Functional changes of endoplasmic reticulum membranes associated with liver regeneration

The fluidity and lipid composition of microsomal membranes have been studied at the earliest stage of liver regeneration in the rat (16 h after partial hepatectomy). The physical properties of the membranes have been measured by electron paramagnetic resonance analysis of freedom of motion of lipid and protein analogue probes. The fluidity of the hydrophobic core and of the microenvironment surrounding membrane proteins appeared to be modified, while no modifications were detectable in the fluidity at the surface or in bulk biochemical composition. The kinetic parameters of two enzymes of the endoplasmic reticulum (3-hydroxy-3-methyl glutaryl coenzyme A reductase and glucose-6-phosphatase) which are differentially localized within the membrane bilayer, were also measured. The temperature dependence of both enzymes was modified in the proliferating system, but these modifications were not consistent with the changes detectable in their specific activity. A model to explain the changes that occur in this proliferating membrane system is presented.     

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.     

Nuclear membrane sphingomyelin-cholesterol changes in rat liver after hepatectomy.

Sphingomyelin and cholesterol play an important role in stabilising the plasma membranes architecture and in many physiological process such as cell growth and differentiation. Degradation of sphingomyelin by exogenous sphingomyelinase induces a decrease of cholesterol due either to an increase of esterification or to a reduced biosynthesis. Variations of sphingomyelin due to the presence of a neutral-sphingomyelinase and of sphingomyelin-synthase have been recently shown in rat liver nuclear membranes. The aim of this research is to study the relation between sphingomyelin and cholesterol in the nuclear membranes following sphingomyelinase activation and during cell proliferation. The nuclear membranes, isolated from liver nuclei, were analysed for their content in protein, nucleic acids, and lipids (sphingomyelin and cholesterol) before and after sphingomyelinase activation and during hepatic regeneration. The activities of nuclear membrane SM-syntase and sphingomyelinase were also determined. The results confirmed that also in the nuclear membranes sphingomyelinase, especially exogenous, causes a strong decrease in cholesterol. The increase observed of sphingomyelin during the first 18 h after hepatectomy followed by a decrease at 24 h, due to the different activity of the enzymes, is accompanied by similar behaviour of cholesterol. This confirms the effect of neutral-sphingomyelinase on cholesterol, due to an increase of esterification process. Changes in cholesterol content modify the nuclear membranes fluidity and, as consequence, mRNA transport as previously shown. It can therefore be concluded that the neutral sphingomyelinase, present in the nuclei, may, across this mechanism, regulate the cell function.     

Studies on regenerating liver and hepatoma plasma membranes--I. Lipid and protein composition

1. Plasma membranes were isolated from normal liver, Morris hepatoma 7288C and regenerating liver, 6, 15, 24, and 48 hr after partial hepatectomy. 2. The cholesterol/phospholipid ratio was lower in regenerating liver 6 hr after partial hepatectomy (0.51) compared to the sham control (0.68), returning to normal after 15 hr. This was accompanied by a small increase in palmitic acid (16:0). There were no other changes in the lipid composition in regenerating hepatocytes in the first 48 hr after partial hepatectomy. 3. Analysis of lipid composition showed a higher cholesterol/phospholipid ratio in the hepatoma plasma membrane compared to normal liver accompanied by an increase in saturation of the fatty acyl groups of the phospholipids. There were also significant changes in the phospholipid classes. 4. There was no change in the two-dimensional electrophoretic profile of membrane proteins in the early stages of liver regeneration, however hepatoma membranes showed significant differences in protein profile. 5. These changes in the lipid composition of the hepatoma plasma membrane would have the effect of decreasing the average fluidity of the membrane and together with the changes in protein composition may be significant in the altered growth of the hepatoma. Changes in the lipid composition of the hepatocyte plasma membrane early in liver regeneration may reflect the onset of renewed cell division.     

