Biliary Lipid Excretion Research Articles

Blocking Sodium-Taurocholate Cotransporting Polypeptide Stimulates Biliary Cholesterol and Phospholipid Secretion in Mice.

Active secretion of bile salts into the canalicular lumen drives bile formation and promotes biliary cholesterol and phospholipid output. Disrupting hepatic bile salt uptake, by inhibition of sodium‐taurocholate cotransporting polypetide (NTCP; Slc10a1) with Myrcludex B, is expected to limit bile salt flux through the liver and thereby to decrease biliary lipid excretion. Here, we show that Myrcludex B–mediated NTCP inhibition actually causes an increase in biliary cholesterol and phospholipid excretion whereas biliary bile salt output and bile salt composition remains unchanged. Increased lysosomal discharge into bile was excluded as a potential contributor to increased biliary lipid secretion. Induction of cholesterol secretion was not a consequence of increased ATP‐binding cassette subfamily G member 5/8 activity given that NTCP inhibition still promoted cholesterol excretion in Abcg8 −/− mice. Stimulatory effects of NTCP inhibition were maintained in Sr‐b1 −/− mice, eliminating the possibility that the increase in biliary lipids was derived from enhanced uptake of high‐density lipoprotein–derived lipids. NTCP inhibition shifts bile salt uptake, which is generally more periportally restricted, toward pericentral hepatocytes, as was visualized using a fluorescently labeled conjugated bile salt. As a consequence, exposure of the canalicular membrane to bile salts was increased, allowing for more cholesterol and phospholipid molecules to be excreted per bile salt. Conclusion: NTCP inhibition increases biliary lipid secretion, which is independent of alterations in bile salt output, biliary bile salt hydrophobicity, or increased activity of dedicated cholesterol and phospholipid transporters. Instead, NTCP inhibition shifts hepatic bile salt uptake from mainly periportal hepatocytes toward pericentral hepatocytes, thereby increasing exposure of the canalicular membrane to bile salts linking to increased biliary cholesterol secretion. This process provides an additional level of control to biliary cholesterol and phospholipid secretion.

Restriction of Food Intake Prevents Postinfarction Heart Failure by Enhancing Autophagy in the Surviving Cardiomyocytes

We investigated the effect of restriction of food intake, a potent inducer of autophagy, on postinfarction cardiac remodeling and dysfunction. Myocardial infarction was induced in mice by left coronary artery ligation. At 1 week after infarction, mice were randomly divided into four groups: the control group was fed ad libitum (100%); the food restriction (FR) groups were fed 80%, 60%, or 40% of the mean amount of food consumed by the control mice. After 2 weeks on the respective diets, left ventricular dilatation and hypofunction were apparent in the control group, but both parameters were significantly mitigated in the FR groups, with the 60% FR group showing the strongest therapeutic effect. Cardiomyocyte autophagy was strongly activated in the FR groups, as indicated by up-regulation of microtubule-associated protein 1 light chain 3-II, autophagosome formation, and myocardial ATP content. Chloroquine, an autophagy inhibitor, completely canceled the therapeutic effect of FR. This negative effect was associated with reduced activation of AMP-activated protein kinase and of ULK1 (a homolog of yeast Atg1), both of which were enhanced in hearts from the FR group. In vitro, the AMP-activated protein kinase inhibitor compound C suppressed glucose depletion-induced autophagy in cardiomyocytes, but did not influence activity of chloroquine. Our findings imply that a dietary protocol with FR could be a preventive strategy against postinfarction heart failure.

