5-Methylcytosine content of nuclear DNA during chemical hepatocarcinogenesis and in carcinomas which result.

The 5-methylcytosine content of nuclear DNA from nuclear hepatocellular tissues was determined during various phases of hepatic regeneration and carcinogenesis. DNA from premalignant nodules and primary hepatocellular carcinomas induced by exposure to acetylaminofluorene, as well as PHC induced by diethylnitrosamine was undermethylated by 20%, 45%, and 32.5% respectively. Since a 12.5% hypomethylation occurred during the DNA synthetic phase of hepatic regeneration, the effect of cell proliferation on DNA-methylation in malignancies was examined in transplantable hepatocellular carcinomas. The DNA from two transplantable hepatocellular carcinoma lines was less methylated than predicted rates of cell division in these tumors. This finding suggested that an aberration in endogenous DNA methylation may occur during neoplastic transformation.     

Hepatic regeneration following glucagon treatment

Liver regeneration was studied as a function of time after suppression of the normal glucagon response. Rats were given a daily subcutaneous injection of 20 micrograms zincglucagon for 14 days, whereafter a 70% hepatectomy was performed. In the glucagon treated rats the rise in plasma glucagon concentration was diminished after hepatectomy. At intervals from 12 to 384 hours after hepatectomy, the gain in liver weight, the hepatic DNA content, and the antipyrine clearance were measured. All 3 variables were found to be significantly higher in animals with diminished glucagon response. The results indicate that prevention of the normal increase in glucagon concentration leads to signs of increased liver regeneration after 70% hepatectomy.     

Changes in activity and F1 content of mitochondrial H+-ATPase in regenerating rat liver

Submitochondrial particles prepared from rat liver during hepatic regeneration exhibit a depressed ATPase activity which is correlated with a decrease in F1 subunit content as shown by SDS-PAGE. Use of an antibody directed against the F1 portion of the H+-ATPase complex demonstrated that there is a definite decrease in the amount of beta-subunit of F1 in both submitochondrial particles and mitochondria from rat liver 24 h after partial hepatectomy.     

Transcriptome atlas of glutamine family amino acid metabolism‐related genes in eight regenerating liver cell types

To explore glutamine family amino acid metabolism of eight liver cell types in rat liver regeneration, eight kinds of rat regenerating liver cells were isolated by using the combination of Percoll density gradient centrifugation and immunomagnetic bead methods, then Rat Genome 230 2.0 Array was used to detect the expression profiles of the genes associated with metabolism of glutamine family amino acid in rat liver regeneration and finally how these genes involved in activities of eight regenerating liver cell types were analysed by the methods of bioinformatics and systems biology. The results showed that in the priming stage of liver regeneration, hepatic stellate cells and sinusoidal endothelial cells transformed proline and glutamine into glutamate; hepatocytes, hepatic stellate cells, sinusoidal endothelial cells and dendritic cells catabolized glutamate to 2‐oxoglutarate or succinate; hepatic stellate cells and sinusoidal endothelial cells catalysed glutamate into glutamyl‐tRNA for protein synthesis; urea cycle, which degraded from arginine, was enhanced in biliary epithelia cells, sinusoidal endothelial cells and dendritic cells; synthesis of polyamines from arginine was enhanced in biliary epithelia cells, sinusoidal endothelial cells, Kupffer cells and dendritic cells; the content of NO was increased in sinusoidal endothelial cells and dendritic cells; degradation of proline was enhanced in hepatocytes and biliary epithelia cells. In the progress stage, biliary epithelia cells converted glutamine into GMP and glucosamine 6‐phosphate; oval cells converted glutamine into glucosamine 6‐phosphate; hepatic stellate cells converted glutamine into NAD; the content of NO, which degraded from arginine, was increased in biliary epithelia cells, oval cells, pit cells and dendritic cells. In the termination stage, oval cells converted proline into glutamate; glutamate degradation, which degraded from arginine, was enhanced in hepatocytes and dendritic cells; the content of NO was increased in oval cells, sinusoidal endothelial cells, pit cells and dendritic cells. The synthesis of creatine phosphate was enhanced in hepatocytes, biliary epithelia cells, pit cells and dendritic cells in both progress and termination stages. In summary, glutamine family amino acid metabolism has some differences in liver regeneration in different liver cells.     

Physicochemical characteristics of hepatic stimulatory factor prepared from cell free supernatant of hepatocyte cultures.

