Phospholipid Secretion Research Articles

Experimental study of relationship of bile composition imbalance with bile duct injury

To investigate the change of bile composition and its role in bile duct injury after orthotopic liver transplantation (OLT). Rats were randomly divided into 3 groups: group A (sham surgery), group B (OLT with 1 h cold preservation), group C (OLT with 12 h cold preservation). The arterialized rat liver transplantation model with biliary extra-drainage was used in group B and C. Animals were sacrificed at posttransplant 1, 3, 5, 7, 10 and 14 day. Routine bile chemistry and pathological assays were performed. Cold preservation/reperfusion injury (CPRI) could repress the secretion of bile salts and phospholipid. However, in contrast with a rapid increase of bile salt secretion, the biliary secretion of phospholipid recovered more slowly, leading to an abnormal high bile salts/phospholipid ratio early after transplantation. Further analysis suggested that the secretion of bile salts correlated strongly with biochemical and histopathological signs of bile duct injury. CPRI can lead to great changes of graft bile composition, which plays a role in the pathogenesis of bile duct injury following liver transplantation.

Mechanism for FGF-1 to regulate biogenesis of apoE-HDL in astrocytes

Fibroblast growth factor-1 (FGF-1) is secreted by astrocytes and stimulates apolipoprotein E (apoE)-HDL biogenesis by an autocrine mechanism to help in recovery from brain injury. In apoE-deficient mouse astrocytes, FGF-1 stimulated cholesterol biosynthesis without enhancing its release, indicating a signaling pathway independent of apoE biosynthesis upregulation. SU5402, an inhibitor of FGF receptor, inhibited FGF-1-induced phosphorylation of MEK, ERK, and Akt, as well as all the apoE-HDL biogenesis-related events in rat astrocytes. LY294002, an inhibitor of phosphatidylinositide 3-OH kinase (PI3K) and of Akt phosphorylation, inhibited apoE-HDL secretion but not cholesterol biosynthesis, whereas U0126, an inhibitor of MEK and of ERK phosphorylation, inhibited cholesterol biosynthesis but not apoE-HDL secretion. Increase of apoE-mRNA by FGF-1 was not influenced by either inhibitor. When rat apoE/pcDNA3.his was transfected to transformed rat astrocyte GA-1 cells that otherwise do not synthesize apoE (GA-1/25), FGF-1 did not influence apoE-mRNA, but did increase the apoE secretion and Akt phosphorylation that were suppressed by LY294002. Lipid biosynthesis was increased by FGF-1 in GA-1/25 cells and suppressed by U0126. FGF-1 upregulates apoE-HDL biogenesis by three independent signaling pathways. The PI3K/Akt pathway upregulates secretion of apoE/apoE-HDL, the MEK/ERK pathway stimulates cholesterol biosynthesis, and an unknown pathway enhances apoE transcription.

Transgenic overexpression of Abcb11 enhances biliary bile salt outputs but does not affect cholesterol cholelithogenesis in mice

Cholesterol gallstone disease is a complex genetic trait and induced by multiple but as yet unknown genes. A major Lith gene, Lith1 was first identified on chromosome 2 in gallstone-susceptible C57L mice compared with resistant AKR mice. Abcb11 encoding the canalicular bile salt export pump in the hepatocyte, is co-located within the Lith1 QTL region and its hepatic expression is significantly higher in C57L mice than in AKR mice. To investigate whether Abcb11 influences cholesterol gallstone formation, we created an Abcb11 transgenic strain on the AKR genetic background and fed them with a lithogenic diet for 56 days. We excluded functionally relevant polymorphisms of the Abcb11 gene and its promoter region between C57L and AKR mice. Overexpression of Abcb11 significantly promoted biliary bile salt secretion and increased circulating bile salt pool size and bile salt-dependent bile flow rate. However, biliary cholesterol and phospholipid secretion, as well as gallbladder size and contractility were comparable in transgenic and wild-type mice. At 56 days of feeding, cholesterol saturation indexes of gallbladder biles and gallstone prevalence rates were similar in these two groups of mice. We conclude that overexpression of Abcb11 augments biliary bile salt secretion, but does not affect cholelithogenesis in mice.

