Suitability of primary monolayer cultures of adult rat hepatocytes for studies of cholesterol and bile acid metabolism

Monolayer cultures of hepatocytes isolated from cholestyramine-fed rats and incubated in serum-free medium converted exogenous [4-14C]cholesterol into bile acids at a 3-fold greater rate than did cultures of hepatocytes prepared from untreated rats. Cholic acid and beta-muricholic acid identified and quantitated by gas-liquid chromatography and thin-layer chromatography were synthesized by cultured cells for at least 96 h following plating. The calculated synthesis rate of total bile acids by hepatocytes prepared from cholestyramine-fed animals was approximately 0.058 micrograms/mg protein/h. beta-Muricholic acid was synthesized at approximately a 3-fold greater rate than cholic acid in these cultures. Cultured hepatocytes rapidly converted the following intermediates of the bile acid pathway; 7 alpha-hydroxy[7 beta-3H]cholesterol, 7 alpha-hydroxy-4-[6 beta-3H] cholesten-3-one, and 5 beta-[7 beta-3H]cholestane-3 alpha, 7 alpha, 12 alpha-triol into bile acids. [24-14C]Chenodeoxycholic acid and [3H]ursodeoxycholic acid were rapidly biotransformed to beta-muricholic acid. 3-Hydroxy-3-methylglutaryl-coenzyme A reductase activity measured in microsomes of cultured hepatocytes decreased during the initial 48 h following plating, but remained relatively constant for the next 72 h. In contrast, cholesterol 7 alpha-hydroxylase activity appeared to decrease during the first 48 h, followed by an increase over the next 48 h. Despite the apparent changes in enzyme activity in vitro, the rate of bile acid synthesis by whole cells during this time period remained constant. It is concluded that primary monolayer cultures of rat hepatocytes can serve as a useful model for studying the interrelationship between cholesterol and bile acid metabolism.     

Effect of chylomicron remnants on cholesterol metabolism in cultured rabbit hepatocytes: production of very low density lipoproteins and bile acids

Primary cultures of rabbit hepatocytes were used to investigate the effect of purified (B-100 free) chylomicron remnants (CR) on lipid and bile acid metabolism. ApoB-100-containing lipoproteins were removed from the CR-enriched plasma fraction by affinity column chromatography on Sepharose 4B conjugated with anti-apoB-100 monoclonal antibodies. CR were shown to stimulate the accumulation of neutral lipids in hepatocytes in a dose-response manner. After 24-hour preincubation of rabbit hepatocytes with 50 micrograms protein/ml CR the cellular neutral lipid content increased: 1.9-4-fold for triglycerides, 1.5-3.7-fold for free cholesterol and 1.5-2.5-fold for esterified cholesterol. This accumulation was accompanied by a decreasing incorporation of [14C] acetate into cholesterol (80-90%) and triglycerides (70-80%). At the same time the incorporation of [14]oleate into triglycerides increased by 50-65%. The inhibited biosynthesis of fatty acids might account for this effect. No effect of CR on cholesterol esterification by [14C]oleate was observed. CR increased the amount of triglycerides and free cholesterol secreted in very low density lipoproteins (VLDL). The secretion of taurocholic acid was decreased. These data confirm our hypothesis that dietary cholesterol is preferentially secreted by hepatocytes within VLDL but is not accumulated as cholesterol esters or oxidized to bile acids.     

Behaviour of phospholipase modified-HDL towards cultured hepatocytes. II. Increased cell cholesterol storage and bile acid synthesis

Human total HDL (hydrated density 1.070-1.210), HDL2 (1.070-1.125), HDL3 (1.125-1.210) or HDL separated by heparin affinity chromatography were treated with or without purified phospholipase A2 from Crotalus adamanteus. Control and treated HDL were reisolated and were then incubated with cultured hepatocytes. 1. Mass measurements evidenced a time-dependent cholesterol enrichment in hepatocytes cultured in the absence of lipoproteins. Addition of HDL2 still enhanced by 25% the cell cholesterol content and down-regulated endogenous sterol synthesis in similar proportions. Conversely, HDL3 slightly decreased the amount of free cholesterol in hepatocytes (-12%). 2. Incubations with phospholipase A2-treated HDL resulted in a 35%-50% increase of both the cellular cholesterol esterification and the cholesterylester accumulation, when compared to cells cultured in the presence of control-HDL. This effect was observed with HDL2, HDL3 and combining the data with all subfractions. 3. Cultured hepatocytes secreted cholic and beta-muricholic acids as major bile acids and HDL2 showed a tendency to stimulate their secretion. Phospholipase treatment of HDL again induced an increased production by hepatocytes of those two bile acids. Thus, whereas HDL2 and HDL3 display different behaviours with respect to cell cholesterol content, neosynthesis and bile acid secretion, their modifications by phospholipases always orientate the cell sterol metabolism in the same direction: increased cholesterylester accumulation and bile acid production.     