We previously reported that the cell free supernatant of hepatocyte cultures contained a hepatic stimulatory factor, which increases survival time and stimulates liver regeneration in the galactosamine induced fulminant hepatic failure animal model. When hepatocytes were microencapsulated in an alginate matrix, the cell free supernatant outside the microcapsuled lacked the above stimulatory effects. However, the cell free content of microcapsules was able to increase both survival time and incorporation of 3H-thymidine in our animal model. This hepatic stimulatory factor has a molecular weight greater than albumin because alginate microcapsules allow the penetration of albumin but not the hepatic stimulatory factor. In this paper we report the molecular weight study and some other physiochemical characteristics of this factor. Using Sephacryl gel chromatography we showed that this factor has a molecular weight of over 110,000 D. This factor loses its hepatic stimulatory effect after heat or trypsin treatment. Although its elution profile on Sephacryl gel does not change after such treatments.     

The nucleolar protein NML regulates hepatic ATP levels during liver regeneration after partial hepatectomy

We previously identified a novel protein complex, eNoSC, which senses intracellular energy status and epigenetically regulates the rDNA locus by changing the ratio between the numbers of active and silent gene clusters. eNoSC contains a novel nucleolar protein, Nucleomethylin (NML), which has a methyltransferase-like domain and binds to Lys9-dimethylated histone H3 at the rDNA locus, along with the NAD⁺-dependent deacetylase SIRT1 and the histone methyltransferase SUV39H. The aim of this study was to determine the role of NML in liver after partial hepatectomy (PHx). We assessed liver regeneration and lipid metabolism after PHx in wild-type (WT) and NML transgenic (NML-TG) mice. Survival rates of NML-TG mice were reduced after PHx. We found that hepatic triglyceride content in NML-TG mice remained elevated 48 h after PHx, but not delayed liver regeneration. Moreover, hepatic ATP levels in NML-TG mice were higher than that in WT 48 h after PHx. These observations suggest that NML may regulate consumption of hepatic triglyceride in liver regeneration after PHx due to storage of excess ATP. The delayed consumption of hepatic triglyceride may be the cause of reduced survival rate in NML-TG mice.     

Regulation of polyamine synthesis in human hepatocytes by hepatotrophic factor augmenter of liver regeneration

Different stages of liver regeneration are regulated by a variety of factors such as the liver growth associated protein ALR, augmenter of liver regeneration. Furthermore, small molecules like polyamines were proven to be essential for hepatic growth and regeneration. Therefore, using primary human hepatocytes in vitro we investigated the effect of ALR on the biosynthesis of polyamines. We demonstrated by HPLC analysis that recombinant ALR enhanced intracellular hepatic putrescine, spermidine, and spermine levels within 9-12h. The activation of polyamine biosynthesis was dose dependent with putrescine showing the strongest increase. Additionally, ALR treatment induced mRNA expression of ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase, both key enzymes of polyamine biosynthesis. Further, ALR induced c-myc mRNA expression, a regulator of ODC expression, and therefore we assume that ALR exerts its liver regeneration augmenting effects through stimulation of its signalling pathway leading in part to enhanced polyamine synthesis.     

The Interactions between the Chronic Exposure to Aluminum and Liver Regeneration on Bile Flow and Organic Anion Transport in Rats

The chronic exposure to Aluminum (Al) may compromise different liver functions, mainly during the hepatic regeneration. The aim of this study is to investigate the interactions between the chronic i.p. exposure to Al and hepatic regeneration (HR) on bile flow and organic anion transport in experimental animals. For this purpose, we studied bile flow and fractional transfer rates for the transport of hepatic organic anions (hepatic uptake, sinusoidal efflux, and canalicular excretion), as well as parameters related with the oxidative stress (OS), on rats chronically treated with Al at 0 and 2 days of HR. The Al treatment and time of HR caused a decrease in the biliary flow and in the hepatic uptake and canalicular excretion constants. In addition, Al and HR increased the lipoperoxidation associated with a reduction of the glutathione content and glutathione peroxidase and catalase enzyme’s activities. Since the effects of Al and HR on biliary flow and transport systems were additive, but not on the oxidative status, different mechanisms might be involved on these alterations. Even though the OS may play a key role on the hepatic deleterious effects, there is no unique cause–effect relationship between OS and liver dysfunction in this experimental animal model.     

