Degradation of extracellular thymidine by cultured hepatocytes from rainbow trout (Salmo gairdneri)

1. Trout hepatocytes cultured as attached monolayers had low rates of [3H]-thymidine ([3H]-TdR) incorporation during replicative or repair synthesis of DNA. 2. Within 2 hr, most [3H]-TdR was metabolized by trout hepatocytes to a major product that eluted in advance of intact [3H]-TdR on Sephacryl S-200 columns. 3. Metabolism of [3H]-TdR by trout hepatocytes rapidly destroyed its ability to label replicating indicator cultures of proliferating rat hepatocytes. 4. These studies demonstrate that [3H]-TdR tracer assays for DNA synthesis cannot be reliably used in cultured trout hepatocytes which catabolize thymidine much more rapidly than do rat hepatocytes.     

Lipid and lipoprotein synthesis in isolated and cultured hepatocytes from lean and obese Zucker rats

Hepatocytes were isolated by EDTA perfusion of livers from lean (Fa/-) and obese (fa/fa) Zucker rats. Triacylglycerol (TG) and sn-glycerol 3-phosphate were increased in fa/fa hepatocytes, but free fatty acids, cholesterol and phospholipid concentrations were similar in both groups. In spite of an identical fatty acid uptake rate, glycerolipid synthesis was higher in obese compared to lean rat hepatocytes, and this difference remained for at least 2-3 days of culture. Triacylglycerol mass secretion was 2-fold higher in obese than in lean rat hepatocytes. This was confirmed by the higher incorporation of labeled glycerol and oleic acid into the medium TG fraction floating at density 1.006 g/ml. Density gradient ultracentrifugation of [14C]oleate-labeled lipoproteins showed that fa/fa hepatocytes secreted more TG-rich lipoproteins, and that 87% of the label was in the VLDL fraction compared with 67% in the medium of Fa/- hepatocytes. Decreased utilisation of leucine for protein synthesis in obese rat compared to lean rat hepatocytes was associated with enhanced leucine oxidation to CO2. [35S]Methionine incorporation showed an identical cell protein synthesis rate. Autoradiography after PAGE separation of secreted apolipoproteins (apoBh, Bl, apoA-VI, apoE, apoA-I, apoC) showed an identical pattern in both cell types.     

The effects of pravastatin,an HMG-CoA reductase inhibitor,on cell viability and dna production of rat hepatocytes

Some metabolites and products of mevalonic acid are involved in various cellular functions, particularly cell growth. In this study, we assessed the effects of pravastatin, a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, on cell viability and DNA production of rat hepatocytes stimulated with epidermal growth factor. Pravastatin (0.1 to 10μM) induced a dose-dependent reduction of DNA synthesis, assessed by ³H-thymidine incorporation in rat hepatocytes, which dropped by approximately 60% al a drug concentration of 10 μM. This suppression of DNA synthesis was nearly reversed by exogenous mevalonic acid, but was not prevented by purified low-density lipoprotein cholesterol. Pravastatin did not affect the mitochondrial reduction of Dimethylthiazolyl-diphenyl-tetrazolium bromide (MTT), but induced apoptotic change as assessed by nuclear chromatin staining. This apoptolic change was also reversed by exogenous mevalonic acid. These results indicate that mevalonic acid metabolites are necessary for DNA synthesis by rat hepatocytes stimulated by epidermal growth factor and for suppressing cell death.     

The effect of EGF and the comitogen, norepinephrine, on the proliferative responses of fresh and cryopreserved rat and mouse hepatocytes

