Effects of protein-deprivation on the regeneration of rat liver after partial hepatectomy.

Rats maintained on a protein-free diet for 3 days have an altered time course of hepatic DNA synthesis during liver regeneration. The delay in DNA synthesis is eliminated by the administration of casein hydrolysate (given as late as 6h after partial hepatectomy), but not by glucose or incomplete amino acid mixtures. Despite the change in the timing of DNA synthesis, the increases in hepatic amino acid pools, which take place at the earliest stages of the regenerative process, occur in a normal pattern in the regenerating liver of rats fed the protein-free diet. Protein-deprived rats have increased protein synthesis and decreased rates of protein degradation in the liver in response to partial hepatectomy, but these adaptations do not prevent a lag in protein accumulation and low protein/RNA ratios. The regenerating livers of these animals show a deficit in the accumulation of cytoplasmic polyadenylated mRNA as well as a smaller proportion of free polyribosomes. It is suggested that the deficit in free polyribosomes found in the regenerating liver of protein-deprived rats might be a consequence of the slow accumulation of mRNA species coding for intracellular proteins.     

Effect of the interferone inducer, polyriboinosinic.polyribocytidylic acid on rat liver regeneration following partial hepatectomy

The administration of the interferone inducer, polyriboinosinic.polyribocytidylic acid, inhibited the rise of activities of thymidylate synthase and thymidine kinase as well as DNA content in 24 h-regenerating rat liver in a dose dependent manner. The immunoblotting assay showed that the decrease of thymidylate synthase activity was due to inhibition of the induction of the enzyme. Co-administration of putrescine did not affect the inhibitory effect of polyriboinosinic.polyribocytidylic acid. Polyriboinosinic acid did not affect DNA synthesis in rat liver regeneration.     

Effect of the activation of macrophages on the course of regeneration of rat liver following partial hepatectomy

Stimulation of the Kupffer cells with E. coli endotoxin (the purified lipopolysaccharide) or with prodigiosan (a polysaccharide from Serratia marcescens) 24 h before partial hepatectomy (resection of 65-70% of the liver) stimulated and intensified the onset of liver regenerative activity (evaluated from changes in liver DNA synthesis, the H5 labelling index and the mitotic activity of the hepatocytes). Liver DNA synthesis increased together with the dose of endotoxin (i.v., from 25 to 1000 micrograms/kg body weight). If E. coli endotoxin was injected during or 3 h after partial hepatectomy, partial inhibition of liver DNA synthesis was observed. In mice stimulated with zymosan (a polysaccharide isolated from yeast), administered 5 days before performing partial hepatectomy, proliferation of the hepatocytes (evaluated from changes in the 3H labelling index and in the mitotic activity of the hepatocytes) was evaluated. The results confirm that proliferation is correlated to the state of reactivity of the Kupffer cells.     

Distribution of pteroylglutamates in rat liver during regeneration after partial hepatectomy.

1. Liver pteroylpolyglutamate distribution was studied during regeneration after partial hepatectomy in rats maintained under controlled feeding conditions. 2. Pteroylhexaglutamate, pteroylpentaglutamate and pteroyltetraglutamate concentrations decrease from 12 to 72 h after operation, then increase and reach normal values at 180 h. Pteroyltriglutamate concentration, already high at 12 h, remains so in the subsequent periods. Pteroyldiglutamate concentration was unchanged. Monoglutamate concentrations at first decrease, and at 180 h exceed normal values. 3. The decrease in polyglutamate derivatives with a high number of glutamate residues, at present considered to be the coenzyme forms of folate, could be related not to a decreased synthesis, but to a greater requirement for these compounds during the early periods of regeneration, when biosynthetic processes are markedly increased. It is indeed probable that the increased availability of the preferred substrate of pteroylpolyglutamate synthetase, i.e. tetrahydrofolate, enhances conversion of folate into coenzyme forms.     

Cellular mechanisms of cirrhotic rat liver regeneration. II. Proliferation, polyploidization and hypertrophy after partial hepatectomy

Using cytofluorimetry and absorptional cytophotometry, hepatocyte DNA and total protein contents were measured in intact and cirrhotic rats in 1, 3 and 6 months after partial hepatectomy (PH). It has been found that within one month of intact rat liver regeneration the level of hepatocyte ploidy rised by 25% to remain elevated for the next 6 months. This was due mainly to reducing the number of cells with diploid nuclei (2c 2-fold, 2c x 2 - 6.6-fold) and to rising the number of octaploid hepatocytes. In cirrhotic animals the ploidy level in hepatocytes increased in 3 months after PH, and decreased by 15% in 6 months. The number of hepatocytes with diploid nuclei (2c and 2c x 2) increased within 3-6 months in both control and cirrhotic rats. The protein content per diploid hepatocyte rised by 30% within 3-6 months of liver regeneration after PH. Special calculations have shown that within 3 months after PH the increase in the liver mass of control and cirrhotic rats was due completely to hepatocyte DNA synthesis, i. e. proliferation and polyploidization. Within the next 3 months of liver regeneration after PH, the contribution of polyploidization to liver mass increase was negative because of depolyploidization of liver parenchyma cell population. At this time hypertrophy was the main process determining the liver mass increase.     

