Growth-associated changes in fatty acid compositions of nuclear phospholipids of liver cells

To know the possible relationships between nuclear phospholipids and cell proliferation, we have extensively analyzed phospholipids extracted from the nuclei of rat hepatic cells at various growth states. The content of phospholipid in nuclei as well as its composition was similar among liver cells tested, i.e., the regenerating rat livers (28 h, post-hepatectomy), sham-operated or non-treated control livers, and rat ascites hepatoma, AH7974 cells. In contrast, the fatty acid compositions of phospholipids differed from each other among these cells. At the 2-position of phospholipids in the regenerating liver nuclei at 28 h after partial hepatectomy, 18:1 (oleic acid) increased transiently at the expense of 20:4 (arachidonic acid) and 22:6 (docosahexaenoic acid), compared with those in the sham-operated control nuclei. This change in fatty acid composition was commonly observed throughout all phospholipids analyzed, i.e., phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI) and phosphatidylserine (PS). On the other hand, the change at 1-position was rather limited: in the regenerating liver nuclei (28 h), 18:1 increased only in PC at the expense of 18:0 (stearic acid). The similar and more marked deviation at the 2-position was observed with AH7974 nuclei it contained approximately 2-times more of 18:1 in PC, PE and PI than regenerating liver nuclei (28 h), and the decreased levels of 20:4 and/or 22:6. It should be noted that there were significant differences in the fatty acid compositions of PE and PS between sham-operated and non-treated controls. So, the sham-operated rat is the appropriate control for proliferation.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

The conversion of orotic acid into uridine 5′-monophosphate by isolated perfused normal and regenerating rat livers

The release of (14)CO(2) from [7-(14)C]orotic acid was measured in isolated perfused normal and regenerating rat livers. With some limitations, the release of (14)CO(2) from [7-(14)C]orotic acid can be used to estimate UMP synthesis in perfused livers. Isolated perfused livers rapidly pick up labelled orotic acid added to perfusate and convert most of it into UMP. Perfused regenerating livers produce approx. 2.5 times as much UMP/g of liver as do perfused normal livers. However, the absolute amount of orotic acid converted into UMP is higher in perfused normal livers than in perfused regenerating livers. Perfused regenerating livers do not differ in their orotic acid uptake and UMP synthesis from livers of comparable size in which regeneration is not taking place. The total amount of orotic acid taken up by the liver (rather than the rate of uptake) and the size of the liver appear to be the determining factors in UMP production. The results suggest that the decrease in liver size caused by partial hepatectomy may be in itself sufficient to account for an increase in the flow of metabolites in the pyrimidine pathway at the early stages of liver regeneration.     

Analysis of hepatocyte cell membrane antigens in regenerating rat liver]

Antigens of plasma membranes in hepatocytes from regenerating rat liver were studied. Immunochemical investigation with polyvalent rabbits antiserum against plasma membrane proteins in hepatocytes from regenerating and normal rat liver have shown that liver regeneration processes are accompanied by the increase of proteins number with molecular weight of--80 kDa, 62 kDa, 40 kDa and 27 kDa. It is not excluded that protein with molecular weight of 27 kDa is the tissue-specific peripheral protein. The influence of antibodies against proteins of hepatocytes plasmatic membranes on histostructure of pathologically changed liver tissue has been studied. The data obtained testify to a possibility of participation of the above mentioned proteins in the regulation of rat liver regeneration processes.     

Fatty acid synthesis in the regenerating liver of the rat.

1. Synthesis de novo of fatty acids in the rat liver, measured per g wet wt. of tissue, was increased by a factor of about two, between 1 and 4 days after partial hepatectomy, compared with rates in sham-operated control rat livers. 2. There were no associated changes in the rates of liver cholesterol synthesis or of adipose-tissue fatty acid synthesis in rats after partial hepatectomy, compared with rates in sham-operated rats. 3. In regenerating livers, perfused under three different conditions, there was no alteration in the capacity for fatty acid synthesis compared with that of control rats. 4. The increased synthesis of fatty acids in regenerating liver was associated with insignificant increases in plasma concentrations of tricylglycerols and free fatty acids, with a decrease in content of liver glycogen, and with no change in hepatic activity of acetyl-CoA carboxylase. 5. The accelerated rate of synthesis of fatty adids in regenerating liver appears not to be due to any intrinsic alteration in hepatic capacity for fatty acid synthesis, but it may be caused by the continuous action on liver of unidentified circulating factors.     