Additive stimulation of hepatic putrescine production by glucagon and insulin after partial hepatectomy in rats

When rats received glucagon or insulin every 2 h after partial hepatectomy (Hx), hepatic putrescine content was increased above control levels at 6 and 12 h, respectively. When the two hormones were combined, the increased levels were additive. Hepatic ornithine decarboxylase activity was above control levels at 12 h after insulin treatment. Hepatic spermidine N1-acetyltransferase activity was enhanced at 6 h only when glucagon was dosed. Putrescine administration from 0 to 4 h or from 6 to 10 h increased hepatic DNA synthesis to similar levels 22 h after Hx. These results suggest that glucagon and insulin additively stimulate hepatic putrescine production after Hx. This may explain the cooperative stimulation of liver regeneration by both hormones.     

重组[B18Ile]人胰岛素的鉴定和特征

突变体「Ｂ１８Ｉｌｅ」猪胰岛素前体经分离纯化,转肽,得到重组「Ｂ１８Ｉｌｅ」人胰素「Ｂ１８Ｉｌｅ」人胰岛素能结晶,其与受体的结合能力为猪胰岛素的８２％,保留了与猪胰岛素基本相同的体内活力,从本文结果和分析表明Ｂ１８Ｖａｌ可能不是胰岛素表现生物功能所必需的。     

Inhibition of alpha-fetoprotein synthesis in hepatocytes of adult mice by dextran sulfate in vivo and in vitro

Embryospecific serum protein alpha-fetoprotein (AFP) is known to be synthesized in the adult liver only during regeneration and development of hepatocellular carcinomas. It was shown that collagenase digestion of hepatic tissue followed by monolayer cell cultivation was a powerful inducer of AFP synthesis, more potent than the liver regeneration in vivo. The treatment of hepatocytes in culture with 50-100 micrograms/ml of dextran sulphate caused a remarkable inhibition of cell proliferation, formation of cord-like multicellular structures and reduction of AFP synthesis. Mouse liver regeneration after CCL4 poisoning was accompanied by a 1000-fold increase in blood AFP levels. Blood AFP levels and the content of AFP-positive cells in the liver tissue were maximum on the 3rd-4th day after poisoning. Injections of 50 micrograms of dextran sulphate per g body weight 3-5 h after poisoning and 24 and 48 h later caused nearly tenfold reduction in AFP blood level and a decrease in the content of AFP-positive cells in the liver on the 3rd day of regeneration.     

Rapid and direct stimulation of hepatic gluconeogenesis by L-triiodothyronine (T3) in the isolated-perfused rat liver

T3 injection into the portal vein of the isolated hypo- and euthyroid liver rapidly stimulated alanine and ¹⁴C-α-amino-isobutyric acid uptake, O₂-consumption, glucose and urea production, and increased the overall, cytoplasmic and mitochondrial energy state. The concentration of the effector molecules long chain acyl CoA, acetyl-CoA, citrat and AMP remained unchanged. After T3 application no alteration in hepatic cAMP content, protein kinase activation, and in the tissue levels of the intermediates of the Embden-Meyerhof-pathway were observed. Our results indicate that T3 rapidly stimulates hepatic glucose production independently of cAMP by increasing amino acid uptake and mitochondrial ATP regeneration and translocation in the cytoplasmic compartment, thus providing both the precursor and energy for gluconeogenesis.     

Compensatory hepatic regeneration after mild, but not fulminant, intraperitoneal sepsis in rats

Sepsis is the leading cause of death in surgical intensive care units. Although both mild sepsis secondary to cecal ligation and single puncture (CLP) and fulminant, double puncture CLP (2CLP) may provoke hepatocyte death, we hypothesize that regeneration compensates for cell death after CLP but not 2CLP. In male Sprague-Dawley rats, hepatic necrosis, as determined by serum alpha-glutathione S-transferase (alpha-GST) levels, was significantly but equally elevated over time after both CLP and 2CLP. Apoptosis, evaluated using both terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling and morphological examination, was minimal after both CLP and 2CLP. Regeneration, assayed by staining tissue for incorporation of exogenously administered bromodeoxyuridine, was present after CLP but not after 2CLP. To further substantiate impaired regeneration, steady-state levels of mRNAs encoding JunB, LRF-1, and cyclin D1 were determined. After 2CLP, the absence of JunB, LRF-1, and cyclin D1 mRNAs confirmed failed activation of the mitogen-activated protein kinase-linked proliferative pathway and progression through the cell cycle. Therefore, failed hepatocyte regeneration may be a manifestation of hepatic dysfunction in fulminant sepsis.     