CETP expression enhances liver HDL-cholesteryl ester uptake but does not alter VLDL and biliary lipid secretion

The aim of this work was to study how CETP expression affects whole body cholesterol homeostasis. Thus, tissue uptake and plasma removal rates of labeled HDL-cholesteryl ester (CE), VLDL secretion rates, and biliary lipid secretion and fecal bile acid content were compared between human CETP transgenic (Tg) and non-transgenic (nTg) mice fed with a standard diet. CETP Tg mice exhibited increased HDL-CE plasma fractional catabolic rate and uptake by the liver, adrenals, adipose tissue and spleen. HDL fractions from both CETP Tg and from nTg mice were removed faster from the plasma of CETP expressing than from nTg mice, suggesting a direct role of CETP in accelerating tissue CE uptake. However, neither hepatic output of VLDL cholesterol and triglycerides nor biliary lipid and fecal bile acid excretion were changed in CETP Tg compared to nTg mice. CETP Tg mice also showed enhanced hepatic cholesterol content. Steady state cholesterol homeostasis was probably preserved through the downregulation of hepatic HMG-CoA reductase and LDL receptor expression. In conclusion, although CETP expression facilitates cholesteryl ester tissue uptake, it does not alter biliary lipid and fecal bile acid excretion, the mandatory final step of the reverse cholesterol transport.

Effects of pravastatin and bezafibrate on biliary lipid excretion and hepatic expression of Abcg5 and Abcg8 in the rat

Multidrug resistance associated gene product 2 (Mdr2) is believed to have a significant role in biliary cholesterol and phospholipid secretions. Both pravastatin and bezafibrate resulted in Mdr2 induction, but increased cholesterol secretion was observed only in pravastatin treatment. To explore the mechanism, the hepatic expression of genes that are responsible for the metabolism of the lipids was studied. Rats were divided into three experimental groups: (i) the control group; (ii) the bezafibrate group, which was fed a diet containing 0.45% bezafibrate for 5 days; and (iii) the pravastatin group, which was fed a diet containing 0.1% pravastatin for 5 days. Serum, hepatic and biliary lipids were measured by colorimetric assays and hepatic mRNA related to lipid metabolism was studied by reverse transcription-polymerase chain reaction (RT-PCR). In the bezafibrate group biliary phospholipid secretion was increased although cholesterol secretion was not increased. In the pravastatin group, biliary cholesterol and phospholipid secretions were significantly increased. The biliary cholesterol/phospholipid ratio was decreased in the bezafibrate group, but the ratio did not change in the pravastatin group. Hepatic Mdr2, Abcg5 and Abcg8 mRNA expression was remarkably increased in the pravastatin group in comparison with the control group (184%, 264% and 247% of control value, respectively). In the bezafibrate group the hepatic gene expression of Mdr2 was increased (157% of control value), but there were no significant changes in hepatic Abcg5 and Abcg8 mRNA expression compared with the control group. Compared with Mdr2, Abcg5 and Abcg8 seem to be more essential transporters for biliary secretion of cholesterol. Pravastatin upregulated Abcg5/Abcg8 while bezafibrate did not, which appears to explain the different effects of these compounds on biliary lipid secretion.

The role of mdr2 in manganese–bilirubin induced cholestasis in mice

The pathogenesis of manganese–bilirubin (Mn–BR) induced cholestasis has only been studied in rats and is associated with alteration in the hepatic homeostasis of cholesterol and phospholipids. Multidrug resistance-2 (mdr2) transporter, which mediates excretion of these lipids, is suggested to be involved in this phenomenon. The present study was undertaken to examine if Mn–BR induced cholestasis is reproducible in mice, then to clarify the role of mdr2 in its pathogenesis, using mice with disrupted mdr2 gene (mdr2 (−/−)). Results showed that Mn–BR combination decreased bile flow in mice. This reduction in bile flow was similar in mdr2 (−/−) and the wild type mdr2 (+/+). Furthermore, the change in biliary lipid excretion was comparable in both genotypes. These data indicate that Mn–BR induced cholestasis is reproducible in mice and provide evidence that mdr2 alteration is not a primary event in this form of cholestasis.

Lack of biliary lipid excretion in the little skate, Raja erinacea, indicates the absence of functional Mdr2, Abcg5, and Abcg8 transporters.