Metabolism of different histone fractions under induction of rat liver regeneration]

Histone metabolism in liver studied within 72-hour period of liver regeneration after partial hepatectomy in 24 hours after the injection of 14C-amino acids in rats. The increase in radioactivity of f2a, f3 and f2b histones and the simultaneous decrease in f1 histone radioactivity was observed in regenerating rat liver as compared with the level of radioactivity estimated for the respective histones in ectomized liver lobes. These changes, which are characteristic for regenerating liver, were not observed after the shame operation and they did not eliminate after the injection of respective unlabelled amino acid. Possible correlation between the increase in specific radioactivity of most nuclear histones under regeneration process and a migration of pre-synthesized labelled histone molecules into nucleus, and also a transformation into histones of other nuclear proteins is discussed.     

Human augmenter of liver regeneration: molecular cloning, biological activity and roles in liver regeneration

The complete amino acid sequence of human augmenter of liver regeneration (hALR) was reported by deduction from nucleotide sequence of its complementary DNA . The cDNA for hALR was isolated by screening a human fetal liver cDNA library and the sequencing of this insert revealed an open reading frame encoding a protein with 125aa and highly homologous (87% ) with rat ALR encoding sequence. The recombinant hALR expressed from its cDNA in transient expression experiments in cos-7 cells could stimulate DNA synthesis of HTC hepatoma cell in the dose-dependent and heat-resistant way. Northern blot analysis with rat ALR cDNA as probe confirmed that ALR mRNA was expressed in the normal rat liver at low level and that dramatically increased in the regenerating liver after partial hepatectomied rat. This size of hALR mRNA is 1.4 kb long and expressed in human fetal liver, kidney and testis. These findings indicated that liver itself may be the resource of ALR and suggested that ALR seems to be an im-portant parac     

Transforming growth factor-beta (TGF-beta) isoforms in rat liver regeneration: messenger RNA expression and activation of latent TGF-beta.

Expression of transforming growth factor-beta s (TGF-beta s) 1-3 was studied in normal liver and during liver regeneration after partial hepatectomy in the rat to determine whether each of these isoforms might be involved in hepatocyte growth in vivo. Expression of the mRNAs for all three TGF-beta isoforms increases in the regenerating liver. In addition, the levels of expression of the mRNAs for several extracellular matrix proteins, including fibronectin, vitronectin, laminin, and collagen, also increase in the regenerating liver. Immunohistochemical staining analysis shows a similar distribution of all three TGF-beta s in normal and regenerating liver; however, in both tissues, the level of expression of TGF-beta 1 is 8- to 10-fold higher than that of TGF-beta 2 as determined by sandwich enzyme-linked immunosorbent assay. Expression of all three TGF-beta mRNAs is restricted to liver nonparenchymal cells. Although hepatocytes from normal and regenerating livers do not synthesize TGF-beta, they are sensitive to inhibition of growth by all three TGF-beta isoforms. Hepatocytes from regenerating livers are capable of activating latent TGF-beta 1 complexes in vitro, whereas normal hepatocytes are not. The different TGF-beta isoforms may function in an inhibitory paracrine mechanism that is activated during liver regeneration and may also regulate the synthesis of extracellular matrix components in the regenerating liver.     

Role of cell-surface modulator of DNA synthesis in liver regeneration

A cell-surface modulator of DNA synthesis by cultured rat hepatocytes was studied in relation to the liver regeneration process. When rat hepatocytes isolated 24 h after partial hepatectomy were cultured, the first burst of DNA synthesis peaked at 5-8 h and declined until 24 h, followed by the second burst. Rat liver plasma membranes added 2 h after plating inhibited only the second burst, while in the case of the normal hepatocytes where the DNA synthesis began to increase after 5 h, this inhibition was observed at 16 h but not at 8 h. The inhibition did not differ when the membranes obtained from regenerating livers 12 h after partial hepatectomy were used. Epidermal growth factor binding to the cultured hepatocytes was not hindered by the membranes. These results suggest that the modulator inhibits hepatocyte proliferation at the early G1-phase of the cell cycle and that its action might be controlled by other factors in the process of liver regeneration.     