Hydroxylation, conjugation and sulfation of bile acids in primary monolayer cultures of rat hepatocytes

Hydroxylation of lithocholic, chenodeoxycholic, deoxycholic and cholic acids was studied in monolayers of rat hepatocytes cultured for 76 h. The majority of added lithocholic and chenodeoxycholic acids was metabolized to beta-muricholic acid (56-76%). A small part of these bile acids (9%), however, and a considerable amount of deoxycholic and cholic acids (21%) were converted into metabolites more polar than cholic acid in the first culture period. Formation of these compounds decreased during the last day of culture. Bile acids synthesized after addition of [4-14C]-cholesterol were almost entirely (97%) sulfated and/or conjugated, predominantly with taurine (54-66%), during culture. Sulfated bile acids were mainly composed of free bile acids. The ability of hepatocytes to sulfurylate bile acids declined with culture age. Thus, rat hepatocytes in primary monolayer culture are capable to sulfurylate bile acids and to hydroxylate trihydroxylated bile acids, suggesting formation of polyhydroxylated metabolites.     

Bile acid synthesis in isolated rat hepatocytes

Normal adult rat hepatocytes were incubated for 48h and the concentration of total and individual bile acids in homogenized samples of the culture was measured at intervals during the incubation, using radiogas chromatography and isotope derivative assay. The net increase in bile acids over the value observed at the start of the culture was taken as synthesis. The results showed that bile acid synthesis was linear up to 24h of incubation, at a rate of 20nmol/g hepatocytes per hour, and that 85% of the newly synthesized bile acid was cholic acid. The bile acid synthesized was mainly conjugated with taurine. These results suggest that isolated hepatocytes cultured in the way described could be a useful in vitro model for the study of bile acid synthesis.     

Foetomaternal relationships of serum bile acid pattern estimated by high-pressure liquid chromatography.

The bile acid patterns in the maternal and umbilical vein and artery serum samples were analysed by a two-step chromatographic method involving group separation by piperidinohydroxypropyl-Sephadex LH-20 and high-pressure liquid chromatography using immobilized 3 alpha-hydroxy steroid dehydrogenase. Glycochenodeoxycholate predominates in the maternal blood and taurochenodeoxycholate in the umbilical blood. In cases where a free bile acid was detected in the maternal blood, the same bile acid was also demonstrated in the corresponding cord blood. The concentrations of taurocholate and taurochenodeoxycholate were found to be significantly higher in the umbilical artery than in the corresponding umbilical vein. Our data suggest that there is a bidirectional placental transfer of free bile acids and that there is a transfer of taurine-conjugated primary bile acids from the foetus to the mother.     

Bile acid synthesis and secretion by rabbit hepatocytes in primary monolayer culture: comparison with rat hepatocytes

Rabbit hepatocytes isolated after liver perfusion with collagenase were maintained in primary monolayer culture for periods up to 96 h. Bile acid synthesis and secretion was measured by capillary gas-liquid chromatography and by a rapid enzymatic-bioluminescence assay. As expected from the bile acid profile of rabbit gallbladder bile, cholic acid was the only bile acid synthesized in detectable amounts and was produced at a linear rate of 170 pmol/h per mg cell protein from 24 to 96 h in culture. Ketoconazole (20 microM) inhibited cholic acid synthesis and secretion by 78%, whereas the bile acids chenodeoxycholic acid (100 microM), deoxycholic acid (100 microM) or lithocholic acid (2 microM) had no effect. When rat hepatocytes were cultured under identical conditions, the rate of bile acid synthesis was found to be only 12 pmol/h per mg cell protein, a value in agreement with previous work. The large difference in rates of bile acid synthesis between rabbit and rat hepatocytes may be due to rapid loss of cytochrome P-450 from rat hepatocytes when placed in monolayer culture. Although reportedly active in cholesterol 7 alpha-hydroxylation, form 4 cytochrome P-450 levels in rabbit hepatocytes did not correlate with rates of bile acid synthesis.     

