Cd36 knockout mice are protected against lithogenic diet-induced gallstones
2017; Elsevier BV; Volume: 58; Issue: 8 Linguagem: Inglês
10.1194/jlr.m077479
ISSN1539-7262
AutoresYan Xie, Vincenza Cifarelli, Terri Pietka, Elizabeth P. Newberry, Susan Kennedy, Amin Khalifeh‐Soltani, Robin D. Clugston, Kamran Atabai, Nada A. Abumrad, Nicholas Davidson,
Tópico(s)Cholesterol and Lipid Metabolism
ResumoThe scavenger receptor and multiligand transporter CD36 functions to promote cellular free fatty acid uptake and regulates aspects of both hepatic and intestinal cholesterol metabolism. However, the role of CD36 in regulating canalicular and biliary cholesterol transport and secretion is unknown. Here, we show that germline Cd36 knockout (KO) mice are protected against lithogenic diet (LD)-induced gallstones compared with congenic (C57BL6/J) controls. Cd36 KO mice crossed into congenic L-Fabp KO mice (DKO mice) demonstrated protection against LD-induced gallstones, reversing the susceptibility phenotype observed in L-Fabp KO mice. DKO mice demonstrated reduced biliary cholesterol secretion and a shift into more hydrophophilic bile acid species, without changes in either BA pool size or fecal excretion. In addition, we found that the mean and maximum force of gallbladder contraction was increased in germline Cd36 KO mice, and gallbladder lipid content was reduced compared with wild-type controls. Finally, whereas germline Cd36 KO mice were protected against LD-induced gallstones, neither liver- nor intestine-specific Cd36 KO mice were protected. Taken together, our findings show that CD36 plays an important role in modifying gallstone susceptibility in mice, at least in part by altering biliary lipid composition, but also by promoting gallbladder contractility. The scavenger receptor and multiligand transporter CD36 functions to promote cellular free fatty acid uptake and regulates aspects of both hepatic and intestinal cholesterol metabolism. However, the role of CD36 in regulating canalicular and biliary cholesterol transport and secretion is unknown. Here, we show that germline Cd36 knockout (KO) mice are protected against lithogenic diet (LD)-induced gallstones compared with congenic (C57BL6/J) controls. Cd36 KO mice crossed into congenic L-Fabp KO mice (DKO mice) demonstrated protection against LD-induced gallstones, reversing the susceptibility phenotype observed in L-Fabp KO mice. DKO mice demonstrated reduced biliary cholesterol secretion and a shift into more hydrophophilic bile acid species, without changes in either BA pool size or fecal excretion. In addition, we found that the mean and maximum force of gallbladder contraction was increased in germline Cd36 KO mice, and gallbladder lipid content was reduced compared with wild-type controls. Finally, whereas germline Cd36 KO mice were protected against LD-induced gallstones, neither liver- nor intestine-specific Cd36 KO mice were protected. Taken together, our findings show that CD36 plays an important role in modifying gallstone susceptibility in mice, at least in part by altering biliary lipid composition, but also by promoting gallbladder contractility. Gallstone disease and its comorbidities continue to represent a healthcare and economic challenge in the developed world (1.Peery A.F. Dellon E.S. Lund J. Crockett S.D. McGowan C.E. Bulsiewicz W.J. Gangarosa L.M. Thiny M.T. Stizenberg K. Morgan D.R. et al.Burden of gastrointestinal disease in the United States: 2012 update.Gastroenterology. 2012; 143Abstract Full Text Full Text PDF PubMed Scopus (1414) Google Scholar). Our understanding of the pathogenesis of cholesterol gallstone disease includes both genetic and environmental risk factors, events that converge on pathways that regulate hepatic cholesterol metabolism and canalicular lipid secretion (2.Di Ciaula A. Wang D.Q. Garruti G. Wang H.H. Grattagliano I. de Bari O. Portincasa P. 