H. Franke

Studies on lipid and lipoprotein metabolism in rat liver cirrhosis induced by different regimens of thioacetamide administration

In female Wistar rats the animal model of TAA-induced liver cirrhosis has been tested for reliability and usefulness for studies on lipid and lipoprotein metabolism in cirrhosis. From our results we draw the following conclusions: Application of 300 mg TAA/l drinking water from the 4th to the 6th month of life leads in all treated rats to liver cirrhosis which is rather uniformly of micronodular surface morphology. Under this treatment the survival rate is about 90 percent. Increasing the administered dose (450 and 600 mg/l) and/or extension of TAA administration time (4 or more months) leads to decreasing survival rates, and to a shift from micronodular towards macronodular cirrhosis. To produce macronodular cirrhosis it is suggested to extent the application time rather than to increase the dose since in the latter case the survival rate is very low. The alterations of lipid and lipoprotein metabolism observed in this animal model, i.e. decrease of pre-beta-lipoproteins, increase of beta-lipoproteins, decrease of serum triglyceride concentration and decrease of hepatic VLDL-TG output into the serum are in good agreement with those observed in human cirrhosis. Thus, the TAA-induced chronic liver injury proved to be a reliable and useful model for studies on lipid and lipoprotein metabolism in liver cirrhosis.

Glutathione synthesis and export in experimental liver cirrhosis induced by thioacetamide: Relations to ultrastructural changes

Micro-and macronodular experimental liver cirrhosis was induced in female rats by administration of 0.03% thioacetamide (TAA) in drinking water for 3 or 6 months, respectively. The glutathione (GSH) status (content, synthesis, export) and ultrastructural changes of liver were investigated 14 d after withdrawal of TAA. The hepatic level of GSH was increased after 6 months TAA treatment. The levels of oxidized glutathione (GSSG) were not changed after 3 months or 6 months TAA administration. The GSH synthesis was not disturbed in the cirrhotic livers; only the ratio between the 2 synthesizing enzymes was changed in macronodular liver cirrhosis. The plasma GSH content was reduced in both cases, independent of the stage of liver cirrhosis. The electron microscopic studies on cirrhotic rat livers revealed a series of characteristic structural changes, such as disorganization and total lack of the microvilli border, appearance of basement membrane-like deposits within the narrowed space of Disse, disappearance of the highly porous endothelial cell lining and partly an intensively detoriated blood supply within the pseudolobules. It is suggested that all these changes may contribute to a disturbance of the GSH export from the hepatocytes into the blood. It is very likely, however, that the alterations of the sinusoidal cell surface play the most important role. 1. The GSH/GSSG redox potential is shifted in favour of the reduced form in this cirrhosis model. This shift seems to be connected with later stages of cirrhogenesis. 2. A GSH export disturbance is responsible for the decreased plasma GSH level in liver cirrhosis.

The visceral yolk sac — an important site of synthesis and secretion of apolipoprotein B containing lipoproteins in the feto-placental unit of the rat

Rat fetuses exhibit a high serum LDL concentration at term. Delivery caused a marked decrease of the LDL apolipoprotein (apo) B concentration independent of whether this occurred on days 21, 22 or 23 of gestation. The interruption of the yolk sac circulation by a ligature in situ for 6 h led to the same alterations of the LDL-apo B concentration as Caesarean section. Immunoelectronmicroscopic studies provided evidence that the epithelial cells of the visceral yolk sac exhibited electron dense LDL-sized and apo B containing particles which were localized over the compartments of the Golgi complexes, endoplasmatic reticulum, secretory vesicles and intercellular spaces, but not over the cell nuclei, mitochondria or lysosomes. ApoB containing LDL-sized particles could be obtained by ultracentrifugation from the disrupted material of the microsomal fraction of yolk sac homogenates. Isolated segments of the yolk sac membranes were capable to secrete apoB containing lipoproteins floating in the d less than 1.020 g/ml as well as in the d = 1.020-1.064 g/ml fraction with a 10-fold higher amount of apoB in the higher density class. Incorporation experiments with [35S] methionine gave evidence that these lipoproteins were at least partially provided with newly synthesized apoB predominantly found in the LDL fraction. The size of the negatively stained particles in the d = 1.020-1.064 g/ml fraction secreted from yolk sac segments corresponded to that of LDL from fetal rat serum. In contrast their acylglycerol content was significantly higher, whereas the percentage contribution of total cholesterol and protein was markedly reduced in comparison with serum LDL of the fetus. In summary, biochemical and ultrastructural studies provide clear cut evidence that the rat yolk sac is able to synthesize and to deliver apo B containing lipoproteins in the density ranges of VLDL, IDL and particular of LDL thus contributing to the supply of serum lipoproteins in the rat fetus. By recalculation of recent tracer kinetic data (Plonné et al. (1990) J. Lipid Res. 31, 747) using a mathematical step function model it was possible to assess the contribution of the rat yolk sac to the LDL influx into the fetal serum.