The effect of cryopreservation on the proliferative response of fresh and cryopreserved (CP) rat and mouse hepatocytes was studied. Of the parameters measured, incorporation of 3H-thymidine and bromodeoxyuridine (BdrU) incorporation were the most sensitive and LDH content was the least sensitive. The optimal seeding density for epidermal growth factor (EGF)-stimulated proliferative response in fresh rat and mouse hepatocytes was 1.8 x 10(4) cells/cm2 and 2.1 x 10(4) cells/cm2, respectively. 3H-thymidine incorporation by fresh rat and mouse hepatocytes was maximal in cultures treated with 10 and 5 ng/ml EGF, respectively. The cell attachment of fresh rat hepatocytes after 48 h was higher (68%) than CP (42%), therefore, the CP hepatocyte seeding density was increased to 7.1 x 10(4) cells/cm2 so that the cell number after 48 h was the same as fresh hepatocytes. Using the adjusted seeding density, the 3H-thymidine and BdrU incorporation into fresh and CP rat hepatocytes was equivalent. The attachment efficiencies of fresh and CP mouse hepatocytes were the same, therefore, no adjustment was needed. The proliferative response (3H-thymidine incorporation and DNA content) to EGF was the same in fresh and CP mouse hepatocytes. The comitogen, norepinephrine (NE), increased the proliferative response to EGF to the same extent in both fresh and CP rat hepatocytes. In summary, cryopreserved rat and mouse hepatocytes retain their ability to proliferate in culture. Adjustment and monitoring of the seeding density is of high importance, especially with rat hepatocytes, which lose some attachment capacity after cryopreservation. The secondary mitogenic effect of NE is also retained by cryopreserved rat hepatocytes, suggesting that these cells retain alpha1-receptor function.     

15—Mt—PGF2α对CCL4所致大鼠离体肝细胞损伤的保护作用

采用大鼠离体肝细胞原代培养２４ｈ,并利用四氯化碳ＣＣｌ４造成急性肝细胞损伤模型,检定１５－甲基－前列腺素Ｆ２α（１５－Ｍｔ－ＰＧＦ２α）对肝细胞损伤的影响。结果表明：（１）１５－Ｍｔ－ＰＧＦ２α可显著降低中毒肝细胞脂质过氧化物水平,抑制肝细胞脂质过氧化,并降低谷丙转氨酶（ＧＰＴ）和谷草转氨酶（ＧＯＴ）水平,稳定脂质膜。（２）显著促进中毒肝细胞ＲＮＡ和ＤＮＡ的合成。（３）超微结构证实１５－Ｍｔ－ＰＧＦ２α能减轻ＣＣｌ４对肝细胞脂质膜,染色质,线粒体,内质网和核蛋白体的损害。     

Biological properties of a hepatocyte growth factor from rat platelets

In an accompanying communication we demonstrated that about half of the potency of rat serum to stimulate DNA synthesis in cultured adult rat hepatocytes resides in a polypeptidelike substance from the platelets. A lysate of rat platelets was able to restore the potency of platelet-poor rat serum, whereas a lysate of human platelets inhibited thymidine incorporation by the hepatocytes. Moreover, addition to these cultures of either highly purified human platelet-derived growth factor (PDGF) or human platelet factor 4 (PF-4) failed to influence DNA synthesis either alone or in the presence of rat or human platelet-poor serum, which is required for expression of PDGF activity. Unlike the human platelet factors, rat platelet lysate (RPL) was moderately active by itself and was augmented equally well by platelet-poor serum from either source. At concentrations below 5%, platelet-poor serum from hypophysectomized rats was as potent as that from normal rats in augmenting RPL activity. This suggests that, unlike PDGF, which is not activated by hypophysectomized rat serum, the hepatotrophic component of RPL does not require the presence of exogenous somatomedins for activity, but interacts instead with other plasma constituents or with somatomedins produced by the hepatocytes in vitro. Rat platelets do, however, appear to contain PDGF or its rat equivalent in addition to the hepatocyte growth factor, since if they are heated to 100 degrees C for 10 min, their ability to stimulate nuclear labeling in confluent BALB/c 3T3 cells is not impaired, while their ability to stimulate DNA synthesis in rat hepatocytes is destroyed. These studies indicate that the hepatocyte growth factor from rat platelets differs from PDGF in its biological as well as physical characteristics, but that rat platelets also contain PDGF or an equivalent substance.     

Growth stimulation of rat fetal hepatocytes in response to hepatocyte growth factor: modulation of c-myc and c-fos expression.

Hepatocyte growth factor, which is a potent growth factor for primary cultured adult hepatocytes, strongly stimulated DNA synthesis of rat fetal (20-day of gestation) hepatocytes. Its mitogenic capacity, measured as (3H)-thymidine incorporation into acid precipitable material was dose dependent, being detectable at 1 ng/ml and maximal at 5 ng/ml. Over 15% of the cells entered into S-phase and mitosis as judged by flow cytometric analysis of the cell cycle. HGF had additive effects with transforming growth factor-alpha, whereas transforming growth factor-beta strongly inhibited DNA synthesis of fetal hepatocytes stimulated by HGF. HGF induced c-fos and c-myc expression in a time-dependent manner, with a maximum at 30 min for c-fos and 8 h for c-myc. These results suggest that HGF may act as a proliferative factor during fetal liver growth.     

