Expression of insulin-like growth factor-I and insulin-like growth factor binding proteins during thioacetamide-induced liver cirrhosis in rats.

OBJECTIVE: The liver plays a central role in insulin-like growth factor (IGF) homeostasis providing the majority of circulating IGF-I and some of its binding proteins (IGFBPs). In liver cirrhosis the IGF axis is severely disturbed, and these alterations are associated with reduced IGF-I, IGFBP-3 but elevated IGFBP-1 serum levels. METHODS: By Northern blotting and in situ hybridization (ISH), hepatic expression of IGF-I and of IGFBP was studied in a rat model of liver cirrhosis induced by thioacetamide. RESULTS: ISH revealed a homogeneous distribution of IGFBP-1, IGFBP-4 and IGF-I mRNA over hepatic parenchyma in normal and cirrhotic liver. Fibrous septa of cirrhotic liver were IGFBP-1 mRNA negative, whereas IGFBP-4 and IGF-I transcripts were detected in single cells. In normal liver, IGFBP-3 mRNA was distributed within nonparenchymal cells of the hepatic lobule and in the wall of the portal vein. In cirrhotic liver, IGFBP-3 transcripts were abundant in mesenchymal cells of fibrous tissue. IGFBP-3 mRNA expression was also prominent in cells at the septal-nodular interface most likely representing monocyte infiltration. IGFBP-3 mRNA expression was reduced in nonparenchymal liver cells located more distantly from the septal-nodular interface in the cirrhotic nodule that correlated with reduced IGFBP-3 mRNA expression observed in Kupffer cells (KC) and sinusoidal endothelial cells (SEC) isolated from macronodular cirrhotic livers. CONCLUSION: Cirrhosis is accompanied by an altered spatial expression of IGFBP-3 in liver tissue, which is characterized by decreased levels of IGFBP-3 mRNA in KC and SEC, but elevated IGFBP-3 expression in myofibroblast-like cells and inflammatory infiltrate.

Postnatal development of hepatocellular apolipoprotein B assembly and secretion in the rat.

In this study, we explored the paradox that in suckling rats the serum concentration of LDL is high although the liver secretes only minimal quantities of VLDL, the presumed precursor of LDL. Freshly isolated hepatocytes and hepatocytes in primary culture obtained from adult (90 days old) and suckling (17 days old) rats were used to investigate the synthesis and secretion of apolipoprotein B (apoB) and lipids as well as the density profile of secreted apoB-containing lipoproteins. Furthermore, the effects of dexamethasone and oleate on apoB biogenesis were investigated in primary cultures of hepatocytes from adult and suckling rats. Hepatocytes from suckling rats were unable to assemble mature VLDL but secreted apoB as primordial lipoprotein particles in the LDL-HDL density range. Intracellular degradation of apoB was also reduced in hepatocytes from suckling rats compared with that in hepatocytes from adults. The immaturity in VLDL assembly and apoB degradation of hepatocytes from suckling rats could be overcome by treating the cultures with dexamethasone plus oleate or dexamethasone alone. The lower microsomal triacylglycerol transfer protein (MTP) mRNA concentrations in hepatocytes from suckling rats in comparison with hepatocytes from adult rats were not reflected in lower MTP activity levels. Furthermore, dexamethasone plus oleate treatment had no effect on MTP activity although VLDL assembly and secretion were clearly stimulated. We conclude that, during the suckling period of the rat, serum LDL is directly produced by the liver. This is a result of impaired hepatic VLDL assembly, which is a consequence of low triglyceride synthesis and an inefficient mobilization of bulk lipids in the second step of VLDL assembly.

Effect of bile acids on the proliferative activity and apoptosis of rat hepatocytes.