Serum alpha-fetoprotein in fulminant hepatitis and hepatic regeneration following partial hepatectomy

The pleomorphism of hepatic regeneration was studied in 10 patients with fulminant hepatitis and 7 with hepatocellular carcinoma, liver cyst, and abscess who underwent partial hepatectomy. Serum AFP levels did not increase significantly following partial hepatectomy. All of four patients who survived fulminant hepatitis had high serum AFP levels with a peak either during or before hepatic encephalopathy. Serum AFP levels decreased rather gradually during the enlargement of the atrophic liver. The observations proposed two kinds of hepatic regeneration, hepatic regeneration following surgical removal of liver and repair of liver damage following virally and probably chemically induced liver deficiency.     

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.     

Effect of α-difluoromethylornithine on polyamine and DNA synthesis in regenerating rat liver: Reversal of inhibition of DNA synthesis by putrescine

The possibility that α-difluoromethylornithine, a specific, irreversible inhibitor of ornithine decarboxylase could be used to prevent the rise in hepatic putrescine and spermidine content following partial hepatectomy was tested. Administration of α-difluoromethylornithine at a dose of 400 mg/kg every 4 h reduced hepatic putrescine to <2 nmol/g, but had only a small effect on the rise in spermidine seen at 28 h after partial hepatectomy. Such treatment also reduced the rise in DNA synthesis produced by partial hepatectomy by up to 70%. The inhibitory effect towards DNA synthesis could be reversed by administration of putrescine which increased the hepatic putrescine content to about 30–40% of that in the regenerating control livers. These results suggest that accumulation of putrescine rather than spermidine is needed for DNA synthesis after partial hepatectomy. They also suggest that part, but not all of the rise in putrescine normally seen in the liver after partial hepatectomy is needed for the enhanced DNA synthesis associated with liver regeneration. Experiments with lower doses of α-difluoromethylornithine showed that a substantial part of the rise in hepatic ornithine decarboxylase activity could be abolished without affecting either the rise in spermidine content or the increase in DNA synthesis after partial hepatectomy.     

小鼠胚胎干细胞分化为肝细胞的基因芯片分析

弄清胚胎肝脏发育的分化调节机制,对指导干细胞在肝再生中的应用以及研究肝分化相关疾病分子机制具有重要意义．胚胎干细胞的全能性使得体外建立肝向分化模型成为可能,采用单层贴壁培养方式,分阶段加入成纤维细胞生长因子(FGF)、肝细胞生长因子(HGF)、制瘤素(OSM)等因子,诱导小鼠胚胎干细胞D3(mESC-D3)的肝向分化．分化细胞在光镜和电镜下呈现肝样细胞形态,RT-PCR、细胞免疫荧光检测以及PAS染色分析表明,这些细胞具有肝细胞特征性的基因表达和生化功能．采用干细胞分化相关基因芯片比较早期肝定向分化前后的基因表达差异,结果显示,48个差异表达基因中(大于2倍),20个上调、28个下调．进一步的生物信息学分析发现,它们集中体现在细胞外基质、细胞连接、FGF、BMP分子及Notch、Wnt信号通路上,提示这些改变可能与胚胎早期的肝向分化密切相关．     

Acute hepatic failure in children.

Many diseases may present as acute hepatic failure in the pediatric age group, including viral hepatitis A and B, adverse drug reactions, both toxic and "hepatitic," and inherited metabolic disorders such as tyrosinemia, alpha 1 antitrypsin deficiency, and Wilson''s disease. Management is primarily supportive, with care taken to anticipate the known complications of hepatic failure. Few "curative" therapies are known, although attempts at stimulating hepatic regeneration may be helpful.     

Emerging role of zinc finger protein A20 as a suppressor of hepatocellular carcinoma

Hepatocellular carcinoma (HCC), the third leading cause of cancer-associated mortality worldwide, is a major public health problem. Zinc finger protein A20 (A20), an acute phase response gene, is a potent inhibitor of NF-κB signaling. A20 serves a critical role in liver protection, including limiting inflammation following hepatic injury, stimulating hepatocyte growth, and preventing hepatic ischemia-reperfusion injury. A20 is also involved in different processes, including tumorigenesis, progression, and metastasis through multiple mechanisms. Accumulated studies have reported the clinical implications and biological relevance of A20 in the development and progression of HCC. The underlying mechanisms of A20 in HCC include inhibition of epithelial–mesenchymal transition, protein tyrosine kinase 2 activation and Rac family GTPase 1 activity. Combining liver protection with tumor inhibition is a unique advantage of A20, which has the potential to be a novel treatment for promoting liver regeneration following liver resection in patients with HCC with liver cirrhosis. This review discusses the hepato-protective effect of A20 on hepatocytes and its potential role in cancer development, particularly its suppressor effect on HCC.