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• W2068210500 abstract "Quantitative trait mapping identified a locus colocalizing with L-Fabp, encoding liver fatty acid binding protein, as a positional candidate for murine gallstone susceptibility. When fed a lithogenic diet (LD) for 2 weeks, L-Fabp−/− mice became hypercholesterolemic with increased hepatic VLDL cholesterol secretion. Seventy-five percent of L-Fabp−/− mice developed solid gallstones compared with 6% of wild-type mice with an increased gallstone score (3.29 versus 0.62, respectively; P < 0.01). Hepatic free cholesterol content, biliary cholesterol secretion, and the cholesterol saturation index of hepatic bile were increased in LD-fed L-Fabp−/− mice. Chow-fed L-Fabp−/− mice demonstrated increased fecal bile acid (BA) excretion accompanied by decreased ileal Asbt expression. By contrast, there was an increased BA pool and decreased fecal BA excretion in LD-fed L-Fabp−/− mice, associated with increased proximal intestinal Asbt mRNA expression, suggesting that intestinal BA absorption was enhanced in LD-fed L-Fabp−/− mice. The increase in biliary BA secretion and enterohepatic pool size in LD-fed L-Fabp−/− mice was accompanied by downregulation of Cyp7a1 mRNA and increased intestinal mRNA abundance of Fgf-15, Fxr, and Fabp6. These findings suggest that changes in hepatic cholesterol metabolism and biliary lipid secretion as well as changes in enterohepatic BA metabolism increase gallstone susceptibility in LD fed L-Fabp−/− mice. Quantitative trait mapping identified a locus colocalizing with L-Fabp, encoding liver fatty acid binding protein, as a positional candidate for murine gallstone susceptibility. When fed a lithogenic diet (LD) for 2 weeks, L-Fabp−/− mice became hypercholesterolemic with increased hepatic VLDL cholesterol secretion. Seventy-five percent of L-Fabp−/− mice developed solid gallstones compared with 6% of wild-type mice with an increased gallstone score (3.29 versus 0.62, respectively; P < 0.01). Hepatic free cholesterol content, biliary cholesterol secretion, and the cholesterol saturation index of hepatic bile were increased in LD-fed L-Fabp−/− mice. Chow-fed L-Fabp−/− mice demonstrated increased fecal bile acid (BA) excretion accompanied by decreased ileal Asbt expression. By contrast, there was an increased BA pool and decreased fecal BA excretion in LD-fed L-Fabp−/− mice, associated with increased proximal intestinal Asbt mRNA expression, suggesting that intestinal BA absorption was enhanced in LD-fed L-Fabp−/− mice. The increase in biliary BA secretion and enterohepatic pool size in LD-fed L-Fabp−/− mice was accompanied by downregulation of Cyp7a1 mRNA and increased intestinal mRNA abundance of Fgf-15, Fxr, and Fabp6. These findings suggest that changes in hepatic cholesterol metabolism and biliary lipid secretion as well as changes in enterohepatic BA metabolism increase gallstone susceptibility in LD fed L-Fabp−/− mice. Gallstone disease is a major health problem in western society, and its attendant complications and comorbidities impose a substantial financial burden on the health care economy (1Sandler R.S. Everhart J.E. Donowitz M. Adams E. Cronin K. Goodman C. Gemmen E. Shah S. Avdic A. Rubin R. The burden of selected digestive diseases in the United States.Gastroenterology. 2002; 122: 1500-1511Abstract Full Text Full Text PDF PubMed Scopus (1174) Google Scholar). Among the factors that predispose to cholesterol cholelithiasis, considerable attention has focused on environmental modifiers, such as obesity, because even in populations at increased genetic risk there appears to be a strong link between gallstone susceptibility and body habitus that may be further modified in a gender-specific manner (2Lyons M.A. Wittenburg H. Cholesterol gallstone susceptibility loci: a mouse map, candidate gene evaluation, and guide to human LITH genes.Gastroenterology. 