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Background & Aims: Reverse cholesterol transport (RCT) is defined as high-density lipoprotein (HDL)-mediated flux of excess cholesterol from peripheral cells to liver, followed by secretion into bile and disposal via the feces. Various steps of this pathway are controlled by the liver X receptor (LXR). We addressed the role of the intestine in LXR-dependent stimulation of fecal cholesterol excretion. Methods: To segregate biliary from intestine-derived cholesterol, wild-type and Mdr2 P-glycoprotein-deficient mice (Mdr2^-^/^-), which are unable to secrete cholesterol into bile, were treated with the LXR agonist GW3965. Results: Treatment with GW3965 increased biliary cholesterol secretion by 74% in wild-type mice but had no effect in Mdr2^-^/^- mice. LXR activation increased fecal neutral sterol excretion 2.1-fold in wild-type mice. Surprisingly, an identical increase was observed in Mdr2^-^/^- mice. Fractional cholesterol absorption was reduced on LXR activation in both strains but was more pronounced in Mdr2^-^/^- mice, coinciding with reduced Npc111 expression. Intestinal gene expression of ATP-binding cassette transporters (Abc) Abca1, Abcg1, Abcg5, and Abcg8 was strongly induced upon LXR activation in both strains, whereas expression of HMGCoA reductase, controlling cholesterol synthesis, remained unaffected. Additionally, LXR activation stimulated the excretion of plasma-derived [^3H]cholesterol into the fecal neutral sterol fraction in Mdr2^-^/^- mice. Conclusions: Increased fecal cholesterol loss upon LXR activation is independent of biliary cholesterol secretion in mice. An important part of excess cholesterol is excreted directly via the intestine, supporting the existence of an alternative, quantitatively important route for cholesterol disposal.

Background & Aims:Diabetes mellitus is associated with changes in bile formation. The aim of our study was to investigate the molecular basis for these changes in rats with experimentally induced diabetes. Methods:Expression of bile canalicular transporters was studied by reverse-transcription polymerase chain reaction, immunoblotting, and immunohistochemistry in control, streptozotocin-diabetic, and insulin-treated diabetic rats. Bile formation was studied under basal conditions and during stepwise increasing intravenous infusion of taurocholate to determine bile salt secretory rate maximum (SRm). Results:In diabetic rats, hepatic gene and protein expression of the multidrug resistance P-glycoprotein type 2 (Mdr2) were increased by 105% and 530%, respectively, associated with increased biliary phospholipid output (+520%) and phospholipid/bile salt ratio (+77%). Protein levels of the canalicular bile salt export pump (Bsep) were unchanged in diabetic rats, but basal biliary bile salt output and the SRm of taurocholate were increased by 260% and 130%, respectively, compared with controls. Alterations in transporter expression and bile formation were partly reversed by insulin administration. The bile salt SRm was strongly correlated with biliary phospholipid concentration (P< 0.001, R = 0.82). Conclusions:Induction of Mdr2expression and biliary phospholipid secretion, rather than Bsepexpression, appears to be responsible for the enhanced capacity of biliary bile salt secretion in experimentally induced diabetes.

Background & Aims: It has been proposed that biliary phospholipids fulfill specific functions in the absorption of dietary fat from the intestine, but the physiological significance has not been established. The aim of this study was to evaluate the role of biliary phospholipids in dietary fat absorption in vivo by using mice homozygous or heterozygous for disruption of the Mdr2 gene (Mdr2^(^-^/^-^), Mdr2^(^+^/^-^)) and control (Mdr2^(^+^/^+^)) mice. Mdr2^(^-^/^-^) mice do not secrete phospholipids and cholesterol into bile, and bile salt secretion is not impaired. Mdr2^(^+^/^-^) mice show only impaired (-40%) phospholipid secretion. Methods: Methods included an analysis of time dependency of intestinal uptake and plasma appearance of intragastrically administered (radiolabeled) triglycerides and measurement of 3-day fecal fat balance with low- and high-fat diets. Results: Intragastric administration of olive oil resulted in a rapid increase in plasma triglycerides in Mdr2^(^+^/^+^) and Mdr2^(^+^/^-^) but not in Mdr2^(^-^/^-^) mice. The ''postprandial response'' of plasma triglycerides could be partially restored in Mdr2^(^-^/^-^) mice by intraduodenal infusion of whole rat bile. After intragastric [^3H]triolein administration in Triton WR1339-pretreated animals, the appearance of ^3H-triglycerides in plasma was reduced by 70% in Mdr2^(^-^/^-^) compared with Mdr2^(^+^/^+^) mice, excluding accelerated lipolysis as the cause of defective triglyceride response in Mdr2^(^-^/^-^) mice. ^3H-triglycerides accumulated in enterocytes in Mdr2^(^-^/^-^) mice. Surprisingly, the efficacy of fat absorption as derived from balance studies was not affected and was only minimally affected in Mdr2^(^-^/^-^) mice fed low (14 energy percent)- and high (35 energy percent)-fat diets, respectively (all >95%). Conclusions: The results show that biliary lipid secretion is necessary for postprandial appearance in plasma of chylomicrons in vivo but not for quantitative absorption of dietary lipids. GASTROENTEROLOGY 2000;118:173-182

