Mice Deleted for Fatty Acid Transport Protein 5 Have Defective Bile Acid Conjugation and Are Protected From Obesity
2006; Elsevier BV; Volume: 130; Issue: 4 Linguagem: Inglês
10.1053/j.gastro.2006.02.012
ISSN1528-0012
AutoresBrian K. Hubbard, Holger Doege, Sandhya Punreddy, Hui Wu, Xueming Huang, Virendar K. Kaushik, Robin L. Mozell, John Byrnes, Alain Stricker‐Krongrad, Chieh Jason Chou, Louis A. Tartaglia, Harvey F. Lodish, Andreas Stahl, Ruth E. Gimeno,
Tópico(s)Metabolism and Genetic Disorders
ResumoBackground & Aims: Fatty Acid Transport Protein 5 (FATP5) is a liver-specific member of the FATP/Slc27 family, which has been shown to exhibit both fatty acid transport and bile acid-CoA ligase activity in vitro. Here, we investigate its role in bile acid metabolism and body weight homeostasis in vivo by using a novel FATP5 knockout mouse model. Methods: Bile acid composition was analyzed by mass spectroscopy. Body weight, food intake, energy expenditure, and fat absorption were determined in animals fed either a low- or a high-fat diet. Results: Although total bile acid concentrations were unchanged in bile, liver, urine, and feces of FATP5 knockout mice, the majority of gallbladder bile acids was unconjugated, and only a small percentage was conjugated. Primary, but not secondary, bile acids were detected among the remaining conjugated forms in FATP5 deletion mice, suggesting a specific requirement for FATP5 in reconjugation of bile acids during the enterohepatic recirculation. Fat absorption in FATP5 deletion mice was largely normal, and only a small increase in fecal fat was observed on a high-fat diet. Despite normal fat absorption, FATP5 deletion mice failed to gain weight on a high-fat diet because of both decreased food intake and increased energy expenditure. Conclusions: Our findings reveal an important role for FATP5 in bile acid conjugation in vivo and an unexpected function in body weight homeostasis, which will require further analysis. FATP5 deletion mice provide a new model to study the intersection of bile acid metabolism, lipid metabolism, and body weight regulation. Background & Aims: Fatty Acid Transport Protein 5 (FATP5) is a liver-specific member of the FATP/Slc27 family, which has been shown to exhibit both fatty acid transport and bile acid-CoA ligase activity in vitro. Here, we investigate its role in bile acid metabolism and body weight homeostasis in vivo by using a novel FATP5 knockout mouse model. Methods: Bile acid composition was analyzed by mass spectroscopy. Body weight, food intake, energy expenditure, and fat absorption were determined in animals fed either a low- or a high-fat diet. Results: Although total bile acid concentrations were unchanged in bile, liver, urine, and feces of FATP5 knockout mice, the majority of gallbladder bile acids was unconjugated, and only a small percentage was conjugated. Primary, but not secondary, bile acids were detected among the remaining conjugated forms in FATP5 deletion mice, suggesting a specific requirement for FATP5 in reconjugation of bile acids during the enterohepatic recirculation. Fat absorption in FATP5 deletion mice was largely normal, and only a small increase in fecal fat was observed on a high-fat diet. Despite normal fat absorption, FATP5 deletion mice failed to gain weight on a high-fat diet because of both decreased food intake and increased energy expenditure. Conclusions: Our findings reveal an important role for FATP5 in bile acid conjugation in vivo and an unexpected function in body weight homeostasis, which will require further analysis. FATP5 deletion mice provide a new model to study the intersection of bile acid metabolism, lipid metabolism, and body weight regulation. See CME Quiz on page 1349. See CME Quiz on page 1349. Fatty acid transport protein 5 (FATP5) (Slc27a5/VLACSR/VLCS-H2) is a liver-specific member of the fatty acid transport protein family.1Hirsch D. Stahl A. Lodish H.F. A family of fatty acid transporters conserved from mycobacterium to man.Proc Natl Acad Sci U S A. 1998; 95: 8625-8629Crossref PubMed Scopus (378) Google Scholar, 2Berger J. Truppe C. Neumann H. Forss-Petter S. A novel relative of the very-long-chain acyl-CoA synthetase and fatty acid transporter protein genes with a distinct expression pattern.Biochem Biophys Res Commun. 