2006; 131: 1943-1970Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). However, despite recent advances in the mechanisms that link hepatic insulin resistance and gallstone susceptibility (3Biddinger S.B. Haas J.T. Yu B.B. Bezy O. Jing E. Zhang W. Unterman T.G. Carey M.C. Kahn C.R. Hepatic insulin resistance directly promotes formation of cholesterol gallstones.Nat. Med. 2008; 14: 778-782Crossref PubMed Scopus (218) Google Scholar), there remain many unanswered questions concerning the pathways by which alterations in hepatic lipid metabolism result in biliary cholesterol supersaturation, leading to cholesterol monohydrate crystal formation and eventually the emergence of gallstones (4Zanlungo S. Rigotti A. Nervi F. Hepatic cholesterol transport from plasma into bile: implications for gallstone disease.Curr. Opin. Lipidol. 2004; 15: 279-286Crossref PubMed Scopus (38) Google Scholar).Our understanding of the genetic factors that predispose to gallstone formation has been advanced through study of inbred murine crosses in which susceptibility loci have been mapped using diet-induced gallstone formation as a quantitative trait. Using this approach, >23 quantitative trait loci have been mapped, allowing formal evaluation of potential candidate genes [reviewed in (2Lyons M.A. Wittenburg H. Cholesterol gallstone susceptibility loci: a mouse map, candidate gene evaluation, and guide to human LITH genes.Gastroenterology. 2006; 131: 1943-1970Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar)]. Among these loci, Liver fatty acid binding protein (L-Fabp) emerged as a positional candidate from a quantitative trait loci near D6Mit123 on chromosome 6 (71.5 Mb, 30 centimorgans) (2Lyons M.A. Wittenburg H. Cholesterol gallstone susceptibility loci: a mouse map, candidate gene evaluation, and guide to human LITH genes.Gastroenterology. 2006; 131: 1943-1970Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 5Lyons M.A. Wittenburg H. Li R. Walsh K.A. Leonard M.R. Korstanje R. Churchill G.A. Carey M.C. Paigen B. Lith6: a new QTL for cholesterol gallstones from an intercross of CAST/Ei and DBA/2J inbred mouse strains.J. Lipid Res. 2003; 44: 1763-1771Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). L-Fabp is an abundant cytosolic lipid binding protein expressed in mammalian enterocytes and hepatocytes with a flexible lipid binding pocket that accommodates FAs and other hydrophobic ligands, including cholesterol, acyl-CoA, and bile acids (BAs) (6Cistola D.P. Sacchettini J.C. Banaszak L.J. Walsh M.T. Gordon J.I. Fatty acid interactions with rat intestinal and liver fatty acid-binding proteins expressed in Escherichia coli. A comparative 13C NMR study.J. Biol. Chem. 1989; 264: 2700-2710Abstract Full Text PDF PubMed Google Scholar, 7Cistola D.P. Sacchettini J.C. Gordon J.I. 13C NMR studies of fatty acid-protein interactions: comparison of homologous fatty acid-binding proteins produced in the intestinal epithelium.Mol. Cell. Biochem. 1990; 98: 101-110Crossref PubMed Scopus (18) Google Scholar). More recent studies in L-Fabp−/− mice imply an important role for this gene in modulating FA trafficking and decreasing hepatic steatosis following fasting (8Newberry E.P. Xie Y. Kennedy S. Han X. Buhman K.K. Luo J. Gross R.W. Davidson N.O. Decreased hepatic triglyceride accumulation and altered fatty acid uptake in mice with deletion of the liver fatty acid-binding protein gene.J. Biol. Chem. 2003; 278: 51664-51672Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar) and in attenuating diet-induced hepatic steatosis and obesity in response to prolonged high-fat feeding (9Newberry E.P. Xie Y. Kennedy S.M. Luo J. Davidson N.O. Protection against Western diet-induced obesity and hepatic steatosis in liver fatty acid-binding protein knockout mice.Hepatology. 