Assay of free and glycine- and taurine-conjugated bile acids in serum by high-pressure liquid chromatography by using post-column reaction after group separation.

An accurate and sensitive method that involves the group separations of serum bile acids (i.e. free and glycine- and taurine-conjugated bile acid fractions) by ion-exchange chromatography on piperidinohydroxypropyl-Sephadex LH-20 is described. Each group was then analysed by high-pressure liquid chromatography by using the post-column reaction technique with immobilized 3 alpha-hydroxy steroid dehydrogenase. The bile acid patterns in the umbilical venous serum samples were analysed by this method. Taurochenodeoxycholate predominated in the umbilical blood.     

Receptor-mediated introduction of pepstatin-asialofetuin conjugate into lysosomes of rat hepatocytes

Pepstatin was linked through a carboxyl group to asialofetuin (PS-ASF). An analysis by separation of hepatocytes from nonparenchymal cells showed that PS-ASF was taken up by hepatocytes, following intravenous injection into rats. After the injection of PS-ASF, pepstatin concentration in the liver reached a maximum at 2 h and then decreased. In an analysis by differential centrifugation of the liver homogenate from rats injected with PS-ASF, pepstatin showed a lysosomal type subcellular distribution pattern. Isolation studies of tritosomes clearly demonstrated the exclusive accumulation of pepstatin within the lysosomes of livers from rats given PS-ASF (at 2 h after administration). Pepstatin contained in tritosomes was in a free form, as determined by column chromatography of Sephadex G-15. The activity of cathepsin D in the livers was markedly inhibited in rats given PS-ASF. However, the treatment of rats with PS-ASF had no effect on the hepatic lysosomal degradation of endocytosed FITC-labeled asialofetuin (FITC-ASF). Introduction of PS-ASF into the hepatocytes was followed by the immediate and time-dependent excretion of free pepstatin into the bile. Quantification of pepstatin excreted into the bile revealed that the biliary excretion route can account for the disappearance of pepstatin from the liver.     

The effect of natural and synthetic antioxidant polyhydroxynaphthoquinones on cholesterol metabolism in cultured rabbit hepatocytes]

Primary cultures of rabbit hepatocytes were used to examine the effect of natural and synthetic antioxidants--polyhydroxynaphthoquinones (PHNQ) and alpha-tocopherol on cholesterol and bile acid synthesis. Histochrome, one of the PHNQ, slightly decreased cholesterol synthesis at concentrations 10-100 microM, whereas alpha-tocopherol stimulated cholesterol synthesis. After administration of histochrome or alpha-tocopherol into culture medium a significant stimulation of bile acid synthesis in dose-dependent manner was observed. The increase of bile acid secretion by histochrome in the presence of physiological concentration of HDL2 was found as well. Since histochrome in contrast to alpha-tocopherol enhanced accumulation of [14C] cholesterol of HDL2 in the hepatocytes, it was concluded that histochrome stimulated bile acid synthesis as a result of increased input of HDL2 cholesterol into hepatocytes. These data suggest that histochrome may exhibit a hypocholesterolemic effect by stimulation of bile acid synthesis and inhibition of cholesterol synthesis.     

Dicer1 敲除对肝脏胆酸代谢和转运功能的影响

目的:微小RNA(microRNAs,miRNAs)在胆固醇的合成,代谢和转运中起着重要作用,而mi RNAs在胆固醇代谢物胆酸的代谢和转运中的作用尚不清楚。Dicer基因是miRNAs生成过程的关键酶。本课题使用肝脏特异的Dicer1基因敲除小鼠,考察肝脏Dicer1基因敲除对C57BL/6小鼠肝脏胆酸代谢和转运的影响。方法:使用白蛋白启动子驱动的Cre重组酶和Loxp系统(Alb-Cre/Loxp)在小鼠肝脏中特异的敲除Dicer1基因;分别收集3~12周龄的小鼠血液和肝脏组织,使用Cobas生化仪检测小鼠血液和肝脏中总胆酸含量;利用实时定量PCR的方法分析肝脏中胆汁酸代谢转运相关基因的表达。结果:实验发现,肝脏Dicer基因敲除后,胆酸在血液和肝脏中明显蓄积,弥漫性肝细胞轻微空泡化,偶见单个肝细胞坏死。检测胆酸代谢和转运相关基因的表达发现,胆酸合成相关基因的表达有轻度升高,但缺乏统计学差异;在肝脏细胞血管侧的胆酸摄取转运体中,Oatp1a1在Dicer1敲除小鼠肝脏中明显下调,Ntcp和Oatp1b2则无明显改变;而肝细胞血管侧胆酸外排转运体的表达均有显著升高,胆管侧的外排转运体中Abcb11表达有明显增加。结论:Dicer基因敲除后,胆酸在血液和肝脏中明显蓄积,肝脏和血液中胆酸总量显著增加。血液中胆酸的蓄积可能与肝脏细胞血管侧摄取转运体的低表达和血管侧外排转运体的高表达有关;而肝脏中胆酸的蓄积可能部分来自于轻度升高的胆酸合成酶,胆酸在肝细胞内运输途径的紊乱可能与肝脏和血液中胆酸总量的显著增加相关。     

