Modulation of bile secretion by hepatic low-density lipoprotein uptake and by chenodeoxycholic acid and ursodeoxycholic acid treatment in the hamster

The effects of both apolipoprotein B,E receptor-dependent and receptor-independent uptake of low-density lipoprotein (LDL) in the liver on bile secretion were studied in bile fistula hamsters. Three groups of animals were studied after 4 wk of feeding either a control, chenodeoxycholic acid-, or ursodeoxycholic acid-containing diet. The hepatic receptor-dependent and receptor-independent uptake of LDL was related to both bile flow and biliary lipid secretion. The correlation with bile flow and biliary lipid secretion was positive for the receptor-dependent, but negative for the receptor-independent uptake of LDL. Although the receptor-mediated LDL uptake appeared to exert a strong influence on bile acid-independent bile flow, the receptor-independent uptake showed a significant relation with biliary bile acid excretion. Differences between the two mechanisms of LDL uptake were also evident in the biliary bile acid-cholesterol coupling, which was significantly stronger during receptor-independent than during receptor-dependent uptake of LDL. The effects of LDL uptake on bile secretion were modulated by the experimentally induced changes in both the content and composition of bile acids in the enterohepatic circulation.     

Effect of chenodeoxycholic and ursodeoxycholic acids on isolated adult human hepatocytes

Chenodeoxycholic and ursodeoxycholic are effective cholelitholytic agents, but differ in their side effects. Chenodeoxycholic acid administration induces diarrhea and a transient rise of GOT, which are virtually nonexistent with ursodeoxycholic acid treatment. Lithocholic acid, a bacterial metabolite of chenodeoxycholic acid, has been implicated as a possible hepatotoxin. In the present investigation, the effect of chenodeoxycholic acid or ursodeoxycholic acid and their glycine and taurine conjugates on isolated human hypatocytes was directly assessed. Chenodeoxycholic acid had drastic effects on isolated human hepatocytes by reducing the number of microvilli and disrupting cell membranes. Pronounced release of GOT was observed. In contrast, ursodeoxycholic acid produced only slight morphological changes and enzyme release. Conjugation of each respective bile acids had a moderating effect.     

The role of Hfe in transferrin-bound iron uptake by hepatocytes

HFE-related hereditary hemochromatosis results in hepatic iron overload. Hepatocytes acquire transferrin-bound iron via transferrin receptor (Tfr) 1 and Tfr1-independent pathways (possibly Tfr2-mediated). In this study, the role of Hfe in the regulation of hepatic transferrin-bound iron uptake by these pathways was investigated using Hfe knockout mice. Iron and transferrin uptake by hepatocytes from Hfe knockout, non-iron-loaded and iron-loaded wild-type mice were measured after incubation with 50 nM (125)I-Tf-(59)Fe (Tfr1 pathway) and 5 microM (125)I-Tf-(59)Fe (Tfr1-independent or putative Tfr2 pathway). Tfr1 and Tfr2 messenger RNA (mRNA) and protein expression were measured by real-time polymerase chain reaction and western blotting, respectively. Tfr1-mediated iron and transferrin uptake by Hfe knockout hepatocytes were increased by 40% to 70% compared with iron-loaded wild-type hepatocytes with similar iron levels and Tfr1 expression. Iron and transferrin uptake by the Tfr1-independent pathway was approximately 100-fold greater than by the Tfr1 pathway and was not affected by the absence of Hfe. Diferric transferrin increased hepatocyte Tfr2 protein expression, resulting in a small increase in transferrin but not iron uptake by the Tfr1-independent pathway. Conclusion: Tfr1-mediated iron uptake is regulated by Hfe in hepatocytes. The Tfr1-independent pathway exhibited a much greater capacity for iron uptake than the Tfr1 pathway but it was not regulated by Hfe. Diferric transferrin up-regulated hepatocyte Tfr2 protein expression but not iron uptake, suggesting that Tfr2 may have a limited role in the Tfr1-independent pathway.     

