Effects of partial and sham hepatectomy on ornithine decarboxylase and thymidine kinase activities and mRNA contents

Ornithine decarboxylase and thymidine kinase are enzymes that increase in activity in regenerating liver. We found that both activities and mRNA levels for these enzymes increase significantly after 70% partial hepatectomy in the rat. After sham hepatectomy (laparotomy) there were significant decreases in activity; however, mRNA content was unaltered. Similar decreases in enzyme activity, without changes in mRNA content, were found with pair-feeding, and additional decreases in activity after starvation. In contrast to previous reports of no change in ornithine decarboxylase and thymidine kinase after sham hepatectomy, the present results indicate that decreases occur. This may be mediated by the decrease in food intake after surgery. Dietary factors may be important in the physiologic regulation of these enzymes in the liver.     

Endogenous plasma lipoprotein lipase activity in fed and fasting rats may reflect the functional pool of endothelial lipoprotein lipase

In this study, a correlation was sought between the circulating lipoprotein lipase activity and nutritional state in the rat. In fed rats, the plasma lipoprotein lipase activity was between 30 and 120 munits/ml, whereas after an overnight fast in restraining cages, the lipoprotein lipase plasma levels were between 280 and 500 munits/ml. The plasma lipoprotein lipase activity was inhibited by a specific high titre goat antiserum to rat lipoprotein lipase. No effect of fasting was seen on the plasma hepatic triacylglycerol lipase. 6 h after fasting, adipose tissue lipoprotein lipase decreased maximally, but plasma lipoprotein lipase was not changed and rose only after 16 h. Thus, it seems that most of the lipoprotein lipase activity in the fasting plasma was related to the 3-fold rise in lipoprotein lipase activity in the heart, which may represent total muscle lipoprotein lipase. The increase in heart lipoprotein lipase was due in part to an increase in the t1/2 of the enzyme from 1.2 to 2.9 h. To determine whether the high plasma levels in the fasting rats might result from impaired clearance of the enzyme by the liver, functional hepatectomy was carried out. 15 min after hepatectomy, plasma lipoprotein lipase rose up to 20-fold in fed and about 6-fold in fasting rats. Lipoprotein lipase activity extracted by the liver was calculated to be 30-60 munits/ml in the fed and 171-247 munits/ml plasma per min in fasting rats. An increase in lipoprotein lipase activity in extrahepatic tissues (heart, lung, kidney, diaphragm and adrenal) occurred 30 min after hepatectomy in fed rats. The increase in heart lipoprotein lipase was due to an increase in heparin-releasable fraction. Since no impairment of hepatic clearance of circulating plasma lipoprotein lipase was found, the high fasting plasma lipoprotein lipase activity may be related to an increase in enzyme synthesis, decreased enzyme turnover and an expansion of the functional pool in tissues such as the heart and probably muscle. The present findings indicate that measurement of endogenous plasma lipoprotein lipase can provide information with respect to the size of the functional pool under normal and pathological conditions.     

Enhancement of neutral phosphatase activity in the cytosol and nuclei of regenerating rat liver: role of endogenous regucalcin.

The role of endogenous regucalcin (RC) in the regulation of neutral phosphatase activity in regenerating rat liver was investigated. The liver weight reduced by a partial hepatectomy (about 70%) was completely restored at 72 h after surgery. Phosphotyrosine, phosphoserine, and phosphothreonine were used as the substrate for the assay of phosphatase activity. Phosphatase activity toward phosphotyrosine in the hepatic cytosol and nuclei was significantly increased at 24-72 h after hepatectomy. Such an increase was not seen in the case of phosphoserine and phosphothreonine. However, the presence of anti-RC monoclonal antibody (200 ng/ml) in the enzyme reaction mixture caused a remarkable elevation of phosphatase activity toward three phosphoaminoacids in the hepatic cytosol at 24 and 48 h after hepatectomy. In the liver nuclei after sham operation or hepatectomy, phosphatase activity toward three phosphoaminoacids was significantly raised by the addition of anti-RC antibody (150 ng/ml). The nuclear phosphatase activity toward phosphothreonine in regenerating liver was significantly enhanced in the presence of anti-RC antibody (100 and 150 ng/ml). The effect of anti-RC antibody to increase phosphatase activity toward three phosphoaminoacids in the cytosol and nuclei of regenerating liver was completely blocked by the addition of exogenous RC (1.0 microM). The present study demonstrates that protein phosphatase activity in the cytoplasm and nuclei is enhanced in regenerating rat liver. This enhancement may be suppressed by endogenous RC.     