2006; 44: 1191-1205Crossref PubMed Scopus (172) Google Scholar).A role for L-Fabp was proposed in modifying the response to cholesterol feeding based on a striking gender-specific phenotype that included enhanced obesity and hepatic steatosis in female L-Fabp−/− mice (10Martin G.G. Atshaves B.P. McIntosh A.L. Mackie J.T. Kier A.B. Schroeder F. Liver fatty acid binding protein gene ablation potentiates hepatic cholesterol accumulation in cholesterol-fed female mice.Am. J. Physiol. Gastrointest. Liver Physiol. 2006; 290: G36-G48Crossref PubMed Scopus (65) Google Scholar). Those findings, however, are at odds with more recent studies in which cholesterol feeding alone (in the absence of high fat feeding) produced generally subtle alterations in hepatic lipid metabolism and no effect on weight gain in L-Fabp−/− mice in a congenic C57BL/6 background (11Newberry E.P. Kennedy S.M. Xie Y. Sternard B.T. Luo J. Davidson N.O. Diet-induced obesity and hepatic steatosis in L-Fabp/mice is abrogated with SF, but not PUFA, feeding and attenuated after cholesterol supplementation.Am. J. Physiol. Gastrointest. Liver Physiol. 2008; 294: G307-G314Crossref PubMed Scopus (60) Google Scholar). However, other studies demonstrated an increase in BA pool size in male L-Fabp−/− mice fed a zero-cholesterol, low-fat diet and a >2-fold increase in gallbladder cholesterol content along with a 4-fold decrease in BA pool size following dietary cholesterol supplementation (12Martin G.G. Atshaves B.P. McIntosh A.L. Mackie J.T. Kier A.B. Schroeder F. Liver fatty-acid-binding protein (L-FABP) gene ablation alters liver bile acid metabolism in male mice.Biochem. J. 2005; 391: 549-560Crossref PubMed Scopus (56) Google Scholar). Differences in the obesity phenotypes notwithstanding, these studies together suggest that gender-specific effects play an important role in modulating the response to cholesterol feeding in L-Fabp−/− mice but left unanswered whether an informative phenotype might emerge when these animals were challenged with a diet known to produce cholesterol cholelithiasis (13Lyons M.A. Wittenburg H. Li R. Walsh K.A. Churchill G.A. Carey M.C. Paigen B. Quantitative trait loci that determine lipoprotein cholesterol levels in DBA/2J and CAST/Ei inbred mice.J. Lipid Res. 2003; 44: 953-967Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar).The current studies were undertaken to examine the importance of L-Fabp as a genetic modifier of diet-induced cholesterol gallstone formation in congenic lines of mice. In particular, we were intrigued by the possibility that the protection against high-fat-induced obesity and hepatic steatosis (9Newberry E.P. Xie Y. Kennedy S.M. Luo J. Davidson N.O. Protection against Western diet-induced obesity and hepatic steatosis in liver fatty acid-binding protein knockout mice.Hepatology. 