1998; 247: 255-260Crossref PubMed Scopus (42) Google Scholar, 3Steinberg S.J. Wang S.J. McGuinness M.C. Watkins P.A. Pevsner J. Human liver-specific very-long-chain acyl-coenzyme A synthetase cDNA cloning and characterization of a second enzymatically active protein.Mol Genet Metab. 1999; 68: 32-42Abstract Full Text PDF PubMed Scopus (48) Google Scholar Similar to other fatty acid transport protein family members, FATP5 mediates the uptake of long-chain fatty acids (LCFAs) when overexpressed in cultured mammalian cells.1Hirsch D. Stahl A. Lodish H.F. A family of fatty acid transporters conserved from mycobacterium to man.Proc Natl Acad Sci U S A. 1998; 95: 8625-8629Crossref PubMed Scopus (378) Google Scholar, 4Doege H. Baillie R.A. Ortegon A.M. Tsang B. Wu Q. Punreddy S. Hirsch D. Gimeno R.E. Stahl A. Targeted deletion of FATP5 reveals multiple functions in liver metabolism alterations in hepatic lipid homeostasis.Gastroenterology. 2006; 130: 1245-1258Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar We recently generated FATP5 knockout mice and showed that primary hepatocytes derived from these mice have a significant reduction in LCFA uptake.4Doege H. Baillie R.A. Ortegon A.M. Tsang B. Wu Q. Punreddy S. Hirsch D. Gimeno R.E. Stahl A. Targeted deletion of FATP5 reveals multiple functions in liver metabolism alterations in hepatic lipid homeostasis.Gastroenterology. 2006; 130: 1245-1258Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar We also found that FATP5 knockout mice have decreased intrahepatic triglyceride levels and show a decreased hepatic fatty acid uptake in vivo with redistribution of lipids from the liver to other LCFA metabolizing organs.4Doege H. Baillie R.A. Ortegon A.M. Tsang B. Wu Q. Punreddy S. Hirsch D. Gimeno R.E. Stahl A. Targeted deletion of FATP5 reveals multiple functions in liver metabolism alterations in hepatic lipid homeostasis.Gastroenterology. 2006; 130: 1245-1258Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar These data are consistent with an important role for FATP5 in hepatic fatty acid uptake in vivo. Beside its role in fatty acid uptake, FATP5 has also been suggested to function in bile acid metabolism as a bile acid-CoA ligase (BAL). Purified rat liver BAL was found to be identical to the rat ortholog of FATP5.5Falany C.N. Xie X. Wheeler J.B. Wang J. Smith M. He D. Barnes S. Molecular cloning and expression of rat liver bile acid CoA ligase.J Lipid Res. 2002; 43: 2062-2071Crossref PubMed Scopus (28) Google Scholar Recombinant rat FATP5, expressed in insect cells, shows bile acid-CoA ligase activity with kinetic properties similar to rat liver BAL.5Falany C.N. Xie X. Wheeler J.B. Wang J. Smith M. He D. Barnes S. Molecular cloning and expression of rat liver bile acid CoA ligase.J Lipid Res. 2002; 43: 2062-2071Crossref PubMed Scopus (28) Google Scholar Furthermore, human FATP5, overexpressed in mammalian cells, confers choloyl-CoA ligase activity.6Steinberg S.J. Mihalik S.J. Kim D.G. Cuebas D.A. Watkins P.A. Wang S.J. McGuinness M.C. Pevsner J. The human liver-specific homolog of very long-chain acyl-CoA synthetase is cholate CoA ligase.J Biol Chem. 2000; 275: 15605-15608Crossref PubMed Scopus (55) Google