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Evidence that the adenosine triphosphate-binding cassette G5/G8-independent pathway plays a determinant role in cholesterol gallstone formation in mice.Hepatology. 2016; 64: 853-864Crossref PubMed Scopus (19) Google Scholar). Other studies, using inbred mouse strains, have led to the identification of quantitative trait loci and corresponding positional candidate genes that predispose to lithogenic diet (LD)-induced gallstones (6.Lyons 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, 7.Lyons 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). Studies examining environmental and genetic risk factors suggest that several distinct metabolic pathways modify gallstone susceptibility. These pathways include those regulated by nuclear hormone receptors involved in sterol and bile acid sensing (8.Moschetta A. Bookout A.L. Mangelsdorf D.J. Prevention of cholesterol gallstone disease by FXR agonists in a mouse model.Nat. Med. 2004; 10: 1352-1358Crossref PubMed Scopus (251) Google Scholar, 9.Cheng S. Zou M. Liu Q. Kuang J. Shen J. Pu S. Chen L. Li H. Wu T. Li R. et al.Activation of constitutive androstane receptor prevents cholesterol gallstone formation.Am. J. Pathol. 2017; 187: 808-818Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar), as well as pathways more broadly involved in hepatic lipid utilization and metabolic channeling. For example, mice with germline deletion of L-Fabp, while protected against diet-induced hepatic steatosis (10.Newberry 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 (173) Google Scholar, 11.Newberry E.P. Kennedy S. Xie Y. Luo J. Jiang H. Ory D.S. Davidson N.O. Phenotypic divergence in two lines of L-Fabp−/− mice reflects substrain differences and environmental modifiers.Am. J. Physiol. Gastrointest. Liver Physiol. 2015; 309: G648-G661Crossref PubMed Scopus (14) Google Scholar) demonstrated increased LD-induced gallstone formation, at least in part through augmented canalicular cholesterol secretion (12.Xie Y. Newberry E.P. Kennedy S.M. Luo J. Davidson N.O. Increased susceptibility to diet-induced gallstones in liver fatty acid binding protein knockout mice.J. Lipid Res. 2009; 50: 977-987Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Similarly, germline deletion of Elovl6 attenuated hepatic steatosis in Ldlr KO mice, yet enhanced gallstone susceptibility (13.Kuba M. Matsuzaka T. Matsumori R. Saito R. Kaga N. Taka H. Ikehata K. Okada N. Kikuchi T. Ohno H. et al.Absence of Elovl6 attenuates steatohepatitis but promotes gallstone formation in a lithogenic diet-fed Ldlr(−/−) mouse model.Sci. Rep. 2015; 5: 17604Crossref PubMed Scopus (18) Google Scholar). Here, we asked whether the multiligand class B scavenger receptor CD36 might play a role in modifying hepatic cholesterol utilization and export. This possibility was in part predicated on findings that mouse hepatic CD36 expression is increased in response to high-fat diet-induced obesity and other findings that adenoviral-mediated hepatic CD36 overexpression increased hepatic FFA uptake and steatosis (14.Koonen D.P. Jacobs R.L. Febbraio M. Young M.E. Soltys C.L. Ong H. Vance D.E. Dyck J.R. Increased hepatic CD36 expression contributes to dyslipidemia associated with diet-induced obesity.Diabetes. 2007; 56: 2863-2871Crossref PubMed Scopus (342) Google Scholar). In addition, CD36 has been shown to regulate cholesterol uptake, utilization, and secretion from both the small intestine (15.Nauli A.M. Nassir F. Zheng S. Yang Q. Lo C.M. Vonlehmden S.B. Lee D. Jandacek R.J. Abumrad N.A. Tso P. CD36 is important for chylomicron formation and secretion and may mediate cholesterol uptake in the proximal intestine.Gastroenterology. 