Bile acids are known to have damaging as well as protective effects on liver cells. A likely candidate for bile acid-mediated hepatocellular injury during cholestasis is glycochenodeoxycholic acid (GCDCA), a hydrophobic bile acid with a direct cytotoxic effect on hepatocytes. In contrast, ursodeoxycholic acid was shown to exhibit protective effects. Our aim was to determine the effect of GCDCA on proliferation, synthesis and secretion of proteins and death processes in cultured rat hepatocytes. Furthermore, it should be studied whether the hydrophilic bile acid tauroursodeoxycholic acid (TUDCA) might be able to protect cells from the damaging effect of GCDCA. Our results demonstrate that GCDCA decreased dose-dependently hepatocellular proliferation, synthesis and secretion of newly synthesized proteins and, at low concentration, induced apoptosis or, at high doses, cytolysis of cultured hepatocytes. TUDCA did not exert cytotoxic effects on the isolated hepatocytes at a wide range of concentrations. However, TUDCA coincubated with GCDCA protected the cells from the damaging effect of GCDCA at all measured parameters except the secretion of newly synthesized protein.

Separation of the intracellular secretory compartment of rat liver and isolated rat hepatocytes in a single step using self-generating gradients of iodixanol.

A novel method is described for the separation on a single gradient of the major intracellular organelles of the secretory pathway, the Golgi, the smooth endoplasmic reticulum, and the rough endoplasmic reticulum. Total microsomes were prepared from rat liver by differential centrifugation and resuspended in 20% iodixanol. The microsomal suspension was then layered between a 30% iodixanol cushion and a layer of 15% iodixanol and centrifuged in a vertical rotor for 2 h. The microsomes distributed in four visible bands. The gradients were collected by upward displacement and were characterized (i) by determination of UDP galactose-galactosyltransferase (Golgi marker) NADPH-cytochrome c reductase (endoplasmic reticulum marker) and RNA (rough endoplasmic reticulum marker); (ii) by immunoblotting for TGN38 (trans-Golgi marker) and GS28 (cis-Golgi marker) and for protein disulfide isomerase (endoplasmic reticulum lumenal marker); (iii) by determination of the lipid composition; and (iv) by electron microscopy. The results suggest that the top band (density 1.045-1. 090 g/ml), which contains 68% of the galactosyltransferase activity, consists of vesicles derived from the Golgi. The second broad band in the middle of the tube (density 1.130-1.160 g/ml), which contains 54% of the NADPH-cytochrome c reductase activity, consists mainly of vesicles derived from the smooth endoplasmic reticulum, overlapped at the top by a small band of Golgi-derived lamellae. The two bands at the bottom of the tube (density 1.130-1.160 and density 1.180-1. 220 g/ml) appear to contain two subfractions of vesicles derived from the rough endoplasmic reticulum.

Chronic liver injury alters basal and stimulated nitric oxide production and 3H-thymidine incorporation in cultured sinusoidal endothelial cells from rats.

BACKGROUND/AIM: Under pathological conditions the nitric oxide synthase (NOS)-mediated nitric oxide production of sinusoidal endothelial cells might be altered. Therefore, studies were performed to evaluate the nitrite formation by cultured sinusoidal endothelial cells from rat livers chronically injured by thioacetamide and the effect of endogenously or exogenously generated nitric oxide on their proliferative activity. METHODS: Basal and stimulated nitrite formation, expression of NOS and DNA synthesis were examined in sinusoidal endothelial cells isolated and cultivated from livers with incipient or advanced chemically-induced cirrhosis. RESULTS: Cultured sinusoidal endothelial cells from injured livers exhibited a reduced basal and an increased lipopolysaccharide-stimulated nitrite production when compared with controls. Western blot analysis revealed a markedly reduced protein expression of endothelial NOS (eNOS) and inducible NOS (iNOS) in sinusoidal endothelial cells from both experimental groups when compared with controls. Lipopolysaccharide stimulated iNOS expression in sinusoidal endothelial cells from control livers only marginally, and from those with cirrhosis more strongly. There was no clear correlation between the amount of enzyme and nitrite formation. Cultured sinusoidal endothelial cells from livers with incipient cirrhosis showed a higher proliferative activity than controls. Endogenously-produced nitric oxide inhibited DNA synthesis in all groups in a cGMP-independent way. Exogenously-generated nitric oxide affected DNA synthesis differently in sinusoidal endothelial cells from controls and injured livers. CONCLUSION: The results provide evidence that cultured sinusoidal endothelial cells from controls and livers with incipient or advanced cirrhosis differ with respect to basal and lipopolysaccharide-stimulated nitrite production. The data can be taken as evidence that in sinusoidal endothelial cells from livers chronically injured by thioacetamide, eNOS and iNOS are aberrantly expressed and differently regulated.