The ABC transporters bile salt export pump (BSEP; encoded by the ABCB11 gene), MDR3 P-glycoprotein (ABCB4), and sterolin 1 and 2 (ABCG5 and ABCG8) are crucial for the excretion of bile salt, phospholipid, and cholesterol, respectively, into the bile of mammals. The current paradigm is that phospholipid excretion mainly serves to protect membranes of the biliary tree against bile salt micelles. Bile salt composition and cytotoxicity, however, differ greatly between species. We investigated whether biliary phospholipid and cholesterol excretion occurs in a primitive species, the little skate, which almost exclusively excretes the sulphated bile alcohol scymnolsulphate. We observed no phospholipid and very little cholesterol excretion into bile of these animals. Conversely, when scymnolsulphate was added to the perfusate of isolated mouse liver perfusions, it was very well capable of driving biliary phospholipid and cholesterol excretion. Furthermore, in an erythrocyte cytolysis assay, scymnolsulphate was found to be at least as cytotoxic as taurocholate. These results demonstrate that the little skate does not have a system for the excretion of phospholipid and cholesterol and that both the MDR3 and the two half-transporter genes, ABCG5 and ABCG8, have evolved relatively late in evolution to mediate biliary lipid excretion. Little skate plasma membranes may be protected against bile salt micelles mainly by their high sphingomyelin content.

Differences in Biliary Lipid Excretion after Major Hepatectomy in Obstructive Jaundiced Rats with Preoperative Internal, External, or No Biliary Drainage

Necessity of preoperative biliary drainage for patients with obstructive jaundice is still controversial. We recently reported that liver regeneration after major hepatectomy was better restored in a rat model of obstructive jaundice with preoperative internal biliary drainage than that without biliary drainage or with external biliary drainage. The aim of this study was to investigate the differences in biliary lipid excretion after hepatectomy in obstructive jaundiced rats with or without preoperative internal or external biliary drainage. After bile duct ligation for 7 days, rats were randomly divided into the three groups; obstructive jaundice-hepatectomy (OJ-Hx), internal biliary drainage-hepatectomy (ID-Hx), and external biliary drainage-hepatectomy (ED-Hx) groups. 70% hepatectomy and internal biliary drainage were carried out 7 days after biliary decompression in the latter two groups and without biliary decompression in the OJ-Hx group. On the day of and on days 1, 2, 3 and 7 after hepatectomy, the liver weight, DNA synthesis rate, biliary lipids excretion rates, and bile acid composition were determined. In the ID-Hx group, the DNA synthesis rate and relative liver weight were significantly higher than those of the OJ-Hx and ED-Hx groups. The excretion rates of biliary lipids were disturbed in the ED-Hx group compared with those in the ID-Hx group and the values in the OJ-Hx group were in-between the ID-Hx and ED-Hx group. The liver regeneration rate was significantly correlated with bile flow and excretion rates of biliary lipids. The maintenance of enterohepatic circulation of biliary lipids before hepatectomy may be important for the liver regeneration.

The effect of ginseng on bile-pancreatic secretion in the rat. increase in proteins and inhibition of total lipids and cholesterol secretion

Ginseng is one of the most popular herbal remedies. Studies were performed in anaesthetized rats to examine the effect of ginseng on bile secretion. Male Sprague–Dawley rats were anaesthetized with intraperitoneal (i.p.) urethane (1.25 g kg −1) and equipped with biliary cannulas inserted into the bile duct through the sphincter of Oddi. Rats were treated with a single i.p. injection of ginseng at 25, 50 or 100 mg kg−1 (1 ml −1) 30 min before bile collection; the control group received i.p. saline only at 1 ml −1volume. The effect of multiple doses of ginseng on bile volume and biliary composition was also studied. Ginseng was given in the higher dose of 100 mg kg −1(1 ml −1, i.p.) every 12 hours for 2 days. Bile was collected in 15 min fractions for 90 min. Bile flow (bile-pancreatic juice), biliary excretion of total proteins, cholesterol and total lipids were measured. The single administration of different doses (25, 50 and 100 mg kg−1 ) of ginseng reduced basal bile secretion in a dose-dependent manner. Single-dose administration of ginseng at 100 mg kg −1caused 32.9% reduction in basal bile flow. Meanwhile, mean basal bile flow was reduced by 15.1% in rats treated with multiple doses of ginseng at 100 mg kg −1for two days. Biliary protein concentrations were significantly increased after single- or multiple-dose administration of ginseng, but protein output was only significantly increased (33%) in rats treated with ginseng (100 mg kg −1) twice a day for 2 days. Biliary total lipids and cholesterol concentration and outputs were significantly reduced after single or multiple administration of ginseng. In conclusion, administration of ginseng in the rat resulted in a reduction of bile flow and in bile secretion of total lipids and cholesterol, while it increased the secretion of proteins in a dose-dependent manner. The precise mechanisms underlying these effects remain to be elucidated. The findings indicate the need for clinical trials for the effect of this herb on bile composition and flow in man in view of a possible modulatory effect for the herb on gallstone formation.