Bezafibrate induces multidrug‐resistance P‐Glycoprotein 3 expression in cultured human hepatocytes and humanized livers of chimeric mice

A decreased function of multidrug-resistance 3 P-glycoprotein (MDR3), limiting the rate of biliary phospholipid secretion, predisposes individuals to cholestasis and/or cholangitis. Fibrates induce the expression of mdr2 (homolog of human MDR3) in rodents. To investigate the effects of bezafibrate (BF) on the expression levels of MDR3 in cultured human hepatocytes and human livers, the amount of protein and subcellular localization of MDR3 was assessed in HepG2 cells treated with BF and humanized livers of BF-treated chimeric mice. In HepG2 cells, while treatment with BF did not increase the protein levels of MDR3, the treatment caused a redistribution of MDR3 in the bile canaliculi. In humanized livers of chimeric mice, oral administration of BF induced a large increase in the protein amount of MDR3 and its redistribution in the bile canaliculi. Moreover, the modulatory effects of BF on key factors involved in hepatic cholesterol and bile acid metabolism in human subjects were traced in the humanized livers of BF-treated chimeric mice. BF causes an induction of MDR3 expression in human livers. This provides a rationale for the beneficial role of BF in improving cholestasis and/or cholangitis associated with defective MDR3 expression and function in various types of cholestatic hepatobiliary diseases.

NF-Y and Sp1 are involved in transcriptional regulation of rat SND p102 gene

SND p102 is a rat liver endoplasmic reticulum cholesterol ester hydrolase recently described as a member of a conserved family of transcriptional coactivators that promotes phospholipid secretion into lipoproteins when overexpressed in hepatocytes. In this work, we report first evidence for a mechanism of transcriptional regulation for the SND p102 (Snd1) gene. Promoter activity of 5′ deletion fragments determined in human HepG2 and rat McA-RH7777 hepatoma cells by luciferase reporter gene assays showed a minimal promoter involving two inverted CCAAT boxes. EMSA demonstrated specific binding of Sp1 to GC boxes in the proximal, highly active promoter region besides that of NF-Y to CCAAT boxes reported earlier. Site-directed disruption of such CCAAT and GC boxes led to reduction in transcriptional activity, confirming the functional implication of NF-Y and Sp1 in SND p102 gene transcription.

Surfactant Protein A without the Interruption of Gly-X-Y Repeats Loses a Kink of Oligomeric Structure and Exhibits Impaired Phospholipid Liposome Aggregation Ability

Pulmonary surfactant protein A (SP-A) belongs to the collectin subgroup of the C-type lectin superfamily. SP-A oligomerizes as an octadecamer, which forms a flower bouquet-like structure. A collagen-like domain of human SP-A consists of 23 Gly-X-Y repeats with an interruption near the midpoint of this domain. This interruption causes a kink, but its role remains unknown. To define the importance of the kink region of SP-A, two mutated proteins were constructed to disrupt the interruption of Gly-X-Y repeats: SP-ADEL, which lacks the Pro47-Cys48-Pro49-Pro50 sequence at the interruption, and SP-AINS, in which two glycines were introduced to insert Gly-X-Y repeats (Gly-Pro47-Cys48-Gly-Pro49-Pro50). Electron microscopy using rotary shadowing revealed that both mutants form octadecamers that lack a bend in the collagenous domain. Electrophoretic analysis under nondenaturing conditions and gel filtration chromatography demonstrated that SP-AINS consisted of a large assembly of oligomers whereas SP-ADEL formed mainly octadecamers. Both SP-ADEL and SP-AINS mutants as well as wild-type SP-A bound to liposomes containing dipalmitoylphosphatidylcholine and galactosylceramide at equivalent levels, but the abilities of the mutants to induce phospholipid liposome aggregation were significantly less developed than that of the wild type. The mutants SP-ADEL and SP-AINS augmented liposome uptake by alveolar type II cells and inhibited secretion of phospholipids from type II cells at a level comparable to that of wild-type SP-A. These results indicate that the interruption of Gly-X-Y repeats in the SP-A molecule is critical for the formation of a flower bouquet-like octadecamer and contributes to SP-A's capacity to aggregate phospholipid liposomes.

