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)     

Course of liver regeneration after partial hepatectomy in rats treated with dialysates obtained 17 hours after partial hepatectomy

Various theories have been put forward to explain the regenerative capacity of liver tissue induce by partial hepatectomy (PH). One of them presumes the existence of humoral factors stimulating proliferation of the liver tissue. We evaluated the course of liver regeneration after 65-70% PH as influenced by dialysates (DIA) of the organs of a rat killed 17 h after PH. In addition to kidney DIA, we were particularly interested in the effect of liver and spleen DIA. The experiments were carried out on rats weighting 310-370 g. Kidney, liver or spleen dialysate was administered subcutaneously and the rats were killed 12 or 24 h later by exsanguination from the abdominal aorta. In further rats, PH was performed 24 h after administering DIA and the rat were killed 18, 24, 30, 48 and 72 h after the operation. The initiation of liver regeneration was stimulated by all the given DIA, but especially by liver DIA. The faster onset of liver regeneration 18 h after PH in rats given spleen DIA is interesting. DIA did not greatly affect the hepatocytes of intact liver, but accelerated the initiation of liver regeneration after PH by synchronizing the cell cycle of proliferating hepatocytes. DIA obtained 17 h after PH contained substances which primarily stimulated liver DNA synthesis. From the changes in inhibition of the migration of spleen macrophages in the medium containing liver antigens, and from the circulating immunocomplex values, we conclude that DIA activation of the immune system, a well as the hepatic stimulator substance contained in the DIA, participates in acceleration of the liver regeneration process.     

Concurrent changes in sinusoidal expression of laminin and affinity of hepatocytes to laminin during rat liver regeneration.

Distribution of fibronectin, laminin, and collagens type I, III, IV, and V in the lobular regions of regenerating rat liver was studied by indirect immunofluorescence. Little or no laminin was detected in sham-operated controls throughout the experimental period, while it was detected in sinusoids of regenerating liver as early as 6 h after partial hepatectomy (PH). After reaching a maximum at 24 h, it decreased and was barely detectable 6 days after PH. Changes in the other extracellular matrix (ECM) proteins were evident 3 days after PH, but not earlier than 24 h. Hepatocytes isolated from regenerating rat livers were tested in a short term assay for attachment to the substrates coated with the ECM proteins. The attachment of hepatocytes to laminin substrates increased 12 h after PH, reached a maximum at 24 h, and decreased to the control level 6 days after PH, while that of the control remained constant. The attachment to fibronectin substrates was not different between regenerating livers and controls at any time point. The attachment to collagen did not change earlier than 24 h after PH, but increased slightly 3 days after PH. Primary rat hepatocytes cultured on the substrates coated with the ECM proteins were determined for replicative DNA synthesis in response to epidermal growth factor. Both in normal liver and in regenerating liver 24 h after PH, laminin was one of the most effective substrates in supporting the responsiveness of hepatocytes to the growth stimulus. Taken together, these results suggest the importance of hepatocyte-laminin interaction during the early stage of liver regeneration possibly in growth stimulation of hepatocytes and/or maintenance of hepatocyte-specific functions.     

Growth-related signaling regulates activation of telomerase in regenerating hepatocytes

Although there have been many reports on the relationship between activation of telomerase and carcinogenesis, the role of telomerase in normal cellular growth is still unclear. In this study, we analyzed the relationship between upregulation of telomerase activity and cell cycle progression during the liver regeneration process by using an in vivo mouse two-thirds partial hepatectomy (PH) model as well as by using in vitro hepatocyte culture systems. Furthermore, we also investigated the effects of growth factors on telomerase activity during liver regeneration and the influence of MAPK pathway inhibitors (MEK inhibitors PD98059 and U0126; p38 MAPK inhibitor SB203580) on the telomerase activity of regenerating hepatocytes in vitro. An upregulation of the telomerase activity was found at 24 h after PH, and thereafter an increase in the S-phase fraction was observed at 36-48 h. There was no remarkable change in the telomere length after PH. Preoperative treatment with EGF and HGF increased the in vivo telomerase activity. In a hepatocyte primary culture, the upregulation of the telomerase activity required the presence of EGF, and this upregulation was accelerated by the addition of HGF. A remarkable activation of p44/42 MAPK was seen but no such activation of p38 MAPK was observed at 48 h after PH. Although SB203580 had no effect on the telomerase activity of regenerating hepatocytes, treatment with MEK inhibitors (PD 98059, U0126) significantly repressed the telomerase activity. In conclusion, the telomerase activity is upregulated before hepatocytes enter the S phase, and both EGF and HGF play important roles in this step. In addition, the activation of the p44/42 MAPK pathway seems to play an essential role in telomerase upregulation during the liver regeneration process.     