Expression of tenascin, fibronectin, and laminin in rat liver fibrogenesis--a comparative immunohistochemical study with two models of liver injury.

The aim of this study was to follow semiquantitatively by immunohistochemical means the alterations of the expression of the hepatic glycoproteins tenascin, fibronectin, and laminin in two different models of chronic liver injury, i.e. thioacetamide-induced liver cirrhosis and fibrosis after bile duct ligation. The tenascin distribution pattern observed during cholostasis-induced liver fibrosis showed some similarities, but also some differences in comparison with the results obtained after TAA intoxication. Most importantly, the data show that tenascin staining was detectable in almost all areas of the chronically injured livers up to 3 and 6 months in bile duct-ligated and chemically-injured livers, respectively. Thus, tenascin does not seem to play only a transient role in the fibrogenetic process as previously suggested. Laminin was strongly stained in proliferating ductules, whereas only a weak continuous distribution was observed along the sinusoidal wall. Furthermore, our findings confirm the role of fibronectin as a pacemaker of fibrosis. Regional differences in the kinetics of the expression of the glycoproteins may reflect local differences in their production by parenchymal or non parenchymal cells or regional patterns of proteolytic activity.

Differential effects of exogenous and endogenously generated H2O2 on phagocytic activity and glucose release of normal and cirrhotic livers.

BACKGROUND/AIMS: Reactive oxygen species play an essential role in necro-inflammatory processes. Therefore, the aim of the present studies was to investigate the effect of exogenous and endogenously produced H2O2 on the phagocytic capacity and glucose release of perfused cirrhotic rat livers in comparison with that on the controls. METHODS: Complete septal cirrhosis was achieved by oral treatment of rats with thioacetamide for 6 months. The phagocytic capacity of the perfused livers was measured by the uptake of colloidal carbon. During the continuous perfusion with colloidal carbon, either H2O2 or benzylamine was added to the perfusion medium for a limited time period. The latter functioned as an endogenous H2O2 donor. RESULTS: In control rats exogenous and endogenously produced H2O2 caused a transient stimulation of the hepatic colloidal carbon uptake as well as of the glucose release. Inhibition of the catalase by aminotriazol doubled the changes evoked by H2O2, whereas blockade of the Kupffer cells by GdCl3 drastically reduced its stimulatory effect. Cirrhotic livers took up less colloidal carbon and released lower amounts of glucose than the controls when stimulated by exogenous H2O2. The inhibition of the nitric oxide synthetase augmented the H2O2-induced effect in controls as well as in the cirrhotic livers by 250% and 620% (colloidal carbon uptake) and 340% and 760% (glucose release), respectively. The blockade of the eicosanoid production by indomethacin and caffeic acid drastically increased the glucose release and the colloidal carbon uptake in controls and, in absolute terms, to a lesser extent in cirrhotic livers. Endogenous H2O2 produced by the addition of benzylamine stimulated the colloidal carbon uptake and glucose release in livers from both groups. The inhibition of the lipoxygenase increased both parameters, whereas different effects were elicited by the addition of superoxide dismutase in controls and cirrhotic livers. CONCLUSION: The maximum uptake of colloidal carbon and glucose release, measured after stimulation by H2O2, was lower in cirrhotic livers than in controls, thus indicating a lowered phagocytic capacity of Kupffer cells and altered glycogenolytic response of the hepatocytes in cirrhotic livers. The use of various effectors provided evidence that superoxide anions, nitric oxide and, possibly, arachidonic acid are involved in the signal transduction between Kupffer cells and hepatocytes when stimulated by exogenous or endogenously produced H2O2. This signalling mechanism seems to be impaired in cirrhotic livers.