Effect of sodium tauroursodeoxycholate on phalloidin-induced cholestasis in rats

We investigated the therapeutic effect of tauroursodeoxycholate on phalloidin-induced cholestasis in rats. Intrahepatic cholestasis was induced by administration of phalloidin (500 μg/kg, i.p.) for 7 days. From the day of the last phalloidin injection, tauroursodeoxycholate (60–360 μmol/kg) was given intravenously twice a day for 4 days. On the next day after the last tauroursodeoxycholate administration, bile flow, serum biochemical parameters and biliary lipid excretion rates were determined. Tauroursodeoxycholate significantly suppressed the decrease in bile flow and increases in serum alkaline phosphatase, leucine aminopeptidase and glutamic pyruvic transaminase activities, cholesterol, phospholipid and bile acid concentrations observed in phalloidin-induced cholestasis in rats. Furthermore, tauroursodeoxycholate significantly improved the biliary cholesterol and phospholipid excretion rates in phalloidin-induced cholestasis in rats. These results demonstrate the usefulness of tauroursodeoxycholate as a therapeutic agent in intrahepatic cholestasis.

Taurolithocholate can inhibit the biliary discharge of lysosomes in the rat

The natural bile salt taurolithocholate (TLC) impairs the biliary excretion of lipids and proteins, which are known to reach the canaliculus via vesicles. In this study we examined whether these observations could be extended to the exocytic discharge of lysosomal contents into bile. The single intravenous injection of a cholestatic dose of TLC, 3 μmol/100 g body wt., markedly inhibited the biliary excretion of the lysosomal enzymes acid phosphatase and β-glucuronidase, despite the excretion of bile salts being normalized after a transient diminution. Under such a condition, TLC did not affect the normal transport to and the processing in lysosomes of the exogenously administered [ 14C]sucrose-labeled horseradish peroxidase. However, the biliary excretion of the radioactive lysosomal metabolites of the protein was significantly reduced. The results indicate that TLC can inhibit the biliary discharge of lysosomes in the rat without altering the functional integrity of these organelles. Possible explanations for these findings are discussed.

Bile Acids in the Diagnosis, Pathology, and Therapy of Hepatobiliary Disease

Bile acids are normally confined in the enterohepatic circulation in which they play an important role in bile formation, biliary lipid excretion, and intestinal lipid absorption. In hepatobiliary diseases, bile acids escape the confinement of the enterohepatic circulation, allowing the measurement of the serum total bile acid concentration as a diagnostic indicator. Accumulation of certain bile acids within the hepatocyte, amplified as a consequence of cholestatic hepatobiliary disease, probably enhances cytotoxicity and leads to secondary pathology. Ursodeoxycholate, a bile acid with atypical physiological effects, may be useful in the treatment of various long-term cholestatic hepatobiliary diseases. Presently, most of the information on the toxicity and therapeutic usefulness of bile acids are based on studies in humans and experimental animals. Further studies, both basic and clinical, are needed to determine the pathologic as well as the therapeutic effects of bile acids in domestic animals.