Scholar, 7Mihalik S.J. Steinberg S.J. Pei Z. Park J. Kim do G. Heinzer A.K. Dacremont G. Wanders R.J. Cuebas D.A. Smith K.D. Watkins P.A. Lu J.F. Braiterman L.T. Kim D.G. Wang S.J. McGuinness M.C. Pevsner J. Participation of two members of the very long-chain acyl-CoA synthetase family in bile acid synthesis and recycling.J Biol Chem. 2002; 277: 24771-24779Crossref PubMed Scopus (93) Google Scholar These data suggest that FATP5 may indeed be a bile acid–CoA ligase. By using our recently generated FATP5 knockout mice,4Doege H. Baillie R.A. Ortegon A.M. Tsang B. Wu Q. Punreddy S. Hirsch D. Gimeno R.E. Stahl A. Targeted deletion of FATP5 reveals multiple functions in liver metabolism alterations in hepatic lipid homeostasis.Gastroenterology. 2006; 130: 1245-1258Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar we decided to examine whether FATP5 is the major bile acid–CoA ligase in vivo and, if so, what the physiological consequences of its deletion are. Figure 1 shows a simplified diagram of bile acid metabolism.8Russell D.W. The enzymes, regulation, and genetics of bile acid synthesis.Annu Rev Biochem. 2003; 72: 137-174Crossref PubMed Scopus (1515) Google Scholar, 9Chiang J.Y. Regulation of bile acid synthesis pathways, nuclear receptors, and mechanisms.J Hepatol. 2004; 40: 539-551Abstract Full Text Full Text PDF PubMed Scopus (407) Google Scholar, 10Hofmann A.F. Chemistry and enterohepatic circulation of bile acids.Hepatology. 1984; 4: 4S-14SCrossref PubMed Scopus (132) Google Scholar The starting point for de novo synthesis of bile acids is acetyl-CoA, which is converted to cholesterol by cholesterol biosynthetic enzymes. Cholesterol is converted to bile acids through the sequential action of bile acid biosynthetic enzymes. Important intermediates in bile acid synthesis are C27 precursors of bile acids, such as 3α,7α,12α-trihydroxy-5β-cholestanoic acid (THCA) and the related molecule 7α,12α-dihydroxy-5β-cholestanoic acid (DHCA). These precursors are activated by addition of CoA to form THCA-CoA and DHCA-CoA, which are subsequently shortened to form the primary bile acids choloyl-CoA and chenodeoxycholoyl-CoA. FATP5 and a related FATP, FATP2, have THCA-CoA ligase activity in vitro and could therefore be involved in de novo bile acid synthesis.7Mihalik S.J. Steinberg S.J. Pei Z. Park J. Kim do G. Heinzer A.K. Dacremont G. Wanders R.J. Cuebas D.A. Smith K.D. Watkins P.A. Lu J.F. Braiterman L.T. Kim D.G. Wang S.J. McGuinness M.C. Pevsner J. Participation of two members of the very long-chain acyl-CoA synthetase family in bile acid synthesis and recycling.J Biol Chem. 2002; 277: 24771-24779Crossref PubMed Scopus (93) Google Scholar CoA derivatives of mature bile acids are conjugated to amino acids, such as taurine, by the bile acid–CoA:amino acid N-acyltransferase (BAT) before secretion into the bile. Bile acids are efficiently reabsorbed from the distal small intestine and reenter the liver to be resecreted into bile, a process that has been termed the enterohepatic recirculation. The recirculation of bile acids is very efficient: under normal conditions, only 5% of bile acids in gall bladder bile is derived directly from de novo synthesis, whereas 95% have been recirculated through the gut at least once.10Hofmann A.F. Chemistry and enterohepatic circulation of bile acids.Hepatology. 1984; 4: 4S-14SCrossref PubMed Scopus (132) Google Scholar While in the gut, a portion of bile acids is deconjugated and/or dehydroxylated through the action of intestinal bacteria resulting in a mixture of conjugated and unconjugated primary and secondary bile acids, which are then reabsorbed.8Russell D.W. The enzymes, regulation, and genetics of bile acid synthesis.Annu Rev Biochem. 