Biliary Lipid Output Research Articles

Ezetimibe prevents the formation of oestrogen-induced cholesterol gallstones in mice.

Oestrogen is an important risk factor for cholesterol cholelithiasis not only in women of childbearing age taking oral contraceptives and postmenopausal women undergoing hormone replacement therapy, but also in male patients receiving oestrogen therapy for prostatic cancer. In women, hormonal changes occurring during pregnancy markedly increase the risk of developing gallstones. We investigated whether the potent cholesterol absorption inhibitor ezetimibe could prevent the formation of oestrogen-induced cholesterol gallstones in mice. Following ovariectomy, female AKR mice were implanted subcutaneously with pellets releasing 17β-estradiol at 6μg/day and fed a lithogenic diet supplemented with ezetimibe in doses of 0 or 8mg/kg/day for 8weeks. Cholesterol crystallization and gallstone prevalence, lipid concentrations and composition in bile, and biliary lipid output were analysed by physical-chemical methods. Intestinal cholesterol absorption efficiency was determined by faecal dual-isotope ratio methods. Ezetimibe inhibited intestinal cholesterol absorption, while significantly reducing hepatic secretion of biliary cholesterol. Consequently, bile was desaturated through the formation of numerous unsaturated micelles and gallstones were prevented by ezetimibe in mice exposed to high doses of oestrogen and fed the lithogenic diet. Ezetimibe did not influence mRNA levels of the classical oestrogen receptors α (ERα) and ERβ, as well as a novel oestrogen receptor the G protein-coupled receptor 30 (GPR30) in the liver. Ezetimibe protects against the oestrogen-mediated lithogenic actions on gallstone formation in mice. Our finding may provide an efficacious novel strategy for the prevention of cholesterol gallstones in high-risk subjects, especially those exposed to high levels of oestrogen.

Acute Hypertriglyceridemia Promotes Intestinal Lymphatic Lipid and Drug Transport: A Positive Feedback Mechanism in Lipid and Drug Absorption

Elevated systemic levels of triglyceride-rich lipoproteins (TRL) are a risk factor for the development of atherosclerosis. In patients with metabolic syndrome (MetS), intestinal TRL overproduction contributes to high systemic TRL levels, and recent studies suggest that systemic changes in MetS such as increases in plasma fatty acids and insulin resistance stimulate intestinal TRL production. The current study has examined whether increases in systemic TRL influence intestinal lipid transport and lipoprotein assembly pathways and evaluates the impact of these changes on the absorption and lymphatic transport of lipids and a model lipophilic drug (halofantrine). Mesenteric lymph-duct or bile-duct cannulated rats were administered IV saline or (14)C-labeled chylomicron (CM) (to increase systemic TRL) and intraduodenal (3)H lipids and drug. Changes to biliary lipid output and lymphatic lipid and drug transport were subsequently examined. Increasing systemic TRL concentrations stimulated a significant increase in lymphatic lipid and drug transport. The increased lipids in lymph were not derived from bile or the intestinal blood supply (fatty acid or IV infused (14)C-CM). Rather, an increase in lymphatic transport of duodenally sourced lipids was evident. Increasing plasma levels of TRL therefore stimulated lipid absorption and lymphatic transport via a positive feedback process. The data also suggest that the changes to intestinal TRL formation that result from raised systemic TRL levels may impact on the absorption of highly lipophilic drugs and therefore the reproducibility of drug treatments.