The lipid peroxidation end product 4-hydroxy-2,3-nonenal up-regulates transforming growth factor beta1 expression in the macrophage lineage: a link between oxidative injury and fibrosclerosis.

An increasing number of reports underscore the frequent association of fibrosclerotic diseases of lung, liver, arterial wall, brain, etc., with the accumulation of oxidatively modified lipids and proteins. A cause-and-effect relationship has been proposed between cellular oxidative damage and increased fibrogenesis based on the fact that experimental treatment with antioxidants either prevents or quenches the fibrotic process. With some peculiarities in the different organs, fibrosclerosis is essentially the result of the interaction of macrophages and extracellular matrix-producing cells. The cross-talk is mediated by fibrogenic cytokines, among which the most important appears to be transforming growth factor beta1 (TGF-beta1). This report describes treatment of different types of macrophage, of both human and murine origin, with 4-hydroxy-2,3-nonenal (HNE) a major aldehyde end product of membrane lipid oxidation found consistently to induce both mRNA expression and synthesis of TGF-beta1. Since increased HNE levels have been demostrated in the cirrhotic liver and in the oxidatively modified low-density human lipoproteins associated with atherosclerosis, the up-regulation of macrophage TGF-beta1 by HNE appears to be involved in the pathogenesis of these and similar diseases characterized by fibrosclerosis.

Histological and biochemical changes induced by total bile duct ligation in the rat.

The aim of the present investigation was to assess in a correlated biochemical and morphological study the dynamics of fibrogenesis after bile duct ligation and to compare the time course of alterations with those occurring in thioacetamide induced liver fibrosis. The data show that, after bile duct obstruction, the deposition of connective tissue elements and formation of ductular proliferates rapidly set in. The index of fibroplasia correlated well with the changes of the OH-proline concentration of the liver. Comparing the biliary fibrosis with the thioacetamide induced liver fibrosis, the progress of the former occurred more rapidly, even though in both cases only a few necroses were observed. Therefore, we suggest that in biliary fibrosis other mechanisms are responsible for the rapid onset of production of extracellular material and proliferative processes than in thioacetamide-induced liver fibrosis.

Macrophages from rat livers with micronodular and macronodular cirrhosis differ with respect to mediator release and DNA-synthesis.

BACKGROUND/AIMS: Liver macrophages play an essential role in necro-inflammatory liver damage which leads to fibrosis and cirrhosis. The aim of the present study was to compare the mediator release and the DNA synthesis of macrophages at an early and at a later stage of liver cirrhosis induced by thioacetamide. METHODS: Liver macrophages were isolated by an enzymic digestion method, followed by elutriation. The release of reactive oxygen species and cytokines, and the synthesis of DNA were measured in cultivated cells. RESULTS: The vitality of isolated macrophages from cirrhotic livers was always higher than 98%. The total yield of macrophages was less in micronodular cirrhotic livers and was markedly higher in macronodular cirrhotic livers when compared with age-matched controls. The cellular granules measured by sideward light scattering showed a shift to larger sizes in macrophages from micronodular cirrhotic livers when compared with the controls and the other experimental group. Macrophages from both cirrhosis groups exhibited a markedly higher unstimulated and lipopolysaccharide-stimulated IL-6 production than the controls. The release of TNF-alpha did not differ between controls and the experimental groups. Macrophages from macronodular cirrhotic livers produced higher amounts of nitric oxide but less superoxide anion radicals than the controls. DNA synthesis was 10-12-fold and 3-10-fold higher in macrophages from micronodular and macronodular cirrhotic livers, respectively, when compared with the age-matched controls. CONCLUSIONS: The data presented provide evidence that it is possible to isolate and to cultivate macrophages from livers with high yield and vitality at different stages of cirrhogenesis. Our results clearly demonstrate functional differences between macrophages from livers with micro- or macronodular cirrhosis; this finding may be important for the pathogenesis or perpetuation of the cirrhogenetic process.