Pluronic L81 enhances triacylglycerol accumulation in the cytosol and inhibits chylomicron secretion

Pluronic L81 (PL81) inhibits fat absorption, and other Pluronic copolymers help overcome drug resistance in cancer cells. To understand how PL81 acts, we synthesized a radiolabeled analog, [14C]PL81, and showed that it was structurally similar to PL81 based on (1)H NMR as well as mass spectrometric analysis. [14C]PL81 inhibited the secretion of chylomicrons (CMs), lipoproteins essential for fat absorption, by differentiated Caco-2 cells similar to PL81. Moreover, PL81 competed with the cellular uptake of [14C]PL81. Thus, [14C]PL81 and PL81 behave similarly in these physiologic assays. Uptake of [14C]PL81 by Caco-2 cells was concentration-, time-, and temperature-dependent and occurred mainly from the apical side. Intracellularly, it was assimilated in the cytosol. Cells excreted PL81 toward the apical side via a pathway partially sensitive to verapamil. Small amounts were secreted toward the basolateral side unassociated with CM, and this secretion was unaffected by the inhibition of CM assembly. Nonetheless, PL81 significantly inhibited the secretion of triacylglycerols (TGs) and phospholipids as part of CM. PL81-treated cells showed decreased activity of microsomal triglyceride transfer protein and accumulated more TGs, but not phospholipids, in their cytosol. We propose that Pluronic copolymers act by interfering with the export of molecules from the cytosol. They inhibit fat absorption by decreasing TG transport to the endoplasmic reticulum and increase drug efficacy against cancer cells by competing for their excretion.

Deletion of the Transmembrane Transporter ABCG1 Results in Progressive Pulmonary Lipidosis

We show that mice lacking the ATP-binding cassette transmembrane transporter ABCG1 show progressive and age-dependent severe pulmonary lipidosis that recapitulates the phenotypes of different respiratory syndromes in both humans and mice. The lungs of chow-fed Abcg1(-/-) mice, >6-months old, exhibit extensive subpleural cellular accumulation, macrophage, and pneumocyte type 2 hypertrophy, massive lipid deposition in both macrophages and pneumocytes and increased levels of surfactant. No such abnormalities are observed at 3 months of age. However, gene expression profiling reveals significant changes in the levels of mRNAs encoding key genes involved in lipid metabolism in both 3- and 8-month-old Abcg1(-/-) mice. These data suggest that the lungs of young Abcg1(-/-) mice maintain normal lipid levels by repressing lipid biosynthetic pathways and that such compensation is inadequate as the mice mature. Studies with A-549 cells, a model for pneumocytes type 2, demonstrate that overexpression of ABCG1 specifically stimulates the efflux of cellular cholesterol by a process that is dependent upon phospholipid secretion. In addition, we demonstrate that Abcg1(-/-), but not wild-type macrophages, accumulate cholesterol ester droplets when incubated with surfactant. Together, these data provide a mechanism to explain the lipid accumulation in the lungs of Abcg1(-/-)mice. In summary, our results demonstrate that ABCG1 plays essential roles in pulmonary lipid homeostasis.