Exosomes from adipose-derived mesenchymal stem cells alleviate liver ischaemia reperfusion injury subsequent to hepatectomy in rats by regulating mitochondrial dynamics and biogenesis

Hepatic ischaemia reperfusion injury (HIRI) is a major factor leading to liver dysfunction after liver resection and liver transplantation. Adipose-derived mesenchymal stem cells (ADSCs) have potential therapeutic effects on HIRI. Exosomes derived from ADSCs (ADSCs-exo) have been widely studied as an alternative of ADSCs therapy. Thus, the aim of this study was to evaluate the potential protective effect and related mechanism of ADSCs-exo on HIRI subsequent to hepatectomy. Rats were randomly divided into four groups: Sham, I30R+PH, ADSCs and ADSCs-exo group. After 24 h of reperfusion, liver and serum of the rats were immediately collected. ADSCs-exo improved liver function, inhibited oxidative stress and reduced apoptosis of hepatocytes in HIRI subsequent to hepatectomy in rats. ADSCs-exo significantly promoted the recovery of mitochondrial function, markedly increased the content of ATP in the liver tissue, and improved the ultrastructure of mitochondria in hepatocytes. Moreover, ADSCs-exo significantly increased the expression of OPA-1, MFN-1 and MFN-2 proteins related to mitochondrial fusion, while DRP-1 and Fis-1 mRNA and protein expression associated with mitochondrial fission were significantly decreased after the treatment with ADSCs-exo. In addition, ADSCs-exo significantly increased the expression of PGC-1α, NRF-1 and TFAM genes and proteins related to mitochondrial biogenesis. ADSCs-exo improves liver function induced by HIRI subsequent to hepatectomy in rats and maintains mitochondrial homeostasis by inhibiting mitochondrial fission, promoting mitochondrial fusion and promoting mitochondrial biogenesis. Therefore, ADSCs-exo may be considered as a potential promising alternative to ADSCs in the treatment of HIRI subsequent to hepatectomy.     

Autocrine expression of activated transforming growth factor-beta(1) induces apoptosis in normal rat liver

The aim of this study was to determine the differential effects of latent and activated transforming growth factor (TGF)-beta(1) in growth control of normal and proliferating hepatocytes in vivo. Rats were injected with adenoviruses expressing control transgenes (Ctrl), latent TGF-beta(1) [TGF-beta(L)], or activated TGF-beta(1) [TGF-beta(A)]. Additional animals underwent two-thirds partial hepatectomy (PH) 24 h after injection. Increased hepatocyte apoptosis was observed in TGF-beta(A)-injected but not TGF-beta(L)-injected animals 24 h postinjection (10.5%) compared with Ctrl animals (0.37%). The percent of apoptotic cells increased to 32.1% in TGF-beta(A)-injected animals 48 h after injection. Furthermore, TGF-beta(A)-injected rats did not survive 24 h after PH. Four hours after PH, 0.25 and 14.1% apoptotic hepatocytes were seen in Ctrl- and TGF-beta(A)-injected rats, respectively. TGF-beta(A)-induced apoptosis in primary rat hepatocytes was blocked with a pancaspase inhibitor. Thus autocrine expression of TGF-beta(A) but not TGF-beta(L) induces hepatocyte apoptosis in the normal rat liver. Rats overexpressing TGF-beta(A) do not survive two-thirds PH due to hepatic apoptosis. Thus activation of TGF-beta(1) may be a critical step in the growth control of normal and proliferating rat hepatocytes.     

Role of melatonin in the oxidative damage prevention at different times of hepatic regeneration