Effects of organic anions and bile acids on biliary lipid excretion in hyperbilirubinemic mutant Sprague-Dawley rats

The effects of organic anions and bile acids on biliary lipid excretion were studied in EHBR, a hyperbilirubinemic mutant Sprague-Dawley rat. A marked delay in the biliary excretion of BSP, cefpiramide, rose bengal and ursodeoxycholate-disulfate was observed in these animals. The marked decrease in the biliary excretion of phospholipids and cholesterol and the uncoupling of biliary bile acids and lipids that occurred after the administration of BSP, cefpiramide and ursodeoxycholate-disulfate in control Sprague-Dawley rats was absent in EHBR. Rose bengal did not change biliary lipid excretion in either the control Sprague-Dawley rats or the EHBR. Although taurocholate markedly increased bile flow and biliary bile acid excretion in both types of rats, the increase in biliary lipid excretion observed in the control Sprague-Dawley rats was absent in EHBR. These findings indicate that EHBR have an impairment of hepatic lipid transfer that is enhanced by bile acids, possibly at the level of intracellular vesicular lipid transport.

Hypolipidemic effects of 6-amino-2-mercapto-5-methylpyrimidine-4-carboxylic acid in rats and tissue culture cells

Abstract6-Amino-2-mercapto-5-methylpyrimidine-4-carboxylic acid (AMMPCA) was found to be a potent hypolipidemic agent at oral closes of 10 and 20 mg/kg/day in rodents. This agent was observed to affect de novo lipid synthesizing enzyme activities in a manner that resulted in lower lipid levels in tissues including the aorta wall. Very low density lipoprotein and low density lipoprotein cholesterol levels were reduced, whereas high density lipoprotein cholesterol levels were significantly increased. AMMPCA interfered with low density receptor activity, suggesting that the drug blocked lipid uptake by peripheral tissue while stimulating binding of lipid to the high density lipoprotein receptor, which should accelerate lipid clearance from the tissues and blood. A second mode of action of the drug is enhanced biliary excretion of lipids, but there was no evidence of a lithogenic effect. Acute toxicity studies in rodents support the fact that AMMPCA is safe in its therapeutic dose range.

Gall-bladder sludge: lessons from ceftriaxone.

Ceftriaxone-associated sludge has been a fascinating story. The occurrence is novel and unique. It has produced a model of gall-bladder sludge in humans. This phenomenon has taught us a great deal about biliary lipid and organic anion excretion by the liver, and the physical chemistry of calcium and calcium sensitive anions. It has added further insights into the pathophysiology of gall-bladder sludge formation. It points to a combination of a hepatic effect where the liver secretes a biochemically abnormal bile, and a gall-bladder effect which provides an environment for precipitation, in order for sludge to develop. The precipitated calcium ceftriaxone has prompted us to re-evaluate the imaging criteria for the diagnosis of gall-bladder sludge versus gallstones. Above all, the rapid onset and rapid disappearance of ceftriaxone sludge has mirrored in a compressed, encapsulated form, the natural history of gall-bladder sludge. It has reminded us that, like gallstones, biliary sludge is usually benign and asymptomatic. However just because it is smaller than gallstones does not mean it cannot cause problems. It can disappear or it can become a calcium ceftriaxone gallstone.

Papaverine inhibits transcytotic vesicle transport and lipid excretion into bile in isolated perfused rat liver.

Papaverine is a nonspecific smooth muscle relaxant and a phosphodiesterase inhibitor. Its effects on biliary excretion of lipids and horseradish peroxidase were investigated in a single-pass isolated perfused rat liver model. A constant infusion of papaverine (1.6 mumol/min; 40 mumol/L) significantly increased bile flow (microliters per minute per gram of liver) before (2.03 +/- 0.09 vs. 1.0 +/- 0.06) and after sodium taurocholate infusion (2.77 +/- 0.10 vs. 1.88 +/- 0.11). However, papaverine significantly and reversibly reduced biliary excretion of phospholipids and cholesterol (nanomoles per minute per gram of liver) after a 1.0 mumol/min sodium taurocholate infusion, from 7.45 +/- 0.83 and 1.42 +/- 0.15 to 1.75 +/- 0.18 and 0.39 +/- 0.06, respectively (p less than 0.01), whereas secretion of bile acids was unaffected. When a 1-min pulse of horseradish peroxidase (25 mg) was infused in isolated perfused rat liver after a continuous infusion of N6,O-2'-dibutyryladenosine 3',5'-cyclic monophosphate (0.25 mumol/min; 6.25 mumol/L), horseradish peroxidase appeared in bile in an early (4 to 6 min) and late (20 to 25 min) peak. Papaverine significantly reduced the late peak, from 1.211 +/- 0.264 to 0.498 +/- 0.107 (p less than 0.01). Papaverine had no significant effects on either cyclic AMP or cyclic GMP in the liver and bile, although it has been reported that papaverine is a phosphodiesterase inhibitor. These findings indicate that papaverine inhibits biliary excretion of lipids but not bile acids, and they suggest that papaverine has an inhibitory effect on transcytotic vesicle transport independent of an increase of cyclic nucleotides in hepatocytes.

