Serum hormone levels following partial hepatectomy in the rat.

Serum levels of insulin, glucagon, growth hormone (somatotrophin) and thyroxine (TT₄) were measured by radioimmunoassay following both sham operation and 70% partial hepatectomy in the rat to evaluate changes in hormone levels during liver regeneration. An eleven fold increase in glucagon was observed (from 112 ± 10 pg/ml to 1500 ± 200 pg/ml) 6 hours following partial hepatectomy but not sham operation. In contrast, insulin levels remained unchanged compared to sham controls for up to 72 hr while growth hormone fell to low levels, 6 to 48 hr after partial hepatectomy. Both total thyroxine and free thyroxine levels also fell 24–72 hours after hepatectomy. These studies suggest that growth hormone, thyroxine and insulin are not primary stimulants of hepatic regeneration although the data suggests that glucagon may modify this growth process.     

Changes in the insulin and glucagon receptors in the regenerating liver following intoxication with carbon tetrachloride.

The response of rat liver plasma membrane adenylate cyclase was studied from one to 14 days after a single dose of carbon tetrachloride (CCl₄). The response to glucagon was diminished to a greater extent than that of fluoride and was due to a deficiency in hormone binding. In contrast, insulin binding increased 300% over control; the change was due to increased number of binding sites. The “affinities” of receptors for either hormone were not altered. The tissue levels of adenosine 3′:5′ -monophosphate increased following CCl₄ poisoning reaching a peak precisely when the adenylate cyclase response to glucagon was at its lowest level. These studies present evidence that receptors for pancreatic hormones change differently when liver is damaged and during its regeneration following CCl₄ intoxication. The change in pattern is remarkably similar to changes reported previously in fetal liver development or following partial hepatectomy of adult rat.     

Sensitivity of liver cells formed after partial hepatectomy to glucagon, vasopressin and angiotensin II

During the active proliferation which follows partial hepatectomy, the sensitivity of liver cells to glucagon is markedly diminished. In hepatocytes obtained from rats partially hepatectomized 3 days before experiments were performed, the dose-response curves to glucagon were shifted to the right by about two orders of magnitude as compared to those of the control cells. Later on (7 days after surgery) the dose-response to glucagon was still shifted to the right but by only one order of magnitude. These data are consistent with the diminution in the number of glucagon receptors in liver plasma membrane during liver regeneration reported by other authors. No stimulation of glycogenolysis, gluconeogenesis or ureogenesis was produced by vasopressin or angiotensin II in hepatocytes from rats partially hepatectomized 3 days before experimentation. However, phosphatidylinositol labeling was stimulated in these cells to a similar extent as in the controls. The ionophore A23187 was also ineffective in stimulating glycogenolysis in these cells. Later, 7 days after surgery, the hepatic responsiveness to vasopressin and angiotensin II was restored. The data suggest that, during the initial stages of liver regeneration, the enzymatic machinery of the hepatocyte is not sensitive to calcium-signalling.     

川芎嗪促进大鼠部分肝切除后修复性再生机制的研究

目的:探讨大鼠部分肝切除后肝功能的变化及川芎嗪对肝修复性再生能力的影响。方法:在相同月龄的动物中按体重均衡的原则随机分组。实验动物共分为5组:设正常对照组(对照组)、青年假手术对照组(假术组)、青年肝切除组(青切组)、中年肝切除组(中切组)和中年肝切除治疗组(切治组),每组动物10只,常规饲养,自由饮水。参照Higgins and Aderson给大鼠施行肝脏70%切除手术,中切组大鼠术前以川芎嗪(200 mg/kg/d)腹腔注射7d,其余组注射生理盐水。假术组大鼠以同样的手术程序打开腹腔但不施行肝部分切除术。各组施行手术动物在切除术后24 h沿腹中线切开动物腹腔,于腹主动脉两髂分支处取血分离血清,切取所有肝脏,待测。采用试剂盒法分别测定各组血清中丙氨转氨酶(ALT)、谷草转氨酶(AST)含量;肝脏匀浆后,采用八木国夫法检测肝组织中丙二醛(MDA)含量,采用western blot法测定肝组织中核增殖抗原(PCNA)和铜锌超氧化物歧化酶(SOD1)、锰超氧化物歧化酶(SOD2)的蛋白表达。结果:与中切组相比手术动物相比,切治组组大鼠血清中ALT、AST水平显著降低(P<0.05),肝细胞中PCNA表达显著升高(P<0.05),肝组织中MDA含量显著降低(P<0.05);肝组织中SOD1、SOD2表达显著增加。结论:肝脏切除70%后,肝功受损,氧化应激增加,但核增殖能力增强。川芎嗪可以抑制肝切手术导致的氧化应激损伤,促进SOD的表达,抑制MDA的升高,降低ALT、AST水平,提高PCNA的表达。提示中年大鼠肝切除后肝功能受损与氧化应激相关,给予抗氧化药物能够促进肝再生修复能力,青年肝切除手术大鼠肝的修复能力强于中年动物。     

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.     