Specific gangliosides increase rapidly in rat liver following partial hepatectomy

Rat liver gangliosides (sialic acid containing glycosphingolipids) were analyzed by HPTLC and HPLC following either partial hepatectomy or sham operation. Analysis of whole liver gangliosides by HPTLC demonstrated that within 6 h after partial (68%) hepatectomy, there was a significant increase in GM1 compared to both sham and control animals. By 48 h, GM1 was further increased and the polysialylgangliosides GD1a, GD1b and GT1b had also risen significantly, whereas changes in GM3 were negligible. Gangliosides associated with the plasma membrane were increased up to 3.5-fold in regenerating liver compared to sham-hepatectomized controls as assessed by HPLC. Although elevations in membrane gangliosides were associated with hepatocyte proliferation, they did not closely follow the growth curve. The time course of changes in ganglioside biosynthesis suggests differential upregulation of GM3 synthase and GD3 synthase in regenerating livers.     

Pulsed electromagnetic fields increase the rate of rat liver regeneration after partial hepatectomy

Pulsed extremely low-frequency electromagnetic fields interact with rat liver regeneration following partial hepatectomy when delivered to the rats immediately after the operation and every 12 hr thereafter. This interaction results first in an increased ornithine decarboxylase activity, an enzyme used as an early marker of cell growth. The rate of labeled thymidine incorporation into DNA is also increased by the treatments with magnetic fields during the early phases of liver regeneration. Glycogen depletion and lipid accumulation, two well-known early peculiar phenomena of liver regeneration following partial hepatectomy, are quantitatively decreased by the treatments with electromagnetic fields. The recovery to normal glycogen and lipid contents is completed within 5 days after surgery, instead of 7 days as found in control rats.     

Oxygenation alters ganglioside expression in rat liver following partial hepatectomy

Gangliosides from livers of weanling rats were analyzed after 15% partial hepatectomy (PH) and different pre- and post-operative hyberbaric oxygenation (pre- and postHBO). Neu5Ac was the predominant ganglioside-derived sialic acid (>85%) compared to Neu5Gc. Almost identical low total sialic acid content (Neu5Ac+Neu5Gc) of the control and operated nonHBO animals opposed a 6.4- to 7.6-fold increase in pre- and postHBO animals (69.26 and 81.64pmol/mg wet weight, respectively). NanoESI-QTOF mass spectrometry combined with HPTLC immunostaining revealed GM3(Neu5Ac) and GM3(Neu5Gc) as major gangliosides, correlating with the respective sialic acid concentrations. Minor neolacto-series gangliosides were enhanced in preHBO and postHBO, but GM1-core gangliosides only in preHBO rats. GM2 and GalNAc-GM1b were clearly detectable in oxygenated rats compared to traces in the control and nonHBO animals. These results point at a functional role of gangliosides in liver growth regulation and reconstitution after PH combined with pre- and post-operative HBO treatment.     

Changes in fructose-induced production of glucose in the rat liver following partial hepatectomy.

The fructose-induced production of glucose in the liver after partial hepatectomy (PH) was evaluated by using the liver-perfusion system. There was no significant difference in plasma glucose level between hepatectomized (HX) and sham-operated (SO) rats at 24 h after surgery, and, thereafter, almost similar levels were obtained in both groups. However, the level of serum free fatty acids (FFA) was significantly higher in HX rats than that in SO rats at 24 and 48 h after surgery. When both groups of rats were given fructose by gavage, the increment of plasma glucose was significantly larger in HX rats than in SO rats. Lactate infusion failed to increase the rate of glucose production in perfused livers of both HX and SO rats and there was no significant difference in the activity of hepatic phosphoenolpyruvate carboxykinase. By contrast, fructose infusion elicited a large increase in glucose production in the perfused livers of HX rats at 24 and 48 h after PH. The increase was closely associated with not the change in fructose 2,6-bisphosphate levels but the increment of the intracellular levels of citrate. Treatment of octanoate or oleate, which supplies acetyl-CoA via fatty acid oxidation, mimicked the fructose-induced increase in glucose production in SO rats with a concomitant increase in hepatic levels of citrate. These results suggest that the oxidation of FFA may play an important role in glucose production induced by fructose administration during the early phase of liver regeneration.     

IL-6 Promotes compensatory liver regeneration in cirrhotic rat after partial hepatectomy

Major hepatic resection in cirrhotic patients is associated with impaired liver regeneration and failure, leading to high peri-operative mortality. In this work, the causes of defective regeneration in cirrhotic liver and the utility of IL-6 treatment were investigated in an experimental model combining cirrhosis and partial hepatectomy in the rat. Relative to normal controls, decompensated cirrhotic animals showed decreased survival, while compensated cirrhotic animals showed similar survival but reduced hepatic DNA synthesis and newly regenerated liver mass amount. Defective liver regeneration was associated with a decrease in STAT3 and NF-kB activation, consistent with an increased accumulation of their respective inhibitors PIAS3 and IkBα, and with a decreased induction of Bcl-xL. Treatment with recombinant IL-6 enhanced survival of decompensated cirrhotic animals, while it did not affect survival of compensated cirrhotic animals but sustained liver regeneration, by restoring STAT3 and NF-kB activation and Bcl-xL induction to the levels found in normal controls. The pro-growth effects exerted by IL-6 treatment in cirrhotic liver were attained also at low, pharmacologically acceptable doses. In conclusion, our results suggest that IL-6 treatment may be therapeutic in major resection of cirrhotic liver.     