2006; 44: 1191-1205Crossref PubMed Scopus (172) Google Scholar) might protect L-Fabp−/− mice from cholesterol gallstone disease. However, our findings revealed that male L-Fabp−/− mice manifest a striking increase in gallstone susceptibility when fed a lithogenic diet, a phenotype coupled to changes in cholesterol metabolism and to alterations in both hepatic and intestinal BA metabolism.MATERIALS AND METHODSAnimals and dietsMale L-Fabp−/− mice (backcrossed onto a C57BL/6 background) and C57BL/6 wild-type mice (Jackson Laboratories, Bar Harbor, ME) were maintained on a 12 h light-dark cycle, in a full-barrier facility. Eight- to ten-week-old mice were fed either a standard rodent chow (PicoLab Rodent Diet 20) or a lithogenic diet (LD) (Research Diet 960393; 18.8% fat, 1.2% cholesterol, and 0.5% cholic acid) for 2 to 4 weeks, as indicated. All animal protocols were approved by the Washington University Animal Studies Committee and conformed to criteria outlined in the National Institutes of Health “Guide for the Care and Use of Laboratory Animals.”Hepatic lipid and serum analysesAnimals were sacrificed after a 4 h fast. Liver and serum were collected and frozen at −80°C until analyzed. Hepatic triglyceride (TG), cholesterol, FFA, phospholipids, and BAs were assayed enzymatically with Wako reagent kits (Neuss, Germany): L-Type Triglyceride H kit (Cat. No. 993-37592, 993-37492), cholesterol E kit (Cat. No.439-17501), Free cholesterol E kit (Cat. No. 435-35801), HR series NRFA-HR2 kit (Cat. No. 995-34691, 995-34791), phospholipid B kit (Cat. No. 990-54009), and total bile acids kit (Cat. No. 431-15001). Serum glucose and alanine aminotransferase were analyzed by Wako Autokit Glucose (Cat. No.439-90901) and Teco ALT set (Cat. No. A526-120). Lipoprotein distribution was examined by fast-protein liquid chromatography (FPLC) (14Xie Y. Newberry E.P. Young S.G. Robine S. Hamilton R.L. Wong J.S. Luo J. Kennedy S. Davidson N.O. Compensatory increase in hepatic lipogenesis in mice with conditional intestine-specific Mttp deficiency.J. Biol. Chem. 2006; 281: 4075-4086Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar).Gallbladder bile and gallstone analysisMice were fasted for 4 h and anesthetized, and gallbladders ligated and punctured at the fundus for collection of gallbladder bile. Bile samples were collected and immediately analyzed by polarizing light microscopy using a visual scale (high power field = 400× magnification) with the following criteria: 0 = absence of cholesterol monohydrate crystals (ChMCs), 1 = small number of ChMCs (<3/high-power field); 2 = many ChMCs (≥3/high power field); 3 = aggregated ChMCs; and 4 = presence of “sandy” light-translucent stones or “solid” light-opaque stones (13Lyons M.A. Wittenburg H. Li R. Walsh K.A. Churchill G.A. Carey M.C. Paigen B. Quantitative trait loci that determine lipoprotein cholesterol levels in DBA/2J and CAST/Ei inbred mice.J. Lipid Res. 2003; 44: 953-967Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar).Biliary lipid and BA species determinationAn external bile fistula was established surgically via the gallbladder fundus. Bile samples collected during the first 15 min were discarded, and bile samples collected for another 60 min were used for further analysis. Hepatic bile volume was determined gravimetrically assuming a density of 1 g·ml−1. Biliary phospholipids, cholesterol, and total BA content were determined enzymatically. Cholesterol saturation indices in hepatic bile were calculated using published parameters (15Carey M.C. Critical tables for calculating the cholesterol saturation of native bile.J. Lipid Res. 1978; 19: 945-955Abstract Full Text PDF PubMed Google Scholar). Individual bile salt concentrations were measured via high-pressure liquid chromatography (16Rossi S.S. Converse J.L. Hofmann A.F. High pressure liquid chromatographic analysis of conjugated bile acids in human bile: simultaneous resolution of sulfated and unsulfated lithocholyl amidates and the common conjugated bile acids.J. Lipid Res. 1987; 28: 589-595Abstract Full Text PDF PubMed Google Scholar). BA standards were purchased from Steraloids (Newport, RI) or Calbiochem (Madison, WI). The