2006; 131: 1197-1207Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 16.Nassir F. Wilson B. Han X. Gross R.W. Abumrad N.A. CD36 is important for fatty acid and cholesterol uptake by the proximal but not distal intestine.J. Biol. Chem. 2007; 282: 19493-19501Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar) and mouse peritoneal macrophages (17.Yu M. Jiang M. Chen Y. Zhang S. Zhang W. Yang X. Li X. Li Y. Duan S. Han J. et al.Inhibition of macrophage CD36 expression and cellular oxidized low density lipoprotein (oxLDL) accumulation by tamoxifen: a peroxisome proliferator-activated receptor (PPAR)gamma-dependent mechanism.J. Biol. Chem. 2016; 291: 16977-16989Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Beyond the metabolic alterations alluded to above, there is a role for altered gallbladder motility in promoting gallstone susceptibility, evidenced by findings in both Cck KO mice and in Cckr1 KO mice (18.Wang D.Q. Schmitz F. Kopin A.S. Carey M.C. Targeted disruption of the murine cholecystokinin-1 receptor promotes intestinal cholesterol absorption and susceptibility to cholesterol cholelithiasis.J. Clin. Invest. 2004; 114: 521-528Crossref PubMed Scopus (82) Google Scholar, 19.Wang H.H. Liu M. Portincasa P. Tso P. Wang D.Q. Lack of endogenous cholecystokinin promotes cholelithogenesis in mice.Neurogastroenterol. Motil. 2016; 28: 364-375Crossref PubMed Scopus (17) Google Scholar). These findings, coupled with findings that CD36-dependent signaling promotes secretin and cholecystokinin (CCK) release from enteroendocrine cells (20.Sundaresan S. Shahid R. Riehl T.E. Chandra R. Nassir F. Stenson W.F. Liddle R.A. Abumrad N.A. CD36-dependent signaling mediates fatty acid-induced gut release of secretin and cholecystokinin.FASEB J. 2013; 27: 1191-1202Crossref PubMed Scopus (75) Google Scholar), raised the question of whether CD36 might function more broadly in modifying gallstone susceptibility, specifically via alterations in gallbladder contractility. Here, we show that germline Cd36 KO mice are protected against LD-induced gallstones via mechanisms including alterations in biliary cholesterol secretion, bile acid composition, and enhanced gallbladder contractility. All animals were maintained in a C57BL6/J background and housed on a 12 h light-dark cycle, in a full-barrier facility. Ten- to 12-week-old male mice were fed either standard rodent chow (PicoLab Rodent Diet 20, fat 4.5%, cholesterol 0.015%) or an LD (Research Diet no. 960393, fat 18.8%, cholesterol 1.2%, and cholic acid 0.5%) for 2–4 weeks, as indicated. L-Fabp−/− mice (12.Xie Y. Newberry E.P. Kennedy S.M. Luo J. Davidson N.O. Increased susceptibility to diet-induced gallstones in liver fatty acid binding protein knockout mice.J. Lipid Res. 2009; 50: 977-987Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar) and Cd36−/− mice (21.Febbraio M. Abumrad N.A. Hajjar D.P. Sharma K. Cheng W. Pearce S.F. Silverstein R.L. A null mutation in murine CD36 reveals an important role in fatty acid and lipoprotein metabolism.J. Biol. Chem. 1999; 274: 19055-19062Abstract Full Text Full Text PDF PubMed Scopus (655) Google Scholar) were used in crosses to generate a line of compound Cd36, L-Fabp double KO mice (hereafter referred to as DKO). Cd36 conditional KO mice were generated from Cd36flox/flox mice (22.Cifarelli V. Ivanov S. Xie Y. Son N.H. Saunders B.T. Pietka T.A. Shew T.M. Yoshino J. Sundaresan S. Davidson N.O. et al.CD36 deficiency impairs the small intestinal barrier and induces subclinical inflammation in mice.Cell. Mol. Gastroenterol. Hepatol. 2017; 3: 82-98Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar) crossed into either Albumin-Cre or Villin-Cre lines (Jax) to generate the compound line of tissue-specific KO (either liver- or intestine-specific) mice. Age-matched and cohoused littermates were used as controls. All animal protocols were approved by the Washington University Animal Studies Committee and followed guidelines outlined by the National Institutes of Health. Animals were euthanized after a 4 h fast. Liver and serum were collected and frozen at −80°C until analyzed. Serum triglyceride and cholesterol were measured by using reagents from Wako (Neuss, Germany), an