Background & Aims: Reduced activity of ferrochelatase in erythropoietic protoporphyria (EPP) results in protoporphyrin (PP) accumulation in erythrocytes and liver. Liver disease may occur in patients with EPP, some of whom develop progressive liver failure that necessitates transplantation. We investigated the mechanisms underlying EPP-associated liver disease in a mouse model of EPP. Methods: Liver histology, indicators of lipid peroxidation, plasma parameters of liver function, and bile composition were studied in mice homozygous (fch/fch) for a point mutation in the ferrochelatase gene and in heterozygous (fch/+) and wild-type (+/+) mice. Results: Microscopic examination showed bile duct proliferation and biliary fibrosis with portoportal bridging in fch/fch mice. PP content was 130-fold increased, and thiobarbituric acid-reactive substances (+30%) and conjugated dienes (+75%) were slightly higher in fch/fch than in fch/+ and +/+ livers. Levels of hepatic thiols (-12%) and iron (-52%) were reduced in fch/fch livers. Liver enzymes and plasma bilirubin were markedly increased in the homozygotes. Plasma bile salt levels were 80 times higher in fch/fch than in fch/+ and +/+ mice, probably related to the absence of the Na^+-taurocholate cotransporting protein (Ntcp) in fch/fch liver. Paradoxically, bile flow was not impaired and biliary bile salt secretion was 4 times higher in fch/fch mice than in controls. Up-regulation of the intestinal Na^+-dependent bile salt transport system in fch/fch mice may enhance efficiency of bile salt reabsorption. The bile salt/lipid ratio and PP content of fch/fch bile were increased 2-fold and 85-fold, respectively, compared with +/+, whereas biliary glutathione was reduced by 90%. Similar effects on bile formation were caused by griseofulvin-induced inhibition of ferrochelatase activity in control mice. Conclusions: Bile formation is strongly affected in mice with impaired ferrochelatase activity. Rather than peroxidative processes, formation of cytotoxic bile with high concentrations of bile salts and PP may cause biliary fibrosis in fch/fch mice by damaging bile duct epithelium. GASTROENTEROLOGY 1999;117:696-705

Background & Aims:mdr2P-glycoprotein (Pgp) deficiency in mice leads to the absence of biliary phospholipids and cholesterol in the presence of normal bile salt secretion. The aim of this study was to evaluate the importance of the biliary pathway in cholesterol homeostasis by determining the effects of mdr2Pgp deficiency on hepatic and plasma lipid levels and cholesterol kinetics in chow-fed mice. Methods:Hepatic lipid content, enzyme activities, plasma lipoprotein levels, and fecal sterol excretion were measured in wild-type (+/+) and mdr2Pgp–deficient (−/−) mice. Cholesterol kinetics were determined using radiotracer techniques. Results:No differences in hepatic lipid content were observed between (−/−) and (+/+) mice. Plasma high-density lipoprotein cholesterol and apolipoprotein A-I levels were strongly reduced in (−/−) mice compared with controls, whereas the apolipoprotein B contents of very-low-density lipoprotein and low-density lipoprotein were increased. Hepatic activity of 3-hydroxy-3-methylglutaryl–coenzyme A reductase was threefold greater in (−/−) mice than in controls; however, compartmental analysis of plasma cholesterol decay showed no differences in cholesterol synthesis between (−/−) and (+/+) mice. A dual isotope approach for estimating cholesterol absorption yielded ~50% lower values in (−/−) mice than in controls. Surprisingly, (−/−) mice showed a fourfold increase in fecal neutral sterol secretion. Conclusions:This study unequivocally establishes the important direct role of biliary lipids in the regulation of plasma lipid levels in mice.