Adult rats prenatally exposed to ethanol have increased gluconeogenesis and impaired insulin response of hepatic gluconeogenic genes.

Rat offspring exposed to ethanol (EtOH rats) during pregnancy are insulin resistant, but it is unknown whether they have increased gluconeogenesis. To address this issue, we determined blood glucose and liver gluconeogenic genes, proteins, and enzyme activities before and after insulin administration in juvenile and adult EtOH rats and submitted adult EtOH rats to a pyruvate challenge. In juvenile rats, basal glucose; peroxisome proliferator-activated receptor-coactivator-1alpha protein and mRNA; and phosphoenolpyruvate carboxykinase enzyme activity, protein, and mRNA were similar between groups. After insulin injection, these parameters failed to decrease in EtOH rats, but glucose decreased by 30% and gluconeogenic enzymes, proteins, and mRNAs decreased by 50-70% in control rats. In adult offspring, basal peroxisome proliferator-activated receptor-coactivator-1alpha protein and mRNA levels were 40-80% higher in EtOH rats than in controls. Similarly, basal phosphoenolpyruvate carboxykinase activity, protein, and mRNA were approximately 1.8-fold greater in EtOH rats than in controls. These parameters decreased by approximately 50% after insulin injection in control rats, but they remained unchanged in EtOH rats. After insulin injection in the adult rats, glucose decreased by 60% in controls but did not decrease significantly in EtOH rats. A subset of adult EtOH rats had fasting hyperglycemia and an exaggerated glycemic response to pyruvate compared with controls. The data indicate that, after prenatal EtOH exposure, the expression of gluconeogenic genes is exaggerated in adult rat offspring and is insulin resistant in both juvenile and adult rats, explaining increased gluconeogenesis. These alterations persist through adulthood and may contribute to the pathogenesis of Type 2 diabetes after exposure to EtOH in utero.     

Effects of hormones, fasting and diabetes on triglyceride lipase activities in rat heart and liver

Male rats were fasted for 3 days, subjected to streptozotocin-diabetes or injected with L-thyroxine, Kenacort-A40 (corticosteroid) and Synacthen (ACTH). Cardiac heparin-releasable lipoprotein lipase (LPL) activity was increased after fasting, experimental diabetes and all hormone treatments. Cardiac neutral lipase activity was decreased during diabetes and enhanced in the fasted state and by L-thyroxine, corticosteroid and ACTH administration. The close correlation between vascular LPL and tissue neutral lipase with cardiac triglyceride content is in agreement with the contention that tissue neutral lipase is similar to LPL (Hülsmann, Stam and Breeman 1982). Myocardial acid lipase activity was reduced during diabetes and L-thyroxine treatment, increased during fasting and corticosteroid administration and not affected by short-term ACTH treatment. Hepatic acid lipase activity was increased during fasting, diabetes and by L-thyroxine and reduced after corticosteroid and ACTH treatment. The alkaline liver lipase activity was depressed by fasting, experimental diabetes, corticosteroid and ACTH treatment, whereas L-thyroxine induced a slight increase in enzyme activity. The possible mechanism underlying the observed changes in acid, neutral, alkaline, and LPL activities in heart and liver are discussed.     

Glucose homeostasis during the early stages of liver regeneration in fasted rats

Kinetic studies with [2-3H]glucose in vivo and gluconeogenic activity measurements in vivo and in vitro were performed in 70% hepatectomized rats submitted to fasting, which represents an extra burden for glucose synthesis but does not impair liver regeneration. Rates of glucose replacement, under steady-state conditions, 14 and 24 h postoperatively, did not differ in partially hepatectomized fasted rats and sham-operated controls. Phosphoenolpyruvate carboxykinase activities increased more rapidly during fasting in remnant livers than in intact livers from controls. Rates of incorporation of 14C from alanine into circulating glucose in hepatectomized rats were already maximal 14 h after surgery, whereas in controls they continued to augment. The maximal rates after partial hepatectomy could not be surpassed by performing the operation in diabetic animals. It is concluded that the relatively high blood sugar levels during fasting in hepatectomized rats do not depend on a reduced peripheral utilization of glucose, but only on a rapid increase in the gluconeogenic activity. The data suggest that hepatocytes in remnant liver can proliferate under conditions of maximal gluconeogenic and low glycolytic activities.     