Sequential changes in alanine metabolism following partial hepatectomy in the rat

After partial hepatectomy, the liver undergoes an array of metabolic changes until regeneration is complete. Since carbons derived from alanine can be incorporated into most metabolic pools, we studied the metabolism of (14)C-labeled alanine during the early phase of regeneration. Sham operated (controls) and partially hepatectomized rats weighing about 200 g each were injected intraperitoneally with 1-[U-(14)C]alanine at 9, 18, and 36 hours after surgery. The animals were killed 2 hours after injection. Compared to the controls, alanine oxidation was markedly depressed (P < 0.05) in the 9- and 18-hour groups, but was restored in the 36-hour group. The specific activity of plasma glucose and hepatic glycogen was elevated 9 and 18 hours after partial hepatectomy. There was a corresponding increase in the activities of fructose-1,6-diphosphatase and phosphoenolpyruvate carboxykinase. Hepatic protein specific activity increased by 30, 74, and 120%, respectively 9, 18, and 36 hours after partial hepatectomy. Hepatic fatty acids followed a similar pattern. In a separate set of experiments, the distribution of radioactivity in glutamic acid was measured. The results showed that alanine carbons enter the citric acid cycle primarily via the acetyl CoA pathway in the controls, but via the oxaloacetate pathway in partially hepatectomized rats. The results demonstrate significant changes in the activities of metabolic pathways of alanine in the early phase of hepatic regeneration.     

Intracellular distribution of transglutaminase activity during rat liver regeneration

Transglutaminase and ornithine decarboxylase activities have been assayed at intervals after partial hepatectomy in regenerating liver cells fractionated to obtain nuclear, cytoplasmic-particulate, and cytoplasmic-soluble fractions. Ornithine decarboxylase activity, localized entirely in the cytoplasmic fractions, undergoes a dramatic induction during the first 4 h after partial hepatectomy and remains elevated. This induction is very sensitive to inhibition by cycloheximide and actinomycin D, as previously reported. Transglutaminase activity is localized in both the cytoplasm and the nucleus with the highest specific activity in the nucleus. Nuclear transglutaminase activity approximately doubles in the first 2 h of liver regeneration, apparently as a result of a translocation of enzyme from the cytoplasm to the nucleus. Inhibitor studies indicate that the translocation is not dependent upon protein or RNA synthesis. In the first 2 h, actinomycin D slightly activates transglutaminase activity in the cytoplasmic-particulate and nuclear fractions. Only at 4 h after the onset of regeneration do actinomycin D and cycloheximide show some inhibition of transglutaminase activity indicating de novo synthesis at this time. A broad increase of transglutaminase activity occurs from hours 12–16 to hour 32 after partial hepatectomy in the nuclear and cytoplasmic-particulate fraction. These data suggest the existence of a function for transglutaminase in the nucleus of rat liver cells.     

Plasma membrane changes associated with rat liver regeneration

The lipid composition and fluidity of plasma membranes have been studied at different stages of liver regeneration (4, 15 and 24 h after surgery). The phospholipid and fatty acid composition is not modified, whereas the cholesterol/phospholipid ratio is lower with respect to control membranes. The modification of the physical properties of the membranes has been studied directly by EPR analysis and indirectly by temperature dependence and cooperativity of some membrane-bound enzymes (Mg2+-ATPase, (Na+ + K+)-ATPase and 5'nucleotidase). Surgical operation or anaesthesia alone causes an early increase in fluidity; such an effect appears to be markedly reduced at a later stage. There seems to be a marked effect of regeneration on plasma membrane fluidity 15 h after partial hepatectomy when several parameters--surface fluidity, cholesterol/phospholipid ratio, and 5'-nucleotidase activity in the presence of concanavalin A -- are modified and indicate an increase in membrane fluidity. It is suggested that this modification of membrane properties could be related to the proliferative process.     

Effect of lipid composition on rat liver nuclear membrane fluidity

Nuclear membrane fluidity is measured in rat liver by use of the fluorescence anisotropy of two probes: diphenylhexatriene and its cationic derivative trimethylammonium-diphenylhexatriene. It has been shown that, in 2-month-old rat liver cells, the bilayer surface is less fluid than the hydrophobic core. The fluidity was higher in 6-day-old rat liver nuclei, in which both the amount of cholesterol and the cholesterol/phospholipid ratio decreased. The influence of the single phospholipids, and in particular of phosphatidylcholine, has been studied by increasing the phosphatidylcholine with a choline base exchange reaction in isolated nuclear membranes. After this reaction, the fluorescence anisotropy of the bilayer surface increased, whereas at the hydrophobic core it decreased. Analysis of fatty acid composition shows an increase of phosphatidylcholine unsaturated fatty acids. The results show that the fluidity of nuclear membranes changes in relation to the lipid content and to the fatty acid composition. The role of nuclear membrane fluidity in cell function is discussed. © 1997 John Wiley & Sons, Ltd.     