Treatment of cholestatic liver diseases; the role of ursodeoxycholic acid]

Ursodeoxycholic acid (UDCA) allows symptomatic treatment of cholestatic liver diseases such as primary biliary cirrhosis, primary sclerosing cholangitis, intrahepatic biliary atresia, and cholestasis of cystic fibrosis. Patients should be treated at an early stage of the disease in order to prevent progression to cirrhosis. Since UDCA has no toxic effects longterm treatment with this substance is possible without the risk of undesired side effects. In patients with primary biliary cirrhosis and rapid progression of the disease, UDCA may be combined with an immunosuppressive substance (i.e. cyclosporin). In primary sclerosing cholangitis, biliary atresia and cholestasis of cystic fibrosis, UDCA at present seems the only treatment of which a benefit for the patients can be expected. In endstage disease liver transplantation is indicated. The role of UDCA in chronic hepatitis and alcohol induced liver disease needs to be clarified in further studies. Whether the improvement of laboratory tests in such patients indicates amelioration of the course of disease, still is unclear.     

Effect of insulin on free fatty acid uptake by hepatocytes in the duck

To elucidate the hypolipacidaemic effect of insulin in ducks, its action on the uptake of free fatty acids (FFA) by duck hepatocytes was determined. At low doses (10 mu./l) insulin stimulated FFA uptake. This effect was not observed with higher doses of insulin (20, 30 and 50 mu./l). Growth hormone at physiological concentrations and corticosterone (14.4 nmol/l) decreased basal activity, probably by reducing glucose metabolism and consequently alpha-glycerophosphate (alpha-GP) supply. Insulin was able to reverse the inhibition induced by GH and corticosterone on both FFA uptake and alpha-GP production. These results therefore suggest that the hypolipacidaemic effect of insulin may be partly mediated by its action on hepatic FFA uptake.     

Utilization of ATP-depleted cells in the analysis of taurocholate uptake by isolated rat hepatocytes.

The usefulness of ATP-depleted rat hepatocytes in transport studies was examined. ATP-depleted hepatocytes were prepared by incubating cell suspensions with 30 microM rotenone. In ATP-depleted hepatocytes, plasma membrane permeability was increased and mitochondrial membrane potential decreased, while both intracellular volume and pH remained normal. Furthermore, in the presence of valinomycin, the initial uptake rates of 3H-tetraphenyl phosphonium (TPP+) with varied medium concentrations of potassium were predicted according to the Goldman-Hodgkin-Katz equation, which demonstrated that a potassium diffusion potential could be produced in this system. Using the thus-characterized ATP-depleted cells, the uptake mechanism of taurocholate was investigated. In the presence of an inwardly directed Na gradient, the taurocholate uptake was markedly stimulated and bile acid was transiently accumulated at a concentration 3-times higher than at equilibrium ('overshoot') in ATP-depleted cells. No overshoot was observed in viable cells, however, which suggests that in ATP-depleted cells the Na gradient, a driving force for taurocholate uptake, decreased with time. In both viable and ATP-depleted cells, the relationship between medium concentrations of Na and the Na-dependent initial uptake rate were sigmoidal, and the Hill coefficients were close to 2. The Na-dependent initial uptake rate of taurocholate was stimulated by a valinomycin-induced inside negative potassium-diffusion potential in ATP-depleted cells, and the movement of a 'one plus' (as a net) charge was revealed by fitting the data to the Goldman-Hodgkin-Katz equation. These results support the hypothesis that sodium-coupled hepatic uptake of taurocholate occuthrough an electrogenic process with the stoichiometry of 2 Na: 1 taurocholate, although this issue is controversial. In the presence of an outwardly directed sodium gradient, efflux of taurocholate from ATP-depleted cells was not stimulated. Consequently, the physiological transport vector of taurocholate from blood to cell is not only due to the direction of the sodium gradient (blood to cell) but also to membraneous orientation of transport carriers. In conclusion, kinetic analysis using ATP-depleted hepatocytes allowed the formulation of a new approach to clarify the as yet unresolved issues concerning transport stoichiometry and the mechanism for vectorial transport of taurocholate.     