Changes in plasma membrane enzyme activities during liver regeneration in the rat.

Changes in activities of plasma membrane enzymes during liver regeneration may be related to the maintenance of hepatic function or to the regulation of cell proliferation. Plasma membranes were isolated from rat livers at various times after partial hepatectomy, and the specific activities of alkaline phosphatase, (Na+ + K+)-ATPase, leucine aminopeptidase, 5'-nucleotidase, and adenylate cyclase (basal and with glucagon or epinephrine) were measured. Alkaline phosphatase and (Na+ + K+)-ATPase activity increased 3.6-fold and 2-fold respectively, during the first 48 h after partial hepatectomy. The time of onset and duration of change suggest that these increases in activity are involved in the maintenance of bile secretion. Decreases in leucine aminopeptidase activity at 48--108 h and in 5'-nucleotidase activity at 12--24 h were observed, which may be involved in the restoration of protein and accumulation of RNA. The basal activity of adenylate cyclase increased after partial hepatectomy. The response of adenylate cyclase to epinephrine showed a transitory increase between 36 and 108 h after surgery, while the response to glucagon was decreased by approximately 50% at all time points through 324 h after surgery. These changes in the hormone responsiveness of adenylate cyclase are similar to those previously observed in fetal and preneoplastic liver.     

Differential expression of apoptosis-associated genes post-hepatectomy in cirrhotic vs. normal rats

Liver regeneration after partial hepatectomy or liver injury is controlled by a wide variety of growth factors that are proven activators or inhibitors of hepatocyte proliferation. Liver regeneration post-hepatectomy has been proven to be decreased and delayed in cirrhotic vs. normal liver. Apoptosis seems to play an important role in cellular proliferation and in liver regeneration. Therefore, this study has analyzed the expression of apoptosis-associated genes following 2/3 hepatectomy in cirrhotic vs. normal rats. Cirrhosis was induced by a weekly intragastric administration of CCl4 for 16 weeks followed by hepatectomy and histological examination of the resected liver. Rats were sacrificed at 6 h, 12 h, 24 h, or 72 h after liver resection. The expression of proapoptotic (Bad, Bak, Bax) and antiapoptotic (Bcl-2, Bcl-XL) genes was analyzed by quantitative RT-PCR. We have observed an early increase in antiapoptotic mRNA levels and a delayed increase in proapoptotic mRNA levels in normal liver following hepatectomy. Before resection, proapoptotic mRNA levels were significantly higher in cirrhotic vs. normal liver. After hepatectomy, apoptotic mRNA levels were decreased and delayed as compared with that observed following hepatectomy in normal liver. These results indicate that apoptosis takes place in liver during CCl4-induced cirrhosis and could participate in the impaired regenerative response observed in cirrhotic liver.     

Hepatotropin mRNA expression in human foetal liver development and in liver regeneration

A 569 bp probe against the β-chain of hepatotropin was used to examine expression of RNA for this growth factor in human adult and foetal liver, foetal kidney and pancreas, and rat liver after partial hepatectomy. Low level expression of a 6kb RNA occurred in human adult and normal rat liver. 70% hepatectomy increased expression, peaking at 10 h and returning to near normal levels 24 h after resection. The 6 kb band was strongly expressed in human foetal liver, as compared with adult, but not in foetal kidney or pancreas, suggesting a major role for hepatotropin in both foetal development and regeneration of the liver.     

Immunocytochemical study of the hepatic innervation in the rat after partial hepatectomy

Summary The autonomic nervous system in rats has been assessed by means of indirect immunofluorescence using monospecific antibodies to neuron-specific enolase, neurofilaments, glial fibrillary acidic protein and S-100 protein (10 days after partial (70%) hepatectomy). Different groups of rats were studied:group A: 70% resection and normal dual blood supply (n=5);group B: 70% resection with only portal blood to the liver remnant (n=5);group C: 70% resection with only arterial blood to the liver remnant (n=5);group D: sham operated controls (n=5).All rats of groups A and D showed normal liver/body weight ratios after 10 days in contrast to groups B and C where liver weights were 50–60% of the preresection weight. In group A the regeneration process was histologically normal and associated with a remarkable increase of autonomic innervation patterns in the portal triad. In contrast, livers of animals in groups B and C showed under the light microscope features of hepatocyte degeneration associated with a decreased autonomic innervation compared to the controls. The changes are identical in groups B and C, and are therefore irrespective of the type of blood deprivation (arterial or portal).These results support the importance of dual blood supply for an optimal regenerative response in liver remnants after liver resection. We suggest that the autonomic nerve supply of the portal triad plays at least an important permissive role in liver regeneration.     