The mechanism of ABCG5/ABCG8 in biliary cholesterol secretion in mice

The main player in biliary cholesterol secretion is the heterodimeric transporter complex, ABCG5/ABCG8, the function of which is necessary for the majority of sterols secreted into bile. It is not clear whether the primary step in this process is flopping of cholesterol from the inner to the outer leaflet of the canalicular membrane, with desorption by mixed micelles, or decreasing of the activation energy required for cholesterol desorption from the outer membrane leaflet. In this study, we investigated these mechanisms by infusing Abcg8(+/+), Abcg8(+/-), and Abcg8(-/-) mice with hydrophilic and hydrophobic bile salts. In Abcg8(-/-) mice, this failed to substantially stimulate biliary cholesterol secretion. Infusion of the hydrophobic bile salt taurodeoxycholate also resulted in cholestasis, which was induced in Abcg8(-/-) mice at a much lower infusion rate compared with Abc8(-/-) and Abcg8(+/-) mice, suggesting a reduced cholesterol content in the outer leaflet of the canalicular membrane. Indeed, isolation of canalicular membranes revealed a reduction of 45% in cholesterol content under these conditions in Abcg8(-/-) mice. Our data support the model that ABCG5/ABCG8 primarily play a role in flopping cholesterol (and sterols) from the inner leaflet to the outer leaflet of the canalicular membrane.

Lipidomics of cellular and secreted phospholipids from differentiated human fetal type II alveolar epithelial cells

Maturation of fetal alveolar type II epithelial cells in utero is characterized by specific changes to lung surfactant phospholipids. Here, we quantified the effects of hormonal differentiation in vitro on the molecular specificity of cellular and secreted phospholipids from human fetal type II epithelial cells using electrospray ionization mass spectrometry. Differentiation, assessed by morphology and changes in gene expression, was accompanied by restricted and specific modifications to cell phospholipids, principally enrichments of shorter chain species of phosphatidylcholine (PC) and phosphatidylinositol, that were not observed in fetal lung fibroblasts. Treatment of differentiated epithelial cells with secretagogues stimulated the secretion of functional surfactant-containing surfactant proteins B and C (SP-B and SP-C). Secreted material was further enriched in this same set of phospholipid species but was characterized by increased contents of short-chain monounsaturated and disaturated species other than dipalmitoyl PC (PC16:0/16:0), principally palmitoylmyristoyl PC (PC16:0/14:0) and palmitoylpalmitoleoyl PC (PC16:0/16:1). Mixtures of these PC molecular species, phosphatidylglycerol, and SP-B and SP-C were functionally active and rapidly generated low surface tension on compression in a pulsating bubble surfactometer. These results suggest that hormonally differentiated human fetal type II cells do not select the molecular composition of surfactant phospholipid on the basis of saturation but, more likely, on the basis of acyl chain length.

Overexpression of SND p102, a rat homologue of p100 coactivator, promotes the secretion of lipoprotein phospholipids in primary hepatocytes

SND p102 belongs to an evolutionarily conserved family of proteins first described as transcriptional coactivators, whose biological function has not yet been defined. High expression levels of homologues of SND p102 in non-nuclear compartments of lipid secretory tissues and in murine liver endoplasmic reticulum suggest a role for SND p102 in lipoprotein secretion in hepatocytes. To address this issue, after ascribing by confocal microscopy and Western blotting a non-nuclear localization of SND p102 in rat hepatocytes, we cloned its full-length cDNA, developed adenoviral vectors encoding the cDNA or a specific antisense sequence, and characterized the lipoprotein particles created and released for 24 h by transfected rat hepatocytes. The cellular ability to secrete apoB and apoA-I was not affected by SND p102 differential expression, nor was that of lipoproteins-triglyceride, -cholesterol and -cholesteryl esters. However, cells overexpressing SND p102 secreted phospholipid-rich lipoproteins. Compared with hepatocytes with basal or attenuated SND p102 expression, they secreted ∼45% and 80% more phospholipid in d < 1.015 g/mL and 1.015 < d < 1.24 g/mL lipoproteins, respectively. Oversecretion affected all phospholipid classes and did not significantly disturb the cellular phospholipid homeostasis. Collectively, the results suggest a specific, positive association of SND p102 and phospholipid release in lipoprotein particles in hepatocytes.