Effect of tryptophan on isolated hepatocytes of rats

The addition of tryptophan to adult rat hepatocyte cultures stimulated DNA synthesis. The increase in DNA synthesis as measured by 3H-thymidine incorporation into DNA was observed on treatment of the cultures with tryptophan for 48 h but also as short as for 6 h in comparison with control cultures. An increase was also apparent at 30 h which was maintained for up to 48 h post treatment with tryptophan. The increase in DNA synthesis by tryptophan cannot be attributed to cell injury or to increased DNA degradation. Of the degradative enzymes added after harvesting the hepatocytes, only DNase decreased incorporation of 3H-thymidine. The observed effect was specific for tryptophan since treatment with kynurenine, isoleucine, methionine or serine failed to show a significant effect. Pretreatment of cultured hepatocytes with hydroxyurea prevented the tryptophan stimulated increase in DNA synthesis suggesting that the latter was due to replicative and not to reparative DNA synthesis. Experiments performed with the addition of diethylnitrosamine also alluded to tryptophan's role in replicative DNA synthesis. The mechanism of tryptophan-induced DNA synthesis is discussed.     

Biosynthesis of gangliosides in primary cultures of rat hepatocytes. Determination of the net synthesis of individual gangliosides by incorporation of labeled N-acetylmannosamine

The ganglioside content of rat hepatocytes increases several-fold during the first 6 days in monolayer culture. To correlate increased levels with rates of de novo synthesis, the incorporation of N-acetyl-[6-3H]D-mannosamine into individual gangliosides was determined. The calculation of synthetic rates was made possible by the simultaneous measurement of the specific radioactivity of the immediate sialic-acid donor, CMP-Neu5Ac. The CMP-Neu5Ac content of hepatocytes was found by HPLC analysis to be 30.5 nmol/g of plated cells. The specific radioactivity of this precursor pool reached a constant plateau 5 h after addition of the labeled N-acetyl-mannosamine and remained constant for at least 70 h. The incorporation into individual gangliosides was measured in primary cultures of rat hepatocytes between 72 and 144 h after seeding. During this period, the increase in ganglioside levels was greatest. The highest rates of incorporation were seen in GD1a followed by GM3, GM1, GD3 and the polysialylated compounds. The following rates of synthesis (nmol per 60 h and mg of protein) were calculated: GD1a 0.68, GM3 0.59, GM1 0.36, GD3 0.13 and GT1 0.02. These values are compared with the net increase of the gangliosides as measured by the resorcinol reaction.     

Glycosylation of apolipoproteins by cultured rat hepatocytes. Effect of tunicamycin on lipoprotein secretion.

Cultured rat hepatocytes were used to measure hepatic synthesis of rat plasma glycoproteins. [3H]Glucosamine was progressively incorporated into the protein of hepatocyte culture media very-low-density lipoprotein, low-density lipoprotein, high-density lipoprotein and the p greater than 1.21 g/ml fraction after 3.5 and 6.5 h incubation. Apolipoproteins B, E and C, as well as transferrin, were identified as glycoproteins. The association of radioactivity with apolipoprotein C of hepatocyte very-low-density and high-density lipoproteins suggests that apolipoprotein C-III-3, the only C apoglycoprotein in the rat, is synthesized de novo by the hepatocytes. Treatment of hepatocytes with tunicamycin, a specific inhibitor of protein glycosylation, resulted in a substantial decrease in [3H]glucosamine incorporation into hepatocyte very-low-density, low-density and high-density lipoproteins and p greater than 1.21 g/ml protein, but had little or no effect on secretion. In the rat, hepatic secretion of lipoproteins and transferrin does not appear to be dependent on prior protein glycosylation.     