2003; 72: 137-174Crossref PubMed Scopus (1515) Google Scholar Purified BAL activates both primary and secondary bile acids5Falany C.N. Xie X. Wheeler J.B. Wang J. Smith M. He D. Barnes S. Molecular cloning and expression of rat liver bile acid CoA ligase.J Lipid Res. 2002; 43: 2062-2071Crossref PubMed Scopus (28) Google Scholar, 11Vessey D.A. Benfatto A.M. Kempner E.S. Bile acid: CoASH ligases from guinea pig and porcine liver microsomes. Purification and characterization.J Biol Chem. 1987; 262: 5360-5365Abstract Full Text PDF PubMed Google Scholar, 12Simion F.A. Fleischer B. Fleischer S. Subcellular distribution of cholic acid:coenzyme a ligase and deoxycholic acid coenzyme a ligase activities in rat liver.Biochemistry. 1983; 22: 5029-5034Crossref PubMed Scopus (20) Google Scholar and is thus able to efficiently reconjugate all bile acid species reentering the liver. What would be the consequences of BAL deletion? It would be expected that conjugated bile acids are synthesized normally via the de novo biosynthetic pathway. However, any bile acid which is deconjugated in the small intestine could not be reconjugated. Over time, because the majority of bile acids in the gall bladder bile have gone through the enterohepatic circulation multiple times, unconjugated bile acids would accumulate. The remaining conjugated bile acids would be mostly primary bile acids, and secondary bile acids would be mostly unconjugated. The physiological consequences of this bile acid conjugation defect might be expected to include hepatic cholestasis and fat malabsorption. In humans, a complete defect in bile acid conjugation, caused by mutation in BAT, leads to increased serum bile acid levels and fat malabsorption.13Carlton V.E. Harris B.Z. Puffenberger E.G. Batta A.K. Knisely A.S. Robinson D.L. Strauss K.A. Shneider B.L. Lim W.A. Salen G. Morton D.H. Bull L.N. Complex inheritance of familial hypercholanemia with associated mutations in TJP2 and BAAT.Nat Genet. 2003; 34: 91-96Crossref PubMed Scopus (267) Google Scholar Because unconjugated bile acids are transported poorly by the bile salt export pump (BSEP),14Gerloff T. Stieger B. Hagenbuch B. Madon J. Landmann L. Roth J. Hofmann A.F. Meier P.J. The sister of P-glycoprotein represents the canalicular bile salt export pump of mammalian liver.J Biol Chem. 1998; 273: 10046-10050Crossref PubMed Scopus (836) Google Scholar BAL deletion has also been predicted to result in a phenotype similar to disruption of BSEP, which in mice causes intrahepatic cholestasis and growth retardation because of nutrient malabsorption.15Wang R. Salem M. Yousef I.M. Tuchweber B. Lam P. Childs S.J. Helgason C.D. Ackerley C. Phillips M.J. Ling V. Targeted inactivation of sister of P-glycoprotein gene (spgp) in mice results in nonprogressive but persistent intrahepatic cholestasis.Proc Natl Acad Sci U S A. 2001; 98: 2011-2016Crossref PubMed Scopus (288) Google Scholar In addition to BAL activity, FATP5 also has TCHA-CoA ligase activity in vitro.7Mihalik S.J. Steinberg S.J. Pei Z. Park J. Kim do G. Heinzer A.K. Dacremont G. Wanders R.J. Cuebas D.A. Smith K.D. Watkins P.A. Lu J.F. Braiterman L.T. Kim D.G. Wang S.J. McGuinness M.C. Pevsner J. Participation of two members of the very long-chain acyl-CoA synthetase family in bile acid synthesis and recycling.J Biol Chem. 2002; 277: 24771-24779Crossref PubMed Scopus (93) Google Scholar If FATP5 is required for activation of DHCA/THCA, one would expect a complete absence of conjugated bile acids, an absence or reduction of mature C24 bile acids, and accumulation of THCA/DHCA. No defects have been described that specifically disrupt DHCA/THCA activation; however, genetic defects in several other bile acid biosynthetic enzymes result in intrahepatic cholestasis, jaundice, and fat malabsorption.16Bove K.E. Heubi J.E. Balistreri W.F. Setchell K.D. Bile acid synthetic defects and liver disease a comprehensive review.Pediatr Dev Pathol. 