The process of regenerating liver is the result of a balance between stimulating factors and inhibitors of hepatocyte proliferation. Melatonin and its metabolites have been found to protect tissues against oxidative damage generated by a variety of toxic agents and metabolic processes. Furthermore, studies in liver of rats showed a decrease in the liver mitochondrial hydroxylation of drugs returning to the normal state after the administration of antioxidants. This study was designed to determine, in experimental animals, whether the administration of an antioxidant agent such as melatonin could prevent cells events leading to tissue injury and hepatic dysfunction after partial hepatectomy (PH). Biliary flow (BF), oxidative stress in hepatic tissue and Na⁺/K⁺ATPase activities in whole plasma membrane were determined. PH decreased the Na⁺/K⁺ATPase activity. PH significantly reduced the BF (36%) and promoted oxidative stress with an increase of lipoperoxidation and decrease of glutathione peroxidase and catalase activities. Treatment with melatonin prevented the decrease of BF in rats with hepatectomy and normalized the Na⁺/K⁺ATPase activity. Moreover, melatonin markedly attenuated oxidative stress produced by PH. This may be the results of the higher efficacy of melatonin in scavenging various free radicals and also because of its ability in stimulating the antioxidant enzymes. We suggest that oxidative stress before and during liver regeneration has a crucial role in cholestasis, apoptotic/necrotic hepatocellular damage and the impairment in liver transport function induced by PH and that melatonin could modulate the degree of oxidative stress and through it prevent the alterations in liver function carrier. Copyright © 2012 John Wiley & Sons, Ltd.     

皮质酮对大鼠再生肝细胞鸟氨酸脱羧酶活性及其基因表达的影响

采用高效液相色谱和原位杂交技术研究了皮质酮对大鼠再生肝细胞鸟氨酸脱羧酶 (ODC)活性及ODCmRNA表达的影响。结果显示 ,大鼠完整肝脏中ODC水平较低 ,2 / 3肝切除 (PH)后 3h ,不同处理组ODC活性开始升高 ,6h达到最高值 ,其中 ,去肾上腺 NaCl组和糖皮质激素受体拮抗剂RU4 86处理组的酶活性高于对照组 (去肾上腺假手术组 ) ,而去肾上腺 皮质酮处理组的酶活性低于对照组 ,36h恢复到肝切除前水平 ;完整肝脏的ODCmRNA水平极低 ,PH后表达量迅速增加 ,5h达到最大值 ,不同处理组mRNA水平的高低顺序与酶活性一致 ,12h降至肝切除前水平 ;在PH前 12h给大鼠注射RU4 86 (10mg/kg体重 ) ,取得了与去肾上腺 NaCl处理鼠相似的结果。以上结果表明 ,在PH诱导的再生肝细胞中 ,ODCmRNA表达量的增加和 /或减少是造成ODC活性改变的原因之一 ,皮质酮对ODC活性及其mRNA的表达具有抑制作用 ,主要表现在肝再生的早期 ,该作用可能是通过受体实现的     

Critical role of exposure time to endogenous oxidative stress in hepatocyte apoptosis

We have previously shown that inhibition of catalase and glutathione peroxidase activities in rat primary hepatocytes by 3-amino-1,2,4-triazole (ATZ) and mercaptosuccinic acid (MS) results in sustained oxidative stress, followed by apoptosis. To examine the effects of duration of oxidative stress, ATZ and MS were removed from culture medium at 3, 6 and 9 h after treatment with both inhibitors. Oxidative stress was induced for periods of time by ATZ and MS exposures in primary hepatocytes. Treatment with ATZ and MS reduced catalase (CAT) and glutathione peroxidase (GPx) activities, and decreased CAT and GPx activities recovered to normal values upon withdrawal. Although oxidative stress of up to 6 h duration did not cause cell death, sustained oxidative stress (over 9 h) induced apoptosis. The increase in the glutathione disulfide/reduced glutathione ratio under oxidative stress up to 6 h was transient and reversible, while that due to sustained oxidative stress was irreversible. These results suggest that irreversible redox shifts resulting from sustained oxidative stress play a critical role in the induction of hepatocyte apoptosis in this experimental system.     

Spatiotemporal changes in Ha-ras p21 expression through the hepatocyte cell cycle during liver regeneration.

