Acceleration of the onset of liver regeneration by carnitine in partially hepatectomized rats

Immediately and 6 h after removal of 70% of the liver tissue, rats were treated with L-carnitine (Carnitene, Sigma-Tau, Italy) and received an injection of 100, 200 or 1,000 mg/kg b.w. into their femoral vein. The control rats were given the same volume of saline solution. The rats were sacrificed 18, 21, 24 or 30 h after the operation. The development of liver regeneration was evaluated from the incorporation of 14C-thymidine into DNA and from the hepatocyte mitiotic activity. In rats given carnitine in a dose of 100 or 200 mg/kg b.w. significantly higher DNA specific activity values were found 18 and 21 h after partial hepatectomoy and higher hepatocyte mitotic activity values after 30 h. In rats given carnitine in a dose of 1,000 mg/kg b.w., DNA specific activity values 21 h after partial hepatectomy were lower than in the control group. We conclude that L-carnitine, in a dose of 100 or 200 mg/kg b.w. has an enhancing effect on the onset of liver regeneration after 70% hepatectomy.     

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.     

Suppressive role of endogenous regucalcin in the enhancement of deoxyribonucleic acid synthesis activity in the nucleus of regenerating rat liver

The role of endogenous regucalcin in the regulation of deoxyribonucleic acid (DNA) synthesis activity in the nuclei of regenerating rat liver after partial hepatectomy was investigated. The addition of regucalcin (0.25 and 0.5 microM) in the reaction mixture caused a significant decrease in the nuclear DNA synthesis activity of normal rat liver. This decrease was also seen in the presence of Ca2+-chelator EGTA (0.4 mM), indicating that the effect of regucalcin is not related to nuclear Ca2+. Nuclear DNA activity was significantly increased in the presence of anti-regucalcin monoclonal antibody (10-50 ng/ml) in the reaction mixture. The effect was completely abolished by the addition of regucalcin (0.5 microM). Nuclear DNA synthesis activity was significantly increased at 24, 48, and 72 h after partial heptectomy. The effect of anti-regucalcin monoclonal antibody (25 ng/ml) in increasing nuclear DNA synthesis activity was significantly enhanced at 24 and 48 h after partial hepatectomy. The presence of staurospone (10(-6) M), trifluoperazine (2 x 10(-5) M), or PD98059 (10(-5) M) in the reaction mixture caused a significant decrease in DNA synthesis activity in the nuclei obtained at 24 after partial hepateactomy. The effect of these inhibitors in the presence of anti-regucalcin monoclonal antibody (25 ng/ml) was greater than that in the absence of the antibody. The present study suggests that endogenous regucalcin plays a suppressive role in the enhancement of nuclear DNA synthesis activity in regenerating liver with cell proliferation after partial hepatectomy in rats.     

Effect of the parenteral administration of energy substrates in different postoperation phases on the initiation and development of liver regeneration in rats subjected to partial hepatectomy

DNA specific activity in the liver, the total DNA content of the liver and the mitotic index of the hepatocytes were studied after the infusion of glucose or lipid emulsions in female laboratory rats with a mean pre-operation weight of 250 +/- 30 g after partial (65-70%) hepatectomy (PH). The infusions were administered in the early prereplication phase (the 1st to 6th hour after the operation), in the late prereplication phase (the 7th to 12th hour after the operation), or continuously from the 1st to the 12th, or the 1st to the 24th, hour after partial hepatectomy. The effect of these parenterally administered energy substrates on the initiation of liver regeneration was evaluated 18 and 24 hours after partial hepatectomy. The results indicate that the infusion of glucose, in any interval after the operation, inhibited the initial phases of liver DNA synthesis (18 h after PH), but not its further development (24 h after PH). Neither the mitotic index of the hepatocytes, nor the total DNA content of the liver differed from the control groups in the case of rats given a glucose infusion. In the experimental groups given lipid emulsions, inhibition of liver DNA synthesis was recorded 18 h after PH only when the infusions were given from the 1st to the 12th or the 1st to the 18th hour after PH. The total DNA content of the liver 18 h after PH was raised in all the experimental groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of branched chain amino acids on liver regeneration after partial hepatectomy

The authors investigated the effect of the enrichment of commercial amino acid solutions with branched chain amino acids on the development of liver regeneration. Partial (65-70%) hepatectomy was performed on male Wistar rats (140-160 g body weight). Starting with the day of the operation, amino acid solutions normally used in clinical practice and the same solutions enriched with branched chain amino acids were administered by stomach tube; 24, 48 and 96 h after the operation the animals were decapitated. The onset of DNA synthesis was found to be more rapid in animals given the enriched solutions. Once regeneration had started, the stimulant effect of an increased supply of branched chain amino acid on liver regeneration was smaller. Nevertheless, even in the later phase after partial hepatectomy branched chain amino acids had a stronger stimulant effect on liver regeneration after partial hepatectomy than an energy supply in the form of sorbitol.     