Identification and characterization of p63 (CKAP4/ERGIC-63/CLIMP-63), a surfactant protein A binding protein, on type II pneumocytes

Surfactant protein A (SP-A) binds to alveolar type II cells through a specific high-affinity cell membrane receptor, although the molecular nature of this receptor is unclear. In the present study, we have identified and characterized an SP-A cell surface binding protein by utilizing two chemical cross-linkers: profound sulfo-SBED protein-protein interaction reagent and dithiobis(succinimidylpropionate) (DSP). Sulfo-SBED-biotinylated SP-A was cross-linked to the plasma membranes isolated from rat type II cells, and the biotin label was transferred from SP-A to its receptor by reduction. The biotinylated SP-A-binding protein was identified on blots by using streptavidin-labeled horseradish peroxidase. By using DSP, we cross-linked SP-A to intact mouse type II cells and immunoprecipitated the SP-A-receptor complex using anti-SP-A antibody. Both of the cross-linking approaches showed a major band of 63 kDa under reduced conditions that was identified as the rat homolog of the human type II transmembrane protein p63 (CKAP4/ERGIC-63/CLIMP-63) by matrix-assisted laser desorption ionization and nanoelectrospray tandem mass spectrometry of tryptic fragments. Thereafter, we confirmed the presence of p63 protein in the cross-linked SP-A-receptor complex by immunoprobing with p63 antibody. Coimmunoprecipitation experiments and functional assays confirmed specific interaction between SP-A and p63. Antibody to p63 could block SP-A-mediated inhibition of ATP-stimulated phospholipid secretion. Both intracellular and membrane localized pools of p63 were detected on type II cells by immunofluorescence and immunobloting. p63 colocalized with SP-A in early endosomes. Thus p63 closely interacts with SP-A and may play a role in the trafficking or the biological function of the surfactant protein.

Spray-dried milk supplemented with α-linolenic acid or eicosapentaenoic acid and docosahexaenoic acid decreases HMG Co A reductase activity and increases biliary secretion of lipids in rats

In our earlier study, we have shown that rats fed spray-dried milk containing α-linolenic acid (LNA 18:3 n-3) or eicosapentaenoic acid (EPA 20:5 n-3) and docosahexaenoic acid (DHA 22:6 n-3) had significantly lower amounts of serum and liver cholesterol. To evaluate the mechanism for hypocholesterolemic effect of n-3 fatty acids containing milk formulation, we fed male Wistar rats with spray-dried milk containing linseed oil (LSO) (source of LNA) or fish oil (FO) (source of EPA + DHA) for 8 weeks. Feeding n-3 fatty acid containing milk formulation lowered the hepatic 3-hydroxy-methylglutaryl coenzyme A (HMG Co A) activity by 17–22% compared to rats given control diet devoid of n-3 fatty acids. The cholesterol level in liver microsomes was found to be decreased by 16% and 20%, respectively, in LSO and FO containing formulation fed rats. The bile flow was enhanced to an extent of 19–23% in experimental groups compared to control animals. The biliary cholesterol and phospholipid secretion was increased to an extent of 49–55% and 140–146%, respectively, in rats fed n-3 fatty acid containing formulation. The increase in the total bile acids secretion in bile was mainly reflected on an increase in the levels of taurine conjugated bile acids. These results indicated that n-3 fatty acid containing spray-dried milk formulation would bring about the hypocholesterolemic effect by lowering HMG Co A reductase activity in liver and by increasing the secretion of bile constituents.