Altered expression of diacylglycerol kinase isozymes in regenerating liver

The liver possesses the capacity to restore its function and mass after injury. Liver regeneration is controlled through complicated mechanisms, in which the phosphoinositide (PI) cycle is shown to be activated in hepatocytes. Using a rat partial hepatectomy (PH) model, the authors investigated the expression of the diacylglycerol kinase (DGK) family, a key enzyme in the PI cycle, which metabolizes a lipid second-messenger diacylglycerol (DG). RT-PCR analysis shows that DGKζ and DGKα are the major isozymes in the liver. Results showed that in the process of regeneration, the DGKζ protein, which is detected in the nucleus of a small population of hepatocytes in normal liver, is significantly increased in almost all hepatocytes. However, the mRNA levels remain largely unchanged. Double labeling with bromodeoxyuridine (BrdU), an S phase marker, reveals that DGKζ is expressed independently of DNA synthesis or cell proliferation. However, DGKα protein localizes to the cytoplasm in normal and regenerating livers, but immunoblot analysis reveals that the expected (80 kDa) and the lower (70 kDa) bands are detected in normal liver, whereas at day 10 after PH, the expected band is solely recognized, showing a different processing pattern of DGKα in liver regeneration. These results suggest that DGKζ and DGKα are involved, respectively, in the nucleus and the cytoplasm of hepatocytes in regenerating liver.     

Delayed liver regeneration after partial hepatectomy in adiponectin knockout mice

We previously demonstrated that adiponectin has anti-fibrogenic and anti-inflammatory effects in the liver of mouse models of various liver diseases. However, its role in liver regeneration remains unclear. The aim of this study was to determine the role of adiponectin in liver regeneration. We assessed liver regeneration after partial hepatectomy in wild-type (WT) and adiponectin knockout (KO) mice. We analyzed DNA replication and various signaling pathways involved in cell proliferation and metabolism. Adiponectin KO mice exhibited delayed DNA replication and increased lipid accumulation in the regenerating liver. The expression levels of peroxisome proliferator-activated receptor (PPAR) α and carnitine palmitoyltransferase-1 (CPT-1), a key enzyme in mitochondrial fatty acid oxidation, were decreased in adiponectin KO mice, suggesting possible contribution of altered fat metabolism to these phenomena. Collectively, the present results highlight a new role for adiponectin in the process of liver regeneration.     

Lack of evidence for a pyruvate kinase isozyme shift in hepatocytes of the regenerating rat liver

1. Pyruvate kinase isozyme shift in a regenerating rat liver was studied. Rats were subjected to a 70% hepatectomy and the liver homogenate or hepatocyte preparations were obtained from the regenerating liver. 2. Using thin layer polyacrylamide gel electrophoresis, liver homogenates from an intact normal rat appeared to contain the L-type isozyme in the greatest number and M2-type to a lesser extent. 3. The ratio of the M2- to L-type increased in the preparations obtained from the regenerating liver. 4. In the hepatocyte preparations prepared from a regenerating rat liver by the conventional method, a small amount of M2-type isozyme was detected. 5. However, the M2-type isozyme was hardly detected in the highly purified hepatocyte preparations prepared using Percoll. 6. Similar results were obtained by separation of the enzyme by DEAE cellulose column chromatography. 7. These results suggest that there is no pyruvate kinase isozyme shift from L- to M2-type in hepatocytes in the course of regeneration. 8. The increased M2-type isozyme in the regenerating rat liver is considered to originate from nonparenchymal cells.     

The nuclear matrix of slowly and rapidly proliferating liver cells.

The nuclear matrix of slowly proliferating rat liver is compared with rapidly proliferating regenerating liver and Zajdela ascites hepatoma cells. While no differences are detected in overall ultrastructure, composition or polypeptide profiles of normal liver versus regenerating liver matrices, significant alterations are observed in the polypeptides of Zajdela hepatoma nuclear matrices.     