Effects of ovariectomy on the composition of fatty acids of phospholipids in Java macaques with cholestasis

Bilateral ovariectomy carried out in Java macaques with simultaneous dosed ligation of common bile duct distally to duodenum in order to induce acalculous hepato-cholecystitis, does not alter the lithogenic index of the bile. Phospholipids present in enterohepatic organs and tissues (hepatocytes, enterocytes, chyme, blood and bile) of gonadectomized animals with hepato-cholecystitis acquire a specific fatty acid pattern which is characterized by prevailing palmitic, stearic and oleic acids, unaffected level of arachidonic acid and traces of essential linoleic acid.     

Bile acid metabolism in isolated rat hepatocytes: studied by gas-liquid chromatography-mass spectrometry-selected ion monitoring

Bile acid contents in isolated rat hepatocytes were determined by gas-liquid chromatography-mass spectrometry-selected ion monitoring with the use of deuterium-labeled internal standards. This allowed us first to monitor the actual amounts of not only major but also minor bile acid components present with sufficient sensitivity and specificity and to follow the changes of individual bile acids in cultured rat hepatocytes simultaneously. In freshly isolated rat hepatocytes, cholic and beta-muricholic acids were the major components, comprising 35 and 46% of the total bile acids, respectively. These two bile acids were found to be most actively synthesized during the first 2 h of incubation and continued to increase thereafter for up to 6 h (the end of the period studied). In contrast, chenodeoxycholic and alpha-muricholic acids, which are the precursors of beta-muricholic acid, showed slight increases only in the first hour of incubation and decreased thereafter. These results suggested that the conversion to beta-muricholic acid from chenodeoxycholic acid via alpha-muricholic acid occurred rapidly in cultured rat hepatocytes. The secondary bile acids such as deoxycholic, hyodeoxycholic, and 3 alpha, 12 beta-dihydroxy-5 beta-cholanoic acids declined steadily from the start of incubation, which supported the findings that further hydroxylation of these dihydroxy bile acids occurs in rat liver.     

Absence of negative feedback control of bile acid biosynthesis in cultured rat hepatocytes

The effect of individual bile acids on bile acid synthesis was studied in primary hepatocyte cultures. Relative rates of bile acid synthesis were measured as the conversion of lipoprotein [4-14C]cholesterol into 4-14C-labeled bile acids. Additions to the culture media of cholate, taurocholate, glycocholate, chenodeoxycholate, taurochenodeoxycholate, glycochenodeoxycholate, deoxycholate, and taurodeoxycholate (10-200 microM) did not inhibit bile acid synthesis. The addition of cholate (100 microM) to the medium raised the intracellular level of cholate 10-fold, documenting effective uptake of added bile acid by cultured hepatocytes. The addition of 200 microM taurocholate to cultured hepatocytes prelabeled with [4-14C]cholesterol did not result in inhibition of bile acid synthesis. Taurocholate (10-200 microM) also failed to inhibit bile acid synthesis in suspensions of freshly isolated hepatocytes after 2, 4, and 6 h of incubation. Surprisingly, the addition of taurocholate and taurochenodeoxycholate (10-200 microM) stimulated taurocholate synthesis from [2-14C]mevalonate-labeled cholesterol (p less than 0.05). Neither taurocholate nor taurochenodeoxycholate directly inhibited cholesterol 7 alpha-hydroxylase activity in the microsomes prepared from cholestyramine-fed rats. By contrast, 7-ketocholesterol and 20 alpha-hydroxycholesterol strongly inhibited cholesterol 7 alpha-hydroxylase activity at low concentrations (10 microM). In conclusion, these data strongly suggest that bile acids, at the level of the hepatocyte, do not directly inhibit bile acid synthesis from exogenous or endogenous cholesterol even at concentrations 3-6-fold higher than those found in rat portal blood.     