3α-6α-Dihydroxy-7α-fluoro-5β-cholanoate (UPF-680), physicochemical and physiological properties of a new fluorinated bile acid that prevents 17α-ethynyl-estradiol-induced cholestasis in rats

3alpha-6alpha-Dihydroxy-7alpha-fluoro-5beta-cholanoate (UPF-680), the 7alpha-fluorine analog of hyodeoxycholic acid (HDCA), was synthesized to improve bioavailability and stability of ursodeoxycholic acid (UDCA). Acute rat biliary fistula and chronic cholestasis induced by 17alpha-ethynyl-estradiol (17EE) models were used to study and compare the effects of UPF-680 (dose range 0.6-6.0 micromol/kg min) with UDCA on bile flow, biliary bicarbonate (HCO(3)(-)), lipid output, biliary bile acid composition, hepatic enzymes and organic anion pumps. In acute infusion, UPF-680 increased bile flow in a dose-related manner, by up to 40.9%. Biliary HCO(3)(-) output was similarly increased. Changes were observed in phospholipid secretion only at the highest doses. Treatment with UDCA and UPF-680 reversed chronic cholestasis induced by 17EE; in this model, UDCA had no effect on bile flow in contrast to UPF-680, which significantly increased bile flow. With acute administration of UPF-680, the biliary bile acid pool became enriched with unconjugated and conjugated UPF-680 (71.7%) at the expense of endogenous cholic acid and muricholic isomers. With chronic administration of UPF-680 or UDCA, the main biliary bile acids were tauro conjugates, but modification of biliary bile acid pool was greater with UPF-680. UPF-680 increased the mRNA for cytochrome P450 7A1 (CYP7A1) and cytochrome P450 8B (CYP8B). Both UDCA and UPF-680 increased the mRNA for Na(+) taurocholate co-transporting polypeptide (NCTP). In conclusion, UPF-680 prevented 17EE-induced cholestasis and enriched the biliary bile acid pool with less detergent and cytotoxic bile acids. This novel fluorinated bile acid may have potential in the treatment of cholestatic liver disease.

24-norUrsodeoxycholic Acid Is Superior to Ursodeoxycholic Acid in the Treatment of Sclerosing Cholangitis in Mdr2 (Abcb4) Knockout Mice

Current therapy for primary sclerosing cholangitis is of limited efficacy. Multidrug resistance gene 2 knockout mice (Mdr2(-/-)) represent a well-characterized model for sclerosing cholangitis. Experiments were performed to test in such mice the therapeutic effects of 24-norUrsodeoxycholic acid, a C(23) homologue of ursodeoxycholic acid with 1 fewer methylene group in its side chain. Mdr2(-/-) mice were fed a diet containing 24-norUrsodeoxycholic acid (0.5% wt/wt) or ursodeoxycholic acid (0.5% wt/wt) as a clinical comparator for 4 weeks; controls received standard chow. Effects on serum liver tests, liver histology, markers of inflammation and fibrosis, and bile acid transport and metabolism were compared. 24-norUrsodeoxycholic acid metabolism was studied in serum, liver, bile, and urine. 24-norUrsodeoxycholic acid markedly improved liver tests and liver histology and significantly reduced hydroxyproline content and the number of infiltrating neutrophils and proliferating hepatocytes and cholangiocytes. 24-norUrsodeoxycholic acid underwent extensive phase I/II metabolism (hydroxylation, sulfation, and glucuronidation), thereby increasing the hydrophilicity of biliary bile acid secretion. There was a coordinated induction of bile acid detoxifying enzymes (Cyp2b10, Cyp3a11, and Sult2a1) and efflux pumps (Mrp3 and Mrp4). Ursodeoxycholic acid, in contrast, increased alanine transaminase and alkaline phosphatase levels, had no significant effects on hydroxyproline content, and induced biliary transporters and detoxification enzymes to a much smaller extent than 24-norUrsodeoxycholic acid. 24-norUrsodeoxycholic acid ameliorates sclerosing cholangitis in Mdr2(-/-) mice. Its therapeutic mechanisms involve (1) increasing the hydrophilicity of biliary bile acids, (2) stimulating bile flow with flushing of injured bile ducts, and (3) inducing detoxification and elimination routes for bile acids.