Effects of ursodeoxycholate, its glucuronide and disulfate and β-muricholate on biliary bicarbonate concentration and biliary lipid excretion

We previously reported that high-dose ursodeoxycholate (UDC) infusion in rats resulted in extensive glucuronidation of UDC, and speculated that the glucuronidation causes bicarbonate-rich hypercholeresis induced by UDC (Takikawa, H., Sano, N., Narita, T. and Yamanaka, M. Hepatology 1990; 11: 743–749). To test this hypothesis, UDC, UDC-3- O-glucuronide, UDC-3,7-disulfate and β-muricholate were separately and intravenously infused into rats (1 μmol/min per 100 g), and biliary bicarbonate concentration was measured. The effects of these bile acids on biliary lipid secretion were also studied. All four bile acids increased bile flow and biliary bile acid excretion. UDC and β-muricholate significantly increased biliary bicarbonate concentration, whereas UDC glucuronide and disulfate did not. Independence of UDC glucuronide excretion and biliary bicarbonate concentration was also confirmed in EHBR, a hyperbilirubinemic mutant Sprague-Dawiey rat. In this case biliary bicarbonate concentration also increased in spite of the absence of UDC glucuronide in the bile after UDC infusion. Biliary phospholipid secretion was increased with UDC, unchanged with β-muricholate, and decreased with UDC glucuronide and disulfate. Biliary cholesterol secretion was increased with UDC, unchanged with β-muricholate and UDC glucuronide, and decreased with UDC disulfate. These data indicate that glucuronidation is not the cause of bicarbonate-rich hypercholeresis induced by UDC but that glucuronidation and sulfation change the effect of UDC on biliary lipid secretion.

Taurolithocholate-induced inhibition of biliary lipid and protein excretion in the rat

Taurolithocholate (TLC), a natural bile salt, induces selective impairment on canalicular membrane of the hepatocyte, which seems to be a major determinant of its cholestatic effect in experimental animals. In order to extend existing studies about the effects of TLC on bile secretion, we examined in TLC-treated rats the biliary excretion of compounds that are transported to canalicular membrane via vesicles, such as lipids and proteins. The single intravenous injection of TLC (3 μmol/100 g body wt.) inhibited transiently the biliary bile salt excretion, while the biliary excretion of lipids (i.e., cholesterol and phospholipids) and proteins remained inhibited even though the biliary excretion and composition of bile salts were normalized. Under such a condition, TLC also inhibited the transcellular vesicular pathway to the exogenous protein horseradish peroxidase entry into bile, without altering the paracellular biliary access of the protein. The hepatic uptake of horseradish peroxidase was unaffected by TLC-treatment. The results indicate that TLC can inhibit the biliary excretion of compounds that reach the canaliculus via a vesicular pathway, such as lipids and proteins, by a mechanism not related to a defective bile salt excretion. Possible explanations for these findings are discussed.

Cholestasis and biliary excretion of lipids induced by ethinylestradiol in rats fed polyunsaturated oils

Pharmacologic doses of ethinylestradiol are known to cause intrahepatic cholestasis in the rat. This is associated with a decrease in liver plasma membranes fluidity and a modification of the activity of membrane-bound enzymes related to bile flow. The intake of a diet containing highly polyunsaturated fats could modify the effects of estrogen, by increasing membranes fluidity due to their incorporation into structural phospholipids. To evaluate this action, rats were fed semipurified diets containing 17% corn ( Zea mais) oil or rose hip ( Rosa moschata Mill) seed oil during 20 days. Following this period, a group of animals were injected with ethinylestradiol during 5 days, while a control group and a pair fed group were included. Cholestasis was similar between corn and rose hip oils fed to ethinylestradiol treated rats. The biliary excretion of cholesterol was higher in rose hip oil compared to corn oil-fed control rats. Biliary cholesterol/phospholipids ratio decreased in estrogen-treated rats compared to control animals. Our results show that the intake of highly polyunsaturated fatty acids did not modify the expected action of ethinylestradiol in rats.