Effect of saline solutions administered parenterally in different postoperation phases on the regeneration of rat liver after partial hepatectomy

Two series of experiments were carried out on female laboratory rats with a mean pre-operation weight of 250 +/- 30 g, fed up to the time of the experiment on a standard laboratory diet with water ad libitum. In the first series the rats were subjected to 65-70% partial hepatectomy (PH) or to laparotomy (LAP) and their serum Na+, K+, Cl- and total calcium concentrations were determined 6, 12, 18 and 24 h after the operation. At given postoperation intervals the serum Na+, Cl- and total calcium concentrations in hepatectomized animals were lower than in the intact controls, while the K+ concentration was higher. In the second series of experiments, the rats were given infusions of physiological saline or Ringer solution at different intervals (1-6, 7-12, 1-12 and 1-24 h) after PH. Specific DNA activity in the liver, the hepatocyte mitotic index, the total DNA content of the liver and other indicators show that physiological saline infusions had an inhibitory, or at most a neutral effect on the initiation of liver regeneration, while the effect of the infusion of Ringer solution on the initiation of liver regeneration, in most of the given intervals, was indifferent. The regeneration response depends on the post-PH phase in which the solution is infused.     

Effect of partial hepatectomy on removal of O6-methylguanine from alkylated DNA by rat liver extracts.

1. The activity of an enzyme catalysing the loss of O6-methylguanine from methylated DNA was increasing during liver regeneration after partial hepatectomy. Activity was increased 3-fold by 24h and was maximal (6-fold increase) over the period 48-72h after operation. 2. This activity could also be induced by chronic treatment with dimethylnitrosamine, but the maximal response amounted to a 2-3-fold change (with the greater effect in male rats) after 4-6 weeks of exposure to daily doses of 2 mg of dimethylnitrosamine/kg. 3. Neither partial hepatectomy nor treatment with dimethylnitrosamine increased the activities of two other enzymes repairing alkylated DNA, DNA (7-methylguanine-)glycosylase and DNA (3-methyladenine-)glycosylase. 4. These results therefore indicate that there is a selective induction of the O6-methylguanine removal system during hepatocyte proliferation. Since this product is known to lead to mutations and its persistence in DNA throughout cell replication has been implicated in tumour initiation, this induction may play a role in resistance to carcinogenesis by alkylating agents.     

Effect of randomly interesterified triacylglycerols containing medium- and long-chain fatty acids on energy expenditure and hepatic fatty acid metabolism in rats

In our previous studies, medium- and long-chain triacylglycerols (MLCT), randomly interesterified triacylglycerols containing medium-chain and long-chain fatty acids in the same glycerol molecule, significantly reduced body fat accumulation in humans and rats. To clarify mechanism(s) for this effect of MLCT, we measured energy expenditure and hepatic fatty acid metabolism in rats by comparison with long-chain triacylglycerols (LCT) or medium-chain triacylglycerols (MCT). MLCT, compared with LCT, showed significantly lower body fat accumulation, higher 24-h energy expenditure and acyl-CoA dehydrogenase activity measured using octanoyl-CoA as a substrate, and similar lipogenic activity. MCT, compared with LCT, showed significantly higher energy expenditure, but fat accumulation was comparable. Additionally, MCT exhibited significantly higher lipogenic activity than the other oils. These data suggest that enhancement of energy expenditure and medium-chain fatty acids (MCFA) oxidation without activating de novo lipogenesis are responsible at least for the lower body fat accumulation in rats fed MLCT. The activation of hepatic lipogenesis by excessive intake of MCFA might counteract their preventive effects on body fat accumulation.     