Effects of tegaserod on bile composition and hepatic secretion in Richardson ground squirrels on an enriched cholesterol diet

BackgroundTegaserod is effective in treating IBS patients with constipation, and does not alter gallbladder motility in healthy individuals or in patients with IBS. However, it is not known if tegaserod affects the biliary tract in gallstone disease, so to this end the effects of tegaserod on bile composition and hepatic secretion of Richardson ground squirrels maintained on an enriched cholesterol diet were examined.ResultsAnimals were fed either a control (0.03%) or enriched (1%) cholesterol diet for 28 days, and treated s.c. with tegaserod (0.1 mg/kg BID) or vehicle. Bile flow, bile acid, phospholipids and cholesterol secretion were measured with standard methods. Tegaserod treatment or enriched cholesterol diet, alone or combination, did not alter body or liver weights. The enriched cholesterol diet increased cholesterol saturation index (CSI), cholesterol concentrations in gallbladder and hepatic duct bile by ~50% and decreased bile acids in gallbladder bile by 17%. Tegaserod treatment reversed these cholesterol-induced changes. None of the treatments, drug or diet, altered fasting gallbladder volume, bile flow and bile salts or phospholipid secretion in normal diet and cholesterol-fed animals. However, tegaserod treatment prevented the decreases in bile acid pool size and cycling frequency caused by the enriched cholesterol diet, consequent to re-establishing normal bile acid to concentrations in the gall bladder. Tegaserod had no effect on these parameters with normal diet animals.ConclusionTegaserod treatment results in increased enterohepatic cycling and lowers cholesterol saturation in the bile of cholesterol-fed animals. These effects would decrease conditions favorable to cholesterol gallstone formation.

Peroxisome Proliferator-Activated Receptor α Antagonism Inhibits Hepatitis C Virus Replication

Hepatitis C virus (HCV) is a global health problem and a leading cause of liver disease. Here, we demonstrate that the replication of HCV replicon RNA in Huh-7 cells is inhibited by a peroxisome proliferator-activated receptor (PPAR) antagonist, 2-chloro-5-nitro-N-(pyridyl)benzamide (BA). Downregulation of PPARgamma with RNA interference approaches had no effect on HCV replication in Huh-7 cells, whereas PPARalpha downregulation inhibited HCV replication. Fluorescence and coherent anti-Stokes Raman scattering (CARS) microscopy demonstrate a clear buildup of lipids upon treatment with BA. These observations are consistent with the misregulation of lipid metabolism, phospholipid secretion, cholesterol catabolism, and triglyceride clearance events associated with the inhibition of PPARalpha. The inhibition of HCV replication by BA may result from disrupting lipidation of host proteins associated with the HCV replication complex or, more generally, by disrupting the membranous web where HCV replicates.

Apical sodium bile acid transporter and ileal lipid binding protein in gallstone carriers

Although a cholesterol supersaturation of gallbladder bile has been identified as the underlying pathophysiologic defect, the molecular pathomechanism of gallstone formation in humans remains poorly understood. A deficiency of the apical sodium bile acid transporter (ASBT) and ileal lipid binding protein (ILBP) in the small intestine may result in bile acid loss into the colon and might promote gallstone formation by reducing the bile acid pool and increasing the amount of hydrophobic bile salts. To test this hypothesis, protein levels and mRNA expression of ASBT and ILBP were assessed in ileal mucosa biopsies of female gallstone carriers and controls. Neither ASBT nor ILBP levels differed significantly between gallstone carriers and controls. However, when study participants were subgrouped by body weight, ASBT and ILBP protein were 48% and 67% lower in normal weight gallstone carriers than in controls (P < 0.05); similar differences were found for mRNA expression levels. The loss of bile transporters in female normal weight gallstone carriers was coupled with a reduction of protein levels of hepatic nuclear factor 1alpha and farnesoid X receptor. In conclusion, in normal weight female gallstone carriers, the decreased expression of ileal bile acid transporters may form a molecular basis for gallstone formation.