Deoxycholic acid activates the c-Jun N-terminal kinase pathway via FAS receptor activation in primary hepatocytes. Role of acidic sphingomyelinase-mediated ceramide generation in FAS receptor activation

We have shown previously that bile acids can activate the JNK pathway and down-regulate cholesterol 7alpha-hydroxylase (CYP7A1), the rate-limiting enzyme in the neutral pathway of bile acid biosynthesis. In this study, the mechanism(s) by which deoxycholic acid (DCA) activates the JNK pathway were examined. FAS receptor (FAS-R) and acidic sphingomyelinase (ASM)-deficient hepatocytes were resistant to DCA-induced activation of the JNK pathway. Activation of the JNK pathway (2-3-fold) in response to tumor necrosis factor-alpha was similar in both wild-type and FAS-R(-/-) hepatocytes. In wild-type and FAS-R(-/-) hepatocytes, ceramide elevation was detected as early as 2 min and peaked at 10 min after DCA treatment. In contrast, ASM(-/-) hepatocytes were defective in DCA-induced ceramide generation. Treatment with DCA resulted in movement of FAS-R to the cell surface, which was blocked upon treatment with brefeldin A. However, brefeldin A failed to block DCA-mediated JNK activation in wild-type hepatocytes. DCA-induced JNK activation was independent of either the epidermal growth factor receptor activation or free radical generation. Addition of ASM to rat hepatocytes activated JNK and down-regulated CYP7A1 mRNA levels. In conclusion, these results show that DCA activates JNK and represses CYP7A1 mRNA levels in primary hepatocytes via an ASM/FAS-R-dependent mechanism that is independent of either the epidermal growth factor receptor or free radical generation.     

Alpha-fetoprotein in the liver of embryonic and newborn rats]

The localization of alpha-fetoprotein in the liver of embryos and newborn Wistar rats was determined by the method of fluorescent antibodies. This protein was found in the cytoplasm of some hepatocytes, endothelium of blood vessels and erythroid blood cells of embryos and new born rats. It was never found in the blood-forming and Kupffer cells of the liver, as well as in the epithelium of bile ducts. The hepatocytes containing this protein were located in the liver lobes near the central veins. Their number and the intensity of fluorescence decreased with the age of animals.     

Mitochondrial genome depletion in human liver cells abolishes bile acid-induced apoptosis: Role of the Akt/mTOR survival pathway and Bcl-2 family proteins

Acute accumulation of bile acids in hepatocytes may cause cell death. However, during long-term exposure due to prolonged cholestasis, hepatocytes may develop a certain degree of chemoresistance to these compounds. Because mitochondrial adaptation to persistent oxidative stress may be involved in this process, here we have investigated the effects of complete mitochondrial genome depletion on the response to bile acid-induced hepatocellular injury. A subline (Rho) of human hepatoma SK-Hep-1 cells totally depleted of mitochondrial DNA (mtDNA) was obtained, and bile acid-induced concentration-dependent activation of apoptosis/necrosis and survival signaling pathways was studied. In the absence of changes in intracellular ATP content, Rho cells were highly resistant to bile acid-induced apoptosis and partially resistant to bile acid-induced necrosis. In Rho cells, both basal and bile acid-induced generation of reactive oxygen species (ROS), such as hydrogen peroxide and superoxide anion, was decreased. Bile acid-induced proapoptotic signals were also decreased, as evidenced by a reduction in the expression ratios Bax-α/Bcl-2, Bcl-xS/Bcl-2, and Bcl-xS/Bcl-xL. This was mainly due to a downregulation of Bax-α and Bcl-xS. Moreover, in these cells the Akt/mTOR pathway was constitutively activated in a ROS-independent manner and remained similarly activated in the presence of bile acid treatment. In contrast, ERK1/2 activation was constitutively reduced and was not activated by incubation with bile acids. In conclusion, these results suggest that impaired mitochondrial function associated with mtDNA alterations, which may occur in liver cells during prolonged cholestasis, may activate mechanisms of cell survival accounting for an enhanced resistance of hepatocytes to bile acid-induced apoptosis.