L-Type Triglyceride M Kit (catalog nos. 465-09791 and 461-09891), and the cholesterol E kit (catalog no. 439-17501). Hepatic or, where indicated, gallbladder lipids were extracted by using chloroform: methanol (2:1) and triglyceride, cholesterol, free cholesterol, free fatty acids, and phospholipids enzymatically with the indicated Wako reagent kits: the L-Type Triglyceride M Kit, the cholesterol E kit, the free cholesterol E kit (catalog no. 435-35801), the HR series NRFA-HR2 kit (catalog nos. 991-34891 and 995-34791), and the phospholipid C kit (catalog no. 433-36201). Animals were fed a LD for 4 weeks as indicated in the relevant figure legends and euthanized after a 4 h fast. Fresh gallbladder bile was immediately analyzed by polarizing microscopy using a visual scale with the following criteria: 0, absence of cholesterol monohydrate crystals (ChMCs), 1, small number of ChMCs (<3 per high power field); 2, many ChMCs (≥3 per high power field); 3 = aggregated ChMCs; and 4 = presence of "sandy" light-translucent stones or "solid" light-opaque stones (7.Lyons 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). Gallbladder bile cholesterol content was measured enzymatically after chloroform:methanol (2:1) extraction. Gallbladder bile phospholipids and bile acid content were analyzed enzymatically by using the total bile acids assay kit (BQ 092A-EALD, BQ Kits, San Diego, CA). Cholesterol saturation indices in gallbladder bile were calculated by using published parameters (23.Carey 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). Mice were fed a LD for 2 weeks and anesthetized after a 4 h fast. An external bile fistula was established surgically, and bile samples collected during the first 15 min were discarded. Bile samples were collected for another 60 min and used for biliary lipid secretion analysis. Hepatic bile volume was determined gravimetrically assuming a density of 1 g·ml−1. Biliary phospholipids, cholesterol, and total bile acid content were determined enzymatically. Biliary lipid secretion values are presented as nmol/min/kg body weight (bw). Individual bile acid species were determined by using HPLC as previously described (12.Xie Y. Newberry E.P. Kennedy S.M. Luo J. Davidson N.O. Increased susceptibility to diet-induced gallstones in liver fatty acid binding protein knockout mice.J. Lipid Res. 2009; 50: 977-987Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar) and with commercially obtained standards. The hydrophobicity index was calculated by using published parameters (24.Heuman 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). Bile acid pool size and fecal bile acids excretion were determined in groups of mice fed a LD for 2 weeks. For bile acid pool size, the liver, gallbladder, and intestine were homogenized together, and bile acid content was measured as previously detailed (12.Xie Y. Newberry E.P. Kennedy S.M. Luo J. Davidson N.O. Increased susceptibility to diet-induced gallstones in liver fatty acid binding protein knockout mice.J. Lipid Res. 2009; 50: 977-987Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). For fecal bile acid excretion, feces from individual animals were collected for up to 72 h. Feces were homogenized, and bile acid content was determined enzymatically. Hepatic total RNA or proximal intestinal RNA was extracted and cDNA was prepared by using an ABI high-capacity cDNA reverse transcription kit with 1 µg of total RNA. Real-time quantitative PCR used cDNAs from four to six animals per group and was performed in triplicate on an ABI Prism7000 sequence detection system using SYBR Green PCR Master Mix (Applied Biosystems) and primer pairs (provided on request) designed by Primer Express software (Applied Biosystems). Relative mRNA abundance is expressed as fold change to WT control after normalization to own GAPDH. To detect hepatic CD36 and L-Fabp protein, 50 mg of liver was homogenized in 0.5 ml of buffer containing 