Unexpected effects of fasting on murine lipid homeostasis – Transcriptomic and lipid profiling

Starvation induces massive perturbations in metabolic pathways involved in energy metabolism, but its effect on the metabolism of lipids, particularly cholesterol, is little understood. A comparative genomic analysis of the gut and the liver in response to fasting was performed, with intestinal perfusion and lipid profiling of the plasma, bile, liver, intestinal tissue, perfusate, and faeces in FVB mice. The expression profiles suggested increased cholesterol trafficking in the liver and decreased trafficking in the small intestine. Plasma cholesterol concentrations significantly increased, and triglycerides decreased in fasting. Surprisingly, in prolonged fasting, the biliary bile salt and lipid output rates increased, with increased hepatic and intestinal lipid turnover, and enhanced trans-intestinal cholesterol excretion. In contrast, faecal sterol loss declined sharply. To investigate whether the increased biliary phospholipid secretion could nourish the intestinal epithelium, we studied the histology of the small intestines upon fasting in multidrug resistant protein 2 deficient mice with scarce biliary phospholipids. Their adaptive biliary response to fasting was lost, while the shortage of biliary phospholipids strongly induced apoptosis and proliferation in the small intestine and increased the number of mucin-producing cells. Even with no dietary fat, lipid levels remain remarkably constant in the murine liver and intestines during prolonged fasting. The biliary system, always assumed to be coupled to the postprandial response, shows a paradoxical increase in activity. We hypothesise that biliary lipids are mobilised to supply the enterocytes with luminal fuel and to stabilise transport systems in the intestine for ensuring a rapid recovery when the food supply resumes.

Effect of Ezetimibe on the Prevention and Dissolution of Cholesterol Gallstones

Cholesterol cholelithiasis is one of the most prevalent and most costly digestive diseases in developed countries and its incidence has increased markedly in Asian countries owing to the adoption of Western-type dietary habits. Because animal experiments showed that high efficiency of intestinal cholesterol absorption contributes to gallstone formation, we explored whether the potent cholesterol absorption inhibitor ezetimibe could prevent gallstones and promote gallstone dissolution in mice and reduce biliary cholesterol content in human beings. Male gallstone-susceptible C57L mice were fed a lithogenic diet and concomitantly administered with ezetimibe at 0, 0.8, 4, or 8 mg/kg/day for 8 or 12 weeks. Gallbladder biles and gallstones were examined by microscopy. Gallbladder emptying in response to cholecystokinin octapeptide was measured gravimetrically. Biliary lipid outputs were analyzed by physical-chemical methods. Cholesterol absorption efficiency was determined by fecal dual-isotope ratio and mass balance methods. Lipid changes in gallbladder biles of gallstone patients vs overweight subjects without gallstones were examined before (day 0) and at 30 days after ezetimibe treatment (20 mg/day). Ezetimibe prevented gallstones by effectively reducing intestinal cholesterol absorption and biliary cholesterol secretion, and protected gallbladder motility function by desaturating bile in mice. Treatment with ezetimibe promoted the dissolution of gallstones by forming an abundance of unsaturated micelles. Furthermore, ezetimibe significantly reduced biliary cholesterol saturation and retarded cholesterol crystallization in biles of patients with gallstones. Ezetimibe is a novel approach to reduce biliary cholesterol content and a promising strategy for preventing or treating cholesterol gallstones by inhibiting intestinal cholesterol absorption.

The lipid-lowering effect of ezetimibe in pure vegetarians

Results of previous studies have shown that ezetimibe (10 mg/day) reduces LDL cholesterol in patients with mild hypercholesterolemia on a normal-cholesterol diet (dietary intake of 200-500 mg/day) by 16-22%. However, the LDL cholesterol-lowering effect of ezetimibe in subjects with an extremely low dietary cholesterol intake (vegetarians) has not been studied. We conducted a randomized, double-blind, placebo-controlled, two-phase crossover study in 18 healthy pure vegetarians to assess the effect of ezetimibe (10 mg/day) on plasma lipids, cholesterol absorption, and its synthesis. Treatment periods lasted 2 weeks each, with an intervening 2 week washout period. Fractional cholesterol absorption was determined using the continuous dual stable isotope feeding method. Mean dietary cholesterol intake in the pure vegetarians was extremely low and averaged 29.4 +/- 16.8 and 31.4 +/- 14.4 mg/day during the placebo and ezetimibe administration phases, respectively. Fractional cholesterol absorption during the placebo phase was 48.2 +/- 8.2% and was decreased by 58% during ezetimibe treatment to 20.2 +/- 6.2% (P < 0.001). This change in intestinal cholesterol absorption was followed by a significant reduction in LDL cholesterol of 17.3%. In individuals with extremely low dietary cholesterol intake, treatment with ezetimibe (10 mg/day) leads to a significant reduction of cholesterol absorption and a clinically relevant decrease of plasma LDL cholesterol, comparable to that of subjects with a normal dietary cholesterol intake. Thus, the lipid-lowering effect of ezetimibe is mediated mainly through a reduction of the absorption of endogenous (biliary) cholesterol.