2004; 7: 315-334Crossref PubMed Scopus (110) Google Scholar Here we use FATP5 knockout mice to show that FATP5 is required for bile acid reconjugation but not de novo synthesis. Despite a reversal of the ratio of unconjugated to conjugated bile acids, we found no evidence of hypercholonemia or overt fat malabsorption. Interestingly, however, FATP5 knockout mice failed to gain weight on a high-fat diet because of decreased food intake and increased energy expenditure. Although the exact mechanism of this unexpected role for FATP5 in body weight regulation remains to be determined, our data together with the data by Doege et al4Doege H. Baillie R.A. Ortegon A.M. Tsang B. Wu Q. Punreddy S. Hirsch D. Gimeno R.E. Stahl A. Targeted deletion of FATP5 reveals multiple functions in liver metabolism alterations in hepatic lipid homeostasis.Gastroenterology. 2006; 130: 1245-1258Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar clearly show that FATP5 functions in both lipid and bile acid metabolism. The generation of FATP5 deletion mice has been described.4Doege H. Baillie R.A. Ortegon A.M. Tsang B. Wu Q. Punreddy S. Hirsch D. Gimeno R.E. Stahl A. Targeted deletion of FATP5 reveals multiple functions in liver metabolism alterations in hepatic lipid homeostasis.Gastroenterology. 2006; 130: 1245-1258Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar Mice for phenotyping were obtained by brother-sister matings of F2 animals. Genotypes were determined by polymerase chain reaction. Mice were fed a low-fat (Standard chow or Research Diets D12450, New Brunswick, NJ) or high-fat diet (Research Diets D12330 and D12492). All experiments were performed on individually housed male animals. Fat mass was determined by dual-energy x-ray absorptiometry (DEXA) by using a PIXImus mouse densitometer (Lunar Corp, Madison, WI). For glucose-tolerance tests, animals were fasted overnight, 2 g/kg glucose was administered IP, and blood glucose levels were determined by using a glucometer (Bayer, Elkhard, IN). For tissue and plasma collection, mice were fasted overnight, glucose was measured by using a glucometer, and animals were euthanized by CO2 asphyxiation. O2 consumption and CO2 production were measured in metabolic chambers (Columbus Instruments Inc, Columbus, OH). Mice either had free access to food (first 24 hours) or no access to food (second 24 hours). The respiratory exchange ratio (RER) was calculated as CO2 produced/O2 consumed. Activity was measured by using the SmartFrame Activity System (Hamilton/Kinder, Poway, CA) consisting of 12 PC-interfaced horizontal frames fitted with photobeams (8LX4W) spaced 5.5 cm apart. Photobeam breaks (basic movement) were recorded for 70 hours and analyzed in 60-minute blocks by using Motor Monitor Software (Hamilton/Kinder). Statistical significance was determined by using 2-way repeated-measures analysis of variance with genotype and time as factors. Rectal temperature was recorded by using a rectal probe (Thermalert TH-5; PhysiTemp, Clifton, NJ). Animals and procedures were approved by the Millennium and the Palo Alto Medical Foundation Research Institute Institutional Animal Care and Use Committees. Bile was collected from gallbladders of ad libitum–fed animals and diluted 1:500 in 50% acetonitrile in water. Ten microliters of the diluent was injected onto an Agilent 1100 capillary HPLC system fitted with a Luna Phenyl-Hexyl 3-μ column (30 × 1 mm; Phenomenex, Torrance, CA) and a single quadrapole mass spectrometer (G1946D SL; Agilent Technologies, Palo Alto, CA). A gradient from 10% to 70% acetonitrile was run at a flow rate of 50 μL/min