25 mM HEPES, 150 mM NaCL, 0.5 mM EDTA, 0.1% SDS, 1% Triton, and protease inhibitors, by using the Bullet BlenderR system (Next Advance) with 1 mm zirconium oxide beads. Aliquots of 50 µg of protein were separated by 4–20% SDS-PAGE gel. Samples of terminal ileum protein were used to prepare total and membrane extracts as described (12.Xie Y. Newberry E.P. Kennedy S.M. Luo J. Davidson N.O. Increased susceptibility to diet-induced gallstones in liver fatty acid binding protein knockout mice.J. Lipid Res. 2009; 50: 977-987Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 25.Dawson 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 (303) Google Scholar) by using the Bullet BlenderR system with 1 mm steel beads. In brief, approximately 50–100 mg of full-thickness ileum was homogenized in 0.8 ml of lysis buffer (250 mM sucrose and 10 mM Triethanolamine-HCL, pH 7.6) containing protease inhibitors (Complete protease inhibitor mixture, Roche Diagnostics). The crude preparation was centrifuged at 2,500 g for 10 min, and the supernatant was recentrifuged at 30,000 g for 60 min at 4°C by using a Beckman MLA-130 rotor. The resulting membrane pellet was resuspended in solubilization buffer [15% SDS, 8 M Urea, 10% (w/v) sucrose, 62.5 mM Tris-HCL, and 5 mM dithiothreitol, pH 6.8]. Membrane proteins (50 µg) were separated on 10% SDS-PAGE gel, transferred to Immobilon®-P membranes (Millipore, Billerica, MA), and immunoblotted with 1:2,000 goat anti-mouse CD36 (16.Nassir F. Wilson B. Han X. Gross R.W. Abumrad N.A. CD36 is important for fatty acid and cholesterol uptake by the proximal but not distal intestine.J. Biol. Chem. 2007; 282: 19493-19501Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar), 1:2,000 rabbit anti-mouse L-Fabp (26.Newberry 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 (230) Google Scholar), 1:300 rabbit anti-mouse Fgf15, and 1:1,000 monoclonal rabbit anti-mouse Asbt antibody (generous gifts from Dr. Paul A Dawson), and quantitated by Kodak Image software. Gallbladders were taken from age- and sex-matched (male) chow-fed WT and Cd36−/− mice. Both surfaces of the excised gallbladder were gently cut and washed with PBS and attached to a force transducer (Radnoti 8-chamber tissue-organ bath system) exactly as described (27.Tharp K.M. Khalifeh-Soltani A. Park H.M. Yurek D.A. Falcon A. Wong L. Feng R. Atabai K. Stahl A. Prevention of gallbladder hypomotility via FATP2 inhibition protects from lithogenic diet-induced cholelithiasis.Am. J. Physiol. Gastrointest. Liver Physiol. 2016; 310: G855-G864Crossref PubMed Scopus (11) Google Scholar). Gallbladders were immersed in warmed, oxygenated buffer for 30 min before the addition of the indicated concentrations of methacholine and KCl. Resting tension of 0.5 g was applied after each treatment as previously described (28.Kudo M. Khalifeh Soltani S.M. Sakuma S.A. McKleroy W. Lee T.H. Woodruff P.G. Lee J.W. Huang K. Chen C. Arjomandi M. et al.Mfge8 suppresses airway hyperresponsiveness in asthma by regulating smooth muscle contraction.Proc. Natl. Acad. Sci. USA. 2013; 110: 660-665Crossref PubMed Scopus (47) Google Scholar). Statistical significance was determined with one-way ANOVA for multiple group comparisons and t-testing as a post hoc test for comparing different pairs using GraphPad Prism 7 software (GraphPad, San Diego, CA). Data are expressed as the mean ± SE unless otherwise noted. Our first objective was to ascertain a role for CD36 in LD-induced gallstone susceptibility. Mice of the indicated genotype (Fig. 1A) were studied after consuming a LD for 4 weeks, with the data showing that Cd36−/− mice (Fig. 1A) exhibited a striking reduction in gallstone incidence with correspondingly reduced gallstone score and reduced cholesterol saturation index (Fig. 1B). Furthermore, when crossed into germline L-Fabp−/− mice, both Cd36−/−and the compound DKO mice (Fig. 1A, B) were protected, as evidenced by the visually clear appearance