Membrane potential measurements during rat liver regeneration

Membrane potential was measured in perfused rat liver and was shown to increase from ?33 ± 1.0 mV in livers from normal rats to ?50 ± 1.1 mV in livers from rats 12 hr after partial hepatectomy. The hyperpolarization of the membrane in regenerating liver was no longer evident after perfusion with 1 mM ouabain for 5 min. Ouabain had a small (4 mV) depolarizing effect on membrane potential in normal liver. The potential measured in normal and regenerating liver decreased as a function of the external potassium concentration above 5 mM; however, the potential was more electronegative in regenerating liver compared to normal liver at all values of external potassium concentration, and the differences in potential between the two kinds of cells did not decrease at higher concentrations of external potassium. Thus, a plot of membrane potential vs external potassium concentration resulted in approximately parallel curves for the two different cell types. We conclude that hyperpolarization of the liver cell membrane is an early event during rat liver regeneration and results from an electrogenic Na-K pump.     

Rates of degradation of glycoproteins from normal and regenerating rat livers: A study using double isotopes

The average decay rates (half-lives) of mixed glycoproteins were measured using double isotopes of fucose and glucosamine and compared to those of mixed overall proteins measured with leucine and NaH¹⁴CO₃ in whole homogenates and plasma membranes from normal and regenerating rat livers. A large reutilization of leucine was observed under both normal and regenerating conditions. Fucose seems to be recycling most predominantly in regenerating liver, whereas glucosamine was found to be very little if not at all reutilized under both conditions. Comparison of the results obtained with NaH¹⁴CO₃ and glucosamine demonstrated that glycoproteins from normal liver homogenate are degraded at a faster rate than mixed proteins. Contrary to that of mixed proteins, the half-life of glycoproteins remains unchanged during liver regeneration, and the use of glucosamine revealed that the degradation of plasma membrane glycoproteins is identical to that found in whole homogenate under both normal and regenerating conditions. Finally, the relative degradation rates of fractionated plasma membrane proteins and glycoproteins were evaluated under the same conditions. During liver regeneration some readjustments are observed in the relative degradation rates of individual species which suggest that the synthesis and degradation of the various surface membrane glycoproteins proceed at rates that are controlled independently.     

Alteration of rat liver proteoglycans during regeneration.

Sepharose CL-6B column chromatography of crude extracts from the slices of regenerating rat livers after partial hepatectomy and sham-operated controls labeled with [35S]sulfuric acid revealed an enhancement of [35S]sulfate incorporation into proteoglycan fractions during regeneration. The 35S-labeled proteoglycans contained heparan sulfate (more than 80% of the total) and chondroitin/dermatan sulfate. The 35S-incorporation into both glycosaminoglycans increased to maxima 3-5 days after partial hepatectomy and decreased thereafter toward the respective control levels. When [35S]sulfuric acid was replaced by [3H]glucosamine, similar results were obtained. These results suggest that the maximal stimulation of proteoglycan synthesis in regenerating rat liver follows the maximal mitosis of hepatic cells 1-2 days after partial hepatectomy. The 35S-labeled proteoglycans from regenerating liver 3 days after partial hepatectomy and control were analyzed further. They were similar in chromatographic behavior on a gel filtration or an anion-exchange column and in glycosaminoglycan composition. Their glycosaminoglycans were indistinguishable in electrophoretic mobility. However, these proteoglycans were slightly but significantly different in their affinity to octyl-Sepharose and in the molecular-weight distribution of their glycosaminoglycans.     

Tumor necrosis factor-alpha increases hepatic DNA and RNA and hepatocyte mitosis.

Tumor necrosis factor-alpha (TNF) has been reported to increase DNA synthesis in normal rat liver. Therefore, we examined the effects of TNF on rat liver regeneration. TNF, 1.5 micrograms ip every 4 h for 5 d, significantly increased hepatic DNA and RNA contents of regenerating and sham operated livers by up to 45%. Mitotic figures in sham operated liver, usually rare, were increased substantially by TNF. ODC mRNA content and enzyme activity were increased in regenerating liver, and were further increased by TNF. These data indicate that TNF, although not specific for regenerating liver, is a potent stimulus for hepatocyte DNA synthesis and mitosis.