Qualitative and quantitative changes in hepatic lipoprotein particles following acute injury of the rat liver induced by thioacetamide

SummaryAcute intoxication of the rat liver with a single dose of 100 mg thioacetamide (TAA)/kg body weight causes within 48 h a fatty liver and a heterogeneous reaction in the hepatocytes. This affects principally the centrilobular liver parenchymal cells (zone 3) and to a lesser extent the periportal ones (zone 1). Ultrastructural analysis was performed to determine to what extent the formation of lipid-carrying particles of the very low density type (VLDL) is changed in affected hepatocytes in zones 1 and 3. Being morphologically the most conspicuous site of VLDL processing, the Golgi complex was chosen for quantitation by measuring its volume, VLDL content and particle size. The concentration and composition of the liver lipids were determined, biochemically. After TAA treatment of the liver the number of Golgi-VLDL particles is significantly reduced to about 50% in both the lobular zones examined. In addition, distinct classes of size-modified Golgi-VLDL particles appear which show an abnormally wide size distribution pattern. In periportal hepatocytes the size distribution of Golgi-VLDL particles shows a clear shift towards smaller particles homogeneous in size (mean diameter 39 nm). In contrast, centrilobular hepatocytes contain particles of very heterogeneous size, the mean diameter of which is nearly doubled (77 nm). The decrease in VLDL particle number and their size modification induced by TAA is not accompanied by significant changes in the volume of the Golgi complex. Biochemical analysis showed that the accumulation of lipids in the TAA-treated liver, mainly evident morphologically as drop-like deposits in the central area of the liver lobules, is due to an increase in triglycerides (TG) by 23 μmol/g liver wet weight, which represents nearly 95% of the accumulated lipids. Despite the striking elevation of the absolute cholesterol ester (CHOL-E) content (2μmol/g liver wet weight), this corresponds to only 5% of the newly accumulated lipids.Our electron optical and biochemical results support the suggestion that, in spite of the markedly different intralobular reaction of TAA-intoxicated hepatocytes, the formation of triglyceride-carrying particles is altered significantly in both lobular zones examined.

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.

Zur elektronenmikroskopischen Struktur der Corticalmembran der Knochenfischeier

Zur elektronenmikroskopischen Struktur der Corticalmembran tier Knoehenfiseheier Die yon ]3ECHER1), WICKLER ~) u n d zah l re ichen ~l teren A u t o r e n au f G r u n d l i ch top t i seher U n t e r s u c h u n g e n ve r t r e t ene Ans ich t , dab die Cor t i ca ln l embran der Knochenf i sche ie r yon feinen, gleichm~iBigen, rad i~rges te l l t en Kan~ilchen du rchse t z t set, i s t du r ch U n t e r s u c h u n g e n von SPEK3), STERBA4), SIEGEIa) u. a. in F rage ges te l l t worden . E l e k t r o n e n m i k r o s k o p i s c h e U n t e r s u c h u n g e n an Radii ira n d T a n g e n t i a l s c h n i t t e n d u r c h die Eihfil len des B u n t b a r s c h e s Civhlasoma festivum (Fig. 1) e r l auben folgende vorl~ufige Fes t s t e l lungen : Die Cor t ica l sch ich t dieser A r t se tz t s ich im Eis t a d i u m I I I u n d IV aus 2 Sch ich ten z u s a m m e n . Die dieke I n n e n sch ich t h a t s c h w a m m g e r f i s t a r t i g e n Charak te r , die Meinen B innen r / i ume yon 0,05 bis 0,2/* D u r c h m e s s e r s ind d imens ions los e iuges t reu t , das Gerf is twerk se lbs t zeigt zahlreiche, unge-

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.

Synthesis, secretion and immunoelectron microscopic demonstration of apolipoprotein B-containing lipoprotein particles in the visceral rat yolk sac.