Lymphatic chylomicron size is inversely related to biliary phospholipid secretion in mice

Biliary phospholipids (PL) stimulate dietary fat absorption by facilitating intraluminal lipid solubilization and by providing surface components for chylomicron (CM) assembly. Impaired hepatic PL availability induces secretion of large very-low-density lipoproteins, but it is unclear whether CM size depends on biliary PL availability. Biliary PL secretion is absent in multidrug resistance protein 2-deficient (Mdr2(-/-)) mice, whereas it is strongly increased in essential fatty acid (EFA)-deficient mice. We investigated lymphatic CM size and composition in mice with absent (Mdr2(-/-)) or enhanced (EFA deficient) biliary PL secretion and in their respective controls under basal conditions and during enteral lipid administration. EFA deficiency was induced by feeding mice a high-fat, EFA-deficient diet for 8 wk. Lymph was collected by mesenteric lymph duct cannulation with or without intraduodenal lipid administration. Lymph was collected in 30-min fractions for up to 4 h, and lymphatic lipoprotein size was determined by dynamic light-scattering techniques. Lymph lipoprotein subfractions were isolated by ultracentrifugation, and lipid composition was measured. Lymphatic CMs were significantly larger in Mdr2(-/-) mice than in Mdr2(+/+) controls either without (+50%) or with (+25%) enteral lipid administration, and molar core-surface ratios were increased [triglyceride (TG)-to-PL ratio: 4.4 +/- 1.4 in Mdr2(-/-) mice vs. 2.7 +/- 0.8 in Mdr2(+/+) mice, P < 0.001]. In contrast, EFA-deficient mice secreted lipoproteins into lymph that were significantly smaller than in EFA-sufficient controls (173 +/- 32 vs. 236 +/- 47 nm), with correspondingly decreased core-surface ratios (TG-to-PL ratio: 3.0 +/- 1.0 in EFA-deficient mice vs. 6.0 +/- 1.9 in EFA-sufficient mice, P < 0.001). CM size increased during fat absorption in both EFA-deficient and EFA-sufficient mice, but the difference between the groups persisted. In conclusion, the present results strongly suggest that the availability of biliary PL is a major determinant of the size of intestinally produced lipoproteins both under basal conditions and during lipid absorption. Altered CM size may have physiological consequences for postprandial CM processing.

Intestinal-fatty acid binding protein and lipid transport in human intestinal epithelial cells

Intestinal-fatty acid binding protein (I-FABP) is a 14–15 kDa cytoplasmic molecule highly expressed in the enterocyte. Although different functions have been proposed for various FABP family members, the specific function of I-FABP in human intestine remains unclear. Here, we studied the role of I-FABP in molecularly modified normal human intestinal epithelial cells (HIEC-6). cDNA transfection resulted in 90-fold I-FABP overexpression compared to cells treated with empty pQCXIP vector. The high-resolution immunogold technique revealed labeling mainly in the cytosol and confirmed the marked phenotype abundance of I-FABP in cDNA transfected cells. I-FABP overexpression was not associated with alterations in cell proliferation and viability. Studies using these transfected cells cultured with [ 14C]oleic acid did not reveal higher efficiency in de novo synthesis or secretion of triglycerides, phospholipids, and cholesteryl esters compared to cells treated with empty pQCXIP vector only. Similarly, the incubation with [ 35S]methionine did not disclose a superiority in the biogenesis of apolipoproteins (apo) A-I, A-IV, B-48, and B-100. Finally, cells transfected with I-FABP did not exhibit an increased production of chylomicrons, VLDL, LDL, and HDL. Our observations establish that I-FABP overexpression in normal HIEC-6 is not related to cell proliferation, lipid esterification, apo synthesis, and lipoprotein assembly, and, therefore, exclude its role in intestinal fat transport.