of the gallbladder, decreased cholesterol saturation index, and reduced gallbladder volume (Fig. 1B). We observed increased abundance of L-Fabp protein in livers from LD-fed Cd36 KO mice compared with WT controls (Fig. 1A), an observation whose underlying mechanisms remain to be elucidated. However, these findings point to a role of CD36 as a modifier of murine gallstone susceptibility in mice either WT or null for the L-Fabp allele. Hepatic lipid content across the genotypes in LD-fed mice indicated no major differences in total or free cholesterol content, but revealed increased bile acid content in both Cd36−/− and DKO mice (Table 1). We next examined the concentration and relative distribution of the most abundant individual bile acid species from newly secreted bile samples in each genotype. These data revealed increased biliary concentration of taurobetamuricholic acid in LD-fed Cd36−/− mice (Fig. 2A) with a shift in relative taurobetamuricholic acid distribution favoring enrichment in both Cd36−/− and DKO mice (Fig. 2B). The net impact of these relatively subtle, albeit significant, differences in bile acid species includes a significant decrease in hydrophobicity index (24.Heuman 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) in both Cd36−/− and DKO mice (Fig. 2C). Daily fecal bile acid excretion (Fig. 2D) and total bile acid pool sizes (Fig. 2E) were comparable across the genotypes, suggesting that there were no major shifts in enterohepatic cycling.TABLE 1Hepatic lipid content in LD-fed mice of the indicated genotypeGenotypeTCFCCEPLFFABAWT (6)159.8 ± 22.927.5 ± 1.3132.3 ± 22.985.6 ± 2.486.6 ± 5.6a21.8 ± 9.1aCd36−/− (6)159.8 ± 10.431.0 ± 4.8128.8 ± 10.992.5 ± 5.085.0 ± 4.9a27.1 ± 15.1aL-Fabp−/− (5)147.5 ± 11.624.0 ± 3.7123.6 ± 12.395.8 ± 4.680.7 ± 9.1a19.6 ± 4.1aDKO (6)159.4 ± 24.932.4 ± 1.9127.0 ± 26.793.9 ± 2.7132.9 ± 10.9b33.2 ± 11.2aMice from four experimental groups (n per group) were fed a LD for 4 weeks. Hepatic lipids were extracted and analyzed (Materials and Methods). Hepatic lipid content is expressed as mean ± SE (µg/mg protein), except FFA (nmol/mg protein). The difference between values associated with different superscript letters for the parameters indicated in each vertical column is statistically significant (P < 0.05). BA, bile acid; CE, cholesteryl ester; FC, free cholesterol; FFA, free fatty acid; PL, phospholipid; TC, total cholesterol. Open table in a new tab Mice from four experimental groups (n per group) were fed a LD for 4 weeks. Hepatic lipids were extracted and analyzed (Materials and Methods). Hepatic lipid content is expressed as mean ± SE (µg/mg protein), except FFA (nmol/mg protein). The difference between values associated with different superscript letters for the parameters indicated in each vertical column is statistically significant (P < 0.05). BA, bile acid; CE, cholesteryl ester; FC, free cholesterol; FFA, free fatty acid; PL, phospholipid; TC, total cholesterol. We further examined biliary lipid secretion rates in the various genotypes, using a shorter (2 week) LD feeding interval. This interval was selected to circumvent the inevitable bile duct obstruction that accompanied the extensive gallstone formation in both WT and L-Fabp−/− mice noted above. Those findings revealed increased biliary cholesterol secretion in L-Fabp−/− mice compared with both WT and Cd36−/− mice (Table 2). There was a trend to decreased biliary cholesterol secretion in Cd36−/− mice (175 nmol/min/kg bw) compared with WT controls (210 nmol/min/kg bw) and in DKO mice (222 nmol/min/kg bw) compared with L-Fabp−/− mice (298 nmol/min/kg bw), without changes in bile flow rate by genotype (Table 2). Taken together, these findings suggest that altered biliary bile acid composition and attenuated cholesterol secretion in LD-fed Cd36−/− mice may in combination contribute to the protection observed against gallstone formation.TABLE 2Biliary lipid secretion in LD-fed