Desensitization of adenylate cyclase and cyclic AMP flux during the early stages of liver regeneration

Liver regeneration is controlled by a complex network of interactions between hormones, growth factors, and a variety of hepatotrophic factors. Transient increases in cAMP in the early stages of liver regeneration that are necessary for DNA synthesis and subsequent mitosis have been reported; however, studies on the mechanisms that control cellular cAMP levels during liver regeneration, namely adenylate cyclase activity, cAMP-dependent phosphodiesterase activity, and cAMP efflux from the cell, have been generally incomplete. In this study we have shown that although there are three peaks in intracellular cAMP levels in the first 24 hours after partial hepatectomy, the adenylate cyclase activity stimulated by glucagon, prostaglandin E2, adrenaline, and fluoride in vitro decreases with time. KD and BMAX of hepatocyte glucagon and beta receptors were similar to the sham controls. Our results are consistent with a mixed homologous/heterologous desensitization of the adenylate cyclase system. There was also a loss of cAMP-dependent phosphodiesterase activity after partial hepatectomy. We speculate that even though the hormone-stimulated adenylate cyclase system has been desensitized, the system retains the ability to respond to the transient pulses of the variety of hormones secreted after partial hepatectomy and thus raise the intracellular concentration of cAMP. The decrease in cAMP-dependent phosphodiesterase may be necessary to prevent rapid breakdown of cAMP.     

Rapamycin-insensitive regulation of 4e-BP1 in regenerating rat liver

In cultured cells, growth factor-induced phosphorylation of two translation modulators, p70 S6 kinase and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), is blocked by nanomolar concentrations of the immunosuppressant rapamycin. Rapamycin also attenuates liver regeneration after partial hepatectomy, but it is not known if this growth-suppressive effect is due to dephosphorylation of p70 S6 kinase and/or 4E-BP1. We found that partial hepatectomy induced a transient increase in liver p70 S6 kinase activity and 4E-BP1 phosphorylation as compared with sham-operated rats. The amount of p70 S6 kinase protein in regenerating liver did not increase, but active kinase from partially hepatectomized animals was highly phosphorylated. Phosphorylated 4E-BP1 from regenerating liver was unable to form an inhibitory complex with initiation factor 4E. Rapamycin blocked the activation of p70 S6 kinase in response to partial hepatectomy in a dose-dependent manner, but 4E-BP1 phosphorylation was not inhibited. By contrast, functional phosphorylation of 4E-BP1 induced by injection of cycloheximide or growth factors was partially reversed by the drug. The mammalian target of rapamycin (mTOR) has been proposed to directly phosphorylate 4E-BP1. Western blot analysis using phospho-specific antibodies showed that phosphorylation of Thr-36/45 and Ser-64 increased in response to partial hepatectomy in a rapamycin-resistant manner. Thus, rapamycin inhibits p70 S6 kinase activation and liver regeneration, but not functional phosphorylation of 4E-BP1, in response to partial hepatectomy. These results indicate that the effect of rapamycin on 4E-BP1 function in vivo can be significantly different from its effect in cultured cells.     

Inhibition of Hedgehog Delays Liver Regeneration through Disrupting the Cell Cycle

Liver regeneration is a complicated biological process orchestrated by various liver resident cells. Hepatic cell proliferation and reconstruction of the hepatic architecture involve multiple signaling pathways. It has been reported that the Hh signal is involved in liver regeneration. However, the signal transduction pathways and cell types involved are ill studied. This study aimed to investigate hedgehog signal response cell types and the specific molecular mechanism involved in the process of liver regeneration. Partial hepatectomy (PH) of 70% was performed on ICR (Institute of Cancer Research) mice to study the process of liver regeneration. We found that the hedgehog signal was activated significantly after PH, including hedgehog ligands, receptors and intracellular signaling molecules. Ligand signals were mainly expressed in bile duct cells and non-parenchymal hepatic cells, while receptors were expressed in hepatocytes and some non-parenchymal cells. Inhibition of the hedgehog signal treated with vismodegib reduced the liver regeneration rate after partial hepatectomy, including inhibition of hepatic cell proliferation by decreasing Cyclin D expression and disturbing the cell cycle through the accumulation of Cyclin B. The current study reveals the important role of the hedgehog signal and its participation in the regulation of hepatic cell proliferation and the cell cycle during liver regeneration. It provides new insight into the recovery of the liver after liver resection.     

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.     

Nonrepetitive DNA transcription in normal and regenerating rat liver.