Monolayer culture of parenchymal rat hepatocytes on collagen-coated microcarriers. A hepatocyte system for short- and long-term metabolic studies

Summary A method is described for the attachment to and monolayer culture of adult rat hepatocytes on collagen-coated or fibronectin-coated microbeads or both in a chemically defined serum-free medium. Protein synthesis measured by the incorporation of [³H]leucine into protein was four-fold higher in the hepatocyte microcarrier cultures than in isolated hepatocyte suspensions. The hepatocyte microcarrier cultures showed acute responsiveness to insulin of fatty acid synthesis, glucose incorporation into glycogen, and decarboxylation of [1-¹⁴C]pyruvate. Microcarrier-cultured hepatocytes have the combined advantages of monolayer culture and suspension systems. They are a potential tool for the study of long-term as well as acute effects of hormones. This work was supported by the British Diabetic Association.     

Stimulation of DNA synthesis and myo-inositol incorporation in mammalian cells

Phosphatidylinositol (PI) synthesis and its role in controlling the cell cycle has been investigated using fibroblasts and liver cells in culture. PI synthesis as measured by incorporation of [³H]-myo-inositol into trichloroacetic acid precipitable material during 0–60 min after serum or growth factor stimulation of serum-starved cells is increased in primary fetal rat liver cells, rat embryo fibroblasts, and 3T3 mouse cells. In contrast, growth stimulation of 3T3 cells and hepatocytes rendered quiescent in G₁ by amino acid starvation is not accompanied by increased incorporation of [³H]-myo-inositol into trichloroacetic acid precipitable material. This suggests that those cells might be arrested at a different point in G₁ than cells arrested by serum depletion. Inhibition of PI synthesis by δ-hexachlorocyclohexane (HCH), a steric analog of myo-inositol, during early times (e.g., 0–4 hr) after growth stimulation, reversibly blocks initiation of DNA synthesis in 3T3 cells. The results support the idea that increased PI synthesis in response to growth stimulation in the cell types studied here is a prerequisite for progression through G₁ and subsequent entry into S phase.     

Effect of an antioxidant complex on the biosynthesis of macromolecules in hepatocytes during acute experimental radiation injury

The radioprotective efficiency of the antioxidant complex of vitamins (AC) has been estimated by incorporation of 3H-thymidine into DNA of irradiated rat hepatocytes after whole-body irradiation. The results obtained indicate that AC is an effective radioprotective drug.     

Effects of amino acids, ammonia and leupeptin on protein synthesis and degradation in isolated rat hepatocytes.

Protein synthesis in isolated rat hepatocytes, as measured by the incorporation of [14C]-valine at constant specific radioactivity, proceeded at a rate of 0.3-0.5%/h in an unsupplemented medium, i.e. only about one-tenth the rate of protein degradation (4%/h). Leupeptin, which inhibits lysosomal protein degradation (previously found to be 75% of the total degradation in hepatocytes), had no effect on protein synthesis, showing that endogenous protein degradation supplied amino acids in excess of the substrate requirements for protein synthesis. The inhibition of protein synthesis by NH4Cl (another inhibitor of lysosomal protein degradation) as well as the stimulation by a physiological amino acid mixture must therefore represent indirect effects, either on general energy metabolism, or on unknown regulatory processes.     

Initiation of altered heparan sulphate on beta-D-xyloside in rat hepatocytes

Inhibition of protein synthesis by cycloheximide 10(-3)M reduced the incorporation of [35S]sulphate into heparan sulphate to about 5% of untreated hepatocytes. Addition of rho-nitrophenyl beta-D-xyloside could partially revert this inhibitory effect. The sulphated material isolated from the cell layer or secretions of hepatocytes grown in presence of cycloheximide and rho-nitrophenyl beta-D-xyloside were shown to be mostly free heparan sulphate chains not bound to core protein. Covalent association of beta-xylosides to the heparan sulphates was demonstrated for heparan sulphate synthetized in the presence of [35S]sulphate, cycloheximide and the fluorogenic 4-methylumbelliferyl beta-D-xyloside. Beta-Xylosides served as an initiator of heparan sulphate chain synthesis in rat hepatocytes only in the absence of protein synthesis. Heparan sulphates primed on artificial beta-xylosides are slightly smaller in molecular size and are more sulphated than chains linked to core protein.     