mice of the indicated genotypeGenotypeTCPLBAFlow rateWT (12)210.5 ± 6.9a646.7 ± 14.9a5103.8 ± 233.7a91.6 ± 1.2Cd36−/− (7)175.1 ± 8.6a603.3 ± 24.1a4827.9 ± 302.6a89.3 ± 1.7L-Fabp−/− (9)298.3 ± 11.9b985.6 ± 39.9b6907.5 ± 426.3b115.9 ± 4.5DKO (4)221.8 ± 8.8ab819.7 ± 31.9a3900.1 ± 589.3a83.2 ± 0.7Mice from four experimental groups (n per group) were fed a LD for 2 weeks. Bile samples were collected after surgical laparotomy for 1 h. Biliary cholesterol (TC), phospholipids (PLs), and bile acids (BAs) were analyzed (Materials and Methods). Biliary lipid secretion is expressed as nmol/min/kg bw. Flow rate is expressed as µl/min/kg bw. The difference between values associated with different superscript letters for the parameters indicated in each vertical column is statistically significant (P < 0.05). Open table in a new tab Mice from four experimental groups (n per group) were fed a LD for 2 weeks. Bile samples were collected after surgical laparotomy for 1 h. Biliary cholesterol (TC), phospholipids (PLs), and bile acids (BAs) were analyzed (Materials and Methods). Biliary lipid secretion is expressed as nmol/min/kg bw. Flow rate is expressed as µl/min/kg bw. The difference between values associated with different superscript letters for the parameters indicated in each vertical column is statistically significant (P < 0.05). We next sought to understand the mechanisms underlying the alterations in biliary lipid secretion noted above by profiling the expression of several mRNAs involved in hepatic cholesterol and bile acid homeostasis. We observed decreased expression of hepatic Cyp7a1 in L-Fabp−/− mice as previously demonstrated (12.Xie Y. Newberry E.P. Kennedy S.M. Luo J. Davidson N.O. Increased susceptibility to diet-induced gallstones in liver fatty acid binding protein knockout mice.J. Lipid Res. 2009; 50: 977-987Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar) and also in both genotypes of Cd36−/− mice, which was accompanied by increased Cyp8b1 expression in LD-fed Cd36−/− and DKO mice (Fig. 3A). The increased expression of hepatic Cyp8b1 mRNA in Cd36−/− and DKO mice was not accompanied by increased biliary taurocholate concentration, as might be predicted (29.Chiang J.Y. Bile acids: regulation of synthesis.J. Lipid Res. 2009; 50: 1955-1966Abstract Full Text Full Text PDF PubMed Scopus (1095) Google Scholar, 30.Kim I. Ahn S.H. Inagaki T. Choi M. Ito S. Guo G.L. Kliewer S.A. Gonzalez F.J. Differential regulation of bile acid homeostasis by the farnesoid X receptor in liver and intestine.J. Lipid Res. 2007; 48: 2664-2672Abstract Full Text Full Text PDF PubMed Scopus (414) Google Scholar), but it bears emphasis that those bile acid distribution data (Fig. 2A and above) reflect mice fed supplemental cholic acid as part of the LD regimen. We also observed decreased expression of HMGR mRNA in Cd36−/− and DKO mice, findings suggesting that de novo hepatic cholesterol production may be decreased, a possibility consistent with the decreased secretion of biliary cholesterol noted above, as well as earlier findings suggesting decreased de novo lipogenesis in Cd36−/− mice (31.Clugston R.D. Yuen J.J. Hu Y. Abumrad N.A. Berk P.D. Goldberg I.J. Blaner W.S. Huang L.S. CD36-deficient mice are resistant to alcohol- and high-carbohydrate-induced hepatic steatosis.J. Lipid Res. 2014; 55: 239-246Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). We next turned to an examination of a panel of intestinal genes involved in cholesterol and bile acid metabolism, in part guided by previous findings of regionally specific adaptive alterations in their expression in germline Cd36−/− mice (16.Nassir F. Wilson B. Han X. Gross R.W. Abumrad N.A. CD36 is important for fatty acid and cholesterol uptake by the proximal but not distal intestine.J. Biol. Chem. 2007; 282: 19493-19501Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). Our findings revea
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