Initial RNA excess hybridization experiments employing total cell RNA and the complete complement of nonrepetitive DNA sequences showed no differences between normal and regenerating rat liver. However, when the DNA from the RNA-DNA hybrids was isolated and then reacted with homologous and heterologous RNAs the sensitivity of the assay was sufficiently improved to reveal that some of the nonrepetitive DNA transcrips present in normal liver are missing at 24 h and 48 h after a 70% partial hepatectomy. Additional experiments showed that while some of the missing sequences were common to both stages of regeneration, some were also different. The data thus suggest both quantitative and qualitative changes during liver regeneration in the population of RNA molecules transcribed from nonrepetitive DNA.     

The effect of triiodothyronine on changes of membrane fluidity in regenerating rat liver.

The increase of the membrane fluidity during the early phase of liver regeneration after partial hepatectomy was described in literature in plasma membrane and in microsomes. We found similar changes also in isolated mitochondria and in crude total membrane fraction of the liver homogenate. The administration of triiodothyronine to rats before partial hepatectomy diminished the increase of the membrane fluidity in the regenerating liver by 50%. Triiodothyronine effect is explained by hormonal modification of lipid metabolism in the regenerating liver.     

Changes in plasma membrane enzyme activities during liver regeneration in the rat

Changes in activities of plasma membrane enzymes during liver regeneration may be related to the maintenance of hepatic function or to the regulation of cell proliferation. Plasma membranes were isolated from rat livers at various times after partial hepatectomy, and the specific activities of alkaline phosphatase, (Na⁺ + K⁺)-ATPase, leucine aminopeptidase, 5′-nucleotidase, and adenylate cyclase (basal and with glucagon or epinephrine) were measured. Alkaline phosphatase and (Na⁺ + K⁺)-ATPase activity increased 3.6-fold and 2-fold respectively, during the first 48 h after partial hepatectomy. The time of onset and duration of change suggest that these increases in activity are involved in the maintenance of bile secretion. Decreases in leucine aminopeptidase activity at 48–108 h and in 5′-nucleotidase activity at 12–24 h were observed, which may be involved in the restoration of protein and accumulation of RNA. The basal activity of adenylate cyclase increased after partial hepatectomy. The response of adenylate cyclase to epinephrine showed a transitory increase between 36 and 108 h after surgery, while the response to glucagon was decreased by approximately 50% at all time points through 324 h after surgery. These changes in the hormone responsiveness of adenylate cyclase are similar to those previously observed in fetal and preneoplastic liver.     

The degree of hepatic regeneration after partial hepatectomy in rats with peritonitis and the role of lipid peroxidation

Bile accumulation in the peritoneal cavity after partial hepatectomy reduces hepatic regeneration. In 70% of hepatectomized rats with bile peritonitis, hepatic DNA synthesis showed a delayed initiation and diminished peak level. Because intraperitoneal bile significantly accelerated lipid peroxidation and decreased energy metabolism in the liver remnant, all hepatectomized rats with bile peritonitis died within 7 days. Subcutaneous administration of exogenous combined antioxidants SOD and catalase dramatically reduced lipid peroxidation and improved the survival rate. Although the slightly elevated serum endotoxin level in rats with peritonitis may play a role in the inhibition of hepatic regeneration, the result suggest that intraperitoneal accumulation of bile components may also directly accelerate lipid peroxidation in the liver remnant, inhibiting the hepatic regeneration.     

The expression of cAMP-dependent protein kinase subunits is differentially regulated during liver regeneration

The levels of the regulatory (RI and RII) and catalytic (C) components of cAMP-dependent protein kinase and of their messages were studied during the first 36 h of liver regeneration after 70% hepatectomy. Both RI alpha mRNA and RII alpha mRNA started to increase 4 h after the resection, reaching peak levels after 9 h. RI mRNA decreased abruptly 9-12 h after resection, whereas RII mRNA stayed elevated. C alpha mRNA was rather constant during the period of study. In accordance with the mRNA data the level of C was constant while RI and RII increased during the prereplicative phase of liver regeneration. RI increased rapidly when its message became elevated. RII, however, increased noticeably only 6-8 h after its mRNA had become elevated. The increased expression of R led to a disproportion between R and C that was most pronounced 14 h after resection, i.e. coinciding with the prereplicative cAMP burst. The increased R/C ratio at that time of regeneration diminished the concentration of active C subunit during the cAMP burst. In that way the otherwise inhibitory effect of high concentrations of active C on the DNA replication may have been decreased. The fractional saturation of RI and RII by endogenous cAMP fluctuated in parallel as a function of liver cAMP levels, although there was a tendency that RI was more highly saturated than RII at high concentrations of cAMP.