Metabolic effects of intravenous LCT or MCT/LCT lipid emulsions in preterm infants

Most lipid emulsions for parenteral feeding of premature infants are based on long-chain triacylglycerols (LCTs), but inclusion of medium-chain triacylglycerols (MCTs) might provide a more readily oxidizable energy source. The influence of these emulsions on fatty acid composition and metabolism was studied in 12 premature neonates, who were randomly assigned to an LCT emulsion (control) or an emulsion with a mixture of MCT and LCT (1:1). On study day 7, all infants received [13C]linoleic (LA) and [13C]alpha-linolenic acid (ALA) tracers orally. Plasma phospholipid (PL) and triacylglycerol (TG) fatty acid composition and 13C enrichments of plasma PL fatty acids were determined on day 8. After 8 days of lipid infusion, plasma TGs in the MCT/LCT group had higher contents of C8:0 (0.50 +/- 0.60% vs. 0.10 +/- 0.12%; means +/- SD) and C10:0 (0.66 +/- 0.51% vs. 0.15 +/- 0.17%) than controls. LA content of plasma PLs was slightly lower in the MCT/LCT group (16.47 +/- 1.16% vs. 18.57 +/- 2.09%), whereas long-chain polyunsaturated derivatives (LC-PUFAs) of LA and ALA tended to be higher. The tracer distributions between precursors and products (LC-PUFAs) were not significantly different between groups. Both lipid emulsions achieve similar plasma essential fatty acid (EFA) contents and similar proportional conversion of EFAs to LC-PUFAs. The MCT/LCT emulsion seems to protect EFAs and LC-PUFAs from beta-oxidation.     

Poly(ADP-ribose) polymerase activity in regenerating liver of hypothyroid rats

The role of PARP, a nuclear enzyme involved in DNA synthesis, repair and cell transformation, was studies during liver regeneration in hypothyroid animals. Hypothyroidism was induced by in vivo administration of propylthiouracil. In regenerating euthyroid animals PARP activity is stimulated showing an early and significant increase at 1.5 h with a maximum at 6 h after partial hepatectomy. Such an increase returns to control values within 18 h preceding the onset of DNA synthesis. A markedly different behavior, with respect to euthyroids, has been evidenced in hypothyroid rats. At first, liver PARP level was about 2-fold higher in non regenerating hypothyroid rats with respect to control euthyroids. During regeneration, PTU-treated animals show a net decrease in PARP activity, with a minimum at 6-9 h after partial hepatectomy. The activity returns to control levels within 24 days. The minimum in PARP activity anticipates, also in this case, the onset of DNA synthesis, which exhibits a maximum at 15-18 h. During liver regeneration PARP activity shows modifications related to the beginning of de novo DNA synthesis. Furthermore, these variations in turn undergo the effects of hypothyroidism.     

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.     

Effect of the parenteral administration of amino acid solutions in different phases after partial hepatectomy on the initiation and development of liver regeneration in rats

Amino acid solutions enriched with branched-chain amino acids or pure branched-chain amino acid solutions were administered parenterally to female laboratory rats (pre-operative weight 230 +/- 30 g) which had been subjected to 65-70% partial hepatectomy (PH), and specific liver DNA activity, the hepatocyte mitotic index and other indicators of the initiation of liver regeneration were studied. Both solutions were infused in an hourly dose of 3.3 ml/kg body weight, during the following postoperative intervals: 1-6, 7-12, 1-12, 1-18 and 1-24 hours. The control rats continued to be fed on the standard laboratory diet after the operation. The results show that the infusion of an amino acid solution enriched with branched-chain amino acids had an inhibitory effect on the onset of DNA synthesis in the liver 18 hours after partial hepatectomy whatever the administration interval. The situation in the case of pure branched-chain amino acid solutions was the same. Twenty-four hours after PH, neither type of solution, irrespective of the infusion interval, was followed by an increase in DNA synthesis compared with the controls fed on the standard laboratory diet. Neither the hepatocyte mitotic index, nor the total liver DNA concentration, showed any changes indicative of stimulation of the initiation of liver regeneration. An infusion stress effect, evaluated from the decrease in the weight of the thymus, was found chiefly in the case of infusions lasting 12 h or longer.     

Effect of triiodothyronine or etiroxate on DNA synthesis in intact and regenerating liver

An increase in liver DNA synthesis (p less than 0.01) was found in rats with an intact liver 24 h after the administration of a single dose of triiodothyronine (200 micrograms/kg i.g.) Statistically significant stimulation of DNA synthesis was also found in rats given triiodothyronine (p less than 0.01) or etiroxate (p less than 0.05) for 3 days at 24-hour intervals. When a single dose of triiodothyronine was administered immediately after partial hepatectomy (65-70% resection of the liver), increased stimulation of DNA synthesis (p less than 0.01) was found 24 h after the operation. Etiroxate partly inhibited DNA synthesis (p less than 0.05). In rats given triiodothyronine at 24-h intervals, starting at the time of partial hepatectomy, DNA synthesis 72 h after the operation was double the value in the control group. Marked stimulation of DNA synthesis by triiodothyronine (p less than 0.01) and an increase in the total DNA content of the liver (p less than 0.05) were likewise found 48 h after partial hepatectomy if the hormone was administered once, 24 h after the operation. The increase in the two indicators after the administration of etiroxate was not statistically significant.     