Fraction from human and rat liver which is inhibitory for proliferation of liver cells

A comparative study was undertaken with human and rat liver of a fraction reported to have growth inhibitory activity when prepared from rat liver. Fractions which were soluble in 70% ethanol and insoluble in 87% ethanol were prepared from liver cytosols. Electrophoretic analysis under denaturing conditions indicated that there were several quantitative or qualitative differences in the fractions from the two species. Fractions from both human and rat liver were found to be inhibitory for the incorporation of 3H-thymidine into DNA of foetal chick hepatocytes. Under conditions in which the rat fraction inhibited precursor incorporation into DNA of rat liver epithelial cells there was not a significant inhibitory effect with the fraction from human liver. DNA synthesis in a rat hepatoma cell line was not significantly inhibited by preparations from either species. The data suggested that corresponding fractions from both rat and human liver could have inhibitory effects on precursor incorporation into DNA but the magnitude of the effects and target cell specificity may differ.     

Enhanced DNA synthesis in rat hepatoma cells by conditioned media from Kupffer cells incubated with supernatants of tumor necrosis factor-alpha-pretreated hepatocytes.

The effects of tumor necrosis factor-alpha (TNF-alpha) on DNA synthesis in AH66 rat hepatoma cells and rat hepatocytes were analysed by means of [3H]thymidine incorporation. DNA synthesis in AH66 cells was suppressed when AH66 cells were directly incubated with TNF-alpha. When primary culture of rat Kupffer cells was incubated with hepatocyte conditioned media pretreated with TNF-alpha (0-200 U/ml), and AH66 cells were then treated with these hepatocyte/Kupffer cell-conditioned media, TNF-alpha used in the pretreatment caused a dose-dependent increase in DNA synthesis in AH66 cells with a maximum effect amounting to a more than 10-fold increase. In contrast, DNA synthesis in primary culture of rat hepatocytes was not stimulated by the TNF-alpha-pretreated hepatocyte/Kupffer cell conditioned media. These results suggest that TNF-alpha-mediated hepatocyte-Kupffer cell interaction selectively promotes proliferation of rat hepatoma cells.     

Effect of D-galactosamine in vitro on [U-14C]palmitate oxidation, triacylglycerol synthesis and secretion in isolated hepatocytes

Isolated rat hepatocytes were used to study in vitro effects of 10 mM D-galactosamine (GalN) on hepatic fatty acids metabolism. At this concentration, membrane integrity and biochemical competence (i.e., gluconeogenesis and ureogenesis) remained unaffected. Protein synthesis and secretion, as measured by the incorporation of [U-14C]leucine into total and medium protein, was significantly inhibited when incubated for more than 2 h. GalN activated the incorporation of [U-14C]palmitate into triacylglycerols and depressed its utilization in the formation of labelled ketone bodies and 14CO2. Hepatocytes isolated from fasted rats exposed to GalN in vitro did not show any variation in prelabelled triacylglycerol secretion. GalN induced a rapid inhibition of prelabelled triacylglycerol secretion by hepatocytes isolated from fed rats in which this secretion occurred to a larger extent than in hepatocytes isolated from fasted rats. The data reported here suggest that GalN induces a rise of triacylglycerol synthesis by inhibiting the palmitate oxidation pathway and a decrease of triacylglycerol secretion through an early derangement of the secretory pathway.     

Minimal requirement of the remnant pancreas for protein and DNA synthesis of cultured hepatocytes prepared from pancreatectomized rats

The incorporation of 3H-thymidine and 3H-leucine into the hepatocytes was studied, using cultured hepatocytes prepared from normal and pancreatectomized rats. (1) In the cultured hepatocytes prepared from 80% pancreatectomized rats, the incorporation of 3H-thymidine and 3H-leucine into hepatocytes remained unchanged compared with those of sham-operated controls. In contrast, in those from totally pancreatectomized rats, the incorporation of 3H-thymidine and 3H-leucine decreased to approximately 67% and 37% respectively of sham-operated controls. However, those returned to near normal in the cultured hepatocytes from totally pancreatectomized rats treated by 0.8 IU/kg of insulin. (2) The addition of insulin (10(-4) M) to the culture medium stimulated the incorporation of 3H-thymidine into cultured hepatocytes prepared from normal rats to 148% of controls. The insulin-stimulated incorporation was inhibited by the addition of glucagon to the culture medium. The combined addition of insulin and glucagon did not synergistically act on DNA synthesis. It is suggested that the portal blood insulin in the presence of more than 20% of the pancreas is imperative for maintaining spontaneous regeneration.