Several compounds, in particular bile acids, undergo enterohepatic circulation (EHC). Limited data are available on the pathophysiologic aspects of this circulation. In the present study we describe a surgical technique in rats that allows a long-term, reversible interruption of the EHC and monitoring of peripheral blood levels without direct surgical intervention. This technique excludes the effects of anesthesia and surgical trauma. The model's validity has been tested extensively. We used this animal model to investigate acute and chronic effects of interruption and subsequent restoration of the EHC on (a) nutritional status, (b) plasma cholesterol levels and hepatic cholesterol synthesis, and (c) biliary bile acid, phospholipid, and cholesterol excretion. Interruption of the EHC resulted in an increased food intake and enhanced fecal energy loss, caused by a less efficient intestinal absorption. Plasma cholesterol concentrations declined immediately after the interruption of the EHC, but returned to almost control values during bile diversion. A marked overshoot followed the subsequent restoration of the EHC. Hepatic cholesterol synthesis showed a fivefold increase after 8 days of bile diversion but returned to control values within 2 days after restoration of the EHC. After interruption of the EHC, bile acid, phospholipid, and cholesterol excretion decreased sharply but stabilized after 3 h at 7.6%, 20%, and 23%, respectively, of their initial values. Bile acid output, representing hepatic synthesis, slowly increased over 4 days, but never exceeded 13% of its value during intact EHC. Subsequent restoration of the EHC could rapidly reverse the observed effects of the interruption. The animal model described in the present study is an excellent tool in studying the acute and chronic effects of disturbances of the EHC.

Altered bile acid metabolism has been claimed to play a role in the etiology of benign recurrent intrahepatic cholestasis (BRIC). Therefore, we studied bile acid metabolism in detail in 10 patients with this syndrome. Pool sizes of both primary bile acids were estimated simultaneously, using deuterated cholic acid and chenodeoxycholic acid. The pool sizes of cholic acid and chenodeoxycholic acid, expressed in micromoles per kilogram body weight, were significantly contracted in BRIC patients during a cholestasis-free period: 8.0 ± 4.2 and 11.7 ± 4.7, respectively, versus 24.1 ± 11.7 and 22.9 ± 7.8 in controls. Fractional turnover rates (per day) for cholic acid and chenodeoxycholic acid were increased: 0.70 ± 0.29 and 0.58 ± 0.27, respectively, versus 0.29 ± 0.12 and 0.23 ± 0.10 in controls. Bile acid pool composition expressed as percentages in BRIC patients was cholic acid 34 ± 17, chenodeoxycholic acid 38 ± 9, deoxycholic acid 27 ± 18, and lithocholic acid 1 ± 1, with a glycine to taurine conjugation ratio of 6.7 ± 4.9. Corresponding values for 32 controls were cholic acid 57 ± 13, chenodeoxycholic acid 29 ± 9, deoxycholic acid 14 ± 9, and lithocholic acid <1, with a glycine to taurine conjugation ratio of 2.4 ± 1.3. Fecal bile acid loss, in micromoles per kilogram body weight per day, was 11.2 ± 9.0 in BRIC patients compared with 2.8 ± 1.4 in controls. The serum 7α-hydroxycholesterol level (nanomoles per liter) was significantly increased in BRIC patients: 326 ± 179 versus 171 ± 90 in controls. These results suggest that in BRIC patients spillover of bile acids into the colon occurs, which leads to increased fecal bile acid loss and a reduced bile acid pool size. Increased serum 7α-hydroxycholesterol is probably indicative of an accelerated bile acid synthesis rate due to increased activity of cholesterol 7α-hydroxylase, the enzyme catalyzing the first step in the major pathway of bile acid synthesis. The results of our study suggest that in BRIC patients a contracted bile acid pool increases the susceptibility of the liver for cholestatic agents.

A number of organic anions has been shown to inhibit biliary phospholipid and cholesterol secretion without affecting bile acid secretion. However, the mechanism of this uncoupling phenomenon is still unclear. This study shows a comparison of the effects of ampicillin (18 μmol/100g body wt), sulfated taurolithocholic acid (0.2 and 1.0 μmol/100 g body wt), and indocyanine green (0.6 μmol/100g body wt) in control and Groningen Yellow-Wistar rats with chronic (8 days) biliary drainage. Groningen Yellow rats have a hereditary defect in hepatobiliary transport of various organic anions. Bile secretion, but not hepatic uptake, of the three organic anions was strongly impaired in Groningen Yellow rats compared with controls. Ampicillin and sulfated taurolithocholic acid caused a strong uncoupling in control rats but had no effect or a much smaller effect on lipid secretion in Groningen Yellow rats. Indocyanine green did not affect lipid secretion, in either control or in Groningen Yellow rats. Gel-filtration chromatography of bile showed a specific coelution of ampicillin and sulfated taurolithocholic acid with the bile acid fraction, whereas indocyanine green coeluted with the phospholipid/cholesterol fraction. This study concludes that the uncoupling by ampicillin and sulfated taurolithocholic acid occurs after their secretion into bile and is caused by interaction of these compounds with bile acids. It is hypothesized that this interaction inhibits the capacity of bile acids to induce secretion of phospholipids and cholesterol into the bile.

BACKGROUND & AIMS: During endotoxemia, expression of inducible nitric oxide synthase (iNOS) and nitric oxide production in the liver is increased. NO has been suggested to have a hepatoprotective function. The aim of this study was to investigate the distribution of iNOS and the effect of different NO synthase inhibitors on liver damage and hemodynamics during endotoxemia.