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.     

Enhanced expression of cytosolic fatty acid binding protein and fatty acid uptake during liver regeneration in rats

BACKGROUND/AIMS: Cytoplasmic liver fatty acid binding protein (L-FABP) has been suggested to be associated with cellular mitotic activity but the changes in L-FABP mRNA and protein levels during liver regeneration following partial hepatectomy (PHx) are not clear. METHODS: In the present study, we determined the time course of L-FABP mRNA expression and L-FABP levels following 70% PHx using Northern and Western blot, respectively. To elucidate one of the roles for L-FABP in PHx, [3H]-palmitic acid clearance in hepatocytes isolated from 24 h post-PHx and control animals was assessed. RESULTS: L-FABP mRNA increased at 30 min, peaked at approximately 1 h (163 +/- 17%; mean +/- SE, n = 5), and returned to control levels 6 h post-PHx. L-FABP level also increased at 1 h but peaked at 24-h (219 +/- 41%; mean +/- SE, n = 5). Hepatocyte [3H]-palmitic acid clearance increased by 29% at 24-h post-PHx, suggesting an increased intracellular transport (or binding) function by L-FABP. Pre-treatment with dexamethasone statistically reduced L-FABP levels (29%) and suppressed the regenerative process (mitotic activity). CONCLUSIONS: L-FABP mRNA increased sharply in response to PHx but the increase was short lived, while L-FABP level increased at a later stage. Both L-FABP content and fatty acid uptake increased significantly during liver regeneration induced by PHx in rats. It is likely that L-FABP is one of the factors responsible for hepatic regeneration.     

Temporal expression profiles indicate a primary function for microRNA during the peak of DNA replication after rat partial hepatectomy

The liver has the unique capacity to regenerate after surgical resection. However, the regulation of liver regeneration is not completely understood. Recent reports indicate an essential role for small noncoding microRNAs (miRNAs) in the regulation of hepatic development, carcinogenesis, and early regeneration. We hypothesized that miRNAs are critically involved in all phases of liver regeneration after partial hepatectomy. We performed miRNA microarray analyses after 70% partial hepatectomy in rats under isoflurane anesthesia at different time points (0 h to 5 days) and after sham laparotomy. Putative targets of differentially expressed miRNAs were determined using a bioinformatic approach. Two-dimensional (2D)-PAGE proteomic analyses and protein identification were performed on specimens at 0 and 24 h after resection. The temporal dynamics of liver regeneration were characterized by 5-bromo- 2-deoxyuridine, proliferating cell nuclear antigen, IL-6, and hepatocyte growth factor. We demonstrate that miRNA expression patterns changed during liver regeneration and that these changes were most evident during the peak of DNA replication at 24 h after resection. Expression of 13 miRNAs was significantly reduced 12-48 h after resection (>25% change), out of which downreguation was confirmed in isolated hepatocytes for 6 miRNAs at 24 h, whereas three miRNAs were significantly upregulated. Proteomic analysis revealed 65 upregulated proteins; among them, 23 represent putative targets of the differentially expressed miRNAs. We provide a temporal miRNA expression and proteomic dataset of the regenerating rat liver, which indicates a primary function for miRNA during the peak of DNA replication. These data will assist further functional studies on the role of miRNAs during liver regeneration.     

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.     

Effect of human fetal liver cells on the rate of DNA and nRNA synthesis in regenerating rat liver

The model of partial hepatectomy was utilized to investigate the influence of human fetal liver cells (FLCs) and of human embryo tissue (PCF) postnuclear cytoplasm fraction injection on the rate of DNA and nRNA synthesis in regenerating rat liver cells. Single infusion of FLCs and PCF into the spleen pulp of rats has been shown to increase the DNA synthesis 24 hours after the operation in 2.5 +/- 0.4 and 3.2 +/- 0.5 times respectively. A group of rats has been identified with no influence of the infusion on the DNA synthesis 24 hours after partial hepatectomy, this process having been even retarding in 48 hours after the operation. Meanwhile the FLCs and PCF infusion enhanced the intensity of nRNA synthesis in 72 hours after the operation in all the animal groups. The effect demonstrated is probably caused by the biologically active substances contained in the fetal tissues.