Biliary Lipid Output in the Early Stage of Acute Liver Failure Induced by 90% Hepatectomy in the Rat

Differences in biliary lipid output were compared in rats after 70% or 90% hepatectomy (Hx) to evaluate a possible index of the early stage of acute liver failure. Male Sprague-Dawley (SD) rats weighing 300 to 350 g were randomly divided into two groups for 70% Hx or 90% Hx, and animals were sacrificed at 0, 6, 24, and 48 h after Hx. Before sacrifice, a polyethylene tube was cannulated into the bile duct and bile was collected for 1 h. Outputs of total bile acids, phospholipid, and total cholesterol in serum and bile were determined. Biliary total cholesterol, bile acid concentrations, and bile acid component levels were determined using gas liquid chromatography. Hepatic microsomal cholesterol 7alpha-hydroxylase and sterol 12alpha-hydroxylase activities were also determined using high performance liquid chromatography. The 3-day survival rate after 90% Hx was 50%. In the 90% Hx group, the serum total bile acid concentration at each point was significantly higher than it was in the 70% Hx group. The bile flow rate and biliary outputs of cholesterol, phospholipid, and bile acids were significantly lower at 6 h after 90% Hx than after 70% Hx. Among bile acid species, cholic and chenodeoxycholic acid outputs into bile were significantly less at 6 h after 90% Hx. The activities of cholesterol 7alpha-hydroxylase and sterol 12alpha-hydroxylase were decreased after 90% Hx. Our results suggest that determinations of the bile flow rate and biliary lipid outputs are supposed to be useful for early detection of hepatic failure after extensive hepatectomy.

The Lymph Lipid Precursor Pool Is a Key Determinant of Intestinal Lymphatic Drug Transport

The influence of the size and turnover kinetics of the enterocyte-based lymph lipid precursor pool (LLPP) on intestinal lymphatic drug transport has been examined. Mesenteric lymph duct-cannulated rats were infused intraduodenally with low (2-5 mg/h) or high (20 mg/h) lipid-dose formulations containing 100 microg/h halofantrine (Hf, a model drug) and 1 microCi/h (14)C-oleic acid (OA) (as a marker for lipid transport) until steady-state rates of lipid(dX(L)/dt)(ss) and drug (dD(L)/dt)(ss) transport in lymph were obtained. After 5 h, the infusion was changed to formulations of the same composition but excluding (14)C-OA and Hf, allowing calculation of the first order rate constants describing turnover of lipid (K(X)) and drug (K(D)) from the LLPP into the lymph from the washout kinetics. The mass of lipid (X(LP)) and drug (D(LP)) in the LLPP was also determined. Biliary-lipid output was determined in a separate group of rats that had been infused with the same formulations. The results indicate that after administration of high lipid doses, lymphatic drug transport is dependent on the mass of exogenous lipid available in the LLPP and the rate of lipid pool turnover into the lymph. In contrast, after administration of low lipid doses, biliary-derived endogenous lipids are most likely to be the primary drivers of drug incorporation into the LLPP and lymph. Therefore, the LLPP size and composition seem to be major determinants of lymphatic drug transport, and formulation components, which increase lipid pool size, may therefore enhance lymphatic drug transport.