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。     

Concentration of liver and kidney fructose-1,6-diphosphatase determined by specific radioimmunoassay

A radioimmunoassay for liver fructose-1,6-diphosphatase (D-fructose-1,6-bisphosphate 1-phosphohydrlase, EC 3.1.3.11) has been developed based on maintenance of its tetrameric structure and immunologic integrity after iodination by the Bolton-Hunter technique. The assay detected as little as 2 ng of standard enzyme. Nonspecific interference by tissue components did not occur. Enzyme concentration (mumol/1000 g tissue wet weight) was measured in tissue extracts of 49 rabbits subjected to a variety of conditions. In animals fed a 'balanced' diet containing 50--60% carbohydrate (by weight), the concentration in liver was 3.4 microM +/- 0.3. After fasts of 48, 72, or 96 h, the concentration in liver increased approximately 1.4-fold. A high-fat diet did not alter the concentration significantly but a high-protein diet caused an increase of 2.1-fold to 7.2 microM +/- 1.4. The greatest concentrations, 8.7 microM +/- 1.9, were observed in the livers of severely diabetic rabbits. The increase paralleled the increasing severity of diabetes and provides one explanation for the augmented gluconeogenesis which occurs in the diabetic state. Changes were less marked in kidney. The greatest apparent incrase, from 2.6 microM +/- 1.1 in the normal fed rabbit to 4.7 microM +/- 2.8, occurred in the severely diabetic animal. However, variation was sufficiently great in kidney to render apparent increases during fasting, protein feefing and diabetes statistically insignificant. For the most part changes in assayable activity followed changes in enzyme concentration except in the rabbits maintained on high-protein diets. In these, liver enzyme concentration increased by 2.4-fold whereas activity increased by only 1.3-fold, and the kidney enzyme concentration increased 1.3-fold whereas activity decreased by 20%.     

Fasting and refeeding cause rapid changes in intestinal tissue mass and digestive enzyme capacities of Atlantic salmon (Salmo salar L.)

Fasting and refeeding effects on gastrointestinal morphology and digestive enzyme activities of Atlantic salmon, held in tanks of seawater at 9 degrees C and 31 per thousand salinity, were addressed in two trials. Trial 1: Fish (mean body mass 1190 g) were fasted for 40 days and intestines sampled at day 0, 2, 4, 11, 19 and 40. Trial 2: Fish (1334 g), fasted for 50 days, were refed and sampled at day 0, 3 and 7. Mass, length, protein, and maltase, lactase, and leucine aminopeptidase (LAP) activities were analyzed for stomach (ST), pyloric caeca (PC), proximal (PI), mid (MI), and distal intestine (DI). PC contributed 50% of gastrointestinal mass and 75% of enzyme capacity. Fasting decreased mass and enzyme capacities by 20-50% within two days, and 40-75% after 40 days. In PC, specific brush border membrane (BBM) maltase activity decreased whereas BBM LAP increased during fasting. Upon refeeding, enzyme capacities were mostly regenerated after one week. The results suggest that refeeding should start slowly with about 25% of estimated feed requirement during the first 3 days, but may then be stepped up rapidly. Investigations of digestive processes of fed fish should only be performed when intestines are feed-filled to avoid bias due to effects of fasting.     

Adenosine aminohydrolase activity in the regenerating rat liver

The specific activity of adenosine aminohydrolase in the regenerating rat liver is significantly increased 12 h after partial hepatectomy. There is a twofold increase in enzyme activity at 48 h, after which the activity begins to decline. However, increased values still persist 7 days postsurgery. The enzyme is located mainly in the soluble supernatant (90-95%) of the cell. The purified enzyme from 48-h regenerating liver and control liver has similar kinetic properties (Km 54-58 microM for adenosine), similar molecular weights (30,000-35,000), and are equally inhibited by an irreversible transition-state analog and a reversible competitive inhibitor. It is concluded that adenosine aminohydrolase in regenerating liver is an integral component of a salvage pathway designed for the reutilization of nucleotides, and thus helps maintain a "growth state" for the regenerating liver.     

