenerating rat liver

Rats entrained to a strictly regulated lighting and feeding schedule have been subjected to partial hepatectomy or a sham operation. In the partially hepatectomised animals the period of liver regeneration is characterised by regular bursts of thymidine kinase activity. Liver microsomes from rats, at times corresponding to maximum thymidine kinase activity, have much reduced rates of lipid peroxidation compared to control preparations: this is due in part to increased levels of lipid-soluble antioxidant at times of maximal DNA synthesis. This temporal relationship between thymidine kinase and lipid peroxidation is consistent with the view that lipid peroxidation is decreased prior to cell division.     

The phosphorylation of nuclear proteins in the regenerating and premalignant rat liver and its significance for cell proliferation.

In liver regeneration or neoplastic transformation, phosphorylation of nuclear proteins is stimulated. In the regenerating liver all main histone fractions are involved in this process. The type of histone phosphorylated seems to be dependent on the position of the partially synchronized cells within the generation cycle. At a time when most cells are exhibiting maximum HnRNA-synthesis, histone F2a2 belongs to those fractions with highly stimulated phosphate incorporation. Phosphorylation of this fraction alone is stimulated by cyclic AMP in parallel to a stimulation of HnRNA-synthesis. The preneoplastic liver is characterized by oscillating phosphorylation and dephosphorylation reactions of nearly all histone fractions during the first days of N-nitroso-diethylamine administration. After 2 months of carcinogen feeding a 50-150% stimulation of the phosphorylation of Fl subfractions is observed. The phosphate content of the other histones, however, has returned to the original level. A series of further proteins, isolated together with the histones, show very similar phosphorylation characteristics. These proteins are mostly of non-histone origin. It is suggested that some of them are responsible for the transport of RNA with messenger properties within the cell.     

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.     

Galactose metabolism in regenerating rat liver.

1. Rats trained on a controlled lighting and feeding schedule were subjected to partial hepatectomy or sham operation. 2. After a large period of about 6h the activity of UDP-galactose 4-epimerase increased threefold, reaching a maximum 4 days after partial hepatectomy, and returned to normal values within a fortnight. 3. The enzyme pattern of the UDP-galactose-glycoprotein galactosyltransferase was biphasic, one peak appearing at 20 h, the second at 72 h after partial hepatectomy. 4. The rise in enzyme activities could be blocked by the injection of actinomycin D, and the Km values for UDP-glucose and UDP-galactose were nearly identical in regenerating and adult liver. It is therefore concluded that the increase in enzyme activity is due to synthesis de novo of enzyme protein.     

Sulphated glycosaminoglycans in regenerating rat liver.

The total weight percentage glycosaminoglycan content of rat liber was found to increase by 50% in the first 30 h after partial hepatectomy. The content returned to near normal by the third day, but then increased again to a second maximum at 5-6 days, only to gradually decline to normal by the ninth day, when regeneration was nearly complete. This biphasic pattern was most marked in the chondroitin sulphate A/C component, with a 6-fold increase by the sixth day. Dermatan sulphate showed the same temporal trend, whereas heparan sulphate remained relatively unaltered. No such changes were detected in the livers of rats subjected to sham operation. The possible molecular mechanisms underlying the apparent link between cellular glycosaminoglycan content and proliferative tendency are discussed.     

Nicotinamide nucleotide synthesis in regenerating rat liver

1. The concentrations and total content of the nicotinamide nucleotides were measured in the livers of rats at various times after partial hepatectomy and laparotomy (sham hepatectomy) and correlated with other events in the regeneration process. 2. The NAD content and concentration in rat liver were relatively unaffected by laparotomy, but fell to a minimum, 25 and 33% below control values respectively, 24h after partial hepatectomy. NADP content and concentration were affected similarly by both laparotomy and partial hepatectomy, falling rapidly and remaining depressed for up to 48h. 3. The effect of injecting various doses of nicotinamide on the liver DNA and NAD 18h after partial hepatectomy was studied and revealed an inverse correlation between NAD content and DNA content. 4. Injections of nicotinamide at various times after partial hepatectomy revealed that the ability to synthesize NAD from nicotinamide was impaired during the first 12h, rose to a peak at 26h and fell again by 48h after partial hepatectomy. 5. The total liver activity of NAD pyrophosphorylase (EC 2.7.7.1) remained at or slightly above the initial value for 12h after partial hepatectomy and then rose continuously until 48h after operation. The activity of NMN pyrophosphorylase (EC 2.4.2.12) showed a similar pattern of change after partial hepatectomy, but was at no time greater than 5% of the activity of NAD pyrophosphorylase. 6. The results are discussed with reference to the control of NAD synthesis in rapidly dividing tissue. It is suggested that the availability of cofactors and substrates for NAD synthesis is more important as a controlling factor than the maximum enzyme activities. It is concluded that the low concentrations of nicotinamide nucleotides in rapidly dividing tissues are the result of competition between NAD synthesis and nucleic acid synthesis for common precursor and cofactors.     

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.