The protein product of the ras oncogene, Ha-ras (p21), is thought to be an important regulator of cell growth. The cytoplasmic relocalization of p21 in the cell during the cell cycle suggests a transient signaling role for this protein in association with its signal transduction function. Because of the importance of this role we examined spatial patterns in vivo of p21 expression at the protein and mRNA levels in hepatocytes during compensatory growth in rat liver following partial hepatectomy. A low level of p21 was immunolocalized on the cytoplasmic membrane of nonregenerating hepatocytes. The level of hepatic p21 increased significantly and without spatial restriction within the liver from 36 to 60 hr after partial hepatectomy (PH). p21 was localized in the cytoplasm of dividing hepatocytes and on the hepatic cytoplasmic membrane. The elevated p21 level decreased and was found mainly on hepatocyte plasma membranes by 96 hr after PH. Immunogold electron microscopy showed p21 localized over mitochondrial membranes and nuclei in nondividing regenerating hepatocytes. Approximately 50% of nonregenerating hepatocytes show nuclear localization of p21. This percentage changes with time following PH. The decrease in nuclear localization was accompanied with an increase in the low number of hepatocytes which demonstrated cytoplasmic localization in nondividing hepatocytes in regenerating liver. Flow cytometric analysis revealed a significant increase of p21 at 36 hr after PH which was 12 hr after the initial induction of ras mRNA. ras mRNA level increased 1.5-fold at 24 hr after PH and a maximum twofold induction was observed at 48 hr. Cell-cycle analysis of regenerating hepatocytes indicated a synchronized first peak of cell division 36-40 hr after PH. Dual parameter flow cytometry revealed that the level of p21 in hepatocytes in S phase and G2/M phase of the cell cycle was significantly higher than that in G0/G1 phase during regeneration. These findings suggest that p21 is important for the progression of regenerating hepatocytes to S phase and then to G2/M phase.     

Critical role of exposure time to endogenous oxidative stress in hepatocyte apoptosis

Abstract

We have previously shown that inhibition of catalase and glutathione peroxidase activities in rat primary hepatocytes by 3-amino-1,2,4-triazole (ATZ) and mercaptosuccinic acid (MS) results in sustained oxidative stress, followed by apoptosis. To examine the effects of duration of oxidative stress, ATZ and MS were removed from culture medium at 3, 6 and 9 h after treatment with both inhibitors. Oxidative stress was induced for periods of time by ATZ and MS exposures in primary hepatocytes. Treatment with ATZ and MS reduced catalase (CAT) and glutathione peroxidase (GPx) activities, and decreased CAT and GPx activities recovered to normal values upon withdrawal. Although oxidative stress of up to 6 h duration did not cause cell death, sustained oxidative stress (over 9 h) induced apoptosis. The increase in the glutathione disulfide/reduced glutathione ratio under oxidative stress up to 6 h was transient and reversible, while that due to sustained oxidative stress was irreversible. These results suggest that irreversible redox shifts resulting from sustained oxidative stress play a critical role in the induction of hepatocyte apoptosis in this experimental system.     

Triiodothyronine stimulates hepatocyte proliferation in two models of impaired liver regeneration

Abstract.  Objectives : Liver regeneration is attenuated in old age and is substantially slower after 90% than after 70% partial hepatectomy (PH). We have previously demonstrated that the proliferative response to a primary mitogen is intact in aged mice, indicating that impaired liver regeneration is not due to loss of proliferative capacity. Here, we have investigated whether mitogenic effects of triiodothyronine (T3) could reverse the impaired regeneration of ageing or 90% hepatectomy, in the rat. Materials and methods : T3 (20 µg/100 g body weight) was administered to 14-month-old rats subjected to 70% PH or to young rats subjected to 90% PH. Cell-proliferative capacity was determined by bromodeoxyuridine incorporation and microscopy and changes of cell cycle-related proteins were analysed by Western blot analysis. Results : Treatment of old intact rats with T3 increased cyclin D₁ expression that was followed by an enhanced proliferative response, the labelling index (LI), being 7.8% versus 1.3% of controls. T3 given before 70% PH stimulated regenerative response (LI was 10.8% versus 2.28%), and expression of cyclin D₁ and proliferating cell nuclear antigen (PCNA) 24 h after PH. Pre-treatment with T3 also improved the regenerative response of the liver after 90% hepatectomy (LI was 27.9% versus 14.2%). Conclusions : These findings show in principle that mitogen-induced hyperplasia could be applied to human therapy in patients with reduced regenerative capacity or massive loss of hepatocytes.     

Molecular dissection of gp130-dependent pathways in hepatocytes during liver regeneration