Insulin inhibits peroxisomal fatty acid oxidation in isolated rat hepatocytes

Inhibition by insulin of long chain fatty acid oxidation in mitochondria is mediated in part by elevating malonyl-CoA levels, which inhibit carnitine palmitoyl-transferase I. Whether insulin alters peroxisomal oxidation has not been studied. We present data which show that insulin inhibits the oxidation of palmitic acid by peroxisomes (IC(50) = 8.5 x 10(-11) M) at hormone concentrations 100-fold less than those needed for mitochondrial inhibition (IC(50) = 1.3 x 10(-8) M). We used a purified peroxisome preparation to study the mechanism of insulin action. Insulin had a direct effect in the peroxisome preparations to decrease oxygen consumption, fatty acyl-CoA oxidizing system activity and acyl-CoA oxidase by approximately 40%, 30% and 15%, respectively. Since insulin degrading enzyme (IDE) is an insulin-binding protein known to be in peroxisomes, we studied the effect of an inhibitory anti-IDE antibody on the ability of insulin to inhibit the fatty acyl-CoA oxidizing system. The antibody eliminated the inhibitory effect of insulin. We conclude that insulin inhibits peroxisomal fatty acid oxidation by a mechanism requiring IDE.     

Monitoring of sodium:proton exchange in isolated hepatocytes by electronic cell sizing

To investigate volume-regulating processes in the hepatocyte, a rapid and precise method of measuring cell volume in isolated hepatocytes was devised which uses a Coulter Counter equipped with a Channelyzer. Isolated hepatocytes exhibit a volume-decreasing mechanism (potassium channel) which is triggered by cell volume increases as small as 10%. Cell volume increases in the hepatocyte may be mediated by activity of the Na:H exchanger. To examine Na:H exchange-mediated cell volume increases, without apparent interference by the volume-decreasing mechanism, acetate was substituted for chloride in the incubation medium. Hepatocytes placed in a medium containing sodium acetate at an acidic pH exhibit a continuous amiloride-sensitive swelling. A simple procedure was devised for estimating Na:H exchanger set-point by electronic cell sizing. In a sodium acetate medium, the internal pH equilibrates with the external pH. By placing cells in sodium acetate medium of various pH values and measuring the rate of amiloride-sensitive swelling, an estimate of Na:H exchanger set-point can be obtained. By this method, the exchanger was estimated to cease activity above an intracellular pH of 7.2. This method could be useful for identification of stimuli that might promote cell enlargement by raising the exchanger set-point. The phorbol ester 12-O-tetradecanoylphorbol-13-acetate tetraacetic acid raises the set-point of the exchanger in the isolated hepatocytes, resulting in exchanger activity at normal cellular pH, and at the same time promotes hepatocyte swelling. Exchanger activation via a kinase C-mediated mechanism is one possible way that hepatocyte enlargement may occur.     

Effect of glucocorticoids on amino acid transport in isolated rat hepatocytes

The effects of glucocoiticoids on neutral amino acid transport were studied in freshly prepared suspensions of isolated hepatocytes from adult rat. Glucocorticoids stimulated the active transport of α-aminoisobutyric acid by increasing the influx. The onset of effect was preceded by a lag period of about 1 h. Cortisol (ED₅₀ ~2 μM) was 10–20 times less potent than synthetic analogs. Glucocorticoids increased the V_max of transport without affecting the K_m. Actinomycin D and cycloheximide virtually abolished this effect. The glucocorticoid effect involved predominantly the A system of transport. The effect was additive with that of glucagon, insulin and catecholamines.     