Influence of subtotal hepatectomy on peroxisomes and peroxisomal enzymes of rat liver and isolated liver cell fractions.

The activities of peroxisomal enzymes of rat liver were followed 1 to 10 days after subtotal (60-70%) hepatectomy in homogenates prepared from regenerating livers and in cell fractions isolated from them. Catalase activity was found to be depressed in the total liver homogenate (H) as well as in the mitochondrial (M) and soluble (S) fractions, while it did not change appreciably in the microsomal (Mc) and lysosomal (L) fractions. Alpha-hydroxyacid oxidase behaved in a similar fashion. In contrast to these enzymes, urate oxidase activity remained unchanged in H, whereas it was decreased in M and increased in L and Mc during the first 5 days after operation. These results agree well with the assumption that microbody proliferation is initiated by the fragmentation of large peroxisomes. The different relations of peroxisomal enzyme activities during regeneration time are discussed with respect to the possible existence of various kinds of peroxisomes with different enzyme equipments and with different turnover rates. Biochemical examinations ions were paralleled to morphological and histochemical studies. An early increase in number of peroxisomes was found to occur during the first day after partial hepatectomy, which is accompanied by decrease in particle size. During the first mitotic wave (24-36 hrs post op.) the number of peroxisomes per cell was reduced to about the half. After this time number and size of the particles began to increase. Positive staining of ribosomes was frequently observed in the vicinity of peroxisomes after the application of the cytochemical catalase reaction (alkaline diaminobenzidine medium). This phenomenon is interpreted to represent rather a diffusion artifact than the cytochemical identification of newly synthesized catalase.     

Effect of realimentation after several days' pure carbohydrate intake on DNA synthesis in regenerating rat liver

The authors studied the effect of realimentation after several days' isolated glucose or fructose intake on DNA synthesis in liver regenerating after partial hepatectomy (PH) (65-70%) or after carbon tetrachloride (CCl4) poisoning 1.5 ml/kg. Two days before PH or the administration of CCl4 and two days after, the experimental rats were given glucose (50% solution) of fructose (50% solution) as the only source of energy. Rats with PH were then fed for one day on a standard laboratory diet (25 cal% protein) or a high protein diet (81 cal% protein). Rats with CCl4 liver damage were fed for one day on the standard laboratory diet only. In the rats given glucose, liver DNA synthesis and the total amount of these nucleic acids in the liver 48 hours after CCl4 administration was lower than in the controls or the rats given fructose. In all the experimental groups (PH and CCl4), stimulation of liver DNA synthesis was observed after one day's realimentation. The total DNA content of the liver of rats with PH rose markedly during realimentation. The experiments indicate that the regenerative activity of damaged liver can be influenced by the nutritional regimen.     

Quantification of liver and kidney phosphofructokinase by radioimmunoassay in fed, starved and alloxan-diabetic rats.

A newly developed specific radioimmunoassay was used to quantify phosphofructokinase protein directly and independently of assayable activity in liver and kidney cytosol of normal fed, starved and alloxan-diabetic rats. In the fed state, liver phosphofructokinase concentration was 0.096 microM and the kidney enzyme was 0.086 microM (mumol/kg of tissue). In the starved state (24h), liver and kidney phosphofructokinase concentrations decreased by 30%. Prolonged starvation up to 72h did not further decrease enzyme concentration. In liver, total enzyme content during starvation declined by more than 50%, secondary also to a decrease in liver weight. In the alloxan-diabetic rats, there was a 22% decrease in enzyme protein concentration in liver and kidney. Total enzyme content per liver actually decreased much more (46%), because diabetes also resulted in a decrease in liver size. In conjunction with assayable activity measurements, the results of the radioimmunoassay allowed us to calculate the apparent specific activity of the enzyme. The specific activity of the kidney enzyme was 2-3 times that of the liver. Little or no change in specific activity of the liver or kidney enzyme occurred as a result of starvation or chemically induced diabetes. Tissue enzyme concentrations of phosphofructokinase unequivocally reconcile the ultimate results of changing rates of synthesis and degradation and are useful data in the design of spectrophotometric, kinetic, aggregation-disaggregation and other studies.     