Antioxidative effect of prostaglandin E2 in thioacetamide-induced liver cirrhosis.

Several studies provided evidence that various prostaglandins exhibited a hepatoprotective effect in vivo as well in vitro the mechanism of which is still in debate. Therefore, the aim of our studies was to examine the effect of PGE2 on some biochemical and morphological alterations in chemically induced liver cirrhosis in rats. A micronodular liver cirrhosis was induced by treatment of rats with thioacetamide for 3 months. Morphologically, the administration of PGE2 for 8 days reduced the extent of vacuolar transformation of the hepatocytes and the density of the nuclear structure without affecting the fibrotic state as assessed by the hepatic hydroxyproline content. The widening of the sinusoids indicated an improved hepatic microcirculation. Administration of PGE2 significantly elevated the percentage portion of arachidonic (20:4) and docosapentaenoic (22:5) acid in the hepatic phospholipids and reduced the ratio 20:3/20:4 fatty acids in comparison to the untreated cirrhotic animals. The hepatic MDA concentration was decreased by 40% in PGE2-treated animals. PGE2 treatment also reduced the content of polar as well as of non-polar carbonyls when compared with the controls. Moreover, treatment with PGE2 lowered iron-induced or iron plus ascorbate-induced MDA production of isolated hepatocytes. From the data it was concluded that the hepatoprotective effect of PGE2 may be related to its antioxidative capacity.

Oxygen radical formation, proliferative activity and phagocytic capacity of cultivated macrophages from cirrhotic rat livers.

A method to isolate and cultivate macrophages from Macronodular-cirrhotic rat livers was developed in order to characterize them biochemically, by comparing various functional parameters in macrophage cell cultures from controls and cirrhotic livers. Cells were prepared from female Wistar rats, made cirrhotic by treatment with thioacetamide, by means of a pronase-collagenase digestion method followed by a nycodenz gradient and elutriation. The yield of macrophages was 8.9 x 10(6) cells/g for controls and 10.6 x 10(6) cells/g for cirrhotic livers. The vitality of the cells was > 95%. Forty-eight hours after cultivation, the purity of the cell fractions amounted to 94% and 91% in controls and in the experimental group, respectively. Nitric oxide synthesis was more markedly stimulated by lipopolysaccharide (LPS) in cultures from cirrhotic livers than in those from controls (25 +/- 4 vs 5.8 +/- 1 nmol/10(6) cells/72 hours). Interferon-gamma (IFN-gamma) induced the nitric oxide synthase more rapidly in macrophage cultures from cirrhotic livers than in controls. The production of superoxide anions by macrophages from cirrhotic livers stimulated by zymosan was significantly lower by about 40% when compared with the controls. Incorporation of 3H-thymidine was increased to 250% in cultivated macrophages from thioacetamide-treated rats in comparison with macrophages from untreated animals. The stimulated phagocytic activity of cultivated macrophages from cirrhotic livers did not differ significantly from that of the controls. The data presented provide evidence that it is possible to isolate and to cultivate macrophages from macronodular-cirrhotic livers with high yield and vitality. They are characterized by enhanced proliferation, reduced formation of superoxide anions, and increased production of nitric oxide.

Dexamethasone enhanced by insulin, but not by thyroid hormones stimulates apolipoprotein B mRNA editing in cultured rat hepatocytes depending on the developmental stage.