Effect of propylthiouracil on liver regeneration in rats after partial hepatectomy.

The effect of propylthiouracil (PTU) on the growth activity of intact liver and liver regenerating after partial (65-70%) hepatectomy (PH) was studied in rats. PTU (Propycil, Kali-Chemie, FRG) was dissolved in drinking water (1 g PTU per litre) and this was given to the rats, as their sole source of fluids, three days before PH and then up to the end of the experiment. In rats given PTU, marked inhibition of liver DNA synthesis and the mitotic activity of hepatocytes was found after PH. This effect was potentiated to some extent by partial inanition of the experimental animals given PTU, as demonstrated in a paired feeding test in control rats. PTU inhibition of DNA synthesis in intact and regenerating liver also took effect in thyroidectomized rats, even with substitution (thyroid hormone) therapy. The experiments demonstrated that the effect of propylthiouracil on DNA synthesis in the liver is mediated primarily by way of its direct effect on the liver.     

Glycosylation and ligand-binding activities of rat plasma fibronectin during liver regeneration after partial hepatectomy

Fibronectin (FN) is a multifunctional glycoprotein present in the extracellular matrix (ECM) and plasma. We previously reported that the glycosylation and ligand-binding of vitronectin (VN) change markedly after partial hepatectomy (PH). Here we show the changes of FN during liver regeneration. The yields of purified sham-operated (SH-) and PH-FN were higher than that of non-operated (NO)-FN, while binding activities of FNs to ECM ligands were changed only slightly by hepatectomy. The carbohydrate concentration of PH-FN decreased to 66% of that of NO- and SH-FN. By using LC/MS(n), eight kinds of complex-type N-glycan structures were found to be present in all FNs, and bi- and trisialobiantennary glycans were the major structures. Fucosylation was markedly increased, while O-acetylation of sialic acid was found to be decreased in PH-FN. The alterations in glycosylation and biological activities of FN after PH are different from those of VN, suggesting that these glycoproteins play different biological functions in tissue remodeling.     

Contribution of cyclooxygenase 2 to liver regeneration after partial hepatectomy.

Partial hepatectomy (PH) triggers a rapid regenerative response in the remaining tissue to reinstate the organ function and the cell numbers. Among the molecules that change in the course of regeneration is an accumulation of prostaglandin E2 in the sera of rats with PH. Analysis of the cyclooxygenase (COX) isoenzymes in the remnant liver showed the preferential expression of COX-2 in hepatocytes. Cultured regenerating hepatocytes expressed significant levels of COX-2, a process that was not observed in the sham counterparts. Maximal expression of COX-2 was detected 16 h after PH with increased levels present even at 96 h. Pharmacological inhibition of COX-2 activity with NS398 shunted the up-regulation of cell proliferation after PH, which suggests a positive interaction of prostaglandins with the progression of the cell cycle. Similar results were obtained after PH of mice lacking the COX-2 gene. The expression of COX-2 in regenerating liver was concomitant with a decrease in CCAAT-enhancer binding protein (C/EBP-a) level and an increase in the expression of C/EBP-b and C/EBP-d. These results suggest a contribution of the enhanced synthesis of prostaglandins to liver regeneration observed after PH.     

Studies on the hyperplasia (''regeneration'') of the rat liver following partial hepatectomy. Changes in lipid peroxidation and general biochemical aspects.

Using the experimental model of partial hepatectomy in the rat, we have examined the relationship between cell division and lipid peroxidation activity. In rats entrained to a regime of 12 h light/12 h dark and with a fixed 8 h feeding period in the dark phase, partial hepatectomy is followed by a rapid regeneration of liver mass with cycles of synchronized cell division at 24 h intervals. The latter phenomenon is indicated in this study by pulses of thymidine kinase activity having maxima at 24 h, 48 h and 72 h after partial hepatectomy. Microsomes prepared from regenerating livers show changes in lipid peroxidation activity (induced by NADPH/ADP/iron or by ascorbate/iron), which is significantly decreased relative to that in microsomes from sham-operated controls, again at 24 h, 48 h and 72 h after the operation. This phenomenon has been investigated with regard to possible underlying changes in the content of microsomal fatty acids, the microsomal enzymes NADPH:cytochrome c reductase and cytochrome P-450, and the physiological microsomal antioxidant alpha-tocopherol. The cycles of decreased lipid peroxidation activity are apparently due, at least in part, to changes in microsomal alpha-tocopherol content that are closely associated in time with thymidine kinase activity.