Interleukin-6 (IL-6) via its signal transducer gp130 is an important mediator of liver regeneration involved in protecting from lipopolysaccharide (LPS)-induced liver injury after partial hepatectomy (PH). Here we generated mice either defective (Delta) in hepatocyte-specific gp130-dependent Ras or STAT activation to define their role during liver regeneration. Deletion of gp130-dependent signaling had major impact on acute phase gene (APG) regulation after PH. APG expression was blocked in gp130-DeltaSTAT animals, whereas gp130-DeltaRas mice showed an enhanced APG response and stronger SOCS3 regulation correlating with delayed hepatocyte proliferation. To define the role of SOCS3 during hepatocyte proliferation, primary hepatocytes were co-stimulated with IL-6 and hepatocyte growth factor. Higher SOCS3 expression in gp130-DeltaRas hepatocytes correlated with delayed hepatocyte proliferation. Next, we tested the impact of LPS, mimicking bacterial infection, on liver regeneration. LPS and PH induced SOCS3 and APG in all animal strains and delayed cell cycle progression. Additionally, IL-6/gp130-dependent STAT3 activation in hepatocytes was essential in mediating protection and thus required for maximal proliferation. Unexpectedly, oncostatin M was most strongly induced in gp130-DeltaSTAT animals after PH/LPS-induced stress and was associated with hepatocyte proliferation in this strain. In summary, gp130-dependent STAT3 activation and concomitant SOCS3 during liver regeneration is involved in timing of DNA synthesis and protects hepatocyte proliferation during stress conditions.     

Ultrastructural zonal heterogeneity of hepatocytes and mitochondria within the hepatic acinus during liver regeneration after partial hepatectomy

BACKGROUND INFORMATION: Partial hepatectomy (70%) induces cell proliferation until the original mass of the liver is restored. In the first 24 h after partial hepatectomy, drastic changes in the metabolism of the remaining liver have been shown to occur. To evaluate changes in hepatocyte ultrastructure within the hepatic acinus during the liver regenerative process, we investigated, by light and electron microscopy observations on specimens taken 0 h, 24 h and 96 h after partial hepatectomy, the hepatocyte structure and ultrastructure in the periportal and pericentral area of the hepatic acinus, with a particular emphasis on mitochondria ultrastructure. Moreover, some biochemical events that could affect the mitochondria ultrastructure and function were investigated. RESULTS: We found that, 24 h after partial hepatectomy, mitochondria with altered ultrastructure were preferentially localized in the periportal area. Periportal hepatocytes showed also an increase in the number of peroxisomes, free ribosomes, lysosomes and autophagosomes. Altered mitochondria showed swelling, an ultrastructural index of increased membrane permeability, a reduction in the number of cristae, and a rarefied, often vacuoled, matrix. Consistently, an increase in the mitochondrial oxidized/reduced glutathione ratio was found as well as calcium release from mitochondria in a manner inhibited by cyclosporin A. Interestingly, light and electron microscopy analysis showed that the hepatocytes in the periportal area were the cells with the major structural attributes to proliferate. At 96 h after partial hepatectomy, the preferential zonation of altered mitochondria was lost and the normal mitochondrial membrane permeability properties were restored. CONCLUSIONS: We suggest that 24 h after partial hepatectomy, a preferential zonation of altered mitochondria in the periportal hepatocytes could be involved in the changes of metabolic and functional heterogeneity of the hepatocytes within the hepatic acinus during the regenerative process.     

Spatiotemporal changes in Ha-ras p21 expression through the hepatocyte cell cycle during liver regeneration

The protein product of the ras oncogene, Ha-ras (p21), is thought to be an important regulator of cell growth. The cytoplasmic relocalization of p21 in the cell during the cell cycle suggests a transient signaling role for this protein in association with its signal transduction function. Because of the importance of this role we examined spatial patterns in vivo of p21 expression at the protein and mRNA levels in hepatocytes during compensatory growth in rat liver following partial hepatectomy. A low level of p21 was immunolocalized on the cytoplasmic membrane of nonregenerating hepatocytes. The level of hepatic p21 increased significantly and without spatial restriction within the liver from 36 to 60 hr after partial hepatectomy (PH). p21 was localized in the cytoplasm of dividing hepatocytes and on the hepatic cytoplasmic membrane. The elevated p21 level decreased and was found mainly on hepatocyte plasma membranes by 96 hr after PH. Immunogold electron microscopy showed p21 localized over mitochondrial membranes and nuclei in nondividing regenerating hepatocytes. Approximately 50% of nonregenerating hepatocytes show nuclear localization of p21. This percentage changes with time following PH. The decrease in nuclear localization was accompanied with an increase in the low number of hepatocytes which demonstrated cytoplasmic localization in nondividing hepatocytes in regenerating liver. Flow cytometric analysis revealed a significant increase of p21 at 36 hr after PH which was 12 hr after the initial induction of ras mRNA. ras mRNA level increased 1.5-fold at 24 hr after PH and a maximum twofold induction was observed at 48 hr. Cell-cycle analysis of regenerating hepatocytes indicated a synchronized first peak of cell division 36–40 hr after PH. Dual parameter flow cytometry revealed that the level of p21 in hepatocytes in S phase and G₂/M phase of the cell cycle was significantly higher than that in G₀/G₁ phase during regeneration. These findings suggest that p21 is important for the progression of regenerating hepatocytes to S phase and then to G₂/M phase.     