hydrophobic index of hepatic bile sample was calculated according to published methods (17Heuman D.M. Quantitative estimation of the hydrophilic-hydrophobic balance of mixed bile salt solutions.J. Lipid Res. 1989; 30: 719-730Abstract Full Text PDF PubMed Google Scholar).BA pool size and fecal BA excretion determinationBA pool size was determined as the sum total BA content of the entire small intestine, gallbladder, and liver, which were homogenized and extracted together in ethanol with [24-14C]taurocholic acid (0.025 μCi) added as an internal recovery standard (18Schwarz M. Russell D.W. Dietschy J.M. Turley S.D. Marked reduction in bile acid synthesis in cholesterol 7alpha-hydroxylase-deficient mice does not lead to diminished tissue cholesterol turnover or to hypercholesterolemia.J. Lipid Res. 1998; 39: 1833-1843Abstract Full Text Full Text PDF PubMed Google Scholar). Total BA mass was determined enzymatically. In addition, fecal BA excretion was determined from stool quantitatively collected from individually housed mice for 72 h. For each determination, 200 mg triplicate aliquots of dried feces were extracted into ethanol as described (18Schwarz M. Russell D.W. Dietschy J.M. Turley S.D. Marked reduction in bile acid synthesis in cholesterol 7alpha-hydroxylase-deficient mice does not lead to diminished tissue cholesterol turnover or to hypercholesterolemia.J. Lipid Res. 1998; 39: 1833-1843Abstract Full Text Full Text PDF PubMed Google Scholar, 19Setchell K.D. Lawson A.M. Tanida N. Sjovall J. General methods for the analysis of metabolic profiles of bile acids and related compounds in feces.J. Lipid Res. 1983; 24: 1085-1100Abstract Full Text PDF PubMed Google Scholar).Cholesterol and fat absorptionCholesterol absorption was measured by a fecal dual-isotope ratio method as described previously (14Xie Y. Newberry E.P. Young S.G. Robine S. Hamilton R.L. Wong J.S. Luo J. Kennedy S. Davidson N.O. Compensatory increase in hepatic lipogenesis in mice with conditional intestine-specific Mttp deficiency.J. Biol. Chem. 2006; 281: 4075-4086Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 20Wang D.Q. Carey M.C. Measurement of intestinal cholesterol absorption by plasma and fecal dual-isotope ratio, mass balance, and lymph fistula methods in the mouse: an analysis of direct versus indirect methodologies.J. Lipid Res. 2003; 44: 1042-1059Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 21Wang D.Q. Paigen B. Carey M.C. Genetic factors at the enterocyte level account for variations in intestinal cholesterol absorption efficiency among inbred strains of mice.J. Lipid Res. 2001; 42: 1820-1830Abstract Full Text Full Text PDF PubMed Google Scholar). Mice were housed individually in metabolic cages and food intake and fecal output measured daily. Fecal fat excretion (percentage) was determined gravimetrically after lipid extraction (9Newberry E.P. Xie Y. Kennedy S.M. Luo J. Davidson N.O. Protection against Western diet-induced obesity and hepatic steatosis in liver fatty acid-binding protein knockout mice.Hepatology. 2006; 44: 1191-1205Crossref PubMed Scopus (172) Google Scholar).Gene expression analysisFor microarray analysis, total liver RNA was pooled from groups of LD-fed (2 weeks) wild-type or L-Fabp−/− mice (n = 4 animals per group). Pooled RNA was hybridized to an Agilent Technologies Mouse Whole Genome microarray (G4122A; Agilent, Palo Alto, CA). Microarray results were confirmed by real-time quantitative RT-PCR (qRT-PCR) using individual cDNA from four or five animals per group. qRT-PCR assays were performed in triplicate on an ABI Prism 7000 Sequence Detection System using SYBR Green PCR Master Mix (Applied Biosystems) and