Role of intestinal sterol transporters Abcg5, Abcg8, and Npc1l1 in cholesterol absorption in mice: gender and age effects

Recent studies have indicated that intestinal cholesterol absorption is a multistep process, which is regulated by multiple genes at the enterocyte level. However, the molecular mechanisms whereby there are gender differences in intestinal cholesterol absorption efficiency and the efficiency of cholesterol absorption increases with age have not yet been fully understood. To explore whether aging increases cholesterol absorption via intestinal sterol transporters, we studied the higher cholesterol-absorbing C57L/J vs. the lower cholesterol-absorbing AKR/J mice at 8 (young adult), 36 (older adult), and 50 (aged) wk of age. To test the hypothesis that estrogen receptor (ER )alpha plays an important regulatory role in cholesterol absorption, we investigated the gonadectomized mice of both genders treated with 17beta-estradiol-releasing pellets at 0, 3, or 6 mug/day and antiestrogenic ICI 182,780 at 125 microg/day. We found that hepatic outputs of biliary cholesterol were significantly increased with age and in response to high levels of estrogen. Aging significantly enhances cholesterol absorption by suppressing expression of the jejunal and ileal sterol efflux transporters [ATP-binding cassette (Abc)g5 and Abcg8] and upregulating expression of the putative duodenal and jejunal sterol influx transporter Npc1l1. Estrogen significantly augmented cholesterol absorption mostly due to an upregulated expression of intestinal Npc1l1, Abcg5, and Abcg8 via the intestinal ERalpha pathway, which can be fully abolished by the antagonist. We conclude that ERalpha activated by estrogen and aging enhances cholesterol absorption by increasing biliary lipid output and mediating intestinal sterol transporters favoring influx of intraluminal cholesterol molecules across the apical membrane of the enterocyte.

Faecal sterol output is increased by arachidyl amido cholanoic acid (Aramchol) in rats.

Fatty acid-bile acid conjugates (FABACs) were shown recently to have important and multiple effects on cholesterol metabolism. In human fibroblasts, they were found to markedly enhance cholesterol efflux by an ATP-binding cassette transporter A1-dependent pathway. In C57L/J mice, they increased CYP7A1 activity and RNA expression, while decreasing moderately 3-hydroxy-3-methylglutaryl-CoA reductase activity. In C57L/J mice and in hamsters, they also decreased serum cholesterol levels, whereas in other animals, this effect was not seen in short-term experiments. In the present study, we investigated potential mechanisms of action of arachidyl amido cholanoic acid (Aramchol), with particular reference to biliary and faecal sterol outputs in rats. Supplementation with Aramchol at a dose of 150 mg x kg(-1) x day(-1) increased neutral sterol output by approx. 50%, while the faecal outputs of bile salts and total sterols increased by almost 2-fold. Biliary lipid outputs were not significantly different between the control and FABAC-supplemented animals. These findings indicate an overall catabolic effect of FABACs on body cholesterol.

Effects of dietary soybean lecithin on plasma lipid transport and hepatic cholesterol metabolism in rats

Dietary lecithin can stimulate bile formation and biliary lipid secretion, particularly cholesterol output in bile. Studies also suggested that the lecithin-rich diet might modify hepatic cholesterol homeostasis and lipoprotein metabolism. Therefore, we examined hepatic activities of 3-hydroxy-3 methylglutaryl coenzyme A reductase “HMG -CoA reductase”, cholesterol 7α-hydroxylase and acyl-CoA: cholesterol acyltransferase “ACAT” as well as plasma lipids and lipoprotein composition in rats fed diets enriched with 20% of soybean lecithin during 14 days. We also evaluated the content of hepatic canalicular membrane proteins involved in lipid transport to the bile (all P-glycoproteins as detected by the C 219 antibody and the sister of P-glycoprotein “spgp” or bile acid export pump) by Western blotting. As predicted, lecithin diet modified hepatic cholesterol homeostasis. The activity of hepatic HMG-CoA reductase and cholesterol 7α-hydroxylase was enhanced by 30 and 12% respectively, while microsomal ACAT activity showed a dramatic decrease of 75%. As previously reported from ACAT inhibition, the plasma level and size of very low-density lipoprotein (VLDL) were significantly decreased and bile acid pool size and biliary lipid output were significantly increased. The canalicular membrane content of lipid transporters was not significantly affected by dietary lecithin. The current data on inhibition of ACAT activity and related metabolic effects by lecithin mimic the previously reported effects following drug-induced inhibition of ACAT activity, suggesting potential beneficial effects of dietary lecithin supplementation in vascular disease.