Comitogenic effects of vasoactive intestinal polypeptide on rat hepatocytes

The primary mitogens such as epidermal growth factor and transforming growth factor-α are known to stimulate DNA synthesis in primary cultures of adult rat hepatocytes. Vasoactive intestinal polypeptide (VIP) was found to amplify DNA synthesis induced by the primary mitogens and thus acted as a comitogen. The comitogenic effect of VIP was specific for the culture medium, suggesting that minor components in the medium were required for hepatocytes to fully respond to VIP. Glutamic acid is probably one of these minor components, although other components present in the nutrient-rich medium were also necessary for the full comitogenic effect. Other comitogens such as insulin, vasopressin, and angiotensin II interacted additively with low concentrations of VIP. The comitogenic effect of VIP was also found in hepatocytes cultured from regenerating rat liver after a partial hepatectomy. In the regenerating hepatocyte cultures, VIP can act as a mitogen even in the absence of the primary mitogen EGF. VIP mRNA was found in several organs including brain, intestine, and liver, and its expression was slightly induced in liver 24 h after a partial hepatectomy. These results suggest that VIP can act as a hepatic comitogen and may play a role in liver cell proliferation. © 1996 Wiley-Liss, Inc.     

Nrf2 Is Involved in Maintaining Hepatocyte Identity during Liver Regeneration

Nrf2, a central regulator of the cellular defense against oxidative stress and inflammation, participates in modulating hepatocyte proliferation during liver regeneration. It is not clear, however, whether Nrf2 regulates hepatocyte growth, an important cellular mechanism to regain the lost liver mass after partial hepatectomy (PH). To determine this, various analyses were performed in wild-type and Nrf2-null mice following PH. We found that, at 60 h post-PH, the vast majority of hepatocytes lacking Nrf2 reduced their sizes, activated hepatic progenitor markers (CD133, TWEAK receptor, and trefoil factor family 3), depleted HNF4α protein, and downregulated the expression of a group of genes critical for their functions. Thus, the identity of hepatocytes deficient in Nrf2 was transiently but massively impaired in response to liver mass loss. This event was associated with the coupling of protein depletion of hepatic HNF4α, a master regulator of hepatocyte differentiation, and concomitant inactivation of hepatic Akt1 and p70S6K, critical hepatocyte growth signaling molecules. We conclude that Nrf2 participates in maintaining newly regenerated hepatocytes in a fully differentiated state by ensuring proper regulation of HNF4α, Akt1, and p70S6K during liver regeneration.     

Growth inhibitory actions of prothrombin on normal hepatocytes: influence of matrix

Most hepatomas have a defect in prothrombin carboxylation, and can secrete under-carboxylated prothrombin or des-gamma-carboxy-prothrombin (DCP), the function of which is unknown. We considered that the prothrombin-DCP axis might also be involved in growth control. Hepatocytes and hepatoma cells were treated with prothrombin and DNA synthesis and cytoskeletal changes were studied. Prothrombin inhibited DNA synthesis in hepatocytes on fibronectin, but not collagen matrix. Hepatoma cell lines were not inhibited. We found that hepatoma cell matrix conferred resistance to hepatocytes. Prothrombin decreased fibronectin but not collagen amounts, but only in the presence of hepatocytes and not hepatoma cells, indicating that it has a differential action on matrix proteins. It also caused changes in cell shape and actin depolymerization. In vivo, there was a decrease in plasma prothrombin activity after a partial hepatectomy (PH), concomitant with the peak of DNA synthesis in the hepatocytes at 24h after PH. Injection of warfarin at the time of PH, further inhibited PT activity and enhanced this 24h peak of DNA synthesis. Furthermore, repeated injection of prothrombin lowered the peak DNA synthesis after PH. The data support the hypothesis that prothrombin can act as a hepatocyte growth inhibitor, likely at the level of fibronectin loss and result in cytoskeletal changes. Hepatomas resist this action, possibly due to their different matrix proteins. This represents a novel mechanism for growth regulation and provides a possible biological significance for the tumor marker DCP.     

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.