over 45 minutes (aqueous phase was 10 mmol/L ammonium acetate pH 9.0). Bile acids and bile acid conjugates were monitored in single ion monitoring mode at their respective mass-to-charge ratios. Taurine-conjugated and unconjugated forms of cholate, chenodeoxycholate, α- and β-muricholate, lithocholate, hyodeoxycholate, and hyocholate (all from Steraloids, Newport, RI) were used as standards. Calibration curves for standards were included in all experiments and were linear. The run-to-run error for individual samples was <5%. Liver microsomes were prepared as described.17Wheeler J.B. Shaw D.R. Barnes S. Purification and characterization of a rat liver bile acid coenzyme A ligase from rat liver microsomes.Arch Biochem Biophys. 1997; 348: 15-24Crossref PubMed Scopus (19) Google Scholar Cholate-CoA ligase activity was determined radiochemically.18Kelley M. Vessey D.A. Determination of the mechanism of reaction for bile acid CoA ligase.Biochem J. 1994; 304: 945-949Crossref PubMed Scopus (19) Google Scholar Briefly, 40 μg protein was incubated in reaction buffer (100 mmol/L Tris, pH 7.5, 10 mmol/L ATP, 5 mmol/L MgCl, 0.2 mmol/L CoA, 20 μmol/L 14C-cholic acid [American Radiochemical Co., St. Louis, MO]) for 40 minutes at 37°C. EDTA and succinic acid was added, and tubes were extracted twice with water-saturated butanol. To determine fecal fat, feces was dried at 70°C for 30 minutes and then extracted with 1:3.3:4 vol of H2O:chloroform:methanol for 1 hour. Organic material was dried, weighed, and assigned as total lipid. To determine fecal free fatty acids, the dried pellet was resuspended in 100% ethanol and free fatty acids were determined by using a diagnostic kit (Sigma, St. Louis, MO) with oleate as a standard. Caloric content of feces was determined by using a Parr 1425 calorimeter (Parr Instrument Company, Moline, IL). Briefly, 60 mg dried feces was wrapped with platinum wire, sealed in a calorimetry bomb, and submerged in a water-filled chamber. The temperature difference in the water before and after combustion of the sample was recorded by using a Parr 1672 calorimeter thermometer and used to calculate the total fecal calories. Commercially available kits were used to determine insulin, leptin (both Crystal Chem, Downers Grove, IL), creatinine (BioAssay Systems, Hayward, CA), cholesterol, glycerol, triglycerides, free fatty acids, β-hydroxybutyrate, and total bile acids (all Sigma, St. Louis, MO). Bile acids and cholesterol were extracted from lyophilized liver homogenate or dried feces by incubating with 75% ethanol at 50°C (bile acids) or with 10 mmol/L potassium hydroxide:methanol (1:9) at 66°C (cholesterol) for 2 hours. Bile acid pool size was measured as described.19Schwarz 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 To determine hepatic lipids, 20 mg lyophilized liver homogenate was extracted with 1 mL chloroform:methanol (2:1) at 60°C, filtered over a 0.2-μmol/L nylon membrane spin column (Schleicher & Schuell, Keene, NH), and re-extracted twice with chloroform:methanol. The organic phase was dried, weighed, and assigned as total lipid. Liver glycogen was determined by digesting liver homogenate with amyloglucosidase and then assaying the amount of glucose in the sample by using a diagnostic kit (Sigma). Gene-profiling data were generated by using the MOE430A chip according to the manufacturer's recommendations (Affymetrix, Santa Clara, CA). Arrays were scanned on an Affymetrix GeneArray scanner and analyzed by using the MAS 5.0 software (Affymetrix). Taqman real-time quantitative PCR was performed by using an ABI Prism 7700 sequence detector (Applied Biosystems, Foster City, CA) with 18S as a control. Gene-specific primers and probes were designed by using the Primer Express software (Applied Biosystems). RNA was digested with DNase A and transcribed to cDNA by using a mixture of random hexamer and oligo dT primers. Relative expression was determined by the Ct method (Applied Biosystems). Average and standard error of the mean are shown. Unless otherwise indicated, statistical significance was determined by using a Student t test. Total bile acid concentrations in gall bladder bile, feces, liver, serum, and urine of FATP5 deletion mice were similar to controls (Table 1). Furthermore, bile acid pool size was similar in FATP5 deletion and wild-type control mice (Table 1). To determine bile acid composition, we used mass spectroscopy coupled to liquid chromatography (Figure 2). In the bile of wild-type mice, taurine-conjugated trihydroxylated (TriOH) bile acids were most abundant (81% of total: tauro-β-muricholic acid 40%, taurocholic acid 32%, and tauro-α-muricholic acid 9%; Figure 2 D). Taurine-conjugated dihydroxylated (DiOH) bile acids (taurohyodeoxycholic acid, taurochenodeoxycholic acid, and taurodeoxycholic acid) together constituted 14% of total bile acids (Figure 2 B), whereas 5% of bile acids was unconjugated cholic acid (Figure 2 C). We were unable to detect lithocholic acid, glycine-conjugated, or glucuronidated bile acids in our samples, and the levels of sulfated bile acids detected were too low to quantitate (data not shown). In summary, in wild-type mice, 95% of the bile acids in gallbladder bile were conjugated and only 5% were unconjugated (Figure 2 H). In contrast, in FATP5 knockout mice, 83% of bile acids were unconjugated, whereas only 17% were conjugated (Figure 2 H). The total amount of mature C24 bile acids was similar in FATP5 knockout and wild-type mice (Figure 2 H) and no masses corresponding to C27 precursors were detected in either background (not shown). The majority of bile acids in FATP5 deletion mice were unconjugated trihydroxylated bile acids (65% of total, cholic acid 26%, and multiple species migrating similar to α- and β-muricholic acid 39%, Figure 2 C and G), whereas taurine-conjugated trihydroxylated bile acids accounted for only 14% of bile acids (taurocholic acid 4%, tauro-α-muricholic 4%, and tauro-β-muricholic 4%; Figure 2 D). This reversal in the abundance of unconjugated versus conjugated bile acid species strongly supports a requirement for FATP5 in bile acid conjugation.Table 1Bile Acid Concentrations in FATP5 Knockout MiceBile Acids in−/−+/+P ValueLiver (μmol/g)0.70 ± 0.060.73 ± 0.01NSFeces (μmol/g)2.59 ± 0.252.22 ± 0.19NSGallbladder (μmol/dL)34.43 ± 1.7031.94 ± 2.27NSSerum (μmol/dL)1.78 ± 0.371.95 ± 0.32NSUrine (nmol/mg creatinine)30.93 ± 1.3133.65 ± 1.55NSBile acid pool (μmol/g tissue)5.8 ± 1.315.6 ± 0.89NSNOTE. Feces and liver were from mice on a high-fat diet for 11 and 20 weeks, respectively. Gallbladder bile, serum, urine, and tissues for determination of bile acid pool size were from 6-month-old mice on a normal diet. n = 3– 5.NS, not significant. Open table in a new tab NOTE. Feces and liver were from mice on a high-fat diet for 11 and 20 weeks, respectively. Gallbladder bile, serum, urine, and tissues for determination of bile acid pool size were from 6-month-old mice on a normal diet. n = 3– 5. NS, not significant. The remaining conjugated bile acids in FATP5 deletion mice could represent newly synthesized bile acids that have not yet undergone deconjugation; alternatively, they could be bile acids that were reconjugated by a bile acid-CoA ligase other than FATP5. To distinguish between these 2 possibilities, we focused our analysis on dihydroxylated taurine-conjugated bile acids (Figure 2 B). Two bile acids in this group, taurohyodeoxycholate and taurodeoxycholate (peak 1 and 3), cannot be produced by de novo synthesis but are