Choleresis and inhibition of biliary lipid secretion induced by piperacillin in the rat.

1. The effects of the administration piperacillin on bile flow and biliary lipid secretion were studied in male Wistar rats. 2. Intravenous injection of piperacillin at doses ranging from 0.3 to 3.0 mmol/kg bodyweight led to an increase in its biliary concentration and excretion rate. Maximal biliary excretion was reached at a dose of 2.0 mmol/kg piperacillin. 3. Excretion of the antibiotic into bile was associated with a marked choleresis. A linear relationship was observed between bile flow and piperacillin excretion, 5.7 micro L bile being produced per micro mol piperacillin excreted into the bile. 4. Continuous i.v. infusion of piperacillin at 2.0 mmol/100 g per min did not result in significant changes in bile acid or cholesterol secretion, but biliary phospholipid secretion was markedly reduced. The inhibitory effect on phospholipid secretion was also present when biliary lipid output had been previously increased by an infusion of taurocholate (200 nmol/100 g per min). Addition of taurocholate did not reverse the impairment of phospholipid secretion induced by piperacillin. 5. These results indicate that acute administration of piperacillin in the rat induces a marked choleresis by stimulating bile acid-independent bile flow. The significant impairment in phospholipid secretion suggests a specific effect on intracellular supply and/or translocation across the canalicular membrane.

The influence of dietary saturated and unsaturated fat on hepatic cholesterol metabolism and the biliary excretion of chylomicron cholesterol in the rat

The biliary excretion of [ 3 H] cholesterol carried in chylomicrons derived from palm oil (rich in long chain saturated fatty acids), olive oil (rich in monounsaturated fatty acids) or corn oil (rich in n-6 polyunsaturated fatty acids was studied in vivo in rats fed the corresponding oil in the diet for 21 days. The secretion of radioactivity into bile as both bile acids and unesterified cholesterol was significantly slower in the animals fed palm oil as compared to those given olive or corn oil, indicating that dietary saturated fat retards the excretion of cholesterol from the diet as compared to mono- or n-6 polyunsaturated fat. In order to investigate the mechanisms underlying these differences, the influence of the three high fat diets on cholesterol esterification, cholesteryl ester hydrolysis and bile acid synthesis in the liver and on biliary lipid output were also measured. The ratio of cholesterol esterification to cholesteryl ester hydrolysis was markedly raised in the olive and corn oil-fed as compared to palm oil-fed animals. Biliary cholesterol secretion was higher in corn oil-fed rats than in those fed olive or palm oil or a low fat diet, and this was associated with a markedly increased lithogenic index in these animals. The activity of cholesterol 7α hydroxylase was higher in the olive and corn oil-fed than in the palm oil-fed animals, although the expression of mRNA for the enzyme was increased only in the olive oil diet group. After 20 h biliary drainage, the rate of bile acid secretion into bile was increased in the rats fed olive and corn oil rather than to palm oil. These findings indicate that feeding rats mono- or n-6 polyunsaturated as compared to saturated fat in the diet promotes the storage of cholesteryl ester in the liver and leads to increased bile acid synthesis, resulting in the more rapid excretion of cholesterol originating from the diet via the bile.