The increase of hepatic apolipoprotein B (apoB) mRNA editing during rat development was not affected by hypothyroidism. Furthermore, the addition of 3,3',5'-triiodothyronine (T3) to cultured hepatocytes taken from fetal, neonatal and adult rats had no effect on apoB mRNA editing. In contrast, dexamethasone markedly stimulated apoB mRNA editing in hepatocytes taken from neonates. This effect was enhanced by the addition of insulin. For the first time our data provide evidence that glucocorticoids together with insulin are important for the regulation of apoB mRNA editing during postnatal development, whereas thyroid hormones are not critical for this process.

Luminol-and lucigenin-amplified chemiluminescence with rat liver microsomes. Kinetics and influence of ascorbic acid, glutathione, dimethylsulfoxide, N-t-butyl-a-phenyl-nitrone, copper-ions and a copper complex, catalase, superoxide dismutase, hexobarbital and aniline.

For the investigation of luminol (LM)-and lucigenin (LC)-amplified chemiluminescence (CL) in rat liver microsomes using both a liquid-scintillation counter (LKB/Wallac 1219 Rackbeta) and a Berthold luminometer (AutoLumat LB 953) optimal incubation mixtures and conditions and basic kinetics have been established. Whereas calibration curves for both LM- and LC-CL are performed with hydrogenperoxide (LC quantum yield is 6.25 fold higher as that of LM), distinct differences were revealed with microsomes, indicating that different reactive oxygen species (ROS) are determined: Both LM- and LC-CL follow the kinetics of enzymatic reactions in terms of dependence on protein and NADPH or NADH concentration, time course, temperature etc., but with differences. LM-CL does not work without addition of Fe2+, whereas LC-CL does. Both copper ions and copper bound in a complex abolish CL, LC-CL being much more sensitive. Isolated cytochrome P-450 (P450) and NADPH P450 reductase from liver of pheno-barbital treated rats alone proved to be inactive in LM-and LC-CL production, whereas te combination 1:1 without and with addition of lipid was highly active in both LM-and LC-CL. Ascorbic acid and glutathione as scavengers diminish both LM- and LC-CL in concentrations higher then 10(5). Dimethyl-sulfoxide (DMSO) was ineffective in LM-CL up to concentrations of 0.2 M, the very high concentration of 2 M diminished LM-CL only to 1/3. LC-CL was diminished starting at concentrations of 100 mM and at 2 M only 10% of maximum LC-CL was observed. The trap substance N-t-butyl-a-phenylnitrone (BNP) also diminished LC-CL more effectively than LM-CL. Clearcut differences were revealed by the addition of catalase and superoxide dismutase: both enzymes diminished LM-CL only, without any influence on LC-CL. Hexobarbital, a potent uncoupler of P450, enhances LM-CL fivefold, whereas LC-CL is barely influenced. Aniline (without uncoupling capability) decreased both LM-and LC-CL increasingly with increasing concentrations. Therefore the conclusion is drawn that LM-CL measures in liver microsomes predominantly superoxide anion radicals, whereas LC-CL is mainly a measure for microsomal hydroxyl radical formation or of reactive organic radicals. With microsomes of phenobarbital and beta-naphthoflavone treated rats CL was much higher but in principle the same kinetic characteristics could be shown. All results on microsomes were obtained uniformly with the liquid scintillation counter and the Berthold luminometer, the letter being much more effective and more sensitive.

Phagocytic function and metabolite production in thioacetamide-induced liver cirrhosis: a comparative study in perfused livers and cultured Kupffer cells.