Decrease of calmodulin and actin in the plasma membrane of rat liver cells during proliferative activation

After proliferative activation of rat liver cells in vivo by a partial hepatectomy a decrease of the calmodulin content in the three plasma membrane domains (blood sinusoidal, canalicular and lateral) was observed. At 24 hours after partial hepatectomy calmodulin was found to be 3 fold lower in the sinusoidal and lateral fractions whereas a 2 fold decrease was detected in the canalicular domain. Decreases on the actin levels have been also detected at 24 hours after a partial hepatectomy. Since at this time after surgery increases on nuclear actin and calmodulin have been reported, these results suggest the possibility that the actin and calmodulin dissociated from the plasma membrane after a partial hepatectomy could subsequently be translocated into the nuclei.     

Characterization and cell cycle kinetics of hepatocytes during rat liver regeneration: in vivo BrdUrd incorporation analysed by flow cytometry and electron microscopy

The incorporation of bromodeoxyuridine (BrdUrd) into newly synthesized DNA has been analysed during hepatocellular regeneration induced by partial hepatectomy in young rats. The kinetic state of the liver has been studied by flow cytometric analysis of the incorporated BrdUrd, while the fine localization of DNA replication sites through the cell cycle has been investigated at the ultrastructural level by the immunogold technique. Eighteen hours after partial hepatectomy flow cytometry revealed an early S phase distribution which corresponded to a specific staining of the interchromatin domains of the hepatocyte nucleus. Thirty-four hours after hepatectomy, on the other hand, when most cells were in late S, a specific staining of heterochromatin domains was observed. The effect of the BrdUrd technique on nuclear aggregation has also been analysed and discussed. The results demonstrate that specific patterns of DNA replication can be recognized during the cell cycle and that flow cytometry and electron microscopy appear to be complementary in the kinetic study of liver regeneration.     

Changes in the nuclear protein kinase activities in the regenerating liver of partially irradiated rat

X rays (4.8 Gy) inhibit both DNA synthesis and phosphorylation of histone H1 in the regenerating liver of the rat. To determine the cause of the inhibition of histone H1 phosphorylation, changes in the nuclear protein kinase activities during the prereplicative phase of regeneration were measured. The cAMP-dependent protein kinase activity was low during regeneration, and the changes in the activity were not statistically significant. The cAMP-independent protein kinase activity increased at 15 h, decreased at 18 h, and increased again at 24 h after partial hepatectomy. X irradiation prior to partial hepatectomy did not inhibit the increase at 15 h, but it did inhibit the increase at 24 h. The activity was not inhibited by isoquinolinesulfonamide inhibitors such as H-7, and it was activated by a commercial preparation of an inhibitor protein of the cAMP-dependent kinase. It was also inhibited by quercetin. The possibility that the radiation-sensitive nuclear protein kinase is a nuclear cAMP-independent protein kinase specific for histone H1 is considered.     

大鼠2/3肝切除72 h后再生肝脏质膜比较蛋白质组学研究

大鼠2/3肝切除模型为研究肝细胞增殖和生理性血管生成提供了一个很好的活体内模型.为了揭示肝再生过程中与肝细胞增殖终止相关及与血管生成启动相关的质膜蛋白质,本研究对大鼠肝2/3部分切除72 h后的肝脏质膜进行了研究：利用两步蔗糖密度梯度离心法对切除组和假手术组的肝脏质膜进行纯化;然后通过双向电泳和质谱技术对肝切除样品进行了比较分析并对几个关键蛋白程序性凋亡相关蛋白-6和丝蛋白-A进行了免疫印迹验证.相对于假手术对照组(Sham组),21种蛋白质在切除后72 h的肝脏中上调,15种蛋白质下调.所鉴定的差异表达蛋白参与了血管生成、细胞分裂增殖和凋亡、细胞分化调控、肝脏组织重新构建、代谢及应急反应.本研究为肝脏再生及其血管生成的研究提供了理论依据.     

Disulfide reductase activity in mouse regenerating liver

Fourteen hours after partial hepatectomy there was a decrease in basal disulfide reductase and glutathione reductase activity in cytosole fraction of proliferating hepatocytes. In nuclear fraction, the activation effect of cAMP and cGMP on the disulfide recovery was replaced by inhibition. Meanwhile the activity of glutathione reductase noticeably increased. Forty-five hours after operation disulfide reductase activity of cytosole appreciably rose during maximal mitotic activity of the regenerating liver. The data obtained provide evidence in favor of the involvement of disulfide reductase enzymes into reparative regeneration of the liver.     