generated from primary bile acids in the small intestine. A third bile acid, taurochenodeoxycholate (peak 2), is produced by de novo bile acid synthesis. Interestingly, conjugated forms of the 2 secondary bile acids are completely absent in the bile of FATP5 knockout mice (Figure 2 B), suggesting an overall requirement for FATP5 in the generation of these bile acids. In contrast, the conjugated primary bile acid, taurochenodeoxycholate, is present in similar concentrations in FATP5 knockout and wild-type animals (Figure 2 B). These data support the notion that de novo synthesis of bile acids in FATP5 deletion mice is largely intact and that the increase in unconjugated bile acids in FATP5 knockout mice is because of a defect in bile acid reconjugation. Surprisingly, unconjugated secondary dihydroxylated bile acids were not increased in FATP5 deletion mice (Figure 2 A). It is possible that unconjugated bile acids are rehydroxylated more readily than conjugated bile acids or are taken up less efficiently by the liver. Interestingly, FATP5 deletion mice had significant amounts of conjugated (2%) and unconjugated (13%) tetrahydroxylated bile acids (Figure 2 E and F). Tetrahydroxylated (TetraOH) bile acids are not present in normal bile but are found in the bile of BSEP knockout mice15Wang R. Salem M. Yousef I.M. Tuchweber B. Lam P. Childs S.J. Helgason C.D. Ackerley C. Phillips M.J. Ling V. Targeted inactivation of sister of P-glycoprotein gene (spgp) in mice results in nonprogressive but persistent intrahepatic cholestasis.Proc Natl Acad Sci U S A. 2001; 98: 2011-2016Crossref PubMed Scopus (288) Google Scholar and in the urine of humans with cholestasis.20Nemeth A. Strandvik B. Urinary excretion of tetrahydroxylated bile acids in children with alpha 1-antitrypsin deficiency and neonatal cholestasis.Scand J Clin Lab Invest. 1984; 44: 387-392Crossref PubMed Scopus (16) Google Scholar The increase in tetrahydroxylated bile acids in FATP5 knockout mice may allow these mice to more efficiently export unconjugated bile acids, resulting in normal levels of intrahepatic bile acids. In summary, our data show that bile acid reconjugation is a major function of FATP5, whereas de novo synthesis of bile acids appears to be intact. To verify the role of FATP5 in bile acid reconjugation, we measured BAL activity in microsomes from wild-type and knockout livers by using 14C-cholic acid as a substrate. BAL activity was significantly decreased in knockout compared with wild-type microsomes (3.4 ± 0.68 pmol/min/mg vs. 37.7 ± 19.5 pmol/min/mg), consistent with FATP5 being the major bile acid–CoA ligase. FATP5 deletion mice survived to adulthood at mendelian ratios, and their body weight and length were similar to controls at 8 weeks of age (data not shown), suggesting normal fat absorption during postnatal development. To examine fat absorption during adulthood, we determined fecal fat and caloric content after feeding a high-fat diet. FATP5 deletion mice showed a subtle increase in the percentage of fat in the feces (Figure 3 A); however, the magnitude of this increase was very small (1.14-fold). Fecal caloric content and fecal free fatty acids were not significantly altered in FATP5 deletion mice (Figure 3 B and C). The amount of cholesterol in the feces of wild-type and FATP5 knockout mice was also similar (10.04 ± 1.32 mg/g and 9.89 ± 1.03 mg/g), and similar amounts of stool were produced by wild-type and knockout mice (data not shown). Thus, although FATP5 deletion mice show a very subtle increase in fecal fat, their fat absorption appears to be largely normal even when high-fat feeding. Although FATP5 deletion mice appeared indistinguishable from controls on a chow d
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