Changes in biliary lipid output after interruption of pravastatin treatment in humans

The use of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (e.g. pravastatin) has gradually increased in the treatment of hypercholesterolaemia. By inhibiting HMG-CoA reductase (the rate-limiting enzyme in cholesterol synthesis) activity, cholesterol synthesis in the liver is reduced and the plasma level of cholesterol, especially low-density lipoprotein (LDL)-cholesterol, is substantially lowered. Simultaneously, inhibition of HMG CoA reductase activity is associated with increased synthesis and accumulation of larger amounts of HMG CoA reductase enzyme protein. The main purpose of this study was to determine if the cessation of pravastatin treatment causes a rapid increase in the synthesis and biliary secretion of cholesterol, a condition which might lead to a temporarily increased cholesterol saturation of bile. Nine patients undergoing surgery for stones in the common bile duct were fitted with T-tubes in the common bile duct peroperatively; the side arm of the T-tube was left open postoperatively, creating a biliary fistula. All patients were given 6 days' treatment with pravastatin (20 mg b.i.d.) following the operation. Bile was collected from the T-tube, in 12-h fractions during this period and for another 3 days after termination of the treatment. Plasma levels of lipoproteins and lathosterol--reflecting cholesterol synthesis--were determined on several occasions. After cessation of pravastatin treatment, the plasma levels of total cholesterol and LDL-cholesterol increased by 21% and 33% respectively. High-density lipoprotein (HDL)-cholesterol did not change. The plasma level of lathosterol was increased two- to fourfold. Outputs of bile acids and phospholipids were significantly increased (23% and 10% respectively) after termination of treatment, whereas the output of cholesterol was not significantly changed. Cholesterol saturation was reduced by 20%, from 175 +/- 37% to 140 +/- 19%, but this change was not significant. The results indicate that, with the present experimental model (biliary diversion), the synthesis and biliary secretion of bile acids seem to be largely dependent on the de novo synthesis of cholesterol in the liver, whereas the biliary output of cholesterol is not.

Regulation of protein secretion into bile: Studies in mice with a disrupted mdr2 p-glycoprotein gene

Background & Aims: Protein is secreted into bile via several independent pathways. The aim of this study was to investigate whether these pathways are influenced by secretion of biliary lipid. Methods: Protein secretion and biliary lipid output were studied in wildtype mice (+/+), heterozygotes (+/−), and homozygotes (−/−) for mdr2 gene disruption. Biliary lipid and protein output were varied by infusion with taurocholate (TC) and tauroursodeoxycholate (TUDC). Results: Exocytosis and transcytosis were unaltered in (−/−) mice. Infusion with TC strongly induced secretion of alkaline phosphatase in (−/−) mice but had little effect in (+/−) and (+/+) mice. Infusion with TUDC had little effect on alkaline phosphatase output. In contrast, both TUDC and TC strongly stimulated secretion of aminopeptidase N and lysosomal enzymes in (+/+) mice but had no effect in (−/−) animals. Aminopeptidase N secretion correlated with phospholipid output, but only at high flux. At low flux, aminopeptidase N was secreted independently from both phospholipid and bile salts. Conclusions: The canalicular membrane enzymes alkaline phosphatase and aminopeptidase N are secreted via separate pathways. Part of alkaline phosphatase output is controlled by bile salt hydrophobicity, whereas at high lipid flux, aminopeptidase N secretion seems to be coupled to phospholipid output. Lysosomal enzymes follow the latter pathway.

P-210 Glucose intolerance in patients with chronic liver diseases — Analysis of insulin secretion and sensitivity

Background & Aims: Protein is secreted into bile via several independent pathways. The aim of this study was to investigate whether these pathways are influenced by secretion of biliary lipid. Methods: Protein secretion and biliary lipid output were studied in wildtype mice (+/+), heterozygotes (+/−), and homozygotes (−/−) for mdr2 gene disruption. Biliary lipid and protein output were varied by infusion with taurocholate (TC) and tauroursodeoxycholate (TUDC). Results: Exocytosis and transcytosis were unaltered in (−/−) mice. Infusion with TC strongly induced secretion of alkaline phosphatase in (−/−) mice but had little effect in (+/−) and (+/+) mice. Infusion with TUDC had little effect on alkaline phosphatase output. In contrast, both TUDC and TC strongly stimulated secretion of aminopeptidase N and lysosomal enzymes in (+/+) mice but had no effect in (−/−) animals. Aminopeptidase N secretion correlated with phospholipid output, but only at high flux. At low flux, aminopeptidase N was secreted independently from both phospholipid and bile salts. Conclusions: The canalicular membrane enzymes alkaline phosphatase and aminopeptidase N are secreted via separate pathways. Part of alkaline phosphatase output is controlled by bile salt hydrophobicity, whereas at high lipid flux, aminopeptidase N secretion seems to be coupled to phospholipid output. Lysosomal enzymes follow the latter pathway.