Formation of superoxide radicals in membranes of subcellular organelles in regenerating liver]

A correlation between the changes in the rates of superoxide radical generation, upsilon, in microsomes, mitochondria, and nuclei and the Cu, Zn- and Mn-SOD activities in rat liver during the first 5 days after partial hepatectomy, has been studied. Level of upsilon in microsomal and mitochondrial membranes in the regeneration process was reduced. The Cu, Zn- and Mn-SOD activities changed in an extreme and antibate manner: the former was characterized by a minimum, whereas the latter-by a maximum with an extreme on the 3rd day after surgery. Analysis of the correlation between the values of upsilon in the nuclear membranes and cell cycle stages (on a literary basis) revealed that the upsilon was decreased 2 times on the stage of DNA synthesis. When mitosis was at maximum, upsilon showed a 4-5-fold increase in comparison with the control, the Cu, Zn-SOD activity being essentially unchanged. A role of SOD and O2-. in cell division is postulated. O2-. is assumed to play a role in gene expression, disassembly, and regeneration of the nuclear membrane; that of SOD is thought to consist in regulation of the proliferative activity.     

Changes in activity and mRNA levels of poly(ADP-ribose) polymerase during rat liver regeneration

ADP-ribosylation of nuclear proteins, catalysed by the enzyme poly(ADP-ribose) polymerase, is involved in the regulation of different cellular processes of DNA metabolism. To further clarify the role of the enzyme during proliferating activity of mammalian cells, we have studied the control of gene expression in regenerating rat liver. The changes in activity and mRNA levels were analysed during the early and late phases of the compensatory model. When enzyme activity was measured in isolated liver nuclei obtained at different times after hepatectomy, two different phases were observed: an early wave occurring before the onset of DNA synthesis, and a second one, starting several hours after the onset of DNA synthesis and returning to control values at later times. The evaluation of the enzymatic level in nuclear extracts and by activity gel analysis showed a more gradual increase starting 1 day after hepatectomy, in concomitance with the peak of DNA synthesis. By using a specific murine cDNA probe, a significant enhancement of mRNA levels for poly(ADP-ribose) polymerase was observed during liver regeneration, slightly preceding the onset of DNA synthesis. The results obtained show that changes in poly(ADP-ribose) polymerase activity, during liver regeneration, are associated both to early events preceding the increase in DNA synthesis and to later phases of the cell proliferation process.     

Calmodulin may decrease cell surface sialic acid and be involved in the expression of fibronectin during liver regeneration

The decrease of sialic acid in plasma membrane glycoproteins and the expression of cell surface fibronectin were studied during the pre-replicative phase of liver regeneration. The aim of this study was to correlate these cell-surface events to the intracellular surge of calmodulin observed a few hours after partial hepatectomy. The fact that calmodulin decreased the specific activity of UDP-N-acetyl-D-glucosamine 2'-epimerase, a key regulatory enzyme in the biosynthesis of glycoprotein sialic acids, and that trifluoperazine prevented the desialylation indicates that the membrane desialylation is a calmodulin-dependent process. On the other hand, Western blotting using anti-rat fibronectin antibody in trifluoperazine-treated animals suggests that calmodulin may also be involved in the surface expression of fibronectin in regenerating hepatocytes.     

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

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

Hepatic pyruvate metabolism during liver regeneration after partial hepatectomy in the rat

Hepatic pyruvate kinase (PK) and pyruvate dehydrogenase (PDHa) specific activities were decreased after partial hepatectomy or sham operation. The decreases were more marked and sustained after partial hepatectomy. These activity changes ensure that hepatic carbon flux after partial hepatectomy is predominantly in the direction of gluconeogenesis. The decrease in PK specific activity observed after partial hepatectomy was associated with a decreased PK activation ratio (activity measured at 0.15 mM PEP: activity measured at 5.0 mM PEP), and hepatic concentrations of PEP were increased. The low hepatic PDHa activity observed at the first day after partial hepatectomy occurred concomitantly with an increased fatty acid concentration. PDHa activity increased after inhibition of lipolysis. The results indicate that carbohydrate utilization is unimportant for hepatic energy supply during liver regeneration. There was no evidence that the control of PK or PDH in the regenerative liver after partial hepatectomy differed from that observed in the liver of the unoperated rat.     

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.