Taurocholate induces pericanalicular localization of C6-NBD-ceramide in isolated hepatocyte couplets

The mechanisms and pathways involved in hepatocellular transport of lipid destined for biliary excretion remain poorly understood. Using fluorescence microscopy of rat hepatocyte couplets in primary culture, we examined the effects of taurocholate (TC) on the intracellular distribution of 6-N-[7-nitrobenz-2-oxa-1,3-diazol- 4-yl]aminocaproyl-sphingosine (C6-NBD-ceramide), a lipid that accumulates in the Golgi apparatus. Microscopic findings were quantified with morphometric and digital image analysis and were correlated with the metabolism of C6-NBD-ceramide in isolated hepatocyte suspensions and the biliary excretion of fluorescent lipid in the isolated perfused liver. After plasma membrane uptake of C6-NBD-ceramide from albumin at 0 degrees C, the lipid was rapidly internalized at 37 degrees C but exhibited only a modest concentration of fluorescence in intracellular organelles. With 17 microM TC in the medium, there was enhanced localization of fluorescence to organelles and significant recruitment of fluorescent lipid to the pericanalicular region of the couples within 30 min. C6-NBD-ceramide was partially metabolized to C6-NBD-sphingomyelin and -glucosylceramide, indicative of transit through the Golgi apparatus. The generation of C6-NBD-sphingomyelin was significantly increased by TC. After a similar loading protocol in the perfused liver, there was little biliary excretion of fluorescent lipid at 37 degrees C under control conditions. However, infusion of TC markedly enhanced the biliary output of fluorescent lipid over the first 30 min, primarily as C6-NBD-sphingomyelin and -glucosylceramide. We conclude that TC modulates the distribution of C6-NBD-ceramide in hepatocytes by inducing translocation of lipid to a pericanalicular location, most likely the Golgi apparatus, before excretion of its metabolites in bile. Our findings support the concept that bile salt-induced biliary lipid excretion is facilitated by the interaction of bile salts with lipids at the level of intracellular organelles.

Circadian rhythms of biliary protein and lipid excretion in rats

To gain insight into the mechanisms by which hepatocytes release lipids and proteins into bile, we studied extended, steady-state secretion of bile, lipids, and lysosomal and canalicular membrane proteins in freely moving, unanesthetized rats with chronic bile fistulas. We found circadian rhythms of biliary secretion for all measured constituents. In the basal state (nocturnal feeding), two distinct secretory patterns emerged: type 1, characterized by a peak at midnight and a nadir at noon; and type 2, characterized by a peak at 8 A.M. and a nadir at 8 P.M. We observed parallel, type 1 circadian rhythms of excretion for bile, biliary lipids (bile acid, cholesterol, phospholipid), and a canalicular membrane enzyme (alkaline phosphodiesterase I). In contrast, a type 2 circadian rhythm was observed for the outputs of two lysosomal enzymes. Hepatic lysosomal enzyme concentrations and the number of pericanalicular lysosomes decreased (P less than 0.05) by 15 and 35%, respectively, at the nadir of their biliary output relative to the time of their peak outputs. In response to daytime feeding, major shifts in the circadian rhythms of excretion of biliary constituents occurred such that secretion of bile, lipids, and the canalicular membrane protein adopted a type 2-like rhythm, whereas the biliary secretion of the lysosomal proteins exhibited a type 1-like pattern. These results indicate that bile flow and biliary excretion of individual lipids and proteins exhibit distinct circadian rhythms that are altered by feeding. Secretory events at the canaliculus that depend on the transmembrane flux of bile acids, such as water and lipid movement or the solubilization of membrane proteins, display a common rhythm.(ABSTRACT TRUNCATED AT 250 WORDS)