primer pairs designed by Primer Express software (Applied Biosystems) (primer sequences are available upon request). Relative mRNA abundance is expressed as fold change compared with mRNA levels in wild-type mice consuming the same diet, normalized to 18S rRNA. Western blotting was performed using anti-L-Fabp antisera (1:2,000) as previously detailed (8Newberry E.P. Xie Y. Kennedy S. Han X. Buhman K.K. Luo J. Gross R.W. Davidson N.O. Decreased hepatic triglyceride accumulation and altered fatty acid uptake in mice with deletion of the liver fatty acid-binding protein gene.J. Biol. Chem. 2003; 278: 51664-51672Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar). Asbt expression was determined on membrane fractions (50 μg protein) prepared from scraped distal intestinal mucosa as detailed using anti-Asbt IgG (1:1,000) (22Dawson P.A. Hubbert M. Haywood J. Craddock A.L. Zerangue N. Christian W.V. Ballatori N. The heteromeric organic solute transporter alpha-beta, Ostalpha-Ostbeta, is an ileal basolateral bile acid transporter.J. Biol. Chem. 2005; 280: 6960-6968Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar).Statistical analysisStatistical significance was determined with an unpaired, two-tailed Student’s t-test. Data are expressed as the mean ± SE unless otherwise noted.RESULTSMetabolic alterations following LD feeding in wild-type and L-Fabp−/− miceBoth genotypes exhibited weight loss following consumption of the LD, with L-Fabp−/− mice demonstrating significantly greater weight loss (Table 1). Serum cholesterol was significantly increased in L-Fabp−/− mice, with a shift into less dense lipoprotein fractions revealed by FPLC (Fig. 1A). This pattern was augmented after 4 weeks of LD feeding (Fig. 1B) and was associated with increased hepatic secretion of cholesterol-rich lipoproteins in L-Fabp−/− mice (Fig. 1C), although VLDL-TG secretion was comparable in L-Fabp−/− and wild-type mice (data not shown). In addition, L-Fabp−/− mice manifested decreased hepatic TG content and an increase in hepatic free cholesterol and phospholipid content (Table 1). L-Fabp expression in wild-type mice showed a tissue-specific divergence in response to LD feeding. There was an ∼3-fold decrease in hepatic L-Fabp mRNA and protein expression with an ∼3-fold increase in intestinal L-Fabp expression (Fig. 1D). These findings collectively point to a range of adaptive alterations in hepatic and serum cholesterol metabolism following 2 weeks of LD feeding in L-Fabp−/− mice, whose functional consequences were further explored.TABLE 1Body weight, liver weight, food consumption, fat absorption, and serum and hepatic lipid profile of LD-fed wild-type and L-Fabp−/− miceParametersWild TypeL-Fabp−/−PBody weight (g)22.7 ± 0.3 (22)20.1 ± 0.5 (21)**Weight gain (g)−0.9 ± 0.2 (22)−1.9 ± 0.3 (21)**Liver weight (g)1.4 ± 0.04 (22)1.3 ± 0.06 (21)*Liver weight (% body weight)6.3 ± 0.2 (22)6.3 ± 0.2 (21)NSFat absorption (%)93.9 ± 0.3 (4)93.6 ± 0.4 (4)NSFeed efficiency−23.7 ± 9.2 (4)−50.6 ± 8.6 (4)NSSerum cholesterol (mg/dl)167.2 ± 8.0 (13)241.3 ± 29.5 (13)*Serum TG (mg/dl)10.1 ± 1.3 (8)12.2 ± 2.2 (8)NSSerum phospholipids (mg/dl)204.3 ± 7.8 (8)208.2 ± 13 (8)NSSerum FFAs (mmol/l)0.24 ± 0.04 (8)0.26 ± 0.09 (8)NSSerum glucose (mg/dl)174.5 ± 8.0 (13)128.7 ± 8.3 (13)**Serum BA (μmol/L)15.9 ± 2.0 (8)33.8 ± 2.0 (8)*Serum ALT (IU/L)74.4 ± 16.4 (13)117.7 ± 13.0 (13)*Hepatic total cholesterol (μg/mg protein)210.2 ± 10.3 (8)232.7 ± 20.3 (8)NSHepatic free cholesterol (μmol/mg protein)31.96 ± 1.73 (8)45.20 ± 3.1 (8)*Hepatic cholesterol ester (μmol/mg protein)178.2 ± 11.1 (8)187.5 ± 21.4 (8)NSHepatic TG (μg/mg protein)120.7 ± 10.8 (8)83.9 ± 6.6 (8)**Hepatic phospholipid (μg/mg