Biliary lipid output by isolated perfused rat livers in response to cholyl-lysylfluorescein

The biliary output of bile acids and lipids is tightly coupled. The ability of the natural bile acid glycocholate to trigger biliary lipid secretion was compared with that of the fluorescent bile acid analogue cholyl-lysylfluorescein (cholyl-lys-F). When administered as a 5 min pulse of 2.5 μmol/min to bile acid-depleted rat livers perfused under recycling conditions, glycocholate produced well-defined peaks of phospholipid and cholesterol output, and of bile flow, which were coincident with the peak of bile acid output. Although cholyl-lys-F did trigger biliary lipid secretion, its time course of appearance was delayed and well-defined peaks of output were not observed. However, the increased biliary output of phospholipid and cholesterol was coincident with that of bile acids and, as judged by phospholipid/bile acid and cholesterol/bile acid ratios, cholyl-lys-F was as effective as glycocholate in triggering biliary lipid output. When administered to livers perfused under single pass conditions, perfusate to bile transfer of glycocholate was > 85% at infusion rates of up to 5 μmol/min whereas transfer of cholyl-lys-F showed saturation at infusion rates of > 0.2 μmol/min; the time course of biliary output of both bile acids was similar. Thus, under recycling conditions, cholyl-lys-F not taken up during first pass will be continually represented for transfer to bile, explaining why bile acid and lipid output did not occur as well-defined peaks.

Short-term feeding of a diet enriched in phospholipids increases bile formation and the bile acid transport maximum in rats

Earlier studies suggested that the secretory rate maximum (SRm) of bile acid and the cholestasis which occurs after the SRm is reached may be determined by the hepatic or extrahepatic biliary phospholipid pool. We therefore investigated whether bile formation and the bile acid SRm could be influenced by feeding a diet enriched in phospholipids. Male rats were fed phospholipid (PLD) or triacylglycerol (TgD) -enriched diet for 3 days, and bile formation as well as biliary lipid output were measured on the 4th day. In other similarly fed groups, cholic acid was infused in stepwise increasing doses to determine the effect of PLD on the SRm of cholic acid. The plasma lipid levels were significantly lower in PLD and TgD diets compared to basal diet. But, while the levels of total cholesterol (CH), HDL-CH, and phospholipid (PH) were not significantly altered by PLD compared to TgD, the triacylglycerol levels were markedly increased by PLD. In the liver of PLD fed rats, triacylglycerol and CH ester contents decreased by 39 and 62%, respectively, while free CH and PH contents were not significantly changed. The PLD significantly augmented spontaneous bile flow, bile acid, PH and CH secretion rates compared to TgD diet (65, 124, 164 and 654%, respectively). The enhanced biliary secretory function was associated with an increase in pericanalicular vacuoles and diverticuli in centrilobular hepatocytes. Compared to TgD fed rats, PLD rats showed a 2-fold decrease in the ratio of cholic acid/chenodeoxycholic acid in bile and a significant decrease in the % contribution of taurine conjugated BA. The PH fatty acids in bile were similar in both groups except that in PLD group the % contribution of C18:2 was higher than in TgD group. No differences were found in plasma membrane CH/PH content or total fatty acid composition. During bile acid infusion, the SRm and the total cholic acid secreted were significantly higher in the PLD than in the TgD rats. Moreover, the cholestatic response observed after high bile acid dose was markedly reduced by PLD. The results show that short-term feeding of PLD induces changes in CH and bile acid metabolism which result in enhanced biliary output of CH and PH. The enhanced pool of biliary lipid may protect plasma membranes from the deleterious effects of high bile acid concentrations.