BACKGROUND/AIMS: The aim of the study presented here was to evaluate the basal and stimulated phagocytic activities and the metabolite production of isolated perfused livers, and also the phagocytic capacity of cultured Kupffer cells from rats with macronodular cirrhosis. METHODS: Rats were made cirrhotic by oral administration of thioacetamide. The phagocytic activity was assessed by the rate of removal of colloidal carbon. The Kupffer cells were prepared by a pronase/collagenase digestion method followed by elutriation. RESULTS: The phagocytic activity and production of glucose, lactate and pyruvate were reduced in cirrhotic livers when calculated per g liver. Due to hyperplastic-regenerative processes the mass of the cirrhotic livers was markedly augmented so that the colloidal carbon uptake calculated per cirrhotic liver was not significantly different from the controls. Colloidal carbon-induced glucose release increased more markedly in the controls than in cirrhotic livers. Isoproterenol considerably stimulated phagocytosis and glucose production in controls, whereas the response was clearly reduced in cirrhotic livers when calculated either per g liver or per total liver weight. The cyclic AMP analogue elicited a marked glycogenolytic response in the controls, whereas there was only a slight increase in glucose production in cirrhotic livers. Phagocytosis of cirrhotic livers was only moderately stimulated by opsonized zymosan when compared with the controls. Freshly isolated Kupffer cells exhibited a reduced phagocytic activity. Stimulation by zymosan was observed only in cell suspensions of the controls. In contrast, Kupffer cells from cirrhotic livers did not differ from controls with respect to basal or zymosan-stimulated phagocytic activity after 48-h cultivation. CONCLUSION: The stimulated phagocytic function was disturbed in perfused macronodular-cirrhotic livers as compared to controls. In contrast, 48-h cultured Kupffer cells from cirrhotic livers exhibited the same basal and stimulated phagocytic capacity as controls. The glucose release from perfused livers, initiated by stimulation of Kupffer cells or hepatocytes, was significantly reduced in cirrhotic livers. Therefore, we postulate an impaired intra- and/or intercellular signalling in macronodular-cirrhotic livers.

The pattern of apolipoprotein B100 containing lipoprotein subclasses produced by the isolated visceral rat yolk sac depends on developmental stage and fatty acid availability.

Explants of visceral rat yolk sacs from gestational days 16, 18 and 22 were used for studying developmental changes of secretion and density distribution of lipoproteins, particularly of those containing apoB. Moreover, the influence of fatty acid supply on the amount and density distribution of secreted apolipoproteins was studied on day 18 of gestation. Active lipoprotein production was observed in yolk sacs taken on days 16 and 18 of gestation. It declined considerably on day 22 of gestation in parallel with the production of total protein, triacylglycerols and cholesterol. On all gestational days, apoB floated mainly in the LDL range ( > or = 70%) with differences in the distribution pattern of LDL subclasses. The lowest density of secreted LDL was found on day 18 of gestation (peak at d = 1.025 g/ml) followed by day 16 (peak at d = 1.035 g/ml) and day 22 of gestation (peak at d = 1.045 g/ml). ApoAIV, apoE and apoAI floated exclusively in the HDL range with a peak at d = 1.089 g/ml independently of the gestational day. After incubation of yolk sacs from the 18th day of gestation with 0.4 mM or 0.8 mM oleate, the density of secreted apoB containing particles was decreased (peaks in the VLDL and IDL density range), whereas palmitate in the same concentrations caused a redistribution of secreted apoB toward higher densities (peaks at d > or = 1.032 g/ml). Taken together, the data provide evidence that the density of LDL subclasses produced by isolated yolk sacs between days 16 and 22 of gestation depended on the gestational stage. Moreover, addition of unsaturated or saturated fatty acids to the organ culture differently affected the secretory rate and the density of lipoproteins delivered by yolk sacs on day 18 of gestation.

Metabolism of leukotrienes is impaired in hepatocytes from rats with thioacetamide-induced liver cirrhosis.