protein)98.5 ± 1.7 (8)123.4 ± 4.9 (8)**Hepatic FFA (nmol/mg protein)33.9 ± 1.6 (8)37.0 ± 3.7 (8)NSValues represent the mean ± SE (n). ALT, alanine aminotransferase; NS, not significant. Feed efficiency is milligrams of weight change per gram of food consumed. *P < 0.05; **P < 0.01. Open table in a new tab Increased gallstone susceptibility in LD-fed L-Fabp−/− miceL-Fabp−/− mice demonstrated increased numbers of aggregated cholesterol monohydrate crystals in gallbladder bile at 2 weeks of LD feeding (Fig. 2A) and a naked-eye appearance of a thickened, opaque gallbladder that contrasted with the findings in wild-type mice (Fig. 2B). The gross appearance of a thickened gallbladder was confirmed by histological examination (Fig. 2C). Gallstones were found in 6/8 L-Fabp−/− mice compared with 1/17 wild-type mice, and the gallstone score (Fig. 2D) was correspondingly higher (3.29 ± 0.53 versus 0.62 ± 0.28 in L-Fabp−/− and wild-type mice, respectively; P < 0.01), demonstrating conclusively that gallstone susceptibility is increased in L-Fabp−/− mice.Fig. 2Biliary cholesterol crystallization, gallbladder inflammation, and gallstone score in LD-fed mice. Wild-type and L-Fabp−/− mice were fed a LD for 2 weeks. A: Fresh gallbladder bile samples were collected and analyzed by polarizing microscopy. A few single ChMCs were present in wild-type gallbladder bile, while aggregated ChMCs were observed in gallbladder bile from L-Fabp−/− mice. B: Gallbladders of wild-type mice appeared transparent, while gallbladders from L-Fabp−/− mice appeared opaque. C: Histological examination of gallbladder mucosal epithelium by hematoxylin and eosin staining. Gallbladder walls of L-Fabp−/− mice were thicker with increased stromal granulocyte infiltration (magnification: 400×). D: Gallstone score (left) and gallbladder volume (right) as described in Materials and Methods. There is an increase in the gallstone score and a trend toward increased gallbladder volume in L-Fabp−/− mice. Data derived from eight animals per genotype; ** indicates P < 0.01.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Biliary lipid secretion is altered in LD-fed but not chow-fed L-Fabp−/− miceBiliary lipid secretion was unaltered in chow-fed L-Fabp−/− mice compared with wild-type controls (Fig. 3A). However, after LD feeding, there was a 2-fold increase in biliary cholesterol secretion in L-Fabp−/− mice (0.27 ± 0.04) versus the wild type (0.13 ± 0.01 μmol/min/kg; P < 0.05) and a significant increase in secretion of phospholipid as well as a trend to increased BA secretion (Fig. 3A). The net effects of these alterations in biliary lipid secretion increased the cholesterol saturation indices of bile (Fig. 3B). There were no changes in the BA species distribution or hydrophobicity index in chow-fed L-Fabp−/− mice (Fig. 4A, C), but the proportion of taurocholate increased in LD-fed L-Fabp−/− mice, which tended to increase the hydrophobicity index (Fig. 4B, D). We also examined the profile of biliary lipid secretion in mice of both genotypes following dietary cholesterol supplementation alone, as other workers reported a 2-fold increase in gallbladder cholesterol and a 4-fold decrease in BA content in cholesterol-fed male L-Fabp−/− mice (12Martin G.G. Atshaves B.P. McIntosh A.L. Mackie J.T. Kier A.B. Schroeder F. Liver fatty-acid-binding protein (L-FABP) gene ablation alters liver bile acid metabolism in male mice.Biochem. J. 2005; 391: 549-560Crossref PubMed Scopus (56) Google Scholar). Our findings show increased biliary cholesterol secretion following dietary cholesterol supplementation (see supplementary Tables I and II) but revealed no differences between the genotypes. In