Effects of niacin on biliary lipid output in the rat.

Effects of niacin on biliary lipid output in the rat.

Effects of niacin on biliary lipid output in the rat

The mechanisms for the hypocholesterolaemic action of niacin (nicotinic acid) were examined in rats administered niacin at a dose of 400 mg/kg body wt/day for either 2 or 4 weeks. Another group of rats were administered diosgenin, an inhibitor of acyl-CoAxholesterol acyltransferase, as a 1% (w/w) supplement in the diet for 7 days. Both agents produced small increases in bile flow rates (up to 40%) and mild hepatotoxicity evidenced by small increases in serum transaminase activities. Niacin treatment for 2 or 4 weeks lowered serum cholesterol concentrations by 13% or 29%, respectively, with the greatest decrease occurring in the low density lipoprotein fraction. This was accompanied by relatively large increases in biliary cholesterol output (114% and 130% after 2 and 4 weeks treatment, respectively) with smaller increases in the biliary output of phospholipid (18% and 45%) and bile acid (26% and 14%). Diosgenin treatment increased serum cholesterol by 29% and increased the biliary output of cholesterol, phospholipid and bile add by 800%, 10% and 45%, respectively. Thus, both agents increased the cholesterol saturation of bile (100% by niacin, 500% by diosgenin). Cholesterol and phospholipid in fistula bile from control rats were present in lamellar and micellar forms. Niacin treatment did not alter the physical form of biliary lipids whilst diosgenin caused the appearance of vesicular lipid in fistula bile. Thus, increased biliary secretion of cholesterol explains, at least in part, the hypocholesterolaemic action of niacin. In addition, since aggregation of biliary vesicles is involved in cholesterol gallstone formation in humans, the non-appearance of vesicular material in fistula bile from niacin-treated rats may be of some importance.

Cholestasis, metabolism and biliary lipid secretion during perfusion of rat liver with different bile salts

The biological effects of bile acids depend largely upon their molecular structure. When bile acid uptake exceeds the maximal biliary secretory rate (SRm) cholestasis occurs. In order to characterize the influence of bile acid structure on its cholestatic potency we systematically studied SRm, maximal bile flow, maximal and cumulative phospholipid and cholesterol secretion with different taurine-conjugated tri-, di- and keto bile acids (Table I) in the isolated perfused rat liver. Bile acids with a high critical micellar concentration (CMC) promoted the greatest bile flow; a positive non-linear correlation between CMC and maximal bile flow was found. 3α-Hydroxylated bile acids with a hydroxyl group in 6α and/or 7β position and lacking a 12α hydroxy group had a high SRm. SRm was not related to CMC or maximal bile flow, respectively. Phospholipids and cholesterol were secreted in a nearly fixed ratio of 12:1; a strong linear relationship could be observed. Cumulative phospholipid secretion over 48 min was significantly lower for non and poor micelle forming bile acids (TDHC and TUC) than for those with comparatively low CMC values (TUDC, TC, THC, THDC, TCDC) (70–140 vs. 210–450 nmol/g liver). At SRm all bile acids with good micelle forming properties showed a similar cumulative biliary lipid output. However, when biliary lipid output was related to 1 μmol bile acid secreted bile acids with a low SRm induced the highest lipid secretion (TCDC, TC). These data (1) demonstrate that a 6α and/or a 7β hydroxy group on the steroid nucleus reduce cholestatic potency if the 12α hydroxy group is absent, (2) suggest that in the case of micelle forming bile acids the total amount of phospholipids secreted in bile (depletion of cellular phospholipids) is associated with the occurrence of cholestasis whereby bile acids with a low SRm deplete the cellular phospholipid content at much lower bile acid concentrations than those with a higher SRm and (3) imply that bile acids with non and poor micelle forming properties (TDHC, TUC) presumably do not cause cholestasis (solely) by depletion of cellular phospholipids.