It is likely that the hepatocellular metabolism of potent mediators of inflammation is impaired in chronic liver injury. Therefore, in this study the degradation of the leukotrienes LTC4, LTE4 and LTB4 was investigated in isolated liver parenchymal cells (LPC) from rats with thioacetamide-induced macronodular liver cirrhosis or after bile duct ligation. The degradation of LTE4 as well as the formation of N-acetyl-LTE4 was significantly delayed in LPC from macronodular cirrhotic rats but not in those from bile duct-ligated rats. LPC from macronodular cirrhotic rats eliminated LTC4 at the same rate as isolated hepatocytes from control animals. The rate of LTB4 degradation was significantly decreased by 35% in LPC from macronodular cirrhotic rats. Furthermore, the rate of LTB4 hydroxylation was significantly lower by 50% in microsomes isolated from hepatocytes of macronodular cirrhotic rats than in those from controls. In summary, one may conclude that the N-acetylation reaction of LTE4 and the hydroxylation reaction of LTB4 is impaired in LPC from rats with thioacetamide-induced macronodular cirrhosis.

Quantitative and qualitative characterization of apolipoprotein B containing lipoproteins produced by the visceral rat yolk sac in two different in vitro systems: organ culture and isolated epithelial cells in suspension culture.

Tissue pieces as well as isolated epithelial cells taken from visceral rat yolk sacs at the 18th day of gestation were able to synthesize and to secrete apo B containing lipoproteins floating in the density ranges of VLDL, IDL and LDL. In all three density classes only the high molecular weight apo B was detectable. VLDL secreted from yolk sac tissue or isolated epithelial cells lacked apo E and apo C. Studies with cycloheximide revealed that about 77% of the particles was delivered from a pool of preformed lipoproteins. Evidence was given that also de novo-synthesis of apo B containing lipoproteins took place in the tissue segments and the isolated epithelial cells. Most of apo B mass (90%) and of apo B radioactivity (60%) secreted by the tissue pieces of the visceral yolk sac floated in the density range 1.020-1.064 g/ml, the remainder being found in the d < or = 1.020 g/ml fraction. In contrast, only 40% of apo B mass and 45% of apo B radioactivity delivered from isolated epithelial cells belonged to the d = 1.020-1.064 g/ml fraction. LDL released from yolk sacs and isolated epithelial cells contained more triacylglycerols (41% vs. 25%) and had a larger mean diameter (24 nm vs. 21.8 nm) than those obtained from fetal rat serum, whereas the comparatively small VLDL produced in vitro (mean diameter = 34 nm) contained less triacylglycerols (46% vs. 60.5%) and more protein (20% vs. 10.2%) in comparison with fetal serum VLDL (mean diameter = 42.3 nm). Incubation experiments with [125I]VLDL led to the conclusion that the lipase secreted by yolk sac tissue into the medium could not be responsible for the conversion of VLDL into LDL, thus supporting our view of a direct LDL secretion by visceral rat yolk sacs.

Zonal differences in lipoprotein formation in the thioacetamide-induced micronodular-cirrhotic rat liver.

The aim of the studies was to answer the question to what extent thioacetamide-induced structural alterations of hepatic architecture leading to fibrosis and micronodular pseudolobuli affect the formation of very low density lipoproteins and the zonation of lipoprotein metabolism observed in normal and acutely injured livers. Therefore, the number of the VLDL particles/Golgi complex and the relative specific volume of Golgi complexes as well as the number and relative specific volume of VLDL-filled vesicles was determined in lobular and nodular zones of normal and the micronodular-cirrhotic livers, respectively. The perinodular and centrinodular regions were morphometrically analysed in nodules with diameters between 0.3 and 0.5 mm. -Generally, in thioacetamide-induced micronodular liver cirrhosis a zonality was observed with respect to the amount of VLDL particles as well as the number and volume of organelles involved in the formation and secretion of hepatic lipoproteins. However, the number of VLDL particles/Golgi complex was significantly reduced to 52% in the centrinodular and to 71% in the perinodular region of cirrhotic livers when compared with the corresponding periportal and perivenous area, respectively. Furthermore, the relative specific volume of the Golgi complexes markedly increased in the perinodular region, thus abolishing the zonal difference observed in the controls.(ABSTRACT TRUNCATED AT 250 WORDS)