addition and again in contrast with those earlier results, our findings demonstrated no effect of cholesterol supplementation alone on BA secretion in either genotype (see Supplementary Table I). These findings demonstrate that LD feeding (but not cholesterol supplementation alone) in male L-Fabp−/− mice alters hepatobiliary cholesterol and BA metabolism and results in increased gallstone formation.Fig. 3Biliary lipid secretion and hepatic bile cholesterol saturation indices on chow or LD. A: Hepatic bile was collected for 60 min and biliary lipid assayed enzymatically. Biliary cholesterol and phospholipid secretion (μmol/min/kg body weight) were similar on chow but increased on LD in L-Fabp−/− mice compared with wild-type controls. There were no differences in BA secretion on either diet between the two genotypes. B: Hepatic biliary cholesterol saturation indices were calculated as described in Materials and Methods. L-Fabp−/− mice demonstrated increased cholesterol saturation indices on LD (right) but not on chow (left) compared with wild-type mice. Bar graphs indicate the mean ± SE. Data were derived from five mice per genotype on chow diet and eight mice per genotype on LD; * indicates P < 0.05.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 4BA species distribution and hydrophobicity indices on chow or LD. Individual BA species were examined in hepatic bile from mice fed chow (A) (n = 5 per genotype) or LD (B) (n = 8 per genotype) by HPLC. Bar graphs represent the mean ± SE. BA profiles were similar on chow, but altered on LD, specifically with increased taurocholate, decreased glycocholate, and taurochenodeoxycholate in L-Fabp−/− mice compared with the wild type. TβMC, tauro-β-muricholate; TUDC, tauroursodeoxycholate; TC, taurocholate; GC, glycocholate; TCDC, taurochenodeoxycholate; TDC, taurodeoxycholate. Bile salt hydrophobicity indices (HI) were comparable in chow-fed animals (C) but showed a trend to increase in L-Fabp−/− mice versus the wild type when fed the LD (D); * indicates P < 0.05.View Large Image Figure ViewerDownload Hi-res image Download (PPT)We also examined the possibility that changes in intestinal cholesterol absorption might contribute to the alterations noted in biliary cholesterol secretion in LD-fed L-Fabp−/− mice. Cholesterol absorption was significantly decreased in chow-fed L-Fabp−/− mice compared with wild-type controls (42 ± 4% versus 55 ± 2%; P < 0.05), but there were no differences observed in LD-fed mice between the genotypes (Fig. 5). In addition, there were no differences in mRNA expression of Npc1L1, Cd36, Fatp4, Abcg5/8 or Srb-1, genes implicated in regulating cholesterol absorption (data not shown).Fig. 5Decreased cholesterol absorption in chow-fed L-Fabp−/− mice, but comparable cholesterol absorption in mice fed LD for 4 or 8 weeks. Cholesterol absorption was measured by the fecal dual isotope ratio method. Bar graphs are mean ± SE, with data from four mice per genotype on chow, nine mice per genotype on LD for 4 weeks, and four wild-type mice and six L-Fabp−/− mice on LD for 8 weeks; * indicates P < 0.05.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Changes in gene expression associated with LD feeding in L-Fabp−/− miceTo pursue the mechanisms by which LD feeding results in such a striking ph" @default.
• W2068210500 created "2016-06-24" @default.
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• W2068210500 date "2009-05-01" @default.
• W2068210500 modified "2023-10-03" @default.
• W2068210500 title "Increased susceptibility to diet-induced gallstones in liver fatty acid binding protein knockout mice" @default.
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