Histone kinase and cell division

1. The activity of the soluble phosphokinase for histone F1 increases in regenerating rat liver during the first period of DNA synthesis after partial hepatectomy. The increase probably represents new enzyme synthesis. 2. A dose of 500rd of gamma-irradiation given early in G1 decreases the amount of histone F1 phosphokinase found 22h after partial hepatectomy by 60-70%. 3. The enzyme preparations also contained a histone F1 phosphatase; the presence together of the kinase and phosphatase caused a disproportion between net (31)P uptake and (32)P incorporation into histone F1. 4. All four subclasses of histone F1 could accept phosphate from ATP. 5. Crude enzyme preparations transferred more (31)P into histone F1 with an initially low phosphate content than into one with a high phosphate content; conversely, more (32)P was transferred into the latter.     

大鼠反复肝切除后再生肝组织的实验形态学研究

９０只雄性ＳＤ大鼠随机分为三组,每组各３０只,以第一次切肝量５０％（Ａ组）,７０％（Ｂ组）,８０％（Ｃ组）为分组标准,均作反复四次肝切除。动态观察其再生肝组织的酶组织化学,组织学及超微结构变化。结果显示：Ａ、Ｂ组三次术后累计存活率（８５％）明显高于Ｃ组（５７％）,Ｐ＜０．０１。Ａ、Ｂ、Ｃ各组三次术后累计切肝率各为１１４．２８％,１２０．７２％,１２５．８１％。Ａ、Ｂ组ＳＤＨ、ＡＴＰａｓｅ、ＬＤＨ活性改变相近,优于Ｃ组。各组ＣＨＥ活性均处于低水平。ＡＣＰ活性随切量,切次增加而增强。肝再生的方式既有小叶数量增多,面积增大,又有细胞肥大,细胞数量增加。电镜显示了增生活跃的肝细胞的核发裂像。所示线粒体结构受损与ＳＤＨ活性下降相对应。研究证明;大鼠反复肝切除是可行的,第一次切肝量,切次多少是影响大鼠耐受反复肝切除的重要因素。     

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.     

Parallel intestinal and liver injury during early cholestasis in the rat: modulation by bile salts and antioxidants

Whereas long-term cholestasis results in intestinal alterations and increased permeability to hepatotoxins, the effect of short-term cholestasis is less known and was investigated in bile duct ligated (BDL) rats. In the intestinal mucosa, at Day 7 BDL, total glutathione and protein sulfhydryl contents had decreased, oxidized glutathione levels increased (P<0.05 vs baseline), and a reduced epithelium thickness with dissolving crypt phenomena was observed in 40% of rats. At Day 10, total protein content, glutathione-related enzyme activities, and the transmural electrophysiological activity had decreased (-50%); by contrast, oxidized proteins doubled (P<0.05), and histological changes were extended to 70% of rats. In vitro exposure to taurodeoxycholate at micellar concentrations determined dysepithelization in normal gut but dissolving crypt phenomena and necrosis in cholestatic bowels. In the liver, ongoing cholestasis was associated with early oxidative changes especially in mitochondria, where protein sulfhydryls were decreased and negatively correlated with glutathione-protein mixed disulfides (r=-0.807, P<0.001). Daily oral administration of tauroursodeoxycholate, a hydrophilic bile salt, and glutathione to BDL rats improved intestinal histology, function, and redox state. In conclusion, short-term cholestasis results in distinctive functional, oxidative, and morphological changes of intestinal mucosa, determined increased vulnerability to toxic injury, and parallel hepatic oxidative damage.     

Possible endocrine control by hepatocyte growth factor of liver regeneration after partial hepatectomy.

Hepatocyte Growth Factor (HGF), which is a most potent growth factor for primary cultured hepatocytes, may act as a trigger for liver regeneration. After 70% of the rat liver was removed, HGF activity in the remnant liver began to increase within 24 h. In parallel with the activity, the HGF mRNA level in the remnant liver increased at 12 h after the operation and reached a maximum at 24 h. Increases in HGF activity and in the mRNA level were much lower and later than those in the liver of rats with hepatitis induced with CCl4. However, the first increase in HGF activity in the plasma of hepatectomized rats was noted 3 h after the resection, that is much earlier than the initial DNA synthesis in the remnant liver. Thus, while HGF production was induced in the remnant liver during regeneration after partial hepatectomy, the initial trigger may not be the liver-derived HGF, rather, it may be HGF derived from extrahepatic organs, via blood circulation.