SummaryElectron microscopic investigations on the involvement of the fetal membranes of the rat (visceral yolk sac) in the lipid metabolism revealed the occurrence of lipoprotein-sized particles located in cisternal Golgi stacks, Golgi vesicles and secretory vesicles of the cells of the visceral yolk sac epithelium as well as in distended areas of the intercellular space between adjacent epithelial cells. Application of the protein A-gold technique with specific anti-apoB antiserum resulted in a specific location of immunogold both over the different compartments of the lipoprotein pathway (RER, Golgi complex, secretory vesicles) as well as over the distended intercellular spaces, thus confirming these particles to be lipoproteins in nature. Isolated visceral epithelial cells prepared by a tryptic digestion method exhibited some ultrastructural alterations, such as a loss of apical brush border, a change from columnar to spherical cell shape, a decrease in phagolysosomes, but an increase in autophagosomal structures after 6 h incubation at a vitality rate of at least 85%. Within this period the epithelial cells secreted measurable amounts of apoB-containing lipoproteins into the medium floating in the density classes d<1.006 g/ml, d=1.006–1.020 g/ml and d=1.020–1.064 g/ml. The production of the lipoproteins was partly inhibited by cycloheximide indicating the secretion of particles with preformed as well as newly synthesized apoB. Negative staining of the particles revealed an average diameter of 34 nm of VLDL, 31 nm of IDL and 24 nm of LDL. In summary, our studies demonstrate that in the feto-placental unit of the rat the fetal membranes are capable of synthesizing and secreting lipoproteins. The cells of the visceral yolk sac epithelium were shown to be the producers of apoB-containing particles.

Biochemical and substructural studies on hepatic and serum lipoprotein metabolism after acute liver injury induced by thioacetamide in rats.

The effect of an acute liver injury induced by thioacetamide (TAA) on hepatic and serum lipoprotein metabolism in rats was studied using biochemical and ultrastructural methods. A single dose of 100 mg TAA/kg body weight caused within 48 h a decrease of hepatic triglyceride (TG) output into the serum by about 50% in comparison to the controls. The steady state of serum TG concentration from 24 to 96 h after TAA treatment implies that the clearance of TG from serum must be diminished to the same extent as the hepatic TG output was found to decrease. Moreover, the TAA treatment caused changes in the electrophoretic mobility as well as in the concentration and composition of circulating serum-lipoproteins. The electrophoresis revealed a decrease in the alpha-band, which can be explained by the decrease in the high density lipoproteins (HDL) total lipid concentration. The pre-beta-migrating band disappeared, whereas a broad beta-mobility band appeared, which most likely consists of a mixture of very low density lipoproteins (VLDL) and low density lipoproteins (LDL) as can be concluded from the changes in concentration and composition of lipids in both fractions. For a better visualization of the VLDL-forming capability in perilobular and centrolobular liver parenchymal cells of the TAA-treated animals the VLDL secretion blocking agent colchicine was used. It was shown that in comparison with colchicine-treated controls the VLDL secretory products are accumulated at a considerably lower rate manifested in a diminution of VLDL clusters, secretory vesicle size and the number of intravesicular VLDL particles.(ABSTRACT TRUNCATED AT 250 WORDS)

Quantitative studies on fatty acid metabolism in isolated parenchymal cells from normal and cirrhotic livers in rats

Parenchymal cells isolated from normal and thioacetamide-induced micronodular cirrhotic rat livers were used to evaluate changes in hepatocellular fatty acid (FA) metabolism in cirrhosis. Exogenous free FA (FFA) are rapidly taken up by hepatocytes obtained either from normal or cirrhotic livers. They are predominantly esterified to triglycerides and accumulate intracytoplasmatically as lipid droplets with increasing cellular FFA uptake. In the parenchymal cells of cirrhotic livers, however, the following changes were observed when compared with controls: (i) decreased cellular output of esterified FA derived both from exogenous sources and from de novo FA synthesis; (ii) increased total ketone body production, mainly as beta-hydroxybutyrate; (iii) decreased cholesterol synthesis; and (iv) enhanced incorporation of newly synthesized FA into phospholipids in spite of an unaffected rate of FA synthesis. In summary, the data provide evidence for intrinsic alterations in the lipid metabolism in the parenchymal cells of cirrhotic livers which are preserved in the isolated hepatocytes under optimum incubation conditions for oxygen and substrate supply.