Effect of insulin on amino acid transport in isolated rat hepatocytes

Summary The effects of insulin on amino acid transport were studied in freshly prepared suspensions of isolated hepatocytes from adult rats. Insulin stimulated the active transport of -aminoisobutyric acid by increasing the influx. The onset of the insulin effect was delayed by thirty to sixty min. Insulin increased the Vmax of transport by about 60% without affecting the Km. Cycloheximide and actinomycin D inhibited hormonal action by 60 to 80%. Only the A system of transport was affected by insulin. Half-maximal stimulation of transport was observed with insulin at 2 to 3nmol/l, a concentration which also occupies about 50% of insulin-specific binding sites at steady state. Insulin did not antagonize the stimulatory effect of glucagon on amino acid transport.     

Effects of ursodeoxycholic acid in esophageal motility and the role of the mucosa. An experimental study

Esophageal motor abnormalities are frequently found in patients with gastroesophageal reflux disease. The role of bile in reflux‐induced dysmotility is still elusive. Furthermore, it is questionable weather mucosal or muscular stimulation leads to motor dysfunction. The aims of this study were to analyze (i) the effect of bile in the amplitude of esophageal contractions; and (ii) the effect of mucosal versus muscular stimulation. Eighteen guinea pig esophagi were isolated, and its contractility assessed with force transducers. Three groups were studied. In group A (n= 6), the entire esophagus was incubated in 100 µmL ursodeoxycholic acid for 1 hour; in group B (n= 6) the mucosal layer was removed and the muscular layer incubated in 100 µmL ursodeoxycholic acid for 1 hour; and in group C (n= 6) (control group) the entire esophagus was incubated in saline solution. In all groups, five sequential contractions induced by 40 mm KCl spaced by 5 minutes were measured before and after incubation. Contractions amplitudes before incubation were 1.319 g, 0.306 g, and 1.795 g, for groups A, B, and C, respectively. There were no differences between groups A and C (P= 0.633), but there were differences between groups A and B (P= 0.039), and B and C (P= 0.048). After incubation amplitude of contraction were 0.709 g, 0.278 g, and 1.353 g for groups A, B, and C, respectively. Only group A showed difference when pre and post‐stimulation amplitudes were compared (P= 0.030). Our results show that (i) bile exposure decreases esophageal contraction amplitude; and (ii) the esophageal mucosa seems to play an important role in esophageal motility.     

Decreased uptake of taurocholate and ouabain by hepatocytes isolated from cirrhotic rat liver

To differentiate between the "intact" and "sick" cell hypothesis explaining decreased clearance of endo- and xenobiotics, we measured uptake of taurocholate and ouabain in hepatocytes isolated from cirrhotic rat liver. Cirrhosis was induced by chronic exposure of male Sprague-Dawley rats to phenobarbital and carbon tetrachloride. Uptake of [14C]taurocholate and [3H]ouabain was measured by a rapid filtration technique. Hepatocytes from cirrhotic liver were as viable as control hepatocytes--as judged by trypan blue exclusion and lactate dehydrogenase release--but consumed 28% less oxygen. Vmax of both taurocholate (3.16 +/- 0.95 vs. 0.40 +/- 0.35 nmoles X min-1 X 10(6) cells-1; p less than 0.001) and ouabain (2.16 +/- 0.78 vs. 0.83 +/- 0.26 nmoles X min-1 X 10(6) cells-1; p less than 0.005) was significantly reduced. These results are compatible with the "sick" cell hypothesis.     

熊去氧胆酸的免疫调节作用

熊去氧胆酸(UDCA)是亲水性胆汁酸制剂,广泛用于各种胆汁淤积性肝脏疾病的治疗.     

熊去氧胆酸在慢性肝病中的应用及机制

熊去氧胆酸(ursodeoxycholic acid,UDCA)为亲水性胆汁酸,具有利胆、细胞保护、抗凋亡、抗氧化和免疫调节等重要作用,是目前唯一被FDA批准用于治疗胆汁淤积性肝病的药物,已广泛地用于临床各种肝病治疗,取得了较好的疗效.